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Purpose of Review Acute respiratory infections of viral etiology (ARIVE) constitute one of the most frequent infectious processes among humans. They cause significant morbidity and mortality every year in all age groups and regions of the world. Their etiology is diverse, and seasonal viruses began their journey, at some point, with an episode of expansion before their annual circulation as seasonal agents. The coronavirus disease 2019 (COVID-19) pandemic is a challenge for Latin America. Understanding dynamics is essential for decision making, to reduce the health, economic, and social impacts of the pandemic. Recent Findings Currently, governments in Latin America have taken measures to mitigate the spread of COVID-19 primarily based on World Health Organization recommendations. However, the potential impact of the virus in Latin America is still unknown. Given the urgency, governments need more accurate estimates of what could happen in Latin America in order to make informed decisions, At the September 20, 2020, cumulative cases 2295 of COVID-19 per 1 million population has been registered in Latin America and the Caribbean. Brazil, Peru, and Chile are the most countries affected by this pandemic, registering a total of cumulative cases per million inhabitants of 21,148, 22,941, and 23,262 respectively. Peru has shown the highest death numbers with 949 per million inhabitants. Summary The Latin American health authorities should make the most beneficial decisions based in scientific facts for the health and life of citizens, both understood in the broadest and most inclusive sense. Once the epidemic is over, Latin America should begin a profound health reform, at a single and universal health system, integrated and coordinated, where the leading role of the Ministry of Health is resumed, to have a national network of modern, integrated, and excellent quality laboratories for the benefit of the entire society.

Background: Blood transfusion safety is based on reliable donor screening for transmissible infections such as the hepatitis C virus (HCV) infection. Study design and methods: A novel HCV core-specific antibody was assayed on random single donations from 2007 first-time blood donors who tested negative for anti-HCV and HCV RNA on routine screening. Sample collection broke the code between donations and donors for ethical reasons. Results: Forty-two donations (2.1%) displayed reactivity in the novel test. The specificity of the reactivity was evaluated by a peptide inhibition assay, and testing against additional nonoverlapping HCV core peptide epitopes and other HCV antigens was performed on these samples. Six donations (14.3%; 0.30% from the total) were considered to contain anti-HCV after such supplemental testing. HCV RNA detection was also performed in peripheral blood mononuclear cells (PBMNCs) and serum or plasma samples from reactive donors after virus concentration by ultracentrifugation. HCV RNA tested negative in all PBMNCs samples, and a very low amount of viral genome was detected in serum or plasma concentrates from three anti-HCV core-reactive donors (7.1%) but not among concentrates from 100 randomly selected nonreactive donors. Sequencing of these polymerase chain reaction products revealed differences between the isolates that excluded partially sample contamination from a common source. Conclusion: These findings argue in favor of an ongoing occult HCV infection among these blood donors and account for some rather low, but perhaps not negligible, infection risk for such donations. Future studies involving larger samples of donations from traceable donors would enlighten the significance of these findings for the viral safety of the blood supply. This work was supported by research grants from DIATERS.A., Madrid, Spain. Sí

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.

Human hepatitis E virus (HHEV) is the proposed name for a diverse group of RNA viruses from the family Hepeviridae that cause acute hepatitis among humans. Waterborne strains are regularly imported into Europe by international travelers, and virus transmission of zoonotic strains via contaminated aliments is involved in autochthonous cases. Therefore, in Europe, hepatitis E displays a unique dual character, having features of both imported and autochthonous infections. Environmental involvement of waterborne and zoonotic diseases puts alimentary safety at risk. In addition, it may lead to serious health problems derived from persistent infection among patients with immune impairment due to organ transplant, cancer, or human immunodeficiency virus infection. Although the European health authorities know at present that HHEV represents a problem worthy of consideration, the actual incidence of the disease in Europe is unknown, and attempts to ascertain the prevalence of the infection is hampered by unresolved technical issues. In order to determine the burden of hepatitis E in Europe, the World Health Organization Regional Office and the European Centre for Disease Prevention and Control should pay specific attention to hepatitis E, and research efforts in the continent should be transnational and collaborative. Development of a specific European network for hepatitis E would help to achieve these goals.

Resumen Fundamento y objetivo Detectar casos asintomaticos relacionados con un brote, valorar la seroprevalencia de hepatitis B (HB) en deportistas de orientacion y establecer recomendaciones. Pacientes y metodo Se realizo un estudio transversal de seroprevalencia entre 116 deportistas de orientacion que habian competido en las categorias implicadas en un brote previo y una muestra estratificada de 166 corredores del resto de otras categorias de competicion. Se analizaron marcadores de HB: antigeno de superficie del virus de la HB (VHB) (HBsAg), anticuerpo contra el antigeno core del VHB (anti-HBc), anticuerpos contra el HBsAg, anti-HBc tipo inmunoglobina M y antecedentes de vacunacion. Los resultados se expresan utilizando pesos ponderados. Resultados La seroprevalencia de HB (anti-HBc positivos) fue del 6,7% (n=12; intervalo de confianza [IC] del 95%: 0,6 a 12,9). No se observo ningun caso de HB aguda o cronica. Todos los marcadores fueron negativos para el 61,1% (n=64; IC del 95%: 46,3 a 75,6), y el 32,3% (n=29; IC del 95%: 18,2 a 46,4) tenia marcadores de inmunidad por vacunacion. Entre los sujetos menores de 25 anos, el 28,4% estaba sin vacunar a pesar de que entraban en el calendario vacunal. Conclusiones Los resultados muestran que la seroprevalencia de HB entre deportistas de orientacion no difiere de la poblacion general. Sin embargo, es necesario reforzar la vacunacion entre adolescentes y adultos jovenes. Se dan recomendaciones generales para la prevencion de HB a las federaciones de orientacion.

Viral hepatitides are satisfactorily diagnosed in the laboratory by immunoassays for either antigen or antibody detection in serum samples. However, the early detection of acute infections during the window period, investigation of occult infections, and issues related to the establishment and follow-up of antiviral therapy in chronic infections pose new challenges that only molecular methods can meet. In addition, full characterization of epidemic outbreaks and surveillance of the emergence of viral variants able to escape from vaccine protection are major public health objectives that can only be achieved through the use of these techniques. As a further attempt to improve the viral safety of blood transfusions, the incorporation of molecular biology techniques into the routine work of transfusion centers has generated new technical and scientific demands on microbiology laboratories, which must in turn incorporate these methods to respond to the challenge. After more than a decade, automatic methods for the detection, quantification, characterization and sequencing of the genomes of these viruses have become a reality for the clinical laboratory, reaffirming the essential role of the microbiologist in the hospital setting.

