| Abstract|| |
Background: Single dose of Nevirapine to prevent mother to child transmission of HIV is the commonest preventive regimen in resource-limited countries. Objectives: The objective of this study was to detect drug-resistant virus after single dose of Nevirapine (sdNVP) provided to delivering HIV seropositive (HIV+ve) women and to evaluate the time taken for its decay. Results: Of the 36 consenting HIV+ve pregnant women enrolled into the study, the mean hemoglobin and total lymphocyte counts were 10.8 g/dl and 1843 cells/mm 3 , respectively. Mean CD4 counts in 64% of women was 363 cells/mm 3 and mean viral load for 16/36 women was 28,143 copies/ml of plasma. Nevirapine-resistance mutations were detected in 28% of women at delivery; using OLA (Oligonucleotide Ligation Assay). K103N mutations were seen in 19.4% of women while the Y181C mutation was seen in 5%. Both the mutations were detected in 2.7% of women. Sequential blood samples collected at delivery, 7-10 days, 6 weeks, 4 months, 6 months and one year postpartum showed that 81% of K103N mutations and 66.7% of Y181C mutations were detected at 6 weeks postpartum . Wild-type virus had replaced the mutants by one year postpartum in all women except one. Conclusion : These observations are relevant for future treatment with antiretroviral therapy in these women for their HIV disease.
Keywords: HIV, mutations, Nevirapine, oligonucleotide ligation assay
|How to cite this article:|
Jacob MS, Durairaj A, Vijayakumari J, Srijayanth P, Sivakumar M R. Detection of HIV drug resistance mutations in pregnant women receiving single dose Nevirapine in south India. Indian J Pathol Microbiol 2011;54:359-61
|How to cite this URL:|
Jacob MS, Durairaj A, Vijayakumari J, Srijayanth P, Sivakumar M R. Detection of HIV drug resistance mutations in pregnant women receiving single dose Nevirapine in south India. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Dec 15];54:359-61. Available from: http://www.ijpmonline.org/text.asp?2011/54/2/359/81637
| Introduction|| |
The simplest of all PMTCT (Prevention of Mother-to-Child Transmission) of HIV drug regimens was tested in the HIVNET 012 trial, in Uganda (1997-1999). This study found that a single dose of Nevirapine given to the mother at the onset of labor and to the baby after delivery roughly halved the rate of HIV transmission. , This is the commonest preventive regimen in resource-limited countries. As it is given only once to the mother and baby, sdNVP is relatively inexpensive and easy to administer. Since 2002, many thousands of babies in India have benefited from this simple intervention, which is still the mainstay of PMTCT program otherwise called the PPTCT (Prevention of Parent to Child Transmission) program in the country.
The Department of Experimental Medicine of the Tamil Nadu, Dr MGR Medical University, initiated the country's first rural PMTCT center in Namakkal district in June 2000 with funds received from Elizabeth Glaser Pediatric AIDS Foundation (USA) at Namakkal District Head Quarter Hospital. Since then single dose Nevirapine is offered to HIV positive pregnant women at the onset of labor and to their infants.
A significant concern about the use of sdNVP is drug resistance. There is strong evidence that a single dose of Nevirapine can make subsequent treatment with non-nucleoside reverse transcriptase inhibitors, such as Nevirapine or efavirenz less effective. , There is also some evidence to suggest that if a mother develops Nevirapine-resistant HIV then this may be passed through breast milk to her infant.  However, recent studies have shown that drug resistance resulting from single dose Nevirapine is usually short lived; if a mother waits for six months before beginning treatment then it is unlikely to fail. 
