Evaluation of the double diffusion, enzyme immunoassay and immunoblotting techniques, for the diagnosis of human hydatid disease in tropical areas

Hydatid disease in tropical areas poses a serious diagnostic problem due to the high frequence of cross-reactivity with other endemic helminthic infections. The enzyme-linked-immunosorbent assay (ELISA) and the double diffusion arc 5 showed respectively a sensitivity of 73% and 57% and a specificity of 84-95% and 100%. However, the specificity of ELISA was greatly increased by using ovine serum and phosphorylcholine in the diluent buffer. The hydatic antigen obtained from ovine cyst fluid showed three main protein bands of 64,58 and 30 KDa using SDS PAGE and immunoblotting. Sera from patients with onchocerciasis, cysticercosis, toxocariasis and Strongyloides infection cross-reacted with the 64 and 58 KDa bands by immunoblotting. However, none of the analyzed sera recognized the 30 KDa band, that seems to be specific in this assay. The immunoblotting showed a sensitivity of 80% and a specificity of 100% when used to recognize the 30 KDa band.

Dot-ELISA-IgM in saliva for the diagnosis of human leptospirosis using polyester fabric-resin as support (Preliminary Report)

In order to improve the diagnosis of human leptospirosis, we standardized the dot-ELISA for the search of specific IgM antibodies in saliva. Saliva and serum samples were collected simultaneously from 20 patients with the icterohemorrhagic form of the disease, from 10 patients with other pathologies and from 5 negative controls. Leptospires of serovars icterohaemorrhagiae, canicola, hebdomadis, brasiliensis and cynopteri grown in EMJH medium and mixed together in equal volumes, were used as antigen at individual protein concentration of 0.2 µg/µl. In the solid phase of the test we used polyester fabric impregnated with N-methylolacrylamide resin. The antigen volume for each test was 1µl, the saliva volume was 8 µl, and the volume of peroxidase-labelled anti-human IgM conjugate was 30 µl. A visual reading was taken after development in freshly prepared chromogen solution. In contrast to the classic nitrocellulose membrane support, the fabric support is easy to obtain and to handle. Saliva can be collected directly onto the support, a fact that facilitates the method and reduces the expenses and risks related to blood processing.

Jungle yellow fever: clinical and laboratorial sudies emphasizing viremia on a human case

The authors report the clinical, laboratorial and epidemiological aspects of a human case of jungle yellow fever. The patient suffered from fever, chills, sweating, headaches, backaches, myalgia, epigastric pains, nausea, vomiting, diarrhea and prostration. He was unvaccinated and had been working in areas where cases of jungle yellow fever had been confirmed. Investigations concerning the yellow fever virus were performed. Blood samples were collected on several days in the course of the illness. Three of these samples (those obtained on days 5,7 and 10) were inoculated into suckling mice in attempt to isolate virus and to titrate the viremia level. Serological surveys were carried out by using the IgM Antibodies Capture Enzyme Linked Immunosorbent Assay (MAC-ELISA), Complement Fixation (CF), Hemagglulinalion Inhibition (HI) and Neutralization (N) tests. The yellow fever virus, recovered from the two first samples and the virus titration, showed high level of viremia. After that, specific antibodies appeared in all samples. The interval between the end of the viremia and the appearance of the antibodies was associated with the worsening of clinical symptoms, including bleeding of the mucous membrane. One must be aware of the risk of having a urban epidemics in areas where Aedes aegypti is found in high infestation indexes.

The relevance of laboratory diagnosis of human cryptosporidiosis and other coccidia

Human infection by Cryptosporidium spp and other coccidia are due to opportunist non-host specific microorganisms. In HIV seropositive patients, the gastrointestinal symptoms accompanying such infections may be serious and prolonged and may include nausea, low-grade fever, abdominal cramps, anorexia and watery diarrhoea. We studied 188 stool samples from 111 patients (84 men and 27 women) with diarrhoea. A modified Ziehl-Nielsen technique for the detection of Cryptosporidium spp and Isospora belli was employed. The mean age of the patients was 31 years. Cryptosporidium spp was seen in 18% (n=20) of the patients, 90% (n=18) of whom were HIV seropositive. Isospora belli was recorded only from HIV seropositive patients (5.4% of all the patients studied and 6.5% of those who were HIV seropositive). These data confirm the good results obtained with this technique for the identification of Cryptosporidium spp and other coccidia and also reaffirm the clinical importance of correctly diagnosing the cause of diarrhoea, particularly in HIV seropositive patients.

