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Contents lists available at ScienceDirect

International Journal of Medical Microbiology

journa l homepage: www.e lsev ier .de / i jmm

enotypic variation of Pneumocystis jirovecii isolates in India based on sequenceiversity at mitochondrial large subunit rRNA

ashmi Guptaa, Bijay Ranjan Mirdhaa,∗, Randeep Guleriab, Sanjay Kumar Agarwalc,yotish Chandra Samantaraya, Lalit Kumard, Sushil Kumar Kabrae, Kalpana Luthra f,ishnubhatla Sreenivasg, Venkateswaran K. Iyerh

Dept. of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, IndiaDept. of Medicine, All India Institute of Medical Sciences, New Delhi, IndiaDept. of Nephrology, All India Institute of Medical Sciences, New Delhi, IndiaDept. of Medical Oncology, Institute Rotary Cancer Hospital (IRCH), All India Institute of Medical Sciences, New Delhi, IndiaDept. of Pediatrics, All India Institute of Medical Sciences, New Delhi, IndiaDept. of Biochemistry, All India Institute of Medical Sciences, New Delhi, IndiaDept. of Biostatistics, All India Institute of Medical Sciences, New Delhi, IndiaDept. of Pathology, All India Institute of Medical Sciences, New Delhi, India

r t i c l e i n f o

rticle history:eceived 3 February 2010eceived in revised form 12 May 2010ccepted 16 May 2010

eywords:neumocystis jiroveciit LSU rRNA genotypes

ndia

a b s t r a c t

Pneumocystis pneumonia (PCP), a common and serious opportunistic infection in immunocompromisedpatients, is caused by Pneumocystis jirovecii (formerly known as Pneumocystis carinii f. sp. hominis). Theaim of the present study was to describe the prevalence and distribution of genotypes of P. jirovecii basedon sequence polymorphisms at mitochondrial large subunit ribosomal RNA (mt LSU rRNA) region in bothHIV and non-HIV immunocompromised individuals with a positive PCR result for PCP in a tertiary healthcare centre in northern India. From January 2005 to October 2008, 50 patients [22 HIV-seropositiveindividuals, 10 post-renal transplant (PRT) recipients, 3 cancer patients, and 15 patients with variousother kinds of immunosuppression] were found to be positive for P. jirovecii using PCR at the mt LSUrRNA gene. Genotyping of the positive samples was performed at the mt LSU rRNA locus. Genotype 2 wasthe most common accounting for 42% of total types. This was followed by the genotypes 3 (24%), 1 (20%),and 4 (8%). Mixed infection was observed in 3 cases (6%). The rates of genotype distribution were similar

in HIV-seropositive individuals, cancer patients, and in patients with other kinds of immunosuppression.In the PRT recipients, genotype 1 was the most prevalent type (80%). This is the first study describingthe prevalence of genotypes in HIV-infected and HIV-uninfected, immunocompromised patients basedon the mt LSU rRNA gene from the Indian subcontinent. The most prevalent genotype observed was type2 in contrast to many studies from other parts of the world where genotype 1 was the most prevalent

hical

type, suggesting geograp

ntroduction

Pneumocystis pneumonia (PCP) caused by the opportunistic fun-al agent Pneumocystis jirovecii (formerly Pneumocystis carinii) isne of the most serious respiratory infections in immunocompro-ised patients, especially amongst human immunodeficiency virus

HIV)-infected individuals. PCP in recent times has also become

Please cite this article in press as: Gupta, R., et al., Genotypic variation of Pmitochondrial large subunit rRNA. Int. J. Med. Microbiol. (2010), doi:10.10

commonly encountered and serious opportunistic infection inIV-uninfected, immunosuppressed individuals, e.g., patients witheoplasia, organ transplant recipients, immunodeficient patients,nd individuals receiving immunosuppressive therapy for a pro-

∗ Corresponding author. Tel.: +91 11 26593508; fax: +91 11 26588641.E-mail address: mirdhabr@hotmail.com (B.R. Mirdha).