Nevirapine, however, is still the only single dose drug available to prevent MTCT. Other "short course" treatments require women to take drugs during and after pregnancy as well as during labor and delivery. This means they are much more expensive and more difficult to implement in resource-poor settings than is Nevirapine, which can be used with little or no medical supervision at all. So, for now, single-dose Nevirapine remains the only practical choice for PMTCT of HIV in areas with minimal medical resources. The objective of this study was to detect the onset and persistence of HIV drug-resistant virus after sdNVP provided to delivering seropositive women and to evaluate the time taken for its decay.
| Materials and Methods|| |
This was a prospective cohort study. HIV-1 infected pregnant women who received 200 mg sdNVP at the time of delivery and who delivered at Namakkal District Headquarter Hospital from January to December 2005 were enrolled into the study after providing written informed consent. Their infants were provided Nevirapine syrup (2 mg/kg body weight) after delivery and were scheduled for post-natal follow-up as part of the PMTCT program at the hospital.
Complete blood counts (using automated cell counter, Transasia), CD4/CD8 Counts (FACScan, BD) and HIV-1 Viral load (Roche Amplicor HIV-1 Monitor test Version 1.5) were performed for the mothers at delivery. CD4 counts were estimated in 23 women and viral load was estimated in 16 women. Oligonucleotide Ligation Assay (OLA) was performed for sequential (longitudinal) whole blood samples collected at delivery, 7-10 days after delivery, 6 weeks, 4th month, 6th months and one year postpartum. OLA estimated the K103N and Y181C mutations and the wild type virus.
Oligonucleotide Ligation Assay
DNA extraction was performed from the whole blood using QIA amp® DNA blood mini kit (Quiagen Gmbh, Germany, Catalog No. 51104). According to the kit protocol, 200 μl of whole blood was used for the DNA extraction and the extracted DNA was stored at -20°C for the PCR amplification. OLA procedure and reaction conditions for detection were carried out as described by Beck and Frenkel,  which was produced and distributed by the NIH AIDS Research and Reference Reagent Program, with more recent development of reagents for non-B subtypes.  K103N and Y181C mutations were evaluated from the NNRTI kit provided in the above kit. All samples and controls provided were tested in duplicate. Optical density (OD) readings were obtained using an ELISA reader for the detection of mutants at 450 nm and for the wild type virus at 490 nm.
Pearson Chi-square was utilized for comparing the prevalence of Nevirapine mutations at different time points at delivery and thereafter.
| Results|| |
In the cohort of 36 women, mean age was 25 years with a range of 19-36 years. The mean hemoglobin for these women was 10.8 g/dl and the mean total lymphocyte count was 1843 cells/mm 3 . Mean CD4 counts estimated for 64% of women (n=23) were 363 cells/mm 3 (range 94-1038) and mean viral load for 16/36 women was 28,143 copies/ml of plasma [Table 1].
Nevirapine-resistance mutations were detected in 28% (10/36) of women. K103N mutations were seen in 19.4% (7/36) of the women while the Y181C mutation was seen in 5% (2/36) of the women. Both the mutations were seen in 2.7% (1/36). The total number of times K103N mutations was detected in the study subjects during the period of study was 11, with the total number of Y181C mutations being 3. Although the mutations were predominantly detected at 6 weeks after delivery, significance was not achieved: 81.8%, (9/11) of K103N mutation (P=0.266) and 66.7% (2/3) of Y181C mutation (P=0.262) were detected at 6 weeks after delivery. In 18.2% (2/10) of women, K103N mutation was detected at 6 months postpartum. Wild-type virus had replaced the mutants by one year postpartum in 90% (9/10) of the women.
| Discussion|| |
In this study of pregnant women at Namakkal, Nevirapine-resistance mutations were detected in 28% of women who received single drug Nevirapine at the time of delivery.
The National AIDS Control Organization's (NACO) current efforts to improve access to universal opt-out testing of pregnant women during the antenatal period, and to enhance the use of integrated counseling and testing centers (ICTCs) throughout the nation can improve efficacy in diagnosis of asymptomatic individuals. 
The HIVNET (HIV Network) 012 trial demonstrating the use of single-dose Nevirapine for PMTCT is regarded as a major breakthrough because it is easy to administer, inexpensive, requires no sophisticated healthcare infrastructure and could potentially be implemented widely and rapidly in even the most resource-constrained setting.