Human enterovirus infection in stray dogs. Some aspects of interest to public health

To investigate the possible role of domestic animals as reservoirs of human enteroviruses, we studied 212 stray dogs captured in different areas of the municipality of São Paulo. The captured animals were divided into 19 groups of 10 to 20 dogs each; faeces of 126 of the 212 dogs were processed for enterovirus isolation. The following viruses were isolated from 12 dogs: poliovirus type 1 (2 dogs), poliovirus type 3 (1 dog), echovirus type 7 (8 dogs) and echovirus type 15 (1 dog). Of the 12 infected animals, four had specific homotypic neutralizing antibody titres > 16. All 212 animals were tested for the presence of neutralizing antibodies to human enteroviruses. The frequency of neutralizing antibodies present in titres of > 16 was 10.3%, 3,8% and 4.3% for vaccinal prototypes of polioviruses 1, 2 and 3 respectively; 1,9%, 1.4% and 1.5% for wild prototypes of the same viruses, 11.3% for echovirus 7, and 2.4% for echovirus 15. The proportion of dogs with neutralizing antibodies varied with the virus studied. Some indication of the susceptibility of dogs to infection with human enteroviruses was demonstrated, and the importance of this fact for the Plan for Global Eradication of the Wild Poliovirus is discussed.

Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.

Cryo-Microtome sections of coproculture larvae of Strongyloides stercoralis and Strongyloides ratti as antigen sources for the immunodiagnosis of human strongyloidiasis

Cryo-microtome sections of larvae of Strongyloides stercoralis and S. ratti respectively obtained from human and rat feces cultures, were used as antigens. Fluoresceinate conjugates against human IgG were employed at the ideal titer of 10 for S. stercoralis and 100 for S. ratti. The sensitivity of the indirect immunofluorescence reaction (IIF) was 94.4% and 92.5% and the specificity 94.2% and 97.1% for the two specific larval antigens, respectively. Sera from 123 persons (54 from carriers of S. stercoralis infections and 69 from controls) were submitted to the reaction. The titers of different sera varied from 20 to 2560. There was a significant linear correlation (r = 0.85 p £ 0.001) between the antibodies from the two species of larval antigens. We conclude that both antigens may be used in the IIF reaction for the diagnosis of human strongyloidiasis. Due to the feasibility of safe and low-cost mass production of S. ratti larvae in the laboratory with a considerable economy of conjugate, their utilization in the serum diagnosis of human strongyloidiasis is recommended

IgM and IgA Antibody Responses in 12 Cases of Human Acquired Toxoplasmosis

The persistence, in some subjects, of specific IgM antibodies to Toxoplasma gondii for several months after the acute phase of infection has complicated the interpretation of serological test results for toxoplasmosis. Several reports have emphasized the value of the detection of Toxoplasma-specific IgA antibodies for the diagnosis of acute toxoplasmosis. In this article, we report the follow-up profiles of Toxoplasma-specific IgM and IgA antibodies in serum samples obtained from 12 patients at various intervals after the onset of the clinical manifestations of infection. IgM antibodies were detected by the indirect immunofluorescence (IIF) test, antibody capture enzyme-linked immunosorbent assay (cELISA) and enzyme-mediated chemilluminescent technique (CmL). IgA antibodies were quantified by the direct ELISA (dELISA) and cELISA procedures. As defined by the manufacturer of the cELISA test for IgA used, most patients with acute toxoplasmosis have antibody levels > 40 arbritary units per ml (AU/ml). At values > 40 AU/ml, the cELISA for IgA detected significant antibody levels for a shorter time than the other techniques used for IgM and IgA detection. However, IgA levels £ 40 AU/ml do not exclude the possibility of acute toxoplasmosis since such levels can be reached very soon after infection with T. gondii. The results obtained in the present study show that the serological diagnosis of acute toxoplasmosis may not be such an easy task. Our data suggest that use of the IgA-cELISA concomitantly with IgM antibody screening could permit, in some circumstances, a more efficient diagnosis of acute acquired toxoplasmosis

Evolution of IgG antibody response against Toxoplasma gondii tissue cyst in acute and chronic human infections

The recognition profile of the tissue cysts antigens by IgG antibodies was studied during acute and chronic human toxoplasmic infection. Thus the IgG response against Toxoplasma gondii was investigated by immunoblotting in two patients accidentally infected with the RH strain as well as in group of naturally infected patients at acute and chronic phase. There was an overall coincidence of molecular mass among antigens of tachyzoites and tissue cysts recognized by these sera, however, they appear not to be the same molecules. The response against tissue cysts starts early during acute infection, and the reactivity of antibodies is strong against a wide range of antigens. Six bands (between 82 and 151 kDa) were exclusively recognized by chronic phase sera but only the 132 kDa band was positive in more than 50% of the sera analysed. A mixture of these antigens could be used to discriminate between the two infection phases. The most important antigens recognized by the acute and the chronic phase sera were 4 clusters in the ranges 20-24 kDa, 34-39 kDa, 58-80 kDa and 105-130 kDa as well as two additional antigens of 18 and 29 kDa. Both accidentally infected patients and some of the naturally infected patients showed a weak specific response against tissue cyst antigens.

