Detection of Drug Resistance Mutations in the Reverse Transcriptase Gene of HIV-1-Infected North Indian Population Failing First-Line Antiretroviral Therapy “A Follow-Up Cohort Study”

Abstract

We aim to characterize the drug resistance mutations in reverse transcriptase gene of HIV-1 subtype C-infected North Indian population in those who are failing first-line antiretroviral therapy (ART) and if these mutations are associated with mortality. We also attempted the assessment of switch over to second-line antiretroviral therapy in these patients. Based on the immunological marker CD4 count (<350 cubic/mm), 192 HIV/AIDS patients were selected and viral load was estimated in those who were enrolled from December 2009 to November 2016. Based on viral load, genotyping was carried out in 57 HIV-1 isolates (VL ≥1,000 copies/mL) by sequencing and drug resistance mutations were examined through the Stanford HIV Drug Resistance Database, USA. Among them, 21 (36.84%) first-line ART failure patients were shifted to second-line ART. These patients were followed for a period wide ranging from 10 months to 11 years. Drug resistance mutation M184V (ATG to GTA) (63.15%) associated with lamivudine and abacavir and K103N (AAG or AAA to AAU) (36.84%) associated with efavirenz and nevirapine were predominantly identified in first-line ART failure patients. During follow-up, it was observed that 3 out of 21 who were in second-line ART died, whereas 9 out of 36 died who were in the first-line ART. No mutation could be associated with mortality although TAM-2 mutations were absent in patients who died. This study indorses the need for a facility for viral load estimation and resistance monitoring in each treatment failure patient and availability of appropriate antiretroviral therapies.

Introduction

Acquired immune-deficiency syndrome (AIDS) is an infectious disease instigated by Human Immunodeficiency Virus-1 (HIV-1).¹ HIV-1 subtype C is the most frequently transmitted subtype in different geographical regions of the world.² HIV-1 subtype C in India start off from the South African lineage.³ Antiretroviral drugs have been used as a preventative tool for HIV-1-infected patients.⁴ The panel of first-line antiretrovirals (ARVs) zidovudine, lamivudine, tenofovir, efavirenz, nevirapine, abacavir, and stavudine and second-line drugs ritonavir, atazanavir, and lopinavir were recommended by National AIDS Control Organization (NACO), Govt. of India, for the treatment of HIV-infected patients.^5
–7

The long-term efficacy of the first-line antiretroviral therapy (ART) in Indian patients in two different clinical settings was evaluated.⁸ Drug resistance mutations were identified in the population of antiretroviral therapy-experienced patient.⁹ The HIV-1 subtype C reverse transcriptase gene sequences from Indian patients failing with a first-line ART d4T/AZT-3TC-NVP/EFV was characterized.¹⁰ The primary HIV drug resistance mutations K219R, L74V, K219N, and Y181C were reported in first-line ART patients in Northwest India.¹¹ The nucleoside reverse transcriptase inhibitors (NRTIs)-associated resistance mutations M184V, T215F, and K219E and nonnucleoside reverse transcriptase inhibitors (NNRTIs)-associated resistance mutations Y188L, K103N, and A98G were emerged in first-line ART failure of East Indian patients.¹² Resistance mutations in HIV-1 subtype C isolates from Indian patients of Mumbai failed to NRTIs and NNRTIs response.¹³ Patients of South Indian regions failing with first-line ART associated with NRTIs and NNRTIs mutations were reported.¹⁴

To monitor the prevalence of transmitted drug resistance mutations, the drug resistance testing among different risk groups is essential.¹⁵ Temporal trends of transmitted drug resistance were observed in the HIV-infected population.¹⁶ Study in a large Tanzanian cohort suggested that switch over to second-line ART helped in a selected group of patients who had shown good adherence. HIV was sheltered in the population experiencing the failure of first-line ART, switched over to second-line ART could prevent the emergence of drug resistance mutations.¹⁷ Several risk factors associated with mortality in treatment with first-line ART have been reported.¹⁸

The objective of the present study is to find out to the drug resistance mutations in reverse transcriptase (RT) gene of HIV-1-infected North Indian population in those who are failing first-line ART and if these mutations are associated with mortality. We also attempted the assessment of switch over to second-line antiretroviral therapy in these patients. We evaluated the virologic outcomes, pattern of regimens, and adherence of the patients, those who were recruited for the first-line ART from 1 year to ≥7 years, in the ART center of Sarojini Naidu Medical College, Agra, India. Various drug resistance mutations that emerged for NRTIs and NNRTIs were identified in the individual antiretroviral therapy users. Patients were followed-up for tracking the switching over to the second-line ART patients and to explore the mortality which might be associated with drug resistance during the therapy.

