Hepatitis A and E among immigrants and refugees in Central Brazil

ABSTRACT OBJECTIVE To estimate the prevalence of hepatitis A virus (HAV) and hepatitis E virus (HEV) among immigrants and refugees in Goiás, Central Brazil. METHODS Overall, 355 individuals were interviewed, and blood samples were tested for anti-HAV and anti-HEV IgG. Anti-HEV-positive samples were similarly tested for HEV RNA. RESULTS All participants were from Latin American countries, most of whom, young adult males. The overall anti-HAV IgG prevalence was 87.4% (95%CI: 83.5-90.4), of whom 94.9%, 75.6%, and 60% were from Haiti, Venezuela, and other Latin American countries, respectively (p < 0.001). Age above 19 years and more than 36 months residing in Brazil were associated with a higher prevalence of previous HAV and HEV infection, respectively. Of the children eligible for HAV vaccination according to the National Immunization Program, only eight (44%) had been vaccinated. The overall anti-HEV IgG prevalence was 6.5% (95%CI: 4.4-9.5). All anti-HEV IgG-positive individuals were Haitians, including a child born in Brazil. HEV RNA was detected in two of the anti-HEV IgG-positive samples. CONCLUSION The survey detected a high prevalence of anti-HAV and anti-HEV IgG among immigrants and refugees, and active HEV infection among some Haitian participants. Prevention measures are urgently required to interrupt enteric virus transmission in this emergent and vulnerable population.

Comparative Analysis of Liver Injury-Associated Cytokines in Acute Hepatitis A and B

PURPOSE: Acute hepatitis A (AHA) and acute hepatitis B (AHB) are caused by an acute infection of the hepatitis A virus and the hepatitis B virus, respectively. In both AHA and AHB, liver injury is known to be mediated by immune cells and cytokines. In this study, we measured serum levels of various cytokines and T-cell cytotoxic proteins in patients with AHA or AHB to identify liver injury-associated cytokines. MATERIALS AND METHODS: Forty-six patients with AHA, 16 patients with AHB, and 14 healthy adults were enrolled in the study. Serum levels of 17 cytokines and T-cell cytotoxic proteins were measured by enzyme-linked immunosorbent assays or cytometric bead arrays and analyzed for correlation with serum alanine aminotransferase (ALT) levels. RESULTS: Interleukin (IL)-18, IL-8, CXCL9, and CXCL10 were significantly elevated in both AHA and AHB. IL-6, IL-22, granzyme B, and soluble Fas ligand (sFasL) were elevated in AHA but not in AHB. In both AHA and AHB, the serum level of CXCL10 significantly correlated with the peak ALT level. Additionally, the serum level of granzyme B in AHA and the serum level of sFasL in AHB correlated with the peak ALT level. CONCLUSION: We identified cytokines and T-cell cytotoxic proteins associated with liver injury in AHA and AHB. These findings deepen the existing understanding of immunological mechanisms responsible for liver injury in acute viral hepatitis.

Molecular characterization of hepatitis A virus strains in a tertiary care health set up in north western India.

Background & objectives: Hepatitis A virus usually causes acute viral hepatitis (AVH) in the paediatric age group with a recent shift in age distribution and disease manifestations like acute liver failure (ALF). This has been attributed to mutations in 5’non-translated region (5’NTR) which affects the viral multiplication. The present study was aimed to carry out the molecular detection and phylogenetic analysis of hepatitis A virus strains circulating in north western India. Methods: Serum samples from in patients and those attending out patient department of pediatric gastroenterology in a tertiary care hospital in north India during 2007-2011 with clinically suspected AVH were tested for anti-hepatitis A virus (HAV) IgM antibodies. Acute phase serum samples were subjected to nested PCR targeting the 5’NTR region followed by sequencing of the representative strains. Results: A total of 1334 samples were tested, 290 (21.7%) were positive for anti-HAV IgM antibody. Of these, 78 serum samples (< 7 days old) were subjected to PCR and 47.4% (37/78) samples showed the presence of HAV RNA. Children < 15 yr of age accounted for majority (94%) of cases with highest seropositivity during rainy season. Sequencing of 15 representative strains was carried out and the circulating genotype was found to be III A. The nucleotide sequences showed high homology among the strains with a variation ranging from 0.1-1 per cent over the years. An important substitution of G to A at 324 position was shown by both AVH and ALF strains. The cumulative substitution in AVH strains Vs ALF strains as compared to GBM, Indian and prototype strain in the 200-500 region of 5’ NTR was comparable. Interpretation & conclusion: Our results showed hepatitis A still a disease of children with III A as a circulating genotype in this region. The mutations at 5’NTR region warrant further analysis as these affect the structure of internal ribosomal entry site which is important for viral replication.