OLAs are rapid, specific and sensitive reactions for the detection of known point mutations. , The observations made above are relevant for the future treatment (non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens) of these women for their HIV disease. In Ugandan women who received sdNVP in the Phase I/II trial HIVNET 006, viral mutations associated with NNRTI resistance could be detected among 20-40% of Nevirapine-exposed women using standard sequencing-based methods.  This study showed, as the others, ,, that Nevirapine-resistance mutations are most frequently detected by 6 weeks after delivery. The lysine (K) to asparagine (N) mutation at codon 103 (K103N) is the most common mutation in this study, which is similar to the results of the HIVNET 012 study. ,
In many resource-limited countries, Nevirapine is the cornerstone of the anti-retroviral therapy (ART) used to treat HIV disease in pregnant and non-pregnant women who need therapy for their own health. Typically, Nevirapine is given in combination with AZT and lamivudine. Development of resistance after exposure to single-dose Nevirapine may limit the mother's response to subsequent treatment with a Nevirapine containing ART for her own health. Furthermore, the mutations that cause resistance to Nevirapine also cause resistance to efavirenz, the other affordable non-nucleoside reverse transcriptase inhibitor used in first-line treatment in resource-limited settings. In this study wild-type virus had replaced the mutants by one-year postpartum in all women except one. This suggests that there is a decay of the resistance viruses over time. However two recent studies suggested that a single dose of Nevirapine can establish antiretroviral resistance within the latent reservoir that may result in potentially lifelong risk for reemergence of Nevirapine-resistant HIV  and may accelerate failure of subsequent antiretroviral treatment. 
The limitation of the study is the small sample size. Another is that Nevirapine-resistance mutations like V106M/A, V108I and G190A could not be detected as the OLA kits did not have the oligonucleotides for the above mutations, however, oligonucleotides for codons V106M and G190A have been developed and evaluated for non-B subtypes (Frenkel, personal communication).
| Conclusions|| |
Despite the small sample size, this is the first study from India to detect Nevirapine-resistant mutations and its decay in sequential samples from women who received sdNVP at the time of delivery. Nevirapine-resistant mutations were detected in 28% of women who received sdNVP at the time of delivery. Majority of the mutations appeared at 6 weeks after delivery. These mutations were not observed by one-year postpartum except in one woman. These observations are relevant for future treatment (non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens) of these women for their HIV disease.
| Acknowledgments|| |
The authors wish to thank Dr Lisa M Frenkel and Dr Ann J Melvin from the University of Washington, Seattle, USA, for reviewing this manuscript. The authors wish to acknowledge NIH AIDS Research and Reference Reagent Program for supplying OLA kits and Dr Lisa Frenkel's laboratory for providing reagents and consumables for OLA testing through the Puget Sound Partners program.
| References|| |
|1.||Guay LA, Musoke P, Fleming T, Bagenda D, Allen Mnone, Nakabiito Cnone, et al. Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: HIVNET 012 randomised trial. Lancet 1999;354:795-802. |
|2.||Jackson JB, Musoke P, Fleming T, Guay LAnone, Bagenda Dnone, Allen M, et al. Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: 18-month follow-up of the HIVNET 012 randomised trial. Lancet 2003;362:859-68. |
