[Prevalence of HIV and sexually transmitted infections and characteristics of men who have sex with men at a community-based center in Yokohama, Japan].

OBJECTIVES: To investigate the characteristics of men who have sex with men (MSM) in a sample of men who sought voluntary testing and counseling (VCT) for human immunodeficiency virus (HIV) and sexually transmitted infections (STIs) in a community-based center in Yokohama, Japan. The prevalence of HIV/STIs and the incidence rate of HIV were also assessed. METHODS: We investigated VCT records of 449 clients who received free anonymous night VCT services between 2008 and 2011. The tests included rapid HIV antibody, Treponema pallidum antibody (TP Ab), and hepatitis B surface antigen (HBs Ag) tests. We also analyzed VCT records of 82 clients who had at least two tests at our center to estimate the HIV incidence rate. RESULTS: The number of MSM who visited the community center for the first time was 423. Those who resided in Kanagawa Prefecture accounted for 78.5% of the sample, and 30.5% had never been tested for HIV previously. The rate of consistent condom use in the past six months was 44.9%. The results revealed that 3.1%, 10.2%, and 1.7% of MSM tested positive for HIV, TP Ab, and HBs Ag respectively. There was no significant difference in age, residence, previous HIV-testing rates, and the rate of consistent condom use in the past six months between subjects who underwent multiple HIV tests at the community center and those who did not undergo any test after the first visit. HIV-positive individuals were all referred to hospitals nearby. Of 82 repeat HIV testers, one was a seroconverter, indicating an incidence rate of 1.00 per 100 person-years (95% confidence interval, 0.00-5.58). CONCLUSION: Our VCT services were accepted by MSM with a high risk of HIV/STIs infection. HIV prevalence was higher than that at local health centers. The HIV incidence rate was equivalent to the previous study that estimated HIV incidence rate from national and sentinel surveillance data. Creating and sustaining alternative VCT venues targeting MSM with high risk of HIV/STIs infection should be considered in other prefectures in Japan to facilitate the early detection and treatment of HIV/STIs.

[Evaluation of a new vesion of the human immunodeficiency virus antigen and antibody combination assay with improved sensitivity in HIV-1 p24 antigen detection].

The performance of a new version of the HIV p24 antigen and antibody combination assays (Genscreen Ultra HIV Ag-Ab) was evaluated by comparing it with three other fourth-generation enzyme immunoassays (Architect HIV Ag/Ab Combo assay, VIDAS HIV DUO Quick and Genscreen Plus HIV Ag-Ab). The assays were examined with 200 HIV positive samples, 1,000 HIV negative samples, 30 samples (28 positives including 24 samples of subtype A, B, B', C, D, F, G, B/D, CRF01_AE in HIV-1 group M, one sample of HIV-1 group O, three samples of HIV-2 and two negatives) of one worldwide HIV performance panel, 59 samples of ten HIV-1 seroconversion panels and the WHO international standard HIV-1 p24 antigen. Both the sensitivity and specificity of Genscreen Ultra HIV Ag-Ab were 100%. All of the 28 positive samples in the worldwide HIV performance panel were positive. The days of the earliest detection in the ten seroconversion panels were the same in three assays (Genscreen Ultra HIV Ag-Ab, Architect HIV Ag/Ab combo assay and VIDAS HIV DUO Quick). Genscreen Plus HIV Ag-Ab which is a former version of the Genscreen Ultra HIV Ag-Ab detected the earliest positive sample one bleed slower than the other three assays in 5 of 10 seroconversion panels. The p24 antigen limit of detection was determined in two ways, using the WHO international standard and three samples from HIV-1 antigen panels; the values obtained were 1IU/mL and 3.5-9.9 pg/mL for Genscreen Ultra HIV Ag-Ab, 1U/mL and 7.1-9.9 pg/mL for Architect HIV Ag/Ab combo assay, 0.5IU/mL and 4.0-7.1 pg/mL for VIDAS HIV DUO Quick, and 32.0-56.5 pg/mL for Genscreen Plus HIV Ag-Ab. In this study, we have shown that Genscreen Ultra HIV Ag-Ab has the sensitivity, specificity and p24 antigen limit of detection that is equal to those of two typical fourth-generation assays. This assay can be considered useful and reliable for HIV screening.

