©2014 International Medical Press ISSN 1359-6535

Limited evolution but increasing trends of primary non-nucleoside reverse transcriptase inhibitor resistance mutations in therapy-naive HIV-1-infected individuals in India

Ujjwal Neogi, Soham Gupta, Riya Palchaudhuri, Shwetha D Rao, Suresh Shastri,Vishal Diwan, Ranbir S Laishram, Ayesha De Costa, Anita Shet Antiviral Therapy 2014; 10.3851/IMP2769 Submission date 2nd January 2014 Acceptance date 9th March 2014 Publication date 4th April 2014 For information about publishing your article in Antiviral Therapy go to http://www.intmedpress.com/index.cfm?pid=12

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Publication: Antiviral Therapy; Type: Short communication

DOI: 10.3851/IMP2769

Short communication

Limited evolution but increasing trends of primary non-nucleoside reverse transcriptase inhibitor resistance mutations in therapy-naive HIV-1-infected individuals in India Ujjwal Neogi1,2*, Soham Gupta3, Riya Palchaudhuri3, Shwetha D Rao3, Suresh Shastri4,Vishal Diwan5, Ranbir S Laishram6, Ayesha De Costa7, Anita Shet3,8 1Hematology Research Unit, Division of Molecular Medicine, St John’s Research Institute, Bangalore, India

2Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm,

Sweden

3Division of Public Health and Infections, St John’s Research Institute, Bangalore, India

4National AIDS Control Organisation, New Delhi, India

5Department of Public Health and Environment, RD Gardi Medical College, Ujjain, India

6Department of Pediatrics, Regional Institute of Medical Science, Imphal, India

7Department of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden

8Department of Pediatrics, St John’s Medical College Hospital, Bangalore, India

*Corresponding author e-mail: ujjwal.neogi@ki.se

Abstract:

Background: After the rapid scale-up of antiretroviral therapy (ART) in resource-limited settings, surveillance of primary drug resistance mutations (DRMs) among ART-naïve individuals has important public health benefits. Although a highly successful national ART-program initiated by the Government of India exists, data on the prevalence of primary-DRMs is scarce. The objective of the study is to estimate the prevalence, pattern and spectrum of population-based primary-DRMs in therapy-naïve HIV-1 infected individuals using clinical strains and database sequences from seven HIV-prevalence states of India.

Methods: Drug resistance genotyping was performed on either plasma RNA or whole blood genomic DNA using a validated in-house method on 170 HIV-1 positive therapy-naïve individuals. An additional 679 database-derived sequences from four other states were included in the analysis. The WHO recommended list of mutations (SDRM_2009) for nucleoside reverse-transcriptase inhibitors (NRTI) and non-nucleoside reverse-transcriptase inhibitors (NNRTI) were used for interpretation of DRMs. Trends of primary DRMs before and after the ART roll-out were studied.

Results: The overall prevalence of primary-DRMs was 2.6% in the selected states of India when clinical isolates as well as database-derived sequences were combined. Common mutations included T69D, D67N (NRTI-mutations) and L100I, K101E, K103N and Y181C (NNRTI-mutations). There was a significant increase in NNRTI-mutations over time.

Conclusions: The overall DRMs prevalence in this study was low. However, an increasing trend in primary NNRTI-resistance was observed during the last decade.

Publication: Antiviral Therapy; Type: Short communication

DOI: 10.3851/IMP2769

Establishment of HIV-drug resistance threshold surveillance will be useful in understanding further trends of transmitted resistance.

