A New Genus and Two New Species of the Subfamily Nesomesochorinae Ashmead (Insecta: Hymenoptera: Ichneumonidae).

Nesomesochorinae is a rather small subfamily of Darwin wasps (Insecta: Hymenoptera: Ichneumonidae), only with three described genera. We recognize a fourth nesomesochorine genus from Perú and describe it as a new genus, Bina Shimizu & Alvarado gen. nov. The new genus is distinguished from previously described genera of Nesomesochorinae by the following combination of character states: mandible parallel-sided proximally and abruptly strongly narrowed ventroapically so that its lower apical tooth is much smaller and sharper than upper tooth; propodeum with lateromedian longitudinal carinae between anterior and posterior transverse carinae developed on more than posterior 0.9 so that the area superomedia is more or less enclosed; thyridium of 2nd metasomal tergite sometimes very large; 1st metasomal tergite stout and arched; body highly shiny. Bina gen. nov. comprises two species also described as new to science: B. huayrurae Shimizu & Alvarado sp. nov. (type species of Bina gen. nov.) and B. nigra Shimizu & Alvarado sp. nov. Identification keys to genera of Nesomesochorinae and to species of Bina gen. nov. are provided.

Helminth antigens (Taenia solium, Taenia crassiceps, Toxocara canis, Schistosoma mansoni and Echinococcus granulosus) and cross-reactivities in human infections and immunized animals.

Helminth antigens were investigated in the search for accessible heterologous antigens capable to discriminate different helminthiases, by the enzyme linked immunosorbent assay (ELISA) and the immunoblot assay (IB). Antigens used were: Taenia solium cysticercus total saline (Tso); Taenia crassiceps cysticercus vesicular fluid (Tcra-VF); T. crassiceps cysticercus glycoproteins (Tcra-GP and Tcra-(18-14)-GP); Toxocara canis larva excretory-secretory (TES); Schistosoma mansoni adult total saline (Sm) and Echinococcus granulosus hydatid fluid (Eg). The assayed sera were from patients with: cysticercosis (n = 18); toxocariasis (n = 40); schistosomiasis (n = 19) and hydatidosis (n = 50) with proven clinical and laboratory diagnosis, and sera from rabbits immunized with Tso, Tcra-VF, TES and Eg. Cross-reactivity occurred mostly between infections caused by Taenia and Echinococcus or in immunized rabbits, by ELISA. Moreover, the cross-reactivity among helminthiases was found with the use of antigens belonging to phylogenetically related parasite species, Eg, Tso and Tcra-VF, by sharing same antigenic components. Lower cross-reactivities were obtained by IB technique, when only peptides were considered as antigens, and the use of T. crassiceps purified glycoproteins demonstrated high sensitivity and specificity in the diagnosis of human cysticercosis, similarly to that using homologous antigen (Tso) by the same technique.

Evaluation of Henes-PCR assay for Mycobacterium detection in different clinical specimens from patients with or without tuberculosis-associated HIV infection.

The need for early diagnosis of tuberculosis, particularly in HIV-infected patients, requires the development of diagnosis methods that have a high sensitivity and specificity, as does the nucleic acid-based technology. With the purpose of improving the detection of mycobacterium in different clinical samples, we proposed and evaluated an assay based on nucleic acid-amplification: heminested-PCR (Henes-PCR). The procedure was designed to identify Mycobacterium spp., M. tuberculosis complex (MTC), and M. avium complex (MAC), although it has the potential to include more primers for the identification of other species. Analytical and clinical evaluation of Henes-PCR was performed by analysis of reference strains and 356 clinical specimens from 246 patients with pulmonary and meningitis tuberculosis and unrelated infections, including 142 HIV-infected individuals. Ninety-three percent (199) positive and 100% (143) negative results were obtained in specimens from patients with tuberculosis and non-tuberculosis infection, respectively. The overall sensitivity of Henes-PCR was 93.4%, specificity was 100%, positive and negative predictive values were 100 and 91.1%, respectively. Sensitivity and negative predictive value of Henes-PCR were significantly higher than culture procedure for microscopy-negative specimens. Even though frequency of HIV infection was higher in patients with tuberculosis, diagnostic parameters of Henes-PCR were similar between HIV-positive and HIV-negative patients. MTB was identified in 194 (98%) specimens while MAC was detected in 5 (2%) specimens. These findings suggest that Henes-PCR is a useful test for rapid detection of mycobacterium in clinically suspected cases of tuberculosis with smear-negative results.