Emergence in Japan of an HIV-1 variant associated with transmission among men who have sex with men (MSM) in China: first indication of the International Dissemination of the Chinese MSM lineage.

A survey of HIV-1 strains circulating in the Tokyo-Kanagawa metropolitan area of Japan during 2004 to 2011 (n = 477) identified six Japanese males (patients 1 to 6), who harbored viruses with genome segments derived from a distinct CRF01_AE variant uniquely found among men who have sex with men (MSM) in China (designated CN.MSM.01-1). These six HIV infections were diagnosed in 2010 and 2011 among MSM (3 of 75) and men with unknown risk factors (3 of 63) and differed from the vast majority of HIV infections among MSM in Japan, which are overwhelmingly characterized by subtype B (239 of 246 [97.2%]). Approximately one-third (91 of 239 [38.1%]) of subtype B strains from MSM in Japan belong to a large monophyletic cluster (designated JP.MSM.B-1). In addition, we identified a smaller subtype B cluster (n = 8) (designated JP.MSM.B-2) that also contains strains from two Chinese MSM living in Japan. Interestingly, patients 5 and 6 were found to be coinfected with CRF01_AE (CN.MSM.01-1) and subtype B (JP.MSM.B-2 or JP.MSM.B-1) variants that are unique to the HIV-1 epidemics among MSM in China and Japan, respectively. Our study demonstrates for the first time the effect of the expanding HIV epidemic among MSM in China on transmission in neighboring countries and shows the ongoing mixing of CRF01_AE and subtype B lineages unique to HIV-1 that cocirculate in MSM populations in East Asia. This finding highlights the importance of strengthening epidemiological surveillance in the region and the need for effective measures to limit transmission among MSM in East Asia.

[Recent progress in HIV screening tests and nucleic acid tests].

HIV testing plays a crucial role in detecting and monitoring HIV infection. Diagnosis of HIV infection is basically made by sequential two tests: a screening test with an enzyme immunoassay(EIA) and a confirmatory test with Western blot. The most recent EIAs, used in commercial laboratories, identify HIV infection earlier because they detect both HIV-1 antibody and antigen. Rapid tests represent another advance for HIV screening. They are widely used in voluntary counselling and testing at public health centers and private clinics. An assay for detection of HIV-1 RNA was approved as a confirmatory test of reactive screening tests to diagnose early infection. These new testing technologies offer more accurate, reliable, and convenient diagnosis of HIV infection.

A human immunodeficiency virus screening algorithm to address the high rate of false-positive results in pregnant women in Japan.

BACKGROUND: Prenatal human immunodeficiency virus (HIV) testing is essential for the prevention of mother-to-child transmission. However, false-positive results of screening testing are a concern as they may cause unnecessary emotional stress to pregnant women waiting for confirmatory test results. In regions with an extremely low prevalence, the positive predictive values of screening are unacceptably low rate. Here, we propose a HIV screening algorithm consisting of serial two fourth-generation enzyme immunoassays to reduce the number of false-positive screening results. METHODOLOGY/PRINCIPAL FINDINGS: When 6461 pregnant women presenting to two maternity hospitals located in the Tokyo metropolitan area of Japan from September, 2004 to January, 2006 were tested using Enzygnost HIV Integral as a first screening test, 27 showed positive reactions. When these positive reaction samples were tested using VIDAS HIV DUO Quick as a second screening test, only one of them had a positive reaction, and the remaining 26 were nonreactive. Confirmatory Western blots and nucleic acid amplification test also showed that one was positive and the remaining 26 were negative; the subject who was positive with the confirmatory tests was identical to the subject who was positive with the second screening test. Thus, by adding the second screening test, the false-positive rate was improved from 0.4% to 0%, and the positive predictive value from 3.7% to 100%, compared with the single screening test. CONCLUSION: By applying our serial screening algorithm to HIV testing in maternity hospitals, many uninfected pregnant women would not need to receive confirmatory tests and be subjected to emotional turmoil while waiting for their confirmatory test results. This algorithm would be suitable for HIV testing of pregnant women living in low prevalence regions such as Japan.

Quantitation of HIV-1 group M proviral DNA using TaqMan MGB real-time PCR.

