Genetic characterization and genotyping of hepatitis B virus (HBV) isolates from donors with an occult HBV infection.

BACKGROUND AND OBJECTIVES: Screening of Thai blood donors has resulted in the detection of donors with an occult HBV infection (OBI), where HBsAg is undetectable, but hepatitis B virus (HBV) DNA is present in serum in low concentrations. This study was designed to determine whether the occurrence of OBI in donors was linked to the HBV genotype and possibly to mutations in the surface (S) and core (C) gene regions. MATERIALS AND METHODS: Mutations in the S and C gene regions in 48 Thai donors with OBI were mapped by sequencing. Genotyping was determined with the INNO-LiPA test and by phylogenetic analysis of sequences from the S and C genes. RESULTS: The majority of OBI samples were genotype C (81·3%) with 6·3% of samples being genotype B. In addition, two genotype I isolates were identified. Mutations in the S region (100%) were found especially in loop 1 of the major hydrophilic loop (MHL) at positions I110L, T114S, T126I and S113T, whereas mutations in the C region (65%) were within the basal core promoter region (position A1762T/G1764A) and precore region (position G1896A). CONCLUSION: The majority of OBI samples were HBV genotype C, although genotype I, which is newly emerging in Thailand, was also detected. The study demonstrated that OBI was probably not associated with a particular HBV genotype or with certain mutations in the S and C gene regions. However, mutations in the C gene region which could potentially impair viral replication and HBsAg production and potentially lead to OBI were identified.

Occurrence of hepatitis E virus infection in acute hepatitis in Thailand.

Hepatitis E virus (HEV) infection is one of the enterically transmitted types of hepatitis. The present study was undertaken to estimate the occurrence of HEV infection in sporadic acute hepatitis in Thailand. Serum samples were obtained from 614 suspected acute hepatitis patients at two large hospitals in Bangkok during 2008, 2009, and 2011. Acute hepatitis E was identified by the presence of anti-HEV IgM (4.8%) using indirect ELISA kits and/or HEV RNA (4.5%) by a semi-nested reverse transcription-polymerase chain reaction assay. HEV IgM was the most common marker for detection (77%) at diagnosis, either by positive HEV IgM alone or together with HEV RNA, whereas HEV RNA alone was detected in 23% of patients. Overall, 4.2% of cases (26 out of 614) were acute HEV infection with the highest attack rate in the elderly age group. In addition, nucleotide sequence analysis of five HEV samples revealed 92.8-99.8% homology. All viruses were clustered into HEV genotype 3 and were similar genetically to swine HEV strains previously detected in the same area. Therefore, the occurrence of HEV infection with closely related to swine genotype 3 was approximately 4-5% of acute hepatitis cases in Thailand. Anti-HEV IgM was the most common marker at diagnosis.

Surveillance of hepatitis A and E viruses contamination in shellfish in Thailand.

AIMS: To survey for hepatitis A virus (HAV) and hepatitis E virus (HEV) contamination in edible bivalve shellfish. METHODS AND RESULTS: A total of 213 shellfish (52 oysters, 69 cockles and 92 mussels) collected from a culture farm and two retailed markets were investigated for HAV and HEV contamination by reverse transcription-polymerase chain reaction (RT-PCR) assay using HA2-HA1 (capsid region) and HE366-HE363 (ORF2/3 overlapping region) primers, respectively. It was found that 3.8% of the shellfish and 2.9 and 6.5% of the cockle and mussel, respectively, showed positive for HAV detection. Nucleotide sequencing of all the 8 HAV-positive shellfish revealed 97-100% similarity to HAV subgenotype IA. Interestingly, viruses were found more frequently in the gills than in digestive tissue (4.5%vs 0.5%, P = 0.045). All the shellfish were negative for HEV. CONCLUSION: Significant contamination of HAV in edible bivalve shellfish was observed. Beside digestive tissue, gills are one of the important samples for viral genome detection. SIGNIFICANCE AND IMPACT OF THE STUDY: HAV-contaminated shellfish can play a role as reservoirs and/or vehicles in faecal-oral transmission in Thailand, and further monitoring of such a contamination is required.

