Multicentre evaluation of a direct agglutination test prototype kit (DAT-LPC) for diagnosis of visceral leishmaniasis.

In this study, we assessed the sensitivity, specificity, and diagnostic accuracy of a previously developed direct agglutination test (DAT) using a freeze-dried antigen derived from Leishmania infantum promastigotes and composed in a prototype kit for visceral leishmaniasis (VL) diagnosis, named DAT-LPC. To evaluate DAT-LPC reproducibility, the kit was used to analyse 207 serum samples from VL patients and 80 serum samples from patients with other parasitic infections or healthy subjects in four laboratories from different public health institutions in Brazil. DAT-LPC showed sensitivity between 96·2 and 99·5% (P = 0·14), specificity ranging from 96·2 to 97·5% (P = 0·95), and diagnostic accuracy ranging from 96·5 to 99% (P = 0·34). The inter-laboratory reproducibility of qualitative results was classified as excellent (κ index: 0·94-0·97). The reproducibility of the end-titre results in relation to the reference laboratory, ranged from 31 to 85%. These results demonstrate an excellent performance of the DAT-LPC, and validate it for the diagnosis of VL that could replace the immunofluorescent antibody test as the routine diagnostic test in the Brazilian public health system.

High prevalence of asymptomatic Leishmania spp. infection among liver transplant recipients and donors from an endemic area of Brazil.

Visceral leishmaniasis is an uncommon disease in transplant recipients; however, if left untreated, the mortality can be high. If an organ donor or recipient is known to be an asymptomatic Leishmania spp. carrier,monitoring is advised. This study proposes to assess the prevalence of asymptomatic Leishmania spp.infection in liver transplant donors and recipients from an endemic area. A total of 50 liver recipients and 17 liver donors were evaluated by direct parasite search, indirect fluorescent antibody test (IFAT), anti-Leishmania rK39 rapid test and Leishmania spp.DNA detection by polymerase chain reaction (PCR).Leishmania spp. amastigotes were not observed in liver or spleen tissues. Of the 67 serum samples, IFAT was reactive in 1.5% and indeterminate for 17.9%, and the anti-Leishmania rK39 rapid test was negative for all samples. The PCR test was positive for 7.5%, 8.9%, and 5.9% of blood, liver and spleen samples, respectively(accounting for 23.5% of the donors and 8% of the recipients). Leishmania infantum-specific PCR confirmed all positive samples. In conclusion, a high prevalence of asymptomatic L. infantum was observed in donors and recipients from an endemic area, and PCR was the most sensitive method for screening these individuals.

Mixed inflammatory/regulatory cytokine profile marked by simultaneous raise of interferon-gamma and interleukin-10 and low frequency of tumour necrosis factor-alpha(+) monocytes are hallmarks of active human visceral Leishmaniasis due to Leishmania chagasi infection.

Considering the complexity of the immunological events triggered during active visceral Leishmaniasis (VL), the relevance of the segregation of the immune response during human VL into type 1 and type 2 still remains unclear. For this purpose, in individuals living in risk areas for VL, we have evaluated especially asymptomatic individuals and patients with active VL, the plasmatic levels of cytokines and reactive nitrogen species under ex vivo conditions. In addition, we have also performed an analysis of intracellular cytokine patterns of circulating leucocytes after short-term culture, particularly in the absence of antigenic-specific stimulation, in order to reflect dynamic events of immune response in vivo during Leishmania chagasi infection. Although asymptomatic individuals and non-infected subjects presented a similar immunological profile, an outstanding inflammatory/regulatory profile, based on higher plasmatic levels of cytokines such as interleukin (IL)-8, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, IL-6 and IL-10, was associated with clinical status observed in active VL. In this context, we hypothesize that IL-10, through its ability to inhibit anti-leishmanial macrophage activation, associated with the lower frequency of TNF-alpha(+) monocytes and ordinary levels of nitrite and nitrate are the major mechanisms associated with disease onset.

Immune response in human visceral leishmaniasis: analysis of the correlation between innate immunity cytokine profile and disease outcome.