A fin de conocer la prevalencia de la infección por el virus de hepatitis B (VHB) en la comunidad indígena Japreira, Venezuela, y determinar las pautas de su adquisición en función de la edad y sexo, se seleccionaron 149 individuos de un total de 300. Se detectó por métodos serológicos la presencia de anticuerpos totales del VHB (anti-HBc total), y en las positivas se investigó la presencia del antígeno de superficie (HBsAg) y del antígeno "e" del VHB (HBeAg). La prevalencia de anti-HBc total fue de 72,9% en el sexo femenino y 81,1% en el masculino, la prevalencia de HBsAg fue elevada (61,1%) en el sexo masculino, sólo cuatro de los portadores de HBsAg fueron positivos para HBeAg. Los resultados obtenidos indican un grado elevado de endemia para esta infección y que su diseminación comienza a temprana edad. El factor involucrado en la adquisición y diseminación del virus seria la vía sexual. Hacinamiento, contacto intercorpóreo con fluidos biológicos, prácticas sociales, y características de la cepa viral circulante podrían estar involucradas en la alta cronicidad observada en la comunidad indígena Japreira.

The hepatitis B virus (HBV) genotypes were studied by a line probe assay (LiPA) and by direct sequencing of a 339 nucleotide fragment from the S region of the viral genome in samples from 269 carriers living in Spain, either native to Spain (231) or immigrants from Africa, Asia, and Eastern Europe (38). The sequences were also used to predict the HBV surface antigen (HBsAg) subtype on the basis of the amino acids specified at selected positions of the HBsAg molecule. Agreement between the two genotyping methods was found in most cases (98.1%) and a HBV genotype could be assigned to all samples. The viral groups D/ayw2 (30.1%), D/ayw3 (28.6%), and A/adw2 (21.2%) were prevalent, with an additional participation of the groups D/ayw4 (4.8%), F/adw4q- (1.9%), A/ayw1 (1.9%), and D/adw3 (0.7%), all of them present among the autochthonous carriers. Strains from genotypes B and C were found exclusively among Chinese immigrants. Genotype E strains were found in immigrants from Central Africa and in one patient native of Spain. Point mutations leading to amino acid changes of residues involved in the expression of the HBsAg subtype determinants were found in 12 samples (4.5%). Some mutations would predict the putative novel genotype-subtype associations A/adw4q+, A/ayr, D/ayr, and E/ayw1, while others would suggest the loss of subtype-specific determinants. The finding of HBV strains characteristic for Africa among the autochthonous carriers confirms the emergence of African HBV strains in Spain.

Sequences of 234 complete genomes and 631 hepatitis B surface antigen genes were used to assess the worldwide diversity of hepatitis B virus (HBV). Apart from the described two subgenotypes each for A and F, also B, C, and D divided into four subgenotypes each in the analysis of complete genomes supported by significant bootstrap values. The subgenotypes of B and C differed in their geographical distribution, with B1 dominating in Japan, B2 in China and Vietnam, B3 confined to Indonesia, and B4 confined to Vietnam, all strains specifying subtype ayw1. Subgenotype C1 was common in Japan, Korea, and China; C2 in China, South-East Asia, and Bangladesh, and C3 in the Oceania comprising strains specifying adrq–, and C4 specifying ayw3 is encountered in Aborigines from Australia. This pattern of defined geographical distribution was less evident for D1–D4, where the subgenotypes were widely spread in Europe, Africa, and Asia, possibly due to their divergence having occurred a longer time ago than for genotypes B and C, with D4 being the first split and still the dominating subgenotype of D in the Oceania. The genetic diversity of HBV and the geographical distribution of its subgenotypes provide a tool to reconstruct the evolutionary history of HBV and may help to complement genetic data in the understanding of the evolution and past migrations of man.

[EN] On the last twenty years, viral hepatitis has emerged as a serious problem in almost all the Amerindian communities studied in the Amazon Basin and in other Amazon-related ecological systems from the North and Center of South America. Studies performed on communities from Bolivia, Brazil, Colombia, Peru and Venezuela have shown a high endemicity of the hepatitis B virus (HBV) infection all over the region, which is frequently associated to a high prevalence of infection by hepatitis D virus among the chronic HBV carriers. Circulation of both agents responds mainly to horizontal virus transmission during childhood through mechanisms that are not fully understood. By contrast, infection by hepatitis C virus (HCV), which is present in all the urban areas of South America, is still very uncommon among them. At the moment, there is not data enough to evaluate properly the true incidence that such endemicity may have on the health of the populations affected. Since viral transmission might be operated by mechanisms that could not be acting in other areas of the World, it seems essential to investigate such mechanisms and to prevent the introduction of HCV into these populations, which consequences for health could be very serious. [ES] A lo largo de los últimos veinte años, las hepatitis víricas se han revelado como un importante problema para las comunidades indígenas de la cuenca amazónica y de otros ecosistemas similares del norte y centro de Sudamérica. Los estudios realizados en comunidades de Bolivia, Brasil, Colombia, Perú y Venezuela han demostrado una alta propensión endémica para la infección por el virus de la hepatitis B, que se asocia con frecuencia a una elevada prevalencia de coinfección con el virus de la hepatitis D entre los portadores crónicos. La circulación de ambos agentes responde a su transmisión horizontal durante la infancia, a través de mecanismos aún poco conocidos. Por el contrario, la infección por el virus de la hepatitis C es aún muy infrecuente entre los indígenas. No existen, por el momento, datos suficientes para evaluar el impacto real que esta endemia pueda tener sobre la salud de esas poblaciones. Considerando que la transmisión de estos agentes podría involucrar mecanismos que quizá no actúen en otras regiones, parece indispensable investigar dichos mecanismos y prevenir cuidadosamente la introducción del virus de la hepatitis C en esas comunidades, ya que las consecuencias para su salud podrían ser muy graves. Sí

