AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 30, Number 12, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/aid.2014.0158
SEQUENCE NOTES
HIV-1 Drug Resistance Mutations Among Infants Born to HIV-Positive Mothers in Busia, Kenya Rency Lel,1,2 Jane Ngaira,2 Raphael Lihana,1,2 and Samoel Khamadi1
Abstract
To determine HIV-1 subtypes and transmitted HIV-1 drug-resistant mutations among HIV-1-positive children born to HIV-positive mothers in Busia County, blood samples were collected from 53 children aged between 6 weeks and 5 years in 2011. Their mothers were HIV-1 positive and on antiretroviral therapy at the time the children were born. The samples were analyzed for HIV-1 drug resistance and subtypes through sequencing of portions of the HIV-1 pol gene. The generated sequences were analyzed for subtype diversity using the REGA and BLAST subtyping tools. HIV-1 drug resistance was determined using the Stanford University HIV database. Of the 53 samples that were successfully amplified and sequenced, 69.8% (37/53) were determined to be HIV-1 subtype A, 22.6% (12/53) were subtype D, 5.6% (3/53) were subtype C, and 1.8% (1/53) were subtype A1C. The prevalence of HIV-1 drug resistance mutations of any kind was 22.6% (12/53).
M
other-to-child transmission (MTCT) of HIV is one of the important means of new transmission of HIV. In 2009 alone, it is estimated that 370,000 infants acquired HIV infection through MTCT, i.e., in utero, during the peripartum period and via breastfeeding.1,2 In Kenya as in other sub-Saharan African countries, efforts have been made to prevent HIV transmission from mothers to children using prophylactic antiretroviral medication.3,4 Reduction in maternal viral load in breast milk and indirect infant prophylaxis by ingestion of antiretrovirals in breast milk are some of the mechanisms used to protect infants from becoming infected with HIV from their mothers. Studies have shown that antiretroviral drugs taken by nursing women are present in breast milk.5,6 Between 25% and 40% of mothers who are not on medication can transmit HIV to their children while those on short-course antiretrovirals during the peripartum period have transmission rates of between 8% and 10%.7 For mothers on triple therapy throughout pregnancy, transmission is less than 5%.8 However, for infants who become infected before or during breastfeeding, the presence of antiretroviral (ARV) drugs in breast milk may induce the development of drug resistance due to viral replication in the presence of low drug concentrations.7 Mutations that occur in the HIV genome and lead to resistance can be classified into primary and secondary mutations. Primary mutations lead to a several-fold decrease in sensitivity to one or more drugs. Secondary mutations may
not result in a significant decrease in drug sensitivity but are associated with increases in viral fitness in the presence of existing drug resistance mutations.9 Transmission of resistant HIV variants has serious implications among infected infants since it might reduce the choices of active ARV drugs. This study was carried out to determine the common transmitted HIV-1 drug resistance mutations in children who were born to HIV-infected mothers in Busia District. This was a retrospective study conducted in Busia County in 2011. After obtaining informed consent from the parents of the children, 5 ml of blood was collected in EDTA tubes and plasma was separated and stored for future analysis. The blood samples were collected from children aged between 1 and 5 years. Scientific and ethical approval was sought from the KEMRI National Ethical Review Committee (ERC). RNA was extracted from 140 ll of plasma samples using the Qiagen RNA extraction kit according to the manufacturer’s instructions. One-step reverse transcriptase polymerase chain reaction (RT-PCR) and nested PCR were performed using in-house primers targeting the protease and reverse transcriptase enzymes in the HIV-1 pol gene as previously described.10 Both the reverse transcriptase and protease genes were amplified and sequenced. The generated sequences were analyzed for subtype diversity using the REGA subtyping tool11 and BLAST tool.12 Drug resistance was determined using the International Aids Society (IAS) algorithm and the Stanford University HIV
1
