Correlation between rotavirus A genotypes detected in hospitalized children and sewage samples in 2006, Córdoba, Argentina.

Routine rotavirus A (RV-A) surveillance is based on clinical cases, so only symptomatic infections are reported. The objective of this study was to determine whether the RV-A genotypes and cold seasonal pattern described in patients with diarrhea is reflected by sewage surveillance, which could be representative of the RV-A genotypes circulating in the population. The genotype distribution of RV-A in effluent samples from a local sewage treatment plant was compared to those from local clinical cases. A total of 52 sewage samples and 70 stool specimens from children with acute non-bacterial diarrhea were collected from January to December 2006. The effluent specimens were concentrated and RNA extracts from concentrated sewage and clinical samples were genotyped for the rotavirus VP7 gene. The proportional distribution of the RV-A G-genotypes in sewage and clinical samples during the cold season was similar: G1 accounted for 26.6% of the typed sewage isolates and 28.8% of the clinical infections; G3 type accounted for 21.9% and 25.8%; G2 type 15.6% and 10.6%; G4 type 17.2% and 21.2%; G8 type 1.6% and 0%; and the G9 type 17.2% and 13.6%, respectively. A similar picture of RV-A genotype detection was obtained in sewage samples collected during the cold and warm seasons. The results indicate that there is a correlation between genotypes of RV-A isolates from human diarrheic patients and of those from sewage samples. In addition, sewage monitoring highlighted the uniform all-year RV-A circulation, which was in contrast to the peak incidence of RV-A infection in the community.

Characterization of liposomes coated with S-layer proteins from lactobacilli.

The stability of liposomes coated with S-layer proteins from Lactobacillus brevis and Lactobacillus kefir was analyzed as a previous stage to the development of a vaccine vehicle for oral administration. The interactions of the different S-layer proteins with positively charged liposomes prepared with soybean lecithin or dipalmitoylphosphatidylcholine were studied by means of the variation of the Z potential at different protein-lipid ratios, showing that both proteins were able to attach in a greater extent to the surface of soybean lecithin liposomes. The capacity of these particles to retain carboxyfluorescein or calcein by exposure to bile salts, pancreatic extract, pH change and after a thermal shock showed that both S-layer proteins increased the stability of the liposomes in the same magnitude. The non-glycosylated protein from L. brevis protects more efficiently the liposomes at pH 7 than those from L. kefir even without treatment with glutaraldehyde.

Intratypic differentiation of poliovirus strains by enzyme-linked immunosorbent assay (ELISA): poliovirus type 2 and poliovirus type 3.

A double-antibody sandwich ELISA was developed for the detection of antigenic differences between wild and vaccine-derived strains of poliovirus type 2 and poliovirus type 3. Antibodies were prepared in rabbits by immunization with purified antigens of vaccine strains (type 2: Sabin P712, and type 3: Sabin Leon) and wild type strains (type 2: MEF, and type 3: Pool 30). Immunoblotting analysis of all antisera demonstrated that the IgG antibodies raised in rabbits have specificity towards the main structural proteins (Vp1, Vp2 and Vp3) of poliovirus. IgG fractions were purified from antisera by affinity chromatography, on a protein A-activated Sepharose 4B column. Purified IgG antibodies were used for coating of microtest plates (catching antibodies). The same reagents labelled with horseradish peroxidase were used as conjugates, after cross-adsorption with antigens of the same type heterologous virus strains (strain-specific conjugates). 29 poliovirus type 2 strains and 73 poliovirus type 3 strains isolated from clinical samples, were differentiated intratypically, as vaccine-derived or wild types, no intermediate strains were found and all samples tested fell in two distinct (vaccine/wild) categories. As little as 40 ng of poliovirus antigens was detected in stool samples from healthy children or from polio patients cultivated in monkey kidney tissue cultures. Preparation of strain specific conjugates did not require large amounts of poliovirus antigens. The developed ELISA, which is economic and capable of (1) detection of low amounts of poliovirus antigens in cultivated clinical samples, and (2) intratypic differentiation of poliovirus antigens as either vaccine-derived or wild type, is therefore well suited for large scale screening of poliovirus isolates.

