Pharmacology, Toxicology and Biomedical Reports (PTB Reports) Editorial Offi ce : Editor-in-Chief...

PTB Reports, Vol 1, Issue 1, Jan-Jun, 2015

Pharmacology, Toxicology and Biomedical Reports (PTB Reports)

PTB Reports A multifaceted peer review journal in the field of Pharmacology, Toxicology and Biology

Dr. R. Raveendran,

Department of Pharmacology,

JIPMER, Puducherry, India

Dr.Bahubali. D. Gane,

Assistant Professor in Pediatrics,

Sri Venkateshwara Medical College,

Pondicherry, India

Dr. B. Balaji,

Department of Pharmacology,

PSG College of Pharmacy,

Coimbatore. India.

Dr. S.E. Damodaran,

Medical Services at Boehringer-Ingel-heim India Pvt. Ltd.,

Chennai, India

Dr. Indulekhaa CL Pillai,

Cardiovascular Research laboratory,

University of California(UCLA),

Los Angeles, USA.

Dr. Kesavan

Department of Pharmacology.

JIPMER, Pondicherry. India.

Dr. K.S. Kesavanarayanan

Faculty of Pharmacy,

UniversitiTeknologi MARA (UiTM),

PuncakAlam Campus,

42300 Selangor DE, Malaysia.

Mrs N. Maithili,

Department of Biochemistry,

JIPMER, Pondicherry 605 006, India

Dr. Mainul Haque

Faculty of Medicine and Health Sciences

Universiti Sultan ZainalAbidin

Kampus Kota, Jalan Sultan Mahmud

20400 Kuala Terengganu, Terengganu, Malaysia.

Dr. Nandha kumar Subbiah,

Dept. of Anatomy & Medical Genetics,

Sri venkateshwaraa Medical College & Research Centre,

Ariyur, Puduchery-605102. India.

Mr. Sam Aaseer Thamby,

Faculty of Pharmacy, AIMST University,

Jalan Bedong - Semeling,

08100 Bedong, Kedah Darul Aman, Malaysia.

Dr. Subhankar Chakraborty,

Mayo Clinic, Division of Gastroenterol-ogy and Hepatology

200 First Street SW, Rochester, Minne-sota 55905, USA.

Dr. Sumanta Kumar Goswami

Hammock Lab,

Department of Entomology,

Briggs Hall, University of California,

Davis, California, USA

Dr S Suresh Kumar

Department of Pharmacology

Karuna Medical College

Vilayodi, Palakkad

Kerala 678103, India.

Dr. R. Suyambu Kesava Vijayan,

Center for Biophysics and Computa-tional Biology,

Dept of Chemistry, Temple University,

SERC, Room 718E,

1835 N. 12th Street,

Philadelphia, PA 19122, USA.

Dr. P. Ramalingam,

Faculty in Medicinal Chemistry,

College of Pharmacy,

Gulf Medical University, Ajman, UAE.

Dr. P. Venkatesh,

Scientist, Natreon Inc.

02-360, Rishitech Park, Action area 1D,

Newtown, Kolkata - 700156. India.

Dr. C. Vijaya,

Professor of Pharmaceutics,

Ultra College of Pharmacy,

Madurai, Tamilnadu. India.

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Dr. D. Krishna Kumar M.Pharm., Ph.D.,

Department of Pharmacy Practice and Doctor of Pharmacy,

C.L.BaidMetha College of Pharmacy,

Chennai.

Dr. A. Surendiran MBBS., MD.,

Assistant Professor of Pharmacology,

JIPMER, Pondicherry, India.

Assistant editorsChakradhara Rao Satyanarayana Uppugunduri, B.Pharm., M.Sc., PhD,

Laboratoire de la fondation CANSEARCH,

Department of Pediatrics (University Hospital of Geneva),

Batiament Tulipe; 64, Avenue De La Roseraie 1205, Geneve.

Dr. H.S. Arun Kumar,DVM, DBA, MVSC, PhD, MEAPCI, MESC.,

Department of Medicine (Cardiovascular Sciences),

Director-Stemcology Dublin, Ireland.

Dr. Melvin George, MBBS., MD., D.M.,

Cardiac Clinical Trials & Research, Dept. of Cardiology,

SRM Medical College Hospital& Research Center, Kattankulatur – 603 203, Kancheepuram (Dt) India.

EDITORIAL BOARD

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Table of Contents

Vol 1 Issue 1 , Jan-Apr 2015

EditorialThe unveiling of Pharmacology, Toxicology and Biomedical Reports (PTB Reports)Subramani ParasuramanDOI: 10.5530/PTB.1.1.1 1

View pointNeed for Extension of Real Time Stability Analysis of Drugs and Quality Control Setup in HospitalsRamalingam Peraman and Mukesh KumarDOI: 10.5530/PTB.1.1.2 2

Review ArticleThe Importance of Pharmacokinetic and Pharmaco dynamic Models in Sustained Release FormulationSelvadurai Muralidharan, Kalaimani Jayaraja KumarDOI: 10.5530/PTB.1.1.3 5

Research ArticleImpact of Microbial Culture Analysis and Prophylactic use of Antibiotics in Acute Severe PancreatitisJagadish babu Dasari, Cristiano Ialongo, Aruna Chandranath Kondakundhi, Sri Harsha Munnamgi, Syed JuiedDOI: 10.5530/PTB.1.1.4 10

Evaluation of Oral Hygiene Practice, Knowledge and Attitude among (10-15 Yrs) School Children in Dharan, Nepal- A Cross- Sectional StudyRoshana Poudyal, Priyata Agrawal, Ashish Shrestha, Mamta Dali, Tarakant Bhagat, Asish ChoudharyDOI: 10.5530/PTB.1.1.5 14

Retrospective Analysis of Musculioskeletal Complications in 10 Patients with TuberculosistHosseinian Amiri Aref, Abedi Siavash, Yazdanian MaryamDOI: 10.5530/PTB.1.1.6 17

Potential Anti-Anxiety Effect of Mucuna pruriens in Experimental Model of Swiss Albino MiceAnjula Sachan, Sarvesh Kumar, Hemant Singh, Pratap Shankar, Dheeraj Kumar, Amod Kumar Sachan, Rakesh Kumar DixitDOI: 10.5530/PTB.1.1.7 2020Influence on Quality of Life Among Patients with HypothyroidismSireesha Paruchuri, Ganesh Pandian Balasubramanian, Parashuram NagulaDOI: 10.5530/PTB.1.1.8 24

Efficacy and tolerability of two intravaginal formulations containing clindamycin plus clotrimazole in women with vaginal infections: A pilot studyDaswani Bharti Ramchand, Naik Shilpa, Palewar Meghana, Ghongane Balasaheb, Sambarey Pradeep Bharadwaj RenuDOI: 10.5530/PTB.1.1.9 27

PTB Reports, Vol 1, Issue 1, Jan-Apr, 2015 i

Case ReportAnti-Snake Venom (ASV) Induced Hypotension: An Emergent Complication of Treatment Yerramalli Roja Ramani, Bandana Rath, Uma Shankar Mishra, Himanshu Bhusan SahuDOI: 10.5530/PTB.1.1.10 35

ii PTB Reports, Vol 1, Issue 1, Jan-Apr, 2015

Contents | Contd...... |

PTB Reports, Vol 1, Issue 1, Jan-Apr, 2015 1


Editorial

The unveiling of Pharmacology, toxicology and Biomedical Reports (PTB Reports)

Dear Professionals colleagues, Scientist and fellow researcher,

It’s my pleasure to introduce the first issue of ‘Pharmacology, Toxicol-ogy and Biomedical Reports’ (Pharmacol Toxicol Biomed Rep/ PTB reports) - an official journal of SciBioMed.org.

PTB reports is an international, peer-reviewed, open access, online journal that publishes research articles, review articles, clinical case reports and recent trends in experimental and clinical pharmacology; toxicology and Biomedicine. PTB reports cover research areas perti-nent to clinical pharmacokinetics, biochemical pharmacology, clinical biochemistry, molecular biology, analytical toxicology, psychophar-macology, neuropharmacology, cardiovascular and renal pharmacol-ogy, and other systemic pharmacology. The official website is http://www.ptbreports.org and Web-based manuscript submission system is available at http://emanuscript.in/submissions/index.php/ptbre-ports/login.

Under the leadership of eminent researchers/ professors from the global scientific community, SciBioMed.org contributes much to scientific field through the publication of research works in various disciplines for more than five years. The main objective of introduc-ing PTB Reports to the scientific society is to provide a rapid publi-cation platform to the researchers for their scientific research with international standards. PTB reports will consider study findings as results to be published without publication and editor bias. Hence, we encourage author’s submission with either positive or negative findings. The submissions will be accessed by one of the editors/ edi-torial board members of the journal, two potential reviewers and the final decision will be provided 30-45 days after initial submission.

Since costs of publication are increasing every year, part of the pub-lication cost (article processing charges) will be collected from the author, taking into consideration other co-charges. Upon receipt of the article processing charges, the provisionally accepted article will be available online in the ‘In Press’ article section. The accepted arti-cles (articles which are in ‘in press’ section) will be published in one of the upcoming PTB issues. Three issues of PTB will be published per year viz., Jan–Apr, May–Aug and Sep–Dec.

PTB reports has a structured editorial board, with experienced and young dynamic editors to initiate rapid review process, and provide better publication service to authors. We believe that 2014 will a vibrant, protective and creative year for those committed to inte-grated research and we expect PTB reports to be the portal that keeps you abreast of cutting edge information in pharmacological, toxicological and biochemical sciences. We welcome your submis-sions, correspondence and suggestions. Members of the editorial team and myself will be happy to discuss the progress and vision of the journal for the future.

Thank you

With regards

Dr. Subramani Parasuraman,Editor-In-Chief

Pharmacology, toxicology and Biomedical Reports Email: [email protected]

DOI: 10.5530/PTB.1.1.1

2 PTB Reports, Vol 1, Issue 1, Jan-Apr, 2015

View pointPTB Reports A multifaceted peer review journal in the field of Pharmacology, Toxicology and Biology

Need for Extension of Real Time Stability Analysis of Drugs and Quality Control Setup in HospitalsPeraman Ramalingam1* and Mukesh Kumar2

1Department of Medicinal and Phytochemistry, College of Pharmacy, Gulf Medical University, Ajman, UAE2Department of Pharmacy practice, College of Pharmacy, Gulf Medical University, Ajman, UAE

About Author : Dr. Peraman RamalingamDepartment of Medicinal and Phytochemistry,College of Pharmacy, Gulf Medical University, Ajman, UAE.E-mail: [email protected]

DOI : 10.5530/PTB.1.1.2

ABSTRACT

Owing to the increased number issues related to medication adherence, efficacy, antimicrobial resistance, tolerance and adverse reaction with unknown ideology, few strategic approaches having been recommended and implemented in treatment procedures. Despite of these clinical oriented strategies, the success rate in eradication of many unwanted therapeutic issues quite questionable. It is believed that the existing clinical and therapeutic oriented issues pertaining the drug, it requires additional strategic implementations that nullify the probability of drug related factors. This strategy can be a drug quality control testing Centre at the hospital premises to ensure the potency and real time stabilityassessment for stability sensitive and dose sensitive drugs. Initially it may be recommended in those hospitals, which conduct clinical research, therapeutic drug monitoring, clinical evaluation of antibiotics and further it can be extrapolated based on outcome. This view invites discussion to bring a fruitful outcome rather than criticism.

Key words: Drug stability, Drugs, Hospitals, Real time testings, Stability sensitive, Quality control.

INTRODUCTIONPotency and stability of drug products or dosage forms are quality attributes to be considered as important factors that influencesthe drug potency related research outcome. In recent years, drug related issues like tolerance, resistance, adverse reactions for existing drugs are continuously increasing.1 But those research outcomes are purely focused on either patient oriented or treatment oriented. It is known that potency, stability and impurity levels would have significant impact on the study outcome. These factors vary with time, storage condi-tion adopted, batch, and manufacturer. In current practice, formula-tions employed in drug administration are based on label and expiry date assigned by the industry. In fact, the potency of the dosage form remains to the therapeutic requirement, only when appropriate stor-age conditions areadopted. After the implementation of ICH guidelines, the impurity levels and stability considerationsare the most important control system for quality pharmaceuticals, which directly relates the efficacy and potency in the treatment.2

Even in the presence of stability testing protocols in industry, many pharmaceutical products and batches have been banned and recalled in recent years. It implies that stability problems and potency deviations have been occurred at the recommended storage conditions itself. In

this juncture, it is well known that many hospitals, inpatient ward; phar-macies maintain their drug products under fluctuated environments. This could trigger the stability issues in product and reduce the expected potency of medication. Apart from noncompliance of proper storage conditions, the potency variation between new products and early expiry products will have statistical significance in patient response.

This concept has been brought into discussion, exclusively for those products which are considered as degradation susceptible dosage form such as injection, infusion, dry powders etc. and for medicament which are belonging to chemical classeslike lactum, lactone, amides, ester, (which can be considered as stability sensitive drugs along with photo-sensitive drugs. Though health careprofessionals have adequate knowl-edge on stability and potency of medication, still assurance on potency and stability for those medications before introducing in clinical research always recommended for significant output.

In this context, there was a report based on the need for stability by the hospital pharmacists3 and also a report based on stability evalua-tion of anticancer drugs in hospitals.4-7 Based on these reports, it is inter-preted that such studies are highly important to understand the impact of drugs potency and stability in therapeutic responses. To support the above cited view, the increased observations on ‘not of quality standard (NQS) reports for drug samples by drugs control departments and mag-istrate issues can also be considered.8 There were many issues on prod-uct recall by pharmaceutical companies due to stability problems too9

The need and extension of real time stability assessment protocolDespite of these stability testing procedure storage and recommenda-tions outlined by the pharmaceutical company as a compliance to reg-


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ulatory guidelines for a particular dosage forms, only the real storage conditions employed at hospital and pharmacy sectors can determines the dug potency and safety. In connection with efficacy and safety requirement, it is also a very important concern.

The search for stability related work for drugs at hospital sectors reveled very few, relatively when compared to stability studies at industry as a part of research. But it is the need for real time analy-sis, by random stability study for the drug, which is stored at hospital after the transportation. Though international conference on har-monization (ICH) has considered the different climatic conditions, while implementing guidelines for stability (ICH Q1), still there is a need for real time analysis of drug after adequate exposure of drugs during transportation and storage at different conditions.

The necessity for real time stability investigation, by sampling from the market may be recommended for those drugs, which are,

• Highly susceptible to temperature• Highly susceptible to hydrolysis in presence of moisture• Highly susceptible to light and undergo photo-oxidation• Producing, highly toxic impurity on storage• Possessing narrow therapeutic window• Responsible for drug resistance in chemotherapy

Table 1: Examples of Few drugs which are highly susceptible to degradation to be considered in Real timestability assessment

Class of drugs Category Expected degradationBeta-lactum antibiotics Penicillin and cephalosporin’s Antibacterial antibiotic Expected to hydrolyze, and produce amino

and their carboxylic acids

Procaine, cinchocaine Local anesthetics Susceptible to hydrolysis and forms corresponding acids

Ergometrine Used in labour Hydrolysis to lysergic acid derivatives

Glutithimide, ethosuccinamide anticonvulsants Hydrolysis of imide bond

Benzodiazepine Anticonvulsants Hydrolyze due to moisture at low pH

Fluroquinolones(ciprofloxacin) Antimicrobials Photo-oxidation

Dihydropyridine(Nifedipine), Timolol, Pindolol, Propranolol Antihypertensive Photo-oxidation

Benzyl penicillin, Streptomycin Antibacterial antibiotic Photo-oxidation

Catecholamine (Adrenaline, Nor adrenaline, isoprenaline) Adrenergic agonists Photo-oxidation to adrenochrome

derivative

Troglitazone Antidiabetic Photo-oxidation

Morphine Opioids Photo-oxidation to dimericpsudomorphine

Tetracycline Antibacterial Photooxidized to quinones

Chloramphenicol Antibacterial Photooxidized to p-nitrobenzaldehyde

Dihydropyridine(Nifedipineetc) Antihypertensive Photo-oxidize to aromatic pyridine

Norethisterone Hormones Photo-oxidize to epoxides

Promethazine, Prochlorperazine, chlorpromazine Antipsychotics Photo-oxidize tosulphoxides

Vitamin D Vitamin Photo-oxidize tosuprasterols and toxisterols

Frusemide Diuretics Photohydrolysis tosalumine

Folic acid haemtonic Oxidative deamination to pteridinecarboxaldehyde and gluamates

Nitrazepam Sedative and hypnotics Photoreduction to dimer derivative

Hydrochlor thiazide Diuretics Photon induced Dechlorination and ring opening

Diazepam Anticonvulsant Photon induced Conversion to benzophenones

• Drug’s chemical structure with imide, amide, ester, lactum, lactone, alkoxy functional groups

• Pro-drugs• Conjugates• There are some examples from literature10 are listed in Table 1.

CONCLUSIONBased on the above note and observations in the current pharma-ceutical health care system, it is proposed to consider for discussion and trace the reality and reliability of real-time stability assessment instability protocol as anextension to the present stability protocols. Based on the outcome, it can also bea recommendation on mainte-nance quality control setup in clinical research oriented hospital set-up. This may nullify the drug related factors in efficacy issues, results in significant effect on quality health care system. This require lot more discussion to come out with fruitful outcome.

CONFILCTS OF INTERESTThe authors declare no conflicts of interest.


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rgREFERENCES1. Stuart BL. Factors impacting on the problem of antibiotic resistance. J Antimicrob Chemother

2002; 4(1): 25-30.2. ICH Harmonized tripartite guidelines, Stability testing of new drug substances and

products, Q1A (R2) guidelines; 1995.3. Per Hortvig H. Drug Stability. Eur J Hosp Pharm. 2012; 19(5): p 427. doi: 10. 1136 /

ejhpharm-2012-00016.74. Sabrina DW, Peter V, Tuong, VNT, Xiaolan D, Isabel S, Schepdael AV. Impact of

temperature exposure on stability of drugs in a real-world out-of-Hospital Setting. Ann Emer Med. 2013; 62(4): 380-7.

5. Bardin C. Guidelines for the practical stability studies of anticancer drugs. A European consensus conference. Ann Pharm Fr. 2011; 69(4): 221–31.

6. Salman D. Evaluation of the solubility and stability of ifosfamide in order to answer practice based challenge. www.bopawebsite.org/posters/view/evaluation-of-the-solubility-and-stability (accessed 9 January 2014).

7. Astier A, Pinguet F, Vigneron J. The practical stability of anticancer drugs: SFPO and ESOP recommendations.www.sfpo.com/IMG/pdf/Recommandation_ Stabilite _EJOP_2010_E3.pdf (accessed 9 January 2014).

8. Surber S, Yaniv A. Analysis of chemotherapy vial waste. J Oncol Pharm Pract. 2013; 17(1): 160.

9. Food drug administration. www.fda.gov/Safety/Recalls/EnforcementReports/ucm310739.htm

10. Bhatnagar S, Kumar A, Ameta SC. Photooxidation of Some Pharmaceutical Drugs by Singlet Molecular Oxygen. Asian J Phar Biol Res. 2011; 1(2): 210-7.



Review Article

The Importance of Pharmacokinetic and Pharmacodynamic Models in Sustained Release FormulationSelvadurai Muralidharan1*, Kalaimani Jaya Raja Kumar2

1Unit of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University, Semeling, 1800-Bedong, Malaysia.2Unit of Pharmaceutical Technology, Faculty of Pharmacy, AIMST University, Semeling, 1800-Bedong, Malaysia.

About Author : Dr. Selvadurai Muralidharan M.Pharm.,M.B.A.,Ph.D.Senior Lecturer, Unit of Pharmaceutical Chemistry,Faculty of Pharmacy,AIMST University, Semeling,Bedong Malaysia.E-mail: [email protected]

DOI : 10.5530/PTB.1.1.3

ABSTRACT

The main role of preclinical pharmacokinetics and pharmacodynamics in drug discovery and development is to optimize candidate selection for the target therapeutic area, taking into consideration the type of agent required, and to predict the dose and dosing regimen for initial clinical trials with due concern to the requirements for effective treatment in the target therapeutic area. In order for this approach to be successful, a clear understanding is required for both the pharmacological target and drug disposition (absorption, clearance and distribution) of new chemical entities. A fundamental tenet in linking the pharmacokinetic and pharmacodynamic phases is that free drug in the systemic circulation is in equilibrium with the receptors. In the pharmacokinetic phase, only free drug can be cleared and drug is reversibly bound to tissues and blood. The pharmacodynamic phase is further subdivided into the interaction with the drug receptor triggering post-receptor events, eventually leading to actual drug effect. In this phase, only free drug can exert pharmacological effect and the free concentration of drug in plasma is in direct equilibrium with the interstitial fluid bathing most cells, since the capillary wall contains sufficient aqueous pores to allow the rapid passage of relatively small molecules, regardless of physicochemistry. Most receptor targets are accessed extracellularly. Therefore, one can expect that all drugs regardless of physicochemistry will be in direct equilibrium, at these targets, with free drug in plasma.