Centre for Virus Research, Kenya Medical Research Institute, Nairobi, Kenya. Institute of Tropical Medicine and Infectious Diseases (ITROMID), Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya. 2
1236
HIV DRUG RESISTANCE MUTATIONS IN CHILDREN IN KENYA
database. Of the 53 samples that were successfully amplified and sequenced, 69.8% (37/53) were determined to be HIV-1 subtype A, 22.6% (12/53) were subtype D, 5.6% (3/53) were subtype C, and 1.8% (1/53) were subtype A1C. The generated sequences were also analyzed for HIV-1 drug resistance mutations. The prevalence of HIV-1 drug resistance mutations of any kind in these children was found to be 22.6% (12/53). Further analysis showed that none of the children had any major protease inhibitor (PI)-related mutations. The only minor mutation that was seen was L10I/V, which was present in 41.5% (22/53) of the children. This is a polymorphic PI-selected accessory mutation that reduces PI susceptibility and may increase the replication of viruses with other PI resistance mutations. Analysis of the reverse transciptase region showed that there were major nucleoside RT inhibitor (NRTI) and nonnucleoside RT inhibitor (NNRTI) resistance mutations present in the children. These are as indicated in Table 1. The results showed that 15% of the children (8/53) had viruses with mutations against the common NRTI drugs such as lamivudine, abacavir, tenofovir, or zidovudine. Twelve (22.6%) of the children had resistance mutations toward NNRTI drugs such as efavirenz and nevirapine. Seven of the children (13%) had mutations against both NRTI and NNRTI drugs as indicated in Table 1.
1237
The findings of this study indicate that HIV-1 subtype A was the most prevalent subtype circulating in the study population with a percentage of 69.8%, with 22.6% being subtype D, 5.6% (3/53) being subtype C, and 1.8% (1/53) being subtype A1C. This mirrors the general picture of HIV-1 subtypes in Kenya where HIV-1 subtype A is predominant. Unlike other areas of Kenya in which HIV-1 subtype A is very dominant, in this population a significant percentage of the viruses was subtype D (22.6%), which is much higher than what has been reported in other parts of Kenya.13 This could be a result of the fact that Busia borders Uganda where there is a significant percentage of HIV-1 subtype D in circulation. The study showed that a significant number of the children (15%) had HIV-1 drug-resistant mutations to NRTI drugs such as lamivudine, abacavir, tenofovir, or zidovudine. Additionally, 22.6% of the children had resistance mutations toward NNRTI drugs, while 13% of the children had mutations conferring resistance to both NRTI and NNRTI drugs. The resistance could have developed in the children during breastfeeding or after they were started on lifesaving ARV therapy.14 It is apparent that some of the mothers were on single-dose nevirapine resulting in incomplete suppression of viral replication. In Kenya, single dose nevirapine has long
Table 1. HIV-1 Drug Resistance-Associated Mutations as Identified from the Children Enrolled in the Study
Sequence ID
NRTI mutations
NRTI mutations drugs
NNRTI mutations
NNRTI mutations drugs
PaedBusia 001
None
None
K103N
Efavirenz, nevirapine
PaedBusia PaedBusia PaedBusia PaedBusia
T215F
None
None
L100I, K103N, V179T
Efavirenz, nevirapine, rilpivirine
K103N, V106I, V108I, P225H, M230L
Efavirenz, Nevirapine, rilpivirine
K103N
Efavirenz, nevirapine Efavirenz, nevirapine Efavirenz, nevirapine Efavirenz, nevirapine Efavirenz, nevirapine Efavirenz, nevirapine
014 019 051 002
PaedBusia 004
M41L, T69N, K70R, V75M, M184V, L210W, T215Y
PaedBusia 005
M41L, T69N, V75M, M184V, T215N, K219N,
PaedBusia 007
M184V
Low level resistance to abacavir, zidovudine, stavudine, didanosine, and tenofovir Lamivudine, abacavir, zidovudine, stavudine, didanosine, emtricitabine, tenofovir Lamivudine, abacavir, zidovudine, stavudine, didanosine, emtricitabine, tenofovir Lamivudine, emtricitabine
PaedBusia 008
M184V
Lamivudine, emtricitabine
K103N, V108I, K238T
PaedBusia 010
Lamivudine, emtricitabine
K101E, G190S
PaedBusia 011
D67G, K70R, M184V, K219Q M184V
Lamivudine, emtricitabine
K103N, P225H
PaedBusia 015
V75M, M184I, T215N
K103N, V106I, M230L
PaedBusia 046
M41LM, M184V
Lamivudine, stavudine, emtricitabine Lamivudine
V106A, F227L
NRTI, nucleotide reverse transcriptase inhibitator; NNRTI, nonnucleotide reverse transcriptase inhibitor.
