Recommendations for the prevention of mother-to-child transmission1,2 include, both for industrialized countries and for resource-limited settings, the administration of triple antiretroviral (ARV) prophylaxis also for women who are not in the need of treatment for their own health. In these cases, the administration of ARV drugs is limited to the end of pregnancy or to the end of breastfeeding when the option of maternal prophylaxis is preferred for the prevention of postnatal transmission.
In both cases, the emergence of viral resistance in women interrupting ARV drugs is considered a potential risk especially for drugs with a low genetic barrier and particularly for nonnucleoside reverse transcriptase inhibitors (NNRTI), because of their long half-life.
This emergence of resistance can have important clinical implications after the demonstration that women previously exposed to single-dose nevirapine (NVP) and harbouring resistance mutations had an inferior virological response to a NNRTI-based regimen compared with a protease inhibitor (PI)-based regimen.3
Partial prevention of resistance emergence at interruption of NNRTI-based regimens can be obtained with either a staggered interruption (continuing the dual nucleoside backbone for a period of time after stopping the NNRTI) or a switched interruption (replacing the NNRTI with a PI for a short period and then interrupting all drugs).4 However, the optimal duration needed to continue either dual nucleosides or the substituted PI-based regimen, after stopping the NNRTI, is not known.
The aimx of our study was to assess the emergence of resistance in a cohort of women in Malawi, receiving ARV prophylaxis for the prevention of breastfeeding transmission and discontinuing ARVs 6 months after delivery, with a staggered interruption for NVP-treated women.
Study Population and Drugs
The study population included treatment-naive HIV-infected pregnant women enrolled in Malawi in an observational study aimed to assess safety and pharmacokinetics of maternal antiretroviral therapy (ART) administration during breastfeeding for the prevention of postnatal transmission [Safe Milk for African Children (SMAC) study]. The study was conducted in 2 study sites (1 in Lilongwe and 1 in Blantyre) within the DREAM (Drug Resource Enhancement against AIDS and Malnutrition) Program of the Community of S. Egidio and was approved by the National Health Research Committee in Malawi. Written informed consent was obtained from all participating women. In the study women not meeting the criteria for treatment (CD4+ count > 350/mm3) were administered a combination of zidovudine (300 mg twice daily), lamivudine (150 mg twice daily), and NVP (200 mg once daily for the first 14 days of treatment and 200 mg twice daily thereafter) starting from week 25 of gestational age (or as soon as possible for the women presenting at a later gestational age) until 6 months postpartum. At the moment of drug interruption a 3-week tail of zidovudine and lamivudine was administered. In women developing NVP-related toxicity, the drug was stopped and substituted with lopinavir/ritonavir (400/100 mg twice daily).
Collection and Processing of Samples
Plasma samples were collected at baseline (before drug administration), then at delivery, and at months 1, 3, and 6 postpartum, while on treatment. For the assessment of viral resistance, initially plasma samples were collected at month 7 (1 month after drug discontinuation and, for NVP-based regimens, approximately 10 days after the interruption of the tail). However, after the first 18 samples, because of the high proportion of samples with undetectable viral load (9 of 18), the protocol was modified and the collection of samples for resistance was moved to month 8 (2 months after drug discontinuation and, for NVP-based regimens, approximately 40 days after the interruption of the tail). The collection of plasma samples was also scheduled for month 12 (approximately 6 months after drug interruption) to evaluate the possible persistence of mutations. At baseline and at month 12, whole blood samples were stored for the assessment of archived resistance.
HIV RNA levels were determined using the Versant kPCR assay (Siemens Healthcare Diagnostics, Deerfield, IL) with a detection limit of 37 copies per milliliter (1.57 log10). Viral resistance was assessed from plasma (when HIV RNA was > 50 copies/mL) by the use of the TruGene HIV-1 Genotyping kit (Siemens Healthcare Diagnostics). The same assay was used to sequence proviral DNA after extraction from whole blood (QIAamp DNA Blood Mini Kit, Qiagen, Hilden, Germany). Resistance mutations were classified according to the International AIDS Society-USA Panel (December 2009 Update).5
Demographic information, ARV drugs exposure, immunological and virological parameters were recorded. Descriptive results are presented as means, medians with interquartile ranges and proportions. Statistical comparisons used the Student t test and the Mann-Whitney test for quantitative continuous variables and the χ2 method or the Fisher exact test for categorical data. Statistical analysis was performed using SPSS, version 17.0 (SPSS, Chicago, IL).