Multicenter evaluation of the Elecsys hepatitis B surface antigen quantitative assay.

The Elecsys hepatitis B surface antigen (HBsAg) II quantitative assay is a new quantitative electrochemiluminescence immunoassay which uses onboard dilution and a simple algorithm to determine HBsAg levels expressed in international units (IU)/ml (standardized against the World Health Organization [WHO] Second International Standard). This study evaluated its performance using routine serum samples from a wide range of HBsAg carriers and patients with chronic hepatitis B (CHB). HBsAg levels were measured in serum samples collected independently by five centers in Europe, Australia, and Asia. Serial dilution analyses were performed to assess the recommended dilution algorithm and determine the assay range free of hook effect. Assay precision was also established. Following assessment of serial dilutions (1:100 to 1:1,000,000) of the 611 samples analyzed, 70.0% and 85.6% of samples tested with analyzers incorporating 1:100 (Elecsys 2010 and cobas e 411) and 1:400 (Modular Analytics E170) onboard dilution, respectively, fell within the linear range of the assay, providing a final result on the first test. No high-dose hook effect was seen up to the maximum HBsAg serum level tested (870,000 IU/ml) using the dilution algorithm. HBsAg levels were reliably determined across all hepatitis B virus (HBV) genotypes, phases of HBV infection, and stages of disease tested. Precision was high across all analyzers (% coefficient of variation [CV], 1.4 to 9.6; HBsAg concentrations, 0.1 to 37,300 IU/ml). The Elecsys HBsAg II quantitative assay accurately and reliably quantifies HBsAg in routine clinical samples. Onboard dilution minimizes retesting and reduces the potential for error.

Comparison of the technical and clinical performance of the Elecsys HBsAg II assay with the Architect, AxSym, and Advia Centaur HBsAg screening assays.

South East Asia has some of the highest prevalence rates of hepatitis B virus (HBV) infection (>or=8%) in the world, and the emergence of hepatitis B surface antigen (HBsAg) mutant strains is a growing problem. Assays with the highest levels of sensitivity, including mutant detection, should be used for routine HBsAg screening. In this large multicenter study, the clinical and technical performance of the fully automated Elecsys HBsAg II assay was compared with the Architect, AxSYM, and Advia Centaur HBsAg assays for HBsAg screening. Nine laboratories (three each from Thailand, Korea, and Singapore) compared the Elecsys HBsAg II assay with their routine HBsAg screening assay against a range of stored and routine clinical samples, including recombinant mutants. The Elecsys HBsAg II assay demonstrated equivalent sensitivity and specificity to the Architect HBsAg assay. However, the Elecsys HBsAg II assay recognized a native mutant sample (L94S, L97V, L98V, T123A) that the Architect HBsAg assay failed to detect. The AxSYM and Advia Centaur HBsAg assays appeared less sensitive for the detection of early HBV infection and also failed to detect some of the recombinant mutant strains. There was almost complete agreement between the Elecsys HBsAg II assay and comparator assays with respect to routine serum samples. The results of this study demonstrate that the Elecsys HBsAg II assay is a highly sensitive and specific screening assay for HBsAg and detects reliably the most important and clinically relevant HBV mutants and genotypes. It is suitable for routine HBsAg screening in Asia.

Multicenter study of a new fully automated HBsAg screening assay with enhanced sensitivity for the detection of HBV mutants.