Key words: Bioavailability, Drug discovery, Formulations, PK-PD models, Sustained release dosage form.

INTRODUCTIONThe cost of bringing a new drug to market is estimated to be between $800 million and $1 billion. Currently, there is a huge gap between the number of candidate drug compounds in testing and the ones that actually get approved. Less than 10% of drugs in phase I clinical trials make it to the approval phase. Two key reasons the drugs fail at late stages are a lack of understanding of the relationship between dose-concentration response and unanticipated safety events. Given this scenario, it is critical to have enabling tools that help pre-dict how a drug will perform in vivo and assist in the success of a clini-cal therapeutic candidate. Pharmacokinetics (PK) characterizes the absorption, distribution, metabolism, and elimination properties of a drug. Pharmacodynamics (PD) defines the physiological and biologi-cal response to the administered drug.

Pharmacokinetic-pharmacodynamic (PK-PD) modelingPK-PD modeling is a scientific tool to help developers for select-ing a rational dosage regimen for confirmatory clinical testing. PK/PD modeling can be executed using various approaches, such as direct versus indirect response models and parametric versus non-parametric models. PK/PD concepts can be applied to individual dose optimization. The limits of PK/PD approaches include the development of appropriate models, the validity of surrogate end-points, and the acceptance of these models in a regulatory envi-ronment. When a PK/PD model is employed, both the time course and variability in the effect versus time relationship can be pre-dicted for different dosage-regimen scenarios. In this way, the PK/PD models help developers to select a rational dosage regimen for confirmatory clinical testing. Failure to determine a safe and effective dosage regimen for use in pivotal clinical trials has been acknowledged as a frequent flaw encountered during the devel-opment of many drugs for humans. It establishes a mathematical and theoretical link between these two processes and helps bet-ter predict drug action. Integrated PK/PD modeling and computer assisted trial design via simulation are being incorporated into many drug development programs and are having a growing impact. PK/PD testing is typically performed at every stage of the drug


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development process. Because development is becoming increas-ingly complex, time consuming, and cost intensive, companies are looking to make better use of PK/PD data to eliminate flawed can-didates at the beginning and identify those with the best chance of clinical success. A major factor in the drug development process is time. Predictive modeling tools can provide invaluable information to better streamline the drug development process. Pharmaceutical companies traditionally perform sequential testing of drug candi-dates by screening and selecting the best performers at every phase of the clinical drug-development cycle. This can take as long as six to 10 years and cost several hundred million dollars. Hence, it is impera-tive for companies to adopt technologies that improve the quality of the drug development process and improve speed to market.

Traditional PK/PD modeling in drug development defines param-eters such as drug dose concentration, drug exposure effects, drug half-life, drug concentrations against time, and drug effects against time. “When used more broadly, quantitative techniques such as drug modeling, disease modeling, trial modeling, and market mod-eling can support the entire development process, which results in better decisions through explicit consideration of risk and better uti-lization of knowledge,”. However, implementing a PK/PD modeling approach can be challenging. “One has to invest time and resources up front in the drug development process. From a modeling stand-point, one needs to identify and build into the trial design frequent measures of clinical efficacy and/or toxicity as well as representative biomarkers for effective modeling”.

To date, methodologies available for PK-PD analysis barely suppose the use of powerful computing resources. Some of these algorithms are able to generate individual estimates of parameters based on population analysis and Bayesian forecasting. Notwithstanding, atten-tion must be paid to avoid over interpreted data from mathemati-cal models, so that reliability and clinical significance of estimated parameters will be valuable when underlying physiologic processes (disease, age, gender, etc.) are considered. PK-PD modeling is the mathematical description of the relationships between PK and PD. PK-PD modeling allows the estimation of PK-PD parameters and the prediction of these derived, clinically relevant parameters as well. PK-PD simulations allow the assessment of the descriptive param-eters as functions of dose and dose rate. These simulations can pro-vide the dose–response curve for onset, magnitude, and duration of effect. This information can be valuable in optimizing dose and dosing regimens.1 Currently, there is growing recognition of the importance of PK-PD studies in all phases of drug development.2–5 In preclinical studies, PK-PD is used to interpret toxicokinetics data and via physi-ological modeling and allometric scaling, it is also used to extrapolate results from animals to humans.6,7 During early clinical testing, PK-PD is used to aid in the interpretation of dose–response and escalation studies. In addition, there are several instances in which PK-PD model-ing has been used by regulatory agencies to recommend a dose and/or regimen not originally studied as part of the clinical program.8 As in the case of pharmacokinetics, methods to measure pharmacologic effects and bio-mathematical models had to be developed to characterize and evaluate pharmacodynamic processes.

Mathematical models can be considered as simplifications of a phe-nomenon described in terms of an algebraic or differential equation. In the case of PK-PD modeling, it is expected to not only describe, but also predict distinct situations, such as scaling between preclinical to clinical trials, multiple dosing schemes, or different routes of admin-istration.9 To choose the most appropriate PK-PD model, it is essen-tial to identify the significance of the biological processes involved in eliciting a drug-induced response. Eventually, PK processes, biophase distribution, drug–receptor interaction, signal transduction, and sec-ondary postreceptor events are factors altering the PD behavior of a drug. If that information is available—although only partially—it is possible to link PK and PD with actual physiologic support instead of only abstract numbers. Then, the model-building process involves fitting the available data and the consideration of possible biological differences that usually are translated into inter and intra variability. In the case of PD variability, it becomes important to identify the use-

ful predictor (covariates) of PD individuality to facilitate individually optimized pharmacotherapy. It is necessary, therefore, to establish very comprehensive patient profiles during the development of stud-ies. Moreover, the study populations must be representative of the target patient population with respect to age, gender, race, and envi-ronmental and pathophysiological characteristics. If these require-ments are absent, the relevance and usefulness of covariates may be questionable.10,11

Because of the multiple factors intervening in a PK-PD study, it then appears adequate to divide the modeling project into the follow-ing two basic blocks such as concerning the clinical or experimen-tal design by itself and the data analysis. Diverse models have been suggested to describe the PK-PD relationship depending upon the nature of drug administration scheme (single doses, multiple doses, long-term infusions, etc.) and the time dependency of PD param-eters. Thus, when the system is kinetically at steady state, the con-centrations of the active moiety at the active site are constant (after long-term infusions or multiple doses), relatively simple models are needed to characterize the PK-PD relationship. Otherwise, after single doses (nonsteady-state condition) and when time variant PD parameters are present, more complex models are needed to account for phenomena involved in the PK-PD relationship. Approaches such as disequilibrium between biophase and plasma compart-ment,12 appearance of active metabolites,13,14 indirect mechanisms of action,15,16 sensitization, and tolerance,17–19 have been proposed to explain the apparent dissociation between time courses of concen-tration and effect. Recently, the combination of powerful nonlinear, mixed effect regression models, statistically robust software tools, and the integration of pharmacokinetic-pharmacodynamic knowl-edge has permitted optimization of the decision process in therapeu-tic management. By incorporation of previous information into these systems, Bayesian forecasting certainly promises the more adequate individualized therapy for a particular patient.

Sustained release dosage formThe sustained release (SR) mode of drug administration has certain features that have an important impact on the magnitude of the pharmacologic response: (a) it minimizes fluctuation in blood drug concentrations (i.e. between peak and trough). However, due to the pronounced non-linear relationship between drug concentration and pharmacologic effect (i.e. pharmacodynamics) the impact of this property differs considerably as a function of the shape of the phar-macodynamic profile and the position of the specific range of con-centrations on the curve of this profile; (b) it produces a slow input rate which tends to minimize the body’s counteraction to the drug’s intervening effect on regulated physiological processes; and (c) it provides a continuous mode of drug administration. This important pharmacodynamic characteristic may produce, in certain cases, an opposite clinical effect than that attained by an intermittent (pulsa-tile) mode of administration of the same drug. For many drugs with non-concentration-dependent pharmacodynamics, the exposure time, rather than the AUC, is the relevant parameter and it can there-fore be optimized by SR preparations. The slow input function may minimize hysteresis in cases where the site of action is not in a rapid equilibrium with the blood circulation. The pharmacodynamics of the desired effect(s) and/or adverse effect(s) may also be influenced by the site of administration, especially in cases where the drug is delivered directly to its site of action. These factors demonstrate the important influence of the mode of administration on the pharma-cological and clinical outcomes. In addition, they highlight the need to include these pharmacodynamic considerations in all stages from drug development to the optimization of their clinical use.

TIME RELEASE TECHNOLOGYIt is also known as sustained-release (SR), sustained-action (SA), extended-release (ER, XR, or XL), time-release or timed-release, con-trolled-release (CR), modified release (MR), or continuous-release (CR) is a mechanism used in pill tablets or capsules to dissolve slowly


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indicate that the dissolution characteristics of the product are not dependent on the product strength, then dissolution profiles in one

medium are usually sufficient to support waivers of in vivo testing. Otherwise, dissolution data in three media (pH 1.2, 4.5, and 6.8) are recommended. We recommend that the f2 test be used to compare profiles from the different strengths of the product. An f2 value > 50 indicates a sufficiently similar dissolution profile such that further in vivo studies are not needed. For an f2 value < 50, further discussions with CDER review staff may help to determine whether an in vivo study is appropriate (21 CFR 320.22(d)(2)(ii)). The f2 approach is not suitable for rapidly dissolving drug products (e.g., > 85% dissolved in 15 minutes or less). For an ANDA, conducting an in vivo study on a strength that is not the highest may be appropriate for reasons of safety, subject to approval by the Division of Bioequivalence, Office of Generic Drugs, and provided that the following conditions are met

• Linear elimination kinetics has been shown over the therapeutic dose range.

• The higher strengths of the test and reference products are pro-portionally similar to their corresponding lower strength.

• Comparative dissolution testing on the higher strength of the test and reference products is submitted and found to be appro-priate.

b. NDAs and ANDAs: Postapproval

Information on the types of in vitro dissolution and in vivo BE stud-ies for immediate-release drug products approved as either NDAs or ANDAs in the presence of specified postapproval changes are pro-vided in an FDA guidance for industry entitled SUPAC-IR: Immediate Release Solid Oral Dosage Forms:

Scale-Up and Post-Approval Changes: Chemistry, Manufacturing, and Controls, In vitro Dissolution Testing, and In vivo Bioequivalence Documentation. For post approval changes, we recommend that the in vitro comparison be made between the prechange and post change products. In instances where dissolution profile comparisons are suggested, we also recommend an f2 test be used. An f2 value of > 50 suggests a sufficiently similar dissolution profile and no further in vivo studies are needed. When in vivo BE studies are called for, we recommend that the comparison be made for NDAs between the prechange and post change products, and for ANDAs between the post change and reference listed drug products.

D. Modified-Release Products

Modified-release products include delayed-release products and extended- (controlled) release products. As defined in the USP, delayed-release drug products are dosage forms that release the drugs at a time later than immediately after administration (i.e., these drug products exhibit a lag time in quantifiable plasma con-centrations). Typically, coatings (e.g., enteric coatings) are intended to delay the release of medication until the dosage form has passed through the acidic medium of the stomach. In vivo tests for delayed-release drug products are similar to those for extended release drug products. We recommend that in vitro dissolution tests for these products document that they are stable under acidic conditions and that they release the drug only in a neutral medium (e.g., pH 6.8). Extended-release drug products are dosage forms that allow a reduction in dosing frequency as compared to when the drug is present in an immediate-release dosage form. These drug products can be developed to reduce fluctuations in plasma concentrations. Extended-release products can be capsules, tablets, granules, pellets, and suspensions. If any part of a drug product includes an extended-release component, the following recommendations apply.

NDAs: BA and BE Studies

An NDA can be submitted for a previously unapproved new molecu-lar entity, new salt, new ester, prodrug, or other noncovalent deriva-tive of a previously approved new molecular entity formulated as a modified-release drug product. We recommend that the first mod-ified-release drug product for a previously approved immediate-release drug product be submitted as an NDA. We also recommend

and release a drug over time. The advantages of sustained-release tablets or capsules are that they can often be taken less frequently than instant-release formulations of the same drug, and that they keep steadier levels of the drug in the bloodstream.20

Today, most time-release drugs are formulated so that the active ingredient is embedded in a matrix of insoluble substance(s) (vari-ous: some acrylics, even chitin; these substances are often patented) such that the dissolving drug must find its way out through the holes in the matrix. Some drugs are enclosed in polymer-based tablets with a laser-drilled hole on one side and a porous membrane on the other side. Stomach acids push through the porous membrane, thereby pushing the drug out through the laser-drilled hole. In time, the entire drug dose releases into the system while the polymer con-tainer remains intact, to be later excreted through normal digestion.

In some SR formulations, the drug dissolves into the matrix, and the matrix physically swells to form a gel, allowing the drug to exit through the gel’s outer surface.

Micro-encapsulation is also regarded as a more complete technology to produce complex dissolution profiles. Through coating an active pharmaceutical ingredient around an inert core, and layering it with insoluble substances to form a microsphere you are able to obtain more consistent and replicable dissolution rates in a convenient for-mat you can mix and match with other instant release pharmaceuti-cal ingredients in to any two piece gelatin capsule.

There are certain considerations for the formation of sustained-release formulation

If the active compound has a long half-life (over 6 hours), it is sus-tained on its own.

If the pharmacological activity of the active compound is not related to its blood levels, time releasing has no purpose.

If the absorption of the active compound involves an active trans-port, the development of a time-release product may be problematic.

Finally, if the active compound has a short half-life, it would require a large amount to maintain a prolonged effective dose. In this case, a broad therapeutic window is necessary to avoid toxicity; otherwise, the risk is unwarranted and another mode of administration would be recommended.

Bioavailability Consideration for Immediate Release Tablets and sustained release TabletsFor product quality21 BA and BE studies, we recommend that where the focus is on release of the drug substance from the drug product into the systemic circulation, a single-dose, fasting study be per-formed. We also recommend that in vivo BE studies be accompa-nied by in vitro dissolution profiles on all strengths of each product. For ANDAs, we also recommend that the BE study be conducted between the test product and reference listed drug using the strength(s) specified in Approved Drug Products with Therapeu-tic Equivalence Evaluations (Orange Book). 2. Waivers of In vivo BE Studies (Biowaivers)

a. INDs, NDAs, and ANDAs: Preapproval When the drug product is in the same dosage form, but in a different strength, and is proportion-ally similar in its active and inactive ingredients to the strength on which BA or BE testing has been conducted, an in vivo BE demonstra-tion of one or more lower strengths can be waived based on dissolu-tion tests and an in vivo study on the highest strength. For an NDA, biowaivers of a higher strength will be determined to be appropriate based on (1) clinical safety and/or efficacy studies including data on the dose and the desirability of the higher strength, (2) linear elimina-tion kinetics over the therapeutic dose range, (3) the higher strength being proportionally similar to the lower strength, and (4) the same dissolution procedures being used for both strengths and similar dis-solution results obtained. We recommend that a dissolution profile be generated for all strengths. If an appropriate dissolution method has been established (see section III.D.), and the dissolution results


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that subsequent modified-release products that are pharmaceuti-cally equivalent and bioequivalent to the listed drug product

be submitted as ANDAs. BA requirements for the NDA of an extended-release product are listed in § 320.25(f). The purpose of an in vivo BA study for which a controlled release claim is made is to determine if all of the following conditions are met:

• The drug product meets the controlled-release claims made for it.

• The BA profile established for the drug product rules out the occurrence of any dose dumping.

• The drug product’s steady-state performance is equivalent to a currently marketed noncontrolled release or controlled-release drug product that contains the same active drug ingredient or therapeutic moiety and that is subject to an approved full NDA.

• The drug product’s formulation provides consistent pharmacoki-netic performance between individual dosage units. As noted in § 320.25(f)(2), “the reference material(s) for such a bioavailability study shall be chosen to permit an appropriate scientific evalua-tion of the controlled release claims made for the drug product,” such as: A solution or suspension of the active drug ingredient or therapeutic moiety

• A currently marketed non controlled-release drug product con-taining the same active drug ingredient or therapeutic moiety and administered according to the dosage recommendations in the labeling

• A currently marketed controlled-release drug product subject to an approved full NDA containing the same active drug ingredient or therapeutic moiety and administered according to the dosage recommendations in the labeling This guidance recommends that the following BA studies be conducted for an extended release drug product submitted as an NDA:

• A single-dose, fasting study on all strengths of tablets and cap-sules and highest

• strength of beaded capsules

• A single-dose, food-effect study on the highest strength

• A steady-state study on the highest strength BE studies are rec-ommended when substantial changes in the components or com-position and/or method of manufacture for an extended-release drug product occur between the marketed NDA dosage form and the clinical trial material.

ANDAs: BE Studies

For modified-release products submitted as ANDAs, the following studies are recommended: (1) a single-dose, nonreplicate, fasting study comparing the highest strength of the test and reference listed drug product and (2) a food-effect, nonreplicate study comparing the highest strength of the test and reference product (see section VI.A).

Because single-dose studies are considered more sensitive in addressing the primary question of BE (i.e., release of the drug sub-stance from the drug product into the systemic circulation), multi-ple-dose studies are generally not recommended, even in instances where nonlinear kinetics are present.

Waivers of In vivo BE Studies (Biowaivers): NDAs and ANDAs

a. Beaded Capsules — Lower Strength

We recommend that for modified-release beaded capsules where the strength differs only in the number of beads containing the active moiety, a single-dose, fasting BE study be carried out only on the highest strength, with waiver of in vivo studies for lower strengths based on dissolution profiles. A dissolution profile can be generated for each strength using the recommended dissolution method. The f2 test can be used to compare profiles from the different strengths of the product. An f2 value of > 50 can be used to confirm that further in vivo studies are not needed.

b. Tablets — Lower Strength

For modified-release tablets, when the drug product is in the same dosage form but in a different strength, when it is proportionally similar in its active and inactive ingredients and when it has the same drug release mechanism, an in vivo BE determination of one or more lower strengths can be waived based on dissolution profile com-parisons, with an in vivo study only on the highest strength. We rec-ommend that the drug products exhibit similar dissolution profiles between the highest strength and the lower strengths based on the f2 test in at least three dissolution media (e.g., pH 1.2, 4.5 and 6.8). We recommend that the dissolution profile be generated on the test and reference products of all strengths.

4. Postapproval Changes

Information on the types of in vitro dissolution and in vivo BE stud-ies for extended release drug products approved as either NDAs or ANDAs in the presence of specified post approval changes are pro-vided in an FDA guidance for industry entitled SUPAC-MR: Modi-fied Release Solid Oral Dosage Forms: Scale-Up and Post-Approval Changes: Chemistry, Manufacturing, and Controls, In vitro Dissolu-tion Testing, and In vivo Bioequivalence Documentation. We rec-ommend that for post approval changes, the in vitro comparison be made between the prechange and post change products. In instances where dissolution profile comparisons are recommended, an f2 test can be used. An f2 value of > 50 suggests a similar dissolution pro-file. A failure to demonstrate similar dissolution profiles may indi-cate an in vivo BE study be performed. When in vivo BE studies are conducted, we recommend that the comparison be made for NDAs between the prechange and post change products, and for ANDAs between the post change product and reference listed drug.

CONCLUSIONPK-PD modeling has emerged as a major tool in clinical pharmacol-ogy to optimize drug use by designing rational dosage forms and dosage regimes. Quantitative representation of the dose–concen-tration–response relationship should provide information for pre-diction of the level of response to a certain level of drug dose. Several mathematical approaches can be used to describe such relationships, depending on the single dose or the steady-state measurements car-ried out. The development of a correlation is based on the scientific principles associated with mathematical modeling, statistical evalu-ation, and numerical deconvolution. The development and validation of a PK/PD is based on the ability of fraction of the drug absorbed versus fraction of the drug-dissolved relationship of various formula-tions.

REFERENCES1. Venitz J. Pharmacokinetic-pharmacodynamic modeling of reversible drug effects. In:

Derendorf H, Hochhaus G, editors. Handbook of Pharmacokinetic/Pharmacodynamic correlation. New York: CRC Press; 1995. p.

2. Van Peer A, Snoeck E, Huang ML, Heykants J. Pharmacokinetic-pharmacodynamic relationships in phase I/phase II of drug development. Eur J Drug Metab Pharmacokinet. 1993; 18(1): 49-59.

3. Steimer JL, Ebelin ME, Van Bree J. Pharmacokinetic and pharmacodynamic data and models clinical trials. Eur J Drug Metab Pharmacokinet. 1993; 18(1): 61-76.

