Azumi Ishizaki

Profile of HIV Type 1 Infection and Genotypic Resistance Mutations to Antiretroviral Drugs in Treatment-Naive HIV Type 1-Infected Individuals in Hai Phong, Viet Nam

We evaluated the prevalence and profile of antiretroviral treatment (ART)-associated resistance mutations among HIV-1 strains in northern Vietnam by genotypically analyzing strains isolated from ART-naive individuals in Hai Phong, a city in which HIV-1 is highly prevalent. Plasma samples were collected from injecting drug users (IDU, n = 760), female sex workers (FSW, n = 91), seafarers (n = 94), pregnant women (n = 200), and blood donors (n = 210), and screened for HIV-1 antibodies. Plasma viral RNA was extracted from HIV-1-positive samples, amplified by reverse transcriptase (RT)-PCR of protease and RT genes, and analyzed for genotypes and ART-associated resistance mutations. HIV-1 prevalence among IDU, FSW, seafarers, pregnant women, and blood donors was 35.9%, 23.1%, 0%, 0.5%, and 2.9%, respectively. Phylogenetic analyses revealed that the most prevalent HIV-1 subtype was CRF01_AE (98.3%), similar to strains prevalent in southern China. Four (1.4%) subtype B strains and one (0.3%) unique recombinant between subtypes B and C were also identified. We found protease inhibitor-associated major resistance mutations in one of the 294 cases analyzed (0.3%; mutation M46I). We found RT inhibitor-associated major resistance mutations in 7/273 cases (2.6%; one occurrence each of L74I, M184I, and K219E; three cases of K103N; and two cases of G190E). One CRF01_AE strain harboring a protease codon 35 insertion was first identified in Vietnam. Thus, monitoring of drug-resistant HIV-1 and establishment of a database are required for the proper selection of ART in Vietnam.

Dilated Cardiomyopathy in an Adult Human Immunodeficiency Virus Type 1–Positive Patient Treated with a Zidovudine-Containing Antiretroviral Regime

We describe an adult woman infected with human immunodeficiency virus type 1 (HIV-1) who developed dilated cardiomyopathy (DCM) with histologically confirmed mitochondrial damage while receiving anti-HIV-1 combination therapy that included nelfinavir, lamivudine, and zidovudine. DCM resolved after discontinuation of the regimen, and cardiac function remained normal after initiation of treatment with nelfinavir, lamivudine, and abacavir, which indicates that DCM was induced by mitochondrial toxicity, most likely caused by zidovudine.

High-risk HPV types in lesions of the uterine cervix of female commercial sex workers in the Philippines.

In order to prevent cervical cancer, vaccines against human papilloma virus types 16 (HPV‐16) and 18 (HPV‐18) have been implemented worldwide. However, the HPV types that cause cancer can differ according to geographical area and ethnicity. In this new era of the HPV vaccine, it is important to elucidate the prevalent HPV types in each area. Therefore, the prevalence of HPV infection and cervical abnormalities among 369 female commercial sex workers in the Philippines were examined. HPV L1 gene was amplified by polymerase chain reaction (PCR) using modified GP5+/6+ primers, and genotyping was performed by sequencing cloned PCR products. HPV DNA was detected in 211 (57.2%) women, among whom 46 HPV types were identified. HPV‐52 was most common and multiple‐type infection was observed in 44.5%. Among 56 women with abnormal cervical cytology (low‐ and high‐grade squamous intraepithelial lesions and adenocarcinoma in situ), HPV‐52 was most common (23.2%), followed by HPV‐16 (19.6%), ‐58 (10.7%), and ‐67 (10.7%). Only 27% of these women were positive for HPV‐16 and ‐18. Multivariate analysis revealed that HPV‐16, ‐39, ‐52, ‐67, and ‐82 were significantly associated with abnormal cytology. Repeated analysis of HPV‐52 single‐positive samples using the original GP5+/6+ PCR primers produced negative results in 57% of cases, suggesting that the prevalence of HPV‐52 infection may have been underestimated in previous studies, and the current vaccines may not be sufficient for preventing infection and the development of premalignant lesions of the cervix in women in the Philippines. J. Med. Virol. 81:545–551, 2009.

Multiple routes of hepatitis C virus transmission among injection drug users in Hai Phong, Northern Vietnam.