En Bolivia no se han realizado estudios específicos sobre los virus de la hepatitis, por lo que su prevalencia y patrones de circulación son prácticamente desconocidos. De 1992 a 1996 se realizó un estudio seroepidemiológico con el fin de adquirir una primera visión de conjunto sobre las prevalencias de las infecciones por virus de la hepatitis B (VHB), C (VHC), D (VHD) y E (VHE) en distintas poblaciones de Bolivia. Sobre la base de los datos obtenidos en otros lugares de América Latina, se prestó atención especial al estudio de las comunidades autóctonas de la región amazónica. En las zonas rurales del altiplano andino, la infección por VHB presentó una prevalencia general que correspondería a una situación de endemia media o baja (11,2%) y no se encontró ningún portador de anticuerpos contra VHC o VHD. En dos poblaciones de alto riesgo de la ciudad de Cochabamba (niños sin hogar y trabajadoras del sexo), la prevalencia de infección por VHB fue similar (11,6%) y podría considerarse baja en comparación con la de otras poblaciones análogas de núcleos urbanos en América Latina. La correspondiente al VHC (un caso positivo, 0,5%) sería parecida a la descrita en esas mismas poblaciones, si bien el escaso número de muestras estudiadas no permite extraer conclusiones más firmes. En concordancia con observaciones anteriores de comunidades similares de zonas tropicales de Suramérica, en las poblaciones autóctonas de la Amazonia boliviana la infección por VHB es sumamente endémica (prevalencia general de 74,0%), pero no se ha detectado la circulación de VHC. Se sabe que la transmisión de VHB es horizontal y tiene lugar desde edades muy tempranas, pero se desconocen los mecanismos de esa actividad. A los 10 años de edad, más de la mitad de la población ya ha experimentado la infección natural que, 10 años más tarde, se habrá difundido a prácticamente toda la población. La tasa muy baja de individuos positivos al HbsAg (1,6%), la ausencia de ADN vírico en las muestras con reactividad aislada a anti-HBc y la alta prevalencia de anti-HBs entre los individuos que presentan marcadores de infección natural (92,4%) excluyen la participación de la transmisión vertical en el mantenimiento de la endemia. Hasta el momento, no se ha documentado ningún brote de infección por VHD en estas comunidades, pero la alta endemia de infección por VHB alerta sobre el riesgo de posibles brotes en el futuro. Los resultados obtenidos con las pruebas de anticuerpos contra VHE sugieren que este virus circula ampliamente en Bolivia y que podría haber producido brotes recientes en el departamento de Cochabamba. Se recomienda vacunar contra VHB en las poblaciones endémicas como medida de corto plazo; buscar activamente en todo el país brotes y casos esporádicos de hepatitis E y continuar realizando estudios que permitan evaluar las repercusiones sanitarias de la situación documentada en este estudio.In Bolivia, no studies have been carried out specifically on hepatitis viruses. Thus, their prevalence and circulation patterns are virtually unknown. A seroepidemiologic study was performed from 1992 to 1996 to generate a preliminary idea of the overall prevalence of infection from hepatitis B, C, D, and E viruses (HBV, HCV, HDV, and HEV, respectively) in different Bolivian population groups. Prompted by the data obtained in other areas of Latin America, the study focused on indigenous communities in the Amazon region. In rural areas of the high Andean plateau, HBV infection showed an overall prevalence compatible with medium to low endemicity (11.2%), and no carriers of HCV or HDV antibodies were found. In two high-risk groups in the city of Cochabamba (homeless children and sexual workers), the prevalence of HBV infection was similar (11.6%) and could be considered low by comparison to that of similar population groups in Latin American urban centers. The prevalence of HCV (one positive case, or 0.5%) was similar to that found in similar population groups, although the small number of samples precludes drawing more definite conclusions. As has been noted previously with similar communities in tropical areas of South America, HBV infection is highly endemic in indigenous populations of the Bolivian Amazon (with an overall prevalence of 74.0%), but circulation of HCV has not been detected. It is a well-known fact that HBV is horizontally transmitted and that transmission can take place very early in life, but the mechanisms involved are unknown. By 10 years of age, more than half the population has already had the natural infection that, in approximately 10 more years will have affected virtually the entire population. The very low rate of positivity to HBsAg (1.6%), the absence of viral DNA in samples showing isolated positivity to anti-HBc, and the high prevalence of anti-HBs among individuals who show markers for natural infection (92.4%) suggest vertical transmission plays no role in persistent endemi city. So far, no outbreak of HDV infection has been documented in these communities, but the high endemicity shown by HBV points to the po ssibility of future outbreaks. Results obtained with tests for the detection of antibodies against HEV suggest that this virus is circulating widely in Bolivia and that it could have caused recent outbreaks in Cochabamba state. Vaccination against HBV in endemic populations is recommended as a short-term measure. Also recommended are actively searching for outbreaks and sporadic cases of hepatitis E in the entire country and performing additional research that will help in assessing the public health consequences of the situation described in this article.