Antigenicity and immunogenicity of rotavirus VP6 protein expressed on the surface of Lactococcus lactis.

Group A rotaviruses are the major etiologic agents of acute gastroenteritis worldwide in children and young animals. Among its structural proteins, VP6 is the most immunogenic and is highly conserved within this group. Lactococcus lactis is a food-grade, Gram-positive, and nonpathogenic lactic acid bacteria that has already been explored as a mucosal delivery system of heterologous antigens. In this work, the nisin-controlled expression system was used to display the VP6 protein at the cell surface of L. lactis. Conditions for optimal gene expression were established by testing different nisin concentrations, cell density at induction, and incubation times after induction. Cytoplasmic and cell wall protein extracts were analyzed by Western blot and surface expression was confirmed by flow cytometry. Both analysis provided evidence that VP6 was efficiently expressed and displayed on the cell surface of L. lactis. Furthermore, the humoral response of mice immunized with recombinant L. lactis was evaluated and the displayed recombinant VP6 protein proved to be immunogenic. In conclusion, this is the first report of displaying VP6 protein on the surface of L. lactis to induce a specific immune response against rotavirus. These results provide the basis for further evaluation of this VP6-displaying L. lactis as a mucosal delivery vector in a mouse model of rotavirus infection.

Rotavirus VP7-gene selection during coinfections in CaCo-2 cells.

A previous rotavirus epidemiological survey in Córdoba, Argentina, revealed an unusually high frequency of mixed G-type infections (41.5%). The genotype distribution of those mixed infections showed that the most prevalent G-type combinations were G1+G4 (65.0%), G1+G2 (12.5%), G2+G4 (3.1%) and G1+G9 (2.5%). In the present study we analyzed the competitive growth in CaCo-2 cell cultures of strains from the most frequent rotavirus G-type coinfections in order to explain some aspect of the dynamic of G-type replacement along the time. Our results indicated that G1-type was preferentially selected compared with G2 and G9-genotypes, meanwhile, G1-G4 coinfections showed an efficient co-amplification of both types. Interestingly, this mirrored the high detection rates of both genotypes as single and mixed infections (G1+G4, 65.0%) in our region. On the other hand, G2-type revealed a better amplification rate with respect to G4-type. Fluctuant rates in the prevalence of different genotypes usually observed along the time could, in part, be explained by successive replacement of strains with different growth characteristics. We hypothesized that one aspect of these different fitnesses can be measured as differential growth in culture of the strains contained in the sample of a mixed infection. Our findings here provide the first data supporting the validity of the competitive replication in vitro to better understand rotavirus G-type circulation patterns.

Molecular epidemiology of group A rotavirus diarrhea among children in Buenos Aires, Argentina, from 1999 to 2003 and emergence of the infrequent genotype G12.

To examine the epidemiology of rotaviruses in Buenos Aires, Argentina, we screened 1,212 stool samples from children with diarrhea in the southern district of Buenos Aires from 1999 to 2003. We identified 187 samples (15.4%) that were positive for group A rotavirus by use of antigen enzyme-linked immunosorbent assay. Among these specimens, 112 were available for typing: 93 (83.0%) were single-type infections, 9 (8.0%) were mixed-type infections with more than one G or P type, and 10 (8.9%) were G and/or P nontypeable. In contrast to the findings in our last study, from 1996 to 1998, genotype P[4], G2 strains were almost completely absent and P[8], G1 and P[8], G4 strains were dominant, representing more than 80% of the G and P types found. Genotypes G2 and G9 were detected in few samples, and type G3 was completely absent. We identified several uncommon genotype G12 strains, representing the first detections outside of Asia and the United States, by sequencing. Using a genotype G12-specific reverse transcription-PCR, we identified eight (6.7%) positive samples for the 1999 to 2003 period. The high degree of sequence identity between recent G12 isolates from Argentina, the United States, and Asian countries suggests a relatively recent introduction(s) of these strains into humans from a common progenitor. The Argentinean G12 strains belonged to genotype P[9], similar to most of the recently described Asian G12 strains. The finding of G12 strains in several other regions of the world raises the possibility that G12 may be emerging globally and suggests that surveillance for this strain should be conducted routinely.