438-4221/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.oi:10.1016/j.ijmm.2010.05.001

variation.© 2010 Elsevier GmbH. All rights reserved.

longed duration. In spite of the significant advances made overthe last few years, much of the basic biology and epidemiol-ogy of P. jirovecii remains poorly understood. This is mainly dueto non-availability of an appropriate in vitro propagation sys-tem for the organism. Thus, molecular methods are employedfor isolation and characterization of P. jirovecii isolates world-wide.

Over the last 2 decades, a number of genes and gene frag-ments have been identified for potential use in typing of P. jiroveciiisolates. Commonly studied genetic loci for typing of P. jirovecii

neumocystis jirovecii isolates in India based on sequence diversity at16/j.ijmm.2010.05.001

include thymidylate synthase (TS) (Latouche et al., 1997a), aromlocus (Tsolaki et al., 1998), mitochondrial small subunit riboso-mal RNA (mt SSU rRNA) (Tsolaki et al., 1998), mitochondrial largesubunit ribosomal RNA (mt LSU rRNA) (Wakefield, 1996; Beardet al., 2000), internal transcribed spacers (ITS) of nuclear riboso-

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al RNA (Lee et al., 1998), and dihydropteroate synthase (DHPS)Helweg-Larsen et al., 1999). A number of typing methods suchs DNA sequencing, restriction fragment length polymorphismRFLP), single-stranded conformation polymorphism (SSCP), and

ajor surface glycoprotein (MSG) expression site typing have beenmployed for genotyping of P. jirovecii. Of all these methods, DNAequence analysis is so far the most common and informativeechnique. Based on these gene loci and by using various typing

ethods, genotypes of P. jirovecii have been described from differ-nt parts of the world.

Molecular diagnosis of P. jirovecii in our earlier studies havehown a prevalence rate of 12–15% (Gupta et al., 2007, 2009),owever, data regarding the genetic heterogeneity of P. jirovecii

solates in India were not available. Thus, in the present study, geno-ypic variations among P. jirovecii isolates were studied based onequence polymorphisms at the mt LSU rRNA region. This locusas considered as it is relatively stable and more easily detected

y PCR than single-copy nuclear genes due to the presence of mul-iple mitochondria in individual organisms. Furthermore, the mtSU rRNA locus has earlier been shown to be very useful to addressarious epidemiological questions related to human pneumocys-osis (Tsolaki et al., 1998; Montes-Cano et al., 2004; Beard et al.,005).

aterials and methods

amples

From January 2005 to October 2008, 395 clinical specimensincluding 174 bronchoalveolar lavage fluid (BALF) samples, 100putum samples (induced and expectorated), 40 tracheal aspiratesTA), 75 nasopharyngeal aspirates (NPA), and 6 gastric aspiratesGA)] were obtained from 323 immunocompromised patients clin-cally suspected of PCP. The patients included individuals bothisiting out-patient departments and those who had been admittedn various in-patient departments including Medicine, Nephrology,nd Transplantation, Pediatrics, and the Institute Rotary Cancerospital (IRCH) of our tertiary health care center. The 323 patients

ncluded in the study were divided into 4 major groups: 113IV-seropositive individuals, 44 post-renal transplant (PRT) recip-

ents, 78 cancer patients, and 88 individuals with various othermmunocompromised conditions (systemic lupus erythematosus,rimary immunodeficiencies, chronic renal failure, prolonged usef immunosuppressive therapy and various other conditions lead-ng to suppression of the immune system). An informed consent

as obtained from all the study participants, and ethical clearanceo conduct the study was obtained from our Institutional Ethicalommittee.