In a multicenter study a new, fully automated Roche Diagnostics Elecsys HBsAg II screening assay with improved sensitivity to HBsAg mutant detection was compared to well-established HBsAg tests: AxSYM HBsAg V2 (Abbott), Architect HBsAg (Abbott), Advia Centaur HBsAg (Bayer) Enzygnost HBsAg 5.0 (Dade-Behring), and Vitros Eci HBsAg (Ortho). A total of 16 seroconversion panels, samples of 60 HBsAg native mutants, and 31 HBsAg recombinant mutants, dilution series of NIBSC and PEI standards, 156 HBV positive samples comprising genotypes A to G, 686 preselected HBsAg positive samples from different stages of infection, 3,593 samples from daily routine, and 6,360 unselected blood donations were tested to evaluate the analytical and clinical sensitivity, the detection of mutants, and the specificity of the new assay. Elecsys HBsAg II showed a statistically significant better sensitivity in seroconversion panels to the compared tests. Fifty-seven out of 60 native mutants and all recombinant mutants were found positive. Among 156 HBV samples with different genotypes and 696 preselected HBsAg positive samples Elecsys HBsAg II achieved a sensitivity of 100%. The lower detection limit for NIBSC standard was calculated to be 0.025 IU/ml and for the PEI standards ad and ay it was <0.001 and <0.005 U/ml, respectively. Within 2,724 daily routine specimens and 6.360 unselected blood donations Elecsys HBsAg II showed a specificity of 99.97 and 99.88%, respectively. In conclusion the new Elecsys HBsAg II shows a high sensitivity for the detection of all stages of HBV infection and HBsAg mutants paired together with a high specificity in blood donors, daily routine samples, and potentially interfering sera.

The prevalence of viral hepatitis among the Hmong people of northern Thailand.

Sera from 269 Hmong people (102 males and 167 females, with mean age 35.4 years, range 16-63 years) were examined in order to determine the seroprevalence of hepatitis virus infection. The seroprevalence rates for HAV (hepatitis A virus), HBV (hepatitis B virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), HGV (hepatitis G virus) and TTV (TT virus) infection were 87.8% (n=140), 76.0% (n=150), 2.0% (n=150), 0.7% (n=150), 6.5% (n=139), 5.3% (n=94) and 25.6% (n=121) respectively. The rate for carriers of HBV (HBsAg) was 13.8% (20.5% in males and 9.6% females) with a peak prevalence in the 21-40 year age group. A high rate of HAV seropositivity was found among the younger subjects. The rate of HEV seroprevalence was low. The prevalence of TTV-DNA was high with no difference between the sexes. HGV-RNA prevalence was low and seen primarily in males. This study indicates that the Hmong people are endemically infected with HAV and HBV infection and should be considered for targeted vaccination. The role of TTV and HGV in producing illness and hepatic disease has yet to be determined in this population.

Application of HIV-1-green fluorescent protein (GFP) reporter viruses in neutralizing antibody assays.

We made reporter HIV-1 DNA constructs carrying green fluorescent protein (GFP) gene and exchangeable env of subtype E. The recombinant constructs were used to produce infectious reporter viruses, which induced infected cells to emit green fluorescent light and rendered them easily detectable at single cell level. Because the env in this construct can be easily exchanged, viruses with different antigenic epitopes can be made. We used these reporter viruses to set up a neutralizing antibody assay based on fluorescence reduction by flow cytometric measurement. The result of this new assay correlated with the standard infectivity reduction assay using primary isolates. Because this new assay is faster and much less costly than the standard assay using a p24 endpoint and can be performed in peripheral blood mononuclear cells (PBMC), it provides a useful tool for analysis of HIV-1 immune responses.

Differential susceptibility of quiescent CD4+ lymphocytes to syncytial-inducing and non-syncytial-inducing isolates of HIV-1.

It is generally believed that quiescent CD4+ T cells are not susceptible to HIV-1 infection. However, infection of unstimulated peripheral mononuclear cells by syncytial-inducing (SI) viruses has been shown to be much more efficient than with non-syncytial-inducing (NSI) viruses. This suggested that SI, CXCR4-tropic viruses may be able to infect quiescent CD4+ T cells. We studied the infection of highly purified quiescent CD4+ T cells by SI and NSI viruses. In this article we show that although NSI viruses failed to significantly infect quiescent cells, SI viruses consistently infected these cells and produced viruses upon cellular activation by interleukin-2, 2 to 7 days after initial infection. To examine whether the difference was the result of viral or host factors, we purified CCR5+ quiescent CD4+ T cells and showed that these cells can be infected by dual tropic (R5X4) but not by R5 virus. This indicated that CCR5+ quiescent T cells were also susceptible to HIV-1 infection, and the failure of NSI, CCR5-tropic viruses to infect quiescent cells may be due to some intrinsic properties of these viruses.