Materials and Methods

Study design

A cohort study was designed on the patients, those who were enrolled in the Antiretroviral Therapy Center, Sarojini Naidu Medical College, Agra, India.

Setting

The ART center is situated on the campus of S.N. Medical College, Agra, and supervised by two nodal officers, one senior medical officer, four counselors, one nurse, one laboratory technician, two data managers, and one pharmacist with a community care coordinator. This center is catering to the residents of Agra city and its periphery who are visiting the center for HIV-related issues. All the clients who were detected as HIV seropositive at Integrated Counseling and Testing Center (ICTC) are referred to ART center for therapy.

Participants

All age group adults and adolescents of 192 first-line ART patients were included in this cohort study.

Ethics approval

The study protocol was approved by the Institutional Ethics Committee. The Institute Ethics Committee is constituted according to the guidelines directed by the Indian Council of Medical Research, New Delhi.¹⁹ Written informed consent was obtained from everyone at the ART clinics.

Period of recruitment

The cohort study was dogged on first-line ART patients who were enrolled from the period of December 2009 to November 2016. Follow-up of the 57 first-line ART failure patients were led from May 2017 to August 2019, and this study is ongoing at the ART Center, Sarojini Naidu Medical College, Agra, India.

Inclusion criteria

In this cohort study, all age group adults and adolescents were included who had been initiated with ART for at least 6 months. Informed consent was obtained from all patients. The main criteria for inclusion were based on the immunological marker, that is, CD4⁺ count (<350/cubic mm). The viral load was estimated in all samples. Out of 192 first-line ART treatment failure patients, 57 patients were found to have higher viral load i:e ≥1,000 copies/mL. Genotyping was conducted on these 57 first-line ART patients with high viral load.

Exclusion criteria

The following patients were excluded from the study: (i) patients whose duration of ART follow-up was less than 6 months and (ii) patients with leprosy, invasive cervical cancer, cryptococcosis, Kaposi's sarcoma, lymphoma, schizophrenia and so on.

Exposure

(i)

Antiretroviral Therapy:

(A) First-Line ART (1) NRTIs/NTRI: Zidovudine, Lamivudine, Tenofovir, Stavudine, and Abacavir

(2)

NNRTIs: Nevirapine and Efavirenz

(B) Second-line ART: Atazanavir and Ritonavir

(ii)

Treatment Policy: The ART center adopted the treatment guidelines by NACO, Govt. of India.^5,20

Outcome (i) Viral load (Copies/mL)

(ii)

Viral Genotypes

(1)

NRTI-associated resistance mutations

(2)

NNRTI-associated resistance mutations

Data collection

A patient information leaflet was used for the collection of sociodemographic and clinical profiles.²¹

Sample size

The study was focused on 192 patients who received first-line ART for at least a period of 1 year up to a maximum of 7 years. After estimation of viral load and genotyping test, 57 first-line ART failure patients having viral load ≥1,000 copies/mL and second-line ART patients were included in the follow-up study.

Sample collection

Ten milliliters of blood sample was collected from each patient, those who were treated with first line ART and whose CD4 counts were <350 cubic/mm. Plasma was isolated by centrifugation at 2,000 rpm for 10 min at 4°C and stored at −80°C. Plasma samples were transported to National Institute for Research in Tuberculosis, Chennai, India, maintaining proper cold chain condition. Viral load and genotyping were performed at NIRT, Chennai.

Viral load

RNA was extracted using the Abbott m2000sp instrument with accessories and reagents. Viral load was done by using the Abbott automated m2000rt instrument stationed at National Institute for Research in Tuberculosis, Chennai. The samples with a viral load ≥1,000 copies/mL were considered for genotyping.

Adherence

The adherence was evaluated by calculating the duration in months from the date of enrollment at ART Center till the date of inclusion in the study upon the total exact duration in months of ART taken by the patient. This is termed as individual adherence of a patient.

Genotyping

The 935 bp protease (PR) and 750 bp RT gene fragments were obtained by first and second round polymerase chain reaction of DNA from viral RNA using the WHO dried blood spot primers and reaction conditions. In second round polymerase chain reaction, the designed primers PR3 start binding on 2,035 position and PR4 start binding on 2,970 position of the HXB2. In second round polymerase chain reaction, the designed primers RT3 start binding on 2,833 position and RT4 start binding on 3,583 position. The PR and the RT gene were sequenced by Sanger sequencing method using WHO dried blood spot protocol 2010 at the National Institute for Research in Tuberculosis, Chennai.²²

The primers, polymerase chain reaction (PCR), and sequencing reaction conditions are given in Supplementary File S1 of Supplementary Tables S1–S3. The reagents are given in the Supplementary File S2.

Analysis of genotyping

The HIV-1 subtype and drug resistance mutation of each sample was identified and interpreted using the Stanford HIV Drug Resistance Database.

