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.

Abdominal ultrasonography in acute clinical schistosomiasis mansoni.

Abdominal ultrasonography was performed on eight children and four adults with acute schistosomiasis mansoni, 12 chronically infected patients, and 12 noninfected individuals from the endemic area, who were paired by age and sex with the acute group. In all acute patients, lymphadenomegaly as well as liver and spleen enlargement were detected. Lymph nodes surrounding the portal vein and the hepatic artery in the hepatic hilus were visualized. In the children, the right lobe of the liver was statistically significantly larger in the acute group than in the noninfected group. The portal and splenic vein diameters were significantly larger in children with acute schistosomiasis than in the chronically infected and negative control groups. The left hepatic lobe and a longitudinal scan of the spleen in acute adult patients were statistically significantly larger than in the chronically infected and negative control groups. Ultrasonography is shown to be a useful tool for the differential diagnosis of acute schistosomiasis mansoni.

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.

The serological differentiation of acute and chronic Schistosoma japonicum infection by ELISA using keyhole limpet haemocyanin as antigen.

Antibodies (immunoglobulin (Ig) G and IgM) that recognize keyhole limpet haemocyanin (KLH), which shares a well defined carbohydrate epitope with the surface of larval schistosomes, were determined by enzyme-linked immunosorbent assay in patients with acute or chronic schistosomiasis japonica. Marked differences in IgG and IgM responses were evident between acute and chronically infected patients at a serum dilution of up to 1:2000. The acute sera had mean (+/- standard deviation) optical density values at 405 nm for IgG and IgM of 0.64 +/- 0.18 and 0.30 +/- 0.19 respectively; the chronic sera had mean readings for IgG and IgM of 0.14 +/- 0.08 and 0.04 +/- 0.03 respectively. Setting the lowest positive limit at 2 standard deviations above the mean value for chronic sera, 41 (98%) of the 42 patients previously diagnosed as having acute schistosomiasis were correctly identified by anti-KLH IgG and 35 (83%) of the 42 patients were correctly identified by anti-KLH IgM detection. Of 17 patients studied longitudinally, IgG optical density values dropped 45% and those of 5 patients fell below the established cut-off level 6 months after treatment. This study supports the use of KLH for rapidly and easily identifying individuals with acute Schistosoma japonicum infection.

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.

Dot-dye-immunoassay and dot-ELISA for the serological differentiation of acute and chronic schistosomiasis mansoni using keyhole limpet haemocyanin as antigen.

Two immunoassays, dot enzyme-linked immunosorbent assay (dot-ELISA) and dot-dye immunoassay (dot-DIA), using soluble egg antigen and keyhole limpet haemocyanin as antigens, were evaluated for the serological differentiation of 25 acute and 37 chronic patients infected with Schistosoma mansoni and 20 non-infected individuals, in comparison with ELISA. Efficiency was 92.7%, 90.0% for ELISA, dot-ELISA and dot-DIA, respectively. Dipstick dot-ELISA and dot-DIA are described and shown to be reliable cheap and simple methods for the serological differentiation of acute and chronic schistosomiasis.

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.

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.

Levels of the schistosome circulating anodic and cathodic antigens in serum of schistosomiasis patients from Brazil.

Serum levels of 2 schistosome circulating antigens, the circulating anodic antigen (CAA) and the circulating cathodic antigen (CAA), were determined in persons infected with Schistosoma mansoni in Brazil. Sensitive monoclonal antibody-based enzyme-linked immunosorbent assays were used to measure levels of the 2 antigens. The study group consisted of 38 individuals with intestinal schistosomiasis, and 20 persons with the hepatosplenic form of the disease. Age and intensity of infection were comparable for the 2 groups. CAA was detected in 65.5% of all patients' sera and CCA was found in the serum of 82.8% of all patients. CAA levels correlated well with the egg output, as determined by duplicate Kato-Katz smears; CCA was significantly positively correlated with egg output in patients with intestinal schistosomiasis only. Whereas no significant difference was found between CAA titre in patients with intestinal schistosomiasis and those with the hepatosplenic form, a significantly higher CCA titre was found in patients with hepatosplenomegaly compared to patients with intestinal schistosomiasis.