1238
been used as the main option for preventing MTCT of HIV. The use of triple-drug ART prophylaxis for pregnant and breastfeeding women has been suggested to be a more efficacious means of preventing MTCT of HIV. However, this has resulted in debates on issues involving treatment interruption, risk of maternal drug toxicities, fetal exposure to multiple drugs, and cost implications of the same.15 All these issues require due consideration and need to be looked at in light of the larger picture of stemming new infections and preventing the development of drug resistance. The greatest limitation of the study resulted from the fact that the samples were collected from children whose mothers did not have a definitive history of the medication that they were taking. As a result we were not able to provide a link between the development of resistance and the type of medication the mother was taking. However, the study was able to show that development of resistance in children born to ART-experienced mothers can be high, especially in resource-limited settings. The 53 sequences generated from this study were submitted to GenBank under accession numbers KM016171– KM016223.
LEL ET AL.
6.
7.
8. 9.
10.
Acknowledgments
We thank all the study participants for consenting to be involved in this study. The study was carried out in Kenya Medical Research Institute, Centre for Virus Research. This work was published with permission from the Director, KEMRI. Author Disclosure Statement
11.
12.
No competing financial interests exist. References
1. Bennett DE, Camach RJ, Otelea D, Kuritzkes DR, Fleury H, Kiuchi M, et al.: Drug resistance mutations for surveillance of transmitted HIV-1 drug resistance: 2009 update. PLoS One 2009;4(3):e4724. 2. Rajesh L, Ramesh K, Hanna LE, Narayanan PR, and Swaminathan S: Emergence of drug resistant mutations after single dose nevirapine exposure in HIV-1 infected pregnant women in south India. Indian J Med Res 2010;132(5): 509–512. 3. Wiktor SZ, Ekpini E, Karon JM, Nkengasong J, Maurice C, et al.: Short-course oral zidovudine for prevention of motherto-child transmission of HIV-1 in Abidjan, Cote d’Ivoire: A randomised trial. Lancet 1999;353(9155):781–785. 4. Jackson JB, Musoke P, Fleming T, Guay LA, Bagenda D, et al.: Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-tochild transmission of HIV-1 in Kampala, Uganda: 18month follow-up of the HIVNET 012 randomised trial. Lancet 2003;362(9387):859–868. 5. Mirochnick M, Siminski S, Fenton T, Lugo M, and Sullivan JL: Nevirapine pharmacokinetics in pregnant women and in
13.
14.
15.
