Dot-ELISA for the detection of IgM and IgG antibodies to Schistosoma mansoni worm and egg antigens, associated with egg excretion by patients

Human schistosomiasis, caused by Schistosoma mansoni, is highly prevalent in Brazil and usually diagnosed by time consuming stool analysis. Serological tests are of limited use in this disease, mainly for epidemiological studies, showing no discrimination between previous contact with the parasite and active infections. In the present study, we standardized and compared a Dot-ELISA for IgM and IgG antibodies against S. mansoni antigens from eggs and worms with a routine IgG and IgM immunofluorescence assay using similar antigens, in the study of sera from 27 patients who had quantified egg stool excretion. The positivity obtained for IgG Dot-ELISA was 96.3% and 88.9% for IgM Dot-ELISA with worm antigen and 92.6% and 90.9% with egg antigen. The IFI presented similar positivities using worm antigen, 92.6% (IgG) and 96.3% (IgM),and lower results with egg antigen, 77.8% (IgG and IgM). The patients studied were divided into two groups according to their egg excretion, with greater positivity of serological tests in higher egg excreters. When comparing the quantitative egg excretion and the serological titers of the patients, we detected a correlation only with IgM Dot-ELISA, with r=0.552 (p=0.0127). These data show that Dot-ELISA can be used for the detection of specific antibodies against S. mansoni in sera from suspected patients or in epidemiological studies and, with further purification of egg antigen and larger samples, IgM Dot-ELISA could be a possible tool for rough estimates of parasite burden in epidemiological studies.

Evaluation of the allergenicity of spore and mycelia extracts of Pisolithus tinctorius

The antigenic and allergenic chemical analysis of spore and mycelia extracts of Pisolithus tinctorius was carried out. The spores were collected from basidiocarps in plantations of Eucalyptus spp and the mycelia from culture in MNM medium. With basis on the fungus growth curve, the mycelia masses were obtained after 10, 20, 30, and 40 days of incubation, which correspond, respectively, to the beginning, middle and end of the log phase, and beginning of the decline phase. The mycelia masses, together with the spores, were submitted to the action of three extractors (Coca, Tris-HCl, and ammonium bicarbonate). The contents of carbohydrates and proteins were determined. The SDS-PAGE electrophoretical analysis revealed separate fractions in these extracts, besides common fractions, in function of cultivation time and extraction methods. The selected extracts for the allergic tests were the ones with the highest number of fractions. The prick-tests were conducted in 374 patients – rural workers, eucalyptus plantation works and college students. The positivity to the "prick test" with the antigenic extract of P. tinctorius was, respectively, 3.78%, 28.20% and 6.40%. Most prick-test positive patients (82.75%) also presented symptoms of respiratory allergy (asthma and rhinitis). There was no reactivity difference when the spore and mycelia extracts were employed. The analysis of the positive patients’ sera revealed the presence of IgE specific to the P. tinctorius antigens. Since Pisolithus tinctorius is found as mycorrhyza of Eucalyptus spp, and this plant is used in reforestation in most countries, the importance of that fungus should be regarded as a possible cause of respiratory allergies, especially in occupationally exposed workers

Entomophthoramycosis by Conidiobolus coronatus. Report of a case successfully treated with the combination of itraconazole and fluconazole

Rhinoentomophthoramycosis caused by Conidiobolus coronatus in a 61-year old woman was unsuccessfully treated during 8 years with all the antifungals available in the Brazilian market, including potassium iodide for 1 month, sulfamethoxazole plus trimethoprim for 2 months, amphotericin B, total dose of 1130 mg, cetoconazole, 400 mg/day for 6 months, fluconazole, 200 mg/day, for at least 2 months and, itraconazole, 400 mg/day for 2 months, followed by 200 mg/day for 4 more months. Complete clinical and mycological cure was achieved using itraconazol 400 mg/day in association with fluconazol 200 mg/day during 24 months. After cure she was submitted to plastic surgery to repair her facial deformation. Today she remains clinically and mycologically cured after 59/60 months (5 years!) without any specific antifungal. We thus suggest the use of the combination of itraconazole and fluconazole as an additional option for the treatment of this mycosis.

Abdominal ultrasound in the evaluation of fibrosis and portal hypertension in an area of schistosomiasis low endemicity

This study was undertaken in the municipality of Bananal, São Paulo, an endemic area for schistosomiasis with a prevalence under 10% and low parasite load among infected individuals. Our objective was to identify the clinical forms of schistosomiasis among 109 patients in whom the disease had been diagnosed through direct fecal analysis and who had been medicated with oxamniquine at the time of the Plan for the Intensification of Schistosomiasis Control Actions (1998-2000). These patients were submitted to an abdominal ultrasonography and fecal analysis by Kato-Katz method, four years, on average, after the end of the Plan. Five patients, whose abdominal ultrasound images were compatible with either peripheral or central periportal fibrosis and portal hypertension, were identified. None of the 109 patients presented Schistosoma mansoni eggs at fecal analysis. Ultrasonography is a sensitive, noninvasive diagnostic method that allows a better identification of the extent of liver involvement in schistosomiasis cases.

Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

High occurrence of Entamoeba histolytica in the municipalities of Ariquemes and Monte Negro, State of Rondônia, Western Amazonia, Brazil

Introduction: Entamoeba histolytica infections were investigated in residents of the Ariquemes and Monte Negro municipalities in Rondônia State, Brazil. Methods: Stool samples of 216 individuals were processed by the spontaneous sedimentation method and analyzed by microscopy for detection of the E. histolytica/E. dispar complex, followed by the immunoassay method using an enzyme-linked immunosorbent assay-based kit for the E. histolytica stool antigen. Results: E. histolytica/E. dispar cysts were present in 61% (50/82) and 44% (59/134) of the samples from Ariquemes and Monte Negro respectively, with a significant difference in the occurrence of infection between the two populations [p < 0.05; χ2 = 5.2; odds ratio = 2.0 (1.1 - 3.6)]. The E. histolytica antigen detection rate was 36.6% (30/82) for stool samples from Ariquemes, and 19.4% (26/134) for stool taken from the residents of Monte Negro. The rate of the occurrence of amoebiasis was significantly higher in the population from Ariquemes [p < 0.05; χ2 = 7.8; odds ratio = 2.4 (1.2 - 4.7)]. Discussion: Due to the high occurrence of E. histolytica infected residents diagnosed in the region and the unavailability in local clinics of a test to distinguish between the two Entamoeba species, physicians should consider treating E. histolytica/E.dispar infections. Conclusion: The results indicate that E. histolytica infection is highly endemic in the studied areas.

Evaluation of the impact of chemical control measures and entomological surveillance on Chagas' disease in the counties of Mambaí and Buritinópolis, Goiás State, Brazil

Epidemiological surveillance activities were implemented in 1980 in Mambaí and Buritinópolis counties, Goiás State. Twenty years later the authors evaluated the impact of these vector control measures on Chagas' disease transmission, based on entomological indicators. Entomological investigation was conducted using the man-hour technique and covering all domiciles. In order to study vector food sources the stomach contents of triatomines were analyzed using the modified precipitins technique. Triatomines were shown to be present in 48 (71.6%) of the 67 locations. Peridomiciliary infestation rates in Mambaí and Buritinópolis were 8.7% and 12.1%, respectively, while intradomiciliary rates were 0.7% and 1.2%. Triatoma sordida was the species identified in 97.3% of all captured specimens. It was also the only species found to be naturally infected with Trypanosoma cruzi. Birds were the most frequent food source (45%) for Triatoma sordida. The most significant result was the complete absence of Triatoma infestans in the two counties.

QBC® for the diagnosis of human and canine american visceral leishmaniasis: preliminary data

"Quantitative Buffy Coat" (QBC®) is a direct and fast fluorescent method used for the identification of blood parasites. Since Leishmania chagasi circulates in blood, we decided to test it in American visceral leishmaniasis (AVL). Bone marrow (BM) and peripheral blood (PB) of 49 persons and PB of 31 dogs were analyzed. QBC® was positive in BM of 11/11 patients with AVL and in 1/6 patients with other diseases. Amastigotes were identified in PB of 18/22 patients with AVL and in none without AVL. The test was positive in 30 out of the 31 seropositive dogs and in 28/28 dogs with Leishmania identified in other tissues. QBC® is a promising method for diagnosis of human AVL, and possibly for the exam of PB of patients with AVL/AIDS, for the control of the cure and for the identification of asymptomatic carriers. Because it is fast and easy to collect and execute, QBC® should be evaluated for programs of reservoir control.

The use of qualitative and quantitative polymerase chain reactions for diagnosis of cytomegalovirus infections in bone marrow and kidney transplant recipients

The purpose of this work was to test a cytomegalovirus qualitative PCR and a semi-quantitative PCR on the determination of CMV load in leukocytes of bone marrow and kidney transplanted (RT) patients. Thirty three BMT and 35 RT patients participated of the study. The DNA was subjected to a qualitative PCR using primers that amplify part of CMV gB gene. CMV load of positive samples was determined by a semi-quantitative PCR using quantified plasmids inserted with part of the gB gene of CMV as controls. The sensitivity of the test was determined to be 867 plasmid copies/µg DNA. CMV loads between 2,118 and 72,443 copies/µg DNA were observed in 12.1% BMT recipients and between 1,246 and 58,613 copies/µg DNA in 22.9% RT recipients. Further studies are necessary to confirm the usefulness of this CMV semi-quantitative PCR in transplanted patients.