4. Lieberman R, McMichael J. Role of pharmacokinetic-pharmacodynamic principles in rational and cost-effective drug development. Ther Drug Monit. 1996; 18(4): 423-8.

5. El-Masri HA, Thomas RS, Benjamin SA, Yang RS. Physiologically based pharmacokinetic/pharmacodynamic modelling of chemical mixtures and possible applications in risk assessment. Toxicology. 1995; 105(2-3): 275-82.

6. van Schaick EA, de Greef HJ, Ijzerman AP, Danhof M. Physiological indirect effect modelling of the antilipolytic effects of adenosine A1-receptor agonists. J Pharmacokinet Biopharm. 1997; 25(6): 673-94.

7. Boxenbaum H, DiLea C. First-time-in-human dose selection: allometric thoughts and perspectives. J Clin Pharmacol. 1995; 35(10): 957-66.

8. 8. Gabrielsson JL, Wei einer DL. Methodology for pharmacokinetic/pharmacodynamic data analysis. Pharm Sci Technolo Today. 1999; 2(6): 244-52. 9.

9. Meibohm B, Derendorf H. Basic concepts of pharmacokinetic/pharmacodynamic (PK/PD) modelling. Int J Clin Pharmacol Ther. 1997; 35(10): 401-13.

10. Levy G. Mechanism-based pharmacodynamic modeling. Clin Pharmacol Ther. 1994; 56(4): 356-8.

11. Levy G. Predicting effective drug concentrations for individual patients: determinants of pharmacodynamic variability. Clin Pharmacokinet. 1998; 34(4): 323-33.


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12. Sheiner LB, Stanski DR, Vozeh S, Miller RD, Ham J. Simultaneous modeling of pharmacokinetics and pharmacodynamics: application to d-tubocurarine. Clin Pharmacol Ther. 1979; 25(3): 358-71.

13. Mandema JW, Tuk B, van Steveninck AL, Breimer DD, Cohen AF, Danhof M. Pharmacokinetic-pharmacodynamic modeling of the central nervous system effects of midazolam and its main metabolite a-hydroxy imidazolam in healthy volunteers. Clin Pharmacol Ther. 1992; 51(6): 715-28.

14. Lee DY, Lee KU, Kwon JS, Jang IJ, Cho MJ, Shin SG, Woo JI. Pharmacokinetic-pharmacodynamic modeling of risperidone effects on electroencephalography in healthy volunteers. Psychopharmacology 1999; 144(3): 272-8.

15. Dayneka NL, Garg V, Jusko W. Comparison of four basic models of indirect pharmacodynamic responses. J Pharmacokin Biopharm. 1993; 21(4): 457-78.

16. Jusko WJ, Ko HC. Physiologic indirect response models characterize diverse types of pharmacodynamic effects. Clin Pharmacol Ther. 1994; 56(4): 406-19.

17. Bauer JA, Balthasar JP, Fung HL. Application of pharmacodynamic modeling for designing time-variant dosing regimens to overcome nitroglycerin tolerance in experimental heart failure. Pharm Res. 1997; 14(9): 1140-5.

18. Lima JJ, Krukemeyer JJ, Boudoulas H. Drug- or hormone-induced adaptation: model of adrenergic hypersensitivity. J Pharmacokin Biopharm. 1989; 17(3): 347-64.

19. Ragueneau I, Laveille C, Jochemsen R, Resplandy G, Funck-Bretano C, Jaillon P. Pharmacokinetic-pharmacodynamic modeling of the effects of ivabradine, a direct sinus inhibitor, on heart rate in healthy volunteers. Clin Pharmacol Ther. 1998; 64(2): 192-203.

20. www.wikipedia.org. Center for Drug Evaluation and Research (CDER); March 2003.


Research ArticlePTB Reports A multifaceted peer review journal in the field of Pharmacology, Toxicology and Biology

Impact of Microbial Culture Analysis and Prophylactic use of Antibiotics in Acute Severe PancreatitisJagadish babu Dasari*1, Cristiano Ialongo1, Aruna Chandranath Kondakundhi2, Sri Harsha Munnamgi3, Syed Juied4

1Department of Molecular biology ad biochemistry, University of RomaTorvergata, Rome, Italy2Department of General Surgery, University of Jinzhou, Liaoning, China.3Department of General Medicine, Yashoda Hospital, Hyderabad, India.4Department of BGS Global Hospital, Bangalore, Karnataka, India.

About Author : Dr. Jagadish babu Dasrai PharmD, PhDDepartment of Molecular Biology and Biochemistry,University of Roma Torvergata Policlinico (Hospital) Rome, Italy.Ph.No: +39-3510 742 543.E-mail: [email protected]

DOI : 10.5530/PTB.1.1.4

ABSTRACT

Objective: The uses of prophylactic antibiotics are beneficiary in acute severe pancreatitis in reducing the infection. The main objective of this study is to compare the effects of antibiotics as prophylaxis and treatment in two groups with and without microbial cultural test, respectively. Methods: The study population consisted of 60 patients treated for acute severe pancreatitis. In group 1 (n=32) patients received prophylactic antibiotics according with the microbial test and were treated accordance with ultra-guided drainage and/or surgical debribment of infected necrosis were performed when the presence of pancreatitis was demonstrated. The primary endpoints were infectious complication rate, incidence of nosocomial infection and mortality rate. In group 2 (n=38), patients received prophylactic antibiotics (ciprofloxacin, metrodinazole) without microbial test. Results: In group 2, 38(54.28%) patients were administrated with prophylactic for >14 days than in group 1, 32(45.71%) the Fine Needle Aspiration and/Ultra Sound guided drainage fluid collections were urine sample 15.62%, blood sample 31.25%, Pus sample 9.37%, peritoneal fluid 12.50% and drain fluid 31.25%. In which 15.62% Escherichia.coli is most commonly grown gram negative bacteria. And carbapenems (meropenam, ertapenam) are most sensitive. We can observe significant difference between groups in mortality and duration of stay in the surgical ward or intensive care unit. Conclusion: In this study, with the microbial analysis the use of the prophylactic antibiotic can reduce the hospital stay, need of surgical management and reduce the frequent use of reoperation.

Key words: Acute severe pancreatitis, Antibiotic prophylaxis, Fine needle aspiration, Microbial cultural test, Nocsocomial infection, Ultra sound guided.

INTRODUCTIONTherapeutic use is an obvious and rational use for antimicrobials in patients with pancreatitis.1 The two factors that are directly related to its morbidity and mortality are organ failure and infection. Organ failure may occur as early as the first week and can cause either early or delayed mortality2,3 and also uncontrolled systemic inflammatory response syndrome is usually associated with high mortality rate.4 Much of the morbidity and mortality accompanying this disease are due to pancreatic and peri-pancreatic infection with reported rates as high as 40% to 70 %.5

Infected pancreatic necrosis is still assumed to indicate that surgi-cal debridement is necessary. However, during recent years, sev-eral studies have reported that conservative treatment such as nonsurgical drainage was successful in some patients.6-8 The clinical importance of pancreatic infection became significant to prevent the infected necrosis could be beneficial9 and prophylactic antibiotics probably have positive effect on the course of the patient with nec-rotizing pancreatitis.10,11 In most of the patients, bacteria complicat-ing acute necrotizing pancreatitis originate from the gastrointestinal tract that includes Escherichiacoli, proteus mirabilis, Enterococcus faecalis, Pseudomonas aeruginosa bacteroides spp and Clostridium spp.12,13

The prophylactic use of antibiotics might be a beneficial treatment option at an early stage of the disease before necrotic areas become infected, since they are capable of penetrating pancreatic tissue and achieve M.I.C. (Maximum Inhibitory Concentrations) levels in serum and pancreatic juice.14 Acute pancreatitis is a disease with extremely different clinical expressions and most of the patients suffer mild and limited disease, but one fifth of the patients develop multiple organ dysfunction syndrome (MODS), accompanied by high mortality rate.


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MODS is not a predictive method but it is a best indicator of acute pancreatitis, its severity and mortality, mainly if it appears early, or persists for more than 48 hours or is multi organic.15

Reducing the complication of acute pancreatitis with the help of prophylactic antibiotics is still a controversy, but in this study the antibiotics plays a significant role, which reduces its complications and further hospital stay. Therefore, we initiated to investigate the effects of antibiotics in both group A and group B in-order to show the rational and knowledge concerning about the prophylactic use of antibiotics in acute pancreatitis. The primary aim of this prospective, randomized and observational study was to evaluate the effective-ness of the prophylactic antibiotics like carbapenams intravenously in delaying and preventing the pancreatic and peri-pancreatic infec-tion in patients. And with this, the beneficiary of microbial test with respect to the patient samples makes wide difference and delaying in pancreatic infection and hospital stay. The main objective of study is to evaluate the effect of prophylactic administration of antibiotics in acute pancreatitis and rationale use of antibiotics can reduce the pancreatic and peri-pancreatic infection.

METHODThis was a prospective, randomized, single center, study from Janu-ary 2014 to November 2014 for 10 months. Approval from the insti-tutional ethics committee was obtained prior to the study. And all patients provided written informed consent form. The culture and sensitivity reports were collected from the records of department of microbiology for study period. A total of 70 patients were enrolled out of 32 patients was analyzed for culture and sensitivity. Prospec-tive data is collected in the department of gastroenterology BGS Global hospital, diagnosed with severe and acute necrotizing pan-creatitis for which the onset of disease occurred within the previous 72 hours.

Inclusion criteria: onset of disease occurred within 72 hours, clini-cal symptoms like abdominal pain, vomiting and nausea, elevation of serum alpha amylase greater than 3 times greater than the normal level and C-reactive protein >110 mg/l, CT-scan performed at 5-7 days after the onset of the disease inorder to assess the severity of acute pancreatitis on admission and during follow up and MODS >7 score. Already debribment of pancreatitis patients are excluded. The study categorize into two different group A and group B with differ-ent treatment strategies utilized in each.

In group 2, 38 patients are admitted to the hospital and were rou-tinely given antibiotic prophylaxis, ciprofloxacin 800 mg/day and metrodinazole 1500 mg/day for 14 days. And patient present within the 72 hours from the onset of the disease CRP>110 mg/l, MODS >7 and necrosis of >35 to 40% as demonstrated on contrast enhanced CT-scan.In this, the only change to the former protocol other than

that to antibiotic prophylaxis was that patients diagnosed with acute pancreatitis were routinely monitored Intra Abdominal Pressure (IAP) as well the growth of microorganism in the respective samples. However, in this group patients were not routinely monitor for the elevated intra abdominal pressure and no bacterial culture examina-tion test were sent.

In group 1, 32 patients were admitted to the hospital with the same criteria, the only change to the former protocol other than that to antibiotic prophylaxis was that patients diagnosed with acute pan-creatitis were routinely monitored Intra Abdominal Pressure (IAP) as well the growth of micro-organism in the respective samples. In this study, the feasibility and effectiveness of the subcutaneous fasciotomy of the anterior rectus abdominal sheath were assessed, as well as the regular check up with the Ultra sound (USG) guided drainage of intra-abdominal and peri-pancreatic fluid collections, as indications to obtain the bacterial cultures from peripancreatic fluid collection, urine, pus, blood, peritonel fluid and ultra guided fine needle aspiration cytology (FNAC) was performed in all patients in whom pancreatic necrosis had been confirmed by CT-scan and also in whom persisting symptoms of MODS score >7 or failure of at least one organ. When severe infection was observed, US-guided drainage or retro-peritonealscopic or surgical debribment (US guided drain-age) of infected necrosis was performed.

Step 1: Percutaneous drainage was performed in all patients.

Step 2: Retro peritoneoscopic or surgical debribment depending on the size and accessibility of the infected collection is considered with patients in whom no improvement was seen after percutaneous drainage.

Step 3: Multi-resistant organism were derived as pathogens, pre-dominantly bacteria, that were resistant to one or more classes of anti-microbial agents those includes Methacillin-Resistant Staphylo-coccus aureus (MRSA), Vancomycin-Resistant Enterococcus (VRE), gram negative bacteria producing ESBL (Extended spectrum beta-lactamase and remaining were resistant to multiple classes of anti-microbial agents.

RESULTSStatistical analysis: Statistics are presented as the mean ± standard deviation and for comparisons between groups student t-test (for normally distributed data) were employed as appropriate. A p-value of <0.5 was considered to indicate statistical significance.

Patients demographic and the etiology of pancreatitis by groups are shown in Table 1. In group 2 38 (54.28%) patients were administrated with prophylactic antibiotics for >14 days as shown in Table 2. Where as in group 1 32 (45.71%) patients were administrated antibiotics according to bacterial culture test, multi-drug resistant micro-organ-ism, requiring the administration of carbepanems were identified

Table 1: Patients Demographic details and its characteristics features of antibiotics (n=70)

Group 1 (n=32) 45.71% Group 2 (n=38) 54.28%Male Female Male Female

Age(Yrs) Mean ± SD 55 ± 1.03 55 ± 1.05

Gender (Mean ± SD) 07.5 ± 04.79 0.5 ± 0.57 08.75 ± 04.11 0.75 ± 0.50

N (%) 30 (93.75) 02 (06.25) 35 (92.10) 03 (07.89)

Etiology n (%)Alcohol 10 (31.25) 13 (34.21)

Biliary 18 (56.25) 20 (52.63)

Idiopathic 04 (12.50) 05 (13.15)

Severity Acute PancreatitisMODS scale 1.25 ± 1.31 1.27 ± 1.32

Necrosis ≥ 30%, n (%) 13 (40.62) 16 (42.10)


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rgPseudomonas and Proteus. In blood samples, 10 (31.25%) were ana-lyzed out of which 03 (30%) were positive and 07 were negative with gram-negative micro-organism is seen. In which 01 of Pseudomonas ESBL, E.coli, Klebsiella and Klebsiella ESBL and 02 E.coli. Bacterial cul-ture analysis shown in Table 3.

Overall, surgical interventions (open necrosectomy, repeated sur-gery and debridement) were more frequently performed in group 1 (p=0.741) and group 2 (p=0.382). The length of stay and clinical out-comes are shown in Table 4.

Antibiotic susceptibility TestEscherichia coli and E.coli ESBL were highly sensitive to meropenam and ertapenam with MIC 0.5, moderate to imipenam and amkacin MIC 1.0. Klebsiella and klebsiella ESBL highly sensitivity to tigecycline MIC 1.0 and ertapenam MIC 0.5 and moderate to cephalosporin’s and fluroquinoles 1.75 MIC. Pseudomonas is highly sensitivity to levofloxacin with MIC 0.25 and Pseudomonas ESBL is sensitivity to colistin with MIC 0.5. Proteus is highly sensitive to ciprofloxacin with MIC 0.5. The overall sensitivity pattern of antibiotics to bacterial culture analysis in acute pancreatitis was the highest to the carbape-nams followed by good sensitivity towards quinolones, polypeptide antibiotic and glycycline antibiotic shown in Table 5.

DISCUSSIONAntibiotic prophylaxis in acute severe pancreatitis (with pancreatic necrosis,organ failure and or/sepsis) has been a controversy over last two decades and various clinical trials have been performed and results were contradictory.16-23 The aim of the current study was to impact and effect of antibiotic prophylaxis and treatment in two dif-ferent groups. The use of prophylactic antibiotics in group 2 had no significant effect on acute severe pancreatitis such as incidence of infectious and mortality rate compared with group 1.

According to the bacterial cultured isolated and antibiotic suscepti-bility test, the antibiotics had a signigicant role in group 1, in which it reduces the infection and length of stay as compared to group 2, this phenomenon should be examined in further treatment prospec-tive. However, surgical interventions and, the absence of nosocomial infection is supported by the use of antibiotic prophylactic in acute severe pancreatitis, which is in accord with expert panel on the man-agement of the acute pancreatitis.24

E.coli was the most common organism in the acute severe pancreati-tis after analyzing bacteriology in acute severe pancreatitis,25 The antibiotic sensitivity pattern showed that most of the bacteria were sensitivity to carbapenams and quinolones. In multi center study22 showed that combination of ciprofloxacin and metrodinazole did not show significant reduction of infected pancreatitis necrosis were determined as 15.75% in group 2 than in group 1 12.56% (p>0.05).

Table 2: Antibiotic Prophylaxis (group 2)

Class No. of patients n (%)Quinolones

Ciprofloxacin 11 (28.94)

PenicillinPiperacillin 06 (15.78)

Cephalosporin’s 3rd GenerationCeftriaxone 16 (42.10)

Amino glycosidesAmikacin 02 (05.28)

NitroimidazoleMetrodinazole 03 (07.89)

Total 38 (100)

Table 3: Bacterial culture analysis

Growth of the Microorganism

No’s Infected (N=32), n (%)

E.coli 05 (15.62)

E.coli ESBL 05 (15.62)

Klebseilla 03 (09.37)

Klebseilla ESBL 03 (09.37)

Pseudomonas 04 (12.50)

Pseudomonas ESBL 01 (03.12)

Proteus 02 (06.25)

No growth 09 (28.12)

Total 32

Table 4: Clinical Outcomes

Outcomes Group 1 (n=32) Group 2 (n=38) p-valueSepsis 02 (06.25) 03 (07.89)

0.217*, 0.0721Infected Pancreatitis 04 (12.56) 06 (15.78)

Severe Acute Pancreatitis 20 (62.50) 18 (47.36)

Major Organ Failure 04 (12.50) 06 (15.78)

Length of stay ICU Ward Surgery Ward ICU Ward Surgery Ward

0.741*, 0.3821≤ 2 weeks 02 01 00 03

≤ 4 weeks 01 03 02 04

≤ 6 weeks 00 00 01 00

DeathsOverall deaths (n),% 03 (09.37) 05 (13.15)*Group-1, 1Group-2

more frequently in group 1 than group 2. of 05 (15.62%) urine sam-ples were analyzed in group 1. Out of which 02 were negative with gram-negative micro-organism, and 03 were positive. Where as in Pus samples 03 (9.37%) were analyzed out of which 01 were nega-tive with gram-negative micro-organism seen. and 02 were positive. Klebsiellla pneumonia is the common gram-negative micro-organism grown in urine and in pus sample.

In group 1, the FNAC and/USG guided drainage of large intra abdom-inal fluid collections were performed. In peritoneal fluid sample in group 1, 04 (12.50%) were analyzed out of 02 were negative with gram negative. Out of which 02 E.coli and 01 E.coli ESBL growth is seen. Out of which 01 was positive. Where as in drain fluid, 10 (31.25%) were analyzed and was negative with gram-negative micro-organism, 02 E.coli, 03 E.coli ESBL and Klebseilla pneumonia and 01


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In 200926 Showed that prophylactic use of ciprofloxacin reduce the secondary risk for pancreatitic infection nor the mortality rate, but in this study it is considered as beneficiary as well as less impact result in group 2. In another study, meta-analysis of eight trails,17 shows sig-nificant reduction in pancreatic and non-pancreatitic infection and in length of stay but not beneficiary towards the mortality rate and need for surgical intervention, similar results can be observed in the current study. In Cochrane review of seven studies,27 found benefit of carbapenams was considered in isolation and significant decrease in pancreatitic infection. Better designed studies are needed, if the use of antibiotic prophylaxis is to be recommended.

The overall resistance pattern of antibiotic are cephalosporins, ampi-cillin, amoxicillin, co-trimazole. This may be due to the wide use of these antibiotics as empirical therapy and not recommended for bac-terial cultural analysis.

CONCLUSIONThe prospective study shows the details of pathogen and their sen-sitivity towards antibiotics pattern. It is clear that E.coli is still most common gram-negative micro-organism grown and carbapenams are the sensitive. The beneficiary of bacterial culture analysis in acute severe pancreatitis makes wide differences and delay in pan-creatitic infection decrease the need for interventional and surgical management. And also prevent the further development of antimi-crobial resistance and it does not affect the mortality rate. Routine monitoring of susceptibility patterns is necessary. This will help in the rational and prophylactic preparation to clinical and also the preparation of antibiotic policy of the individual institute.

CONFLICTS OF INTERESTThe authors declare no conflicts of interest.

ACKNOWLEDGEMENTSThe authors would like to thank the BGS global hospitals and staffs members of the department of microbiology for their kind support and co-operation during the study.

Table 5: Sensitivity towards the antibiotics (group 1)

Growth of the microorganism

Sensitivity of the drug

MIC (Minimum Inhibitory

Concentration)E.coli Meropenam* 0.5

E.coli ESBL Ertapenam* 0.5

Klebseilla Tigecycline1 1.0

Klebseilla ESBL Ertapenam* 0.5

Pseudomonas Levofloxacin3 0.25

Pseudomonas ESBL

Colistin2 0.5

Proteus Ciprofloxacin3 0.5*Carbapenams, 1Glycylcycline antibiotic, 2polypeptide antibiotic, 3Quinolones.