We investigated the cytokine profile of cells of the innate immune response and its association with active (ACT), asymptomatic (AS) and cured (CUR) human visceral leishmaniasis (VL), as well as noninfected (NI) subjects. The frequency of cytokine-producing cells was determined after short-term in vitro incubation of whole peripheral blood samples with soluble Leishmania antigen (SLA). Our data demonstrated a predominant type 2 cytokine profile in NI and ACT. In NI, we observed an increase of IL-4+ neutrophils, IL-10+ eosinophils besides a decrease of tumour necrosis factor (TNF)-alpha+ eosinophils/monocytes. Yet in ACT, we observed an increase of IL-4+ neutrophils and natural killer (NK) cells and IL-10+ monocytes, a reduced frequency of IL-12+ and IFN-gamma+ eosinophils and lower levels of TNF-alpha+ and IL-12+ monocytes. AS presented a mixed profile, characterized by an increase of IFN-gamma+ neutrophils/eosinophils and NK cells, of IL-12+ eosinophils/monocytes, as well as increase of IL-4+ neutrophils and NK cells and IL-10+ eosinophils/monocytes. In contrast, CUR was characterized by a type 1 response with an increase of IFN-gamma+ neutrophils/eosinophils and NK cells, associated with an increase in IL-12+ monocytes. In conclusion, we show a correlation between innate immune cytokine patterns and clinical status of VL, suggesting that these cells, in addition to other factors, may contribute to the cytokine microenvironment in which Leishmania-specific T cells are primed and to disease outcome.

Leishmania/HIV co-infection in Brazil: an appraisal.

Given the epidemiology of human leishmaniasis and AIDS in Brazil, numerous cases of Leishmania/HIV co-infection might be expected. Relatively few Brazilian cases have been reported, however, even from regions where the overall incidences of HIV and Leishmania infection are both relatively high. Many cases of co-infection probably go undetected because of a lack of awareness among clinicians or limited access to appropriate diagnostic methods. In contrast to the situation in Europe, intravenous-drug users do not predominate among those exposed to HIV infection in Brazil. The success of the Brazilian programme for the free and universal distribution of antiretroviral drugs has decreased the prevalences of the commoner opportunistic infections among HIV-positives and increased the longevity of AIDS cases. Recent changes in the epidemiological patterns of HIV and Leishmania infections are likely to lead to a greater degree of overlap and a greater risk of co-infection and they justify increased alertness. This review of the co-infection in Brazil addresses three main topics: the current situation, in terms of the epidemiology of AIDS and Leishmania infection; the related epidemiological trends and their likely impact on the co-infection; and the co-infection cases reported in Brazil by June 2003.

Brain abscess caused by Cladophialophora (Xylohypha) bantiana in a renal transplant patient.

Infectious disease is the most significant cause of morbidity and mortality in allotransplantation because of heavy immunosuppression. Brain abscesses caused by melanized fungi have been found occasionally and are an example of this complication. In this paper, we describe a case in a 61-year-old black man, who received a cadaveric kidney transplantation in December 1993, followed by triple therapy with cyclosporine, azathioprine, and prednisone. The patient developed right hemiparesis at the beginning of April 1998. A computed tomography scan showed a mass in the left parieto-temporal region of the brain. The patient underwent surgery and a brown-colored encapsulated brain abscess was resected. Histology of the tissue revealed a large number of pigmented fungal hyphae. Culture in a Sabouraud dextrose medium with cyclohexamide and chloramphenicol at 25 degrees C resulted in the growth of dark-green colonies. The fungus identified was Cladophialophora bantiana, based on characteristic microscopic features and on growth at 40 degrees C. The abscess recurred in spite of treatment with fluconazole. The patient was submitted to a second brain surgical procedure and was treated with amphotericin B in addition to fluconazole. Ten days later the patient's blood cultures became positive for Escherichia coli. After 3 days the patient died due to septic shock.

Detection of circulating Leishmania chagasi DNA for the non-invasive diagnosis of human infection.