Comparison of 3 Automated Immunoassays for Detection of Anti-Hepatitis A Virus Immunoglobulin M in a Tertiary Care Hospital

Three automated immunoassay kits for anti-Hepatitis A Virus (HAV) IgM-Architect, (Abbott Laboratories, USA), Elecsys (Roche Diagnostics, Germany), and ADVIA Centaur (Siemens Healthcare Diagnostics Inc., USA)-were compared. We included 178 consecutive samples, for which an anti-HAV IgM test was requested at Seoul National University Hospital from September 2009 to January 2010. Reviewing of medical records, reverse transcription (RT)-PCR for HAV RNA, or total anti-HAV assay were performed on 16 (9.0%) samples with discrepant results. The percent agreements (kappas) of the Architect and ADVIA Centaur, Architect and Elecsys, and ADVIA Centaur and Elecsys kits were 96.6% (0.91), 96.6% (0.92), and 97.8% (0.94), respectively. Eight out of 16 discrepant samples showed gray-zone values in Architect but were nonreactive in the others. Slightly earlier seroconversion was suspected in Elecsys. The 3 assays showed comparable performances with excellent agreements in a tertiary care hospital setting.

Multiplex polymerase chain reaction test for the diagnosis of acute viral hepatitis A

BACKGROUND/AIMS: The early diagnosis of acute hepatitis A (AHA) is hindered because serum IgM against hepatitis A virus (HAV) can yield false-negative results during the window period. This study evaluated the diagnostic accuracy of a polymerase chain reaction (PCR) kit for HAV RNA for the diagnosis of AHA. METHODS: Samples were collected from 136 patients with acute severe hepatitis at their admission to Asan Medical Center between June 2010 and July 2010. Samples were analyzed for serum IgM anti-HAV using an immunoassay test and for qualitative HAV RNA using the Magicplex HepaTrio PCR test kit. The diagnostic accuracies of these methods were tested on the basis of clinical and laboratory diagnoses of AHA. RESULTS: The concordance rate and kappa value between IgM anti-HAV and HAV RNA PCR were 88.2% and 0.707, respectively. For the diagnosis of AHA, the sensitivity and specificity of IgM anti-HAV were 90.7% and 100%, respectively, when an "equivocal" result was regarded as positive; and 79.1% and 100%, respectively, when an "equivocal" result was regarded as negative. The sensitivity and specificity of HAV RNA PCR were 81.4% and 100%, respectively. All four patients with negative IgM anti-HAV and positive HAV RNA PCR results and all four patients with equivocal IgM anti-HAV RNA and positive HAV RNA PCR results were eventually diagnosed with AHA. CONCLUSIONS: The qualitative HAV RNA PCR test has an equivalent diagnostic accuracy for AHA compared to IgM anti-HAV and may be more sensitive during the window period.

Investigação do silenciamento gênico produzido por pequenos RNAs de interferência no ciclo replicativo do vírus da hepatite A / Investigation of gene silencing produced by small interfering RNAs in the replication cycle of hepatitis A virus

O vírus da hepatite A (HVA) possui genoma RNA de fitas simples de 7,5 Kb e polaridade positiva. Este vírus pertence à família Picornaviridae, gênero Hepatovirus, com propriedades biológicas únicas, em particular, o crescimento lento em cultura celular com ausência de efeito citopático. O HAV causa uma infecção autolimitada normalmente com pouco ou nenhum sintoma em pacientes jovens (abaixo de 5 anos). Já em pacientes adultos, sintomas graves podem aparecer com 1por cento dos casos podendo evoluir para uma hepatite fulminante. A inibição da replicação viral pode retardar a infecção pelo vírus prevenindo uma hepatite fulminante. Pequenos RNAs de interferência (siRNAs) que agem com base na degradação sequência-específica do genoma, pode constituir uma nova estratégia para a inibição específica de vários tipos de vírus. A interferência por siRNA é um processo onde fitas duplas de RNA (dsRNA) são capazes de silenciar as funções específicas de um gene alvo. A via de siRNA pode ser induzida em células por transfecção de oligômeros sintéticos de 21-23 nucleotídeos. O objetivo deste estudo foi avaliar o silenciamento produzido por três sequências especificas de siRNAs, uma para região 2C (helicase), e duas para 3D (protease) do genoma do HAV. Vinte e quatro horas antes da infecção, células FRHK-4 foram cultivadas em placas de 24 poços, com meio de 199 e densidade de 105 células por poço...