REFERENCES1. De Waele Jan J. Rational Use of Antimicrobial is in Patients with Severe Acute Pancreatitis.

Seminars in Respiratory and Critical Care Medicine 2011; 32(2): 174-80.2. Beger HG, Rau B, Mayer J, Pralle U. Natural course of acute pancreatitis. World J Surg

1997; 21(2): 130-5.3. Parraga HB. Antibiotic prophylaxis in acute pancreatitis: Yes. Rev Col Gastroenterol. 2010;

25(4): 263-6.4. Ignatavicius P, Vitkauskiene A, Pundzius J, Dambrauskas Z, Barauskas G. Effects of

prophylactic antibiotics in acute pancreatitis. HPB. 2012; 14(6): 396–402.5. Dellinger EP, et al. Early Antibiotic Treatment for Severe Acute Necrotizing Pancreatitis:

A Randomized, Double-Blind, Placebo-Controlled Study. Annals of Surgery. May 2007; 245(5): 674-83.

6. Dubner H, Steinberg W, Hill M, et al. Infected pancreatic necrosis and peripancreatic fluid collections: serendipitous response to antibiotics and medical therapy in three patients. Pancreas 1996; 12(3): 298–302.

7. Adler DG, Chari ST, Dahl TJ, et al. Conservative management of infected necrosis complicating severe acute pancreatitis. Am J Gastroenterol. 2003; 98(1): 98–103.

8. Runzi M, Niebel W, Goebell H, et al. Severe acute pancreatitis: nonsurgical treatment of infected necroses. Pancreas 2005; 30(3): 195–199.

9. Isenmann, et al. prophylactic Antibiotic Treatment in Patients with Predicted Severe Acute Pancreatitis: A Placebo-Controlled, Double-Blind Trial. Gastroenterology 2004; 126(4): 997–1004

10. Sharma VK, Howden CW. Prophylactic antibiotic administration reduces sepsis and mortality in acute necrotizing pancreatitis: A meta-analysis. Pancreas 2001; 22(1): 28 –31.

11. Golub R, Siddiqi F, Pohl D. Role of antibiotics in acute pancreatitis: a meta-analysis. J Gastrointest Surg.1998; 2(6): 496 –502.

12. Balzan S, de Almeida Quadros C, de Cleva R, Zilberstein B, Cecconello I. Bacterial translocation: overview of mechanisms and clinical impact. J Gastroenterol Hepatol. 2007; 22(4): 464–71.

13. Cicalese L, Sahai A, Sileri P, Rastellini C, Subbotin V, Ford H, et al. Acute pancreatitis and bacterial translocation. Dig Dis Sci. 2001; 46: 1127–32.

14. Bradley EL. Antibiotics in acute pancreatitis. Current status and future directions. Am J Surg. 1989; 158(5): 472-8.

15. Cruz-Santamaria DM, Taxonera C, Giner M. Update on pathogenesis and clinical management of acute pancreatitis. World J Gastrointest Pathophysiol. 2012; 3(3): 60-70.

16. Manes G, Uomo I, Menchise A, Rabitti PG, Ferrara EC, Uomo G. Timing of antibiotic prophylaxis in acute pancreatitis: a controlled randomized study with meropenem. Am J Gastroenterol. 2006; 101(6): 1348–53.

17. Xue P, Deng LH, Zhang ZD, Yang XN, Wan MH, Song B, et al. Effect of antibiotic prophylaxis on acute necrotizing pancreatitis: results of a randomized controlled trial. J Gastroenterol Hepatol. 2009; 24(5): 736–42.

18. Jafri NS, Mahid SS, Idstein SR, Hornung CA, Galandiuk S. Antibiotic prophylaxis is not protective in severe acute pancreatitis: a systematic review and meta-analysis. Am J Surg. 2009; 197(35): 806–13.

19. Xue T, Cai Q. Prophylactic antibiotic treatment in acute necrotizing pancreatitis: results from a meta-analysis. Scand J Gastroenterol. 2008; 43(10): 1249–58.

20. Heinrich S, Schafer M, Rousson V, Clavien PA. Evidence-based treatment of acute pancreatitis: a look at established paradigms. Ann Surg. 2006; 243(3): 154–68.

21. Manes G, Rabitti PG, Menchise A, Riccio E, Balzano A, Uomo G. Prophylaxis with meropenem of septic complications in acute pancreatitis: a randomized, controlled trial versus imipenem. Pancreas. 2003; 27(4): 79–83.

22. Isenmann R, Runzi M, Kron M, Kahl S, Kraus D, Jung N, et al. Prophylactic antibiotic treatment in patients with predicted severe acute pancreatitis: a placebo-controlled, double-blind trial. Gastroenterology. 2004; 126(4): 997–1004.

23. Maravi-Poma E, Gener J, Alvarez-Lerma F, Olaechea P, Blanco A, Dominguez-Munoz JE. Early antibiotic treatment (prophylaxis) of septic complications in severe acute necrotizing pancreatitis: a prospective, randomized, multicentre study comparing two regimens with imipenem–cilastatin. Intensive Care Med. 2003; 29(11): 1974–80.

24. Pezzilli R, Zerbi A, Di CV, Bassi C, Delle Fave GF. Practical guidelines for acute pancreatitis. Pancreatology 2010; 10(5): 523–35.

25. Noor MT, Radhakrishna Y, Kochhar R, Ray P, Wig JD, Sinha SK, et al. Bacteriology of infection in severe acute pancreatitis. JOP. 2011; 121(1): 19–25.

26. Garcia-Barrasa A, Borobia FG, Pallares R, Jorba R, Poves I, Busquets J, et al. A double-blind, placebo-controlled trial of ciprofloxacin prophylaxis in patients with acute necrotizing pancreatitis. J Gastrointest Surg. 2009; 13(4): 768–74.

27. Villatoro E, Mulla M, Larvin M. Antibiotic therapy for prophylaxis against infection of pancreatic necrosis in acute pancreatitis. Cochrane Database Syst Rev. 2010; 5(1): CD002941.



Evaluation of Oral Hygiene Practice, Knowledge and Attitude among (10-15 Yrs) School Children in Dharan, Nepal- A Cross-Sectional StudyRoshana Poudyal1, Priyata Agrawal2, Ashish Shrestha3, Mamta Dali4*, Tarakant Bhagat5, Asish Choudhary6

1,2Dental Surgeon, BPKIHS, CODS, Dharan, Nepal3Additional Professor and Head, Department of Public Health Dentistry, BPKIHS, CODS, Nepal4Department of Pedodontics and Preventive Dentistry, CODS, Dharan, Nepal5Assistant Professor, Department of Public Health Dentistry, BPKIHS, CODS, Nepal.6Assistant Professor, Department of Public Health Dentistry, BPKIHS, CODS, Nepal

About Author : Dr. Mamta DaliAssistant professor,Department of Pedodontic and Preventive Dentistry,BPKIHS, CODS, Dharan, Nepal.E-mail: [email protected]

DOI : 10.5530/PTB.1.1.5

ABSTRACT

Background: The study is aimed to evaluate oral hygiene practice, knowledge and attitude among (10-15 yr) school children in Dharan. Materials and Methods: This is a cross sectional study which included 200 school students of age group (10-15 years) from five different schools of Dharan. The school and the students were selected by random sampling method. The consent for the participation of school children were obtained from the head of the school verbally. A pre-tested close- ended questionnaire was used for the study which was adopted from different conducted research. The questions were in English language. Results: The result of this study show that only 15% of the students knew that gum bleeding means gingivitis. 83% reported that use of fluoride strengthens teeth and 81% knew that healthy teeth is strong and caries free teeth. 64.5% participant reported the reason for dental visit was general dental check up while 36% visited dentist only when dental pain and 76% felt the necessity of regular dental visit. 96% of the respondents used toothbrush and toothpaste as their brushing material and 83% of them brushed twice a day. Among all the students surveyed 80.5% had the habit of rinsing their mouth after eating. Results of this study prove that oral hygiene habits, oral health awareness and knowledge level among school children in Dharan is satisfactory. The participants had conducive oral health behavior, sufficient knowledge, positive attitude and practice regarding oral health.

Key words: Attitude, Awareness, Knowledge, Oral hygiene, Practice.

INTRODUCTIONWhile the eyes may be the window to the soul, our mouth is a window to our body’s health.1 The state of your oral health can offer lots of clues about your overall health. Oral health may be defined as a standard of health of the oral and related tissues which enables an individual to eat, speak and socialize without active disease, discomfort or embar-rassment and which contributes to general well being.2 Childhood is the period of greatest change in life. It is widely recognized that good oral health practices are necessary from a young age to ensure posi-

tive long term dental health and hygiene. The oral health of children is important towards their overall well being.3 Oral hygiene is the practice of keeping the mouth healthy and clean by brushing and flossing to prevent dental decay and gum diseases. With developing country like Nepal, Dental hygiene is poor with inadequate and improper brush-ing of teeth, no washing of mouth after intake of sweets, wide-spread substance abuse and addiction, hyper-acidity, increased consumption of refined sugar and sweetened foods. Use of toothbrush in underdevel-oped areas is grossly limited and Neem twigs are traditionally utilized for dental cleaning. Regular brushing of teeth after principal meals is not practiced universally.4 The children tend to be more vulnerable to dental diseases due to social, economic and demographic factors like lack of awareness, lack of transportation, limited access to professional dental care, lack of perceived need for dental care.5

Little is known about oral health attitudes and behaviors of children from developing countries as comparison with developed countries,6


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although such knowledge is an indication of the efficacy of applied dental health education programs.

Aims and ObjectivesEvaluate the awareness and knowledge among school children (10-15 yrs) on dental health problems, their oral hygiene practices and pattern of practices.

Determine the methods by which we can improve the awareness about oral hygiene among school children.

Generate awareness about the importance of oral hygiene practice and the necessity of regular dental check up.

MethodologyThe study was a cross-sectional survey which included 200 school students of age group (10-15 years) from five different schools of Dharan. The school and the students were selected by random sam-pling method. The consent for the participation of school children were obtained from the head of the school verbally.

A pre-tested dental health questionnaire from various researches was adopted in this study and distributed to all subjects. The ques-tionnaires were designed to collect information on knowledge about oral disease and methods of prevention, attitudes towards preven-tive methods of periodontal disease, dental health behaviour in rela-tion to periodontal disease, and sources of information about dental health.

Data analysisThe data was analyzed using the SPSS version 10.0 software. The individual scores were summed up to yield a total score and given in subtitles for each questions. Frequency distribution, number and percentage were calculated and was tabulated.

Results of the SurveyVarious questions were asked regarding the knowledge and aware-ness on oral health, such as meaning of gum bleeding, role of fluoride on teeth, healthy teeth means etc.

Table 1 reported that approximately 85% of the students did not know about bleeding gums or gave wrong answers such as gingival bleeding reflects healthy gingiva or injury to the gums. Only 15% of the study population was aware that gingival bleeding reflects swol-len gums (gingivitis).

Table 2: Attitude toward professional dental care

Visit to adentist

No. of students

Percentage of students

a. Every 6 months 68 34%

b. Every 12months 9 4.5%

c. Occassionally 25 12.5%

d. Only when dental pain 72 36%

e. Never visited 26 13%

Reason for visita. Tooth decay 49 28.1%

b. Bleeding gums 12 6%

c. Dental check up 139 64.5%

Necessity of regular dental visita. Yes 152 76%

b. No 33 16.5%

c. Don’t know 15 7.5%

TOTAL 200 100%

Table 1: Knowledge and awareness on Gum bleeding, role of fluoride and healthy teeth

Gum bleeding means No. of students


a. Swollen gums (gingivitis) 30 15%

b. Healthy gums 17 8.5%

c. Injury to gums 138 69%

d. Don’t know 15 7.5%

Using fluoride strengthens teetha. Yes 166 83%

b. No 34 17%

Healthy teeth isa. White and shiny teeth 32 16%

b. Strong and caries free teeth

162 81%

c. Don’t know 6 3%

TOTAL 200 100%

When subjects were asked about the role of fluoride on teeth, 83% of the students were aware that fluoride strengthens the teeth, whereas 17% had no knowledge about the effect of fluoride on teeth. Majority of the students (81%) believed that healthy teeth is strong and caries free teeth, while (16%) said that healthy teeth is white and shiny teeth and (3%) had no awareness about healthy teeth.

Table 2 showed that about 36% of the respondents reported that they would only visit the dentist when they have dental pain and 13% stated that they have never visited a dentist. Of 174 (87%) reported to visit the dental clinic whereby 64.5% were for dental checkup, 28.1% of those were due to tooth decay and only 6% were due to bleeding gums. When questioned on the necessity of regular dental visit, approximately 76% of the students had the positive atti-tude that regular dental visit is necessary and 16.5% did not feel the necessity of regular dental visit.

Table 3 revealed that all participants brushed their teeth regularly. Most of individuals (83%) brushed twice a day, (12%) brushed only once a day and only (10%) brushed occasionally. A greater percent-age of the students (96%) practiced brushing with toothbrush and toothpaste and few used brush & tooth powder (1.5%), finger & tooth powder (1.5%), Dattiwon and others (1%). 80.5% answered that they always rinse their mouth after eating while 14.5% children rinse sometimes and 5% rinse never.

DISCUSSIONThis study presented a comprehensive overview of the oral health behavior, knowledge and attitude among school children of Dharan, Nepal. The present study cannot be exactly compared with the other studies but careful observations can be made with the other studies.

Concerning knowledge, most of subjects did not know the meaning of gingival bleeding and very few reported that gingival bleeding means gingivitis, however, more than two-third of school students were aware that fluoride prevent dental caries and healthy teeth means strong and caries free strengthen teeth. This shows that there is lack of awareness regarding gingival bleeding as an indica-tor of poor periodontal condition among school children whereas the knowledge of fluoride and healthy teeth is quite adequate, could be through the media like television, radio, newspaper and various advertisements.

In relation to attitude toward professional dental care, majority of the participants were found that they visit dental clinic only when they have dental pain however comparatively a larger no of partici-


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rgThe result of the practice questionnaire showed that major group (96%) of children used toothpaste and toothbrush to clean their teeth and had habit of rinsing mouth after eating. This accounts for the awareness the participants have regarding the efficacy of tooth brush and toothpaste over toothpowder, finger, dattiwan (Neem sticks) and others. Though, a small percentage of children did indicate the use of both Toothpaste and Toothpowder. This could be due to low socio-economic factor or lack of proper knowledge on brushing aids.10

Since the study was conducted on children of considerably prudent age group who are inclined to make a positive impression about them, hence the results of the oral health practices could be biased especially the response to the frequency of brushing and mouth rinsing.The change to healthy attitude and practice can be occurred by giv-ing adequate information, motivation and practice of the measures to the subjects.11 Results of this study prove that oral hygiene habits, oral health awareness and knowledge level among rural schoolchil-dren is poor and needs to be improved. Parents and teachers need to be informed, motivated about dental care so that their attitudes change. Based upon these findings, the establishment of a school-based oral health education program in school children, including parents and teachers is recommended.

CONCLUSIONResults of this study prove that oral hygiene habits, oral health awareness and knowledge level among school children in Dharan is satisfactory. The participants had conducive oral health behavior, sufficient knowledge, positive attitude and practice regarding oral health. The information given to the parents and teachers, the role the media played in raising awareness helped the children to change their attitude and practice. The findings of this study suggested that the 10-15 years age group students would be the appropriate tar-get group to receive the first organized intervention leading towards improving their oral health and reducing prevalence of dental dis-ease.


ACKNOWLEDGEMENTThe authors wish to thank Department of Public Health Dentistry for their help in conducting this survey.

pants (34%) has reported that dental check up should be in every six months interval and 76% of the students had the positive attitude that regular dental visit is necessary whereas 16.5% did not feel the necessity of regular dental visit. Pain was the main reason for visiting the dentist and agrees with other study.7 proved in their study that pain is the main driving factor for children to visit the dentist. Rea-sons for this drastic behaviour might be lack of oral health education programe and indicates the necessity of awareness among parents of school children for regular dental visit to prevent future dental diseases.

It is very encouraging and satisfactory to know that majority of the stu-dents reported to brush their teeth twice a day (83%) and not a single student reported who did not brush his\her teeth. This can be because the children of this present study come from a very medium or high socioeconomic background and affordability plays an important role. This result can be compared.8 it was 44.4 percent and with another study of Chinese school children where only 22% of the 12 year old group brushed at twice a day, 62% brushed once a day and 16% never brushed or brushed less frequently.9

Table 3: knowledge on oral hygiene practices

Frequency ofbrushing

No. of students


a. No brushing 0 0%

b. Occassionally 10 5%

c. Once a day 24 12%

d. Twice a day 166 83%

Brushing materialsa. Brush+ tooth powder 3 1.5%

b.Brush + tooth paste 192 96%

c. Finger + tooth powder 3 1.5%

d. Dattiwon(Neem stick)and others

2 1%

e. Not any 0 0%

Mouth rinsing after eatinga. Never 10 5%

b. Sometimes 29 14.5%

c. Always 161 80.5%

TOTAL 200 100%

REFERENCES1. http: // www.wm.edu / offices / hr / benefits / commonhealth / oralhealth / index.php.

Accessed on 09.01.20102. Department of health. An oral health strategy for England, London: dept. of health; 1994.3. Mathur A, Gupta T. oral health attitude knowledge behavior and consent towards dental

treatment among school children. Journal of orofacial research 2011; 1(1): 6-10.4. Mistry KM. Factors related to the promotion of oral health in developing countries. J

Indian Dent Assoc. 1992; 63(1): 59-63.5. Oral Health Status in rural child population: Promotional & Interventional Strategies. A

GOI-WHO Collaborative Programme 2006-07.www.whoindia.org/en/Section 30_1453.htm.

6. Al- Omiri MK, Board J, Al-Wahadni AM, Saeed KN. Oral health attitudes, knowledge and behavior among school children in North Jordan. Journal of Dental Education 2005; 70 (2): 179- 87.

7. Rajab LD, Petersen PE, Bakeen G, et al. Oral health behaviour of school children and parents in Jordan. Int J Pediatr Dent. 2002; 12(3): 168-76.

8. Zhu L, Petersen PE, Wang HY, Bian JY, Zhang BX. Oral health knowledge, attitudes and behaviour of adults in China. Int Dent J. 2005; 55(4): 231-41.

9. Peterson PE, Esheng Z. Dental caries and oral health behavior situation in children and school children in Wuham, People Republic of China. Int Dent J. 1998; 48(1): 210-6.

10. Punitha et al. oral hygiene status, knowledge, attitude and practices of oral health among rural children of kanchipuram district. Indian Journal of Multidisciplinary Dentistry 2011; 1(2): 115-8.

11. Smyth E, Caamano F, Fernandez-Riveiro P. Oralhealth knowledge, attitudes and practice in 12-year-old schoolchildren. Med Oral Patol Oral Cir Bucal. 2007; 12(8): 614-20.



Research Article

Retrospective Analysis of Musculioskeletal Complications in Patients with TuberculosisHosseinian Amiri Aref1, Abedi Siavash2, Yazdanian Maryam3

1Department of rheumatology, Imam Khomeini hospital, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran2Department of pulmonology, Imam Khomeini hospital, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran3MA Educational Psychology Student, Sari Azad university, Mazandaran, Sari, Iran.

About Author : Mr. Aref Hosseinian AmiriDepartment of RheumatologyImam Khomeini hospital, Faculty of Medicine,Mazandaran University of Medical Sciences,Sari, Iran.Ph.No: +151-3543088, Fax: +151-3543087E-mail: [email protected]

DOI : 10.5530/PTB.1.1.6

ABSTRACT

Background: In adults, Osteoarticular involvement with M. tuberculosis, usually occurs from either a quiescent pulmonary focus or an extrapulmonary site. The definitive diagnosis is made by the demonstration of M.tuberculosis in tissue or synovial fluid. Common presentations of osteoarticular tuberculosis are spinal TB, Infection at peripheral joint especially weight-bearing joints, tendons, bursae, or bones and Reactive arthritis (Poncet’s disease). Aim: The aim of this study was analysis of 10 adult patients with tuberculosis to determine the pattern of musculoskeletal complications during 2 years. Subjects and Methods: This is a retrospective study of cases of musculoskeletal complications among patients with tuberculosis during two years that refers to rheumatologic clinic of mazandaran university of medical sciences at January 2011 to December 2013. Data were analyzed using the SPSS version 20. Variables analyzed include age, sex and kind of musculoskeletal complications. Results: There were 10 patients with tuberculosis refers in our rheumatologic clinic for musculoskeletal complications. All of patients were adults between 18 to 48 years old with median age of 30.8 years old. Seven (70%) patients were male and 3(30%) patients were female. The most common musculoskeletal complication was spondylodiscitis in 6 (60%), arthritis in 5 (50%), osteomyelitis in 2 (20%), reactive arthritis in 2 (20%), soft tissue abcess in 2 (20%) and dactilitis in 1 (10%) of patients. Conclusion: Musculoskeletal complications are very important and must be considered in patients with tuberculosis. Spondylodiscitis (pott’s disease) and arthritis of weight bearing large jonts are the most common rheumatologic complications of this disorder.

