J. A. López

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.

Prevalencia de las infecciones por virus de las hepatitis B, C, D y E en Bolivia

En Bolivia no se han realizado estudios especificos sobre los virus de la hepatitis, por lo que su prevalencia y patrones de circulacion son practicamente desconocidos. De 1992 a 1996 se realizo un estudio seroepidemiologico con el fin de adquirir una primera vision 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 America Latina, se presto atencion especial al estudio de las comunidades autoctonas de la region amazonica. En las zonas rurales del altiplano andino, la infeccion por VHB presento una prevalencia general que corresponderia a una situacion de endemia media o baja (11,2%) y no se encontro ningun portador de anticuerpos contra VHC o VHD. En dos poblaciones de alto riesgo de la ciudad de Cochabamba (ninos sin hogar y trabajadoras del sexo), la prevalencia de infeccion por VHB fue similar (11,6%) y podria considerarse baja en comparacion con la de otras poblaciones analogas de nucleos urbanos en America Latina. La correspondiente al VHC (un caso positivo, 0,5%) seria parecida a la descrita en esas mismas poblaciones, si bien el escaso numero de muestras estudiadas no permite extraer conclusiones mas firmes. En concordancia con observaciones anteriores de comunidades similares de zonas tropicales de Suramerica, en las poblaciones autoctonas de la Amazonia boliviana la infeccion por VHB es sumamente endemica (prevalencia general de 74,0%), pero no se ha detectado la circulacion de VHC. Se sabe que la transmision de VHB es horizontal y tiene lugar desde edades muy tempranas, pero se desconocen los mecanismos de esa actividad. A los 10 anos de edad, mas de la mitad de la poblacion ya ha experimentado la infeccion natural que, 10 anos mas tarde, se habra difundido a practicamente toda la poblacion. La tasa muy baja de individuos positivos al HbsAg (1,6%), la ausencia de ADN virico en las muestras con reactividad aislada a anti-HBc y la alta prevalencia de anti-HBs entre los individuos que presentan marcadores de infeccion natural (92,4%) excluyen la participacion de la transmision vertical en el mantenimiento de la endemia. Hasta el momento, no se ha documentado ningun brote de infeccion por VHD en estas comunidades, pero la alta endemia de infeccion 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 podria haber producido brotes recientes en el departamento de Cochabamba. Se recomienda vacunar contra VHB en las poblaciones endemicas como medida de corto plazo; buscar activamente en todo el pais brotes y casos esporadicos de hepatitis E y continuar realizando estudios que permitan evaluar las repercusiones sanitarias de la situacion 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 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.

Use of Overlapping Synthetic Peptides to Characterize Samples from Blood Donors with Indeterminate Results to Hepatitis C Virus Core Antigen

Background and Objectives: Despite recent improvements in supplemental assays, isolated reactivity to the hepatitis C virus (HCV) core antigen continues as one of the main problems in the confirmation of anti-HCV in blood donors. Reactivity against individual peptides from the c22-3 HCV recombinant antigen has been described as a useful tool for anti-HCV confirmation and donor counseling in such cases. Materials and Methods: We used a previously described set of overlapping peptides spanning the entire sequence of the c22-3 antigen to study 87 single serum samples from blood donors with reactivity for c22-3 antigen alone in second generation recombinant immunoblot assay (RIBA-2). All of them had been previously studied by RIBA-3, anti-HCV E2 EIA and HCV PCR. Results: 66 of the 87 samples studied could be classified as positive or negative for anti-HCV core using the multipeptide assay and such classification correlated well with the results obtained with RIBA-3 and the anti-E2 EIA. However, some discrepancies were found. The epitopes located along the N-terminal half of the molecule were mainly responsible for the specific antibody recognition but those enclosed within amino acids 1–15 were frequently involved in nonspecific reactivity. Some 38% of samples were considered to have specific antibody to the c22-3 antigen and a further 9% reacted for both anti-E2 and single core peptides that were often involved in specific antibody recognision. Conclusion: Testing of blood donor samples indeterminate to the HCV c22-3 antigen for reactivity against individual core peptides can confirm the presence of specific antibody and recognize nonspecific reactivity with certain cross-reacting epitopes. Third generation supplemental tests have reduced such false reactivity, but confirmation of samples with anticore alone is still necessary. Single reactivity to the HCV core antigen is likely to reflect prior exposure to the virus, but rarely active infection, either acute or chronic.