Detection of group a human rotavirus G9 genotype circulating in Córdoba, Argentina, as early as 1980.

The incidence of human rotavirus G types was determined over a 25-year period (1979-2003) by using reverse transcription-PCR (RT-PCR) to examine 519 stool specimens found to be positive for rotavirus by enzyme linked immunosorbent assay (ELISA) or polyacrylamide gel electrophoresis (PAGE). These stool samples were obtained from children under 3 years old who had been treated for acute diarrhea at public hospitals in Córdoba, Argentina. The present study describes the continued circulation of the common human G types G1 (53.8%), G2 (10.2%), G3 (4.4%), and G4 (27%), and also the detection of the unusual types G8 (0.5%) and G9 (4.2%). Genotype G9 was detected during the 1980-1988 and 1997-2003 periods at relatively low rates. Rotavirus G types distribution was independent of age (1-18 months), gender or out-patient or in-patient status. Unexpectedly, 44.6% of mixed infections were detected, involving common and unusual genotypes. Overall, 95.4% of the typed strains belonged to the most prevalent human serotypes (G1-G4) but the detection of G9 infection throughout this study period highlights the importance of this serotype as a human pathogen.

Serological diagnosis of mumps and parainfluenza type-1 virus infections by enzyme immunoassay, with a comparison of two different approaches for detection of mumps IgG antibodies.

Two solid-phase enzyme-linked immunosorbent assays (ELISA) for detection of mumps IgG antibodies, viz., indirect ELISA and catching-antibodies (C.A.)-ELISA, are described and the results obtained with both assays are compared with each other and with the conventional complement-fixation (CF) test. In the indirect method, mumps antigens are used for coating the wells of the microtest plates, whereas in the C.A.-ELISA method mumps antigens are selectively bound to rabbit anti-mumps antibodies coated surfaces. A positive correlation was found between the optical density (O.D.) values given by both ELISA assays and CF-antibody titers. The ELISA assays showed improved sensitivity compared to the CF test, since 54% (C.A.-ELISA) and 33% (Indirect-ELISA) of additional positive reactions were detected by these assays. In terms of specificity, however, only the C.A.-ELISA was superior to CF, since significant rises of IgG-antibodies were detected only in paired sera of mumps patients. Conversely, when the indirect method was used significant IgG antibody rises were demonstrated in paired sera from mumps patients and in serum pairs of six patients with parainfluenza type-1 virus infection. With the CF test, heterologous antibodies responses were demonstrated in 2 of these patients. Absorption experiments of mumps sera with mumps and parainfluenza virus strains demonstrated that the IgG antibodies detected by the C.A.-ELISA are specific for mumps virus and therefore interference due to heterologous antibody responses were not observed. Results with purified mumps virus proteins demonstrated that the antigen-antibody reactions that partake in the C.A.-ELISA are mainly associated with the nucleoprotein antigen. Detection of IgG-antibodies to parainfluenza virus type-1 was assessed by ELISA (paraflu-T1-ELISA) using only the indirect approach. The results obtained with this assay showed improved sensitivity compared to a paraflu T1-CF test, since 47% of additional positive reactions were demonstrated by ELISA. In terms of specificity, however, heterologous antibody responses were detected by both the ELISA and the CF test in 4 out of 20 patients with mumps infections.

Prevalence of IgG-antibodies to mumps and measles virus in non-vaccinated children.

The prevalence of mumps and measles IgG antibodies in a randomly selected population of children was determined by an enzyme-linked immunosorbent assay (ELISA) before routine measles-mumps-rubella (MMR) vaccination was introduced in Denmark. Testing of sera from about 2,520 Danish children between one and 17 years of age showed that mumps antibodies were acquired at an early age. The peak acquisition rate was between the ages of four and five; before the age of 15, 90% of children had antibodies to mumps. Immunity to measles occurred at an even earlier age; more than 50% of four-year-old and nearly all (98%) nine-year-old children had IgG antibodies to measles virus. The study showed that about 10% of the young adult Danish population was still susceptible to mumps infection whereas only about 1% of individuals at age 17 had not acquired immunity to measles virus.