One-step heminested PCR for amplification of Neisseria meningitidis DNA in cerebrospinal fluid.

A one-step polymerase chain reaction (Heminested-PCR) was designed to target the 16S rRNA fragment simultaneously using a set of primers for the universal bacterial group and a Neisseria meningitidis species-specific sequence for diagnostic purposes. The diagnostic features of the Heminested-PCR were evaluated in the study of 168 cerebrospinal fluid (CSF) specimens from 84 patients with a N. meningitidis infection, meningitis caused by unrelated bacteria and other etiologies (57 patients), or suspicious cases (27 patients) with clinical symptoms of bacterial meningitis but with negative results from bacteriological procedures. About 90% of patients with bacterial meningitis, including those suspicious cases, had prior antibiotic therapy. The sensitivity, specificity, positive, and negative predictive values found in relation to culture and/or microscopy were 91.7, 100, 100, 100, and 90.5%, respectively. In patients suspected of having bacterial meningitis, the Heminested-PCR revealed 51.9% (14 patients) positive for N. meningitidis infection and 40.7% (11 patients) positive for unrelated bacterial infections. The agreement of the Heminested-PCR with culture and/or microscopy was high and ranked as almost perfect (kappa indices > 0.856), in contrast to its agreement with other techniques. These findings speak in favor of the molecular diagnosis of meningococcal meningitis in patients who are culture- and/or microscopy-negative, due to their prior antibiotic treatment.

Detection of specific antibody isotypes and subtypes before and after treatment of American visceral leishmaniasis.

Sera from patients with American visceral leishmaniasis (AVL) were studied before and after treatment based on their antibody isotypes and subtypes. The study was comprised of 33 Brazilian patients with well-defined diagnosis of AVL and 39 clinically healthy individuals. Antileishmanial antibody isotypes and subtypes were observed in almost all patients, except IgA that was detected in about 63% of them. The sensitivity and specificity of the immunofluorescence assay in the detection of antibody isotypes (IgG and IgM) and subtypes (IgG1, IgG2, IgG3, and IgG4) were high with no statistical difference, ranging from 0.937 to 1.000 and from 0.954 to 1.000, respectively. All IgG antibodies and its subtypes had their levels reduced after treatment. However, the IgG4 had an early decay and its conversion to negative was significantly high in children. Moreover, the profile of IgG4 before treatment corresponded to a unimodal curve that shifted to a patent bimodal curve after treatment, indicative of therapeutic success. Thus, the IgG4 shows to be a suitable immunological marker for the assessment of chemotherapy in AVL patients or communities. Our findings suggest that IgG4 correlates with IL-4 that also decreases after therapy.

Urinary tract infection: detection of Escherichia coli antigens in human urine with an ELIEDA immunoenzymatic assay.

Escherichia coli is the most common causative agent of urinary tract infection (UTI), and diagnosing this infection usually relies on bacteriologic methods. Nevertheless, screening methods can be useful for a rapid presumptive diagnosis even though some of these screening methods have low sensitivity or are expensive. To investigate a possible new alternative approach, an antigen-based immunoassay--enzyme-linked immunoelectrodiffusion assay (ELIEDA)--was standardized for screening for this bacterial infection. Combining counter-immunoelectrophoresis with an immunoenzymatic assay, the ELIEDA requires concentrated urine specimens, a cellulose acetate membrane, polyclonal antibodies to E. coli raised in rabbits, and peroxidase-labeled sheep antibodies to rabbit immunoglobulin G (IgG). This ELIEDA technique was evaluated using 244 urine specimens, 76 of them with E. coli, 47 with heterologous bacteria, and 121 without bacteria. In comparison to bacteriologic methods, the sensitivity, specificity, and positive and negative predictive values for the ELIEDA were 93.4%, 98.2%, 95.9%, and 97.1%, respectively. The data obtained suggest that this assay is useful for routine diagnostic screening for UTI caused by E. coli. In addition, since the ELIEDA stained membranes can be stored, this assay makes retrospective studies possible.

Improvement of the indirect hemagglutination test for the detection of antibodies to Streptococcus pyogenes.