Key words: Musculoskeletal complications, Spondylodiscitis, Tuberculosis, Peripheral arthritis, Poncet's disease.

INTRODUCTIONMycobactral tuberculosis Infection usually acquired by inhalation, and cause nonspecific pneumonitis, followed by lymphatic and hema-togenous spread to other organs such as musculoskeletal system. Reactivation of bacilli in dormant foci may occur during a period of diminished host immunity, and spread via lymphatics or blood.1

Overally osteoarticular involvement occurs in about 5% of patients with tuberculosis,1 with estimated percentages ranging from about 2% in the United States to more than 6% in developing countries.1

In adults, bone infection usually occurs from either a quiescent pul-monary focus or an extrapulmonary site. The classic presentation of musculoskeletal tuberculosis is spondylodiscitis or Pott’s disease.

Peripheral joint arthritis especially in weight-bearing joints, ten-dons and bursae involvement and osteomyelitis also occur. Reactive arthritis (Poncet’s disease) has been reported.2 Thoracic vertebrae are involved most frequently, followed by lumbar, and, less com-monly, cervical and sacral vertebrae.3 Tuberculosis nfection often begins in the anterior portion of the vertebral bodies, with sub-sequent disc involvement and destruction of vertebral end plates causing the characteristic gibbus deformity.4 Sometimes infection extends to adjoining discs or vertebrae, or to distant sites. Localized soft tissue inflammation such as paravertebral or psoas abscesses or sinus tracts may cause neurologic injury. Spinal TB can mimic ver-tebral osteomyelitis caused by pyogenic bacteria.5 Radiography of spine show disc space narrowing with vertebral collapse and para-spinous abscess.4

Peripheral joint arthritis often occurs as monoarticular arthritis affecting weight bearing joints as hip or knee, but may involve other joints.6 Most patients in tuberculous arthritis are middle-aged or older and often with underlying medical disorders and the onset of disease is typically insidious.7 Articular TB is usually due to reacti-vation of a hematogenously seeded focus and sometimes can also spread from adjacent osteomyelitis. Tuberculous osteomyelitis can occur without joint involvement and metaphysis of long bones are


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rgmost common site. Tuberculous dactylitis may involves hands and feet especially in childrens.8 Poncet’s disease is a form of reactive arthritis occurring during active TB and often involve hands and feet.9

The diagnosis of tuberculous arthritis is best made by histologic and microbiologic examination of synovium. Histology may demonstrate caseating or noncaseating granulomas. Tuberculous arthritis usually responds to combination chemotherapy. Surgery may be needed for synovectomy, debridement, joint stabilization, or removal of infected prostheses.1,6,8

SUBJECTS AND METHODSThis retrospective study was performed at the TOOBA referral rheu-matologic clinic of Mazandaran University of medical sciences during two years from January 2011 to December 2013. Ten patients with tuberculosis refer to clinic for musculoskeletal disorders during this period for evolution. Their medical history were retrieved from the hospital’s medical records department and each patient examined by a rheumatologist and pulmonologist. Patients included in this study were 18 to 48 years old with tuberculosis that diagnosed by radio-logic findings, PPD test and the definitive diagnosis is made by the demonstration of M.tuberculosis in tissue or synovial fluid. Cases with who any kind of traumatic or congenital musculoskeletal abnormali-ties and other inflammatory and infective disorders or incomplete data or unclear diagnosis were excluded. Spondylodiscitis or Potts disease confirmed by clinical findings and radiography of spine and confirmed by biopsy of tissue. Diagnosis of the vertebra collapse and stress fractures confirmed by physical examination and radio-graphic finding. Osteomyelitis, on the other hand was diagnosed as a relapsing and persistent infection that evolves over months to years characterized by low-grade inflammation, fever, presence of dead bone (sequestrum), new bone apposition, and fistulous tracts. Septic arthritis was diagnosed by aspiration of joint and culture of organism and in patients with negative culture, biopsy of synovial tissue or PCR of fluid were performed. Reactive arthritis of TB or poncet's disease were confirmed by presence of arthritis in joint without isolation of organism from the involved joint and presence of active disease in another site in the body. Dactilitis and tenosyno-vitis confirmed by clinical findings and imaginary studies including radiography and sonography of involved organ and confirmed by biopsy of tissue. Objective of this study was to determine the age, sex, kind and pattern of musculoskeletal complications of tubercu-losis in these adult patients. Data were analyzed by using the SPSS version 20 soft-wares. Data were expressed as the mean _ SD (range) or number (percentage). Means were compared using the Mann-Whitney rank sum test.

All probabilities were 2-sided, with P values less than 0.05 consid-ered statistically significant.

RESULTSA total of 10 patients with tuberculosis refer to our clinic for mus-culoskeletal disorders during 2 years from January 2011 to Decem-ber 2013 for further evaluation. All of the patients were adults with ages between 18 to 48 years old (median: 30.8 years old). Seven of patients (70%) were males and three of them were female (30%). Table 1 depicts the demographic profile of the patients. The most common musculoskeletal finding was spondylodiscitis or pott's dis-ease with painful axial pain specially in thoracolumbar region in six (60%) of cases. Most of patiens were male (4 patients) and two cases was female. Stress fracture and vertebral collapse occurred in two patients that one of them was male and another one was female. Peripheral joint arthritis was diagnosed in five patient (50%) and the most common involved joints were knees that occurred in four patients and hip joint in one case. All of patients were male. Other musculoskeletal complications was osteomyelitis that occurs in 2 (20%) of patients that were female. One region of osteomyelitis was in 5th rib at the right side of the chest and another site was in the shaft of left hip. Reactive arthritis or poncet's disease occurred in 2 (20%) of patients that all of them were male. Saroilitis joint of right side and arthritis of small joint of hand was another side of involvement. Dactilitis occurred in one patient that occurs in third fingers of right hand. This patient was female and had spondylodiscitis concordantly. In another one (10%) of patients soft tissue abcess occurred that was in psoas muscle of left side. This patient had spondylodiscitis concor-dantly.

DISCUSSIONMusculoskeletal tuberculosis refers to tuberculous involvement of the bones, joints and musculature.10 Skeletal tuberculosis accounts for 10 to 35 percent of cases of extrapulmonary tuberculosis and almost 2 percent of all TB cases.11 According the study of Malaviya AN et al, Most patients in tuberculous arthritis are middle-aged or older.7 In our study, the median age of patients was 30.8 (18-48) years old. In this study 10 patients with tuberculosis musculoskel-etal disorders refers during 2 years from January 2011 to December 2013 for further evaluation.

According to study of Chapman M et al, the spine is the most com-mon site of involvement in skeletal tuberculosis and accounting for approximately half of musculoskeletal TB cases.11 According the study of Jellis JE et al, between 48% and 67% of lesions occur in the lower thoracic and thoracolumbar spine in HIV-negative patients, whereas the lumbar spine is most commonly involved in HIV-positive patients.12 In our study, the most common site of involvement was spine that occurs in 6 (60%) of cases and the most common site of involvement was in lower thoracic region that occurred in 4 (40%)patients and in another two cases occurred in lumbar spine. All of patients were HIV negative.

Table 1: Characteristics of musculosketal disorders in 10 adult patients with tuberculosis

Case no

Age(years) sex Potts disease

(spondylodiscitis) Osteomyelitis arthritis dactilitis Poncet'sdisease

Soft tissueabcess

1 18 M + _ _ _ _ +

2 26 M _ _ + _ _ _

3 19 F + + _ _ _ _

4 33 M _ _ + _ + _

5 42 F + _ _ + _ _

6 29 M + _ + _ _ _

7 30 M _ _ + _ _ _

8 28 F _ + _ _ _ _

9 48 M + _ _ _ + _

10 35 M + _ + _ _ _


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According study of Evanchick CC et al, dactylitis may also occur in children and represented 19% of bone and joint TB and 15% of cases of osteomyelitis of hematogenous origin.23 In this stud , two (20%) patients had poncet,s disease that occurred in Saroilitis joint of right side and arthritis of small joint of hand was another side of involve-ment.

The reason for variations at different studies could be due to differ-ence of age of cases, geographical and sample size.

CONCLUSIONThe most common musculoskeletal complications in adults with tuberculosis are insidious onset of spondylodiscitis that commonly occurred in thoracolumbar resion of spine and peripheral joint arthritis that commonly occurred in lower weight bearing joint and especially in knee joint. Other complications including osteomyelitis, dactilitis and reactive arthritis or poncet's disease. In comparison with another studies of adult patients with tuberculosis, the spinal involvement was similar at thoracolumbar region involvement. In other study, the hip join was involved more frequently but at this study knee joint involved more frequently. Other findings were simi-lar to another studies. The small sample size and inability to explore other risk factors also limited this work and its generalizability.


ACKNOWLEDGEMENTSThe researchers would like to appreciate cooperation of the hon-orable staff of Infectious diseases department of Razi Hospital of Ghaemshahr and Imam Hospital of Sari.

The next most common form of musculoskeletal tuberculosis is monoarthicular arthritis.13

The typical pattern is arthritis that involves the large joints, most commonly the hip and knee.14 According the study of Evanchick CC et al, Of 23 cases of musculoskeletal TB, 9 involved the spine, 1 the hip, and the remaining 13 the peripheral joints. Most patients were men older than 50 years. In our study, 5 (50%) of patients had periph-eral arthritis and most common joint was knee joint that involved in 4 patients and in another ones hip joint was involved.

Tuberculous osteomyelitis occurs in both children and adults and can be involves the long bones. Bone lesions begin with hematogenous implantation of organisms in the medullary area and lesions are typically destructive.15 The femur and the tibia are most commonly affected.16 In this study, osteomyelitis occurred in 2 (20%) patients and the site of involvement were in 5th rib at the right side of the chest and another site was in the shaft of left hip.

Other clinical manifestations of Musculoskeletal tuberculous is soft tissue mass that can occurs as extraspinal soft tissue mass or as a psoas abscess.17 Formation of soft tissue mass or cold abscess is uncommon, although according of study of Polly P et al in South African series it was described in 16 of 98 cases.18 In our study one patients (10%) had soft tissue abcess. The most commn sign of soft tissue mass are local pain associated with muscle spasm and rigidity with Constitutional symptoms such as fever and weight loss.19

Poncet disease or tuberculous reactive arthritis is an acute symmet-rical polyarthritis that occurs with active extrapulmonary, pulmo-nary, or miliary TB. In general there is inflammation of the involved joints but no objective evidence of active TB infection.20 Poncet disease is relatively rare and occurs in large and small joints and the pathogenesis is probably immune-mediated and the arthritis gen-erally resolves within a few weeks of initiation of antituberculosis therapy.21,22

REFRENCES1. Leonard MKJ, Blumberg HM. Musculoskeletal tuberculosis. In: Schlossberg D, ed.

Tuberculosis & nontuberculous mycobacterial infections. 5th ed. New York: McGraw-Hill; 2006; 242–63.

2. Martini M, Ouahes M. Bone and joint tuberculosis: a review of 652 cases. Orthopedics 1988; 11(6): 861–6.

3. Cormican L, Hammal R, Messenger J, Milburn HJ. Current diffi culties in the diagnosis and management of spinal tuberculosis. Postgrad Med J 2006; 82(963): 46–51.

4. Ridley N, Shaikh MI, Remedios D, Mitchell R. Radiology of skeletal tuberculosis. Orthopedics 1998; 21(11): 1213–20.

5. Perronne C, Saba J, Behloul Z, et al. Pyogenic and tuberculous spondylodiskitis (vertebral osteomyelitis) in 80 adult patients. Clin Infect Dis 1994; 19(4): 746–50.

6. Garrido G, Gomez-Reino JJ, Fernandez-Dapica P, Palenque E, Prieto S. A review of peripheral tuberculous arthritis. Semin Arthritis Rheum 1988; 18(2): 142–9.

7. Malaviya AN, Kotwal PP. Arthritis associated with tuberculosis. Best Pract Res Clin Rheumatol. 2003; 17(2): 319–43.

8. Dall L, Long L, Stanford J. Poncet’s disease: tuberculous rheumatism. Rev Infect Dis. 1989; 11(1): 105–7.

9. Daniel TM, Bates JH, Downes KA. History of tuberculosis. In: Tuberculosis: Pathogenesis, Protection, and Control, Bloom BR (Ed), American Society for Microbiology, Washington; 1994. p. 13.

10. Watts HG, Lifeso RM. Tuberculosis of bones and joints. J Bone Joint Surg Am. 1996; 78(2): 288.

11. Chapman M, Murray RO, Stoker DJ. Tuberculosis of the bones and joints. Semin Roentgenol 1979; 14(4): 266–82.

12. Jellis JE. Human immunodeficiency virus and osteoarticular tuberculosis. Clin Orthop Relat Res. 2002; 398(3): 27–31.

13. Vohra R, Kang HS, Dogra S, et al. Tuberculous osteomyelitis. J Bone Joint Surg Br. 1997; 79(4): 562.

14. Babhulkar S, Pande S. Tuberculosis of the hip, Clin Orthop Relat Res. 2002; 398(1): 93–9. 15. Tsay MH, Chen MC, Jaung GY, et al. Atypical skeletal tuberculosis mimicking tumor

metastases: report of a case. J Formos Med Assoc. 1995; 94(7): 428–31. 16. Shih HN, Hsu RW, Lin TY. Tuberculosis of the long bone in children. Clin Orthop Relat

Res. 1997; 335(1): 246–52. 17. Girdlestone GR, Somerville EW. Tuberculosis of Bone and Joint, 2nd ed, Oxford

University Press, London; 1952.18. Polley P, Dunn R. Noncontiguous spinal tuberculosis: incidence and management. Eur

Spine J. 2009; 18(8): 1096. 19. Hodgson SP, Ormerod LP. Ten-year experience of bone and joint tuberculosis in

Blackburn 1978-1987. J R Coll Surg Edinb 1990; 35(4): 259. 20. Isaacs AJ, Sturrock RD. Poncet’s disease--fact or fiction? A re-appraisal of tuberculous

rheumatism. Tubercle 1974; 55(2): 135. 21. Sood R, Wali JP, Handa R. Poncet’s disease in a north Indian hospital. Trop Doct. 1999;

29(1): 33.22. Babhulkar SS, Pande SK. Unusual manifestations of osteoarticular tuberculosis. Clin

Orthop Relat Res. 2002; 398:114–120. 23. Evanchick CC, Davis DE, Harrington TM. Tuberculosis of peripheral joints: an often

missed diagnosis. J Rheumatol. 1986; 13(1): 187–9.



Potential Anti-Anxiety Effect of Mucuna pruriens in Experimental Model of Swiss Albino MiceAnjula Sachan1, Sarvesh Kumar1, Hemant Singh2, Pratap Shankar1*, Dheeraj Kumar1, Amod Kumar Sachan1, Rakesh Kumar Dixit1

1Department of Pharmacology & Therapeutics, King George’s Medical University, Lucknow, Uttar Pradesh, 226003, India.2Department of Surgery, ELMC, Lucknow, India.

About Author : Mr. Pratap ShankarDepartment of Pharmacology & Therapeutics,King George’s Medical University, Lucknow,Uttar Pradesh, 226003, India.Ph.No: +91-9454691251E-mail: [email protected]

DOI : 10.5530/PTB.1.1.7

ABSTRACT

The usage of benzodiazepines, the major class of anxiolytic drugs is invariably accompanied by many side-effects like sedation and muscle relaxation leading to incoordination of movements. Search for novel anxiolytic agents have identified flavonoids as potential compounds devoid of these adverse effects. Mucuna pruriens commonly known as cowhage plant has been claimed to possess various beneficial effects like anti-parkinsonian, anti-tumor, neuroprotective, antioxidant, anti-diabetic and antimicrobial activities. Previous studies have reported that Mucuna pruriens contains LDOPA and 5-hydroxy tryptophan (5-HT) as a major constituent with higher concentration in seeds. Present study was designed to evaluate the anxiolytic activity of Mucuna pruriens extract in Swiss albino mice. The study was conducted on 30 male Swiss albino mice. Three doses of Mucuna pruriens (100, 200, 400 mg/kg, p.o.) and standard dose of diazepam (2 mg/kg, i.p.) were used for treatment. The pharmacologically validated model elevated plus maze (EPM) was used to take as a measure of antianxiety effect. Mucuna pruriens at the doses of 200 mg/kg and 400 mg/kg significantly reduced the time spent and no. of entries in closed arm, increased the time spent and entries into open arm in elevated plus maze (p<0.05) as compared to control group. Present study demonstrates the anxiolytic activity of Mucuna pruriens in Swiss albino mice.

Key words: Anxiolytic, Diazepam, Elevated plus maze, Mucuna pruriens, Side effects, Swiss Albino Mice.

INTRODUCTIONAnxiety usually refers to the experience of fear, apprehensiveness, nervousness, panic, restlessness, tension, and agitation. Manifest symptoms include trembling, fainting, headaches, and sweating, pos-sibly elevated blood pressure, and changes in other psychophysiolog-ical indices such as heart rate, muscle tone, and skin conductance.1 Anxiety affects most of the population nearly one-eighth of the total population world-wide.2 Benzodiazepines are the major class of com-pounds commonly prescribed for treating anxiety. However, their use is associated with side effects like psychomotor impairment, potentiating of other central depressant drugs and addiction liabil-ity.3 A number of plants are being investigated in complementary alternative medicines for anxiety.4,5 Research has been conducted in the search of an alternate, more specific and cost-effective therapy.6

Neurotransmitters involved in anxiety generation include serotonin, dopamine, noradrenaline, GABA, Corticotropin releasing factor (CRF), Melanocyte stimulating hormone (MSH), neuropeptides and neurosteroids.7 The recognition of anxiolytic effects of non-benzo-diazepine azapirones agents, which act as 5HT1A partial agonists and their therapeutic role in clinical anxiety and mood disorders has further focused attention on the 5-HT1A receptor.8 Previous studies have reported that Mucuna pruriens contains L-DOPA and 5-hydroxy tryptophan (5-HTP) as a major constituent with higher concentra-tion in seeds.9 The recognition of anxiolytic effects of non-benzo-diazepine azapirones agents (buspirone, gepirone, and ipsapirone), which act as 5HT1A partial agonists and their therapeutic role in clinical anxiety and mood disorders has further focused attention on the 5-HT1A receptor. Although the azapirones interact with other neurotransmitter systems, such as the dopaminergic and noradren-ergic, they display nanomolar affinity for 5HT1A receptor sites.10 Buspirone appears to only interact with the dopaminergic system with reasonable potency and exhibits properties of both a dopamine agonist and a dopamine antagonist. This suggests that dopamine is implicated in the etiology and expression of anxiety. Based on earlier studies it has been postulated that dopaminergic agents may play an important role in the pharmacotherapy of anxiety.11

Mucuna pruriens is a popular medicinal plant of India, which has long been used in Ayurvedic system of medicine. Numbers of studies have


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shown beneficial effects of Mucuna pruriens as aphrodiastic, anti-parkinsonism, hypoglycemic, antioxidant, antibacterial, antifungal, and anticancer agents.12-18 Phytochemical evaluation on the seeds revealed the presence of 5-indolic compounds, especially trypt-amine and 5-hydroxytryptamine, alkaloids like flavonoids, mucunine, mucunadine, prurine and prurienine.19,20 Flavonoids are an important group of polyphenolic compounds derived from nature. These com-pounds have been shown to possess many useful biological activities like antioxidant, anti-inflammatory, cytotoxic21 and anti-nociceptive properties.22 Some of the naturally occurring flavonoids and their synthetic derivatives have been reported to selectively bind to the central benzodiazepine receptors and to exert anxiolytic and other benzodiazepine like effects in animals.23 Therefore present study was planned with the objectives to evaluate anti-anxietyeffect of Mucuna pruriensusing elevated plus maze in mice.