A polymerase chain reaction (PCR) assay for the detection of Leishmania spp. DNA in peripheral blood was optimized and evaluated for the diagnosis of human visceral leishmaniasis (VL) in Brazil during May 2001 to December 2002. Optimization of the technique resulted in a detection limit of 1.65 fg of purified L. (L.) chagasi DNA, equivalent to 1.65 x 10(-2) parasites. Leishmania DNA was detected in the blood of 48 of 53 patients with parasitologically-confirmed VL, which corresponds to a sensitivity of 91%. No DNA was detected in the peripheral blood of 15 healthy, non-exposed volunteers, giving a specificity of 100%. We conclude that detection of parasite DNA in peripheral blood offers a non-invasive, sensitive and rapid method for the detection of VL caused by L. (L.) chagasi.

Antibody subclass profile and avidity during acute and chronic human Schistosoma mansoni infection.

The avidity of IgA, IgM, IgG and IgG subclass antibodies against Schistosoma mansoni soluble egg antigen (SEA) was determined by ELISA in serum samples collected in 1995 from individuals in Brazil with acute (n = 36) and chronic (n = 40) schistosomiasis. Fifteen individuals at the acute phase were also evaluated 6 months after clinical diagnosis. Predominance of low-avidity IgG, IgG1, IgG2, IgG3 and IgA characterized the acute phase. IgG4 was detected in only 2 individuals with acute disease (5.6%). Levels of anti-SEA IgM were similar between the study groups. IgG1 avidity showed the strongest association with the chronological evolution of the infection, presenting 100% of low avidity during the acute infection and reaching 100% of high avidity 6 months after. It is suggested that distinct anti-Schistosoma egg antigens subclass profile and antibody avidity characterize the clinical phases of S. mansoni infection. In particular, determination of anti-SEA IgG1 offers a new tool for the laboratory analysis of the disease.

Serum antigliadin antibody levels as a screening criterion before jejunal biopsy indication for celiac disease in a developing country

The objective of the present study was to determine the efficacy of detection of antigliadin immunoglobulins G and A (IgG and IgA) for the diagnosis of celiac disease in a developing country, since other enteropathies might alter the levels of these antibodies. Three groups were studied: 22 patients with celiac disease (mean age: 30.6 months), 61 patients with other enteropathies (mean age: 43.3 months), and 46 patients without enteropathies (mean age: 96.9 months). Antigliadin IgG and IgA ELISA showed sensitivity of 90.9 and 95.5 percent, respectively. With the hypothetical values of prevalence ranging from 1:500 to 1:2000 liveborns, the positive predictive value varied from 8.5 to 2.3 percent for IgG and from 4.8 to 1.1 percent for IgA. Considering the patients without enteropathies, specificity was 97.8 and 95.7 percent for IgG and IgA, respectively. In patients with other enteropathies, specificity was 82.0 and 84.1 percent, respectively. When patients with and without other enteropathies were considered as a whole, specificity was 88.8 and 91.6 percent, respectively. The specificity of positive IgG or IgA was 93.5 percent in children without enteropathies and 78.7 percent in the presence of other enteropathies. The negative predictive value for hypothetical prevalences varying from 1:500 to 1:2000 liveborns was 99.9 percent. Thus, even in developing countries where the prevalence of non-celiac enteropathies is high, the determination of serum antigliadin antibody levels is a useful screening test prior to the jejunal biopsy in the investigation of intestinal malabsorption

Serum antigliadin antibody levels as a screening criterion before jejunal biopsy indication for celiac disease in a developing country.

The objective of the present study was to determine the efficacy of detection of antigliadin immunoglobulins G and A (IgG and IgA) for the diagnosis of celiac disease in a developing country, since other enteropathies might alter the levels of these antibodies. Three groups were studied: 22 patients with celiac disease (mean age: 30.6 months), 61 patients with other enteropathies (mean age: 43.3 months), and 46 patients without enteropathies (mean age: 96.9 months). Antigliadin IgG and IgA ELISA showed sensitivity of 90.9 and 95.5%, respectively. With the hypothetical values of prevalence ranging from 1:500 to 1:2000 liveborns, the positive predictive value varied from 8.5 to 2.3% for IgG and from 4.8 to 1.1% for IgA. Considering the patients without enteropathies, specificity was 97.8 and 95.7% for IgG and IgA, respectively. In patients with other enteropathies, specificity was 82.0 and 84.1%, respectively. When patients with and without other enteropathies were considered as a whole, specificity was 88.8 and 91.6%, respectively. The specificity of positive IgG or IgA was 93.5% in children without enteropathies and 78.7% in the presence of other enteropathies. The negative predictive value for hypothetical prevalences varying from 1:500 to 1:2000 liveborns was 99.9%. Thus, even in developing countries where the prevalence of non-celiac enteropathies is high, the determination of serum antigliadin antibody levels is a useful screening test prior to the jejunal biopsy in the investigation of intestinal malabsorption.

