Hepatitis E virus infection in Latin America: a review.

Data reported during recent years reveal the complex picture of the epidemiology of hepatitis E virus (HEV) infection in Latin America. Whereas in countries like Argentina and Brazil is almost identical to the characteristic of most countries from North America and Europe, HEV in the Caribbean and Mexico involves the water-borne, non-zoonotic viral genotypes responsible for epidemics in Asia and Africa. Nevertheless, Latin America has been considered a highly endemic region for hepatitis E in the scientific literature, a generalization that ignores the above complexity. In addition, reports from isolated Amerindian communities, which display well known, important and very specific epidemiological features for hepatitis B and D virus infections are neither taken into account when considering the epidemiology of hepatitis E in the region. This review updates compilation of the available information for the HEV infection, both among humans and other mammals, in Latin America, discusses the strengths and the weaknesses of our current knowledge, and identifies future areas of research.

Prevalence of specific antibody to hepatitis E virus in the general population of the community of Madrid, Spain.

Hepatitis E virus (HEV) is an infectious agent causing hepatitis among humans. Although hepatitis E has been reported from many European countries, its incidence in Europe is largely unknown, and the prevalence of the HEV infection is also unknown for most countries of the region. Antibody to HEV (anti-HEV) was tested on 2,305 serum samples from the general population of the Community of Madrid (Spain) collected in the year 2008 among people aged 2-60 years. Total anti-HEV was tested by enzyme-immunoassay (EIA), and reactive samples were retested separately for anti-HEV IgG and IgM by recombinant immunoblot test (RIBT). Fifty samples (2.17%) displayed reactivity for total anti-HEV after EIA testing, and anti-HEV IgG was confirmed by RIBT in 25 (1.08%). The frequency of RIBT-confirmed anti-HEV ranged from 0.97% among the youngest to 3.61% among the oldest, and displayed a statistically significant trend to increasing with age. The rate of RIBT confirmation was also significantly higher among the individuals aged above 20 years old than among those younger of 21 years. HEV infection would be less frequent in the Community of Madrid than in Catalonia or the United Kingdom, and contact with HEV would be very uncommon among children and adolescents of the region. Confirmation of EIA-reactive samples by RIBT reduced the final numbers of anti-HEV testing as much as 50%, and some findings of this study suggest that such testing protocol would reflect better the real prevalence of anti-HEV in settings of low endemicity than the single testing by EIA.

Imported and autochthonous hepatitis E virus strains in Spain.

Hepatitis E virus (HEV) causes hepatitis E, an acute liver disease displaying diverse epidemiological patterns that correlate with the genetic diversity of the virus. Only a few strains have been characterized to date from cases of hepatitis E in Spain. Using three sets of new, HEV-specific primers, viral genome fragments were amplified from serum samples from 13 patients with acute hepatitis in different regions of Spain. Direct sequencing of these fragments and analysis of sequences lead to identify six genotype 1, six genotype 3, and one genotype 4 viral strains. Genotype 1 sequences were found in the clade with subtype 1a strains, and were amplified from travelers from India and Bangladesh, and from an African immigrant. Genotype 3 sequences were found in the clade with subtype 3f strains, were always amplified from patients who did not travel abroad recently, and were closely related to sequences from swine strains isolated in Spain. Patients infected by these strains lived in different regions and were mainly men aged above 50 years. The single genotype 4 sequence detected was amplified from a traveler returning from Vietnam. Hepatitis E is both an imported and an autochthonous disease in Spain, and closely related HEV genotype 3f strains are responsible for infections acquired locally in different regions of the country within a given time. Studies involving a significant number of human, swine, and environmental viral strains collected prospectively are, however, required in order to confirm a swine origin for autochthonous HEV genotype 3 human infections.

Full coding hepatitis E virus genotype 3 genome amplification method.

