Short Communication: Drug Resistance Mutations in the HIV Type 1 Protease and Reverse Transcriptase Genes in Antiretroviral-Naive Vietnamese Children

I n the developed world, newly acquired pediatric HIV infections have been virtually eliminated; however, pediatric HIV infection remains a significant public health problem in developing countries, including Vietnam. The number of children infected with HIV who seek treatment at health facilities has been increasing, especially at Children Hospital 1, which is a central pediatric hospital for the treatment of children in southern Vietnam. Anti-HIV drugs have recently become available for the treatment of children infected with HIV in Vietnam; however, the genetic background of HIV-1 drug resistance in antiretroviral-naive children has yet to be studied. To provide a better understanding of the molecular characteristics of the HIV-1 strains that have been isolated from Vietnamese children infected with HIV and the effect of these characteristics on antiviral therapy, this study aimed to describe the drug resistance mutations (DRMs) of the protease and reverse transcriptase (RT) genes of HIV-1 strains that were isolated from children infected with HIV-1 who were hospitalized in Children Hospital 1 in Ho Chi Minh City, Vietnam, from 2004 to 2005.

In total, 79 HIV-1 strains were used in the amplification and sequence analyses of the protease and RT genes. The corresponding samples were selected randomly from 104 stored extracted proviral DNA samples, which were known to be positive for HIV-1 subtype CRF01-AE based on the results from our previous study. ¹ Ethical approvals were obtained from the Scientific Research Board of Children Hospital 1 in Vietnam and from the Ethical Committee of the University of Tokyo in Japan. The original whole blood samples were collected from patients infected with HIV who were hospitalized at Children Hospital 1 in Ho Chi Minh City, Vietnam, from October 2004 to September 2005. The HIV-1 protease and RT regions were amplified by nested PCRs using previously described primers. ^2,3 Sequence analyses were performed, and the detected mutations were compared with the World Health Organization (WHO) recommended drug resistance mutations for surveillance (“WHO list”) ^4,5 and the mutations on the International AIDS Society (IAS) HIV drug resistance (HIVDR) mutations list (December 2010). ⁶

Of the 79 infants and children who were enrolled in the study, 37 (46.8%) were male and 42 (53.2%) were female. The age distribution ranged from 1.5 months to 5 years; however, more than half of the patients were infants less than 6 months of age (41/79, 51.9%). Regarding geographic distribution, the patients were from provinces or cities located in southern Vietnam (data not shown). None of the patients was treated with antiretroviral drugs during their hospitalization due to the lack of availability of anti-HIV drugs. No information on the use of drugs to prevent mother-to-child transmission of HIV was recorded. It was not determined whether HIV infection occurred perinatally or postnatally.

In the protease gene, the following minor mutations were found: L10I (detection rate, 8.7%), I13V (60.8%), G16E (41.8%), M36I (100%), D60E (3.8%), I62V (6.3%), L63P (5.1%), I64V (2.5%), H69K (100%), V82I (6.3%), and I93L (24.1%). No major mutations in the protease gene were observed at positions 30, 32, 33, 46–48, 50, 54, 58, 76, 82, 84, 88, or 90, which are important sites of drug resistance mutations according to the IAS list (data not shown). None of the changes that were found is on the WHO list for the surveillance of primary or transmitted mutations.

Regarding the RT gene, 12 (15.2%) of the 79 samples tested had mutations associated with HIVDR: V75I was found in one sample, V75M in one, V106I in three, V179D in two, Y181C in two, and M184I was found in three samples. Of these six RT mutations, V75I, V106I, and V179D are not on the WHO list of recommended mutations for use in HIVDR surveillance. The M184I mutation causes resistance to emtricitabine and lamivudine, both of which are nucleoside analog reverse transcriptase inhibitors (NRTIs). The V75M mutation conferred low-level resistance to the NRTIs didanosine and stavudine. The Y181C mutation caused resistance to efavirenz, etravirine, and nevirapine, which are non-NRTIs. The M184I, V75M, and Y181C mutations may represent primary or transmitted mutations.

In this study, we analyzed HIV-1 subtype CRF01-AE strains that were isolated from antiretroviral-naive children. Amino acid changes were found in the protease gene in all of the samples tested, with the M36I and H69K mutations being especially common; however, all of the changes were considered to be polymorphisms because they are not on the WHO list for HIVDR surveillance. Additionally, the V75I, V106I, and V179D mutations in the RT gene are not on the WHO list. In the RT gene, three major mutations, V75M, Y181C, and M184I, were found in one (1.3%), two (2.5%), and three (3.8%) samples, respectively. The prevalence of primary or transmitted HIVDR to all of the drugs and drug classes that were evaluated was 7.6%. The following mutations were present in four patients less than 6 months of age: V75M in one patient, M184I in one, and Y181C in two. In addition, the M184I mutation was found in two patients greater than 6 months of age. The prevalence rates were 9.8% for patients <6 months of age and 5.3% for patients >6 months of age. There was no significant difference between the two groups with respect to the prevalence rate (p>0.05).

The three HIV-1 DRMs that were found in infants and children have been reported previously in Vietnamese adult patients. The M184I mutation was detected in Haiphong, northern Vietnam, by Ishizaki et al. in 2009. ⁷ The Y181C mutation was found in three other studies in northern Vietnam. ^{8 –10} The V75M mutation was reported in a multicenter study across Vietnam. ¹⁰ In addition, the V75M mutation was found in pregnant antiretroviral-naive Vietnamese women, but the M184I and Y181C mutations were not. ¹¹ The prevalence of HIV-1 DRMs found in pregnant antiretroviral-naive Vietnamese women, by Ayouba et al. in 2008, was lower than that in infants and children in this study. ¹¹ The HIV-1 DRMs that were identified in the viruses from the infants and children in this study may have been primary or transmitted from mothers who had primary or transmitted DRMs or who acquired DRMs from unrecorded antiviral treatments.

In comparison with the prevalence of primary or transmitted HIVDR in this study, the prevalences in adult antiretroviral-naive individuals in Thailand and Cambodia were lower (less than 5%). ^{12 –15} In addition, the prevalence rates that were reported previously in northern Vietnam were less than 5%. ^{7 –9} However, a prevalence rate of 6.3% was recently reported this year in Haiphong. ¹⁶ In addition, a multicenter study conducted in five major Vietnamese cities, two in the north and three in the south, showed a prevalence of 7.6% in commercial sex workers and injecting drug users. ¹⁰ Taken together with previous findings, this study suggests that the prevalence of HIVDR in Vietnam may have recently increased.

In conclusion, this is the first report of DRMs in the HIV-1 protease and RT genes in antiretroviral-naive Vietnamese children. This study provides a useful background for antiretroviral therapy in Vietnam and contributes reference data for HIVDR surveillance around the world. The monitoring of HIVDR in Vietnam is necessary.

Sequence Data

The nucleotide sequences of the strains that were studied have been assigned the following GenBank accession numbers: GQ161848–GQ161853, GQ161855–GQ161866, GQ161868–GQ161913, and HQ876613–HQ876706.