[ES] En Bolivia no se han realizado estudios específicos sobre los virus de la hepatitis, por lo que su prevalencia y patrones de circulación son prácticamente desconocidos. De 1992 a 1996 se realizó un estudio seroepidemiológico con el fin de adquirir una primera visión de conjunto sobre las prevalencias de las infecciones por virus de la hepatitis B (VHB), C (VHC), D (VHD) y E (VHE) en distintas poblaciones de Bolivia. Sobre la base de los datos obtenidos en otros lugares de América Latina, se prestó atención especial al estudio de las comunidades autóctonas de la región amazónica. En las zonas rurales del altiplano andino, la infección por VHB presentó una prevalencia general que correspondería a una situación de endemia media o baja (11,2%) y no se encontró ningún portador de anticuerpos contra VHC o VHD. En dos poblaciones de alto riesgo de la ciudad de Cochabamba (niños sin hogar y trabajadoras del sexo), la prevalencia de infección por VHB fue similar (11,6%) y podría considerarse baja en comparación con la de otras poblaciones análogas de núcleos urbanos en América Latina. La correspondiente al VHC (un caso positivo, 0,5%) sería parecida a la descrita en esas mismas poblaciones, si bien el escaso número de muestras estudiadas no permite extraer conclusiones más firmes. En concordancia con observaciones anteriores de comunidades similares de zonas tropicales de Suramérica, en las poblaciones autóctonas de la Amazonia boliviana la infección por VHB es sumamente endémica (prevalencia general de 74,0%), pero no se ha detectado la circulación de VHC. Se sabe que la transmisión de VHB es horizontal y tiene lugar desde edades muy tempranas, pero se desconocen los mecanismos de esa actividad. A los 10 años de edad, más de la mitad de la población ya ha experimentado la infección natural que, 10 años más tarde, se habrá difundido a prácticamente toda la población. La tasa muy baja de individuos positivos al HbsAg (1,6%), la ausencia de ADN vírico en las muestras con reactividad aislada a anti-HBc y la alta prevalencia de anti-HBs entre los individuos que presentan marcadores de infección natural (92,4%) excluyen la participación de la transmisión vertical en el mantenimiento de la endemia. Hasta el momento, no se ha documentado ningún brote de infección por VHD en estas comunidades, pero la alta endemia de infección por VHB alerta sobre el riesgo de posibles brotes en el futuro. Los resultados obtenidos con las pruebas de anticuerpos contra VHE sugieren que este virus circula ampliamente en Bolivia y que podría haber producido brotes recientes en el departamento de Cochabamba. Se recomienda vacunar contra VHB en las poblaciones endémicas como medida de corto plazo; buscar activamente en todo el país brotes y casos esporádicos de hepatitis E y continuar realizando estudios que permitan evaluar las repercusiones sanitarias de la situación documentada en este estudio. [EN] In Bolivia, no studies have been carried out specifically on hepatitis viruses. Thus, their prevalence and circulation patterns are virtually unknown. A seroepidemiologic study was performed from 1992 to 1996 to generate a preliminary idea of the overall prevalence of infection from hepatitis B, C, D, and E viruses (HBV, HCV, HDV, and HEV, respectively) in different Bolivian population groups. Prompted by the data obtained in other areas of Latin America, the study focused on indigenous communities in the Amazon region. In rural areas of the high Andean plateau, HBV infection showed an overall prevalence compatible with medium to low endemicity (11.2%), and no carriers of HCV or HDV antibodies were found. In two high-risk groups in the city of Cochabamba (homeless children and sexual workers), the prevalence of HBV infection was similar (11.6%) and could be considered low by comparison to that of similar population groups in Latin American urban centers. The prevalence of HCV (one positive case, or 0.5%) was similar to that found in similar population groups, although the small number of samples precludes drawing more definite conclusions. As has been noted previously with similar communities in tropical areas of South America, HBV infection is highly endemic in indigenous populations of the Bolivian Amazon (with an overall prevalence of 74.0%), but circulation of HCV has not been detected. It is a well-known fact that HBV is horizontally transmitted and that transmission can take place very early in life, but the mechanisms involved are unknown. By 10 years of age, more than half the population has already had the natural infection that, in approximately 10 more years will have affected virtually the entire population. The very low rate of positivity to HBsAg (1.6%), the absence of viral DNA in samples showing isolated positivity to anti-HBc, and the high prevalence of anti-HBs among individuals who show markers for natural infection (92.4%) suggest vertical transmission plays no role in persistent endemicity. So far, no outbreak of HDV infection has been documented in these communities, but the high endemicity shown by HBV points to the possibility of future outbreaks. Results obtained with tests for the detection of antibodies against HEV suggest that this virus is circulating widely in Bolivia and that it could have caused recent outbreaks in Cochabamba state. Vaccination against HBV in endemic populations is recommended as a short-term measure. Also recommended are actively searching for outbreaks and sporadic cases of hepatitis E in the entire country and performing additional research that will help in assessing the public health consequences of the situation described in this article. Sí

The adw4 subtype of hepatitis B virus (HBV) belongs to a unique genomic group (genotype F) representing the original HBV strains from the New World. Data regarding the prevalence of this subtype among HBV carriers in South America are, however, scarce, and those concerning HBV genotype F are based on only a few samples from Latin America. In this study, serum samples were obtained from 141 hepatitis B surface antigen (HBsAg) carriers from Amerindians and urban populations from Venezuela. The HBsAg subtype was identified with monoclonal antibodies in 105 samples, and the HBV genotype was identified by reverse-phase hybridization with DNA fragments in 58 samples. The adw4 subtype was highly prevalent in the population studied (75%); among the Amerindians, the prevalence was 97%. The adw2 subtype was also present (10%), while other subtypes (ayw3 and ayw4) were only occasionally found. The HBV subtype was associated with the expected genotype in most cases (80%), and thus genotype F was highly prevalent. Sequencing of viral strains that gave genotypes unpredicted by the HBsAg subtyping confirmed seven of them as belonging to not previously described genotype-subtype associations: namely, adw2 and ayw4 within genotype F.

Chronic hepatitis E virus infection with rapid progression to cirrhosis is reported in 2 human immunodeficiency virus (HIV)-infected patients with severe immunosuppression. Monotherapy with ribavirin led to temporary viral response and marked improvement of liver damage. Chronic hepatitis E should be regarded as another opportunistic event within HIV infection.

Cerebrospinal fluid (CSF) samples from 46 patients with encephalitis were studied for the presence of herpes simplex virus (HSV) types 1 and 2 and/or varicella zoster virus (VZV)-specific DNA sequences by the polymerase chain reaction (PCR) assay. Patients were studied because of detection of intrathecal production of IgG antibody to HSV alone (10 patients, Group A) or to both HSV and VZV (11 patients, Group B) or because of the presence of specific anti-HSV IgG in CSF without evidence of intrathecal antibody production (25 patients, Group C). CSF samples taken between days 1 and 10 from onset of encephalitis were available from all patients, and follow-up samples (taken after 10 days from onset) were obtained from some of them. Positive PCR results were obtained in a total of 13 patients. Four patients (three from Group A and one from Group B) gave amplification of HSV type 1 DNA alone, two patients (both from Group B) showed amplification of VZV DNA alone, and seven patients (all from Group B) gave dual amplification of both HSV type 1 and VZV DNA sequences in CSF. All CSF samples from patients in Group C were negative by PCR. Ten patients with CSF samples positive by PCR lacked a prior history of herpetic cutaneous lesions. In seven patients, serum antibody tests (specific IgM detection and specific IgG avidity assays) identified both primary and recurrent infections. The results suggest that the dual presence of IgG antibody to both HSV and VZV in CSF from patients with encephalitis may reflect in some cases a dual infection of the central nervous system caused by both agents.