Statistical methods

The sociodemographic and clinical data collected from patient information form and questionnaire were tabulated in the excel sheet. Statistical analysis was performed using Stata SE v11.0 (Stata Corp, College Station, TX). Comparative analyses of continuous variables between drug-resistant and drug-respondent patients were performed using Student's t-test. Mutation frequencies in alive and patients who died were comparatively analyzed using the Pearson chi² (χ ²) test. Strength of association was expressed as odds ratio with 95% confidence interval. The duration of regimen of study groups (alive and dead patients) was comparatively analyzed using Student's t-test. p < .05 was considered statistically significant for all analyses. The registration number of Institute Human Ethics Committee was ECR/257/Inst/ UP/ 2013. The ethics committee meeting was held on 27.5.2016 at National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra-282004 for the project entitled “Characterization of drug resistant HIV-1 mutants of Agra region, India by genomic and proteomic approaches”.

Results

Sociodemographic, immunological, and clinical profiles

Among the 192 patients enrolled in this the study, patients were classified into two categories based on RNA copies, that is, drug-resistant patients (first-line ART failure) (RNA copies ≥1,000/mL, N = 57) and drug-respondent patients (RNA copies ≤1,000 to <40 copies/mL to target not detected level, N = 135). There was reaction failure in six patients. The male to female ratio was significantly higher in drug responders than in drug-resistant patients (Chi² = 3.97, p = .04). There was no difference in the mean age between these two categories. The major mode of transmission was the heterosexual mode. The marital status as well as educational profile among subjects was similar in both the study groups, that is, drug-resistant and drug-respondent (p > .05) groups. The average baseline CD4 count was similar in both groups. The mean viral load was 123,510.4 RNA copies/mL in the drug-resistant group and 77.94 RNA copies/mL in the drug-respondent group. Adherence to the regimens was significantly different ( = 0.01) in these two groups, the adherence being higher in the drug-respondent group (mean = 97.47 months) and 96.06351 months in the drug-resistant group. The average months of first-line ART of 192 patients was 42.25 months. The average months of first-line ART of the drug-resistant patients were 45.36 months and the drug-respondent patients were 41.16 months.

The details of the sociodemographic, immunological, and clinical profiles of the study subjects are given in Tables 1–3.

Table 1.

Demographic Details with Mode of Transmission, Marital, and Education Status of Drug-Resistant and Drug-Respondent Patients

Characteristics	Drug-resistant patients (57)No. (%)	Drug-respondent patients (135)No. (%)	Pearson chi-squared test (χ²)	p value
Male	40 (70.18)	112 (82.96)
Female	17 (29.82)	23 (17.04)	3.97	.04^*
Age (Mean ± SD)	35.56 ± 10.84	37.21 ± 9.11		.27 (NS)
Range (Min-Max)	(14–70)	(19–65)
Mode of transmission
Heterosexual	40 (70.18)	98 (72.59)
Blood transfusion	5 (8.77)	5 (3.70)
Homosexual	2 (3.51)	3 (2.22)	3.17	.67 (NS)
Unknown	7 (12.28)	22 (16.30)
Mother to child transmission	1 (1.75)	1 (0.74)
Probable unsafe injection	2 (3.51)	6 (4.44)
Marital Status:
Married	44 (77.19)	101 (74.81)
Unmarried	7 (12.28)	14 (10.37)	1.57	.66 (NS)
Widow	6 (10.53)	17 (12.59)
Divorce	0 (0.00)	3 (2.22)
Education
Illiterate	25 (43.86)	54 (40.00)
Primary school	14 (24.56)	48 (35.56)	2.41	.49 (NS)
Secondary school	13 (22.81)	24 (17.78)
Graduate	5 (8.77)	9 (6.67)

Pearson chi-squared (χ ²) test was carried out for gender, mode of transmission, marital status, and education, Student's t test was carried out for age. p value with ^* signifies significant and (NS) = nonsignificant.

Table 2.

Social Habits of Drug-Resistant and Drug-Respondent Patients

Characteristics	Drug-resistant patients (57)No. (%)	Drug-respondent patients (135)No. (%)	Pearson chi-squared test (χ²)	p value
Habit of alcohol
Never	39 (68.42)	81 (60.00)
Past	8 (14.04)	24 (17.78)	1.25	.74 (NS)
Social	8 (14.04)	23 (17.04)
Habitual	2 (3.51)	7 (5.19)
Habit of smoking:
Never	43 (75.44)	81 (60.00)
Past	8 (14.04)	30 (22.22)	4.18	.12 (NS)
Current	6 (10.53)	24 (17.78)
Habit of Tobacco:
Never	49 (85.96)	94 (69.63)
Past	3 (5.26)	19 (14.07)	9.06	.02^*
Social	1 (1.75)	0 (0.00)
Current	4 (7.02)	22 (16.30)

Pearson chi-squared (χ ²) test was carried out. p value with ^* signifies significant and (NS) = nonsignificant.

