|Year : 2015 | Volume
| Issue : 2 | Page : 225-230
Emergence of HIV-1 drug-resistant variants in women following antiretroviral prophylaxis for the prevention of mother to child transmission
M Mani1, VV Ramalingam1, J Lionel2, SA Christina2, J Sachithanandham1, A Peedicayil2, R Kannangai1
1 Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Obstetrics and Gynecology, Christian Medical College, Vellore, Tamil Nadu, India
|Date of Submission||17-Feb-2014|
|Date of Acceptance||07-Oct-2014|
|Date of Web Publication||10-Apr-2015|
Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Purpose: Emergence of drug resistance following HIV prophylaxis has an important impact on ART program. Objective: To investigate the emergence of drug resistance in HIV-1 infected pregnant women. Materials and Methods: Fifty-three HIV-1 infected pregnant women who had received 4-12 weeks of antenatal AZT followed by Nevirapine during delivery and Combivir [AZT + 3TC] for 1 week postpartum (group-1, n = 48) or who come at the time of delivery and received Nevirapine followed by Combivir for 1 week (group-2, n = 5) were recruited. Samples were collected prior to the start of the prophylaxis and 5-8 weeks postpartum. In addition, a third sample was collected between 26-65 weeks postpartum from 7 women. Amplification of HIV-1 pol gene and drug resistance analysis was done. Result: Two (3.8%) women in group-1 showed transmitted drug resistance and they continued to show this even at 6 weeks postpartum. One (2%) woman from group-1 showed a mutation after 6-8 weeks of prophylaxis. Among the samples collected between 26-65 weeks postpartum, 3/7 (43%) showed mutations and all these women belong to group-1. Women belonging to group-2 didn't show mutation prior to or following prophylaxis. Conclusion: In contrast to the available data among pregnant women with ART prophylaxis, our data showed reduced frequency of mutations following 5-8 weeks of postpartum but an emergence of mutation later (26-65 weeks). The addition of Combivir with the single dose Nevirapine during delivery and the early stage of the disease with higher CD4 counts could be the reasons for this.
Keywords: ART, HIV-1, India, mother to child transmission
|How to cite this article:|
Mani M, Ramalingam V V, Lionel J, Christina S A, Sachithanandham J, Peedicayil A, Kannangai R. Emergence of HIV-1 drug-resistant variants in women following antiretroviral prophylaxis for the prevention of mother to child transmission. Indian J Med Microbiol 2015;33:225-30
|How to cite this URL:|
Mani M, Ramalingam V V, Lionel J, Christina S A, Sachithanandham J, Peedicayil A, Kannangai R. Emergence of HIV-1 drug-resistant variants in women following antiretroviral prophylaxis for the prevention of mother to child transmission. Indian J Med Microbiol [serial online] 2015 [cited 2019 Dec 16];33:225-30. Available from: http://www.ijmm.org/text.asp?2015/33/2/225/154860
| ~ Introduction|| |
HIV is one of the highly infectious blood borne viruses that has caused a pandemic since its discovery in the year 1983.  One of the routes of acquiring this infection especially among children is by mother to child transmission (MTCT) while in utero, during labour or through breast milk.  In most of the developed countries, the MTCT has been reduced to less than 2% and reported cases of paediatric infections have been gradually reduced.  This has been possible due to the availability of HAART, which is given to most of HIV 1 infected pregnant women as prophylaxis. Even though preventive efficacy of Zidovudine (AZT) has been established in 1994 MTCT of HIV remains a major health problem worldwide. 
There are several studies in which nucleoside reverse transcriptase inhibitors like Zidovudine (AZT), Lamivudine (3TC), and the non-nucleoside reverse transcriptase inhibitor Nevirapine (NVP), either alone or in combination, have been shown to reduce MTCT of HIV effectively.  Compared to placebo, either a short- or a long-term treatment with AZT alone has shown 26% to 68% of efficacy in reducing the transmission.  Due to the ease of administration and scientific evidence of effectiveness, the single dose of NVP prophylaxis is being implemented in developing world to prevent MTCT of HIV-1 especially in resource-constrained settings.  This has reduced the transmission rate to 8% at 6 weeks after delivery.  There are reports in which the efficacy of the different antiretroviral regimens is compared in reducing the MTCT risk. , In comparison with NVP, only the longest regimen of AZT + 3TC was found to be significantly more effective. This suggested an equivalence of choice between single-dose NVP and short-course AZT, and confirms the greater efficacy of AZT + 3TC than with any single antiretroviral drug.  One of the drawbacks of prophylaxis is the acquisition of drug resistance. , There are very few data related to the emergence of drug resistance following ART prophylaxis among pregnant women from India. , The aim of this study was to investigate the emergence of polymorphisms in the HIV-1 RT gene indicating drug resistance in HIV-1 infected pregnant women following anti-retroviral prophylaxis for prevention of MTCT.