Two polymerase chain reaction (RT-PCR) assays were developed to allow rapid detection of enteroviral RNA in cerebrospinal fluid samples (CSF). Primers homologous to the conserved 5′ noncoding region of the enterovirus genome were designed. The RT-PCR product size was-500 bp (479 bp for Poliovirus, 500 bp for Cox-sackievirus) and was visualized using ethidium bromide-stained gels. Assay 1 utilized Moloney Murine Leukaemia Virus Reverse Transcriptase (MMLV-RTase) for reverse transcription and Taq polymerase for subsequent PCR. Assay 2 utilized a thermoactive DNA polymerase of Thermus thermophilus (rTth enzyme) for both reverse transcription and DNA amplification. In addition, in Assay 2 reverse transcription and PCR wer eaccomplished within the same reaction tube. Both assays detected between 1 and 0.02 TCID50 of prototype strains of Polio and Coxsackie type B viruses propagated in VERO cell and spiked in a pooled preparation of CSF samples from patients with noninfective neurological disorders. However, Assay 1 was 10-fold more sensitive than Assay 2 when applied to the detection of enteroviral RNA in CSF samples from patients with etiologically well characterized acute aseptic meningitis. © Wiley-Liss, Inc.

International audience; Amino acid changes within the major antigenic determinant of the hepatitis B virus (HBV) surface antigen (HBsAg) may eventually modify the antigenic properties of the protein and may have impact on the sensitivity of diagnostic assays. Modifications in the design of an assay can, however, improve significantly its capability to detect these mutants. One-hundred and forty-seven clinical samples containing HBsAg variants, and 54 supernatants of cells expressing recombinant HBsAg mutants were tested by two generations of a commercial HBsAg test (Enzygnost® HBsAg 5.0 and 6.0, Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany), and results were compared. A significant improvement was demonstrated for the second test by comparing mean and individual sample/cut-off values, as well as by the rescue of several samples displaying amino acid changes in residues 120 and 145 of the HBsAg that were recorded negative by the former test. Results showed that modifications in design introduced in the last generation improved very much the ability of the test to detect HBsAg mutants, and that troubles in detecting such HBV variants should not be expected from the routine use of the test in diagnostic laboratories and in blood transfusion centres.

The sensitivity of immunoassays for hepatitis B virus (HBV) surface antigen (HBsAg) detection may be hampered by the presence of mutants involving the major antigenic determinant of the protein. The performance of the VITROS HBsAg Assay has been shown to be affected by mutations comprising amino acid changes at residues 143, 144, and 145 of the HBsAg molecule. Sixty-seven serum samples from HBV carriers containing major populations of natural HBsAg mutants assayed previously by that assay were tested by the new VITROS HBsAg ES Assay. Samples displayed either single or multiple amino acid substitutions between positions 112 and 145 of the HBsAg, including changes in relevant residues such as 118-120, 125-127, and 143-145. Testing of undiluted samples by the current assay gave rise to false negative results in two samples displaying the single substitutions 145A and 145R, and in one additional sample displaying a dual mutation 118A + 145A. Unusually weak reactivity (

BACKGROUND: Mathematical models predict that, in Spain, a significant number of blood units will be obtained during the window period of the hepatitis B virus (HBV) infection. Routine nucleic acid testing (NAT) on individual blood units may provide experimental data to evaluate such a theoretical risk. STUDY DESIGN AND METHODS: Between February and July 2005, a total of 34,631 individual units were screened for HBV DNA by a multiplex transcription-mediated amplification (TMA) test. Units that repeatedly reacted in the test, but did not react for HBV surface antigen (HBsAg), were submitted to additional testing by both molecular and conventional assays, and the donors were recalled for follow-up studies and the collection of clinical and epidemiologic data. RESULTS: Confirmatory testing and follow-up studies identified 2 blood units donated during the HBV infection window period (1/17,316 units studied). Sequencing of amplification products obtained by nested polymerase chain reaction (n-PCR) revealed two HBV strains from genotypes D/ayw3 and F/adw4q-, but did not identify HBsAg mutants. The HBV DNA concentration in the index donations was estimated to be below the n-PCR detection level (180 IU/mL), in both cases. One donor developed acute hepatitis 2 months after donating blood, but the other remained asymptomatic and displayed normal alanine aminotransferase levels at follow-up. CONCLUSIONS: The HBV infection window period is a real issue in the setting of Spanish blood transfusions. NAT of individual units by TMA would make a significant contribution to improving the safety of the blood supply in Spain. Additional studies involving a larger number of units and longer periods of time are required, however, to ascertain the true incidence of the problem in this country.

Hepatitis B virus (HBV) is a human DNA virus, which replicates through an RNA intermediate because of the reverse-transcriptase (RT) activity of its DNA polymerase. As a result, the mutation rate for HBV is higher than the rate observed for most DNA viruses. HBVs are classified into genotypes based on genomic sequencing, and antigenic subtypes based on the antigenic properties of its major surface glycoprotein, the HBV surface antigen (HBsAg). Subgenotypes have been identified within most of the HBV genotypes. The HBV groups defined by the different genotype-HBsAg subtype associations found over the world display characteristic geographical distributions, reflecting the movements of human populations and other epidemiologically significant events. Such HBV groups constitute genetically stable viral populations sharing a common evolutionary history, but additional stable changes, originating from mutation and mutant selection, are observed within all of them. These viral sub-populations are known as the HBV variants, and some of which have medical and public health relevance. Pre-core (pre-C) defective variants have been shown to make HBV infection much less susceptible to interferon treatment, and treatment failures with other antiviral drugs have been associated with selection of resistant variants that display specific mutations in the genome region encoding the viral RT activity. Since the RT region of the genome overlaps the sequence encoding the HBsAg molecule, selection of drug resistant variants involves, in some cases, the indirect selection of HBsAg variants. Viral variants displaying changes in HBsAg seem to be very common among chronic HBV carriers; and some of these variants may emerge under the pressure of the neutralizing antibody response, leading to vaccine resistance and resistance to immunotherapy. Mutations conferring resistance to immunotherapy are noted often among liver transplant recipients and among babies born to HBV-carrier mothers. In addition, some of these HBsAg variants have been associated with lack of detection by HBsAg tests used for the diagnosis of HBV infection, for the identification of chronic carriers, for screening of blood donations for transfusion, and in the manufacture of therapeutic blood products.