Accepted 9 March 2014, published online 4 April 2014

Short Title: Prevalence of primary DRMs in India

Introduction

The management of human immunodeficiency virus (HIV) infection has progressed significantly since the

introduction of antiretroviral therapy (ART). Administration of ART durably suppresses HIV-replication and is

known to reduce both morbidity and mortality associated with HIV-infection. A roadblock in the success of

the ART is the emergence of drug resistance. Transmission of HIV-1 isolates with drug resistance mutations

(DRM) to the next generation of patients can negatively impact prevention programs and therapeutic

strategies. Thus surveillance of primary DRMs has important public health benefits since the rapid scale-up

of ART was associated with increase in transmitted DRMs (TDRMs). Since the inception of the National

AIDS Control Program in the year 2004 to provide free ART, India has observed a rapid scale-up of ART

access. In resource rich settings, routine primary drug resistance testing is standard practise for those who

are initiating therapy. However this is not a part of routine standard of care in resource limited settings like

India. Previous studies from India report a variable prevalence of first-line nucleoside reverse-transcriptase

inhibitors (NRTI) and non-nucleoside reverse-transcriptase inhibitors (NNRTI) related DRMs in therapy naïve

patients ranging between 1.6% and 38% [1–8]. As observed by our group and others, the use of different

DRM lists to evaluate the DRM can result in differing prevalences and can sometimes report falsely elevated

prevalence of DRM [9,10]. Additionally, the other factors were the regional variance of the sampling,

subtypes under study and naturally occurring polymorphisms in non-B subtypes. A decade after the ART roll-

out in India, there is still no archetypal data on primary-DRMs in therapy-naïve individuals.

In India, it is not practical to get large number of samples from newly diagnosed cases spanning

across the different geographical corners of the nation. Hence, we aimed to estimate the prevalence, pattern

and spectrum of population-based primary-DRMs in therapy-naïve HIV-1 infected individuals by using clinical

strains and sequences obtained from a secondary database. Compiling this information would be useful

nationally for the purpose of TDR surveillance [11].

Methods

Between 2010 and 2012, blood samples were collected from HIV-1 seropositive antiretroviral-naïve subjects

(n=178) hailing from four different sites in three states of India, including Karnataka (Bangalore and Mysore;

n=103), Manipur (Imphal; n=50) and Madhya Pradesh (Ujjain; n=25). Majority of the patients (64.6%) were

newly diagnosed within 3 months at the time of sample collection and 58% of the patients were initiating first-

line of therapy. Plasma viral RNA (for Karnataka samples) and whole blood genomic DNA (for Madhya

Pradesh and Manipur samples) was extracted from the blood fraction. In view of non-availability of cold chain

transport, whole blood genomic DNA was used for Madhya Pradesh and Manipur samples. Our previous

study has shown a high concordance in the primary DRM profile in both the plasma viral RNA and whole

blood genomic DNA in therapy-naïve patients. We therefore believe that samples from both compartments

can reliably detect DRM in therapy-naïve individuals.

Publication: Antiviral Therapy; Type: Short communication

DOI: 10.3851/IMP2769

Partial pol gene was amplified and sequenced and subjected for drug resistance genotyping using a

validated in-house method as described by us previously [9]. Subtyping was carried out using maximum

likelihood (ML) phylogenetic tree with GTR+G+I model using the Molecular Evolutionary Genetics Analysis

software version 5.05 (MEGA 5.05) [12] with the reference sequences of different subtypes (n=170)

downloaded from HIV Los Alamos Database (“LANL”, www.hiv.lanl.gov).The sequences that were not

clustered with pure subtypes were further analysed for recombination in Recombination Identification

program version 3 (RIP 3.0) (www.hiv.lanl.gov).

Partial pol sequences comprising at least 41 to 235 amino acids of reverse transcriptase (RT) region

from therapy-naïve individuals from previous studies [1–3,6,7,13–16] were downloaded from the LANL. Only

non-identical sequences (n=679), without any frame shift, stop codons and APOBEC restrictions were

included in the analysis. Database sequences belonged to individuals from four states of India including

Tamil Nadu (n=307), Andhra Pradesh (n=47), Maharashtra (n=280), Delhi (n=61).

Drug resistance genotyping were interpreted using mutation lists provided by World Health

Organization (WHO) recommendations for Surveillance of Drug Resistance Mutations updated in 2009

(SDRM_2009) [17]. Based on our recent finding of low level prevalence of T215S and L74I mutations in

therapy failure patients, we have excluded these two minor nucleoside reverse transcriptase inhibitors

(NRTI) mutations [9].