Table 3.

Immunological and Clinical Profiles of Drug-Resistant and Drug-Respondent Patients

Characteristics	Drug-resistant patients (57)No. (%)	Drug-respondent patients (135)No. (%)	Pearson chi-squared test (χ²)	p value
CD4 counts
Mean baseline CD4 ± SD	178.82 ± 118.41	163.17 ± 111.42		.38 (NS)
Mean Total CD4 ± SD	228.25 ± 76.94	232.99 ± 76.14		.69 (NS)
Viral load in copies/mL
Mean Viral load ± SD	125310.4 ± 198312	77.94 ± 131.79		.00^*
Mean Adherence ± SD	96.06 ± 3.92	97.47 ± 3.03		.007^*
Opportunistic infections
Tuberculosis	15 (26.31)	37 (27.40)
Candidiasis	0 (0.00)	1 (0.74)
Ulcer	0 (0.00)	1 (0.74)	4.13	.53 (NS)
Herpes	0 (0.00)	1 (0.74)
Fever	1 (1.75)	1 (0.74)
First-line ART duration in months
Mean ± SD	44.68 ± 20.87	40.98 ± 18.18		.22 (NS)

Pearson chi-squared (χ ²) test was carried out for opportunistic infections. Student's t test was carried out for CD4 counts, drug adherence, and duration of first-line ART, p values with ^* signify significant and (NS) = nonsignificant.

Characteristics of patients living with first-line ART failure and the group who died after a duration of ART. After genotyping of 57 first-line ART failure patients, the drug resistance mutations of individual patients were analyzed. Some patients were transferred to second-line ART after consultation with the physician. Follow-up of these first-line ART failure patients and switched over to second-line ART patients is being continued at present. Among the 57 patients, 45 patients are still alive, continuing with the first-line and second-line ART and 12 patients died during first-line and second-line ART. At present 27 and 18 patients have been continuing with the first-line and second-line ART, respectively. The life span of the first-line and second-line ART patients was 28.48 months and 10.97 months, respectively. Total CD4 count was observed to be higher in alive than patients who died. This difference was nearly significant (p = .06). There was no significant difference in these two groups for all the other variables. The details of the comparative characteristics of the first-line ART failure alive and dead patients are given in Tables 4–6.

Table 4.

Comparative Analysis of Life Span and Risk Factors of HIV/AIDS Patients Who Failed First-Line Antiretroviral Therapy (Alive) and Who Died After a Certain Period with Antiretroviral Therapy (Dead)

Characteristics	Patients alive N = 45No. (%)	Patients dead N = 12No. (%)	Pearson chi-squared test (χ²)	p value
Gender
Male	3015 (66.67)	10 (83.33)	1.25	.26 (NS)
Female	15 (33 33)	02 (16.67)
Age in Years
Mean Age ± SD	35.55 ± 10.29	35.58 ± 13.24		.99 (NS)
Range (Min-Max)	20–70	14–62		.99 (NS)
Life span in months
Mean ± SD	28.48 ± 9.94	10.97 ± 6.81		.00^*
Range (Min-Max)	2.07–35.8	2.87–24.53		.00^*
Mode of transmission:
Heterosexual	32 (71.11)	8 (66.67)	15.48	.008^*
Blood transfusion	5 (11.11)	0 (0.00)
Homosexual	1 (2.22)	1 (8.33)
Unknown	7 (15.56)	0 (0.00)
Mother to child transmission	0 (0)	1 (8.33)
Probable unsafe injection	0 (0)	2 (16.67)

Pearson chi-squared (χ ²) test was carried out for comparison of gender, mode of transmission. Student's t test was carried out for comparing age and life span in months. p values with ^* signify significant and (NS) = nonsignificant.

Table 5.

Comparative Analysis of Clinical and Immunological Features with Drug Regimens of HIV/AIDS Patients Who Failed First-Line Antiretroviral Therapy (Alive) and Who Died After a Certain Period with Antiretroviral Therapy (Dead)