Evaluation of the Virus-elimination Efficacy of Nanofiltration (Viresolve NFP) for the Parvovirus B19 and Hepatitis A Virus / 대한진단검사의학회지

BACKGROUND: The safety of plasma derivatives has been reinforced since 1980s by variable pathogen inactivation or elimination techniques. Nucleic acid amplification test (NAT) for the source plasma has also been implemented worldwide. Recently nanofiltration has been used in some country for ensuring safety of plasma derivatives to eliminate non-enveloped viruses such as parvovirus B19 (B19V) and hepatitis A virus (HAV). We evaluated the efficacy of nanofiltration for the elimination of B19V and HAV. METHODS: To verify the efficacy of nanofiltration, we adopted a 20 nm Viresolve NFP (Millipore, USA) in the scaling down (1:1,370) model of the antithrombin III production. As virus stock solutions, we used B19V reactive plasma and porcine parvovirus (PPV) and HAV obtained from cell culture. And 50% tissue culture infectious dose was consumed as infectious dose. The methods used to evaluate the virus-elimination efficacy were reverse-transcriptase polymerase chain reaction for B19V and the cytopathic effect calculation after filtration for PPV and HAV. RESULTS: B19V was not detected by RT-PCR in the filtered antithrombin III solutions with initial viral load of 6.42x10(5) IU/mL and 1.42x10(5) IU/mL before filtration. The virus-elimination efficacy of nanofiltration for PPV and HAV were > or =10(3.32) and > or =10(3.31), respectively. CONCLUSIONS: Nanofiltration would be an effective method for the elimination of B19V and HAV. It may be used as a substitute for NAT screening of these viruses in source plasma to ensure safety of plasma derivatives in Korea.

Investigation of a hepatitis A outbreak from Shimla Himachal Pradesh.

Background & objective: Hepatitis A is an enterically transmitted viral disease, highly prevalent in India and mainly presents as a paediatric sporadic disease. This study investigated an outbreak of viral hepatitis at Shimla, Himachal Pradesh, India, during January-March 2007. Methods: Eighty seven blood samples, 3 water samples and 2 sewage samples were collected. Serum samples were tested for IgM and IgG anti HAV and IgM and IgG anti HEV antibodies. Serum, sewage and water samples were tested for HAV-RNA by nested RT- PCR. Nearly complete full genome (excluding extreme 5’ end) was amplified from one serum sample. Results: The hepatitis cases were mainly seen among children and young adults and 63.2 per cent (55/88) were positive for anti-HAV IgM. These cases were reported from the areas getting water supply from Ashwani Khud water supply system. This water purification system received water from a natural stream in which treated sewage water was let into 4 km upstream the collection point since one year. HAV-RNA present in serum, sewage and water samples showed 100 per cent sequence homology. Phylogenetic analysis based on 5’ non coding (5’ NC) and nearly complete genome showed the evidence of HAV genotype IIIA in all the samples. Interpretation and conclusion: The aetiological agent of the present outbreak was hepatitis A virus which is emerging in an outbreak form in India, emphasizing a definite need for formulating vaccination / control strategies.