The level of human immunodeficiency virus type 1 (HIV-1) proviral DNA is likely to be an important marker of the long-term effectiveness of highly active antiretroviral therapy. A new method was developed for quantifying HIV-1 group M proviral DNA using TaqMan real-time PCR, in which degenerate primers and an MGB probe were used to resolve the difference in amplification efficiencies among different subtypes. The present assay provided good linearity and accuracy in the range of 4-5000 copies of proviral DNA in 0.5microg of cellular DNA. The intra-assay and inter-assay coefficients were <31.6% and <30.1%, respectively. In 19 HIV-1 clinical isolates of six subtypes (A, B, C, CRF01_AE, F, and G), quantitation values by the real-time PCR assay matched closely those by Poisson distribution analysis of PCR results at endpoint dilution (R(2)=0.988). This assay is characterized by the use of degenerate primers and having been validated by comparing with a Poisson distribution-based assay. The present real-time PCR assay is highly sensitive, linear, reproducible, accurate, and independent of group M subtypes. The assay will be useful for studying the relationship between HIV-1 proviral loads and the long-term efficacy of antiretroviral therapy for subtype B as well as non-B subtype strains.

[Testing services for sexually transmitted infections in Japan, public funding for HIV testing compared to public funding for other STI testing].

HIV and other STIs testing services of public funded setting have not been integrated in Japan. Public health centers and other public funded testing sites provide free anonymous HIV test. This has been playing an important role to confirm almost half of asymptomatic patients. Early diagnosis is an essential intervention for personal health, and critical for preventative strategies of public health. However, the role of public health centers and other public funded testing sites are very limited for other STIs. The symptomatic patients visit private clinic/hospital for diagnosis and treatment, but it is difficult for asymptomatic person to visit such medical facilities. The prevalence of genital chlamydia in young women in Japan remains very high compared to other developed countries. So, I think public funding of testing for genital chlamydia and other asymptomatic STIs should be expanded and integrated with HIV testing programs in Japan. Recently, there is the problem of the shortage of OB/GY doctors and clinics. This might influence the accessibilities of STIs testing and treatment opportunities for women. This is a new problem of STI testing in Japan.

[Evaluation of new fourth-generation human immunodeficiency virus antigen and antibody detection assay with enzyme-linked fluorescent immunoassay].

We evaluated the fourth-generation HIV screening assay VIDAS HIV DUOII (DUOII) based on ELFA for simultaneous detection of anti-HIV-1 and anti-HIV-2 antibodies and HIV-1 p24 antigen through comparison with other HIV antigen-antibody detection assays. Materials were 1228 HIV-negative specimens, 95 HIV-antibody-positive specimens, and HIV commercial panels. The specificity of DUOII was 99.8% and sensitivity 100%, detecting all of HIV-1 group M subtype A, B, B', C, D, A/E, F, G, B/D, HIV-1 group O, and HIV-2. The sensitivity test to HIV-1 p24 antigen was 5pg/ mL, higher than other assays. DUOII was equivalent to or superior in detecting results earlier than other assays in an evaluation using 10 commercial HIV-1 seroconversion panels of primary infection. DUOII detects anti-HIV IgM antibody, so no negative sample was found in the second window between p24 antigen disappearance and raised anti-HIV IgG antibody. DUOII has sufficient specificity and sensitivity for HIV screening, and detects primary infection sooner than other assays. These results indicate that DUOII is useful and reliable in HIV screening.

Drug-resistant HIV-1 prevalence in patients newly diagnosed with HIV/AIDS in Japan.

The increasing prevalence of drug-resistant HIV transmission has become a critical epidemic in the world today. Studies in developed countries reported 8-27% of newly diagnosed HIV/AIDS patients are infected by drug-resistant strains. To determine the prevalence of drug-resistant HIV-1 among newly diagnosed cases in Japan, eight HIV/AIDS clinical centers, three public health laboratories and the National Institute of Infectious Diseases conducted a nationwide survey. Between January 2003 and December 2004, 575 newly diagnosed HIV/AIDS patients with both acute and chronic infections were enrolled in the study. Twenty-three cases, including three recently infected patients, were infected with HIV-1 having major drug-resistance mutations, including M41L, D67N, L100I, K103N, V106A, M184I, M184V, L210W, and revertant mutations at the 215 codon in reverse transcriptase and M46I in protease encoding regions. In this newly diagnosed population, we also clarified the prevalence of hepatitis virus coinfection, which was 8.8% for HBV and 4.3% for HCV. In conclusion, the drug-resistant transmission rate was 4.0% in Japan. Although this rate is significantly lower than that of other developed countries, this rate almost reaches the threshold at which baseline genotypic resistance testing would be cost-effective for all infected persons before initiating therapy.