their infants after in utero exposure. Pediatr Infect Dis J 2001;20(8):803–805. Moodley D, Moodley J, Coovadia H, Gray G, and McIntyre J: A multicenter randomized controlled trial of nevirapine versus a combination of zidovudine and lamivudine to reduce intrapartum and early postpartum mother-to-child transmission of human immunodeficiency virus type 1. J Infect Dis 2003;187:725–735. Zeh C, Weidle PJ, Nafisa L, Lwamba HM, Okonji J, Anyango E, Bondo P, Masaba R, Fowler MG, Nkengasong JN, Thigpen MC, and Thomas T: HIV-1 drug resistance emergence among breastfeeding infants born to HIV-infected mothers during a single-arm trial of triple antiretroviral prophylaxis for prevention of mother-to-child transmission: A secondary analysis. PLoS Med 2011;8(3):e1000430. Dabis F and Ekpini ER: HIV-1/AIDS and maternal and child health in Africa. Lancet 2002;359(9323):2097–2104. Mirochnick M, Thomas T, Capparelli E, Zeh C, and Holland D: Antiretroviral concentrations in breast-feeding infants of mothers receiving highly active antiretroviral therapy. Antimicrob Agents Chemother 2009;53:1170–1176. Lwembe R, Ochieng W, Panikulam A, Mongoina CO, Palakudy T, Koizumi Y, Kageyama S, Yamamoto N, Shioda T, Musoke R, Owens M, Songok EM, Okoth FA, and Ichimura H: Anti-retroviral drug resistance-associated mutations among non-subtype B HIV-1-infected Kenyan children with treatment failure. J Med Virol 2007;79(7):865–872. Alcantara LCJ, Cassol S, Libin P, Deforche K, Pybus OG, Van Ranst M, Galvao-Castro B, Vandamme A-M, and de Oliveira T: A standardized framework for accurate, highthroughput genotyping of recombinant and non-recombinant viral sequences. Nucleic Acids Res 2009;37:W634–642. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, and Lipman DJ: Gapped BLAST and PSIBLAST: A new generation of protein database search programs. Nucleic Acids Res 1997;25(17):3389–3402. Kageha S, Lihana RW, Okoth V, Mwau M, Okoth FA, Songok EM, Ngaira JM, and Khamadi SA: HIV type 1 subtype surveillance in central Kenya. AIDS Res Hum Retroviruses 2012;28(2):228–231. Giaquinto C, Rampon O, and De Rossi A: Antiretroviral therapy for prevention of mother-to-child HIV transmission: Focus on single-dose nevirapine. Clin Drug Invest 2006;26(11):611–627. Mofenson LM: Prevention in neglected subpopulations: Prevention of mother-to-child transmission of HIV infection. Clin Infect Dis 2010;50(Suppl 3):S130–148.
Address correspondence to: Samoel Khamadi Centre for Virus Research Kenya Medical Research Institute P.O. Box 54840 00200 Nairobi Kenya E-mail: [email protected]
SEQUENCE NOTES
HIV-1 Drug Resistance Mutations Among Infants Born to HIV-Positive Mothers in Busia, Kenya Rency Lel,1,2 Jane Ngaira,2 Raphael Lihana,1,2 and Samoel Khamadi1
Abstract
To determine HIV-1 subtypes and transmitted HIV-1 drug-resistant mutations among HIV-1-positive children born to HIV-positive mothers in Busia County, blood samples were collected from 53 children aged between 6 weeks and 5 years in 2011. Their mothers were HIV-1 positive and on antiretroviral therapy at the time the children were born. The samples were analyzed for HIV-1 drug resistance and subtypes through sequencing of portions of the HIV-1 pol gene. The generated sequences were analyzed for subtype diversity using the REGA and BLAST subtyping tools. HIV-1 drug resistance was determined using the Stanford University HIV database. Of the 53 samples that were successfully amplified and sequenced, 69.8% (37/53) were determined to be HIV-1 subtype A, 22.6% (12/53) were subtype D, 5.6% (3/53) were subtype C, and 1.8% (1/53) were subtype A1C. The prevalence of HIV-1 drug resistance mutations of any kind was 22.6% (12/53).