Hepatitis E virus (HEV) genotype 3 produces zoonotic infection associated with the consumption of infected animals. HEV infections can become chronic in immunocompromised (IC) patients. The viral genome has three well defined open reading frames (ORF1, ORF2 and ORF3) within which various domains and functions have been described. This paper (i) describes a new method of complete sequencing of the HEV coding region through overlapping PCR systems, (ii) establishes a consensus sequence and polymorphic positions (PP) for each domain, and (iii) analyzes the complete coding sequence of an IC patient. With regard to the consensus, a high percentage of PP was observed in protease (PP=19%) and the X domain (PP=22%) within ORF1, the N-terminal region of the S domain (PP=22%) in ORF2, and the P1 (PP=35%) and P2 (PP=25%) domains in ORF3. In contrast, the ORF1 Y, ORF2 S, ORF2 M and ORF3 D1 domains were conserved in the reference sequences (0.40, 1, 0.70 and 0% of PP, respectively). The sequence from the IC patient had more mutations in the RpRp (D1235G, Q1242R, S1454T, V1480I, I1502 V, K1511R, G1373 V, E1442D, V1693 M), the terminal ORF2 S- domain (F10L, S26T, G36S, S70P, A105 V, I113 V), the X domain (T938 M, T856 V, S898A) and the helicase (S1014N, S975T, Q1133 K).

Multicenter clinical evaluation of the new 3rd generation assay for detection of antibodies against hepatitis C virus on the VIDAS(®) system.

BACKGROUND: Detection of antibodies (anti-HCV) against hepatitis C virus (HCV) is indispensable for screening and diagnosis of viral hepatitis and for the viral safety of blood, tissue or organ donations. It gains additional importance by the new HCV drugs which improve the therapeutic possibilities dramatically. OBJECTIVE: To evaluate the performance of a newly developed immune assay for anti-HCV based on the well-established VIDAS platform. STUDY DESIGN: The assay was evaluated with samples from anti-HCV negative blood donors and from patients with or without HCV markers in six centres in France, Spain and Egypt. The status of the samples was determined by using CE-marked immune assays (Architect, AxSym, Prism, Vitros), two immunoblots (RIBA, Inno-Lia) and/or HCV RNA results. RESULTS: Specificity was 99.67% in 10,320 French blood donors without anti-HCV, 99.5% in 200 anti-HCV negative hospitalized European patients and 99.0% in 198 negative patients from Egypt. Sensitivity was 99.7% in 1054 patients pretested positive by other assays; 345 patients with known genotype had genotype 1-6; 61 patients were co-infected with HIV. VIDAS was reactive in 78% of 91 patients with uncertain or very weak anti-HCV. It became on average positive at day 37 with seroconversion panels. CONCLUSIONS: This multicentric, international study with >12,000 samples show that the new VIDAS anti-HCV assay is very suitable for screening and confirmation of HCV infection. Sensitivity, specificity and recognition of seroconversion compare favorably with well-established CE-marked tests and help to clarify discrepant results obtained with other assays.

Screening of antibodies to varicella-zoster virus in leukemic patients: comparison of commercial methods of enzyme-immunoassay and fluoroimmunoassay.

Commercial methods of enzyme immunoassay and fluoroimmunoassay for the detection of varicella-zoster antibodies were compared using serum samples from individuals in a program of bone marrow transplantation. The correlation was 93.4%. Enzyme immunoassay showed to be more sensitive. Both methods are applicable to the detection of varicella-zoster antibodies. However, when fluoroimmunoassay is used, we recommend the confirmation of negative results by a sensitive enzyme immunoassay.

Evaluation of commercial methods of enzyme immunoassay (EIA) for the measurement of rubella-specific IgM.