|3.||Morris L, Pillay Cnone, Chezzi Cnone, Lupondwana Pnone, Ntsala Mnone, Levin Lnone, et al. Low frequency of the V106M mutation among HIV-1 subtype C- infected pregnant women exposed to Nevirapine. AIDS 2003;17:1698-700. |
|4.||Jourdain G, Ngo-Giang-Huong N, Le Coeur S, Bowonwatanuwong Cnone, Kantipong Pnone, Leechanachai Pnone, et al. Intrapartum Exposure to Nevirapine and Subsequent Maternal responses to Nevirapine-Based Antiretroviral Therapy. N Engl J Med 2004;351:229-40. |
|5.||Lee EJnone, Kantor R, Zijenah Lnone, Sheldon Wnone, Emel Lnone, Mateta Pnone, et al. Breast-Milk Shedding of Drug-Resistant HIV-1 Subtype C in Women Exposed to Single-Dose Nevirapine. J Infect Dis 2005;192:1260-4. |
|6.||Lockman S, Shapiro RL, Smeaton LM, Wester C, Thior I, Stevens L, et al. Response to Antiretroviral Therapy after a Single Peripartum Dose of Nevirapine. N Engl J Med 2007;356:135-47. |
|7.||Beck IA, Frenkel LM. Genotyping Kits for the Detection of HIV-1 pol Drug-Resistance Mutations by an Oligonucleotide Ligation Assay, Protocol Version 1.2: NIH AIDS Research and Reference Reagent Program; 2003. |
|8.||Beck IAnone, Crowell Cnone, Kittoe Rnone, Bredell Hnone, Machaba Mnone, Willamson Cnone, et al. Optimzation of the oligonucleotide Ligation Assay, a rapid and inexpensive test for detection of HIV-1 Drug resistance mutations for Non-North American Variants. J Acqir Immuno Defic Syndr 2008;48:418-27. |
|9.||Dandona L, Kumar SG, Ramesh YK, Rao MC, Marseille E, Kahn JG, et al. Outputs, cost and efficiency of public sector centres for prevention of mother to child transmission of HIV in Andhra Pradesh, India. BMC Health Serv Res 2008;8:26. |
|10.||Jackson JBnone, Becker-Pergola Gnone, Guay LAnone, Musoke Pnone, Mracna Mnone, Fowler MGnone, et al. Identification of the K103N resistance mutation in Ugandan women receiving nevirapine to prevent HIV-1 vertical transmission. AIDS 2000;14:F111-F5. |
|11.||Loubser Snone, Balfe Pnone, Sherman Gnone, Hammer Snone, Kuhn Lnone, Morris Lnone. Decay of K103N mutants in cellular DNA and plasma RNA after single-dose nevirapine to reduce mother-to-child HIV transmission. AIDS 2006;20:995-1002. |
|12.||Johnson JA, Li JFnone, Morris Lnone, Martinson Nnone, Gray Gnone, McIntyre Jnone, et al. Emergence of drug-resistant HIV-1 after intrapartum administration of single-dose nevirapine is substantially underestimated. J Infect Dis 2005;192:16-23. |
|13.||Palmer S, Boltz V, Maldarelli F, Martinson N, Gray G, McIntyre J, et al. Persistence of NNRTI-resistant variants after single dose nevirapine in HIV-1 subtype C-infected women. 2005 Twelfth Conference on Retroviruses and Opportunistic Infections, Boston, February [abstract 101]. |
|14.||Eshleman SH, Guay LAnone, Mwatha A, Brown ERnone, Cunningham SPnone, Musoke Pnone. Characterization of nevirapine resistance mutations in women with subtype A vs. D HIV-1 6-8 weeks after single-dose nevirapine (HIVNET 012). J Acquir Immun Defic Syndr 2004;35:126-30. |
|15.||Eshleman SH, Mracna M, Guay LA, Desevve M, Cunningham S, Mirochnick M, et al. Selection and fading of resistance mutations in women and infants receiving nevirapine to prevent HIV-1 vertical transmission (HIVNET 012). AIDS 2001;15:1951-7. |
|16.||Wind-Rotolo Mnone, Durand Cnone, Cranmer Lnone, Reid Anone, Martinson Nnone, Doherty Mnone, et al. Identification of nevirapine-resistant HIV-1 in the latent reservoir after single-dose nevirapine to prevent mother-to-child transmission of HIV-1. J Infect Disnone 2009;99:1301-9. |
|17.||Gadhamsetty S, Dixit NM. Estimating frequencies of minority nevirapine resistant strains in chronically hiv-1 infected individuals naïve to nevirapine using stochastic simulations and a mathematical model. J Virol 2010;84:10230-40. |
Mini S Jacob
Department of Experimental Medicine, The Tamil Nadu Dr. MGR Medical University No. 69, Anna Salai, Guindy, Chennai
Source of Support: None, Conflict of Interest: None