NA extraction and PCR amplification

The clinical samples were transported to the laboratory, andNA was extracted from the samples using commercially availableNA extraction kit by Qiagen (DNeasy tissue kit, Qiagen, USA). Weave earlier reported a high sensitivity of nested mt LSU rRNA PCR

or the detection of P. jirovecii (Gupta et al., 2009). Thus, detectionas carried out using nested PCR amplification of mitochondrial

arge subunit rRNA.The primers pAZ102E (5′-GATGGCTGTTTCCAAGCCCA-3′) and

AZ102H (5′-GTGTACGTTGCAAAGTACTC-3′) were used in the

Please cite this article in press as: Gupta, R., et al., Genotypic variation of Pmitochondrial large subunit rRNA. Int. J. Med. Microbiol. (2010), doi:10.10

xternal round (Wakefield et al., 1990). In the nested round, primersAZ102X (5′-GTGAAATACAAATCGGACTAGG-3′) and pAZ102Y (5′-CACTTAATATTAATTGGGGAGC-3′) were used (Wakefield, 1996).or the external round PCR, 2.5 �l of DNA were added to the reac-ion mixture containing 50 mM KCL, 10 mM Tris, 1.5 mM MgCl2,

PRESSical Microbiology xxx (2010) xxx–xxx

0.20 mM dNTPs, 0.4 �M primers, and 1.8 U Taq polymerase. PCRassay was carried out by an initial denaturation at 94 ◦C for 5 minfollowed by 35 cycles of 94 ◦C for 1 min, 56 ◦C for 1 min, and 72 ◦Cfor 1 min in ABI 2720, thermocycler (Applied Biosystems). For thenested round, 2 �l of 10 times diluted external round product wasadded into the reaction mixture containing 50 mM KCl, 10 mMTris, 1.5 mM MgCl2, 0.20 mM dNTPs, 0.4 �M primers, and 1.8 U Taqpolymerase. The PCR conditions for the nested round were initialdenaturation at 94 ◦C for 5 min followed by 25 cycles of 94 ◦C for1 min, 61 ◦C for 1 min, and 72 ◦C for 1 min. The sizes of the first roundand second round product were 346 bp and 267 bp, respectively.

To prevent cross contamination, stringent necessary measureswere taken. These included performing DNA extraction and pre-PCR and post-PCR procedures in different areas of the laboratory orin different rooms. The handling and DNA extraction of samples wasdone in a laminar flow cabinet. For all kinds of reagent transfers,filter barrier pipette tips were used. In each PCR run, multiple neg-ative controls (ultra-purified distilled water) and a positive control(P. jirovecii DNA obtained from one of our patients with PCP) wereincluded. In all positive cases, DNA extraction and PCR procedureswere repeated to check the reproducibility of results.

Clinical data

Varied demographic and clinical information were collectedfrom the patients. This included age, gender, symptoms at the timeof presentation, history of any previous episodes of PCP, currentPCP prophylaxis, chest X-ray findings, laboratory results includ-ing CD4+T cell counts in HIV-infected individuals, arterial bloodgas (ABG) analysis, other organisms identified in the specimen,treatment against other infections, anti-PCP treatment, and clinicaloutcomes.

Sequencing of P. jirovecii

The PCR-amplified products were purified using a PCR purifica-tion kit (QIAquick PCR purification kit, Qiagen) and in some casesby gel extraction kit (QIAquick gel extraction kit, Qiagen). Thepurified products were directly sequenced bidirectionally usingABI cycle sequencing kit. For each reaction, 3 �l of PCR-purifiedproducts, 2 �l of terminator ready mix, and 3.2 pm of primerwere added. The extension products were purified by ethanol andsodium acetate precipitation procedure to remove excess dye ter-minators. Each sample pellet was resuspended in 20 �l of Hidiformamide and denatured at 95 ◦C and snap-chilled. Electrophore-sis was carried out on the ABI 3130 XL Genetic Analyser (AppliedBiosystems, USA) in accordance with manufacturer’s recommen-dations. The sequences were analyzed using chromas lite software(Technelysium, Pty, Tewantin, Australia). Genotypes of P. jiroveciiwere distinguished by identifying polymorphisms at position 85and 248 of the mt LSU rRNA and were numbered using the methoddescribed by Beard et al. (2000). The sequences were aligned amongthemselves and with the prototype using Clustal W method ofsequence alignment using MEGA version 4.1 (Tamura et al., 2007).The sequence submitted by Sinclair et al. (1991) was used as pro-totype sequence.

neumocystis jirovecii isolates in India based on sequence diversity at16/j.ijmm.2010.05.001

Statistical analysis

STATA version 9.2 was used for all the analysis. The Chi-squareor Fisher’s exact tests were used to investigate the associationbetween qualitative variables at a significance level of 0.05.