MATERIAL AND METHOD

Selection of AnimalsThe study was conducted in the Department of Pharmacology, King George’s Medical University (KGMU), Lucknow. Prior permission was sought from the Institutional Animal Ethics Committee (IAEC) for conducting the study [Project no. 47/IAEC/2013]. Adult healthy male Swiss Albino mice, of similar physical constitution (in terms of age, body weight), weighing 20-30 g had been use dinstudy. Animals had been obtained from animal house of Indian Institute of Toxicol-ogy Research, Lucknow, which is certified by Committee for the Pur-pose of Control and Supervision of Experiments on Animals (CPCSEA) for breeding and housing of animals. The animals were housed in our Institutional Animal facility under temperature, humidity and light and dark cycle-controlled environment [25 ± 2°C, 70%, 12 hrs. cycle) and were given standard pellet diet and water ad libitum. The maintenance of the animals and the experimental procedures were in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ (Lates-trevisionin 2011) and the guiding principles of IAEC which strictly adhered to the guidelines of CPCSEA.24

Drugs, Dosage and Treatment GroupsMucuna pruriens (MP) seed extract was procured from Himalaya drug company, Bangalore, India. The standard drug Diazepam (NEON Laboratories) was purchased from government authorized medical store. It was administered in a dose of 2 mg/kg, i.p.25 MP dissolved in distilled water and administered per-orally (p.o.). Total 30 mice were divided randomly into control and experimental groups (n=6). Group 1 received the Distilled water and served as the control group, group 2 received the standard drug diazepam (2 mg/kg) i.p., groups 3, 4 and 5 received the test drug (MP) in doses of 100, 200 and 400 mg/kg, per-orally.

ELEVATED PLUS MAZEThe elevated plus maze model is well established animal model for testing anxiolytic drugs.26 The ratio of enteries, time spent and rear-ing behaviour in open arms to close arms reflects the safety of close arms with relative fearfulness of open Arms. Exploring behaviour in the open arm is anatural tendency when a mouse is exposed to a new environment. The plus maze apparatus constructed of wood consist-ing of two open arms (16x5 cm) and two closed arms (16 x 5 x 12 cm) and a central platform (5×5 cm), arranged in such a way that the two arms of each type were opposite to each other to give the apparatus a plus sign appearance. The entire maze was elevated to a height of 25 cm above the floor.27 Animals were brought to the testing room 1 hr. prior to testing. Each mouse was placed at the centre of the elevated plus maze with its head facing the open arms. The dose administration schedule was so adjusted that each mice was having its turn on the elevated plus maze apparatus 30 min after Diazepam (2 mg/Kg i.p.) and 60 min after the oral administration of the extracts doses and vehicle. Each mouse was observed for a total of 5 minutes at approximately 2 m distance from the apparatus. Entry into an arm was defined as the point when the animal places all four paws into the arm.

STASTISTICAL ANALYSISAll result are expressed as mean ± S.D. Statistical analyses were car-ried out using the software package SPSS (Version 17.0). Data were analyzed using one way analysis of variance (ANOVA), to assess the comparability of the groups assigned to the treatment groups fol-lowed by Tukey’s multiple comaparison tests. P values <0.05 were considered significant.

RESULTIn EPM distilled water treated animals the time spent in the open and closed arms, and entries in the open and closed arms were compared with Mucuna pruriens extract at the dose of 100 mg/kg, 200 mg/kg and 400 mg/kg & also Diazepam (2 mg/kg). MPE at the dose of 200 mg/kg and 400 mg/kg showed significant (p<0.05) increase in the time spent in the open arms and at the dose of 400 mg/kg it showed significant (p<0.05) increase in number of entries in open arm (Graph 2). Furthermore, MPE 200 and 400 mg/kg had decrease in time spent and number of entries in closed arm (Table 1) as compared to control group showed a significant (p<0.05) in elevated plus-maze.

MPE has shown dose dependent increase in time spent and no. of entries in open arm. Among the groups which received different doses of MPE, the maximum time spent in open arm is seen at dose of 400 mg/kg (Table 1 and Figure 1&2), followed by dose of 200 mg/kg and then at 100 mg/kg. As compared to control group, it is significant at the dose 200 mg/kg and 400 mg/kg of MPE and with diazepam.

Table 1: Effect of MPE on EPM in mice

GROUPTime spent in seconds

(Mean±SD)No. of entries

(Mean±SD)Open arm Close arm Open arm Close arm

Distilled water 17.67±1.51 200.83±13.58 3.50±1.05 14.67±2.38

Diazepam (2 mg/kg) 91.33±8.76** 135.67±6.10** 14.00±1.37** 7.67±1.63**

Mucuna pruriens(100 mg/kg) 25.00±4.37 186.60±9.50 4.00±.90 12.50±1.87

Mucuna pruriens(200 mg/kg) 30.08±4.26** 182.15±7.79* 5.17±1.47 11.33±1.51*

Mucuna pruriens(400 mg/kg) 78.83±8.79** 137.50±5.96** 10.50±1.38** 8.33±0.82**

p-value < .01## < .01## < .01## < .01##

# (p< .05) ## (p< .01) (ANOVA), * (p< .05), ** (p< .01), (Tukey’s multiple comparison test).


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Figure 1: Effect of Mucuna pruriens (MP) and Diazepam(2 mg/kg) in EPM.The column represents the mean of the time spent in open arm recorded in a 5 min observation period.*(p< .05), **(p< .01), (Tukey’s multiple comparison test), compared with control group.

Figure 2: Effect of Mucuna pruriens (MP) and Diazepam(2 mg/kg) in EPM.The column represents the mean of no. of entries in open arm recorded in a 5 min observation period.*(p< .05), **(p< .01), (Tukey’s multiple comparison test), compared with control group.

DISCUSSIONTraditional Indian Medicines are widely beneficial to mankind. Num-bers of studies have shown beneficial effects of Mucuna pruriens. How-ever, studies pertaining to antianxiety action of Mucuna pruriens are lacking so far. Toxicity study to determine the safe oral dose of Mucuna pruriens was not done in our study because acute toxicity tests, as per OECD 423 guidelines (2010) have already been conducted in many pre-vious studies, reported that extract of Mucuna pruriens did not produce any toxicity, or significant behavioural change, or mortality upto an oral dose of 2000 mg/kg in albino mice.28

In the present study we have evaluated the anti-anxiety effect was assessed by using elevated plus maze. In the elevated plus maze, rodents tend to avoid entry into open arm. The animal hence prefers to spend more time and shows normal rearing behaviour in the closed arm. Anxiolytics tend to show an increase in the number of entries, time spent and rears in the open arms. They also increase the ratio of open arm to total arm entries. Hence, there is dose dependent increase in effect of drugs. Other workers have also reported similar results regard-ing same drug but at different doses.

MPE at the dose of 200 mg/kg and 400 mg/kg showed significant (p<0.05) increase in the time spent in the open arms and at the dose of 400 mg/kg it showed significant (p<0.05) increase in number of entries in open arm (Figure 2). Furthermore, MPE 200 and 400 mg/kg had decrease in time spent and number of entries in closed arm (Table 1) as compared to control group showed a significant (p<0.05) in elevated plus-maze. Antianxiety effect of MPE at the dose of 400 mg/kg is com-parable to the antianxiety effect of diazepam.

Previous studies have reported that Mucuna pruriens contains L-DOPA and 5-hydroxy tryptophan (5-HTP) as a major constituent with higher concentration.29 GAB Aergic and serotoninergic systems are most frequently associated with anxiety. Earlier studies have pos-tulated that dopamine also plays an important role in the pharmaco-therapy of anxiety.30 The presence of L-DOPA and 5-hydroxy tryptophan (5-HTP) as a major constituent in Mucuna pruriens may suggest the role of dopaminergic and/or serotonergic pathways in its anxiolytic activi-

ties. Anxiety disorders can also be due to free radical induced damage to GABA ergic and serotoninergic system. Recent studies have shown that patients with anxiety disorders have higher activity levels of the enzymes like superoxide dismutase and glutathione peroxidase as well as higher lipid peroxidation activity.31 Hence oxidative metabolism is also regarded as a plausible pathway that can affect the regulation of anxiety.

Phytochemical screening of Mucuna pruriens confirms the presence of flavonoids.32,33 Flavonoids are known for their antianxiety effects. Ben-zodiazepine receptors activation by flavonoids has been proposed for the antianxiety responses produced by different flavonoids.34 Therefore we hypothesize that flavonoids present in the extracts of Mucuna pru-riensmay have acted through benzodiazepine GABA chloride channel receptors. However further studies are needed to know the exact mech-anism responsible for antianxiety activity.

CONCLUSIONDue to wide benefits of the traditional Indian medicine to the mankind, many studies have shown the importance of the Mucuna pruriens. The Phytochemical screening of Mucuna pruriens confirms the pres ence of flavonoids and their antianxiety effects. Here we hypothesize that flavonoids present in the extracts of Mucuna pruriens may have acted through benzodiazepine GABA chloride channel receptors. However further studies are needed to know the exact mechanism responsible for antianxiety activity.


ACKNOWLEDGEMENTSWe are thankful to Head of Department of Pharmacology & Therapeu-tics, King George's Medical University, Lucknow, India for his kind sup-port to study conduct.

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6. Adnaik RS, Pai PT, Sapkal VD. Anxiolytic activity of Vitexnegunda Linn. International Journal of Green Pharmacy 2009; 3(3): 243-47.

7. Roy CM, Kulkarni SK. Anti-anxiety profile of ondansetron, a selective 5-HT3 antagonist, in a novel animal model. Exp Clin Pharmacol 1997; 19(2): 107-11.

8. Kunovac JL, Stahl SM. Future directions in anxiolytic pharmacotherapy. Psychiatr Clin N Am. 1995; 18(4): 895–909.

9. Daxenbichler ME, VanEtten CH, Hallinan EA, Earle FR, Barclay AS. Seeds as source of L-dopa. Journal of Medicinal Chemistry 1971; 14(5): 463-5.

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Original ArticlePTB Reports A multifaceted peer review journal in the field of Pharmacology, Toxicology and Biology

Influence on Quality of Life Among Patients with Hypothyroidism

Paruchuri Sireesha*1, Balasubramanian Ganesh Pandian2, Nagula Parashuram3

1Department of Pharmacy Practice, Vaagdevi College of Pharmacy, Warangal, India.2Unit of Clinical Pharmacy & Pharmacy Practice, Faculty of Pharmacy, AIMST University, Semeling, Kedah Darul Aman, Malaysia.3Department of Ear, Nose & Throat, Kakatiya Medical College/MGM Hospital, Warangal, India.

About Author : Ms. Sireesha PDepartment of Pharmacy Practice,Vaagdevi College of Pharmacy, Warangal, India.E-mail: [email protected]

DOI : 10.5530/PTB.1.1.8

ABSTRACT

Aim: The study is to analyze the influence of quality of life with the patients suffering from hypothyroidism. Methods: This is a prospective observational study conducted for 6 months at endocrinology OPD of a tertiary care teaching hospital, India. Patients diagnosed with hypothyroidism were included in our study. Parameters such as TSH, tT3 and tT4 levels were monitored and their QOL were measured by using short form 36 (SF-36) questionnaires during their baseline and review visit. This study was approved by the human ethical committee. Influence of thyroid parameters over QOL scales were measured statistically using stepwise multiple linear regression and paired ‘t’ test was used to measure the significant difference with the treatment. Results and Discussion: A total of 13 patients met the inclusion criteria of our study among 30 patients irrespective of gender, significant change in thyroid parameter with treatment with L-thyroxine was observed with TSH (p=0.0169*) and further the influence of QOL have shown some significant values, which shows the need for considering the QOL while treating hypothyroidism. Conclusion: The influence of hypothyroidism on the quality of life was observed significantly correlating with few QOL subscales.

Key words: Correlating factors, Hypothyroidism, SF-36, Thyroid function test, QOL, Patient reported outcome.

INTRODUCTIONQuality of life (QoL) is defined as an individual’s perception of his/her position in life, in thecontext of the culture and value systems, in which he/she lives, in relation to his/her goal, expectation, standards and concerns.1 Patients who have chronic diseases, in which a long term survival is not at risk, may be more concerned about QoL than longevity.2,3 Thyroid disorders are chronic which are highly prevalent even though it is less life threatening. Hypothyroidism is a condition in which the thyroid gland does not make enough thyroid hormones, characterized by a reduction in metabolic rate. The main symptoms of hypothyroidism are fatigue, weakness, increased sensitivity to cold, constipation, unexplained weight gain, dry skin, hair loss or coarse dry hair, muscle cramps and depression. However, most symptoms take years to develop. The slower the metabolism gets, the more obvious the signs and symptoms will become. If hypothyroidism goes untreated, the signs and symptoms could become severe, such as a swollen thyroid gland (goitre), slow thought processes, or demen-tia.3-5 Influence of the QOL in thyroid disease is already published in

various studies which have significant importance of it, the patients with hypothyroidism are also in need to check for their depression and anxiety status. Hypothyroid patients may feel discomfort with their neck enlargement which will stress them with the physical appearance; the impact or influence of the disease state in patients living quality is less studied.5,6

MATERIALS AND METHODSThis is a prospective observational study conducted for a time period of 6 months at the out-patient department of ENT and Endocrinology, Mahatma Gandhi Memorial Hospital, Warangal, Andhrapradesh which is a 1000 bedded multidisciplinary government & tertiary care teach-ing hospital. This study was approved by the human ethical commit-tee. Patients diagnosed with hypothyroidism for first time irrespective of gender and with 12–40 years of age were included in this study. Patients those who are already on thyroid treatment and on treatment with lipid lowering agents, pregnant & lactating women, with a history of thyroid disease were not included in our study. Study participants those who are not following the treatment and fail to make follow-up were also excluded from the study. Parameters such as TSH, tT3 and tT4 levels during baseline and on review after three months were col-lected from the patient’s lab report. Study participants were explained and given SF-36 questionnaire on baseline and review visit, those who were unable to fill the questionnaire due to cognition intact were not


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Tab

le 1

: Q

OL

sco

rin

gs

ove

rall

QO

L su

bsca

leB

asel

ine

(Mea

n ±

SD)

Rev

iew

(Mea

n ±

SD)

Phys

ical

func

tioni

ng93

.33

± 22

100

± 0

Rol

e lim

itatio

ns d

ue to

ph

ysic

al fu

nctio

ning

61.9

± 4

610

0 ±

0

Rol

e lim

itatio

ns d

ue to

em

otio

nal p

robl

ems

50 ±

50

100

± 0

Vita

lity

57.8

3 ±

2166

.5 ±

14

Men

tal h

ealth

56.2

± 2

467

.38

± 13

Soci

al fu

nctio

ning

58.7

5 ±

1669

.23

± 13

Bod

ily p

ain

73.9

1 ±

2975

.3 ±

18

Gen

eral

hea

lth45

± 1

560

± 1

4

Tab

le 2

: In

flu

ence

of

thyr

oid

par

amet

ers

ove

r Q

OL

QO

L Sc

ale

BA

SELI

NE

REV

IEW

TSH

tT3

tT4

TSH

tT3

tT4

R2

pR

2p

R2

pR

2p

R2

pR

2p

Phy

sica

l fu

nctio

ning

0.07

89

0.35

23

0.23 23

0.09 53

0.21 56

0.10

99

0.14 51

0.19 91

0.23 23

0.09 53

0.00 12

0.90

77

Rol

e lim

itatio

ns

due

to p

hysi

cal

func

tioni

ng

0.01

41

0.69

85

0.01 97

0.64 71

0.20 86

0.11

67

0.00 84

0.76 58

0.08 09

0.34 61

0.01 72

0.66

91

Rol

e lim

itatio

ns

due

to e

mot

iona

l pr

oble

m

0.00

46

0.82

42

0.05 94

0.42 22

0.13 51

0.21

67

0.01 13

0.72 90

0.02 52

0.60 39

0.43 23

0.01

46*

Ene

rgy

/ fat

igue

6.99

3e-0

070.

997

80.

14 670.

19 640.

15 540.

182

60.

16 110.

17 410.

12 920.

22 770.

10 850.

271

7

Em

otio

nal

wel

lbei

ng0.

351

70.

032

7*0.

01 280.

71 230.

01 430.

202

10.

06 160.

41 350.

04 340.

49 460.

16 320.

171

0

Soc

ial

func

tioni

ng0.

079

10.

351

70.

00 010.

97 410.

36 080.

029

9*0.

00 220.

87 670.

01 320.

70 760.

00 440.

829

0

Pai

n 0.

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90.

628

80.

09 440.

30 720.

35 740.

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9*0.

07 230.

37 440.

16 360.

17 050.

06 070.

416

8

Gen

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80.

890

10.

06 510.

40 020.

11 910.

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10.

01 010.

74 290.

00 300.

85 870.

04 020.

510

8


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rgprovided with the questionnaire. Scorings were given according to the answers selected by them. The SF-36 questionnaire includes one multi-item scale that assesses eight health status dimensions: 1) Physical func-tioning: limitations in physical activities because of health problems; 2) Social functioning: limitations in social activities because of physical or emotional problems; 3) Role physical: limitations in usual role activities because of physical health problems; 4) Pain: bodily pain; 5) Mental health: general mental health (psychological distress and well-being); 6) Role emotional: limitations in usual role activities because of emo-tional problems; 7) Vitality: feeling of energy and fatigue; and 8) General health: general health perceptions. For each of the eight dimensions, item scores were recorded, summed up, and transformed, using a scor-ing algorithm, into a scale ranging from 0 to 100, with higher scores rep-resenting better subjective perception of physical and mental health.Significant difference in thyroid parameters with the treatment is cal-culated using paired ‘t’ test and the influence of the disease state (thy-roid parameters) over the study participants QOL during baseline and review visits was found by stepwise multiple linear regression.

RESULTS AND DISCUSSIONThe purpose of this study is to evaluate the relation between the dis-ease state and their quality of life before and after treatment. A total of 30 patients (29 female and 1 male) were included in our study, but only 13 have met with the inclusion criteria. The mean age of the study population was 26.9 ± 8.1 years. Among the study population patients were diagnosed with sub-clinical hypothyroidism (66.66%) and overt hypothyroidism (33.33%).The patients diagnosed with hypothyroidism were treated with (L-thyroxine) and there were significant difference (p=0.0169*) in TSH levels with its treatment. Few study have shown there is no significant change in TSH levels. Patients living quality was assessed by using SF-36 questionnaire which contains eight subscales

for measuring physical, psychologicaland social functioning, scorings were given in percentile value. Overall mean score of study participants for both the baseline and review is represented in (Table 1) which clearly indicates the subjects QOL with hypothyroidism and with its treatment.

Further the influence of disease parameters over the study partici-pant’s quality of living was calculated (Table 2), which shows that TSH is significantly influencing emotional wellbeing (p=0.0327*), emotional well being indicates the mental health of any subjects where the least scoring indicating any nervous feeling and depression all of the time and the highest scoring indicating peacefulness, happy and calm all of the time felt by any person. tT4 levels influencing social functioning (p=0.0299*) and pain (p=0.0309*) scales during baseline. During review after 3 months tT4 levels were found significantly correlating with role limitations due to emotional problem (p=0.0146*).

CONCLUSIONFrom our study we found that very few QOL scales were significantly influencing with the thyroid parameters during their first and second visit, this is mainly because of the less sample size which requires con-ducting this study in a large population. Further, QOL is also a major concern.


ACKNOWLEDGEMENTSWe would like to thank the Superintendent, MGM Hospital for permit-ting to conduct this study and all the study participants for actively par-ticipating in this study.

REFERENCES1. Crevenna R, Zetting G, Keilani M, Posch M, Schmidinger M, Pirich C, Nuhr M, Wolzt

M, Quittan M, Fialka-Maser V, Dudczak R. Quality of life inpatients with non-metastatic differentiated thyroid cancerunder thyroxine supplementation therapy. Suppor Care Cancer 2003; 11: 597-603.

2. McNeil BJ, Weichselbaum R, Pauker SG. Speech and survival: Tradeoffs between quality and quantity of life in laryngeal cancer. N Engl J Med 1981;305:982-7.

3. Vadiveloo T, Donnan PT, Cochrane L, Leese GP. The Thyroid Epidemiology, Audit, and Research Study (TEARS): the natural history of endogenous subclinical hyperthyroidism. J ClinEndocrinolMetab. 2011; 96:E18.

4. Ware JE, Snow KK, Kosinski M, Gandek B. SF-36 Health Survey: Manual and Interpretation Guide.Boston: The Health Institute, New England Medical Centre. 1993.

5. Meyerovitch J, RotmanPikielny P, Sherf M, et al. Serum thyrotropin measurements in the community: fiveyearfollowup in a large network of primary care physicians. Arch Intern Med. 2007 Jul 23;167:15338.

6. Tsigos C, Chrousos GP. Hypothalamicpituitaryadrenal axis, neuroendocrine factors and Clin Endocrinol Metab. 2011;96:E18.



Research Article

Efficacy and tolerability of two intravaginal formulations containing clindamycin plus clotrimazole in women with vaginal infections: A pilot studyDaswani Bharti Ramchand1, Naik Shilpa2, Palewar Meghana3, Ghongane Balasaheb1, Sambarey Pradeep2 Bharadwaj Renu3

1Department of Pharmacology, BJ Government Medical College, Pune. India.2Department of OBGY, BJ Government Medical College, Pune. India.3Department of Microbiology, BJ Government Medical College, Pune. India.