Four commercial EIA methods for measuring rubella-specific IgM (three indirect tests and one anti-mu capture test) were evaluated, using sucrose gradient centrifugation and hemagglutination inhibition as the reference method. Evaluation was conducted with the aid of four serum panels, including 53 primary rubella cases, 30 healthy pregnant women, 21 sera positive for rheumatoid factor(s) (RF) and 35 sera from 29 cases of heterophil-positive infectious mononucleosis with EBV-specific IgM detected by immunofluorescence. All EIA methods were more sensitive than the reference method when applied to very early samples (1-5 days post-exanthema) and no differences in sensitivity were found between them. On the other hand, we observed a significant incidence of false-positive results if an indirect EIA method is applied to RF-positive samples. False positivity is significantly reduced, but not totally eliminated, when samples are preabsorbed with anti-human IgG serum and, in all cases, the absorbance values obtained were low. In contrast, there were no false-positive results using an anti-mu capture method, even in sera from cases of infectious mononucleosis. The basis for choosing between an indirect method and an anti-mu capture method for the diagnosis of congenital and post-natal rubella virus infection is discussed.

Detection and typing of human herpesviruses by multiplex polymerase chain reaction.

A new approach to simultaneous detection and typing of related agents by the multiplex polymerase chain reaction (PCR) is described. The reaction was been applied to human herpesviruses by nested amplification of fragments of the DNA polymerase genes. During the first amplification, primers were used as two equimolar mixtures of non-degenerate oligonucleotides, aligning the 3'-ends with selected consensus regions, and their 5'-ends with the non-related sequences of each herpesvirus to be amplified. The specific fragments obtained were the substrate for a second, multiplex reaction for which primers were designed to produce different-size fragments for each related virus. The results showed high specificity for the detection and typing of the human herpesviruses with known sequences and no amplification of human DNA, in spite of the presence of the same consensus regions within human DNA polymerase alpha. It is concluded that this new approach would be useful for the differential diagnosis of herpesviruses, as well as for other groups of agents with conserved regions in their genomes and causing similar syndromes.

Detection of enteroviral RNA and specific DNA of herpesviruses by multiplex genome amplification.

A reverse transcription (RT) multiplex polymerase chain reaction (PCR) assay was developed to allow rapid, sensitive and simultaneous detection of enteroviral RNA and herpesviral DNA specific sequences in a single tube. The method involves a reverse transcription step followed by a multiplex nested PCR in which the combination of primers amplifies cDNA from enteroviruses and specific herpesviruses DNA. Nested amplification utilises primers designed to anneal into the amplification product from the first reaction. Individual viruses were then detected and differentiated by the size of their PCR products determined using ethidium bromide stained agarose gels. To exclude false negatives due to sample inhibitors an internal amplification control, a cloned fragment of DNA from Pseudorabies virus (PRV DNA) was included in the reaction mixture. Detection levels between 0.01 and 0.001 TCID50 of prototype strains of Polio and Coxsackie type B viruses and between 1 and 100 molecules of cloned-DNA of herpesviruses prototype strains were achieved. The RT multiplex PCR method proved capable of detecting enteroviral RNA or herpesviral DNA in cerebro spinal fluid (CSF) samples from patients with aetiologically well characterized encephalitis or aseptic meningitis.

Multiplex polymerase chain reaction for the simultaneous detection and typing of polyomavirus JC, BK and SV40 DNA in clinical samples.

A novel multiplex nested PCR (nPCR) method was developed for detecting and differentiating simultaneously the DNA of polyomaviruses JC, BK and SV40 in a single tube. In the first amplification step the same set of primers were used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV and SV40. The second round of multiplex nPCR was carried out using a set of primers designed to render products of different size for each related virus. The thermocycling parameters and concentration of each reaction component were optimised systematically to achieve optimal specificity and sensitivity for the nPCR assay. The sensitivity of the method ranged between one and 10 copies of polyomavirus genome. Cerebrospinal fluid (CSF) was examined from AIDS patients with clinical and neuroradiological evidence of progressive multifocal leukoencephalopathy (PML) and CSF from AIDS patients with other neurological alterations. Urine specimens from bone marrow transplant recipients affected by haemorrhagic cystitis were also tested. The results obtained suggest that the assay is a good tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and in other studies in order to define better the role of polyomaviruses in human disease.

Detection of specific IgM antibody in the investigation of an outbreak of pneumonia due to Legionella pneumophila serogroup 1.