[Introduction of rapid HIV tests to a public health center in Japan and evaluation of its effects].

OBJECTIVE: As an attempt to make HIV testing more accessible to many people, rapid HIV tests were introduced to a public health center. We evaluated logistic issues and effects of the introduction. METHODS: In January 2003, the Tochigi Prefectural Kennan Public Health and Welfare Center introduced rapid HIV tests simultaneously with conventional HIV tests. We studied the number and types of the clients before and after its introduction as well as comparing them with those of other public centers. Information on rapid HIV test practices was uploaded on the website, "HIV Kensa-Soudan Map" and its effects were also evaluated. RESULTS: The public health center had performed 130 conventional HIV tests in 2002 prior to the introduction of rapid HIV tests but performed 453 HIV tests in 2003 (250% increase). Of those, 404 clients (94%) preferred the rapid tests. Out of the 404 rapid HIV tests performed, five were tested positive but only one was confirmed to be positive (false positive rate 1%). The introduction of rapid HIV tests had a negative impact on other sexually transmitted infection (STI) tests. The access rates to TP antibody tests (rapid tests) and Chlamydia antibody tests (non rapid tests) offered to HIV test recipients declined from 77% to 63% and from 76% to 33%, respectively. However, the actual number of these STI tests increased due to the increased number of HIV tests. Sixty-one per cent of those who came to take the HIV tests referred to the website "HIV Kensa-Soudan Map". During the same time period, the increase rate in the number of those who took the non rapid HIV tests provided at other public health centers in Tochigi prefecture was 0% and the increase rate nationwide was 20%. CONCLUSIONS: The introduction of these tests to public health centers greatly increases access to HIV testing. It was found that the website "HIV Kensa-Soudan Map" was a useful information source for HIV testing. However, because of high false positive rate (about 1%), it is important to establish an effective pre- and post-test counseling system. It is also necessary to prevent the number of tests for other STIs from declining when rapid HIV tests are implemented simultaneously.

Analysis of a long-term discrepancy in drug-targeted genes in plasma HIV-1 RNA and PBMC HIV-1 DNA in the same patient.

Drug-resistance genotypes were investigated in a patient under treatment with anti-HIV drugs. Since the drug resistance-associated mutations in plasma HIV-1 RNA and proviral DNA in peripheral blood mononuclear cells (PBMCs) were inconsistent, changes were followed over time, and the discrepancy was shown to persist for a long period. In plasma HIV-1 RNA, D67N, K70R, T215Y, and Y188L were present in the reverse transcriptase (RT) region, and two primary mutations, I84V and L90M, were noted in the protease (Pro) region. In contrast, in proviral DNA, no drug resistance-associated mutations were found in the RT region, and mutations such as L90L/M were only infrequently present in the Pro region. This situation persisted for more than 3 years. In addition, sequencing analysis of the V3 loop in the envelope gene showed that non-syncytium-inducing/macrophage-tropic viruses contribute to acquisition of drug resistance. In this study, drug-resistant viruses were produced primarily at macrophages, and drug-sensitive viruses were maintained in PBMCs as a reservoir.

Identification of attenuated variants of HIV-1 circulating recombinant form 01_AE that are associated with slow disease progression due to gross genetic alterations in the nef/long terminal repeat sequences.

We identified an unusual case of human immunodeficiency virus type 1 (HIV-1) infection in a patient (GM43) who exhibited a persistently low antibody response and undetectable viral load during a 5-year follow-up period. GM43 harbored HIV-1 circulating recombinant form 01_AE with gross deletions in the nef/long terminal repeat (LTR) region. The sizes of the deletions increased progressively from 84 to >400 bp during the 5-year period. GM43 appeared to have acquired defective variants from her husband. The genetic alterations in the nef/LTR region were remarkably similar to those that have been reported in slow progressors (such as the slow progressors in the Sydney Blood Bank Cohort). The present study is the first report of slow disease progression due to gross genetic alterations in the nef/LTR region in a person infected with an HIV-1 non-subtype B strain.