Background Recent data suggest that the prevalence of genotype 4 HCV strains among Spanish carriers is increasing. Objective To assess changes in the prevalence of HCV genotypes in Spain during the last nine years. Methods HCV RNA was amplified by the polymerase chain reaction from 3161 serum samples from unselected, anti-HCV-positive individuals, and the HCV genotype was identified by a reverse hybridisation assay (line probe assay, LiPA). Samples came from 17 different regions of Spain and were obtained between January, 1996 and December, 2004. Results The overall prevalence of HCV genotypes was: 1b, 41.3%; 1a, 24.1%; 3, 19.6%; 4, 11.6%; 2, 3.1%; and 5, 0.3%. The prevalence of genotypes 1a, 3 and 4 increased significantly among patients born after 1950, and that of genotype 1b decreased among them. These significant differences in regard to age were also observed among patients lacking notified high-risk factors. A main switch-up in prevalence of genotypes 1a and 3 was found when patients born in 1941-1950 were compared with those born in 1951-1960, but the same finding in genotype 4 was delayed to patients born in 1961-1970. Conclusions Two separate epidemics of HCV seem to have occurred in Spain during the last 30 years. The former one involved the spread of HCV genotypes 1a and 3. The second was more recent, and involved the spread of genotype 4.

Until recently, the lack of suitable type specific assays has hampered the serological diagnosis of herpes simplex virus (HSV) infections, due to the high crossreactivity between types 1 and 2. The aim of the present paper is the evaluation of new commercial methods for the detection of HSV-1 and HSV-2-specific IgG using glycoprotein G as antigen (BioElisa HSV-1 and BioElisa HSV-2), in their application to viral diagnosis and seroepidemiological studies. Eighty two serum samples from HSV recent infections (30 samples from 13 cases), normal children (28 samples), and patients attended in clinics for sexually transmitted diseases (STD) (24 samples from 20 patients) were studied by such methods and the results compared with those obtained by a conventional indirect ELISA, and by the complement fixation test. The methods gave a type specific identification of the antibody response in nine of the 13 HSV patients. Positive results for anti-HSV-2 IgG were obtained in four cases among the STD patients but in none among the normal children. Nineteen of the former and seven of the latter were positive for anti-HSV-1. Only one sample was reactive in the HSV-1 assay, and negative by the indirect ELISA. Type specific HSV-1 and HSV-2 assays may help the serological diagnosis of HSV infections, since they allow the correct characterization of the antibody response. Bearing in mind the high specificity of the method for HSV-2 IgG, it might be useful in screening of populations for anti-HSV-2 and especially in prevention of the neonatal HSV-2 infection.

Ana Avello´n* and Jose´ M. EchevarriaHepatitis Laboratory, Diagnostic Microbiology Service, National Centre of Microbiology,Instituto de Salud Carlos III, Majadahonda, Madrid, SpainThe prevalence in the population of hepatitisB virus (HBV) surface antigen (HBsAg) variantsthat may impair diagnosis, or allow the virus toescape vaccine-induced immunity or passiveimmunoglobulin therapy is unknown. A genomefragment encoding HBsAg amino acids 112–212was amplified and sequenced from the sera of272 unselected DNA-positive, HBV-chronic car-riers from Spain. The genotype and the HBsAgsubtype were predicted from the sequences.Analysis of amino-acid positions 112–157revealed single or multiple substitutions in 39%of the carriers studied. Mutations were notdetected for residues 121, 135, 137, 139, 140,141, 142, 146, 147, 148, 149, 151, 152, 153, 155,156, and 157. Substitutions reported previouslyto be in association with failures of diagnostictests or with vaccine or immunoglobulin therapyescape were found in 12.5%, 6.6%, and 9.2%of carriers, respectively. Met133Thr (2.2%);Gln129His, Met133Ile, Phe/Tyr134Asn (1.8%);Phe/Tyr134Leu,Gly145Ala(1.5%),andPro120Thr(1.1%) were the most frequent. Other substitu-tions, including Gly145Arg (0.4%), were found ata frequency of less than 1%. Samples containingHBV mutants were tested with three commercialassays for HBsAg screening. Almost all themutants reacted to the upper cut-off values ofthe assays, but six samples with weak reactivitywithoneormoreofthemethodswerealsofound.Thus, HBV mutants with a potential impact onclinical and public health issues are moderatelyfrequent among chronic carriers from Spain,although their influence on the performance ofdiagnostic tests seems to be slight. J. Med.Virol. 78:24–36, 2006.

Background and objectives Compliance with current regulations regarding the prevention of hepatitis C virus (HCV) transmission in the blood transfusion setting requires the use of sensitive assays for HCV antibody (anti-HCV) detection, which should, ideally, identify any donor having had prior contact with the virus. Therefore, low-level anti-HCV positive blood units should be detected by the screening assays, even those reflecting a past and resolved infection. To assess the sensitivity of two versions of an automated chemiluminescent microparticle immunoassay (CMIA) for anti-HCV screening (ARCHITECT ® Anti-HCV), 113 single serum samples containing low levels of anti-HCV, assessed by two immunoblot tests, were selected from 3686 samples received for confirmation of HCV infection by a reference laboratory over a 2-year period. Materials and methods The panel included 17 samples with HCV RNA detected by the polymerase chain reaction (PCR) and 96 PCR negative samples with either positive or indeterminate (anti-Core and anti-NS3 alone) results by immunoblot. Results All but 13 specimens (100/113, 88.5%) were detected by the current version of the ARCHITECT ® Anti-HCV assay and 10 additional samples (110/113, 97.3%) tested positive in a modified version of the test. Conclusion The results showed that the modification introduced in the ARCHITECT ® Anti-HCV assay achieves a significant sensitivity improvement including samples with low-level anti-HCV which are either PCR positive or negative.