Microbial killing activity of peracetic acid.

In vitro killing activity of peracetic acid (Perasafe) at a concentration of 0.26 per cent w/v was tested against Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Salmonella paratyphi A, Acinetobacter baumannii, Sternotrophomonas maltophilia, Enterococcus faecium, Enterococcus faecalis, methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis spore, Mycobacterium tuberculosis and human immuno-deficiency virus type I. Exposure to Peracetic acid (0.26% w/v) for 10 minutes resulted in massive killing of all the aforementioned organisms and spore.

Anti-HIV-1 antibody testing using modified gelatin particle agglutination: a large field study.

Anti-HIV testing using gelatin particle agglutination (GPA) assay was investigated in parallel with ELISAs from routine service at Siriraj Hospital. In the first strategy, 174,032 sera from a patient population with an HIV-1 seroprevalence of 13.72 per cent were assayed using reduced volumes of GPA reagents, giving a cost reduction of 40 per cent. In the second strategy, 90,560 pregnant women and 48,936 emigrant workers with an HIV-1 seroprevalence of 2.2 per cent and 0.3 per cent, respectively, were tested in pools of 4 sera using the manufacturer's recommended volumes, giving a cost saving of 67 per cent. Overall, the sensitivity and specificity were almost identical with standard methods. Thus, parallel use of either modified GPA might be considered appropriate when testing large numbers of samples. However, both modified versions of GPA are not recommended as the first assay for diagnostic or blood bank screening especially in high prevalence of HIV infection.

Role of maternal humoral immunity in vertical transmission of HIV-1 subtype E in Thailand.

The significance of the maternal humoral immune response in relation to vertical transmission of HIV-1 was investigated in 123 mothers infected with subtype E from Thailand. Antibody binding titers to HIV-1 env domains (monomeric gp120, the CD4/gp120 binding site [BS], V3 loop, and gp41) and antibody-mediated neutralization of primary and T-cell line-adapted (TCLA) subtypes B and E HIV-1 isolates were investigated. No correlation between maternal anti HIV-1 antibodies at delivery and vertical transmission of HIV-1 subtype E was found. However, a trend to higher titer antibody-mediated cross-neutralization of a heterologous subtype B TCLA isolate, HIV-1MN, was observed in nontransmitting mothers postpartum. The HIV-1-specific antibody titers in these infected mothers increased significantly from delivery to 6 months postpartum (p < .05), but this was only partially attributable to hemodilution and an additional factor or factors appear to affect humoral immunity to HIV-1 during late pregnancy.

Characterization of sera from subjects infected with HIV-1 subtypes B and E in Thailand by antibody binding and neutralization.

The range and specificity of the humoral immune response to HIV-1 subtypes B and E was investigated in Thai samples. Sera from HIV-1-positive subjects, consisting of subtypes B (n = 24) and E (n = 138), were characterized in relation to the neutralization of primary isolates and T-cell line-adapted (TCLA) strains and binding to monomeric gp120, the CD4/gp120 binding site (BS), and V3 peptides. A subtype-specific pattern of antibody binding was observed with the exception of the CD4/gp 120MN BS. Neutralization of TCLA strains (n = 4) was strongly type-specific (p = .002); however, neutralization of primary isolates (n = 8) was weak and group specific. Thus, the subtype specificity of B and E sera in the neutralization of TCLA strains, but not primary isolates, supports the dominance of the V3 region in TCLA virus neutralization but does not support the distinction of subtypes B and E as discrete neutralization serotypes in Thailand.