The role of nutritional status and insulin-like growth factor in reduced physical growth in hepatosplenic Schistosoma mansoni infection.

The influence of nutritional status and hormonal growth activity on the impaired somatic development of adolescents with the hepatosplenic clinical form of Schistosoma mansoni infection (HS), the intestinal form with high (IH) or low (IL) egg output and non-infected (NI) individuals was evaluated (in Comercinho, Minas Gerais State, Brazil, in 1996-97) by measuring body mass index (BMI), insulin-like growth promoting factor (IGF-I) and its carrier protein (IGFBP-3). BMI, IGF-I and IGFBP-3 concentrations were significantly lower in the HS group compared with the IH and the NI groups, irrespective of age. BMI did not remain associated with the clinical form in the bi-variate model that included IGF-I and BMI or IGFBP-3 and BMI, suggesting that in these groups IGF-I and IGFBP-3 levels were related to the clinical form but independent of nutritional status. It is suggested that physical growth impairment in hepatosplenic S. mansoni infection results from the synergistic action of both hepatic damage and nutritional restriction.

[Leishmaniasis urbanization and low diagnosis capacity in the Metropolitan Region of Belo Horizonte]. / A urbanização das leishmanioses e a baixa resolutividade diagnóstica em municípios da Região Metropolitana de Belo Horizonte.

During the period from 1994 to 1999 cutaneous leishmaniasis was reported in 32 (89%) out of 36 municipalities in the Metropolitan Region of Belo Horizonte, Brazil, of which one (2,8%) municipality was classified as a very high risk area, 16 (44,5%) as high risk, seven (19,4%) as moderate risk areas and 12 (33,3%) as low risk. From 1994 to 1995, visceral leishmaniasis was reported in six (16%) municipalities whereas in 1998 - 1999 this number increased to 15 (42%). Annual numbers of cases during 1994 to 1999 were 30, 53, 64, 53 and 84, respectively. In 19 (61.3%) municipalities no reference center for the diagnosis of the infection was available, so that most of the patients (80%) were referred to Belo Horizonte. Twelve (39%) municipalities have a center for leishmaniasis evaluation, however in only eight (67%) of these basic specific diagnostic tests were available. Rapid and extensive increase of leishmaniasis associated with low diagnosis capacity has been observed in the metropolitan area of Belo Horizonte.

A urbanização das leishmanioses e a baixa resolutividade diagnóstica em municípios da Região Metropolitana de Belo Horizonte / Leishmaniasis urbanization and low diagnosis capacity in the Metropolitan Region of Belo Horizonte

During the period from 1994 to 1999 cutaneous leishmaniasis was reported in 32 (89%) out of 36 municipalities in the Metropolitan Region of Belo Horizonte, Brazil, of which one (2,8%) municipality was classified as a very high risk area, 16 (44,5%) as high risk, seven (19,4%) as moderate risk areas and 12 (33,3%) as low risk. From 1994 to 1995, visceral leishmaniasis was reported in six (16%) municipalities whereas in 1998 - 1999 this number increased to 15 (42%). Annual numbers of cases during 1994 to 1999 were 30, 53, 64, 53 and 84, respectively. In 19 (61.3%) municipalities no reference center for the diagnosis of the infection was available, so that most of the patients (80%) were referred to Belo Horizonte. Twelve (39%) municipalities have a center for leishmaniasis evaluation, however in only eight (67%) of these basic specific diagnostic tests were available. Rapid and extensive increase of leishmaniasis associated with low diagnosis capacity has been observed in the metropolitan area of Belo Horizonte.