FETO-PLACENTARY PATHOLOGY IN HUMAN PARVOVIRUS B19 INFECTION

In view of the scarce references concerning the histological data in congenital parvovirus human B19 infection, we intend to provide a description of the pathological features observed in six autopsies.The virus was detected by DNA hybridization (ISH-DBH),PCR and electronmicroscopy (EM) in paraffin-embedded feto-placentary tissues.These cases constitute a subset from 86 Non Immunologic Hydrops Fetalis (NIHF) cases, in which a systemic complex of inflammatory/degenerative lesions of unknown etiology was visualized by optical microscopy. In one case a syphilitic process was detected, typefying a double infection. All fetuses showed a similar pathology - hydrops, hepato-splenomegaly, lung hypoplasia and erythroblastemia, the specific histological feature being the presence of intranuclear inclusions in the erythroid progenitors, in the erythropoietic visceral tissue and in blood marrow. Complex cardiopathy allied to abnormal lung lobulation and polisplenia were observed once; in 2 cases endocardial fibroelastosis was diagnosed. The pulmonary lesions were represented by dysmaturity allied to interstitial mononuclear infiltration. The hepatic consisted of cholestasis, portal fibrosis, canalicular proliferation, hemossiderosis, focal necroses and giant cell transformation. The central nervous system lesions were predominantly anoxic although the autolysis impaired a correct diagnosis.

Efficiency of indirect immunofluorescence assay as a confirmatory test for the diagnosis of human retrovirus infection (HIV-1 and HTLV-I/II) in different at risk populations

We compared the indirect immunofluorescence assay (IFA) with Western blot (Wb) as a confirmatory method to detect antibodies anti retrovirus (HIV-1 and HTLV-I/II). Positive and negative HIV-1 and HTLV-I/II serum samples from different risk populations were studied. Sensitivity, specificity, positive, negative predictive and kappa index values were assayed, to assess the IFA efficiency versus Wb. The following cell lines were used as a source of viral antigens: H9 ( HTLV-III b); MT-2 and MT-4 (persistently infected with HTLV-I) and MO-T (persistently infected with HTLV-II). Sensitivity and specificity rates for HIV-1 were 96.80% and 98.60% respectively, while predictive positive and negative values were 99.50% and 92.00% respectively. No differences were found in HIV IFA performance between the various populations studied. As for IFA HTLV system, the sensitivity and specificity values were 97.91% and 100% respectively with positive and negative predictive values of 100% and 97.92%. Moreover, the sensitivity of the IFA for HTLV-I/II proved to be higher when the samples were tested simultaneously against both antigens (HTLV-I-MT-2 and HTLV-II-MO-T). The overall IFA efficiency for HIV-1 and HTLV-I/II-MT-2 antibody detection probed to be very satisfactory with an excellent correlation with Wb (Kappa indexes 0.93 and 0.98 respectively). These results confirmed that the IFA is a sensitive and specific alternative method for the confirmatory diagnosis of HIV-1 and HTLV-I/II infection in populations at different levels of risk to acquire the infection and suggest that IFA could be included in the serologic diagnostic algorithm.

IgG recognizing 21-24 kDa and 30-33 kDa tachyzoite antigens show maximum avidity maturation during natural and accidental human toxoplasmosis

We describe the avidity maturation of IgGs in human toxoplasmosis using sequential serum samples from accidental and natural infections. In accidental cases, avidity increased continuously throughout infection while naturally infected patients showed a different profile. Twenty-five percent of sera from chronic patients having specific IgM positive results could be appropriately classified using exclusively the avidity test data. To take advantage of the potentiality of this technique, antigens recognized by IgG showing steeper avidity maturation were identified using immunoblot with KSCN elution. Two clusters of antigens, in the ranges of 21-24 kDa and 30-33 kDa, were identified as the ones that fulfill the aforementioned avidity characteristics.