Typing of hepatitis C virus antibody with specific peptides in seropositive blood donors and comparison with genotyping of viral RNA

Background and objectives: Serotyping of antibody 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 (RIBA™ 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 of the viral RNA in 82 viremic 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.

Detection of Antibody to Hepatitis C Virus E2 Recombinant Antigen among Samples Indeterminate for Anti‐HCV after Wide Serological Testing and Correlation with Viremia

The detection of antibody to the second envelope protein (E2) of the hepatitis C virus (HCV) has been hampered by the lack of suitable antigens. A previously described E2 recombinant antigen (CHO‐E2) expressed as a non‐fused, highly glycosylated protein in mammalian cells was used to detect specific antibody (anti‐E2) in samples from blood donors and viraemic patients showing positive or indeterminate results for anti‐HCV after a wide serological study. Anti‐E2 was detected in 50–75% of the donors positive for anti‐HCV, 80% of viraemic immunocompetent patients with anti‐NS3 alone and 28% of non‐viraemic donors with anticore alone. In donors with anti‐NS3 (15 samples) or anti‐NS4 (51 samples) alone, anti‐E2 was found occasionally (3 cases). Moreover, two anti‐E2‐positive samples from viraemic patients were misidentified by some commercial assays for screening anti‐HCV These results suggest that testing for anti‐E2 may be useful for improving the performance of the current assays for anti‐HCV screening and confirmation.

Serological Studies and Hepatitis C Virus RNA in Serum Samples from Blood Donors with Indeterminate Results in Second-Generation Recombinant Immunoblot Assay

The prevention of post-transfusional hepatitis C virus (HCV) infections is based on thescreening of blood donors for aspecific antibody against HCV (anti-HCV) in serum and exclusion of reactive donors. Further studies, using a second-generation recombinant immunoblot assay (RIBA2) for antiHCV and amplification of HCV-RNA sequences by the polymerase chain reaction (PCR), have shown that more than half of these donors are truly positive for anti-HCV and, in most cases, are viremic and able to transmit the infection [1-4]. The finding of indeterminate results in RIBA2 (i. e., isolated reactivity to a single recombinant HCV antigen) affects, however, a significant proportion of the reactive samples, giving rise to a serious problem in counseling the donors. Since most of the RIBA2-confirmed, anti-HCV-reactive donors are found to be viremic when tested by PCR, it has recently been suggested that the amplification and detection of HCV-RNA sequences in serum by PCR can be used to decide whether or not a sample showing indeterminate results in RIBA2 presents, in fact, anti-HCV, thus being highly useful in counseling the donor and for clinical orientation [5]. However, we have obtained evidence that anti-HCV is truly and frequently present among samples with indeterminate results in RIBA2 which are negative by PCR, which also suggests that additional tests to detect antibody against individual HCV antigens should be used prior to PCR testing in the study of these cases. Table 1. Classification of 294 samples indeterminate for anti-HCV (RIBA2 and neutralization for anti-C100-3) after study by three additional supplemental tests (InnoLIA, RIBA3, UBI Multi-EIA)

ARE SYNTHETIC PEPTIDES SENSITIVE ENOUGH FOR SCREENING ANTI-HEPATITIS C VIRUS AT BLOOD BANKS ?