About Author : Dr. Daswani Bharti RamchandDepartment of Pharmacognosy,BJ Government Medical College,Pune, India.E-mail: [email protected]

DOI : 10.5530/PTB.1.1.9

ABSTRACT

Aims: To compare the effectiveness and tolerability of soft gelatine capsule versus Extended-release tablet containing clindamycin with clotrimazole in vaginal infections. Subjects and Methods: Following baseline vaginal examination, 66 women having clinical diagnosis of vaginal infection were randomized to receive three doses [once daily] of an immediate release (SG-group) or an extended release combination of clindamycin with clotrimazole (ER-group) and followed up for assessing resolution of clinical and microbiological evidence of vaginal infection by 8th day; maintenance of clinical and microbiological cure at 29th day; and occurrence of side effects. Results were presented using descriptive statistics. Qualitative data was analyzed by Fischer’s Exact test Unpaired t-test was used for quantitative data. Results: 27 women from SG-group and 30 women from ER-group completed the study. In SG-group, 69.23% had complete cure and 7.69% had partial remission at 8th day of which 88.89% maintained remission, while in ER-group 73.68% women had a complete cure of which 85.71% maintained remission on 29th day. Delayed remission was observed in 25% women from SG-group and 60% women from ER-group, while none of the women experienced intolerable adverse effects. Conclusion: Both formulations containing clindamycin plus clotrimazole were effective, as empiric therapy, in inducing and maintaining microbiological as well as clinical remission in women with clinical diagnosis of vaginal infection to a similar extent but should not to be recommended for cases specifically identified to have trichomoniasis. An adequately powered study using a larger population should be conducted to further explore differences between these two formulations.

Key words: Clindamycin, Clotrimazole, Vaginal formulation, Vaginal infections.

INTRODUCTIONVaginal discharge, being the most common reason for non-preg-nancy, non-routine gynaecologist visits, is often due to infections that may lead to gynecologic and obstetric complications, huge health care costs, low success and frequent recurrences.1,2 Hence, a timely diagnosis and appropriate treatment is essential. Prompting the clinician to make a presumptive diagnosis based on symptoms, nature of discharge and signs. Therefore, WHO introduced the con-cept of ‘Syndromic management’ of reproductive tract infections3,4

whereby diagnosis and treatment is not directed to specific disease

based on testing, but rather towards syndromes and treatment is generally given for most of the diseases that could cause that syn-drome. Furthermore, microbiological diagnosis barely correlates with clinical diagnosis5,6 and 30% cases have infections of polymicro-bial nature7 giving rise to need for treatment that covers all common vaginal pathogens. Since, most commonly encountered aetiologies of vaginal discharge are trichomoniasis, moniliasis, bacterial vagi-nosis, chlamydial infection and gonorrhoea, according to the princi-ples of syndromic management, treatment in clinical set-up should be directed against all these diseases. Current syndromic manage-ment for vaginal discharge in patients without evidence of cervical discharge or abdominal pain consists of Secnidazole/ Metronidazole: 2 g single oral dose STAT (or Tab. Tinidazole 500 mg orally, twice daily for 5 days) and Fluconazole 150 mg single oral dose STAT (or topical Clotrimazole 500 mg).8 A Cochrane review of 24 randomized controlled trials (RCTs) in bacterial vaginosis showed that clinda-mycin is as effective as metronidazole9 and six RCTs showed topical and oral antibiotic preparations to be equally effective.10 Similarly, a


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rgCochrane review of 19 RCTs reported no statistically significant dif-ferences in clinical cure rates of anti-fungals administered by the oral and intra-vaginal routes for the treatment of uncomplicated vaginal candidiasis.11 Thus, currently CDC recommends oral or topical met-ronidazole or clindamycin for bacterial vaginosis, oral fluconazole or any azole topically for Vulvovaginal candidiasis and oral metronida-zole for trichomoniasis.12

As topical therapies are minimally absorbed and associated with fewer side effects, coupled with equal effectiveness as compared to systemic therapies, their availability has obviated the need for systemic therapy in many patients with bacterial vaginosis and vul-vovaginal candidiasis. Thus, combinations of various topical agents are being tested and marketed for empiric syndromic management of vaginal infections. One such combination contains clotrimazole along with clindamycin.

In addition to its activity against candida,13 clotrimazole has also documented a weak activity against T. vaginalis.14 Though CDC does not currently recommend the use of clotrimazole for treatment of trichomoniasis, some medical practitioners consider clotrimazole suppositories for patients with trichomoniasis when nitroimidazoles cannot be used due to hypersensitivity or in first trimester of preg-nancy, whereby clotrimazole mainly offers symptomatic treatment but may cure as many as 50% of infections.15,16 On the other hand, clindamycin is effective against anaerobes and Gram-positive aer-obes 17 including organisms like group B streptococci and, S.aureus, involved in aerobic vaginitis, or mixed aerobic-anaerobic infection that responds poorly to metronidazole.18-21 Thus, such a combination of clotrimazole and clindamycin may provide empiric coverage for majority of the organisms involved in vaginal infections.

Two different intravaginal formulations, ClinSupV3 (immediate release soft gelatin vaginal capsule) and ClinSupV3-ER (extended-release vaginal tablet that releases the drugs over eight hours), each containing a combination of clindamycin and clotrimazole, are avail-able commercially and are commonly prescribed as empiric therapy for vaginal infection. Though the drugs contained in these two for-mulations are same, they differ in terms of release patterns which could possibly result in a difference in efficacy. Rapid release of drugs from vaginal formulations has theoretical limitations like greater sys-temic absorption [toxicity], leakage and wastage of drug, messiness and low residence time giving high local concentration of drug only transiently. As against this, the extended-release tablet avoids fluc-tuations in drug concentrations and provides continued presence for longer period. Secondly, therapeutic efficacy requires a drug to be present in right amounts, at desired site, for appropriate duration making pharmacokinetic-pharmacodynamic correlation of drug for-mulations quite pertinent for selecting appropriate dosage-forms. In case of clindamycin-clotrimazole combination, both drugs inhibit growth of respective organisms.13,17 This static effect suggests that inhibition would last only as long as the drugs are present in vicin-ity of the organism, giving a theoretical advantage to an extended-release formulation.

Hence, the present pilot study compares these two formulations to gather clinical and microbiological evidence for making a choice.

OBJECTIVESTo compare effectiveness and tolerability of three day treatment with ClinSupV3 versus ClinSupV3-ER in women with clinical diagno-sis of bacterial, trichomonal, candidial or mixed vaginitis.

SUBJECTS AND METHODSThis was a two-arm, randomized, comparative, prospective, single center pilot study, conducted at Sassoon General Hospitals, Pune, India between April 2011 and September 2011.

The study was open-labeled with reference to the clinician and study subjects, but the microbiologist evaluating vaginal swabs was blinded to study group allocation of the respective subjects.

The protocol was approved by Institutional Ethics Committee and informed written consent obtained from participants.

Inclusion Criteria The study included women attending gynaecology OPD with symp-toms of vaginal discharge and a clinical diagnosis of vaginal infection [based on symptoms and per speculum signs], age at least 18 years, agreeing to abstain from intercourse for eight days from start of treatment, agreeing not to douche or use intravaginal products dur-ing study period.

Exclusion Criteria Exclusion criteria were post-menopausal state, menstruating at diagnosis, pregnancy, presence of intrauterine device, use of antifun-gal or antibacterial during previous 14 days or immunosuppressants within 4 months, presence of vaginal/vulval ulcer, medical condition or treatment that might confound response, inability to attend fol-low-up visits, hypersensitivity to clotrimazole/clindamycin, regional enteritis, ulcerative colitis or ‘antibiotic associated’ colitis, significant disease or acute illness that could complicate the evaluation.

Study drugBoth dosage forms, ClinSupV3 (intravaginal soft gelatin cap-sule) [Softesule Pvt Ltd, Mumbai, India; Mfg. date 12/2010; Exp.Date 05/2012] and ClinSupV3-ER (intravaginal extended-release tablet) [Cure Medicines(I) Pvt Ltd, India; Mfg. date 02/2011; Exp. Date 07/2012] contained clotrimazole 200 mg plus clindamycin phosphate equiva-lent to 100 mg clindamycin and were supplied by Resilient Cosme-ceuticals Pvt Ltd, India.

Drug AdministrationAfter baseline vaginal examination and sample collection, the eligi-ble women were allocated to receive ClinSupV3 [SG-group] or Clin-SupV3-ER [ER-group] using computer-based randomization codes.

First dose was inserted per vagina by the gynaecologist (Day 1). The procedure for drug administration was repeated by the women for two consecutive days (Day 2 and Day 3). All women were asked to refrain from strenuous activities for four hours after insertion of intravaginal formulations, and abstain from sexual intercourse and douching for eight days from initiation of drug administration.

Follow-upThe women were reassessed on Day 8 of the study [second visit] and Day 29 [third visit]. A flexibility of -2 days to +7 days was permis-sible for the second visit and -7 days to +7 days for the third visit for women unable to report on the expected day of follow-up.

At each visit, the women filled a subjective evaluation form for ques-tions related to vaginal discharge, and itching whereby they were asked to rate their Vaginal discharge quantity as Nil=0, little=1, moderate=2, profuse=3; Vaginal discharge odour as Nil=0, malodor-ous=1; and Itching as Nil=0, little=1, moderate=2, severe=3. Simi-larly, at visit 2, tolerability of the test formulation was evaluated by the asking the women about occurrence of various adverse effects such as vaginal irritation, burning micturition etc and rating them as absent, mild, moderate and severe.

Sample CollectionAt each visit, sample of vaginal discharge was collected from lateral wall of vagina for Whiff test and pH [using pH-paper]. Additionally, one swab was sent in 2 ml normal saline, for microbiological diagno-sis (wet mount, culture on Sabourauds medium, and Gram stain). The smear was scored from Grade 0 to 4 using Hay/Ison criteria.22


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clinical and microbiological diagnoses and an apparently unequal distribution of microbiological findings between the two groups, despite randomization, we additionally analyzed microbiological findings and clinical responses for individual type of infection sepa-rately. This, however, was not a pre-specified outcome measure.

Unpaired t-test was used to compare the quantitative data in both groups. Descriptive data of the two groups were analyzed using Fisher exact test. Effect size was expressed as Relative risk and 95% Confidence interval. All statistical tests were performed using Ope-nEpi: Open Source Epidemiologic Statistics for Public Health, Ver-sion 2.3.1, (Updated 2010/19/09).

RESULTS66 women were recruited for the study of whom, 32 women were randomized to SG-group and 34 women to ER-group. Finally, 27 women from the SG-group and 30 women from the ER-group com-pleted the study (Figure 1). Mean age of the study volunteers was 31.39 + 7.5 years.

Of the 27 women in SG-group, 13 had microbiologically proven infection with isolated abnormal vaginal bacterial flora (ABVF) in three, candidiasis in five, trichomoniasis in one and mixed infec-tion [ABVF+candidiasis] in four women while in the ER-group, 19 women had microbiologically proven infection with isolated ABVF in nine, candidiasis in five and mixed infection in five women [ABVF+candidiasis- three; ABVF+trichomoniasis- two]. Thus, the study participants randomized to the two groups were comparable with respect all relevant parameters though relatively more women from ER-group had grade3 ABVF (Table 1).

Primary outcome measure for global effectivenessIn the SG-group 69.23% women had complete cure while 7.69% women had partial remission as compared to 73.68% women with complete cure in the ER-group (Table 2).

Secondary outcome measure for global effectiveness at 29th day In the SG-group, 88.89% women maintained remission while 11.11% women relapsed at third visit. Of the women who did not have com-plete cure at second visit, 25% had delayed cure. The ER-group had

OUTCOME MEASURES

Primary outcome measure for Global effectivenessIn women with microbiologically proven infection at baseline, if the vaginal swab was negative for bacterial, trichomonal and candidial vaginitis at second visit, with substantial improvement in clinical symptoms and regression of signs, it was considered as ‘Global com-plete cure’. Partial or complete regression of signs and symptoms with partial microbiological remission [conversion from Grade 3 to Grade 2] was considered as ‘Global partial remission’. Persistence of more than one symptom or sign of infection or microbiological evidence of infection was considered as ‘Global failure of therapy’.

Secondary outcome measures for Global effectivenessIn women with microbiologically proven infection at baseline and global complete cure/global partial remission at the second visit, if vaginal swab remained negative for bacterial, trichomonal and can-didial vaginitis even at third visit and she remained free of signs and symptoms of vaginal infection, it was considered as ‘Global mainte-nance of remission’.

If a woman who had global partial remission or global failure of ther-apy at second visit, met criteria for complete remission [vide primary outcome measure-‘complete cure’] at the third visit, she was consid-ered to have ‘Global delayed cure’. Women with clinical or microbio-logical evidence of persistence of infection at second and third visit were considered as ‘Global total failure of therapy’.

Conversion of global complete cure or global partial remission at second visit to global partial remission [in case of complete remission] or frank microbiological or clinical evidence of infection at the third visit was considered as ‘Global relapse’.

Secondary outcome measures for tolerabilityIf the woman completed the three day course without any break and did not experience intolerable side effects, this was considered as treatment success referring to our secondary endpoint for toler-ability.

STATISTICAL ANALYSISThe results for primary and secondary outcome measures were ana-lyzed on per-protocol basis. Considering the discrepancy between

Figure 1: Subject enrollment and follow-up



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rgTable 1: Baseline demographic data of women who were eligible for final analysis

SG groupn (%)

ER groupn (%) p

Number of women 27 30 _

Age [yrs] (mean + SD) 32.07 ± 7.95 30.77 ± 7.17 0.5869

Past history of similar illness [Yes/ No] 12/15 16/14 0.6860

Other Medical illness-Diabetes mellitus 2 0 0.4398

Clinical Diagnosis-Bacterial vaginosisCandidiasisMixed Infection

1 (3.70)7 (25.93)

19 (70.37)

1 (3.30)9 (30)

20 (66.67)

>0.9990.9648 0.9896

Length of duration for visit 2 (days) 8.56 + 2.81 8.21 + 5.09 0.734

Length of duration for visit 3 (days) 28.13 + 1.76 28.47 + 1.58 0.422

pH>4.5 22 (81.48) 22 (73.33) 0.6806

Malodorous discharge 6 (22.22) 5 (16.67) 0.8437

Itching at baseline 16 (59.26) 18 (60) >0.999

Microbiological findings [Mixed classified according to respective pathogens]:Abnormal vaginal bacterial flora 7 (25.93) 14 (46.67) 0.1774

Grade 2 4 (14.81) 2 (6.67) 0.5706

Grade 3 3 (11.11) 12 (40.00) * 0.0271

Pus cells 11 (40.74) 8 (26.67) 0.3988

Candidiasis 9 (33.33) 8 (26.67) 0.7944

Candida albicans 7 (25.93) 4 (13.33) 0.3865

Non-albicans Candida 2 (7.41) 4 (13.33) 0.7749

Trichomoniasis 1 (3.70) 2 (6.67) >0.999

Table 2: Effect on Primary and Secondary outcome measures for effectiveness at 8th day [Visit 2] and 29th day [Visit 3] of the study

Treatment group

Visit 1 Visit 2 Visit 3Total cases with

proven infection

Global complete

curen (%)

Global partial

remission n (%)

Global failure of

therapy n (%)

Global maintenance of remission

n (%)

Global delayed

cure n (%)

Global relapse n (%)

Global complete failure of therapy n

(%)SG group 13 9 (69.23) 1 (7.69) 3 (23.1) 8 (88.89) 1 (25) 1 (11.11) 3 (23.08)

ER group 19 14 (73.68) 0 (0) 5 (26.3) 12 (85.71) 3 (60) 2 (14.29) 2 (10.53)

Relativerisk(95% CI)

- 0.940( 0.598-1.475) - 0.877

( 0.252-3.047)1.037

(0.757-1.421)0.417

(0.066-2.63)0.778

(0.082-7.376)2.192

(0.424-11.35)

p (Fisher exact) - >0.999 0.8125 >0.999 >0.999 0.7143 >0.999 0.6334

maintenance of remission in 85.71% women, relapse in 14.29% and delayed remission in 60% (Table 2).

Of all women with microbiologically proven infection at baseline, there was total failure of therapy in 23.08% of the 13 women in SG-group and 10.53% of the 19 women in ER-group.

Secondary outcome measure for tolerability None of the women complained of intolerable side effects or discon-tinued treatment because of side effects.

The two treatment groups did not differ statistically with respect to primary or secondary outcome measures.

MICROBIOLOGICAL FINDINGSOverall complete microbiological cure was seen in 76.47% women in SG-group and 83.33% women in ER-group at second visit and 82.35% versus 83.33% respectively at third visit.

On analyzing individual infections, it was observed that, at second study visit, 85.71% women with ABVF in both treatment groups showed complete microbiological cure. A similar proportion of women from both treatment groups had complete microbiological cure at third visit. One woman from SG-group developed de novo ABVF and one with partial cure at second visit showed relapse at third visit. In the ER-group, there was one woman with relapse and one with delayed cure.

Soft gelatin capsule produced cure in 77.78% women with candi-diasis, which was maintained upto third visit. The extended-release tablet produced cure in 100% of women with candidiasis at second visit, but 25% of them relapsed providing net cure rate of 75% at third visit.

None of the women with trichomoniasis showed recovery at second visit but both the groups had a 100% cure at third visit

Thus, the two treatment groups did not differ with respect to the microbiological cures for various types of vaginal infections. Simi-larly, the two treatments did not differ with respect to delayed cure, relapse, new infection, and abolition of lactobacilli, grade 4 vaginal flora or effect on pus cells (Table 3, Figure 2).

CLINICAL FINDINGSAt second visit, there was complete remission of clinical manifesta-tions in 59.26% and partial remission in 40.74% women of SG-group.



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Table 3: Microbiological effects on vaginal bacterial flora

SG groupn (%)

ER groupn (%)

Relative risk(95% CI)

p(Fisher exact)

VISIT 2Worsening of bacterial flora [Grade 2 to Grade 3] 0 (0.00) 1 (50.00) - 0.667

Non-responding [no change in micro diagnosis + worsening]

0 (0.00) 2 (14.29) - 0.867

Cases showing Abolition of lactobacilli [Grade 0] 10 (37.04) 15 (50.00) 0.7407

(0.403, 1.361) 0.474

Abolition of lactobacilli in patients with proven BV on 1st visit

3 (42.86) 6 (42.86) 1(0.351, 2.851) >0.999

Recent Abolition of lactobacilli in patients with no BV on 1st visit

7 (35.00) 9 (56.25) 0.622(0.298, 1.3) 0.3488

Grade 4 [BV cured but Gram stain showed aerobic vaginitis flora]

1 (3.70) 1 (3.33) 1.111(0.073, 16.91) >0.999

Pus cells persisted 2 (18.18) 4 (50.00) 0.364(0.087, 1.523) 0.3313

New appearance of pus cells 2 (12.50) 1 (4.55) 2.75

(0.272, 27.77) 0.759

VISIT 3Delayed cure 0 (0.00) 1 (7.14) - >0.999

Relapse 1 (14.29) 1 (8.33) 1.714(0.126, 23.32) >0.999

De novo abnormality of bacterial flora [Grade 2] 1 (5.00) 0 (0.00) - >0.999

Non-responding [no change in micro diagnosis + worsening + relapse]

1 (14.29) 2 (14.29) 1(0.108, 9.228) >0.999

Sample showing GRADE 0- [TOTAL] 6 (22.22) 9 (30.00) 0.741

(0.303, 1.808) 0.718

Persistent abolition of lactobacilli 4 (40.00) 4 (26.67) 1.5

(0.484, 4.651) 0.786

Delayed abolition of lactobacilli at 3rd visit 2 (11.76) 5 (33.33) 0.353

(0.08, 1.559) 0.298

Pus cells persisted after second visit 1 (25.00) 1 (20.00) 1.25

(0.109, 14.34) >0.999

8th Day [Visit 2] 29th Day [Visit 3]

Figure. 2: Microbiological cure at second and third visit - percentage of infections



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SG groupn (%)

ER groupn (%)

Relative risk(95% CI)

p(Fisher exact)

VISIT 2Discharge reduced by atleast 1 unit 24 (88.89) 24 (80.00) 1.111

(0.889, 1.389) 0.583

pH normalized [<4.5] 14 (63.64) 16 (72.73) 0.875(0.583, 1.314) 0.747

Discharge malodor treated 6 (100.00) 5 (100.00) 1(1,1) >0.999

Itching/ Pruritis reduced by atleast 1 unit 15 (93.75) 15 (83.33) 1.125

(0.883, 1.433) 0.695

Adverse effect [patients]0.667

0.830

3 (11.11) 5 (16.67) (0.176, 2.53)

LeakageMild vaginal irritationMild lower abdominal painMild burning/ irritation during micturition

1110

1112

VISIT 3

Clinical Relapse 2 (7.41) 1 (3.70) 2(0.193, 20.77) >0.999

pH normalized [<4.5] by end of visit 3 15 (68.18) 16 (72.73) 0.938

(0.639, 1.375) >0.999

pH remained normalized [<4.5] from visit 2 12 (85.71) 11 (68.75) 1.247

(0.841, 1.848) 0.512

Discharge reduced by atleast 1 unit by end of visit 3

27 (100.00) 26 (86.67) 1.154(1.003, 1.328) 0.139

Discharge remained reduced by atleast 1 unit from visit 2

24 (100.00) 22 (91.67) 1.091(0.967, 1.231) 0.489

Discharge malodor treated 6 (100.00) 5 (100.00) 1(1,1) >0.999

Itching/ Pruritis reduced by atleast 1 unit 16 (100.00) 18 (100.00) 1

(1,1) >0.999

Figure 3: Clinical response at second and third visit- percentage of subjects

In ER-group, complete clinical remission was seen in 53.33% and par-

tial remission in 36.67% women.