A case of symptomatic primary HIV infection.

A 30-year-old homosexual Japanese man had fourteen days of fever, malaise, appetite loss, sore throat, and four days of diarrhea and slightly congested eyes before he developed a skin eruption. He presented with measles-like exanthems on his face, trunk, and extremities. Deep red enanthems were seen on his left buccal mucosa opposite the premolar teeth, and whitish enanthems were seen on the buccal and gingival mucosa. HIV RNA was detected at the high concentration of 5.8 x 10(6) copies /ml in his serum. Cerebrospinal fluid examination revealed aseptic meningitis with 5,488 copies /ml of HIV RNA. Anti-HIV 1 antibodies against Gp160 and p24 tested by Western blot assay showed seroconversion on day 5 of his admission, seven days after he developed the skin eruptions. The fever lasted for three weeks from the initial onset, and the skin eruptions lasted for twelve days. Histopathologically, a mononuclear cell infiltration was seen mainly in the upper dermis surrounding small vessels and sweat ducts, with CD8+ cytotoxic T lymphocytes predominant. Additionally, CD1a+ putative interdigitating dendritic cells had also infiltrated perivascularly, and were surrounded by CD8+ and CD4+ T cells. In situ hybridization study failed to detect HIV products in skin biopsy specimens. Our findings suggested that CD8+ T cells and their interaction with CD1a+ dendritic cells in the skin may be important in inducing skin manifestations in acute HIV infections.

Current state and need for improvement of system for antibody testing and counseling for HIV infection at public health centers in Japan.

BACKGROUND: Public Health Centers (PHCs) throughout Japan have been playing a role in preventing HIV in Japan. The number of HIV seropositive persons and AIDS patients is increasing from year to year in Japan. METHODS: A survey regarding the HIV antibody test was sent out to 594 PHCs between January 1997 and December 2001. The response rate was 73.9%. In order to assess the HIV testing system at PHCs, Pearson's correlation coefficient, an analysis of variance (ANOVA) and multiple regression analysis were used. RESULTS: There was a difference in the demand for HIV antibody tests according to area. The change in number of HIV antibody tests per 100,000 persons was high in metropolitan areas during the 5-year period. It was influenced by two factors; population density and ratio of daytime population to nighttime population. Rate of examinees who did not return to receive their test results was influenced by the area where the PHC was located. CONCLUSIONS: PHCs need to establish an HIV testing system which reflects the characteristics of the area and meets the needs of people who want to get tested.

[Evaluation of flow-through immunoassay for rapid detection of influenza A and B viruses].

We evaluated a flow-through immunoassay for rapid detection of influenza A and B viral antigens, RapidTesta FLU AB (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan), by using 507 specimens collected from patients with influenza-like symptoms during the 2002/2003 influenza season in Japan. The specimens consisted of 239 nasal swabs and 268 nasal aspirates; 374 specimens were collected from pediatric patients (under 16 years of age) and 133 from adult patients. RapidTesta FLU AB was compared with cell culture and nested reverse transcription-polymerase chain reaction (RT-PCR). Cell culture detected influenza virus from 66.7% of the 507 specimens (influenza AH3: 44.0%, B: 22.7%). For nasal swabs, it had a sensitivity of 81.9% (77/94), a specificity of 97.9% (142/145) and an efficiency of 91.6% (219/239) for influenza A virus as well as a sensitivity of 80.0% (52/65), a specificity of 98.3% (171/174) and an efficiency of 93.3% (223/239) for influenza B. For nasal aspirates, RapidTesta FLU AB had a sensitivity of 83.2% (109/131), a specificity of 98.5% (135/137) and an efficiency of 91.0% (244/268) for influenza A as well as a sensitivity of 82.7% (43/52), a specificity of 97.7% (211/216) and an efficiency of 94.8% (254/268) for influenza B. RapidTesta FLU AB is characterized by high specificity, low false positive rate, and 10-minute detection of influenza virus. These advantages suggest that RapidTesta FLU AB is a useful kit to assist physicians in making a diagnosis of influenza on candidates for antiviral therapy.

[Evaluation of an immunochromatography test using enzyme immunoassay for rapid detection of influenza A and B viruses].