M
other-to-child transmission (MTCT) of HIV is one of the important means of new transmission of HIV. In 2009 alone, it is estimated that 370,000 infants acquired HIV infection through MTCT, i.e., in utero, during the peripartum period and via breastfeeding.1,2 In Kenya as in other sub-Saharan African countries, efforts have been made to prevent HIV transmission from mothers to children using prophylactic antiretroviral medication.3,4 Reduction in maternal viral load in breast milk and indirect infant prophylaxis by ingestion of antiretrovirals in breast milk are some of the mechanisms used to protect infants from becoming infected with HIV from their mothers. Studies have shown that antiretroviral drugs taken by nursing women are present in breast milk.5,6 Between 25% and 40% of mothers who are not on medication can transmit HIV to their children while those on short-course antiretrovirals during the peripartum period have transmission rates of between 8% and 10%.7 For mothers on triple therapy throughout pregnancy, transmission is less than 5%.8 However, for infants who become infected before or during breastfeeding, the presence of antiretroviral (ARV) drugs in breast milk may induce the development of drug resistance due to viral replication in the presence of low drug concentrations.7 Mutations that occur in the HIV genome and lead to resistance can be classified into primary and secondary mutations. Primary mutations lead to a several-fold decrease in sensitivity to one or more drugs. Secondary mutations may
not result in a significant decrease in drug sensitivity but are associated with increases in viral fitness in the presence of existing drug resistance mutations.9 Transmission of resistant HIV variants has serious implications among infected infants since it might reduce the choices of active ARV drugs. This study was carried out to determine the common transmitted HIV-1 drug resistance mutations in children who were born to HIV-infected mothers in Busia District. This was a retrospective study conducted in Busia County in 2011. After obtaining informed consent from the parents of the children, 5 ml of blood was collected in EDTA tubes and plasma was separated and stored for future analysis. The blood samples were collected from children aged between 1 and 5 years. Scientific and ethical approval was sought from the KEMRI National Ethical Review Committee (ERC). RNA was extracted from 140 ll of plasma samples using the Qiagen RNA extraction kit according to the manufacturer’s instructions. One-step reverse transcriptase polymerase chain reaction (RT-PCR) and nested PCR were performed using in-house primers targeting the protease and reverse transcriptase enzymes in the HIV-1 pol gene as previously described.10 Both the reverse transcriptase and protease genes were amplified and sequenced. The generated sequences were analyzed for subtype diversity using the REGA subtyping tool11 and BLAST tool.12 Drug resistance was determined using the International Aids Society (IAS) algorithm and the Stanford University HIV
1
Centre for Virus Research, Kenya Medical Research Institute, Nairobi, Kenya. Institute of Tropical Medicine and Infectious Diseases (ITROMID), Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya. 2
1236
HIV DRUG RESISTANCE MUTATIONS IN CHILDREN IN KENYA
database. Of the 53 samples that were successfully amplified and sequenced, 69.8% (37/53) were determined to be HIV-1 subtype A, 22.6% (12/53) were subtype D, 5.6% (3/53) were subtype C, and 1.8% (1/53) were subtype A1C. The generated sequences were also analyzed for HIV-1 drug resistance mutations. The prevalence of HIV-1 drug resistance mutations of any kind in these children was found to be 22.6% (12/53). Further analysis showed that none of the children had any major protease inhibitor (PI)-related mutations. The only minor mutation that was seen was L10I/V, which was present in 41.5% (22/53) of the children. This is a polymorphic PI-selected accessory mutation that reduces PI susceptibility and may increase the replication of viruses with other PI resistance mutations. Analysis of the reverse transciptase region showed that there were major nucleoside RT inhibitor (NRTI) and nonnucleoside RT inhibitor (NNRTI) resistance mutations present in the children. These are as indicated in Table 1. The results showed that 15% of the children (8/53) had viruses with mutations against the common NRTI drugs such as lamivudine, abacavir, tenofovir, or zidovudine. Twelve (22.6%) of the children had resistance mutations toward NNRTI drugs such as efavirenz and nevirapine. Seven of the children (13%) had mutations against both NRTI and NNRTI drugs as indicated in Table 1.