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Table 1Frequency of various mt LSU rRNA genotypes observed in the study.

Genotype Number (%)

1 10 (20%)2 21 (42%)3 12 (24%)4 4 (8%)

Mixed 3 (6%)– (type 1 + type 3)– (type 1 + type 2)

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sputum were obtained, one individual showed type 2 in sputumand type 1 in BALF while the other individual showed the same

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mplification with specific primers

The mt LSU rRNA nested primers amplified a 267-bp product in0 (15.5%) of 323 patients enrolled in the study. Of these 50 patients,2 were HIV-infected, and 28 HIV-uninfected immunosuppressedatients. The HIV-uninfected immunosuppressed patients included0 PRT recipients, 3 patients with malignancies, and 15 patientsith various other kinds of immunosuppressive conditions. Theatients with various other kinds of immunosuppression com-rised of 3 patients with primary immunodeficiency, 2 each withystemic lupus erythematosus, Crohn’s disease, and prolonged usef steroids, and one each with myasthenia gravis, Cushing’s syn-rome, nephrotic syndrome, malnutrition, chronic liver disease,nd primary alveolar proteinosis. Altogether, 57 samples werebtained from 50 patients, with more than one clinical sampleeing obtained from 6 patients.

enotype frequency

At the mt LSU rRNA locus, all the genotypes were distin-uished on the basis of polymorphisms at nucleotide positions 85nd 248. Four unique genotypes were observed (Table 1). Geno-ype 2 (85A/248C) was most common accounting for 42% of allamples. The second most common genotype was genotype 385T/248C) (24%), followed by genotype 1 (85C/248C) (20%). Geno-ype 4 (85C/248T) was the least frequent type observed with a

Please cite this article in press as: Gupta, R., et al., Genotypic variation of Pmitochondrial large subunit rRNA. Int. J. Med. Microbiol. (2010), doi:10.10

requency of 8%. Mixed infection with 2 types was observed in 3ases (6%).

able 2t LSU rRNA genotypes observed among repeat samples from patients.

Patient isolate No. of samples positive Time interval T

PJ4 2 5 days BB

PJ77 3 2 days BBB

PJ123 2 2 days SB

PJ133 2 3 days BS

PJ232 2 2 months SS

PJ247 2 2 days SS

Fig. 1. Frequency (%) of mt LSU rRNA genotypes among different groups of patients(PRT: post-renal transplant recipients). Numbers above the bars indicate percent-ages.

Distribution of genotypes in different groups of patients

When distribution of genotypes was analyzed in differentgroups of patients (Fig. 1), it was observed that among the HIV-seropositive individuals, type 2 was the most prevalent genotype(40.9%), followed by genotype 3 (36.4%), and genotype 4 (9.1%).Genotype 1 was observed in only one HIV-positive patient. Mixedinfection (types 1 and 2, types 2 and 3) was observed in 2HIV-seropositive individuals. The same trend of prevalence ofgenotypes was observed in patients with various other kinds ofimmunocompromising conditions (Fig. 1). In case of PRT patients,it was observed that type 1 was the most frequent genotype,accounting for 80% of the total population. The presence ofgenotype 1 was significantly higher in PRT recipients (P < 0.05).All the 3 patients with malignant conditions showed genotype2.

No significant association could be established between mt LSUrRNA genotypes and the severity of disease (as measured by partialpressure of O2 < 60 mm Hg) (P = 0.493) or the outcome of the patient(P = 0.45).