An indirect hemagglutination test for a seroepidemiological survey of Streptococcus pyogenes infection was standardized. This is an improved modification of the indirect hemagglutination test which utilizes an unstable reagent prepared with fresh blood cells. Two types of bacterial antigens represented by extracellular products and purified streptolysin O were assayed, but only the former antigen gave good results. Pretreatment of the bacterial antigen with 0.15 M NaOH and neutralization to pH 5.5, as well as postfixation of sensitized red cells with 0.1% glutaraldehyde at 56 degrees C for 30 min were found to be essential to give long stability to the reagent in liquid suspension, at least 9 months at 4 degrees C. A total of 564 serum samples with high, moderate and low anti-streptolysin O antibodies as determined by the neutralization assay were studied by the indirect hemagglutination test using the new reagent. The sensitivity, specificity, efficiency, positive predictive value and negative predictive value of the test in relation to the neutralization assay were 0.950, 0.975, 0.963, 0.973, and 0.955, respectively. The kappa agreement index between the two techniques was high (0.926) and ranked as "almost perfect". Antibody levels detected by both techniques also presented a high positive correlation (rs = 0.726). Five reagent batches successively produced proved to be reproducible. Thus, the improved indirect hemagglutination test seems to be useful for public health laboratories.

Improvement of the indirect hemagglutination test for the detection of antibodies to Streptococcus pyogenes

An indirect hemagglutination test for a seroepidemiological survery of Streptococcus pyogenes infection was standardized. This is an improved modification of the indirect hemagglutination test which utilizes an unstable reagent prepared with fresh blood cells. Two types of bacterial antigens represented by extracellular products and purified streptolysin O were assayed but only the former antigen gave good results. Pretreatment of the bacterial antigen with 0.15 M NaOH and neutralization to pH 5.5, as well as postfixation of sensitized red cells with 0.1 per cent glutaraldehyde at 56 degrees Celsius for 30 min were found to be essential to give long stability to the reagent in liquid suspension, at least 9 months at 4 degrees Celsius. A total of 564 serum samples with high, moderate and low anti-streptolysin 0 antibodies as determined by the neutralization assay were studied by the indirect hemagglutination test using the new reagent. The sensitivity, specificity, efficiency, positive predictive value and negative predictive value of the test in relation to the neutralization assay were 0.950, 0.975, 0.963, 0.973, and 0.955, respectively. The kappa agreement index between the two techniques was high (0.926) and ranked as "almost perfect". Antibody levels detected by both techniques also presented a high positive correlation (r(s)=0.726). Five reagent batches sucessively produced proved to be reproducible. Thus, the improved indirect hemagglutination test seems to be useful for public health laboratories.

Immunosuppression and parasitic diseases: experimental Schistosomiasis mansoni.

Cyclosporin A is an immunosuppressive agent of clinical relevance and also possesses a potent antiparasitic effect. In organ transplants and tissue grafts, this agent is frequently used in combination with hydrocortisone. Thus the reciprocal effects of these immunosuppressants on experimental Schistosomiasis mansoni were studied. Mice were subcutaneously inoculated with Schistosoma mansoni cercariae, and infected animals which were treated or not with oxaminiquine were subsequently immunosuppressed or not. Potentially fatal exacerbations of parasitemia and parasitism were observed in immunosuppressed animals, in contrast to control animals, suggesting that in transplanted patients an adverse Schistosomiasis may be evolved. Despite the prominent immunomodulation effect, these drugs showed a moderate antiparasitic effect, complementing the schistosomicidal activity of oxamniquine. This effect also seems favorable in the antischistosomal treatment of transplanted patients with S. mansoni infection.

Serological diagnosis of Chagas' disease: a potential confirmatory assay using preserved protein antigens of Trypanosoma cruzi.

The diagnosis of Chagas' disease relies mostly on data provided by immunologic tests, but inconclusive results often require elucidation, especially in blood banks. When six different types of Trypanosoma cruzi epimastigote antigens were studied by an immunoblotting assay (IBA), a preserved protein antigen (Ag PP) was found to present the most interesting immunochemical features because of its high reactivity with anti-T. cruzi antibodies. Thus, the IBA with Ag PP (PP IBA) was assessed with panels of coded and noncoded serum samples prepared in different laboratories, including the Brazilian Reference Laboratory for Chagas' Disease. It was found that serum samples from patients proved (clinically, eletrocardiographically, serologically, and epidemiologically) to have Chagas' disease consistently recognized 12 bands (140, 100, 85, 78, 59, 57, 46, 35, 27, 23, 20, and 18 kDa) of Ag PP. In contrast, sera from nonchagasic patients, including patients with mucocutaneous leishmaniasis, were negative or reacted weakly, and one serum sample did not have more than five different bands. These bands were 78, 57, 46, 35, 27, 23, 20, or 18 kDa. A criterion was adopted to interpret the results obtained in the PP IBA. The criterion considered positive a serum sample recognizing all 12 bands and considered negative a serum sample that did not recognize any of the bands except the eight nonspecific bands mentioned above. The PP IBA indicated maximum sensitivity and specificity as well as high positive and negative predictive values. The data demonstrate that the PP IBA discriminates chagasic from nonchagasic infections and seems to be applicable as a confirmatory assay for elucidating inconclusive results obtained by standard serology.