Characteristics	Patients alive N = 45	Patients dead N = 12	Patients dead N = 12
CD4 counts
Base line (Mean ± SD)	184.75 ± 122.37	156.58 ± 103.88	0. 46 (NS)
Total (Mean ± SD)	1,923.84 ± 1,252.53	1,195.41 ± 994.96	.06 (NS)
Viral load in Copies/mL
Mean ± SD	117,866.3 ± 211,183	153,225.6 ± 144,026	.58 (NS)
Adherence in months
(Mean ± SD)	96.39 ± 3.77	94.81 ± 4.37	.21 (NS)
Combination of Regimen
No., (Avg. Months ± SD) taken)
ZLE	13 (16.13 ± 13.96)	3 (13.05 ± 19.03)	.74 (NS)
ZLN	23 (27.76 ± 20.36)	3 (22.72 ± 23.12))	.69 (NS)
TLN	23 (22.60 ± 57.05)	5 (15.93 ± 12.99)	.79 (NS)
TLE	30 (22.70 ± 11.83)	11 (28.06 ± 16.61))	.25 (NS)
SLN	11 (9.95 ± 5.61)	2 (7.16 ± 1.43)	.51 (NS)

Mean value of each variable was compared using Student's t test and p-value was represented.

NS, nonsignificant.

Table 6.

Comparative Analysis of Duration of Antiretroviral Therapy Taken Between the Study Groups of HIV/AIDS Patients Who Failed First-Line Antiretroviral Therapy (Alive) and Who Died After a Certain Period with Antiretroviral Therapy (Dead)

Characteristics	Patients alive N = 45	Patients deadN = 12	p value
Total months in first-line ART
Mean ± SD	44.26 ± 20.36	46.24 ± 23.55	.77 (NS)
Total months in second-line ART
Mean ± SD	10.17 ± 9.18	10.87 ± 16.60	.91 (NS)
Proportion of patients in first-line ART till follow-up
No, (%)	27 (60)	9 (75)
Proportion of patients in second-line ART till follow-up
No, (%)	18 (40)	3 (25)
Types of second-line ART switch over	SL/ATV/RTV	TL/ATV/RTV
	TL/ATV/RTV	ZL/ATV/RTV
	ZL/ATV/RTV	AL/ATV/RTV

Mean total months in ART was compared between two groups by Student's t test and p-value was represented. NS, nonsignificant.

First-line ART: ZLE = Zidovudine +Lamivudine +Efavirenz, ZLN = Zidovudine +Lamivudine +Nevirapine, TLN = Tenofovir +Lamivudine +Nevirapine, TLE = Tenofovir +Lamivudine +Efavirenz, SLN = Stavudine +Lamivudine +Nevirapine.

Second-line ART: SL/ATV/RTV = Stavudine+ Lamivudine/Atazanavir/Ritonavir, TL/ATV/RTV = Tenofovir +Lamivudine/Atazanavir/Ritonavir, ZL/ATV/RTV = Zidovudine +Lamivudine/Atazanavir/Ritonavir, AL/ATV/RTV = Abacavir +Lamivudine/Atazanavir/Ritonavir.

Genotypic results

Genotyping data of 57 first-line ART failure patients (VL ≥1,000 copies/mL) were analyzed. Invariably, all 57 subjects were of the HIV-1 subtype-C. There was a total of 13 different mutations associated with resistance to a panel of NRTIs and 19 different mutations associated with resistance to two NNRTIs were identified in these patients. The list of NRTIs- and NNRTIs-associated mutations are given in the Tables 7 and 8, respectively. No mutation was detected in 10 (17.54%) and 5 (8.77%) of the study subjects associated with any of the NRTIs and NNRTIs resistance, respectively. The mutation M184V (ATG to GTA) associated with lamivudine and abacavir was identified in 36 (63.15%) first-line ART failure patients and was found to be the most prevalent. The mutation K103N (AAG or AAA to AAU) associated with efavirenz and nevirapine was identified in 21 (36.84%) first-line ART failure patients. The thymidine analog mutations-1 (TAM-1), M41L and T215Y, were present in 30.19%, whereas thymidine analog mutations-2 (TAM-2), D67N, K70R, T215F, and K219E/Q, were present in 56.12% of these patients. There were no TAMs-2 mutations associated with the group who died. After genotypic analysis, few patients were shifted to second-line ART. The average follow-up of second-line ART was 10.52 months. To analyze the possible association of the emergence of the NRTIs and NNRTIs drug resistance-associated mortality, all 57 subjects were categorized based on living or death status. The frequency of mutations M184V, K65R, and M41L associated with resistance to NRTIs in 45 alive patients was (63.15%), (21.05%), and (15.78%), respectively, whereas these frequencies were M184V (58.33%), K65R (16.66%), and M41L (16.66%) in 12 patients who died subsequently. The mutations V106M, G190A, Y181C, K103N, and V108I associated with resistance to NNRTIs were (15.55%, 22.22%, 33.33%, 40%, and 13.33%), respectively, in alive patients, whereas these frequencies were (16.66%, 8.33%, 8.33%, 0.00%, and 16.66%), respectively, in patients who died subsequently. The frequencies of prevalent NRTI and NNRTI-associated mutations were compared between these two categories using the Pearson chi square test. The K103N mutation was observed to be more frequent in patients who were alive and no mutation was detected in those patients who died during the study period (p = .007).