Molecular characterization of hepatitis A virus isolates from Goiânia, Gioás, Brazil

Hepatitis A virus (HAV) infection is a public health problem worldwide and the virus has been classified into six genotypes. In Brazil, the only genotype that has been found is genotype I, predominately from subgenotype IA. Here, the HAV genotypes were analyzed of 18 isolates circulating between 1996-2001 in Goiânia, state of Goiás, Brazil. Viral RNA was extracted from 18 serum samples and amplified (RT-PCR/nested-PCR), followed by the genomic sequencing of the VP1/2A junction region of the HAV genome. Sequences of 168 nucleotides were compared and analyzed using the BLAST N, Clustal X and PAUP v. 4.10b programs. All samples were classified as genotype I, with 10 belonging to subgenotype IA and eight to subgenotype IB. The subgenotype IA isolates showed greater diversity than the subgenotype IB isolates at the nucleotide level. Elevated identity values were found between isolates obtained in this study and those from other regions of the world, including Brazil, highlighting the high conservation among different isolates of this virus. However, changes in the HAV subgenotype circulation could also be observed during the evaluated period.

Genetic variability of hepatitis A virus strain HAF-203 isolated in Brazil and expression of the VP1 gene in Escherichia coli

The hepatitis A virus (HAV) HAF-203 strain was isolated from an acute case of HAV infection. The primary isolation of HAF-203 in Brazil and its adaptation to the FRhK-4 cell lineage allowed the production of large amounts of viral particles enabling molecular characterization of the first HAV isolate in Brazil. The aim of our study was to determine the nucleotide sequence of the HAF-203 strain genome, compare it to other HAV genomes and highlight its genetic variability. The complete nucleotide sequence of the HAF-203 strain (7472 nucleotides) was compared to those obtained earlier by others for other HAV isolates. These analyses revealed 19 HAF-specific nucleotide sequence differences with 10 amino acid substitutions. Most of the non-conservative changes were located at VP1, 2C, and 3D genes, but the 3B region was the most variable. The availability of HAF-203 complementary DNA was useful for the production of the recombinant VP1 protein, which is a major determinant of viral infectivity. This recombinant protein was shown by enzyme-linked immunoassay and blotting, to be immunogenic and resemble the native protein, therefore suggesting its value as a reagent for incorporation into diagnostic tests.

Co-circulation of genotypes IA and IB of hepatitis A virus in Northeast Brazil

The Northeast region is the location of most cases of acute hepatitis A virus (HAV) in Brazil. In the present study, the genotypes of HAV strains from Pernambuco State, one of most populous states in the Northeast region, were characterized. Blood samples positive for anti-HAV IgM from 145 individuals (mean age = 29.1 years), collected during 2002 and 2003, were submitted to nested RT-PCR for amplification of the 5'non-translated region (5'NTR) and VP1/2A regions of the HAV genome. The VP1/2A and 5'NTR regions were amplified in 39 and 21 percent of the samples, respectively. Nucleotide sequencing was carried out in 46 percent of VP1/2A and in 53 percent of 5'NTR isolates. The identity in nucleotide sequence of the VP1/2A region ranged from 93.6 to 100.0 percent. Phylogenetic analysis of the VP1/2A sequences showed that 65 percent belong to sub-genotype IA and 35 percent to sub-genotype IB. Co-circulation of both sub-genotypes was observed in the two years studied. Distinct clusters of highly related sequences were observed in both sub-genotypes, suggesting endemic circulation of HAV strains in this area. In the 5'NTR isolates, 92.7-99.2 percent identity was observed and two isolates presented one deletion at position 413. Phylogenetic analysis showed that genotype IA strains cluster in the tree in the same way as genotype IB strains, but one IIIA isolate from Spain clusters with genotype IB strains. These results do not allow us to state that 5'NTR could be used to genotype HAV sequences. This is the first report of co-circulation of sub-genotypes IA and IB in this region, providing additional information about the molecular epidemiology of HAV strains in Brazil.

Molecular characterization of hepatitis A virus from a large outbreak from Kerala, India.