PIP: The range and specificity of the humoral immune response to HIV-1 subtypes B and E were investigated in sera and plasma collected from 168 infected patients from Thailand in 1990-94. Specifically, samples were examined for the presence of binding antibody to env regions within monomeric gp120, the CD4/gp120 binding site, and the V3 domain as well as neutralizing antibodies to T-cell line-adapted (TCLA) and primary HIV-1 isolates from subtypes B and E. A subtype-specific pattern of antibody binding was observed with the exception of the CD4/gp120MN binding site. Although neutralization of TCLA strains was highly type-specific, neutralization of primary isolates was weak and group-specific. This finding supports the dominance of the V3 region in TCLA virus neutralization but fails to confirm the distinction of subtypes B and E as discrete neutralization serotypes in Thailand.

Neutralization of primary and T-cell line adapted isolates of human immunodeficiency virus type 1: role of V3-specific antibodies.

The role of the third variable domain (V3) of gp120 in the neutralization of primary and T-cell line adapted (TCLA) strains of human immunodeficiency virus type 1 (HIV-1) by serum from HIV-1-infected individuals was investigated. A primary virus isolate, M2424/4, when adapted to H9 cells, was more sensitive to neutralization on MT2 cells than the same stock passaged in PBMC. Neutralization of the PBMC-passaged stock by V3-specific MAbs was abrogated by addition of V3 (MN) peptide. However, exogenous V3 (MN) peptide failed to reduce the neutralization of this isolate on PBMC, or MT2 cells, by high titre anti-HIV-1 polyclonal human sera in contrast to the extensive reduction of neutralization by the same sera on MT2 cells using the prototype MN strain (4- to > or = 24-fold) and the TCLA M2424/H9 isolate (2- to 8-fold). These results indicate that the neutralization of primary virus isolates by serum from HIV-1-infected individuals is not significantly mediated by V3-specific antibodies.

The reference range of CD4+ and CD8+ T-lymphocytes in healthy non-infected infants born to HIV-1 seropositive mothers; a preliminary study at Siriraj Hospital.

The results of CD4+, CD8+ T-lymphocyte values as percentage, number, and ratio were studied in infants aged 1 to 29 months. The 283 subsequent blood samples from 89 infants born to HIV-1 seropositive mothers were investigated. From 208 sequential samples of 70 healthy non-infected infants, the reference values of CD4+ and CD8+ T-lymphocytes have been established and compared to Caucasian reference values. The results were analysed in 4 difference age groups (1-5, 6-11, 12-17 and > or = 18 months). At age 12 months, CD4 number and percentage declined significantly while CD8 percent increased. At age 6 months CD4/CD8 ratio decreased. Of 19 infected infants CD4+ percentage and number as well as CD4/CD8 ratio declined at age 6 months and showed significant differences from uninfected infants. A significantly elevated CD8 percentage was demonstrated in infected infants at age of 12 months. In 9 infants who showed symptoms at age 6-18 months, the CD4 and CD8 values were different from the reference range and 6 of 9 patients showed lower CD4 percentage, CD4 number and reversed CD4/CD8 ratio before the symptoms appeared. In 10 infants who were asymptomatic at age 18 months, there was no evidence of immunosuppression at age 6 months or before. After age 6 months, lymphocyte subset values of some asymptomatic infected children were beyond the reference range. These preliminary findings should be very useful for monitoring children born to HIV infected mothers. The results of CD4+ and CD8+ T-lymphocytes in uninfected infants could be used as reference values for the Thai and other Southeast Asian pediatric populations.

Langerhans cell tropism of human immunodeficiency virus type 1 subtype A through F isolates derived from different transmission groups.

To test the hypothesis that some subtypes of human immunodeficiency virus type 1 (HIV-1), especially subtype E, are more likely to infect mature Langerhans cells (mLC), we titrated a panel of 26 primary HIV-1 isolates of subtypes A through F on peripheral blood mononuclear cells (PBMC) and mLC. The majority of HIV-1 isolates from heterosexually infected patients did not show a preferred tropism for mLC compared to homosexually transmitted HIV-1 isolates. Only 6 of 26 isolates, 2 from patients infected by homosexual contact and 4 from patients infected by heterosexual contact, showed a higher infectivity for mLC than for PBMC. Both syncytium-inducing and non-syncytium-inducing isolates were able to infect mLC which express mRNA for the chemokine receptors CCR3, CCR5, and CXCR4.