To identify hepatitis C virus (HCV) transmission routes among injection drug users in Northern Vietnam, plasma samples were collected from 486 drug users in Hai Phong. Plasma viral RNA was extracted from 323 (66.5%) samples that were positive for anti‐HCV antibodies. Portions of the HCV 5′‐untranslated (5′UTR)‐Core and NS5B genes were amplified by reverse‐transcriptase polymerase chain reaction, sequenced directly, and genotyped in 194 and 195 specimens, respectively. Both regions were genotyped in 137 specimens. In the 5′UTR‐Core region, genotype 6a was predominant (32.5%), followed by genotype 1a (23.7%), genotype 1b (20.6%), and genotype 6e (14.4%). In the NS5B region, genotype 1a was predominant (42.6%), followed by genotype 1b (24.1%), genotype 6a (14.4%), genotype 3b (7.2%), and genotype 6e (5.1%). Of the 137 specimens with both regions genotyped, 23 (16.8%) showed discordant genotyping results between the two regions, suggesting possible recombination and/or dual infection. Phylogenetic analysis revealed close associations between Hai Phong strains and strains from Southern China: the Yunnan province for genotype 3b; the Guangxi province for genotype 6e; the USA for genotype 1a; and Southern Vietnam for genotypes 1a and 6e. The human immunodeficiency virus (HIV) infection rate among HCV‐infected injection drug users was 52.6–55.4% and did not differ significantly by HCV genotype. Most drug users infected with HIV‐1 [98.8% (171/173)] were co‐infected with HCV. These results suggest multiple routes of HCV transmission among injection drug users in Northern Vietnam that may also be HIV transmission routes. J. Med. Virol. 82:1355–1363, 2010.

Rapid detection of human immunodeficiency virus type 1 group M by a reverse transcription-loop-mediated isothermal amplification assay.

Abstract A rapid one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the pol-integrase gene was developed to detect human immunodeficiency virus type 1 (HIV-1) group M. This HIV-1 RT-LAMP assay is simple and rapid, and amplification can be completed within 35min under isothermal conditions at 60°C. The 100% detection limit of HIV-1 RT-LAMP was determined using a standard strain (WHO HIV-1 [97/656]) in octuplicate and found to be 120 copies/ml. The RT-LAMP assay was evaluated for use for clinical diagnosis using plasma samples collected from 57 HIV-1-infected and 40 uninfected individuals in Cameroon, where highly divergent HIV-1 strains are prevalent. Of the 57 samples from infected individuals, 56 harbored group-M HIV-1 strains, such as subtypes A, B, G, F2, and circulating recombinant forms (CRFs) _01, _02, _09, _11, _13; all were RT-LAMP positive. One sample harboring group-O HIV-1 and the 40 HIV-1-uninfected samples were RT-LAMP negative. These findings indicate that HIV-1 RT-LAMP can detect HIV-1 group-M RNA from plasma samples rapidly and with high sensitivity and specificity. These data also suggest that this RT-LAMP assay can be useful for confirming HIV diagnosis, particularly in resource-limited settings.

Prevalence of HBV infection among different HIV-risk groups in Hai Phong, Vietnam

Hepatitis B virus (HBV) infection in Hai Phong, northern Vietnam, was characterized by analyzing the prevalence and genotype distribution of HBV as well as co‐infection with human immunodeficiency virus type 1 (HIV‐1) among five different risk groups for HIV infection. Plasma samples were collected from intravenous drug users (n = 760, anti‐HIV‐1 antibody positive rate: 35.9%), female sex workers (FSWs; n = 91, 23.1%), seafarers (n = 94, 0%), pregnant women (n = 200, 0.5%), and blood donors (n = 210, 2.9%) in 2007 [Ishizaki et al. (2009): AIDS Res Hum Retroviruses 25:175–182]. Samples were screened for the hepatitis B surface antigen (HBsAg) and anti‐HBs antibody and analyzed genetically. The cumulative HBV incidence rate (HBsAg + anti‐HBs) was 53.2% (10.7 + 42.5%) in intravenous drug users, 51.6% (11.0 + 40.6%) in FSWs, 54.3% (9.6 + 44.7%) in seafarers, 50.5% (12.5 + 38.0%) in pregnant women, and 51.0% (18.1 + 32.9%) in blood donors; there was no significant difference among these groups. Of 163 HBsAg‐positive samples, 113 could be analyzed genetically. Phylogenetic analysis, based on the preS1 region, revealed genotype B4 was most prevalent (90/113; 79.6%), followed by C1 (17.7%), I1 (1.8%), and B2 (0.9%). There was no significant difference in HBV genotype distribution among different HIV infection‐risk groups. The prevalence of HBsAg was 10.3% (31/301) in HIV‐1‐infected individuals and 12.5% (132/1,054) in non‐HIV‐1‐infected individuals, which was not significant. In addition, no significant difference in HBV genotype distribution was observed between HBV/HIV‐1 co‐infected and HBV mono‐infected groups. These results suggest that, although HBV and HIV‐1 share modes of transmission, major transmission routes of HBV have been different from those of HIV‐1 in Hai Phong, Vietnam. J. Med. Virol. 83:399–404, 2011.

Characterization of HIV Type 1 Genotypes and Drug Resistance Mutations Among Drug-Naive HIV Type 1-Infected Patients in Northern Vietnam

To evaluate HIV-1 drug resistance-associated mutations among drug-naive HIV-1-infected patients in Northern Vietnam, we performed sequence analysis of HIV-1 pol-PR and pol-RT in samples collected from 206 (161 men and 45 women) consenting patients in 2008. From these 206 samples, we successfully sequenced 173 pol-PR and 155 pol-RT genes. Phylogenetic analysis revealed that all patients were infected with HIV-1 CRF01_AE. Major protease inhibitor resistance mutations, such as L33F, M46I, and M46L, were found in three patients (1.7%). Major reverse-transcriptase inhibitor (RTI) resistance mutations were found in seven patients (4.5%), four of whom had single mutations: A62V (nucleoside RTI resistance mutation) in two cases and K103N and Y181C (nonnucleoside RTI resistance mutation) in one case each. Three patients had multiple RTI resistance mutations: two, three, and seven, respectively. Thus, monitoring for drug-resistant HIV-1 and performing drug resistance testing before initiating antiretroviral therapy (ART) are recommended to facilitate selection of the appropriate ART and better clinical outcomes in Vietnam.