BACKGROUND & OBJECTIVES: Hepatitis A is highly prevalent in India and mainly presents as a sporadic disease. This study investigated an outbreak of viral hepatitis at Medical College Hospital area, Kottayam, Kerala state, India during January 2005. METHODS: Blood (133), faecal (1), sewage (4), and water samples (13) were collected. Sera were tested for IgG- and IgM-anti-HAV and IgM antibodies against hepatitis E (IgM-anti-HEV). Sewage, faeces and water samples were tested for HAV RNA in nested RT-PCR and HAV RNA positive samples were further processed for RNA quantitation using Real Time PCR. RESULTS: Of the 1180 total cases, 540 were reported from Medical college area. Two deaths were reported among doctors. Patients from the community gave a previous history of visit to medical college hospital area. The sewage treatment plant at the campus was non-functional since 1990 and the untreated sewage was constantly overflowing and getting mixed with a canal. At the time of the study, all the water sources were superchlorinated. HAV RNA was present in the faeces of hepatitis A patient (1.36 x 10(7) copies/ml), sewage tank (2.57 x 10(3) copies/ml and the canal (<100 copies/ml). None of the 13 water samples concentrated 10,000-fold and the soil sample showed presence of HAV RNA. Phylogenetic analysis based on 5'-non-coding and P2 regions showed HAV-genotype IIIA in all samples. INTERPRETATION & CONCLUSION: The aetiological agent of the present outbreak was found to be HAV. Epidemic hepatitis A (genotype-IIIA) is emerging in Indian adults, emphasizing the need for definite policy for control.

Early infection and asymptomatic spread of hepatitis A virus in a public child care center in Rio de Janeiro, Brazil: should attending children under two years of age be vaccinated?

A cross-sectional study was conducted in order to identify hepatitis A virus (HAV) serological markers in 418 individuals (mean age, 16.4 years; range, 1 month-80 years) at a public child care center in Rio de Janeiro, Brazil, as well as to analyze risk factors and determine circulating genotypes. Serum samples were tested using an enzyme immunoassay. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect and characterize HAV RNA, and sequencing was performed. Anti-HAV antibodies and IgM anti-HAV antibodies were detected, respectively, in 89.5 percent (374/418) and 10.5 percent (44/418) of the individuals tested. Acute HAV infection in children was independently correlated with crawling (p < 0.05). In 56.8 percent (25/44) of the IgM anti-HAV-positive individuals and in 33.3 percent (5/15) of the IgM anti-HAV-negative individuals presenting clinical symptoms, HAV RNA was detected. Phylogenetic analysis revealed co-circulation of subgenotypes IA and IB in 93.3 percent (28/30) of the amplified samples. In present study, we verify that 79 percent (30/38) of children IgM anti-HAV-positive were asymptomatic. In child care centers, this asymptomatic spread is a more serious problem, promoting the infection of young children, who rarely show signs of infection. Therefore, vaccinating children below the age of two might prevent the asymptomatic spread of hepatitis A.

Prevalence of hepatitis A virus infection in Goiânia, Goiás, Brazil, by molecular and serological procedures, 1995-2002

In this study, a total of 865 serum samples were collected between 1995 and 2002 from individuals living in Goiânia, Central Brazil, and clinically suspected of hepatitis. After exclusion of 162 samples which were positive for hepatitis B virus or hepatitis C virus, 703 samples were tested for anti-hepatitis A virus (anti-HAV) IgM antibodies by enzyme immunoassay. In addition, 588 of these samples and 22 fecal samples were analyzed by reverse transcription-nested PCR for HAV RNA detection, with positivity indices of 13.1 percent (77/588) and 54.5 percent (12/22), respectively. A similar index of viral RNA detection in anti-HAV-IgM positive or negative samples was observed in serum samples. HAV infection is a public health problem worldwide and this study underscores the extent of HAV circulation in our region.

Comparison of four extraction methods to detect hepatitis A virus RNA in serum and stool samples

The efficiency of extraction methods for hepatitis A virus (HAV) RNA in clinical samples is of great importance for molecular diagnosis, especially in regions endemic for HAV, such as Brazil. We compared the efficiency of four different extraction techniques in serum and stool samples for the detection of hepatitis A virus by reverse transcription PCR (RT-PCR). We used PCR to analyse serum and stool samples of 12 patients who were referred to the Brazilian Reference Center for Viral Hepatitis (BRCVH) in Rio de Janeiro. The methods tested were Proteinase K, Silica, TRIzol and Guanidine isothiocyanate. Proteinase K extraction was the best method for serum samples; it detected the HAV-RNA in 11 of the 12 samples. The guanidine isothiocyanate method was the most effective for stool samples, detecting HAV-RNAs in 9 of the samples. The TRIzol« method worked best with serum samples, and the silica method was unsatisfactory with both serum and stool samples. The RNA extraction method affected the outcome. The use of appropriate RNA extraction methods is a critical step for successful and valid PCR studies on clinical samples. We recommend that RNA extraction techniques be carefully selected for their efficiency with each type of specimen

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.