A solid-phase, polyclonal IgM-RF binding assay for circulating immune complexes.

A polyclonal IgM-RF-binding assay (pRF-BA) for the detection of circulating immune complexes (IC) is described. The method is based on the competitive binding of heat-treated iodinated IgG (deltaIgG) and naturally occurring IC to solid phase IgM-RF. The sensitivity limit of the assay was 300--400 ng deltaIgG/ml dilute serum. The coefficient of variation for the assay varied from 6 to 12% of the total binding when deltaIgG concentrations up to 1 microgram/ml were measured. One hundred and six patient sera were examined for IC occurrence and significant differences (p less than 0.01) were observed between 30 normal control sera and sera from SLE, sarcoidosis and glomerulonephritis patients. About 40% of patients suffering from acute myocardial infarction (AMI) gave IC-positive reactions with samples taken 5 to 10 days after the infarction. The kinetics of IC appearance was studied in AMI patients by the pRF-BA and three complement-dependent assays. IC appearance was registered in the RF assay 5 to 12 days after the rise in ASAT enzyme values and the IC reactivity corresponded to complexes ranging from 2 to 5 x 10(6) in molecular weight.

Monoclonal antibodies for the direct detection of influenza-A virus by ELISA in clinical specimens from patients with respiratory infections.

BACKGROUND: Monoclonal antibody technology provides antibody reagents of known specificity, high titres and unlimited availability, that form ideal reference antibodies for use in specific viral antigen-detection methods. OBJECTIVES: To produce mouse monoclonal antibodies against antigenic sites of influenza-A virus, and evaluate their use as diagnostic reagents in a sandwich ELISA. STUDY DESIGN: (1) Production and characterization of monoclonal antibodies against influenza-A virus; (2) application of these antibodies in an ELISA method for direct antigen detection; and (3) evaluation of the ELISA as routine procedure. RESULTS: Four monoclonal antibodies (A1-A4) from mice immunized intranasally with influenza-A virus were selected according to their specific reactivity with either nucleoprotein or matrix protein antigens as demonstrated by Western blot analysis. These antibodies lacked haemagglutination inhibition and neutralization properties and recognized both H1N1 and H3N2 strains of influenza-A virus equally. A sandwich ELISA using unlabelled antibodies for antigen capture and biotin-labelled antibodies for antigen detection was used to analyse nasopharyngeal secretions or nasal swabs from culture-confirmed influenza-A-infected patients and comparable specimens from patients with other viral respiratory infections. Only influenza-A virus (strains H1N1 and H3N2) could be detected in samples from patients with known influenza-A and influenza-B infections, and also after re-isolation of such viruses in conventional cultures of MDCK cells or embryonated hens' eggs. The antigen-detection assay showed a diagnostic sensitivity of 100% and a specificity of 98.3% compared with conventional culture methods. CONCLUSION: The reported ELISA appears to be a rapid and inexpensive method for diagnosis and epidemiological studies of influenza-A infections.

High incidence of beta-2-microglobulin containing macromolecular complexes in sarcoidosis sera.

In thirteen out of fourteen sarcoidosis patients with elevated serum levels of immune complexes beta-2-microglobulin (beta 2m) was detected in immune complex-enriched fractions prepared by precipitation with 3% polyethylene glycol. Beta-2-microglobulin was also contained in complexes isolated by means of solid-phase Clq. The levels of free beta 2m in sarcoidosis sera did not differ from the concentrations in sera from healthy donors.

Intratypic differentiation of poliovirus strains by enzyme-linked immunosorbent assay (ELISA): poliovirus type 1.