No período de 1994 a 1999, foram notificados casos de leishmaniose tegumentar em 32 (89%) dos 36 municípios da Região Metropolitana de Belo Horizonte. Em um (2,8%) município o risco de adquirir a doença foi considerado muito alto, em 16 (44.5%), médio em sete (19,4%) e baixo em 12 (33.3%). Leishmaniose visceral foi notificada em seis (17%) dos 36 municípios, nos anos 94 e 95, elevando-se para 15 (42%) no biênio 98/99. O total de casos de leishmaniose visceral notificados anualmente no período 94 a 99 foi 30, 53, 64, 60, 53, 84, respectivamente. Não há serviços referenciados para atendimento da doença em 19 (61,3%) de 31 municípios, sendo 80% dos pacientes encaminhados para Belo Horizonte. Em 12 (39%) municípios com serviços referenciados, somente oito (67%) dispõem de testes diagnósticos específicos para leishmaniose. Verificou-se rápida e extensa expansão das leishmanioses na região metropolitana de Belo Horizonte e baixa capacidade de resolução diagnóstica pelos seus municípios.

Oral fluids for the immunodiagnosis of Schistosoma mansoni infection.

Saliva and oral transudate were evaluated for their potential as human specimens in the detection of IgG antibodies against soluble Schistosoma mansoni egg antigen (SEA). Preliminary laboratory testing of 49 subjects, 37 with parasitological proven infection and 12 negative controls, displayed 100% sensitivity in ELISA using serum and oral transudate and 94.6% using saliva. The specificity of the ELISA with serum was 100% versus 91.7% with both oral fluids. Significant Spearman rank correlations of anti-SEA IgG levels with egg counts were observed for serum, oral transudate and saliva (P < 0.05). The sensitivity of dot-ELISA was 100% for serum, 89% for transudate and 81% for saliva, and specificity was 100% for all 3 samples. The immunodiagnostic value of ELISA for the detection of anti-SEA IgG antibodies in oral transudate was further evaluated in 197 individuals from an endemic area of Brazil. The ELISA using serum and oral transudate showed sensitivities of 98.8% and 100% respectively and specificities of 67.8% and 64.3% respectively. Use of oral fluids for the diagnosis of S. mansoni infection was equivalent to sera with respect to test efficacy, offering an alternative to blood collection.

Seroconversion for Helicobacter pylori in adults from Brazil.

We studied, prospectively, seroconversion for Helicobacter pylori in adults from a developing country and investigated risk factors for the acquisition of the microorganism in this population. A group of 213 volunteers of low socioeconomic level from a district in the metropolitan area of Belo Horizonte, south-east Brazil was evaluated. Anti-H. pylori IgG antibodies were measured by ELISA using Cobas Core anti-H. pylori EIA (Roche) in serum samples collected in 1992 and in 1997. The subjects were interviewed and sociodemographic data were collected. A total of 174 (81.7%) subjects presented anti-H. pylori antibodies on the occasion of the first visit. During 56 months of follow-up, 2 of 39 seronegative adults converted to seropositive with an annual infection rate of 1.1%, and 2 of 174 seropositive subjects reverted to seronegative (0.2%/year). The prevalence of infection increased significantly with age and an inverse association was observed between prevalence of infection and educational level. In conclusion, the results of the present study demonstrate that in a developing country there is a low but continuous risk of H. pylori infection in adulthood.

Serodiagnosis of Trypanosoma cruzi infection using the new particle gel immunoassay--ID-PaGIA Chagas.

The ID-Chagas test is a particle gel immunoassay (PaGIA). Red coloured particles are sensitised with three different synthetic peptides representing antigen sequences of Trypanosoma cruzi: Ag2, TcD and TcE. When these particles are mixed with serum containing specific antibodies, they agglutinate. The reaction mixture is centrifuged through a gel filtration matrix allowing free agglutinated particles to remain trapped on the top or distributed within the gel. The result can be read visually. In order to investigate the ability of the ID-PaGIA to discriminate negative and positive sera, 111 negative and 119 positive, collected in four different Brazilian institutions, were tested by each of the participants. All sera were previously classified as positive or negative according to results obtained with three conventional tests (indirect immunofluorescence, indirect hemaglutination, and enzyme linked immunosorbent assay). Sensitivity rates of ID-PaGIA varied from 95.7% to 97.4% with mean sensitivity of 96.8% and specificity rates varied from 93.8 to 98.8% with mean specificity of 94.6%. The overall Kappa test was 0.94. The assay presents as advantages the simplicity of operation and the reaction time of 20 min. In this study, ID-PaGIA showed to be highly sensitive and specific.