Evaluation of proteinuria in an area of Brazil endemic for schistosomiasis using a single urine sample.

A two-step protocol was designed to evaluate the frequency of proteinuria related to Schistosoma mansoni infection in an endemic area, as measured by the protein/creatinine ratio (P/C R). A pre-test on 32 in-patients with renal disease and 20 healthy individuals showed a high correlation (r = 0.948) between the classical measurement of protein excretion per square metre of body surface during 24 h and the P/C R. The P/C R was then used to evaluate the frequency of proteinuria in 189 individuals in an endemic area in the northeast of Minas Gerais state, Brazil, with a schistosomiasis prevalence of 49.3% and 3.4% with the hepatosplenic form. The low prevalence of proteinuria (1.06%) in the studied population can be attributed to the accuracy of the method used and to the low prevalence of the hepato-splenic form of schistosomiasis. The P/C R is a reliable and appropriate method for the measurement of proteinuria in field studies.

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.

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.

Analysis of anti-keyhole limpet haemocyanin antibody in Brazilians supports its use for the diagnosis of acute schistosomiasis mansoni.

Antibody (immunoglobulin (Ig) G) to the haemocyanin of the keyhole limpet (KLH) (Megathura crenulata), which shares a well defined carbohydrate epitope with the surface of schistosomula of Schistosoma mansoni, was determined by enzyme-linked immunosorbent assay (ELISA) in the sera of Brazilians with acute schistosomiasis. Of 53 such individuals tested, 51 had a level of KLH reactivity in excess of the mean +2 standard deviations of that exhibited by chronically infected individuals. This difference in reactivity allowed the acute cases to be readily identified by visual inspection of ELISA plates. The levels of IgG in patients with hepatointestinal and hepatosplenic schistosomiasis, as well as in non-infected, seropositive residents of endemic areas and infected children from endemic areas, were not statistically different from those of intestinal patients. Significant levels of anti-KLH IgG were not detected in patients with leishmaniasis, Chagas disease, ancylostomiasis or ascariasis. The results support the use of KLH as a means of rapidly and easily identifying individuals with acute schistosomiasis.

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.

Stool examination and rectal biopsy in the diagnosis and evaluation of therapy of schistosomiasis mansoni.

From each of a group of 217 adult males selected through enzyme-immunoassay or skin-test (Group A), six stool samples were examined by both the Lutz/Hoffman, Pons & Janer (Lutz/HPJ) and Kato/Katz methods. In addition, one oogram of the rectal mucosa was performed. By these methods, schistosomiasis was detected in 44.7%, 47.5% and 40.1% of the individuals respectively. To evaluate the methods in the assessment of cure, the last 40 patients from group A, treated with a single oral dose of oxamniquine at 15 mg/kg were followed up for six months (Group B). The criteria for parasitological cure included three stool examinations by Kato/Katz and Lutz/HPJ methods, one, three and six months post-treatment and a rectal biopsy between the fourth and sixth months post-treatment. The examinations were negative in 87.5%, 90% and 95% of the patients, respectively. The efficacy of oxamniquine was 82.5% when the three methods were considered together and there was no statistically significant difference between the sensitivity of the individual methods.

[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.

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.

Diagnosing schistosomiasis.

The ideal diagnostic method for schistosomiasis detection seems to be still far from available. Paucity of egg output in low prevalence situations, low levels of circulating antigens in individuals with low intensity of infection and inadequate specificity of antibody detection systems outline pieces of a puzzle that challenges scientific efforts. Estimated prevalence, financial resources and operational reality must be taken into account when deciding the diagnostic method to be used. A combination of a screening step, using a fast strip test for antibody detection with a parasitological ratification step such as Kato-Katz repeated stool examination may serve as a diagnostic approach for a previously untreated low level endemic area. However, when eradication is the aim, and high financial investment is available, re-treatment may be based on the association between multiple stool examination and circulating antigen detection. Ethical aspects as well as cost-benefit rates between treatment and diagnosis approaches lead to the conclusion that in spite of the recent advances in simple administered and relatively safe drugs, treatment should only be performed when supported by appropriated diagnosis.