A double antibody sandwich-ELISA has been developed for the detection of antigenic differences between wild and vaccine derived strains of Poliovirus type 1. Poliovirus strains antibodies were prepared in rabbits by immunization with virus suspensions of: Sabin LSc2ab (vaccine derived) and Brunhilde and Mahoney (wild types). IgG fractions were purified from antiserum by precipitation with ammonium sulphate and DEAE-Sephadex A50 chromatography. Purified IgG antibodies were used for coating of microtest plates (catching antibodies). The same reagents labeled with horseradish peroxidase were used as conjugates (detecting antibodies). Detecting antibodies were made strain specific by cross-absorption with the heterologous virus strain. Absorbed and non-absorbed detecting antibodies were subsequently used for detection and quantitation of the poliovirus antigen(s) bound to IgG-coated surfaces. Poliovirus laboratory strains and isolates from sixty-six individuals were differentiated intratypically as vaccine derived or wild types when the ELISA was performed using absorbed conjugates. No intermediate strains were found, and all clinical samples tested fell in two distinct categories. Conversely, when detecting antibodies were used before absorption a high degree of homology between wild and vaccine strains was demonstrated and the differentiation between the two groups was poorly achieved. The ELISA has been optimized in terms of specificity and sensitivity. Less than 10 ng of poliovirus antigens could be detected by non-absorbed detecting antibodies whereas 18 ng was the minimal amount detected by the same antibodies after absorption. Preparation of strain specific antibodies did not require a previous concentration of the poliovirus suspension used for the absorption. It is proposed that the developed ELISA is capable of: 1) detection of low amounts of poliovirus antigens in clinical samples, and 2) intratypic differentiation of poliovirus antigens as either vaccine derived or wild types.

Detection of specific immunoglobulin M to mumps virus in serum and cerebrospinal fluid samples from patients with acute mumps infection, using an antibody-capture enzyme immunoassay.

An immunoglobulin M (IgM) antibody-capture immunoassay using peroxidase-labelled mumps antibodies is described and its suitability for the practical diagnosis of acute mumps infection is evaluated. All 45 patients with confirmed mumps infection showed specific mumps IgM antibodies in their sera. Similarly, mumps IgM antibodies were detected in cerebrospinal fluid samples and sera from 12 patients with meningitis. Among these patients 8 had positive mumps virus isolates from the cerebrospinal fluid and 4 had not. Conversely, mumps IgM antibodies were detected neither in 418 serum samples from adult patients expected to be negative for mumps IgM, nor in 184 cerebrospinal fluid specimens from patients with various central nervous system symptoms, nor in sera from 10 patients with high levels of IgM-rheumatoid factor. Among 20 patients with respiratory illness caused by parainfluenza type-1 virus none showed an IgM mumps antibody response. In 99% of cases with acute mumps infection the IgM-ELISA gave a positive result in the first available serum samples most of which were negative for CF and mumps IgG antibodies. The virus proteins which take part in the IgM-ELISA test immune reaction are mainly the nucleoprotein and to a lesser extent the membrane protein. The IgM-ELISA method, which can be performed in 6 hours, offers a reliable, sensitive and rapid alternative to routine methods for the diagnosis of acute mumps infection.

Subclinical measles infection in vaccinated seropositive individuals in arctic Greenland.

Measles vaccination was performed in the arctic district of Scoresbysund, Greenland in 1968, which had never been exposed to natural measles. More than 90% of the total population was vaccinated and a 94-100% seroconversion was obtained. During a serological survey to examine the immunity status of the vaccinees, it was discovered that a temporary increase in measles antibodies took place in the majority of the population 2-4 years after the vaccination. This was not accompanied by clinically observed measles. Most likely, it was due to an inapparent measles infection in a population considered highly immune after vaccination.

Estimation of immune complexes by a microplate-adapted C1q-Protein A enzyme-linked-immunosorbent-assay (C1q-PA-ELISA).