BACKGROUND: The aim of the European Sero-Epidemiology Network (ESEN2) is to harmonise the serological surveillance of vaccine-preventable diseases in Europe. OBJECTIVE: To allow comparison of antibody prevalence in different countries by standardising results into common units. STUDY DESIGN: For varicella zoster virus (VZV), a reference laboratory established a panel of 148 samples, characterised by indirect enzyme-immunoassay (ELISA), indirect immunofluorescence, and complement fixation test. Fifty-seven samples were also studied by the fluorescence antibody to membrane antigen test. The geometric mean of the antibody activity (GMAA) obtained from four ELISA determinations was used to characterise each sample of the panel as positive (GMAA: >100 mIU/ml), equivocal (GMAA: 50-100 mIU/ml) or negative (GMAA

Background: Exanthematic diseases are a group of syndromes mainly caused by acute viral infections. Aim: To obtain information about the viruses that cause exanthematic diseases in our region. Patients and methods: During 1998, 267 serum samples from patients with an acute rash or patients presenting a febrile syndrome accompanied by enlarged lymph nodes, headache and other symptoms, were collected. Specific antibody of the IgM class (anti-IgM) against Rubella, Measles, Dengue types 1-4 and Cytomegalovirus (CMV) were measured by immunoenzymatic assay (EIA). Epstein-Barr virus (EBV) antibodies were measured by immunofluorescence. Results: An etiologic agent was detected in 208 cases (77.9%). Cases due to Dengue (40,6%) and Rubella (21%) viruses predominated, but the frequency of other agents was also high in specific age groups. The agreement between the clinical suspicion and the laboratory findings varied broadly, from a 100% for suspected Dengue to just a 14,8% for cases of suspected CMV infection. Conclusions: Dengue was the most common viral exanthematic disease in the Zulia State during 1998 (Rev Méd Chile 2004; 132: 1078-84

Introduccion El tratamiento de la hepatitis B cronica con lamivudina se ve frecuentemente afectado por la emergencia de mutaciones en el motivo YMDDV de la ADN polimerasa viral que confieren resistencia a la droga. Este fenomeno acostumbra a aparecer tras varios meses de tratamiento, pero a veces sucede en momentos tempranos del mismo. Este hecho, junto con otros hallazgos realizados en Japon y Francia, sugiere que las variantes resistentes circulan entre la poblacion y originan infecciones en las que existe resistencia primaria al tratamiento. Metodos Se utilizo una prueba de hibridacion reversa en tira (line probe assay, LiPA) para estudiar la presencia de mutaciones asociadas a resistencia a lamivudina o famciclovir en fragmentos del genoma del virus de la hepatitis B (VHB) amplificados del suero de 79 portadores cronicos mediante la reaccion en cadena de la polimerasa. Las muestras fueron remitidas desde diferentes centros sanitarios espanoles para deteccion y cuantificacion del ADN viral entre enero de 2001 y mayo de 2002, incluyendose en el estudio una muestra, elegida al azar, de las que resultaron positivas en ese periodo. Se recogieron datos sobre tratamiento antiviral previo al momento de la toma de la muestra en pacientes seleccionados. Resultados Se detecto alguna de las mutaciones estudiadas en muestras procedentes de 10 pacientes. Tras revisar los datos disponibles y solicitar informacion adicional, se encontro que tres de ellos (3,8% de los 79 pacientes estudiados) carecian de antecedente de tratamiento antiviral. En 2 casos, se hallo mezcla de secuencias salvajes y de las mutaciones rtm204l (m552i) y rtV207l (v555i), respectivamente. En el restante, se detecto la mutacion rtV207l en ausencia de niveles detectables de la secuencia salvaje. Conclusiones Los hallazgos sugieren que un porcentaje significativo de los portadores cronicos de VHB que existen en nuestro medio sufren infecciones que involucran variantes potencialmente resistentes al tratamiento con lamivudina famciclovir. Toda vez que cabe esperar que el tratamiento con esos farmacos sea ineficaz en presencia de variantes resistentes, estas observaciones llevarian a tomar en consideracion la posibilidad de estudiar rutinariamente la presencia de tales mutaciones antes de seleccionar la pauta terapeutica mas adecuada para cada paciente.

Background and objectives: Serotyping ofantibody to hepatitis C virus (anti-HCV) with specific peptides has been developed as an alternate method for typing HCV infections. The method does not require a prior amplification of viral RNA sequences from the sample. Identification of the viral genotype may be relevant for prognosis and clinical management. Materials and methods: We used a previously described HCV serotyping assay (RlBA TM HCV Serotype SIA kit, Chiron Corp.) to investigate the serotype in 191 samples from blood donors selected for anti-HCV patterns (positive and indeterminate), ALT levels, and the presence or absence of viral RNA. The serotypes were compared with the genotypes obtained from typing the 5'-noncoding region ofthe viral RNA in 82 virem-ic samples. Results: We were able to obtain the viral serotype in 85% (114/134) of samples positive for anti-HCV but in only 3.5% (2/57) of the indeterminates. Lack of anti-NS4 in the sample was significantly associated with both untypable results and the presence of HCV serotypes other than serotype 1. The overall correlation with genotyping was 78% (64/82), rising to 95.5% (64/67) if only samples that could be both genotyped and serotyped were considered. The assay was easy to perform, gave reactivity patterns easy to interpret, and performed with high proficiency on anti-HCV-positive samples lacking detectable levels of viral RNA. Conclusions: This is a practical and useful method for typing HCV infections in the clinical setting. The poor ability of the Core peptides to give the serotype in samples lacking anti-NS4 and the lack of specific peptides to recognize HCV types other than 1, 2, and 3 are, however, some aspects of the method that need improvement in the future.