The study was approved by Institutional Ethical Review Board, St. John’s Medical College Hospital,

Bangalore, India. Written informed consent was obtained from all the study participants prior to recruitment.

Results

Of the 178 patient samples successful amplification and sequencing was done in 170 individual samples,

[Bangalore (n=51), Mysore (n=51), Imphal (n=47) and Ujjain (n=21)]. Patient characteristics are presented in

table 1. Among the primary clinical sequences, HIV-1 subtype C has the highest prevalence with 90%

(153/170) of the infection followed by subtype B (4.7%; 8/170); recombinant strains (URF_BC) (4.7%; 8/170)

and subtype A1 (0.6%; 1/170).

The overall prevalence of DRMs in the clinical samples from the three states was 3.5% (6/170).

Baseline DRM prevalence in Karnataka (Bangalore and Mysore), Manipur (Imphal) and Madhya Pradesh

(Ujjain) was 3.9%, 2.1% and 4.8% respectively (Table 2). The prevalence of NRTI-mutations was 1.2%

(2/170), consisting of T69D and D67N mutations. The prevalence of NNRTI-mutations was 2.3% (4/170) with

K101E, K103N, Y181C and L100I+K103N mutations (Table 2).

For DRM analysis we used the modified SDRM_2009 mutation list; which excluded the mutations

T215S and L74I, as these two mutations were present at <0.5% in failing patients in our study [9]. If we

included the T215S mutation (observed in two samples), this resulted in the prevalence of primary DRM in

the study population still below the threshold level of <5% (4.7%, 8/170).

We observed no significant association of baseline DRMs with individual clinical and demographic

factors. Analysis of the database-derived sequences (n=679), also identified low prevalence of NRTI-related

DRMs (1%; 7/679) and NNRTI-related DRMs (1.3%; 9/679) with an overall prevalence of 2.4% (16/679)

which was broadly uniform across the country.

Publication: Antiviral Therapy; Type: Short communication

DOI: 10.3851/IMP2769

Combining both the clinical and database-derived sequences, the prevalence of primary DRMs was

2.5% (22/849) with 1.1% (9/849) NRTI-mutations and 1.5% (13/849) NNRTI-mutations (Table 2). The overall

prevalence in all the seven states studied is below 5% (Figure 1A). Temporal analysis of the sequences

based on the timing of sample collection within specific time periods of 2004 (before the national ART roll-

out became widespread), 2005-2009 and 2010 (after rapid scale up of ART) years revealed a significant

increase in NNRTI mutations: from 0.9% (2005-2009) to 3.6% (2010) (p<0.05) (Figure 1B). The prevalence

of NRTI mutations was observed to be stable through these time periods.

Discussion

The results from our study indicate that despite an overall increase in primary DRM in naïve-patients from

1.5% to 4.5% over a period of 10 years, the overall prevalence was found to be low (<5%) in all the seven

states of the country under study including the five high prevalence states. In the absence of a nationwide

surveillance data of TDRMs in India, we can extrapolate that the national prevalence of TDRMs is below 5%

based upon our current data on primary DRM among the therapy-naïve population as suggested by a recent

study [11]. It is also to be noted that 65% of the individuals included in the study were newly diagnosed.

A recent review showed that the prevalence of TDRM is greater in resource-rich settings such as

Europe and North America (10.9% to 12.9%) compared to resource-limited settings (4.2% to 6.3%), where

ART was introduced later [18]. A meta-regression analysis estimated that in East Africa there was an

increase of TDRMs from 0.9% to 7.4%, 8 years after ART roll-out [19]. In India, previous studies on drug

resistance surveillance studies among recently diagnosed individuals from Kakinada, Mumbai and Northern