We evaluated the performance of an improved version of Espline Influenza A & B-N (Fujirebio Inc., Japan), an immunochromatography test using enzyme immunoassay for rapid diagnosis of influenza A and B. The test produced positive results for four strains of influenza viruses and thirty-one influenza viral antigens and negative results for all of thirty strains of other respiratory viruses that were tested. The detection limit of this test was 5.8 x 10(2) to 5.8 x 10(3) pfu/assay, which is more sensitive than the old version of Espline. Furthermore, 715 respiratory specimens collected from the patients (children, 79.4%; adults, 18.5%; unknown, 2.1%) with influenza-like illnesses during the 2002/2003 influenza season in Japan were tested as part of a clinical evaluation of this test. The relative performance of this test compared to cell culture and nested RT-PCR results were examined. In the cell cultures, influenza viruses were detected in 488 of the 715 specimens (overall, 68.3%; AH3, 41.7%; B, 26.4%; AH3 and B, 0.1%). For influenza A, the sensitivity of this test was 95.4% (125/131) for nasal aspirates, 96.8% (92/95) for nasal swabs, and 85.1% (63/74) for throat swabs. For influenza B, the sensitivity of this test was 91.2% (52/57) for nasal aspirates, 88.1% (59/67) for nasal swabs, and 71.6% (48/67) for throat swabs. The new test exhibited a remarkably higher sensitivity to influenza A in throat swabs than the old version of Espline. Only two false positive results were obtained out of a total of 223 virus negative specimens; the specificity of the test was 100% (88/88) for nasal aspirates, 97.6% (81/83) for nasal swabs, and 100% (52/52) for throat swabs. We conclude that the new Espline Influenza A&B-N rapid diagnostic test is easy to use and has a high sensitivity and specificity, especially for influenza A.

Transfusion-transmitted hepatitis E caused by apparently indigenous hepatitis E virus strain in Hokkaido, Japan.

BACKGROUND: In industrialized countries, sporadic cases of hepatitis E have been reported in individuals who have never been in an endemic area. Hepatitis E virus (HEV) infection commonly occurs via the fecal-oral route but a potential risk of transfusion transmission route has been suggested. STUDY DESIGN AND METHODS: A 67-year-old Japanese male patient who had never been abroad received a transfusion of blood from 23 voluntary donors and developed acute hepatitis with unknown etiology after transfusion. His blood samples were tested for viral markers of hepatitis viruses. RESULTS: HAV, HBV, HCV, CMV, and EBV were ruled out as causative agents in this case. The patient's blood sample in the acute phase contained HEV RNA as well as IgM and IgG anti-HEV. HEV RNA was also detected in one of the FFP units transfused. The donor had no history of traveling abroad and had a normal ALT level at the time of donation. The PCR products from the patient and the donor showed complete identity for two distinct regions of HEV within open reading frame 1. CONCLUSION: The patient was infected with HEV via transfused blood from a volunteer donor. A potential risk of posttransfusion hepatitis E should be considered even in nonendemic countries.

Human immunodeficiency virus in Uzbekistan: epidemiological and genetic analyses.

This study investigates the molecular epidemiology of HIV in Uzbekistan--a former Soviet Union (FSU) country located in central Asia. A total of 18,910,370 subjects were involved in an HIV serological examination through a population survey conducted in 1987-2002. Rapid changes in epidemiological dynamics and transmission modes have been observed since 1999: incidence rose from 25 newly HIV-infected subjects per year to more than 100 new cases per month within the first half of 2002, and the rate of intravenous drug use (IVDU)-associated HIV infection increased to 75% per year during the same period. Thirty HIV-1 strains, isolated from specimens obtained in 1999-2000, were directly sequenced in the env region. Phylogenetic analysis revealed a relationship to genotype A in 56.7%, and to 03_CRFAB in 13.3%; both variants have been previously reported in the FSU. The majority (85.7%) of these strains were isolated from IVDUs. The demographic history of the most prevalent HIV strain in Uzbekistan was inferred from reconstructed molecular phylogenies; exponential growth of the viral population size was thus observed to occur after the mid-1990s. In summary, detectable HIV seroprevalence remains low in the general population of Uzbekistan. However, the current study demonstrates a substantially increasing number of new infections, in association with IVDU, and an exponentially growing effective population size of the IVDU-associated HIV strain.