Prevention of the transmission of the hepatitisC virus (HCV) to recipients of blood products is currently based on the screening of blood units for specific antibody to HCV (anti-HCV). Either recombinant HCV antigens or synthetic peptides, as well as mixtures of both, are used in enzyme immunoassays (EIA) useful for this purpose. Most EIA methods using mixtures of recombinant antigens containing epitopes encoded by both structural and nunstructural regions of the HCV genome (second-generation recombinant EIAs) are known to he sensitive for antiHCV detection and suitable for screening of blood units. False-positive results, arising from the presence of antibody to the carrier protein or to residual antigens of the microorganism used for cloning and production of the recombinants, are, however, moderately frequent with such assays. Anti-HCV EIA methods using synthetic peptides avoid these nonspecific rcactions, hut some concerns rcmain, however, regarding thcir sensitivity. I n a previous report [ I ] , we could not find major differences between recombinant and synthctic peptidc anti-HCV EIA in their pcrforinance using a panel of positive samples confirmed by RIBA-2. However, recombinant tests were able to detect reactivity in a group of 4 Samples with indeterminate results in RIBA-2, whereas synthetic peptide methods gave negative results in all ofthem. Thus, we concluded that further invcstigations, using a wide group of such samples, should he donc before synthetic peptides could hc recommended for use at blood hanks. Now, we have done this study and obtained results supporting the use of synthetic peptides for this purpose. Five hundred and fifty-six sainplcs with indeterminate results i n RIBA-2 wcre found in a multicentrc study involving 3,987 antiHCV-reactive blood donors in Spain. These donors wcre selectcd in the collaborating blood hanks mainly with second-generation recombinant EIA tests. A group ofthese saniples wcrc fiirther characterized for anti-HCV using the following supplementary assays: RIBA-3 (Chiron Corp.), InnoLlA (Innogcnetics Inc.), Matrix-HCV (Ahhott Labs.), UBI multi-EIA (United Biomedical Inc.) and Deciscan HCV (Sanofi Diagnostics Pasteur), which include both recombinant antigens, diffcrent from those involved in RIBA-2, and synthetic peptides from the core and nonstructural (NS3, NS4 and NSS) regions ofthe HCV genome. Aftcr these additional studies, SO samples werc considcrcd to he anti-HCV positive, because of their reactivity to antigens derived from, at least, two different rcgions of the HCV genoinc (table I ) . Then, these S O anti-HCV-positive, selccted samples were tested by four recombinant, second-generation anti-HCV tests and six methods using synthetic peptides as antigens in the solid phase. As can he seen in table 2, no significant diffcrences could he found concerning thc reactivity ofthc samples and the nature of the antigens, although each method showed its own ability to detect anti-HCV among thesc sainplcs. The results ohtaincd previousTable 1. Anti-HCV patterns among S O samples from blood donors indeterminate i n RIBA-2 hut positive after study with additional supplementary assays

HBsAg subtype distribution among different populations of HBsAg carriers in Spain

Data concerning the HBsAg subtype distribution in Spain are out-of-date and confined to a restricted geographical area. Furthermore, the complex distribution observed in the countries surrounding Spain prevents any prediction. To obtain further data on HBsAg subtype distribution among Spanish HBsAg carriers, subtyping analysis (d andy determinants) was performed in 670 samples from subjects belonging to various epidemiological risk groups and coming from different geographical areas of the country. Similar frequencies were found for both mutually exclusived/y subtype determinants among non-risk, normal HBsAg carriers from almost all geographical areas studied. In contrast, the ay subtype was clearly predominant (79–87%) among intravenous drug users, irrespective of their geographical origin. Thirteen different institutions for mentally retarded patients behaved as closed communities for HBV circulation, showing independent subtype distributions. Thus, no significant geographical variations were found for HBsAg subtype distribution in Spain. The prevalence of each particular subtype is mainly dependent on the epidemiological characteristics of the carriers studied. Subtype distribution was independent of the presence of HBeAg or HDV infection serum markers when homogeneous groups were considered separately. Atypical HBsAg phenotypes, either with coexistence or absence of both subtype determinants, were found in some cases.

Sensitivity of seven commercial assays in the detection of hepatitis B virus Type 2-like infection

Two hundred serum samples taken from patients with hepatitis B virus type 2-like infections were tested with seven commercial enzyme immunoassay methods for detection of HBsAg. Positive results were obtained with all methods in some samples, the rate of detection of HBsAg ranging from 9 % to 90 % for the different methods. A direct correlation was found between the analytical sensitivity of a method and its ability to yield positive results. It is suggested that these findings should be taken into consideration when selecting HBsAg detection methods in blood banks in order to avoid transmission of the HBV2 agent to recipients of blood or blood products.