A total of 84 women had samples available for resistance testing after drug discontinuation. Five women had HIV RNA < 50 copies per milliliter (in 4 cases samples were collected 1 month after drug discontinuation only and subsequent samples were not obtained; in 1 case, HIV RNA was undetectable although the sample was collected 60 days after drug discontinuation). In one case, the sample was collected 65 days after NVP discontinuation but the HIV RNA was = 188 copies per milliliter and the sequence was not obtained. Sequences were therefore available for a total of 78 women. Of these 78 women, 8 had switched from a NVP-based regimen to a lopinavir/ritonavir-containing prophylaxis because of grade ≥2 skin rash. The treatment change occurred in 7 cases during pregnancy (at a median time of 4 weeks after starting ART); and in 1 case, 22 days after delivery.
Overall, the median duration of ART during pregnancy was 63 days, and the total duration of ART administration was 240 days. The median time from ARV prophylaxis cessation to genotypic resistance testing was 60 days (interquartile range: 58-64). For NVP-treated women, the median time from the interruption of all drugs (including the dual nucleosides tail) and testing was 39 days. A total of 5 women of 78 (6.4%, 95% confidence interval: 0.97 to 11.85) had NNRTI mutations after drug discontinuation; all of them were among those receiving NVP-based ART (the proportion was 7.1%, 95% confidence interval: 1.11 to 13.17, considering only NNRTI-receiving women).
Women with or without mutations did not differ with respect to pretreatment CD4 cell count, pretreatment viral load, duration of ART, viral load at delivery or time from treatment cessation to genotypic resistance testing (Table 1). The HIV RNA level at the moment of drug discontinuation was significantly higher (P = 0.02) in women subsequently developing mutations. Considering only NVP-receiving women, these findings did not change (data not shown).
All plasma samples with HIV RNA >50 copies per milliliter during treatment were also evaluated for the presence of resistance. During ART administration, mutations were present in 6 of 78 patients (7.7%). The proportion was 60% (3 of 5) among women with resistance after drug discontinuation and 4.1% (3 of 73) among women who subsequently showed a wild-type virus (P = 0.002).
Details of women harbouring resistance mutations after drug discontinuation are reported in Table 2. Two women had the same mutation archived in baseline HIV DNA. Both of them had a previous pregnancy, however, they denied any previous use of NVP. The other 3 women had, at least once, a detectable viral load and presence of mutations during treatment, as reported above. Six months after drug discontinuation (month 12), there was persistence of mutations only when the mutations were previously archived in baseline HIV DNA.
The mutations present during treatment in women who, after drug discontinuation, had a wild-type virus were the K103N + G190A in one woman, the K101E in another, and the K103N + Y188C mutations in the third woman, all of them at month 6.
None of the infants born to these women was infected with HIV.
Emergence of resistance at discontinuation of regimens for the prevention of breastfeeding transmission has not been previously studied. Our results demonstrate that the risk of developing resistance mutations in compliant women who receive ARV prophylaxis and interrupt drugs 6 months after delivery is limited.
Presence of mutations after drug discontinuation was associated with the previous emergence of resistance during treatment, underlying the importance of drug adherence in these patients. Only 2 women with undetectable viral load during treatment had presence of resistance after drug discontinuation. These women had archived resistance at baseline, which is known to possibly re-emerge during treatment interruptions.6 Archived resistance was systematically evaluated only in patients with mutations after drug discontinuation, so, unfortunately, we cannot determine the impact of archived resistance on the entire cohort. The presence of archived resistance in this population is intriguing. Although we cannot exclude a possible undisclosed prior therapy (these women denied any previous use of single-dose NVP), these data may suggest transmission of drug-resistant variants that should be further investigated.
Overall, however, the rate of emergence of resistance at discontinuation of a NVP-based regimen in our study (7.1%) is lower compared with reports of other cohorts of pregnant women, showing rates between 13% and 25%.7,8 Only one of these studies 7 included the administration of a 5-day tail with 2 nucleoside reverse transcriptase inhibitors. Because in a previous study we have shown that detectable levels of NVP may be present for longer than 2 weeks after discontinuation of a chronic therapy,9 we hypothesize that the staggered interruption performed in our study (a 3-week tail of zidovudine and lamivudine) may have contributed to the low risk of resistance.