Seasonal profile and level of CD4+ lymphocytes in the occurrence of cryptosporidiosis and cystoisosporidiosis in HIV/AIDS patients in the Triângulo Mineiro region, Brazil

Patients with AIDS are particularly susceptible to infection with intestinal coccidia. In this study the prevalence of infections with Cryptosporidium sp and Cystoisospora belli were evaluated among HIV/AIDS patients in the Triângulo Mineiro region, Brazil. Between July 1993 and June 2003 faecal samples from 359 patients were collected and stained by a modified Ziehl-Neelsen method, resulting in 19.7% of positivity for coccidian (8.6% with Cryptosporidium sp, 10.3% with Cystoisospora belli and 0.8% with both coccidian). Patients with diarrhoea and T CD4+ lymphocyte levels < 200 cells/mm3 presented higher frequency of these protozoans, demonstrating the opportunistic profile of these infections and its relationship with the immunological status of the individual. It was not possible to determine the influence of HAART, since only 8.5% of the patients positive for coccidian received this therapy regularly. Parasitism by Cryptosporidium sp was more frequent between December and February and thus was characterised by a seasonal pattern of infection, which was not observed with Cystoisospora belli.

Geographic analysis on the occurrence of human and canine leptospirosis in the city of Maringá, state of Paraná, Brazil

INTRODUCTION: Leptospirosis is a zoonotic disease, the primary hosts of which are wild, synanthropic, and household animals. Humans behave as terminal and accidental hosts. The prevalence of leptospirosis depends on carrier animals that disseminate the agent, on the environmental survival of this agent, and on the contact of susceptible individuals. Each serovar has one or more hosts with different adaptation levels. The focuses of leptospirosis are infected, sick, and asymptomatic animals, which are considered to be sources of environmental infection. This study aimed to determine the risk areas for leptospiral infection in stray dogs and patients diagnosed with leptospirosis from 2006 to 2008 in Maringá, State of Paraná, Brazil. METHODS: Three hundred and thirty-five stray dogs and 25 patients were studied. Serum from both animals and patients was examined by the microscopic serum agglutination test to study anti-leptospiral antibodies. To determine the risk areas and the spatial distribution of the disease, thematic maps were designed. RESULTS: Forty-one (12.2%) dogs positive for one or more leptospire serovars were observed, the most frequent serovars being Pyrogenes (43.9%), Canícola (21.9%), and Copennhageni (19.5%). Among the humans, 2 (8%) were positive for serovars Pyrogenes and Hardjo Prajitno and for Pyrogenes and Cynopteri. CONCLUSIONS: Spatial analysis showed that the risk for dogs and humans in the City of Maringá to become infected with leptospires exists in both the central and the peripheral areas, a fact that reinforces the relevance of this study and of continuous epidemiological and environmental surveillance actions to control the disease in animals and in humans.

Interpretation of the presence of IgM and IgG antibodies in a rapid test for dengue: analysis of dengue antibody prevalence in Fortaleza City in the 20th year of the epidemic

INTRODUCTION: The diagnosis of dengue and the differentiation between primary and secondary infections are important for monitoring the spread of the epidemic and identifying the risk of severe forms of the disease. The detection of immunoglobulin (Ig)M and IgG antibodies is the main technique for the laboratory diagnosis of dengue. The present study assessed the application of a rapid test for dengue concerning detection of new cases, reinfection recognition, and estimation of the epidemic attack rate. METHODS: This was a retrospective, cross-sectional, descriptive study on dengue using the Fortaleza Health Municipal Department database. The results from 1,530 tested samples, from 2005-2006, were compared with data from epidemiological studies of dengue outbreaks in 1996, 2003, and 2010. RESULTS: The rapid test confirmed 52% recent infections in the tested patients with clinical suspicion of dengue: 40% detected using IgM and 12% of new cases using IgG in the non-reactive IgM results. The positive IgM plus negative IgG (IgM+ plus IgG-) results showed that 38% of those patients had a recent primary dengue infection, while the positive IgG plus either positive or negative IgM (IgG+ plus IgM+/-) results indicated that 62% had dengue for at least a second time (recent secondary infections). This proportion of reinfections permitted us to estimate the attack rate as >62% of the population sample. CONCLUSIONS: The rapid test for dengue has enhanced our ability to detect new infections and to characterize them into primary and secondary infections, permitting the estimation of the minimal attack rate for a population during an outbreak.