Identification of Toxoplasma gondii antigens involved in the IgM and IgG indirect hemagglutination tests for the diagnosis of toxoplamosis

Extratos brutos de Toxoplasma gondii constituem materia prima antigenica para o preparo de reagentes empregados em diferentes testes sorologicos para o diagnóstico da toxoplasmose, incluindo entre estes as reaçöes de hemaglutinaçäo indireta para a detecçäo de anticorpos IgM (HA IgM) e IgG (HA IgG). Até o presente momento, moleculas antigenicas do parasita que realmente estäo envolvidas na interaçäo com anticorpos aglutinantes, anti-T. gondii, näo säo ainda bem conhecidas. O processo de absorçäo de soros de pacientes com toxoplasmose, utilizando o reagente de HA IgG (HA-toxo-G), possibilitou a demonstraçäo de que hemacias deste reagente estavam sensibilizadas com antigeno do parasita associado as bandas de massa molecular relativa de 39, 35, 30, 27, 22 e 14 kDa...

[Detection of Neisseria meningitidis group B antigens by MB-Dot-Elisa test in patients with meningitis]. / Detección de antígenos de Neisseria meningitidis del grupo B con la prueba MB-Dot-ELISA en pacientes con meningitis.

Infection with Neisseria meningitidis group B has been difficult to detect, partly because this bacterial group's polysaccharide is a weak immunogen. This article describes work carried out to test a new procedure (MB-Dot-ELISA) employing a high-titered horse antiserum for detection of N. meningitidis group B antigens. The study assayed cerebrospinal fluid samples from 585 subjects, 574 with suspected meningitis cases and 11 with neurologic disorders. The results of the assay indicated a sensitivity of 0.991 and a specificity of 0.826. These results were superior to those obtained with latex agglutination and in substantial agreement with the results of counterimmunoelectrophoresis and bacteriologic methods. Overall, the MB-Dot-ELISA was found to be sensitive, inexpensive, and suitable for public health laboratory investigations.

Decay of antibody isotypes against early developmental stages of Schistosoma mansoni after treatment of schistosomiasis patients.

Antibodies to a number of parasite antigens are found in schistosomiasis patients, and antibodies to early developmental stages were demonstrated to be efficient immunologic markers for the diagnosis of schistosomiasis. In the present study, decay patterns of IgM and IgG antibodies against cercariae and schistosomula were investigated, in comparison to antibodies against worms and eggs in schistosomiasis patients after chemotherapy, for an investigation of seroepidemiologic aspects. Data obtained in the study of 359 serum samples from patients with Schistosoma mansoni infection, noninfected individuals, and patients followed-up for a period of 12 to 15 months after treatment provided the basis to postulate a general pattern for the kinetics of antibody decay. Before treatment, the antibody pattern was represented by a unimodal curve, which shifted to a bimodal curve after treatment, and ended with a unimodal curve similar to that for the noninfected group. Different types of antibodies were classified into four categories according to their decay features, and anti-schistosomulum IgM was classified into the moderate-decay category, whereas other antibodies to early parasite stages were classified into the slow-decay category. The present methodology permits the identification of the most suitable antibodies to be detected in field control programs for schistosomiasis or other parasitoses.

Detection of N. meningitidis group B antigens by MB-Dot-ELISA in patients with meningitis.

Infection with Neisseria meningitidis group B has been difficult to detect, partly because this bacterial group's polysaccharide is a weak immunogen. This article describes work carried out to test a new procedure (MB-Dot-ELISA) employing a high-titered horse antiserum for detection of N. meningitidis group B antigens. The study assayed cerebrospinal fluid samples from 585 subjects, 574 with suspected meningitis cases and 11 with neurologic disorders. The results of the assay indicated a sensitivity of 0.991 and a specificity of 0.826. These results were superior to those obtained with latex agglutination and in substantial agreement with the results of counterimmunoelectrophoresis and bacteriologic methods. Overall, the MB-Dot-ELISA was found to be sensitive, inexpensive, and suitable for public health laboratory investigations.

Humoral immunity patterns based on antibody reactivity to rotavirus antigens in Brazilian children under 5 years of age.