India showed <5% TDRM prevalence [1–3]. However, level of primary DRMs among the therapy naïve

individuals from different parts of India reported the prevalence as moderate (5 to 15%) [5–7,13–15]. In our

study, the overall prevalence of reverse transcriptase DRM for Indian strains from naïve patients was

determined to be 2.5% and most of our samples and sequences originated from the high HIV-prevalent

states of India. There was a gradual increase in DRM in therapy-naïve patients over the last decade yet the

overall prevalence was below the WHO threshold level of drug resistance (<5%). Our study also showed that

there was significant rise in NNRTI-mutations and not in the NRTI-mutations during the course of DRM

evolution over the past decade. Similar results have been obtained in another low and middle income

country, Argentina, where the NNRTI mutations was increased from 2.1% in 2003–2005 to 5.9% in 2010–

2011. The rise in NNRTI-mutation might be due to use of drug nevirapine (NVP) which is considered to be

the drug-of-choice in India in the past decade. Our previous study in this setting also proposed in favour of

efavirenz (EFV) as a first-line NNRTI choice in the country while re-examining the use of NVP. This is

because four out of ten individuals failing on NNRTI-based therapy, acquired cross-resistance to second

generation of NNRTIs like etravirine (ETR) probably due to the usage of NVP in our setting [20]. The higher

ART exposure in the period after the rapid scale up of ART in 2005 may also have led to the apparent higher

prevalence seen among the sequences obtained between 2005 and 2009.

Our study has some inadvertent limitations. First, in the present study the samples are restricted only

to seven high prevalent states of India. Excluding Manipur and Madhya Pradesh, majority of the

samples/sequences used in this study were obtained from urban-settings. The reasons for this being limited

logistic challenges and availability of the drug resistance genotyping in the big cities. Further, the therapy-

Publication: Antiviral Therapy; Type: Short communication

DOI: 10.3851/IMP2769

naïve status of the patients was as per the self-report by the patients. Thus the risk of undisclosed prior ART

use and can have potential impact on HIVDR prevalence estimates. Nevertheless, primary DRM was

observed to be <5%. And finally, there might be an inherent selection bias of the samples, specifically

between the recently diagnosed patients and chronically infected patients. However the strength of our study

are noteworthy, and trump the limitations. This study represents the first nationwide compilation of data on

primary DRM profile. Additionally for the first time we reported DRM profile from Manipur and Ujjain, Madhya

Pradesh, remote areas bereft of sophisticated laboratory facilities

In conclusion, our study demonstrates a significant increase in NNRTI-mutations over a decade

despite low (2.5%) baseline prevalence of drug resistance. Although the prevalence is low and reassuring,

the successful rapid scale-up of access to first-line ART in India warrants an urgent need to establish an on-

going system of HIVDR surveillance as per WHO guidelines for HIVDR “Early Warning Indicators”. A

capacity building program for regional drug resistance monitoring should be prioritised to implement the drug

resistance genotyping in the country. This will not only help us to track the level of TDR at the population

level, but will also assist in the optimisation of future therapeutic regimens.

Acknowledgements

Authors would like to thank staff of the respective sites for their support in the recruitment of participants in this study. A special thanks to the HIVIND study team and the patients. Authors would like to thank National AIDS Control Organization (NACO) and Karnataka State AIDS Prevention Society (KSAPS) for their generous support. The authors are also grateful to the Manipur Network of Positive People in northeast India for their support. The study has been partially supported by European Union Framework Program 7 (EU FP7).

Conflict of interest

None

References

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Figure Legends:

Figure 1. Prevalence and trends of baseline DRM in India.

A. The prevalence distribution in study states was entered and a choropleth map was generated using ArcGIS® software (ESRI, NY, USA). (B). Trends of transmitted drug resistance over a period of 10 years. A total 849 patient samples and

database sequences collected in different time periods were included in the analysis. They were categorized based on

the year of sampling; 2004 (n=197), 2005-2009 (n=430) and 2010 (n=222). The DRM in the three time periods was 1.5%, 2.1% and 4.5% respectively, while NRTI was 1%, 1.2% and 0.9% respectively. The NNRTI mutations were 0.5%,

0.9% and 3.6% in 2004, 2005-2009, 2010. The p values between each time points were mentioned.