OBJECTIVE: Management of outbreaks of pneumonia due to Legionella pneumophila serogroup 1 (SG1) infection requires rapid and accurate diagnostic tests. Current serologic approaches, based on detection of seroconversion for total antibody, do not fulfil this requirement. METHODS: A diagnostic test based on detection of IgM antibody to L. pneumophila SG1 by indirect immunofluorescence was developed and used to evaluate serum samples from patients involved in a community outbreak of L. pneumophila SG1 pneumonia that occurred in Spain. RESULTS: Testing of samples from serologically proven, sporadic cases of pneumonia due to L. pneumophila SG1 (14), cases of atypical pneumonia due to other infectious agents (16) and healthy controls (100) supported the sensitivity and specificity of the assay. On samples from the outbreak, the IgM assay recognized five of six cases with isolation of L. pneumophila SG1 from respiratory secretions or lung tissue and more than 70% of cases with confirmed or presumptive diagnosis as determined by the current serologic criteria. In addition, the IgM assay was positive in 23-70% of patients who fulfilled the clinical and epidemiologic criteria of case definition but did not display diagnostically significant serologic results or who lacked a detectable antibody response in the routine assay. Among cases confirmed by the current criteria, detection of specific IgM was occasionally achieved before the conventional serology gave significant results. CONCLUSION: Incorporation of IgM antibody detection in the current diagnostic criteria for L. pneumophila SG1 infection may help to improve the management of outbreaks of pneumonia due to this agent.

Evaluation of an automated complement-fixation test (Seramat) for diagnosis of acute respiratory infections caused by viruses and atypical bacteria.

The complement-fixation test (CFT) permits low-cost screening of serum samples for different agents within a single assay, and is a useful tool for the serological diagnosis of acute respiratory infections. This study evaluated the automated Seramat CFT system with 160 paired serum samples taken from 80 patients with acute respiratory infection in comparison with in-house CFTs against a panel of agents, including influenza A and B, adenovirus, respiratory syncitial virus, cytomegalovirus, Mycoplasma pneumoniae, Coxiella burnetti and Chlamydia spp., and in comparison with indirect immunofluorescence (IIF) against Legionella pneumophila. Overall, the Seramat system identified 75 (88.2%) of the 85 seroconversions recognised by in-house CFTs or IIF. In comparison to the in-house CFTs, the correlation was 89.2% (66/74). For L. pneumophila, the Seramat system detected nine (81.8%) of the 11 cases diagnosed by IIF. The Seramat system also identified eight additional seroconversions that were not detected by the in-house assays; none of these seroconversions was detected by the in-house assay on retesting. The Seramat system represents a significant technical improvement that may enable many clinical laboratories to use the CFT as a routine diagnostic tool.

[Myelitis associated with varicella-zoster virus in the absence of cutaneous zoster]. / Mielitis asociada al virus varicela zoster en ausencia de zoster cutáneo.

INTRODUCTION: The spectrum of neurological complications associated with the infection by varicella-zoster virus (VVZ) is very broad. The diagnosis, usually based on clinical findings and their temporal relationship with cutaneous herpes zoster should be confirmed by serological and/or virological techniques. However, there are an increasing number of cases compatible with this diagnosis in the absence of a skin rash. CLINICAL CASE: We describe the case of a previously healthy woman of 27 who developed a neurological condition of subacute-chronic course, not preceded by a skin rash and compatible with the diagnosis of myelitis. She had had varicella at the age of 13. The MR of the medulla showed two hyperintense lesions in potentiated sequences in T2 at the level of the cervical medulla (segments C3-C4 and C6). Studies made to rule out other causes of myelopathy were normal or negative. After the first lumbar puncture there was an increase in the number of cells seen (up to 50/mm3) mainly mononuclear with oligoclonal bands, raised tibling index, antibodies (ab) IgG to VVZ and the indexes showing specificity to these abs and their intrathecal production were positive. Treatment with acyclovir produced no change in either her clinical condition or in the cerebrospinal fluid findings. CONCLUSION: One should consider the possibility of the association with VVZ in patients of any age, whether immunodeficient or not, who present any neurological syndrome for which no other aetiology has been found, whether or not it is preceded by a typical skin rash. The improvement of serological and virological methods permits precise diagnosis of the disorder.