Outbreaks of human-herpes virus 6 (HHV-6) infection in day-care centers in Belém, Pará, Brazil

A total of 730 children aged less than 7 years, attending 8 day-care centers (DCCs) in Belém, Brazil were followed-up from January to December 1997 to investigate the occurrence of human-herpes virus 6 (HHV-6) infection in these institutional settings. Between October and December 1997 there have been outbreaks of a febrile- and -exanthematous disease, affecting at least 15-20% of children in each of the DCCs. Both serum- and- plasma samples were obtained from 401 (55%) of the 730 participating children for the detection of HHV-6 antibodies by enzyme-linked immunosorbent assay (ELISA), and viral DNA amplification through the nested-PCR. Recent HHV-6 infection was diagnosed in 63.8% (256/401) of them, as defined by the presence of both IgM and IgG-specific antibodies (IgM+/IgG+); of these, 114 (44.5%) were symptomatic and 142 (55.5%) had no symptoms (p = 0.03). A subgroup of 123 (30.7%) children were found to be IgM-/IgG+, whereas the remaining 22 (5.5%) children had neither IgM nor IgG HHV-6- antibodies (IgM-/IgG-). Of the 118 children reacting strongly IgM-positive ( > or = 30 PANBIO units), 26 (22.0%) were found to harbour the HHV-6 DNA, as demonstrated by nested-PCR. Taken the ELISA-IgM- and- nested PCR-positive results together, HHV-6 infection was shown to have occurred in 5 of the 8 DCCs under follow-up. Serological evidence of recent infections by Epstein-Barr virus (EBV) and parvovirus B19 were identified in 2.0% (8/401) and 1.5% (6/401) of the children, respectively. Our data provide strong evidence that HHV-6 is a common cause of outbreaks of febrile/exanthematous diseases among children attending DCCs in the Belém area.

Association between human parvovirus B19 and arthropathy in Belém, Pará, north Brazil

A total of 220 patients with arthropathy were selected in Belém, Pará between January 1994 and December 2000, and screened for the presence of human parvovirus B19 IgM and IgG antibodies by enzyme-linked immunosorbent assay (ELISA). A subgroup (n = 132) of patients with high levels of antibodies (either IgM+/IgG+ or IgM-/IgG+) were examined for the presence of DNA by polymerase chain reaction/nested PCR. Recent/active infection (detection of IgM and/or IgG-specific antibodies and presence of viral DNA) was identified in 47.7% of the 132 individuals with arthropathy. In our study, women were significantly more affected (59.7%) than men (35.4%) (P = 0.0006). The age group of 11-20 years (84.6%), among female patients, and 21-30 years (42.1%), among male, were those with the highest incidence rates. The analysis of the temporal distribution of B19-associated arthropaties showed a cyclic pattern, with peak incidence rates occuring at 3-5 year intervals. Significant diference (P = 0.01) was observed when comparing both the highest (39.0%) and the lowest (11.0%) seropositivity rates for the years of 1995 and 2000, respectively. The interfalangial joints of hands and feet were mostly affected, with 50.0% and 48.0% of cases among both women and men, respectively. In a smaller proportion, other joints such as those of knee, ankle, pulse and shoulder were affected. As for the duration, symptoms lasted 1 to 5 days in 54.0% of the individuals, whereas in 46.0% of them the disease lasted 6-10 days, if considered the subgroup (n = 63) of patients with recent/active infection by parvovirus B19. In our study, joint clinical manifestations occurred symmetrically. Our results indicate that B19 may be an important agent of arthropathies in our region, and this underscores the need for specific laboratory diagnosis when treating patients suffering from acute arthropathy, mainly pregnant women.

Visceral leishmaniasis caused by Leishmania (Viannia) braziliensis in a patient infected with human immunodeficiency virus

The current article reports the case of a 19-month-old-girl, from the state of Minas Gerais, Brazil, with visceral leishmaniasis, by Leishmania (Viannia) braziliensis, and Human Immunodeficiency Virus (HIV) co-infection. The child's mother and father, aged 22 and 27 years old, respectively, were both HIV positive. The child was admitted to the General Pediatric Center, in Belo Horizonte, presenting high fever, fatigue, weight loss and enlargement of liver and spleen. Indirect immunofluorescent test revealed a titer of 1:320 for Leishmania. Such result was confirmed by the presence of amastigotes in bone marrow aspirate samples and culture of promastigote forms. Parasites were identified as being Leishmania (Viannia) braziliensis through PCR, using a L. braziliensis complex primer and a generic primer, followed by hibridization. Specific leishmaniasis therapy (GlucantimeÒ antimonial) was intravenously administered.