Publication: Antiviral Therapy; Type: Short communication

DOI: 10.3851/IMP2769

Table 1. Socio-demographic and clinical parameters of individuals included in this study

Bangalore (n=51)

Mysore (n=52)

Ujjain (n=25)

Imphal (n=50)

Age, mean (SD), years 40.5 (9.5) 35 (6) 30.5 (10.5) 35 (10.6)

Gender, No. (%) Male Female

33 (64.7%) 18 (35.3%)

33 (63.5%) 19 (36.5%)

13 (52%) 12 (48%)

44 (88%) 6 (12%)

Route of transmission, No. (%) Heterosexual Homosexual IVDU Vertical

51 (100%)

0 0 0

52 (100%)

0 0 0

22 (88%)

1 (4%) 0

2 (8%)

5 (10%)

0 44 (88%)

1 (2%)

Duration of sero-diagnosis, Median (IQR), days

18 (13-58) 14 (10-17) 66 (5-358) NA

CD4 cell count, Median (IQR), cells/mm

3

124 (71-238) 147 (83-228) 397 (191-566) 367 (298-550)

Viral Load, Mean (SD), Log copies/mL

5.62 (0.68) 5.63 (0.79) NA NA

Genotyping performed, No. (%) 51 (100%) 51 (98%) 21 (84%) 47 (94%)

HIV Subtype, No. (%) HIV-1C HIV-1 B HIV-1A1 Recombinants

51 (100%)

0 0 0

50 (98%)

0 1 (2%)

0

21 (100%)

0 0 0

31 (66%) 8 (17%)

0 8 (17%)

*NA- Not available

Publication: Antiviral Therapy; Type: Short communication

DOI: 10.3851/IMP2769

Table 2. Drug resistant mutation profile in clinical cohorts (Karnataka, Manipur and Madhya Pradesh) and database derived sequences (Andhra Pradesh, Tamil Nadu, Maharashtra and Delhi)

Mutations

Clinical Cohorts Database Sequences Total

Karnataka Manipur Madhya Pradesh

Andhra Pradesh

Tamil Nadu

Maharashtra New Delhi (n=849)

(n=102) (n=47) (n=21) (n=47) (n=303) (n=273) (n=56)

NRTI-Mutations 2 (1.9%) 0 0 0 2

(0.7%) 5 (1.8%) 0 9 (1.1%)

M41L 0 0 0 0 2

(0.7%) 0 0 2 (0.2%)

D67N 1 (1%) 0 0 0 0 1 (0.4%) 0 2 (0.2%)

T69D 1 (1%) 0 0 0 0 0 0 1 (0.1%)

M184V 0 0 0 0 0 2 (0.7%) 0 2 (0.2%)

K219R 0 0 0 0 0 1 (0.4%) 0 1 (0.1%)

M41L+M184V+T215Y

0 0 0 0 0 1 (0.4%) 0 1 (0.1%)

NNRTI-Mutations 2 (1.9%) 1 (2.1%) 1 (4.8%) 1 (2.1%) 2

(0.7%) 5 (1.8%)

1 (1.8%)

13 (1.5%)

K101E 1 (1%) 0 0 1 (2.1%) 0 0 0 2 (0.2%)

K103N/S 0 1 (2.1%) 0 0 0 2 (0.7%) 0 3 (0.4%)

V106M 0 0 0 0 0 2 (0.7%) 0 2 (0.2%)

Y181C 1 (1%) 0 0 0 1

(0.3%) 0 0 2 (0.2%)

G190A 0 0 0 0 1

(0.3%) 0

1 (1.8%)

2 (0.2%)

L100I+K103N 0 0 1 (4.8%) 0 0 0 0 1 (0.1%)

K103N+V106M 0 0 0 0 0 1 (0.3%) 0 1 (0.2%)

Overall 4 (3.9%) 1 (2.1%) 1 (4.8%) 1 (2.1%) 4

(1.3%) 10 (3.7%)

1 (1.8%)

22 (2.6%)