Circulating antigens levels in different clinical forms of the Schistosoma mansoni infection.

With the aim to evaluate the circulating cathodic antigen (CCA) levels in relation to the different clinical phases of Schistosoma sp. infection a sandwich ELISA using monoclonal antibody 5H11 was performed. The sera of three groups of 25 Brazilian patients with acute, intestinal and hepatosplenic forms of S. mansoni infection were tested and compared to a non-infected control group. Patients and control groups were matched for age and sex and the number of eggs per gram of feces was equally distributed among the three patient groups. Sensitivity of 100%, 72%, 52% of the assay was observed for the intestinal, hepatosplenic and acute toxemic groups respectively. The specificity was 100%. Intestinal and hepatosplenic groups presented CCA levels significantly higher in comparison to those observed for acute patients (F-ratio = 2,524; p = 0.000 and F-ratio = 6,314; p = 0.015 respectively). There was no significant difference of CCA serum levels between hepatosplenic and intestinal groups (F-ratio = 1,026; p = 0.316).

Flow cytometric study of blood leucocytes in clinical forms of human schistosomiasis.

The aim of this study was to examine three distinct groups of schistosomiasis patients and to determine whether cell phenotype profiles could be correlated with the different clinical forms of the disease. The data obtained indicate that Schistosoma mansoni infected patients have a lower percentage of CD3+ T cells than do non-infected individuals. Interestingly, infected patients presented more than twice the mean percentage of circulating activated T cells (CD3+HLA-DR+) when compared to the control group. Examination of T lymphocyte subpopulations showed that patients with the severe hepatosplenic form (HS) of the disease had lower levels of both CD8High+ and CD8Low+ cells when compared to the other groups of patients. All infected individuals had a higher percentage of circulating B cells, with an increase in the CD5+ B cell population that was more evident in the HS group. The data presented here are evidence to support a relationship between the hepatosplenic form of the disease, a decrease on the CD8+ cell population and an elevation on CD5+ B cells.

Daily fluctuation of levels of circulating cathodic antigen in urine of children infected with Schistosoma mansoni in Brazil.

The fluctuation of circulating cathodic antigen (CCA) levels in urine was studied in 69 Brazilian school-children infected with Schistosoma mansoni and compared to egg counts. Faeces and urine samples were simultaneously collected at 7 times during a period of 2 weeks. CCA was determined by enzyme-linked immunosorbent assay and could be detected in 96% of the urine samples; the individual mean CCA level ranged from 609 to 350,700 pg/mL. 90% of the faecal samples contained S. mansoni eggs and the individual mean egg output ranged from 9 to 5510 eggs/g. The Spearman rank correlation coefficient between these individual means was 0.69. Kendall's coefficient of concordance (W) was 0.88 for CCA levels and 0.80 for egg counts.

Humoral immune responses in patients with acute Schistosoma mansoni infection who were followed up for two years after treatment.

Eighteen patients with acute Schistosoma mansoni infection were followed up for 2 years after treatment with praziquantel or oxamniquine. Cure rates, clinical features, abdominal ultrasonographic findings, and specific humoral responses were determined at 2-, 6-, and 24-month follow-ups. Fourteen patients (77.8%) were considered parasitologically cured. Levels of IgA antibody to soluble egg antigen (SEA) and IgM antibody to keyhole limpet hemocyanin (KLH) became negative or decreased to the cutoff level for chronic infection 2 months after treatment, while levels of IgG antibody to KLH declined between 12 and 24 months after treatment. Levels of IgM and IgG antibodies to saline worm adult protein as well as IgM and IgG antibodies to SEA remained positive during the follow-up period. Discrete lymph node enlargement and hepatomegaly were still present in six of the eight cured children 2 years after treatment, while complete regression was observed in adults. In our group of patients, in addition to presenting with more intense clinical manifestations, children were cured less often and had slower abatement of symptomatology after treatment than adults.