Our findings also confirm previous data on the lack of emergence of resistance at interruption of PI-containing regimens.8,10
Although NVP-based regimens are not recommended in the World Health Organization guidelines for the prevention of mother-to-child transmission, when women do not need ART for their own health,2 we believe that our data can still be relevant. In fact, the new guidelines recommend, for women not meeting the criteria for treatment, either a lopinavir-based or an efavirenz-based regimen or 3 NRTIs. It is hypothesizable that the efavirenz-based regimens will be the most widely used for reasons of efficacy, costs, and formulation, especially compared with the lopinavir-based regimens. Our data on the emergence of resistance at NNRTI-interruption can therefore be important, particularly because they suggest the need of a long dual nucleoside tail to prevent resistance.
Limitations of our study include the use of population sequencing which allows the detection of mutations only when the mutation is present in 20%-25% of the entire virus population. Also, the study may have underestimated the number of mutations because of the relatively long interval between drug discontinuation and testing and the possibility that the virus have reverted to wild-type (especially for M184V which is known to disappear quickly in the absence of selective drug pressure).11 However, a previous study, which examined the utility of genotyping assays during treatment interruptions, showed that resistance testing was of value up to 3 months after the interruption because it was observed that the probability to detect a mutation up to 3 months was comparable with up to 1 month after the interruption.12
In conclusions, in our study, women receiving a 3-week tail with 2 NRTIs who had controlled viremia during treatment had a limited risk of developing mutations at interruption of a NNRTI-based regimen administered for the prevention of breastfeeding transmission. Further data on this topic are needed to define the best strategies to prevent emergence of resistance in this setting.
We acknowledge Jean-Baptiste Sagno, MD, and Martin Maulidi, MSc, DREAM Program, Community of S. Egidio, Malawi, for the clinical care of the enrolled patients, David Chimwaza, DREAM Program, Community of S. Egidio, Malawi, and Maria Grazia Mancini and Roberta Amici, Istituto Superiore di Sanità, Rome, Italy, for their support in the laboratory analysis, Alessandra Mattei, Istituto Superiore di Sanità, for her administrative support.
1. Panel on Treatment of HIV-Infected Pregnant Women and Prevention of Perinatal Transmission. Recommendations for Use of Antiretroviral Drugs in Pregnant HIV-1-Infected Women for Maternal Health and Interventions to Reduce Perinatal Transmission in the United States
. May 24, 2010: 1-117. Available at: http://aidsinfo.nih.gov/ContentFiles/perinatalGL.pdf
. Accessed March 11, 2011.
3. Lockman S, Hughes H, McIntyre J, et al. Antiretroviral therapies in women after single-dose nevirapine exposure. N Engl J Med
4. Fox Z, Phillips A, Cohen C, et al. Viral resuppression and detection of drug resistance following interruption of a suppressive non-nucleoside reverse transcriptase inhibitor-based regimen. AIDS
5. Johnson VA, Brun-Vezinet F, Clotet B, et al. Update of the drug resistance mutations in HIV-1. Top HIV Med
6. Palmisano L, Giuliano M, Bucciardini R, et al. Determinants of virologic and immunologic outcomes in chronically HIV-infected subjects undergoing repeated treatment interruptions; The Istituto Superiore di Sanità-Pulsed Antiretroviral Therapy (ISS-PART) Study. J Acquir Immune Defic Syndr
7. Lyons FE, Coughlan S, Byrne CM, et al. Emergence of antiretroviral resistance in HIV-positive women receiving combination antiretroviral therapy in pregnancy. AIDS
8. Paredes R, Cheng I, Kuritzkes DR, et al, Women and Infants Transmission Study (WITS) Group. Postpartum antiretroviral drug resistance in HIV-1-infected women receiving pregnancy-limited antiretroviral therapy. AIDS
9. Gingelmaier A, Eberle J, Kost BP, et al. Protease inhibitor-based antiretroviral prophylaxis during pregnancy and the development of drug resistance. Clin Infect Dis
10. Pirillo M, Palmisano L, Pellegrini M, et al. Nonucleoside reverse transcriptase inhibitor concentrations during treatment interruptions and the emergence of resistance: a substudy of the ISS-PART trial. AIDS Res Hum Retrovir
11. Deeks S, Hoh R, Neilands TB, et al. Interruption of treatment with individual therapeutic drug classes in adults with multidrug-resistant HIV-1 infection. J Infect Dis
12. Chilton D, Dervisevic S, Pillay D, et al. Determinants of HIV drug resistance mutations in plasma virus after treatment interruptions. AIDS