The age distribution of antibody to simian rotavirus (SA-11) was studied in serum specimens obtained from 399 children aged to 5 years and living in the city of Recife (PE), located in the north eastern region of Brazil. Sera were examined for group-specific rotavirus antibody using a blocking enzyme immunoassay (bELISA) and a hemagglutination inhibition antibody (HIA) test, and for anti-VP2, anti-VP4, anti-VP6, and anti-VP7 antibodies using an immunoblotting assay (IBA). Antibody prevalence was similar in all bELISA and HIA assays, showing a steep rise in the 6-to 17-month-old age groups. The results indicate early acquisition of antibody to rotavirus. The majority of children aged 2 to 4 years had bELISA (50% to 60%) and HIA (70% to 81%) antibodies. There was an association in prevalence data obtained by HIA and bELISA with immunoblotting (IBA), revealing four serologic profiles. Children with profiles I and II (60%) respectively had HAI and ELISA antibody or HAI antibody alone and all had immunoprotective antibodies to VP4 and/or VP7. These children were regarded as "immune," resembling convalescent patients with a rotavirus infection. Children with profile III (4%) had no HIA antibody and only non-protective anti-VP6 and/or VP7 antibody, and were considered to be "partially immune." Children with profile IV (36%) had no detectable antibody and were classified as "nonimmune." These children should be considered to be susceptible to rotavirus infection, with the risk of developing clinically severe diarrhea.

Detection of antibody isotypes to streptolysin O by dot ELISA.

The presence of antibody isotypes (IgG, IgA and IgM) to streptolysin O was determined by dot ELISA in 222 serum samples from patients with different levels of anti-streptolysin O (SLO) antibodies as measured by the neutralizing assay (NA), from patients with diseases not related to nonsuppurative complications of Streptococcus pyogenes infection, and from clinically healthy individuals. Immunoglobulin G antibodies were found in 72% of sera from patients with SLO antibodies higher than 333 Todd units (TU), and IgA antibodies were also detected in 53%, but no IgM antibodies were demonstrable. High copositivity (0.94), conegativity (0.97), and positive (0.96) and negative (0.96) predictive values were observed when IgG and IgA findings were combined. The dot ELISA gave highly reproducible results. The present data suggest that the assay may be of practical value for routine detection of SLO antibodies when employed with an anti-human immunoglobulin light chain peroxidase conjugate.

Detection of antibody isotypes to streptolysin O by dot ELISA

The presence of antibody isotypes (IgG, IgA and IgM) to streptolysin O was determined by dot ELISA in 222 serum samples from patients with different levels of anti-streptolysin O (SLO) antibodies as measured by the neutralizing assay (NA), from patients with diseases not related to nonsuppurative complications of Streptococcus pyogenes infection, and from clinically healthy individuals. Immunoglobulin G antibodies were found in 72 per cent of sera from patients with SLO antibodies higher than 333 Todd units (TU), and IgA antibodies were also detected in 53 per cent, but no IgM antibodies were demonstrable. High copositivity (0.94), conegativity (0.97), and positive (0.96) and negative (0.96) predictive values were observed when IgG and IgA findings were combined. The dot ELISA gave highly reproducible results. The present data suggest that the assay may be of practical value for routine detection of SLO antibodies when employed with an anti-human immunoglobulin light chain peroxidase conjugate.

Improvement of the slide hemagglutination test for rapid Chagas' disease screening in epidemiological surveys.

A slide hemagglutination test, here called SHAT, which is practical and economical for seroepidemiological surveys was standardized. This is an improved modification of the rapid hemagglutination test (RHA) which utilizes a short-lived reagent prepared with fresh blood cells. The reagent and conditions of the test were considerably modified and, most importantly, an alkaline-solubilized Trypanosoma cruzi epimastigote antigen reagent is proposed. The stability of the SHAT reagent was at least one year at 4 degrees C, in an appropriate liquid suspension. The SHAT was applied to 71 serum samples from patients with Chagas' disease and from 235 clinically healthy blood donors. Sensitivity, specificity and positive and negative predictive values for the selected cutoff titer corresponding to 1:4 dilution were 0.972 (0.903-0.992), 0.983 (0.957-0.993), 0.945 (0.867-0.979) and 0.991 (0.969-0.998), respectively. These values were comparable to those found for the RHA, immunofluorescence (IFT), indirect hemagglutination (IHAT) and complement fixation (CFT) tests. These data suggest that the SHAT should be useful for seroepidemiological surveys conducted at public health laboratories in developing countries.