A microplate-adapted enzyme linked immunosorbent assay (ELISA) for detection of C1q-binding immune complexes (IC) and aggregated IgG (delta IgG) is described. Purified human C1q was adsorbed to the wells of flat-bottomed microtiter plates and EDTA-treated serum samples were subsequently introduced. Bound IC was measured by use of alkaline phosphatase-labelled Protein A followed by the substrate para-nitro-phenyl-phosphate. A dose response was found for both delta IgG and BSA anti-BSA complexes, while variations in the concentration of monomer IgG did not affect the optical density. Elevated levels of IC were found in the majority of sera from patients with rheumatoid arthritis and SLE. The described C1q-PA-ELISA is a simple and inexpensive method for detection of C1q-binding immune complexes. The reproducibility is acceptable and the sensitivity is higher than for most IC-methods based on C1q-binding.

Immune complexes in the sputum of patients with cystic fibrosis suffering from chronic Pseudomonas aeruginosa lung infection.

12 cystic fibrosis (CF) patients chronically infected with mucoid P. aeruginosa and presenting multiple precipitins in serum against this bacterium and 12 patients without P. aeruginosa infection were examined for occurrence of soluble immune complexes in their sputum sol phase by a complement consumption assay and a solid phase rheumatoid factor binding assay. The correlation between the results obtained in the two assays was significant (r = 0.625, p less than 0.01). The patients chronically infected with P. aeruginosa showed a significantly (p less than 0.01) higher frequency of immune complex activity in their sputum sol phase, as compared to the patients without P. aeruginosa lung infection. These findings point to the possibility that chronic lung infection with mucoid P. aeruginosa in CF may be an immune complex disease.

Circulating immune complexes, free antigen and alpha 1-antitrypsin in levels in sarcoidosis patients.

Consecutive serum samples from 26 sarcoidosis patients were examined for circulating immune complex (IC) activity. Fifteen (58%) gave IC-positive reactions in a complement consumption (CC) test and a significant difference as regards anticomplementary was observed when comparing patients in clinical stages 2 and 1 respectively (P less than 0.01) The serum alpha 1-antitrypsin levels were not correlated (r=0.36) to the results of the CC-assay. The duration of IC-occurrence was studied, by two IC-assays, over 1 1/2 to 2 years. The majority of the positive reactions were registered in patients in clinical stage 2. Isolated IC was used for immunization of rabbits. Absorbed immune sera produced a single precipitate of postalbumin mobility with seven out of 36 sarcoidosis sera. In two cases a tailing of the recipitate suggested that the antigen was in complex formation. Immunoelectrophoresis indicated identity between the antigen detected in different sarcoidosis sera. No precipitates were observed using 130 sera from other patients, and screening of 100 blood donors revealed one positive reaction. The antigen, which eluted in the first protein peak on Sephadex G-200, has not been identified serologically.

Monoclonal antibodies for the rapid diagnosis of influenza-B virus infections by ELISA: production and characterization.

BACKGROUND: Monoclonal antibodies directed against conserved epitopes of viral proteins have substantially improved the accuracy of several immunochemical methods in diagnostic virology. OBJECTIVES: To characterize mouse monoclonal antibodies directed against structural protein antigens of influenza-B virus and evaluate their use as diagnostic reagents for the direct detection of such antigens in clinical specimens from patients with respiratory infections of unknown aetiology. STUDY DESIGN: (a) Production and characterization of monoclonal antibodies against influenza-B viral antigens, and (b) their use in two different ELISA systems for detecting influenza-B antigen either directly in clinical specimens or after confirmation by rapid culture in MDCK cells. RESULTS: Four monoclonal antibodies were selected for their specificity for the nucleoprotein antigen as demonstrated by Western blot analysis. The specificity of these antibodies for different epitopes of the nucleoprotein was demonstrated by competition experiments, using unlabelled and biotin-labelled purified antibodies in a sandwich assay. All four antibodies belong to the mouse IgG(2a) isotype, lack haemagglutination inhibition and neutralization properties and exhibit titres as high as 10(-6) in ELISA with as little as 30 ng purified influenza-B virus. ELISA methods using these antibodies detected only influenza-B viral antigens in direct testing of clinical specimens from patients with known influenza-B or influenza-A infections, or after reisolating virus from such specimens in tissue culture of MDCK cells. CONCLUSION: The antibodies were suitable for the direct detection and typing of influenza-B virus in clinical specimens or for use in rapid confirmation cultures.