Varicella-zoster virus (VZV) is a cause of neurologic disease among humans. Both primary infection and recurrence may lead to neurologic infection and disease. Neurologic syndromes associated with acute VZV infection are caused by abnormal immune responses, the most frequent manifestation being cerebellar ataxia. Those associated with recurrences are often due to the direct effect of viral replication in the nervous tissue. VZV reaches the nervous system either through the bloodstream or by direct spread from sensory ganglia where it remains latent. Postherpetic neuralgia, acute encephalitis, aseptic meningitis and myelitis are the most frequent diseases and have been recorded both in association with herpes zoster and in the absence of a cutaneous rash. Early diagnosis may be established by detecting virus-specific DNA sequences in the cerebrospinal fluid (CSF) after amplification by the polymerase chain reaction and then confirmed by detection of intrathecally produced, specific IgG antibody. Virus isolation from CSF and antibody testing in serum are unsuitable for diagnosis. Early acyclovir therapy is recommended in immuno-compromised patients and those with serious disease.

Hepatitis C virus (HCV) is transmitted mainly by the parenteral route after percutaneous exposure to virus-infected products or body fluids. Thus, HCV shares with hepatitis B and D (HBV, HDV) viruses this common transmission route. The prevalence of antibody against HCV (anti-HCV) was studied in 1155 serum samples from individuals at risk of infection by bloodborne or sexually transmitted agents, as well as from others lacking such risk factors, from the city of Maracaibo, Venezuela. Anti-HCV and serological markers of infection by HBV and HDV were also studied in further 550 samples taken from Bari Indians living in different communities in the Perija mountains, State of Zulia, Venezuela. The results obtained showed that recipients of blood or blood products are at increased risk of HCV infection in Maracaibo, whereas sexual transmission plays only a minor role if any. Both HBV and HDV infections were highly prevalent among Bari Indians (64.4% positive for anti-HBc; 11.1% of HBsAg carriers; 15.3% positive for anti-HDV among HBsAg carriers). No anti-HCV positive samples were, however, detected among them, thus suggesting either that HCV has not still reached this population or that HBV and HDV are transmitted by routes unshared by HCV. Anti-HCV was also absent among samples from mentally retarded patients from Maracaibo, thus confirming similar findings from other countries and supporting the existence of specific transmission mechanisms for HBV and HDV which are not working for HCV.

Hepatitis B viral serum markers suggestive of infection by hepatitis B virus type 2 were found in 354 patients tested in the laboratory during a 2.5 year period of study. Confirmation of the HBsAg reactivity by neutralization and subtyping analysis and determination of serum HBV-DNA by molecular hybridization were carried out on selected samples from these patients. Clinical and epidemiological data were obtained from 234 patients and serological follow-up was done in 70 cases. The results obtained from the confirmatory tests and DNA assays indicated that HBsAg reactivities were specific, being frequently associated to low levels of HBV-DNA. The incidence rates obtained for HBV2 serological patterns among patients at different level of risk for HBV infection, as well as the finding of a high rate of coinfection with hepatitis A virus among those showing acute hepatitis, suggested that the HBV2 agent could be transmitted by the oral route. An hypothesis considering the so called HBV2 infections as resulting from infections by an HBV variant strain which has an improved ability for non-sexual, non-parenteral transmission is suggested. Specific recommendations for detection and confirmation of such cases in blood banks are also outlined.

IgG antibodies to varicella-zoster virus (VZV) were detected by indirect enzyme-immunoassay (EIA) in CSF of two patients with acute aseptic meningitis syndrome (AAMS) not associated with evident cutaneous lesion or recent history of zoster infection. Their characteristic features and serological data are compared with those observed in two patients with AAMS and zoster cutaneous lesions, and in 13 patients with AAMS of unknown or other etiology. According to several indexes applied to assess the origin of the detected antibodies, it is concluded that VZV IgG antibodies are of intrathecal production. The possible etiologic relationship between the neurological syndrome and the detection of VZV antibodies in CSF is discussed. Routine serological procedures are proposed for the diagnosis of CNS acute infections of probable viral etiology that should provide additional data on these rarely described cases.

The levels of Varicella-zoster virus (VZV)-specific IgG, IgM, and IgA antibodies produced in 22 cases of varicella, 22 cases of cutaneous zoster, and 12 cases of acute aseptic meningitis due to VZV in the absence of cutaneous lesions, were measured by indirect enzyme immunoassay (EIA) and compared with those observed in a group of 34 age-matched controls. The definition of cutoff titres for each serological marker and combinations of them allowed early diagnosis of infection in 82% of varicella patients and 91% of patients with acute aseptic meningitis lacking cutaneous lesions on a single serum sample, the specificity being over 90%. The system was as sensitive as the demonstration of intrathecally produced IgG antibodies for the early diagnosis of the infection in 22 cases of neurological disease due to VZV. A working protocol for the serological diagnosis of VZV infections, using currently available EIA reagents, is described.

A commercially available reverse transcription (RT)-PCR method (AMPLICOR EV; Roche Diagnostic Systems, Inc., Branchburg, N.J.) was evaluated for detection of enteroviruses in cerebrospinal fluid from patients with neurological disease. This assay was compared with virus isolation in cell culture and an in-house RT-PCR method designed with a nonoverlapping region of the enteroviral genome. A panel of 200 cerebrospinal fluid specimens prospectively collected from patients with a wide variety of neurological symptoms, including 50 patients involved in three different outbreaks of acute aseptic meningitis, was assayed. A second panel of 97 archived cerebrospinal fluid specimens, stored for 2 to 5 years, from patients with aseptic meningitis associated with several enterovirus outbreaks was also studied. From the first panel, enteroviruses were detected in 13 of 50 specimens by cell culture (26%), in 43 of 50 specimens by AMPLICOR EV (86%), and in 46 of 50 specimens by the in-house assay (92%) from patients with aseptic meningitis associated with outbreak and 1 of 29, 3 of 29, and 4 of 29 specimens, respectively, from sporadic cases of aseptic meningitis. The remaining 121 cerebrospinal fluid specimens from patients with other neurological syndromes were negative by all tests. From the second panel, enteroviral RNA was detected by the AMPLICOR test (31 of 97 specimens, 32%) and the in-house assay (39 of 97 specimens, 40%). According to our results, patients with aseptic meningitis should be analyzed for enteroviral infection in cerebrospinal fluid by RT-PCR methods, and the AMPLICOR EV test is a suitable tool for performing such studies. Archival cerebrospinal fluid specimens are less suitable for evaluation of the performance of RT-PCR methods designed for enterovirus detection.