By third visit, overall complete clinical cure was observed in 88.89%

women from SG-group and 86.67% women from ER-group; while

partial remission was observed in 3.7% women from SG group and

10% women from ER-group. Two women from the SG-group and one woman from the ER-group showed clinical signs suggestive of relapse. Both treatment groups showed similar effectiveness at nor-malizing vaginal discharge, pruritis, malodor and vaginal pH (Table 4, Figure 3). Thus, the two treatment groups did not differ with respect to clinical cure in women at second or third visit.



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DISCUSSIONIn the present study, there was a significant discrepancy in the clinical and microbiological diagnoses. According to clinical diagnosis only 3.5% cases had bacterial vaginosis, 28.07% had candidiasis; none had trichomoniasis and 68.42% had mixed infection, while according to microbiological diagnosis 37.5% women had abnormal vaginal bac-terial flora, 31.25% had candidiasis; 3.13% had trichomoniasis and 28.13% had mixed infection. Furthermore, 32 women [56.14%] had a microbiological diagnosis of infection while the rest had symptoms but did not have microbiological evidence of vaginal infection. The clinical diagnosis matched with microbiological diagnosis in only 10 of 57 women. These findings are in concurrence with reports show-ing that microbiological diagnosis can’t be reached using diagnostic approaches in 10-58% of the women with vaginal discharge5 and classifications like candidiasis, trichomoniasis, and bacterial vagino-sis are insufficient to explain all symptoms. In a study by Donders et al, a group of women with symptoms of vaginitis were found to have abnormal vaginal findings characterized by clue cell-negative, lactobacillus-negative, abundant pus cells, foul smelling vaginal dis-charge negative for Whiff test and pH> 6, in whom metronidazole was highly ineffective.18 These women have aerobic vaginitis which is not detected by Amsel’s criteria23 or Nugent’s score24 resulting in a diagnostic dilemma. In our study, 19 out of 57 women had pus cells in the vaginal smear at baseline. The presence of pus cells, though nor-mally suggestive of bacterial infection, was unrelated to presence of microbiological evidence of bacterial vaginosis and was thus indica-tive of some other infection.

In SG-group these pus cells disappeared in nine out of 11 cases [81.82%] but there was new appearance of pus cells in two more cases. One out of these four cases [25%] having pus cells at second visit showed persistence of pus cells at the third visit.

In ER-group these pus cells disappeared in four out of eight cases [50%] and there was one case with de novo appearance of pus cells. One out of these five cases [20%] with pus cells at second visit showed persistence of pus cells even at third visit.

Thus, in addition to comparing the two dosage forms, we also tried to find out whether empirical treatment of such women with an intra-vaginal combination of clindamycin and clotrimazole could provide clinical as well as microbiological cure.

The observations suggest that both intra-vaginal dosage forms con-taining clindamycin and clotrimazole combination produced clinical and microbiological cure in substantial number of women with clini-cal diagnosis of vaginitis and were able to maintain remission when used as empirical therapy. However, both the dosage forms caused marked abolition of lactobacilli. These findings are consistent with reports of inhibitory effects of clindamycin on lactobacilli in vitro.25 Furthermore, this study did not find evidence that the two formula-tions differed with respect to their effects on microbiological find-ings, overall clinical cure, or individual symptoms and signs.

However, a closer look at the microbiological findings suggests a dif-ference of greater than 20% in some parameters that did not reflect in the statistical tests, probably because of the small number of patients in subgroup analyses.

In case of women with grade 3 ABVF, extended-release tablet showed a complete microbiological cure in 91.67% (11 of 12) women against

66.67% (2 of 3) women receiving soft gelatin capsule [NNT=4]. On the contrary, in women with grade2 ABVF, the extended-release tablet produced complete microbiological cure in only 50% (1 of 2) women compared to 100% (4 of 4) women in the SG-group [NNH=2] and was associated with a relatively greater chance of worsening from grade 2 to grade3 ABVF as well as abolition of lactobacilli in case of women who did not have ABVF at baseline [NNH=4.7]. Therefore, it appears that the extended-release tablet, though more rapidly effective in women with grade3 ABVF, should be used judiciously. Further, the extended-release formulation was relatively slow in clearing pus cells.

In both groups, trichomoniasis showed no response at second visit but complete cure at final visit. This is probably because neither clindamycin nor clotrimazole have strong anti-trichomonal action or that the anti-trichomonal action, if any, follows a lag period. Also, as trichomonads may reside in the urethra, perivaginal glands and the crypts of the vagina, it is reasonable that topically applied antimicro-bials may not work as well as the systemic treatments.12

CONCLUSIONEmpirical treatment with both intra-vaginal dosage forms contain-ing clindamycin with clotrimazole produced clinical and microbio-logical cure as well as maintained remission in substantial number of women with clinical diagnosis of vaginal infection. Amongst indi-vidual infections, trichomoniasis showed least response. Neither of the two formulations could treat trichomoniasis by end of first week, though there was a delayed response of similar magnitude with both formulations. Thus, a clinical or microbiological diagnosis of tricho-moniasis should essentially prompt systemic treatment of both sex-ual partners with metronidazole or tinidazole.

Secondly, although this study did not find evidence that the two formulations differed with respect to efficacy or tolerability, in the context of treatment of candidiasis, abnormal vaginal bacterial flora, and abolition of lactobacilli, the magnitude of difference (in terms of percentage) was large enough to be clinically relevant.

Being a pilot study, with a small sample size, it is not possible to draw valid conclusions regarding the differences between clini-cal and microbiological efficacy of the two formulations. Hence, an adequately powered study using a larger population should be con-ducted to further explore differences between soft gelatin capsule and extended-release tablet on these parameters.


ACKNOWLEDGEMENTSThe authors of this study are thankful to Resilient Cosmeceuticals Pvt Ltd, Pune and their Medical Advisor, Dr Kedar Joshi for supplying the drugs gratis, paying conveyance at the rate of Rupees 100/- per visit to all the study participants and providing HPLC consumables required for studying the in vitro release pattern. However, they had no role in preparing or submitting the manuscript for publication.

REFERENCES1. Bradshaw CS, Morton AN, Hocking J, Garland SM, Morris MB, Moss LM, et al.

High recurrence rates of bacterial vaginosis over the course of 12 months after oral metronidazole therapy and factors associated with recurrence. J Infect Dis. 2006; 193(11): 1478-86.

2. Boris J, Påhlson C, Larsson PG. Six years observation after successful treatment of bacterial vaginosis. Infect Dis Obstet Gynecol. 1997; 5(4): 297–302.

3. International Planned Parenthood Federation IPPF. International Medical Advisory Panel IMAP. International Planned Parenthood Federation (IPPF). Statement on sexually transmitted diseases and reproductive health. IPPF Med Bull. 1988; 22(6): 3-4.

4. World Health Organization Guidelines for the management of sexually transmitted infections, Geneva; WHO. Available from http://whqlibdoc.who.int/publications/2003/9241546263.pdf Published 2003. Accessed September 20, 2012.

5. Rekha S, Jyothi S. Comparison of visual, clinical and microbiological diagnosis of symptomatic vaginal discharge in the reproductive age group. Int J Pharm and Biomed Res. 2010; I(4): 144-8.

6. Schwiertz A, Taras D, Rusch K, Rusch V. Throwing the dice for the diagnosis of vaginal complaints? Ann Clin Microbiol Antimicrob. 2006; 5(1): 4.

7. CepickÃ P, Malina J, LÃ-balovÃ Z, Kuzelov ÃM. Mixed and miscellaneous vulvovaginitis: diagnostics and therapy of vaginal administration of nystatin and nifuratel. Ceska Gynekol. 2005; 70(3): 232-7.

8. National Guidelines on Prevention, Management and Control of Reproductive Tract Infections including Sexually Transmitted Infections Produced and published by National AIDS Control Organisation Ministry of Health & Family Welfare, Government of India August 2007.



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rg9. Oduyebo OO, Anorlu RI, Ogunsola FT. The effects of antimicrobial ther-apy on

bacterial vaginosis in non-pregnant women. Cochrane Database Syst Rev. July 2009; 8(3): CD006055.

10. Barry Hainer L, Maria Gibson V. Vaginitis: Diagnosis and Treatment. Am Fam Physician. 2011; 83(7): 807-15.

11. Nurbhai M, Grimshaw J, Watson M, Bond C, Mollison J, Ludbrook A. Oral versus intra-vaginal imidazole and triazole anti-fungal treatment of uncomplicated vulvovaginal candidiasis (thrush). Cochrane Database Syst Rev. October 2007; 17(4): CD002845.

12. Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines 2010. Diseases characterized by vaginal discharge. http://www.cdc.gov/std/treatment/ 2010/toc.htm.

13. John Bennett E. Antifungal agents. In: Laurence L. Bruntun, Bruce A Chabner, Bjorn C. Knollmann, editors. Goodman & Gilman’s The Pharamcological Basis of Therapeutics. 12th ed. Mc Graw Hill Medical Publication; 2011. p. 1571-92.

14. du Bouchet L, Spence MR, Rein MF, Danzig MR, McCormack WM. Multicenter comparison of clotrimazole vaginal tablets, oral metronidazole, and vaginal suppositories containing sulfanilamide, aminacrine hydrochloride, and allantoin in the treatment of symptomatic trichomoniasis. Sex Transm Dis. 1997 Mar; 24(3): 156-60.

15. Darvin Scott Smith, Natalia Ramos, Burke A Cunha. Trichomoniasis http:// emedicine.medscape. com/article/230617 Updated: Jun 29, 2012]

16. Stover KR, Riche DM, Gandy CL, Henderson H. What would we do without metronidazole? Am J Med Sci. 2012; 343(4): 316-9.

17. Conan MacDougall, Henry Cahmbers F. Protein Synthesis Inhibitors and miscellaneous antibacterial agents In: Laurence L. Bruntun, Bruce A Chabner, Bjorn C. Knollmann,

editors. Goodman & Gilman’s The Pharamcological Basis of Therapeutics. 12th ed. Mc Graw Hill Medical Publication; 2011. p. 1521-48.

18. Donders GG., Vereecken A, Bosmans E, Dekeersmaecker A, Salembier G, Spitz B. Definition of a type of abnormal vaginal flora that is distinct from bacterial vaginosis: aerobic vaginitis. BJOG. 2002; 109(1): 34–43.

19. Donders GG. Definition and classification of abnormal vaginal flora. Best Pract Res Clin Obstet Gynaecol. 2007; 21(3): 355-73.

20. Líbalová Z, Cepický P, Malina J, Stanslický K, Kuzelová M, Medalová Z, Sosnová K. Vulvovaginitis. Occurrence and importance of mixed and unclassifiable pictures. Ceska Gynekol. 2007; 72(1): 32-7.

21. Donders GG, Van Calsteren K, Bellen G, Reybrouck R, Van den Bosch T, Riphagen I, et al. Predictive value for preterm birth of abnormal vaginal flora, bacterial vaginosis and aerobic vaginitis during the first trimester of pregnancy. BJOG. 2009; 116(10): 1315-24.

22. Sherrard J, Donders G, White D, Jensen JS. European (IUSTI/WHO) guideline on the management of vaginal discharge. Int J STD AIDS. 2011; 22(8): 421–9.

23. Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, Holmes KK. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am J Med. 1983; 74(1): 14–22.

24. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol. 1991; 29(2): 297–301.

25. Aroutcheva A, Simoes JA, Shott S, Faro S, The inhibitory effect of clindamycin on lactobacillus in vitro. Infect Dis Obstet Gynecol. 2001; 9(4): 239–44.



Case Report

Anti-Snake Venom (ASV) Induced Hypotension: An Emergent Complication of TreatmentYerramalli Roja Ramani, Bandana Rath, Uma Shankar Mishra, Himanshu Bhusan Sahu

Department of Pharmacology, Assistant Professor (Pharmacology), MKCG Medical College, Berhampur Ganjam Odisha, India

About Author : Dr. Yerramalli Roja RamaniAssistant Professor (Pharmacology),Department of Pharmacology,MKCG Medical College,Berhampur Ganjam Odisha, India.E-mail: [email protected]

DOI : 10.5530/PTB.1.1.10

ABSTRACT

Increased incidence of snake bites has been found, especially in coastal regions where tropical cyclones are very frequent. Anti-snake venom (ASV) is the only effective antidote and a snake bite victim is always vulnerable to its associated adverse reactions. It has to be used only in patients in whom the benefits of treatment are considered to exceed the risks of reactions. The present case was appropriate candidate for ASV administration and showed signs of improvement initially as seen in most such cases, but developed hypotension 24 hours later which persisted till the next 24 hours in spite of prompt management with dopamine and continuation of ASV. But it was seen that, following withdrawal of ASV on the third day the patient recovered. Therefore, such emergent reactions like hypotension can be avoided by cautious use and continuous monitoring of a patient on ASV.

Key words: Adverse reactions, Anti-snake venom, Coastal regions, Hypotension, Tropical cyclones.

INTRODUCTIONIncreased incidence of animal and insect bites has been found, espe-cially in coastal regions where tropical cyclones are very frequent.1 Snake bite is one of the frequently devastating aftermaths in these regions. It is a major public health problem with an estimated 1000 deaths per annum,2 and in India alone the mortality is suggested to be around 30,000.3 Anti-snake venom (ASV) is the only effective antidote which neutralizes the circulating venom. Even though only 20% of bites result in significant envenoming and need ASV therapy,4

its indiscriminate use in these areas commonly make the patient vul-nerable to related adverse effects. Here we present a case of hypo-tension induced by ASV.

CASE REPORTA 60-year-old woman was admitted to our hospital with diagnosis of snake bite on right lower limb. She came to the OPD 6hrs following the bite with a piece of cloth tightly tied to the limb. She was con-scious and complained of pain at the bite site. There was no history of use of any other medication, presence of any systemic disease or

allergy. On examination around 5 cm of bite site was swollen, ery-thematous. There were blisters all over the oral mucosa with swollen lips; peripheral pulses well palpable, heart rate 90/minute, respira-tory rate 40/minute and blood pressure of 90/60 mm Hg. On admis-sion, investigations like complete blood count, Hb%, blood urea nitrogen (BUN), serum creatinine, sodium, potassium, urine micros-copy, ECG and 20-minute-wholeblood clotting tests were done. Supportive treatment along with anti-snake venom (10 ml/ vial) by slow IV drip was administered. Following 10 vials of ASV injection on first day, the patient improved with all her vitals returning to normal. But 24 hours following improvement she suddenly developed signs of shock and her BP was 90/60 mm Hg. The patient was managed with dopamine (5 mcg/kg/minute) infusion along with ASV (8 vials). In spite of rigorous management, her condition detoriated and BP was found to be 80/60 mm Hg. Next day, the ASV was withdrawn and managed with fluids and injection dopamine. In the next 24 hours, the patient recovered from hypotension and was discharged on fifth day when her BP was 140/90 mm of Hg.

DISCUSSIONA most important decision in the management of a snake-bite victim is whether or not to administer ASV. The patient has to be assessed for the degree and severity of local or systemic envenoming. It is rec-ommended that it should be used only in patients in whom the ben-efits of treatment are considered to exceed the risks of Antivenom reactions.5 Prior to the present case, we the authors had encoun-tered three such cases with similar findings which went unreported. In the present case, initially the patient presented with swelling and


Ramani et al.: Anti-snake venom induced hypotension w

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rgerythema confined to the bite site, mild respiratory distress, hypo-tension with no significant neurotoxic signs, spontaneous systemic bleeding or renal failure. Investigation findings revealed mild leu-kocytosis, Hb 9 gm%, BUN 12 mg/dL (normal, 7-20 mg/dL), serum creatinine 1.0 mg/dL (normal, 0.6-1.2 mg/dL), sodium 145 mmol/L (normal, 135-155 mmol/L), potassium 2.7 mmol/L (normal, 3.5-5.5 mmol/L). 20-minutes-whole-blood clotting test and ECG were nor-mal. WHO recommends the initial dose of Indian polyvalent ASV as 100 ml.6 Accordingly, the patient received the appropriate dose of ASV initially and showed signs of improvement as seen in most such cases, but developed hypotension 24 hours later which persisted till the next 24 hours in spite of prompt management with dopamine and continuation of ASV. Here ASV was readministered on the 2nd day assuming that redistribution of snake venom from tissue into vascu-lar space might have occurred as a result of antivenin therapy. It has been noticed that, sudden removal of tourniquet can lead to a mas-sive surge of venom, leading to paralysis, hypotension, etc. Keeping in mind such consequences in the present case care was taken while removing the cloth tightly secured to the limb with which the patient had come to the hospital.

Antivenom is an immunoglobulin usually pepsin refined F(ab) frag-ments of IgG purified from the serum or the plasma of a horse or sheep that has been immunized with the venom of one or more species of snakes. Each ml of polyvalent ASV produced in India neutralizes 0.6 mg dried Indian cobra venom, 0.45 mg dried common krait venom, 0.6 mg of dried Russell’s viper venom and 0.45 mg of dried Saw-scaled viper venom.5 In the present case, lyophilized form of ASV was reconstituted with 10 ml water. Generally adverse reactions due to ASV mostly occur because of the foreign proteins, to preliminary sensitization of the patient to horse serum, or to the presence of immune complex. They are mostly of the following three types.4 Early anaphylactic reactions usu-ally develop within 10-180 minutes of starting antivenom. Pyrogenic (endotoxin) reactions like shaking chills (rigors), fever, vasodilatation and a fall in blood pressure usually develop 1-2 hours after treatment,

which may be due to pyrogen contamination during the manufactur-ing process. Late (serum sickness type) reactions develop 1-12 (mean 7) days after treatment. The persistent hypotension seen in present case does not fall into any of the above mentioned categories.Early hypoten-sion in snake bite cases is usually due to pooling of blood in the pulmo-nary and splanchnic vascular beds. In later stages, hemolysis and loss of intravascular volume into soft tissues may play important roles. Most cases respond to prompt fluid resuscitation with isotonic saline as seen in the present case of clinical shock. Vasopressors (e.g., dopamine) can be added if tissue perfusion fails to respond to volume resuscitation and antivenom administration should generally be continued as needed until the victim shows definite improvement (e.g., stabilized vital signs, reduced pain). As the patient did not respond to the above measures finally ASV was withdrawn following which she recovered.

CONCLUSIONRational use of ASV has to be practised in hospitals where there is regu-lar demand and resources are scarce. Continuous evaluation of a snake bite victim is essential from the time of admission. Emergent reactions like hypotension following ASV can be avoided by being vigilant and practicing its cautious use.

CONFLICTS OF INTERESTThe author(s) declare that there is no conflict of interest.

ACKNOWLEDGEMENTAuthors would like to thank the Department of Medicine for their coop-eration in collecting necessary information about the present case.

REFERENCES1. Patra Madhusmita. Tripathy Swarnamayee, Jena Indramani. Health Hazards by Sea

Cyclones in Odisha,the Supercyclone and the Phailin. Odisha Review. 2013; VOL. LXX (4): 30-7.

2. Jena I, Sarangi A. Snakes of Medical Importance and Snakebite Treatment. Ashish Publishing House, New Delhi, Behuria B, OdishareSaap (in Odia). and Venomous Snakes of Odisha; 1993.

3. Warrell DA. Injuries, envenoming, poisoning, and allergic reactions caused by animal. In: Warrell DA, Cox TN, Firth JD, Benj J Jr, editors. Oxford Textbook of Medicine. Oxford: Oxford University Press; 2003. pp. 923–45.

4. Simpson ID, Norris RL. Snake antivenom product guidelines in India. The Devil is in the Details. Wilderness Environ. Medicine. 2007; 18(3): 163-8.

5. Warrel, David A. Guidelines for the management of snake-bites. A SEARO Publication. WHO. 2010.

6. Mohapatra BN, Mohanty CBK. Guidelines for Anti Snake Venom Therapy. Medicine Update. 2010; 20(2): 426-30.

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