| ~ Materials and Methods|| |
The study proposal was approved by the Institutional Review Board (IRB). The study population was drawn from pregnant women who come to a tertiary care hospital in south India for antenatal or perinatal care and found to be positive only for HIV-1 by NACO testing algorithm (strategy III) and those who were ART naive. Fifty-three HIV infected women who met these criteria were recruited for the study. Among the 53 women, 48 belonged to group-1, i.e. HIV-1 infected pregnant women who received 4-12 weeks of antenatal AZT followed by a single dose of NVP during delivery and Combivir (AZT + 3TC) for 1 week postpartum and 5 women belonged to group-2 i.e. women who came to the hospital at the time of delivery and received a single dose of NVP followed by AZT + 3TC for a week.
Samples were collected prior to the start of the prophylaxis and 5-8 weeks following delivery. In addition to this, a third sample was collected between 26-65 weeks postpartum from 7 women. Six millilitres of whole blood was collected in K 2 EDTA coated tubes for HIV-1 RNA estimation and HIV-1 drug resistance assay. The blood samples were centrifuged at 2000 rpm at 4 ° C for 10 minutes and the plasma samples were aliquot in multiple vials and stored at -70°C. Three millilitres of whole blood was collected in another K 2 EDTA coated tube for CD4 + T cell count estimation.
Absolute CD4 + T cell counts were estimated by the BD FACS Count machine (Becton Dickinson [San Jose, CA, USA]) as per the manufacturer's instruction. 
HIV 1 -RNA testing
HIV-1 viral load was estimated using real time PCR, Rotor gene 3000 (Corbett Research Scientific, Australia) with artus HIV 1RG RT PCR assay (Qiagen GMVH, Germany).  The manufacturer's instructions were followed for the extraction of RNA and for the reverse transcriptase PCR. The real time PCR system was based on the TaqMan principle. The assay contains reagents and enzymes for the reverse transcription and specific amplification of a 73 bp region of the LTR region in the HIV 1 genome. This PCR makes use of two channels; cycling A FAM and cycling A JOE, the former is used for the direct detection of the specific amplicon and the later to identify possible PCR inhibition (detected as internal control). The quantitation is based on the four external positive standards (HIV 1 RG QS 1- 4) representing 1 × 10 4 copies/ml - 1 × 10 1 copies/ml. The analytical sensitivity of the artus HIV 1 RG RT PCR assay was determined using clinical specimen by the manufacturers and the analytical detection limit for HIV 1 RNA was consistently 72 copies/ml.
Drug resistance testing
The genotypic resistance assay was carried out by amplification of pol gene followed by sequencing.  The HIV-1 pol gene was amplified by a nested PCR from extracts of plasma RNA using QIAamp viral RNA extraction kit (QIAGEN GmbH, Hilden, Germany). Initially QIAGEN one step RT PCR was carried (cDNA synthesis and first round PCR) out followed by a second round PCR for a pol gene product of 1800 bp. The cycling condition for the one step RT PCR was as follows: 50 ° C for 30 minutes, 95 ° C for 15 min for activation of the hot start Taq enzyme followed by 40 cycles of 94 ° C for 45 sec, 60 ° C for 45 sec and 72 ° C for 2 minutes followed by a final extension at 72 ° C for 7 minutes.
For the second round PCR, the cycling conditions was as follows: 95 ° C for 15 min for activation of the hot start Taq polymerase enzyme followed by 23 cycles of 94 ° C for 45 sec, 55 ° C for 45 sec and 72°C for 2 min followed by a final extension at 72°C for 7 min. Products from the PCR was first analyzed on an ethidium bromide-stained agarose (2%) gel to check for the specific 1800 bp size amplicon using the gel documentation system Geldoc 2000 (Bio-Rad, California, USA) using the software quantity one version 4.1.1 (Bio-Rad, California, USA). The sequencing of the amplified products was done with 3 sets (forward and reverse) of the sequencing primers using Big dye terminator assay with the ABI PRISM 310 genetic analyzer (PE Applied Bio-System, California, USA). The sequences were aligned using the Bioedit software (www.mbio.ncsu.edu/bioedit/bioedit.html) and subsequently submitted to the Stanford HIV drug resistance database (http://hivdb.stanford.edu) for analysis and interpretation. 
Quality Control of the Assays
CD4+/CD3 + T cell count
Every day as part of internal quality control, two samples tested on the previous day were included, one with the highest count and the other with lowest count. A variation of less than 20% was taken as acceptable. The laboratory is also participating in an external quality control program with satisfactory performance.