In 2 patients with PCP, the same genotype was observed inrepeat BALF samples (Table 2). In 2 patients where both BALF and

neumocystis jirovecii isolates in India based on sequence diversity at16/j.ijmm.2010.05.001

type in both samples. There was no difference in genotypes of the 2sputum samples obtained from a patient at an interval of 2 months

ypes of samples Types observed

Bases at 85 and 248 Mt LSU rRNA type

AL CT Type 4AL CT Type 4

AL AC Type 2AL AC Type 2AL AC Type 2

putum AC Type 2AL CC Type 1

AL TC Type 3putum TC Type 3

putum AC Type 2putum AC Type 2

putum AC Type 2putum AC Type 2

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uring a single episode of PCP. In another patient, the same typeas observed in 2 sputum samples obtained at an interval of 2ays.

Fifty-seven samples analyzed from 50 PCR-positive patientsonsisted of 34 BALF, 19 sputum, and 4 TA. Among the 34 BALF, 1029%) had genotype 1, 12 (35%) had genotype 2, 5 (15%) had geno-ype 3, 5 (15%) had type 4, and mixed types were observed in 2 (6%)amples. Among the 19 sputum samples, type 2 was observed inhe majority of samples (63%), type 3 was observed in 6 (32%) sam-les, and type 1 was observed in just one sample (5%). Among theTA, type 3 was observed in 2 samples (50%), type 2 in one (25%),

nd mixed infection in one sample (25%).

iscussion

The present study was carried out to find out the genotype dis-ribution of P. jirovecii in our population by sequence analysis ofhe mt LSU rRNA region. Based on polymorphisms at 2 positions85 and 248), our study identified a high frequency of genotype 285A/85C) with a rate of 42%. Our findings are similar to those ofstudy carried out in the United States (Beard et al., 2005), whereenotype 2 accounted for 42.3% of total types. Another study con-ucted on 324 samples from 5 different cities of the United StatesBeard et al., 2000) showed more or less equal prevalence of geno-ype 1 (38%) and genotype 2 (36.7%). However, most of the studiesrom Europe reported genotype 1 to be the most prevalent typeFig. 2) (Miller et al., 2003, 2005; Montes-Cano et al., 2004; Estevest al., 2008). Similarly, a recent study from Australia showed thatenotype 1 accounted for 87% of all types (van Hal et al., 2009).n our study, genotype 1 accounted for only 20%. In contrast tohe above, genotype 3, the second most common type (24%) inur study, has been identified as the most frequent type (57%) in

Please cite this article in press as: Gupta, R., et al., Genotypic variation of Pmitochondrial large subunit rRNA. Int. J. Med. Microbiol. (2010), doi:10.10

study from Africa (Miller et al., 2003). The different prevalenceates of P. jirovecii types observed in different parts of the worldndicate that the epidemiological factors inherent to a particularrea (geographical and climate characteristics) may influence theistribution, circulation, and transmission of different P. jirovecii

f the world. The numbers inside the bar indicate percentages.

types. In an earlier study (Beard et al., 2000), allelic frequency geo-graphical distribution patterns of P. jirovecii isolates were found tobe associated with the place of diagnosis rather than the place ofbirth. The association with geographical distribution is consistentwith recent acquisition of clinical infections, although the actualsource of the infection is still difficult to ascertain. Thus, identifi-cation of a specific environmental reservoir for P. jirovecii, if oneexists, would help to elucidate the disease transmission as well asto improve efforts towards prevention.

We observed a similar pattern of genotype distribution inHIV-seropositive individuals and in patients with other immuno-suppressive conditions. This provides support to the concept ofa common source of infection. These results are in agreementwith earlier studies that suggested interindividual transmission inhumans (Totet et al., 2003) and in an animal model (Dumoulinet al., 2000). Furthermore, possible person-to-person transmis-sion of P. jirovecii has been strongly suggested by many studiesdescribing clusters of PCP cases (Rabodonirina et al., 2004; Höckeret al., 2005; de Boer et al., 2007; Schmoldt et al., 2008). In con-trast to HIV-infected and other immunocompromised patients, P.jirovecii isolates from post-renal transplant recipients showed ahigh frequency of genotype 1 (80%) in our study. Similar findings ofhigh prevalence of a particular genotype within a particular groupof patients have been reported. A high frequency of genotype 3has been described in colonized patients with severe chronic pul-monary disease (Montes-Cano et al., 2004), in immunocompetentinfants from USA (Beard et al., 2005), and in cystic fibrosis patientsfrom Spain (Montes-Cano et al., 2006, 2007). Beard et al. (2005)suggested that the differences in the genotype frequency observedbetween adults with AIDS and immunocompetent infants maybe caused by an independent acquisition cycle. However, a studyconducted on cystic fibrosis patients (Montes-Cano et al., 2007)