1237
The findings of this study indicate that HIV-1 subtype A was the most prevalent subtype circulating in the study population with a percentage of 69.8%, with 22.6% being subtype D, 5.6% (3/53) being subtype C, and 1.8% (1/53) being subtype A1C. This mirrors the general picture of HIV-1 subtypes in Kenya where HIV-1 subtype A is predominant. Unlike other areas of Kenya in which HIV-1 subtype A is very dominant, in this population a significant percentage of the viruses was subtype D (22.6%), which is much higher than what has been reported in other parts of Kenya.13 This could be a result of the fact that Busia borders Uganda where there is a significant percentage of HIV-1 subtype D in circulation. The study showed that a significant number of the children (15%) had HIV-1 drug-resistant mutations to NRTI drugs such as lamivudine, abacavir, tenofovir, or zidovudine. Additionally, 22.6% of the children had resistance mutations toward NNRTI drugs, while 13% of the children had mutations conferring resistance to both NRTI and NNRTI drugs. The resistance could have developed in the children during breastfeeding or after they were started on lifesaving ARV therapy.14 It is apparent that some of the mothers were on single-dose nevirapine resulting in incomplete suppression of viral replication. In Kenya, single dose nevirapine has long
Table 1. HIV-1 Drug Resistance-Associated Mutations as Identified from the Children Enrolled in the Study
Sequence ID
NRTI mutations
NRTI mutations drugs
NNRTI mutations
NNRTI mutations drugs
PaedBusia 001
None
None
K103N
Efavirenz, nevirapine
PaedBusia PaedBusia PaedBusia PaedBusia
T215F
None
None
L100I, K103N, V179T
Efavirenz, nevirapine, rilpivirine
K103N, V106I, V108I, P225H, M230L
Efavirenz, Nevirapine, rilpivirine
K103N
Efavirenz, nevirapine Efavirenz, nevirapine Efavirenz, nevirapine Efavirenz, nevirapine Efavirenz, nevirapine Efavirenz, nevirapine
014 019 051 002
PaedBusia 004
M41L, T69N, K70R, V75M, M184V, L210W, T215Y
PaedBusia 005
M41L, T69N, V75M, M184V, T215N, K219N,
PaedBusia 007
M184V
Low level resistance to abacavir, zidovudine, stavudine, didanosine, and tenofovir Lamivudine, abacavir, zidovudine, stavudine, didanosine, emtricitabine, tenofovir Lamivudine, abacavir, zidovudine, stavudine, didanosine, emtricitabine, tenofovir Lamivudine, emtricitabine
PaedBusia 008
M184V
Lamivudine, emtricitabine
K103N, V108I, K238T
PaedBusia 010
Lamivudine, emtricitabine
K101E, G190S
PaedBusia 011
D67G, K70R, M184V, K219Q M184V
Lamivudine, emtricitabine
K103N, P225H
PaedBusia 015
V75M, M184I, T215N
K103N, V106I, M230L
PaedBusia 046
M41LM, M184V
Lamivudine, stavudine, emtricitabine Lamivudine
V106A, F227L
NRTI, nucleotide reverse transcriptase inhibitator; NNRTI, nonnucleotide reverse transcriptase inhibitor.
1238
been used as the main option for preventing MTCT of HIV. The use of triple-drug ART prophylaxis for pregnant and breastfeeding women has been suggested to be a more efficacious means of preventing MTCT of HIV. However, this has resulted in debates on issues involving treatment interruption, risk of maternal drug toxicities, fetal exposure to multiple drugs, and cost implications of the same.15 All these issues require due consideration and need to be looked at in light of the larger picture of stemming new infections and preventing the development of drug resistance. The greatest limitation of the study resulted from the fact that the samples were collected from children whose mothers did not have a definitive history of the medication that they were taking. As a result we were not able to provide a link between the development of resistance and the type of medication the mother was taking. However, the study was able to show that development of resistance in children born to ART-experienced mothers can be high, especially in resource-limited settings. The 53 sequences generated from this study were submitted to GenBank under accession numbers KM016171– KM016223.
LEL ET AL.
6.
7.
8. 9.
10.
Acknowledgments
We thank all the study participants for consenting to be involved in this study. The study was carried out in Kenya Medical Research Institute, Centre for Virus Research. This work was published with permission from the Director, KEMRI. Author Disclosure Statement
11.
12.