[Infection by hepatitis virus among the indigenous populations of South America: a review of the problem]. / La infección por los virus causantes de hepatitis en poblaciones indígenas de Suramérica: una revisión del problema.

After the report of the epidemic outbreak of delta hepatitis among the Yukpa amerindians in the early 80s, the viral hepatitis arose as an important health problem in all the Amerindian communities from the north of South America and the Amazonian Basin. Despite the few data available, the results obtained in different communities from Venezuela (Yukpa, Barí, Yanomami) have shown a high endemicity of hepatitis B and D virus infections and a significant prevalence of hepatitis E virus-specific antibody among their members. By contrast, the infection by hepatitis C virus, which is present in all the urban areas from South America, seems uncommon, or even absent among some Amerindian populations. At the moment, a satisfactory explanation for this findings has not yet been arised. However, it could be possible that the margination of these populations regarding the health care system has been keeping them free of an infection largely linked worldwide to iatrogeny. Vaccination of Amerindian populations against hepatitis B should be taken as a priority of the health care programs. Moreover, such programs should consider the iatrogenic transmission of the HCV as a matter of concern regarding such populations, since parenterally transmitted hepatitis viruses seems to spread quickly among their members once they are introduced, giving rise to serious health problems.

[Epidemic outbreak of hepatitis B from the tattoo in gypsy families]. / Brote epidémico de hepatitis B por tatuaje en familias de étnia gitana.

BACKGROUND: To document an outbreak of Hepatitis B in a gypsy community in the Upper Aragón region, as well as the control measures adopted. METHODS: Documented study of Hepatitis B cases and families, including an epidemiological survey and the determining of hepatitis B viral indicators (MVHB) using immunoenzymatic methods. RESULTS: 84.8% participation (39/45). During the months of February and March 1988, 5 cases of Hepatitis B were detected in a gypsy community in the Upper Aragon region (12.8% attack rate, 5/39), with an average age of 13.0 + 7.3, (4 women and one man). Four of the cases detected had previously undergone tatooing. The fifth case was due to direct transmission from mother to a recently born child. The MVHB study of families showed a further two cases. MVHB rate being 17.9% (7/39). Vaccinations were given to all persons susceptible to the disease. CONCLUSIONS: It is suggested that tatooing could be a significant factor to be considered in relation to the transmission of Hepatitis B in gypsy communities. Due to the high rate of incidence of the disease in this ethnic group, general vaccination is prescribed.

Diagnosis of acute hepatitis E by antibody and molecular testing: a study on 277 suspected cases.

BACKGROUND: Acute hepatitis due to hepatitis E virus (HEV) infection is both indigenous and imported to Europe. Few studies provide information about the role of HEV as an agent for acute hepatitis in Spain. OBJECTIVES: To investigate the frequency of the HEV infection among patients displaying acute hepatitis of unexplained origin in Spain, comparing the performance of two different diagnostic approaches. STUDY DESIGN: Specific IgM antibody and HEV RNA tests were used to study samples from 277 patients with acute hepatitis of unknown aetiology received during a six-year period. Samples were sent by 52 hospitals from almost all regions of Spain. RESULTS: Evidence of acute infection by HEV was obtained for 30 patients in total (10.8%), and 16 cases were unrelated to recent international travel. On samples from 158 patients tested for both anti-HEV IgM and HEV RNA at admission, the yield of IgM antibody testing (11.4%) was higher than the yield of HEV RNA testing (9.5%). CONCLUSIONS: HEV could be responsible in Spain of about 11% of cases of acute hepatitis of unknown origin overall, and of about 8% of cases unrelated to international travel or immigration. India and neighbour countries represent the highest risk for import of epidemic HEV strains into Spain. Both antibody assays and molecular tests are required to optimise the final yield of laboratory diagnosis.