neumocystis jirovecii isolates in India based on sequence diversity at16/j.ijmm.2010.05.001

excluded this hypothesis and suggested that a different underly-ing condition might determine the presence of specific P. jiroveciimtLSU type.

Mixed infections (with more than one genotype in a sample)in Pneumocystis have been reported by several authors (Hauser et

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l., 1997, 2001; Beard et al., 2000; Miller et al., 2003). Hauser et al.2001) reported mixed infection with 2 types in 50% of the isolatesy SSCP analysis of 4 different genomic regions. The rate of mixed

nfections based on sequence analysis of the mt LSU rRNA locus haseen quite variable, ranging from 3.7% in Spain (Montes-Cano et al.,004) to 10.2% in USA (Beard et al., 2000). Mixed infections were

dentified in 6% of our isolates. The presence of mixed genotypesay indicate (i) coinfection, (ii) heterozygosity of diploid organ-

sms, or (iii) multi-copy gene with genotypic variations betweenhe copies. However, it has been shown through the analysis ofomplex SSCP patterns (Nahimana et al., 2000) that the presence ofeveral alleles in a single specimen was neither due to heterozygos-ty of diploid organisms nor due to the presence of multiple copiesf the genes, but most likely due to coinfection. Thus, the findingsndicate that P. jirovecii infection may not necessarily be clonal.

Among repeat specimens obtained from 5 patients, no changen the genotype at the mt LSU rRNA was observed in our study.his probably suggests that P. jirovecii does not mutate fre-uently in human hosts during an episode of PCP. Our resultsre in agreement with previous reports in Britain (Miller et al.,005) and France (Latouche et al., 1997b), in which no change

n genotype was observed in repeat BALF samples. However, webserved different genotypes in BALF and sputum samples takenrom one patient during an episode of PCP. In a study by Ma etl. (2002), genotype differences were observed in the upstream-onserved sequence (UCS) repeat region as well as in the mt LSURNA gene between sputum and BALF samples or between rightnd left lung BALF obtained from the same patient. This findingupports the hypothesis of heterogeneity and compartmentaliza-ion of P. jirovecii genotypes within the lungs (Helweg-Larsent al., 2001). These observations suggest that the interpretationf genotype data is needed to be made cautiously. Genotypingith respiratory samples cannot be assumed a priori to repre-

ent all genotypes present within the lungs (Helweg-Larsen et al.,001).

Similar to one of the earlier studies (Miller et al., 2005), we didot find any significant association between mt LSU rRNA geno-ypes and the severity of disease or the outcome in our patients.uch a lack of association further reinforces the fact that other lociight be involved in determining the virulence of this organism andarrants further studies. To our knowledge, this is the first studyescribing the prevalence of different genotypes on the basis of thet LSU rRNA gene from the Indian subcontinent. The study pro-

ides a step towards understanding the epidemiology of P. jiroveciinfection in a large country like India, and further investigation tostablish a potential relationship between genotypes and clinicalisease will be of help in the future.

cknowledgements

We thank all the patients who participated in the study. Finan-ial support for conducting the study was provided by Council ofndustrial and Scientific Research (CSIR), and the Department ofcience and Technology (DST), Government of India. We wouldlso like to acknowledge the Indian Council for Medical ResearchICMR), Department of Health Research, Government of India, forroviding fellowship to the first author, Mrs. Rashmi Gupta.

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