Sequence analysis of an HIV type 1 env subtype E isolate from Thailand with discordant V3-loop serotyping.

PIP: HIV-1 genetic subtypes B and E are currently circulating in Thailand. Subtype E accounts for more than 90% of heterosexual transmission nationwide, while subtype B is transmitted mainly among IV drug users. This paper reports an HIV-1 Thai E isolate which yielded discordant results in serotyping and genotyping assays. An HIV-1-infected mother enrolled in Bangkok in a perinatal transmission study was identified independently as subtype B by V3 serotype by St. Mary's and HIV/AIDS Collaboration laboratories using different in-house assays. Her plasma was also screened for other HIV-1-specific immune responses, yielding a pattern of antibody reactivity similar to other subtype E sera. The genetic subtype of the isolate was identified by heteroduplex mobility assay (HMA) to further characterize it. Analysis results suggest that the woman was infected with HIV-1 subtype E. The env region encoding C2-V3 was subsequently sequenced to clarify the discordant results between the V3 serotype B and the genetic subtype E.^ieng

Immune status in congenital infections by TORCH agents in pregnant Thais.

A cross-sectional, sero-epidemiological survey of the prevalence of antibodies to TORCH agents during various stages of gestation revealed an overall rate of 13-15 percent having antibodies to Toxoplasma gondii; 85-87 percent, to rubella ; 79-81 percent, to herpes simplex virus (HSV); 100 percent, to cytomegalovirus (CMV); 82-86 percent, to human herpes virus type 6 (HHV-6); 1-2 percent, to hepatitis C virus (HCV). None of human T lymphotropic virus type I (HTLV-I) antibody was detected, and a prevalence of hepatitis B surface antigen (HBsAg) was 6 percent. Although a tendency was noted towards an increase of antibody detection to each TORCH agent as gestation progressed, a statistically significant increase in antibodies titer and specific IgM antibody was found with regard to CMV. These results suggest an increase in CMV infection or reactivation during pregnancy whereas an increase in the other TORCH infections was not obvious.

V3 peptide enzyme immunoassay for serotyping HIV-1 infected pregnant Thais.

Previous molecular epidemiological studies show that at least 2 subtypes of HIV-1 circulate in Thailand. HIV-1 subtype B or Thai genotype B was associated with an early epidemic and was prevalent in intravenous drug users. Meanwhile, HIV-1 subtype E or Thai genotype A was becoming widespread among heterosexuals. We studied the HIV subtypes of 161 HIV-1 seropositive pregnant women. Of these, 143 pregnant patients (88.8%) tested positive for subtype E alone and 8 women (5.0%) had evidence of infection with subtype B alone. There was serologic evidence of infection with a mixture of subtypes in 7 women while the infecting subtype could not be identified in the remaining 3 women. This result agrees with previous information that subtype E predominates in Thai heterosexuals.

Detection of HIV-1 proviral DNA by polymerase chain reaction: a preliminary study in Bangkok.

Nested polymerase chain reaction (nested PCR) was used to separately amplify part of gag, pol, and env genes of human immunodeficiency virus type 1 (HIV-1) to evaluate that primer specific to either gag (SK380/390&SK38/39), pol (JA17/18&JA19/20), or env (JA9/10&JA11/12) genes is suitable for HIV-1 PCR based diagnosis in Thailand. The positive PCR results in 70 HIV-1 infected adults are 100, 97, 89 per cent and in 75 HIV-1 infected infants are 100, 94, 74 per cent by gag, pol, env primer, respectively. The specificity of all three primer sets is 100 per cent. The unamplified samples by pol and env primers were identified as HIV-1 subtype E by PELISA method. False negative in HIV-1 PCR based diagnosis caused by high genetic variation of HIV-1 can be overcome by using several primer sets as shown in this study.