Table 7.

Drug Resistance Mutations Associated with Nucleoside Reverse Transcriptase Inhibitors in HIV/AIDS Patients Who Failed First-Line Antiretroviral Therapy (Alive) and Who Died After a Certain Period with Antiretroviral Therapy (Dead)

S.No.	DRAM	Total No. (%)N = 57	Alive No. (%)N = 45	Dead No. (%)N = 12	Pearson chi-squared test (χ²) (D.F.)	OR (95% CI)	p value
1	M41L	9 (15.78)	7 (15.5)	2 (16.66)	0.01 (1)	1.08 (0–5.47)	.92 (NS)
2	T215Y	5 (8.77)	3 (5.55)	2 (16.66)
3	M184V	36 (63.15)	29 (64.44)	7 (58.33)	0.15 (1)	0.77 (0.21–2.69)	.69 (NS)
4	D67N	5 (8.77)	5 (13.88)	0 (0.00)
5	K70R	7 (12.28)	7 (15.55)	0 (0.00)
6	T215F	6 (10.52)	6 (13.33)	0 (0.00)
7	K219E	4 (7.01)	4 (8.88)	0 (0.00)
8	K219Q	3 (5.26)	3 (6.66)	0 (0.00)
9	K65R	12 (21.05)	10 (22.22)	2 (16.66)	0.18 (1)	0.70 (0–3.38)	.67 (NS)
10	Y115F	3 (5.26)	3 (6.66)	0 (0.00)
11	K70G	1 (1.75)	1 (2.22)	0 (0.00)
12	M184I	2 (3.50)	1 (2.22)	1 (8.33)
13	K70E	1 (1.75)	1 (2.22)	0 (0.00)

N, number; DRAM, drug resistance-associated mutations; OR, odds ratio; D.F., degree of freedom; CI, confidence interval Pearson chi-squared test (χ ²) was carried out for frequencies of DRAM between HIV/AIDS patients who failed first-line ART (alive) and who died after a certain period with ART (dead).

NS, nonsignificant.

Table 8.

Drug Resistance Mutations Associated with Non-Nucleoside Reverse Transcriptase Inhibitors in HIV/AIDS Patients Who Failed First-Line Antiretroviral Therapy (Alive) and Who Died After a Certain Period with Antiretroviral Therapy (Dead)

S.N	DRAM	Total No. (%)N = 57	Alive No. (%)N = 45	Dead No. (%)N = 12	Pearson chi-squared test (χ²), (D.F.)	OR (95% CI)	p value
1	V106M	9 (15.78)	7 (15.55)	2 (16.66)	0.01 (1)	1.08 (0–5.47)	.92 (NS)
2	G190A	11 (19.29)	10 (22.22)	1 (8.33)	1.17 (1)	0.31 (0–2.2)	.27 (NS)
3	Y181C	16 (28.07)	15 (33.33)	1 (8.33)	2.93 (1)	0.18 (0–1.23)	.08 (NS)
4	H221Y	5 (8.77)	4 (8.88)	1 (8.33)
5	K103N	18 (31.57)	18 (40.00)	0 (0.0)	7.23 (1)		.007^*
6	A98G	5 (8.77)	5 (11.11)	0 (0.0)
7	G190S	3 (5.26)	2 (4.44)	1 (8.33)
8	Y318F	1 (1.75)	1 (2.22)	0 (0.00)
9	V108I	8 (14.03)	6 (13.33)	2 (16.66)	0.09 (1)	1.3 (0–6.7)	.76 (NS)
10	P225H	6 (10.52)	4 (8.88)	2 (16.66)
11	K101H	2 (3.50)	1 (2.22)	1 (8.33)
12	V179D	4 (7.0)	3 (6.66)	1 (8.33)
13	M230L	2 (3.50)	2 (4.44)	0 (0.00)
14	Y188L	2 (3.50)	1 (2.22)	1 (8.33)
15	K101E	4 (7.01)	3 (6.66)	1 (8.33)
16	G190E	1 (1.75)	0 (0.00)	1 (8.33)
17	K103S	2 (3.50)	2 (4.44)	0 (0.00)
18	L100I	1 (1.75)	1 (2.22)	0 (0.00)
19	F227L	2 (4.25)	1 (2.22)	1 (8.33)

K103N mutation was absent in HIV/AIDS patients who failed first-line ART and died.

N, number; DRAM, drug resistance associated mutations; OR, odds ratio; D.F., degree of freedom; CI, confidence interval Pearson chi-squared test (χ ²) was carried out for frequencies of DRAM between HIV/AIDS patients who failed first-line ART (alive) and who died (dead).

NS, nonsignificant.

