Comparative study of adenoviruses with monoclonal antibodies

The obtainment of monoclonal antibodies for adenovirus species 4(Ad4) is described.The specificities of selected monoclonal antibodies were determined by means of viral neutralization test in cell culture, immunofluorescence and Enzyme-Linked Immunosorbent Assay (ELISA), in the presence of the following species of human adenovirus: 1, 2, 5 (subgenus C), 4 (subgenus E), 7 and 16 (subgenus B) and 9 (subgenus D). Two monoclonal antibodies species specific to adenovirus 4 (1CIII and 3DIII) and one monoclonal antibody that cross reacted with adenovirus species 4 and 7 (2HIII) were obtained.

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.

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.

Concomitant rotavirus serotypes 1 and 4 infections in a diarrhoeic child from Belém, Brazil

Concomitant serotypes 1 and 4 infections were detected in a 15-month old female child with community-acquired diarrhoea which lasted 7 days and coursed with moderate dehydration. The evidence for dual rotavirus infection was offered by the following findings: a) enzyme-linked immunosorbent assay (ELISA) positive reactions to both 1 and 4 serotypes; and b) extra-migrating bands at electro-phoresis of RNA in polyacrylamide gel (PAGE). These results suggest that children living under poor sanitation conditions are heavily exposed to rotavirus infections; in addition, the co-circulation of different serotypes in the same setting sustains the current concept that a rotavirus vaccine should be rnultivalent, in order to protect children against the four epidemiologically important rotavirus G serotypes.

Polyvinyl alcohol-glutaraldehyde as solid-phase in Elisa for Schistosomiasis

Soluble adult Schistosoma mansoni antigen preparation (SWAP) was covalently fixed onto polyvinyl alcohol-glutaraldehyde discs and an enzyme linked-immunosorbent assay (ELISA) was set up. The best conditions for the assay were established and it was found that small amount of antigen such as 1.5 µg was required. A comparison between this procedure and the conventional ELISA was proceeded. A reliable method of antigen immobilization was achieved and the low prices of the employed reagents are economically attractive

Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

Visceral larva migrans (VLM) is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA) using the larval excretory-secretory antigen of T. canis (TES), the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa). Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

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

Anti-phenolic glycolipid-I (PGL-I) determination using blood collection on filter paper in leprosy patients

The authors studied 70 leprosy patients and 20 normal individuals, comparing the traditional sera collection method and the finger prick blood with the conservation on filter paper for specific antibodies against the native phenolic glycolipid-I (PGL-I) from Mycobacterium leprae. The finger prick blood dried on filter paper was eluated in phosphate buffer saline (PBS) containing 0.5% gelatin. The classical method for native PGL-I was performed for these eluates, and compared with the antibody determination for sera. It was observed that there is a straight correlation comparing these two methods; although the titles found for the eluates were lower than those obtained for serology. This blood collection method could be useful for investigation of new leprosy cases in field, specially in contacts individuals.

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.

Evaluation of anti-Schistosoma mansoni igG antibodies in patients with chronic schistosomiasis mansoni before and after specific treatment

The circumoval precipitin test (COPT), enzyme-linked immunosorbent assay (ELISA) and the immunoblotting anti-adult worm antigen (AWA) and soluble egg antigen (SEA) tests were applied to 17 chronically schistosome-infected patients for the detection of anti-Schistosoma mansoni antibodies before and on four occasions after oxamniquine administration over a period of six months. Compared to a control group, schistosomiasis patients showed high levels of IgG antibodies in AWA and SEA-ELISA. A decrease in IgG levels was observed six months after treatment, although negative reactions were not obtained. Significant decreases in IgG1, IgG3 and, mainly, IgG4, but not anti-SEA IgG2 levels were observed six months after treatment, again without negativity. Analysis of anti-AWA IgG antibodies by immunoblotting before treatment showed a 31 kDa strand in 14 patients (82%) which disappeared in three cases up to six months after treatment; furthermore, anti-SEA IgG antibodies showed the same band in nine patients (53%) before treatment, which disappeared in only four cases up to six months after treatment.

Frequency of serum anti-cysticercus antibodies in the population of a rural Brazilian community (Cássia dos Coqueiros, SP) determined by ELISA and immunoblotting using Taenia crassiceps antigens

Considering the impact of cysticercosis on public health, especially the neurologic form of the disease, neurocysticercosis (NC), we studied the frequency of positivity of anti-Taenia solium cysticercus antibodies in serum samples from 1,863 inhabitants of Cássia dos Coqueiros, SP, a municipal district located 80 km from Ribeirão Preto, an area considered endemic for cysticercosis. The 1,863 samples were tested by enzyme linked immunosorbent assay (ELISA) using an antigenic extract from Taenia crassiceps vesicular fluid (Tcra). The reactive and inconclusive ELISA samples were tested by immunoblotting. Of the 459 samples submitted to immunoblotting, 40 were strongly immunoreactive to the immunodominant 18 and 14 kD peptides. Considering the use of immunoblotting as confirmatory due to its high specificity, the anti-cysticercus serum prevalence in this population was 2.1%.

