J. M. Echevarría

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 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.

Viral Diagnosis of Neurological Infection by RT Multiplex PCR: A Search for Entero- and Herpesviruses in a Prospective Study

The diagnosis of a wide range of different neurological syndromes was established by a reverse transcription multiplex PCR assay. The presence of enterovirus and herpesviruses was studied in cerebrospinal fluid samples collected prospectively from 200 patients hospitalized with neurological diseases suspected of viral infection. Positive PCR results for enterovirus and neurotropic herpesvirus (herpes simplex, HSV, and varicella zoster, VZV) were obtained among the immunocompetent patients (55/156, 35%) who presented aseptic meningitis or encephalitis. Among immunocompromised patients the yield of positive PCR results was 41% (18/44), predominantly lymphotropic herpesviruses (15/44, 34%). Cytomegalovirus (CMV) DNA was detected in patients with several clinical syndromes, including encephalitis, chronic meningitis, retinitis, ventriculitis, polyradiculomyelitis, and myeloradiculitis. Epstein‐Barr (EBV) and VZV‐specific DNA sequences were detected in patients with either encephalitis, aseptic meningitis, and chronic meningitis. Dual infections of CMV and HSV or CMV and EBV were established in two AIDS patients with encephalitis and polyradiculomyelitis, respectively. The applications of this RT multiplex PCR assay are extensive and may prove to be particularly valuable for the rapid and sensitive diagnosis of neurological diseases in both immunocompetent and immunocompromised patients. J. Med. Virol. 57:145–151, 1999.

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. J. Med. Virol. 81:1743–1749, 2009.

Acute meningitis due to Toscana virus infection among patients from both the Spanish Mediterranean region and the region of Madrid

Toscana virus (TOSV) is a member of the genus Phlebovirus that is transmitted to humans by two different species of sand fly and causes acute aseptic meningitis (AAM) and meningoencephalitis in Central Italy. Fifteen cases of AAM due to TOSV have been found at the Spanish province of Granada, but no data regarding the presence of TOSV-related disease in other regions of Spain have been still reported. A collection of 88 serum and 53 cerebrospinal fluid (CSF) samples taken from 81 selected patients with AAM of unknown aetiology, residing at Madrid or at the southern Mediterranean coast of Spain, was retrospectively studied for presence of TOSV-specific antibodies from both IgG and IgM classes. Anti-TOSV IgG was also investigated in 457 serum samples from healthy individuals, aged 2-60 years, residing at the south of the Region of Madrid. Specific IgM in serum and/or intrathecally produced anti-TOSV IgG were detected in seven patients, three residents from the Mediterranean region and the remainder four from the Region of Madrid. The overall prevalence of anti-TOSV among the healthy population studied was 5%. These results confirm the role of TOSV as an agent causing AAM in the Spanish Mediterranean coast, extend these findings to the central region of the country and suggest that TOSV might be producing infection and neurological disease in every area of Spain harbouring significant populations of the viral vectors.

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. J. Med. Virol. 85: 1037–1045, 2013.

Differential diagnosis of hepatitis E virus, cytomegalovirus and Epstein-Barr virus infection in patients with suspected hepatitis E

BACKGROUND The accuracy of the diagnosis of hepatitis E in the clinical setting relies mainly on the performance of assays for hepatitis E virus (HEV)-specific IgM (anti-HEV IgM) testing in serum. OBJECTIVES Identification of factors influencing the specificity of the results obtained with these assays is an important issue in regard to the accuracy of the diagnosis. STUDY DESIGN Anti-HEV IgM and HEV RNA were studied in samples from 153 patients with acute hepatitis of unknown aetiology received during a two-year period. Fifteen patients were positive for anti-HEV IgM, and eight of them were also positive for HEV RNA. Investigation of CMV and Epstein-Barr virus (EBV) infection markers among the remaining seven patients, and of HEV infection markers among 18 patients with infectious mononucleosis, was performed. RESULTS The results obtained showed that acute infection by CMV or EBV may cause false reactivity for anti-HEV IgM, likely because of polyclonal B-cell stimulation. CONCLUSIONS Since infection by these herpesviruses may produce acute hepatitis, such event can cause diagnostic mistakes and should be investigated in patients positive for anti-HEV IgM and negative for HEV RNA.

Comparative sensitivity of commercial tests for hepatitis E genotype 3 virus antibody detection

Hepatitis E virus (HEV) acute infection is often diagnosed only by anti‐HEV IgM ELISA methods, whose sensitivity varies, according to different reports. Reports assessing the specificity of commercial assays for anti‐HEV IgG testing are scarce, and estimates of sensitivity and specificity are both controversial. The aim of this work is to assess the sensitivity of different commercial techniques for HEV genotype 3 antibody (anti‐HEV) IgM and IgG detection in entirely specific sample panels including both high and low antibody concentrations. The anti‐HEV IgM and IgG ELISA methods compared were: DSI, Mikrogen, Wantai, Euroimmun, MP, and Dia.pro. The rapid test All Diag was also included in the anti‐HEV IgM comparison. Our results show that low anti‐HEV IgM concentrations were better detected by DSI, Mikrogen, and All Diag, these tests being the most sensitive in our study. Euroimmun, MP and Dia.pro gave concordant results, showing lower sensitivity than the others. Regarding anti‐HEV IgG our results revealed similar anti‐HEV IgG sensitivity. Furthermore, there was a striking overall lack of concordance among the results. We present a thorough review of previous comparative reports, with particular reference to the anti‐HEV IgG comparison, since published results differ from ours. This discrepancy may be related to the improved versions of the tests for MP and Dia.pro that we employed. J. Med. Virol. 87:1934–1939, 2015.

Diagnosis ofMycoplasma pneumoniae infection by microparticle aggluination and antibody-capture enzyme-immunoassay

The performance of two new commercial assays for the serological diagnosis ofMycoplasma pneumoniae infection (microparticle agglutination and antibody-capture enzyme-immunoassay) was studied using a panel of 169 serum samples from patients withMycoplasma pneumoniae pneumonia and a control group. Both assays were shown to be sensitive and specific for diagnosis. The performance of the capture immunoassay, however, decreased in older patients, probably due to its inability to detect cases of reinfection without IgM antibody response.

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.