Follow up observations

The second-line ART (SL/ATV/RTV, TL/ATV/RTV, and ZL/ATV/RTV) are being continued by 18 patients who are alive on an average for 10.17 months, whereas the average month of second-line ART (TL/ATV/RTV, ZL/ATV/RTV, and AL/ATV/RTV) was 10.87 months for those who died. The frequency of mutations was statistically analyzed between alive and patients who died using a Pearson chi-squared test. There was no significant difference in these two groups for NRTI-associated mutations. The association between the number of persons taken the particular therapy and the number of persons with respective drug resistance-associated mutations was evaluated. It was found that 21% of nevirapine therapy user had Y181C mutation, whereas 60% of nonuser had this mutation (p = .013). Similarly, K103N mutation was found in 26% of nevirapine user and 60% in persons who are not using nevirapine therapy (p = .03).

The details of genotype results and frequency are given in the Tables 7 and 8.

Discussion

Accumulation of high genetic diversity, including drug resistance mutations, has been addressed earlier.²³ The emergence of HIV drug resistance is a risk to the epidemic and is a threat to the population.²⁴ To control the drug resistance threat, HIV-1 drug resistance testing and analysis have been advocated in HIV-infected populations.²⁵ We conducted a cohort study with the first-line ART failure patients who attended the ART center, Sarojini Naidu Medical College, Agra, India. To characterize the polymerase gene of HIV-1-infected patients enrolled in first-line ART (1 to ≥7 years), in-depth sequencing analysis of PR and RT gene of HIV-1 was conducted. The NRTIs- and NNRTIs-associated drug resistance mutations were assessed in individual patients. The characterization of strains, drug resistance mutations, and types of first-line and second-line ART in individual patients have not yet been studied in ART users of the North Indian population. The origin and evolution dynamics of HIV-1 subtype C revealed a single South African lineage in the middle of the 1970s. Since the middle of 1970s, the subtype C is geographically distributed in different regions of this country.³ Here, we observed the incidence of HIV-1 subtype C-infected population in all drug-resistant patients. However, the other subtypes were reported from its neighboring regions like Aligarh, New Delhi, and so on.^26,27 The resistance mutations associated to NRTIs, NNRTIs, and PR inhibitors, etc. through genotyping have been carried out in several countries of the world.^8,28

–32

The point mutation M184V (ATG to GTA) associated with NRTIs resistance and K103N (AAG or AAA to AAU) associated with NNRTIs resistance was reported to be more frequent in patients with treatment failure.^33,34 Three nucleoside analogs (Abacavir, lamivudine, and tenofovir) are known to select K65R resistance mutation.³⁵ This K65R mutation was observed in 21.05% of treatment failure patients in our study. Therefore, the optimal antiretroviral therapy may be required to attenuate the emergence of the K65R mutation. The HIV drug resistance mutations in Indian adults treated with first-line ART were analyzed previously and a rising trend of K65R mutation was observed.³⁶

The frequency of M184V mutation associated with resistance to NRTIs and K103N mutation associated with resistance to NNRTIs was reported from South India.³⁷ In agreement with their observation, we also confirmed that the M184V and K103N were the most frequent mutations in HIV-1 subtype C-infected individuals in first-line ART failure patients in the North Indian population. As these patients were treated with lamivudine, tenofovir, abacavir, and stavudine (1 to ≥7 years), this K65R mutation was observed in 21.05% patients with ART for ≥7 years. This K65R mutation was also observed in HIV-1 subtype C-infected western Indian population.³⁸ Drug resistance study has been widely conducted in the different regions of India.^{11,12,38
–40} Mutations M184V and K103N in HIV-1 subtype C-infected South African patients receiving first-line ART were reported to be predominant.⁴¹ Thus, the frequent mutations in subtype C observed by us are probably of South African lineage. The major NNRTIs-associated resistance mutations V106M, Y181C, and K103N (minor) were reported in HIV-1 subtype C coinfected tuberculosis patients in South India.⁴² In our study, some patients had tuberculosis during ART. When we compared our NNRTIs-associated resistance mutation finding with South Indian patients, we found the major emerged NNRTIs-associated mutations V106M, Y181C, and K103N persevere in the North Indian population. High rates of drug resistance mutations in the HIV-infected population are a serious problem, and strategic switching to medications against HIV has been recommended by the experts.⁴³ Our study cohort is being followed by us and we are closely observing these patients who are switching over to the second-line ART. To know the impact of these mutations on the life span of first-line ART failure patients, we statistically analyze the major NRTIs- and NNRTIs-associated resistance mutations in live patients and patients who died. K103N resistance mutation associated with efavirenz and nevirapine were present in 40% of patients who were alive, whereas no mutation was there who died after some period. Although, it is inexplicable to know the impact of these mutations, death could have occurred due to other AIDS-defining conditions. When we correlated the major NRTIs and NNRTIs resistance mutations with the associated drugs, the Y181C resistance mutation was associated with nevirapine nonusers (p = .013) and the K103N resistance mutation was found to be associated with efavirenz and nevirapine nonusers (p = .037). Although at this point of time it is not possible to explain the reason, we found that efavirenz and nevirapine therapy did not suppress the virus in alive and dead patients. When we followed these patients, we found that 21 patients switched to second-line ART without efavirenz (Table 6) and nevirapine and 3 patients died among them. Possibly, the change in NNRTIs will be effective to controlling the viral load and live longer.