No competing financial interests exist. References
1. Bennett DE, Camach RJ, Otelea D, Kuritzkes DR, Fleury H, Kiuchi M, et al.: Drug resistance mutations for surveillance of transmitted HIV-1 drug resistance: 2009 update. PLoS One 2009;4(3):e4724. 2. Rajesh L, Ramesh K, Hanna LE, Narayanan PR, and Swaminathan S: Emergence of drug resistant mutations after single dose nevirapine exposure in HIV-1 infected pregnant women in south India. Indian J Med Res 2010;132(5): 509–512. 3. Wiktor SZ, Ekpini E, Karon JM, Nkengasong J, Maurice C, et al.: Short-course oral zidovudine for prevention of motherto-child transmission of HIV-1 in Abidjan, Cote d’Ivoire: A randomised trial. Lancet 1999;353(9155):781–785. 4. Jackson JB, Musoke P, Fleming T, Guay LA, Bagenda D, et al.: Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-tochild transmission of HIV-1 in Kampala, Uganda: 18month follow-up of the HIVNET 012 randomised trial. Lancet 2003;362(9387):859–868. 5. Mirochnick M, Siminski S, Fenton T, Lugo M, and Sullivan JL: Nevirapine pharmacokinetics in pregnant women and in
13.
14.
15.
their infants after in utero exposure. Pediatr Infect Dis J 2001;20(8):803–805. Moodley D, Moodley J, Coovadia H, Gray G, and McIntyre J: A multicenter randomized controlled trial of nevirapine versus a combination of zidovudine and lamivudine to reduce intrapartum and early postpartum mother-to-child transmission of human immunodeficiency virus type 1. J Infect Dis 2003;187:725–735. Zeh C, Weidle PJ, Nafisa L, Lwamba HM, Okonji J, Anyango E, Bondo P, Masaba R, Fowler MG, Nkengasong JN, Thigpen MC, and Thomas T: HIV-1 drug resistance emergence among breastfeeding infants born to HIV-infected mothers during a single-arm trial of triple antiretroviral prophylaxis for prevention of mother-to-child transmission: A secondary analysis. PLoS Med 2011;8(3):e1000430. Dabis F and Ekpini ER: HIV-1/AIDS and maternal and child health in Africa. Lancet 2002;359(9323):2097–2104. Mirochnick M, Thomas T, Capparelli E, Zeh C, and Holland D: Antiretroviral concentrations in breast-feeding infants of mothers receiving highly active antiretroviral therapy. Antimicrob Agents Chemother 2009;53:1170–1176. Lwembe R, Ochieng W, Panikulam A, Mongoina CO, Palakudy T, Koizumi Y, Kageyama S, Yamamoto N, Shioda T, Musoke R, Owens M, Songok EM, Okoth FA, and Ichimura H: Anti-retroviral drug resistance-associated mutations among non-subtype B HIV-1-infected Kenyan children with treatment failure. J Med Virol 2007;79(7):865–872. Alcantara LCJ, Cassol S, Libin P, Deforche K, Pybus OG, Van Ranst M, Galvao-Castro B, Vandamme A-M, and de Oliveira T: A standardized framework for accurate, highthroughput genotyping of recombinant and non-recombinant viral sequences. Nucleic Acids Res 2009;37:W634–642. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, and Lipman DJ: Gapped BLAST and PSIBLAST: A new generation of protein database search programs. Nucleic Acids Res 1997;25(17):3389–3402. Kageha S, Lihana RW, Okoth V, Mwau M, Okoth FA, Songok EM, Ngaira JM, and Khamadi SA: HIV type 1 subtype surveillance in central Kenya. AIDS Res Hum Retroviruses 2012;28(2):228–231. Giaquinto C, Rampon O, and De Rossi A: Antiretroviral therapy for prevention of mother-to-child HIV transmission: Focus on single-dose nevirapine. Clin Drug Invest 2006;26(11):611–627. Mofenson LM: Prevention in neglected subpopulations: Prevention of mother-to-child transmission of HIV infection. Clin Infect Dis 2010;50(Suppl 3):S130–148.
Address correspondence to: Samoel Khamadi Centre for Virus Research Kenya Medical Research Institute P.O. Box 54840 00200 Nairobi Kenya E-mail: [email protected]