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.

Anti-Taenia solium metacestodes antibodies in serum from blood donors from four cities of Triângulo Mineiro area, Minas Gerais, Brazil, 1995

Serological survey was performed to detect IgG antibodies anti-Taenia solium metacestodes in blood donors of Hemocentro Regional de Uberlândia, Minas Gerais, Brazil. A total of 1133 sera from blood donors coming from four cities of Triângulo Mineiro area were analyzed by the indirect fluorescence antibody test (IFAT) and the enzyme linked immunosorbent assay (ELISA). Specific IgG antibodies were found in 5.6% of the studied population, showing differences in the positive rates according to their origin: Araguari (13.5%), Tupaciguara (5.0%), Monte Alegre de Minas (4.8%) and Uberlândia (4.7%). The results indicate the probable endemicity of cysticercosis in this population.

Prevalence of human herpesvirus 8 antibodies in the population of Belém, Pará, Brazil

Serum samples from 497 children and adults inhabiting two neighbourhoods (Guamá and Terra Firme) in Belém, Pará, North Brazil were screened for the presence of human herpesvirus 8 (HHV-8) antibody using an enzyme-linked immunosorbent assay. An overall 16.3% prevalence was found for these urban communities. Taken both genders together, prevalence rates of HHV-8 antibody increase gradually, across age-groups, ranging from 12.0% to 33.3%. When seroprevalence is analysed by gender, similar rates are found for female (18.4%) and male (14.0%) individuals. In the former gender group, seroprevalence rates increased from 10.3%, in children £ 10 years of age, to 30.0% in adults 41-50 years of age. Conversely, among male subjects, the prevalence of HHV-8 antibodies decreased from 13.3% in children/young adults aged £ 10 to 20 years of age to 6.1% in adults aged 21-30 years. From the 31-40 year-old group male onwards, seropositivity rates increased gradually, ranging from 8.3% to 66.7%. A significant difference in seropositivity rates was noted when comparing 21-30 age groups for female and male subjects: 23.3% and 6.1%, respectively (P = 0.03). Geometric mean optical densities were found to increase slightly from the lower to the higher age-groups. Our data suggest that transmission of HHV-8 occurs frequently in the general urban population of Belém, and that prevalence of antibody seems to increase with age.

Evidence of active herpesvirus 6 (variant-A) infection in patients with lymphadenopathy in Belém, Pará, Brazil

A total of 323 patients with lymphadenopathy were selected in Belém, Brazil, between January 1996 and December 2001, and screened for the presence of human herpesvirus-6 (HHV-6) IgM- and- IgG antibodies by enzyme-linked immunosorbent assay (ELISA). When seroprevalence is analyzed by gender, similar rates are found for female (60.6%) and male (55.7%) individuals. Seventy-seven (23.8%) patients were HHV-6-IgM-and- IgG-positive (IgM+ subgroup), with positivity rates of 29.7% and 17.7% (p = 0.0007) for female- and male individuals, respectively. Sera from a subgroup (n = 120) of these subjects, with high HHV-6 antibody levels (either IgM+ or IgG+ reactivities), were subsequently processed for the presence of HHV-6 DNA by polymerase chain reaction (PCR)/nested PCR. Active infections (IgM+ and/or IgG+ high levels specific antibodies plus detection of viral DNA) were diagnosed in 20/77 (20.0%) and 8/43 (18.6%); subgroup of the 120 individuals suspected of having HHV-6 suggestive recent infection. All (n = 28) cases of active infection were found to be associated with HHV-6 variant-A (HHV-6A), as detectable by PCR/nested PCR, using variant-specific primer that amplify regions of 195 base pairs (bp) (HHV-6A) and 423 bp (HHV-6B). Rates of HHV-6 DNA detection between female and male patients were similar (p > 0.05) in the IgM+ and IgG+ groups: 20.4% versus 35.7% and 25.0% versus 13.0%, respectively. HHV-6 DNA was detected across < 5 through 41-50-year age-groups for patients whose serum samples were IgM+, with rates ranging from 7.7% (female subjects aged < 5 years) to 80.0% (male, 11-20 years). Among patients whose serological status was IgG+, HHV-6 DNA was detected in < 5, 6-10, 21-30 and > 50 age-groups at rates that ranged from 15.4% (male, < 5 years of age) to 100.0% (female aged 11-20 years). Swelling cervical lymph nodes were the most common sign, accounting for 9 (32.0%) cases in each gender group. Among patients (n = 28) with active infection by HHV-6A variant, duration of symptoms lasted 1-5 days in 35.7% of subjects, whereas in 64.3% of them the disease lasted 6-20 days. Our data suggest that it is worth seeking for HHV-6 infection whenever a patient (infant or adult) presents with lymphadenopathy as a prominent symptom in the course of an acute febrile illness.