The TAM-2 mutations were observed in patients who are alive, whereas no TAM-2 mutations were observed in patients who died. The dynamic genotypic changes in the HIV-1-infected long-term treatment failure patients were reported in Yunnan province, China.⁴⁴ The TAM-2 mutation observed in the study group who are alive suggested the production of resistance to other NRTIs in these patients. This could be due to the therapy with zidovudine and stavudine for a long time in this group. TAM clustering phenomenon is probably happening in patients who are alive compared to those who died. The presence of TAM-2 in these patients was probably due to their regimen containing stavudine earlier. Although, the stavudine was phased out by the World Health Organization in the year 2013, the similar mutations persist in the alive patients at present.

In this study, the disease progression was observed to be variable in HIV-1 subtype C-infected patients, which could be due to acquirement of NRTI- and NNRTI-associated mutations. In both the groups the CD4 counts always declined ≤350 cubic/mm with high viral load (≥1,000 copies/mL) with acquired NRTI- and NNRTI-associated mutations. It was reported earlier that the rapid accumulation of the drug resistance mutations occurs when ART is continued in patients despite the virologic failure.⁴⁵ The accumulation of NRTI- and NNRTI-associated mutations in our study groups could be due to the changing of multiple first-line ART during the period of 1 year to over 6 years. Our findings also suggested the virological assessment in the first-line ART failure patients in the ART centers for better management of these patients.

No mutation was detected in the PR gene of 56 first-line ART failure North Indian patients. However, in another study, the PR gene mutation was observed in HIV-1-infected first-line ART naive populations from North India.²⁶ To the best of our knowledge, such kind of study has not been published from India in the long-term ART patients with follow-up second-line ART. This finding has a high value to focus on NNRTIs in the North Indian Population. The United Nations Program on HIV/AIDS (UNAIDS) collecting information from global, regional, and country levels for progress against 90-90-90 targets (90% of people living with HIV (PLHIV), 90% of people on treatment to know the status, 90% of people to suppress the virus on treatment).⁴⁶ This study findings on people living with HIV in the North Indian population may address the UNAIDS for 90-90-90 target.

Conclusion

Various NRTIs- and NNRTIs-associated mutations were observed in first-line ART failure HIV/AIDS patients with high viral load during 2–7 years of therapy. These patients were followed up. Among them 12 patients died. Although TAM-1 was present in both the groups, TAM-2 was present only in patients who were alive. No mutation could be associated with mortality. These findings would enhance our understanding of HIV drug resistance in this region.

This study emphasizes the need for a facility for viral load estimation for each patient on first-line and second-line ART and resistance monitoring in each treatment failure patient and for implementation of optimal ART combinations, which could cease the mortality rate in HIV-1-infected patients.

Gene Bank accession number

The sequences of 57 isolate from first-line ART failure AIDS patients were deposited in gene bank, NCBI. The gene bank accession numbers obtained are as follows: MG788697 to MG788753.

Footnotes

Acknowledgment

Indian Council of Medical Research, New Delhi, India Govt. of India is acknowledged.

Authors' Contributions

S.K.B. article write-up, reviewed article, sample collection and experiments, viral load and genotyping, data collection and data analysis; A.K.B. given idea on primary mutation data analysis; P.S.M. reviewed article; S.P.T. conception of the project and reviewed article; L.E.H. reviewed article; R.K. HIVDR data analysis; S.P. viral load and genotyping; T.P.S. patient follow-up; R.T. patient follow-up; S.T. patient counseling; S.J. reviewed article; S.A.P. reviewed article; K.K.M. supervision of experiment and data, reviewed article, data analysis, article write-up, and editing.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

The project “Characterization of drug-resistant HIV-1 mutants of Agra region, India by genomic and proteomic approaches” funded for senior research fellowship of Mr. Sushanta Kumar Barik (File No. 80/990/2015-ECD-I). Mr. M. M. Alam, JALMA, ICMR and Mr. Rakesh Kumar Mishra, ART Center, S.N. Medical College is acknowledged for providing technical help in the collection of blood samples.

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary File S1

Supplementary File S2

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