Changes in the HIV Type 1 Envelope Gene from Non-Subtype B HIV Type 1-Infected Children in Kenya

A switch of coreceptor usage from CCR5 to CXCR4 occurs in about half of HIV-1-infected individuals in the natural course of infection. To investigate whether antiretroviral therapy (ART) enhances the coreceptor switch of HIV-1, we genotypically analyzed the env-V3 amino acid sequences from 81 HIV-1-infected children in Kenya whose plasma samples were obtained between 2000 and 2007. Of 41 children on ART, 35 had HIV-1 using CCR5 as a coreceptor at baseline. In 7 (20%) of them HIV-1 switched the coreceptor usage during the follow-up period. The mean duration of ART to the time of coreceptor switch was 2.6 years (range: 0.5-5.2). Of the remaining 40 children without ART, 32 had HIV-1 using CCR5 as a coreceptor at baseline and in 3 (9.4%) HIV-1 switched the coreceptor usage. The mean age of the children with HIV-1 coreceptor switch with and without ART was 7.3 and 9.7 years, respectively. The difference in the rate and age of coreceptor switch between treated and untreated children was not significant (p = 0.38 and 0.31, respectively). Of the HIV-1-infected children, 10 started ART by the age of 5 years (rapid progressors) and 23 did not need ART by the age of 10 years (slow progressors). The rate of coreceptor switch was strongly higher in rapid progressors (40%) than slow progressors (8.7%) (p = 0.053). These results suggest that switching of coreceptor usage from CCR5 to CXCR4 among HIV-1-infected children is not influenced by ART, but by factors responsible for rapid disease progression.

Infection with high-risk HPV types among female sex workers in northern Vietnam

Vaccines against two high‐risk human papillomavirus (HPV) types, HPV‐16, and HPV‐18, are in use currently, with high efficacy for preventing infections with these HPV types and consequent cervical cancers. However, circulating HPV types can vary with geography and ethnicity. The aim of this study was to investigate the prevalence of HPV types and the association between HPV types and abnormal cervical cytology among female sex workers in Northern Vietnam. Cervical swabs and plasma samples were collected from 281 female sex workers at two health centers in Hanoi and Hai Phong in 2009. The HPV L1 gene was amplified by PCR using original and modified GP5+/6+ primers. Amplified PCR products were genotyped by the microarray system GeneSquare (KURABO) and/or clonal sequencing. Of the 281 women, 139 (49.5%) were positive for HPV DNA. Among the HPV‐positive samples, 339 strains and 29 different types were identified. Multiple‐type and high risk‐type HPV infections were found in 85 (61.2%) and 124 (89.2%) women, respectively. The most common genotype was HPV‐52, followed by HPV‐16, HPV‐18, and HPV‐58. Abnormal cervical cytology was detected in 3.2% (9/281) of the women, and all of these samples were positive for HPV‐DNA. Age ≤25 years and infection with human immunodeficiency virus were associated positively with HPV infection among the women while ever smoking was associated negatively. These results show that HPV‐52 is most prevalent among female sex workers in Northern Vietnam, most of whom had normal cervical cytology. This information may be important for designing vaccination strategies in Vietnam. J. Med. Virol. 85:288–294, 2013.

Two-year outcome of first-line antiretroviral therapy among HIV-1 vertically-infected children in Hanoi, Vietnam.

A retrospective analysis of 86 HIV-1 vertically-infected Vietnamese children with a follow-up period >24 months after initiating antiretroviral therapy (ART) was performed from 2008 to 2012, to assess the outcome of first-line ART in resource-limited settings. Of the 86 children, 68 (79.1%) were treated successfully (plasma HIV-1 viral load [VL] <1000 copies/ml), and 63 (73.3%) had full viral suppression (VL <400 copies/ml) after 24 months of ART. No significant difference between successfully treated patients and failure groups was observed in VL, CD4+ T-cell count or clinical stage at baseline; age at ART start; or ART regimen. All 14 children with VL >5000 copies/ml, one of four children with VL 1000–5000 copies/ml and none with VL <1000 copies/ml developed reverse transcriptase inhibitor (RTI)-resistance mutations by 24 months of ART. Y181C and M184V/I were the most dominant non-nucleoside and nucleoside RTI-resistance mutations, respectively (13/15, 86.7%). These findings suggest that VL testing after 24 months of ART can be used to efficiently differentiate ART failures among HIV-1 vertically-infected children in resource-limited settings.