HIV-1 viral load estimation
An IQC (close to 5000 copies/ml) and multiple negative controls were included in the assay. A variation of <0.5 log copies was acceptable. We were also participating in the UKNEQAS external quality control programme and all the results till date were within acceptable range.
Drug resistance testing
Along with the study samples, we included samples from HIV-infected individuals on ART and who presented with clinical failure. All these samples showed mutations related to the drug regimen they were on. We also carried out regular audit sample testing and the results were concordant.
The HIV-1 sequences identified from 30 pregnant women prior to ART prophylaxis and 8 sequences following prophylaxis were submitted to GenBank database (accession numbers KF890214 - KF890251).
The univariate and multivariate analysis of data comparison with HIV-1 viral load level, CD4 counts and duration of AZT to see any association with the emergence of mutations were performed using STATA 10.0 (Stata Corp, College Station, TX).
| ~ Results|| |
In our study group, majority (91%) were on the complex prophylaxis based on the WHO PMTCT guidelines' recommendation of AZT monotherapy during pregnancy, NVP-SD at labour onset, AZT + 3TC during labour and for one week after delivery. The mean age of the women in the study group was 26.7 years (range 20-36 years). Prior to delivery, all the women in group-1 received AZT for an average period of 10 weeks (range, 4-14 weeks).
None of the HIV-1 infected pregnant women belonging to the group-2 showed any mutations prior to or following prophylaxis. 2 out of 48 women belonging to the group-1 showed mutations prior to anti-retroviral prophylaxis. Among the two women, one had two mutations (V106M, G190A) that can lead to high level resistance to NVP and Efavirenz (NNRTI) and the other women had one mutation (M41L) that can lead to low level resistance to AZT and Stavudine (NRTI). In both these individuals, the mutations continued to exist even in a postpartum sample collected following ART prophylaxis. This data shows that the incidence of transmitted drug resistance in the study population is 3.8%. One other woman showed mutation (K103N) that can lead to high level resistance to NVP and Efavirenz (NNRTI), after 6-8 weeks of prophylaxis. However, for this individual the viral RNA was undetectable during the first visit. The mean, median and standard deviation of CD4 + and CD3 + T cell counts, CD4/CD3 ratio, HIV-1 viral load level and the details of mutations observed prior to ART prophylaxis and 6-8 weeks of postpartum in these 53 women (one women did not have drug resistance data prior to treatment) are shown in [Table 1].
|Table 1: The mean and median of CD3+/CD4+T cells, HIV-1 RNA level and drug resistance mutations prior to prophylaxis (1st visit) and 6 - 8 weeks postpartum (2nd visit) |
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Among the 7 (13%) individuals in whom a third sample was collected at 26-65 weeks postpartum, 3 (43%) showed mutations such as K103N, K101E, and E138K, which can reduce the susceptibility to NNRTI drugs. The data on these 7 individuals are shown in [Table 2]. The number of total women in the two study groups and the number of women showed mutations that can lead to HIV-1 drug resistance is shown in [Figure 1].
|Figure 1: The number of women in the two study groups and the number of women showed mutations that can lead to HIV-1 drug resistance following ART prophylaxis|
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|Table 2: The CD4+/CD3+T cell count, HIV - 1 viral load and the mutations observed in the 7 individuals who had been followed up for 26-65 weeks |
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Among the 53 women in the study, 4 (7.5%) had undetectable HIV-1 viral load and another 14 (26.4%) showed > 0.5 log copies reduction of viral load at 6-8 weeks postpartum following prophylaxis while 13 (24.5%) had > 0.5 log copies increase in the RNA level. The absolute CD4 + T cell count showed an elevation among 42 (79.2%) women following prophylaxis. However, when we compared the CD4/CD3 percentage only 12 (22.6%) had a significant increase of >3%. Majority of the study participants showed an elevated CD3 + T cell count during the 2 nd visit (6-8 weeks postpartum). Among the 53 individuals, 4 (7.5%) had undetectable HIV-1 viral load and another 14 (26.4%) showed >0.5 log copies reduction of viral load at 6-8 weeks postpartum following prophylaxis while 13 (24.5%) had >0.5 log copies increase in the RNA level. However, when we compared the CD4/CD3 percentage only 12 (22.6%) had significant increase of >3%. Majority of the study participants showed an elevated CD3 + T cell count during the 2 nd visit (6-8 weeks postpartum). Overall, there was no significant (P = 0.86 and P = 0.65, respectively) difference between the viral load and CD4 + T cell/CD3 + T cell values prior to and following treatment.
A multivariate and univariate analysis was carried out with viral load level, CD4 counts and duration of AZT to see any association with the emergence of mutations, and it failed to show any significant association (P = <0.05). When we analyzed the CD4 and viral load level of individuals who developed mutation following ART prophylaxis (n = 4), all had a drop in the CD4 + T cell count and increase in HIV-1 viral load compared to the values prior to ART [Figure 2].
|Figure 2: Association of emergence of mutation (K103N, K101E and E138K) in three individuals with the CD4+ T cell count and HIV-1 RNA level among the seven individuals who had followed up for 26-65 weeks|
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| ~ Discussion|| |
Several ART regimens were available in the past for the prevention of mother to child HIV transmission.  Irrespective of these regimens used, these women developed drug resistance following ART prophylaxis. , The emergence of drug resistance is associated with incomplete suppression of HIV-1 viral load in mothers who are taking ART prophylaxis. In our study group, majority [91%] of the women were on a complex prophylaxis based on the WHO PMTCT (prevention of MTCT) guidelines' recommendation of AZT monotherapy during pregnancy, NVP-SD at labour onset, AZT + 3TC during labour and for one week after delivery.  As reported earlier, a small proportion of women in our study population also showed the emergence of HIV-1 drug-resistant variants. The study reported here used the population-based sequencing technique that detects mutated virus, only when 10-20% of the total population. hauser et al. (2012) has compared the utility of population sequencing and the allele-specific PCR for the detection of minor variants up to 1% in women who were given the complex prophylaxis regimen.  They showed the emergence of HIV-1 drug resistant variants in 20 out of 50 (40%) women by the allele-specific PCR. Mutation to AZT were found in 11 (22%), NVP-resistant mutation in (18%) and 3TC-resistant mutation in 4 women (8%). However, when the population sequence-based findings were analyzed in the same group the frequency of drug resistance emergence was only 4% in both 4-6 and 12-16 weeks postpartum.  In our study group, the emergence of drug resistance was 2% at 6-8 weeks and at 26-65 weeks, postpartum drug resistance was detected in 3 more additional individuals out of the 7 tested.
Compared to the frequency of mutation reported in other studies with different drug regimens, our study had a low frequency of drug resistance. Advanced disease stage and low CD4 cell counts have been shown to be associated with a higher frequency of AZT resistance. , It is noted that the emergence of mutation is high when the CD4 count is low while starting the prophylaxis. The low frequency of emergence of drug resistance with the complex treatment prophylaxis in our study may be because of the high CD4 + T cell counts (Median: 534 cells) observed in the women of our study population. There are studies, which showed that certain resistance mutations appear only at lower CD4 cell count levels (median: 390 cells/mm 3 ) compared to the relatively high CD4 + T cell count (median: 500 cells/mm 3 ). , We also observed that the individuals who developed drug resistance in the study population had an increase in viral load after the initiation of prophylaxis.
A study from South India showed NVP resistance mutations in 28% of women delivering.  The most common mutations observed were K103N and Y181C mutations and the maximum were detected at 6 weeks of postpartum. It is also observed that wild-type virus had replaced the mutants by one year postpartum in all women except one. In contrast to this, the 5 individuals (group-2) who received single dose NVP during delivery and AZT + 3TC combination for 1 week in our study did not show any mutations 6 weeks postpartum. This may be because of the addition of given for 1 week.
The WHO has recently modified the guidelines on the initiation of prophylaxis to prevent transmission of HIV infection from mother to child in developing countries. As per the guidelines the prophylaxis could be started at any time after 14 th week with a tenofovir-based regimen with three drugs, which has a high potency and genetic barrier for viral resistance.  This treatment regimen may lead to complete suppression of the HIV-1 viral RNA and may further reduce the emergence of drug resistance.
The frequency of transmitted drug resistance in our study population was 3.8% (2/53) and was similar to the earlier report from the same region.  Among which one woman had two NNRTI mutations (V106M, G190A) and the other woman had one NRTI mutation (M41L), respectively. These two women continued to show the same mutation(s) even during postpartum follow up. In addition, two other women showed polymorphic accessory mutation (V90I), which had no impact on the drug susceptibility.
In conclusion, in contrast to the available data on drug résistance following ART prophylaxis among pregnant women, our data showed reduced frequency of mutations following ART prophylaxis. The addition of antenatal AZT (4-12 weeks) and 1 week of postpartum Combivir (AZT + 3TC) with the single dose NVP during delivery and the early stage of the disease could be the reason for this decreased frequency of mutations and also the delay in the emergence apart from high CD4 count observed during prophylaxis. It is ideal to carry out more sensitive assays like allele-specific PCR for the detection of drug resistance in these situations as this may identify even mutated variants with less than 1% population.
| ~ Acknowledgements|| |
We gratefully acknowledge the funding received from Indian Council of Medical Research (ICMR/No. HIV/50/101/2007/ECD-II), New Delhi, India. The authors report that they have no conflicts of interests.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]