A simple approach improving the performance of urine reagent strips for rapid diagnosis of urinary schistosomiasis in Nigerian schoolchildren

In Nigeria, schistosomiasis, caused predominantly by the species Schistosoma haematobium, is highly endemic in resource-poor communities. We performed a school-based survey in two rural communities in Osun State (Southwestern Nigeria) and assessed macrohaematuria, microhaematuria and proteinuria as indirect indicators for the presence of disease. Urine samples were inspected macroscopically for haematuria and screened for microhaematuria and proteinuria using urine reagent strips. The microscopic examination of schistosome eggs was used as the gold standard for diagnosis. In total, 447 schoolchildren were included in this study and had a 51% prevalence of urinary schistosomiasis. The sensitivity of microhaematuria (68%) and proteinuria (53%) for infection with S. haematobium was relatively low. In patients with a heavy infection (>500 eggs/10 mL), the sensitivity of microhaematuria was high (95%). When the presence of macrohaematuria and the concomitant presence of microhaematuria and proteinuria were combined, it revealed a sensitivity of 63%, a specificity of 93% and a positive predictive value of 91%. Macrohaematuria also showed high specificity (96%) and a positive predictive value of 92%, while sensitivity was < 50%. These data show that combining urine reagent strip tests (presence of proteinuria and microhaematuria) and information on macrohaematuria increased the accuracy of the rapid diagnosis of urinary schistosomiasis in an endemic rural West African setting. This simple approach can be used to increase the quality of monitoring of schistosomiasis in schoolchildren.

Comparison of tests for the detection of circulating filarial antigen (Og4C3-ELISA and AD12-ICT) and ultrasound in diagnosis of lymphatic filariasis in individuals with microfilariae

Significant advances were made in the diagnosis of filariasis in the 1990s with the emergence of three new alternative tools: ultrasound and tests to detect circulating antigen using two monoclonal antibodies, Og4C3 and AD12-ICT-card. This study aimed to identify which of these methods is the most sensitive for diagnosis of infection. A total of 256 individuals, all male and carrying microfilariae (1-15,679 MF/mL), diagnosed by nocturnal venous blood samples, were tested by all three techniques. The tests for circulating filarial antigen concurred 100% and correctly identified 246/256 (96.69%) of the positive individuals, while ultrasound detected only 186/256 (73.44%). Of the circulating antigen tests, ICT-card was the most convenient method for identification of Wuchereria bancrofti carriers. It was easy to perform, practical and quick.

Use of a saline gradient for the diagnosis of schistosomiasis

The development of novel methods for parasitological diagnosis that are both highly sensitive and low in cost has been strongly recommended by the World Health Organization. In this study, a new technique for diagnosis of schistosomiasis mansoni is proposed based on the differential sedimentation of eggs when subjected to a slow continuous flux of 3% saline solution through a porous plaque. This influx suspends low-density faecal material, effectively cleaning the sample. The remaining sediment covering the porous plaque surface is then transferred to a glass slide and examined under a bright field microscope. Twelve Kato-Katz slides were used for comparison in the present study. Our results suggest that the saline gradient method detects a signifi-cantly higher number of eggs than the 12 Kato-Katz slides (p < 0.0001). We also found microscopic inspection to be quicker and easier with our newly described method. After cleaning the sample, the obtained sediment can also be conserved in a 10% formaldehyde solution and examined for at least 45 days later without statistically significant egg count differences.

Serological diagnosis of Chagas disease: evaluation and characterisation of a low cost antigen with high sensitivity and specificity

In spite of evident progress in the serology of Chagas disease, the requirement for new diagnostic antigens persists. We have evaluated different antigens obtained from Trypanosoma cruzi grown in medium rich in nutrients or under nutrient stress, autoclaved or sonicated and fractionated by differential centrifugation. The resulting antigens were evaluated for diagnosis of Chagas disease using ELISA. Immunofluorescence of the parasites demonstrated that nutrient stress induced changes in the distribution and density of antigens recognised by a pool of sera from experimentally infected mice. When evaluated using ELISA, it was evident that most fractions had good sensitivity but poor specificity. Surprisingly, the best specificity and sensitivity was observed with parasites cultured under nutrient stress and autoclaved. Furthermore this antigen had low cross reactivity with sera from other parasitic diseases, Leishmaniasis in particular. Western blot analysis demonstrated that autoclaving seems to non-specifically eliminate cross-reactive antigens. In conclusion, autoclaving epimastigotes of T. cruzi, after nutrient stress, allowed us to obtain an antigen that could be used in the serological diagnosis of Chagas disease.

Genus-specific kinetoplast-DNA PCR and parasite culture for the diagnosis of localised cutaneous leishmaniasis: applications for clinical trials under field conditions in Brazil

The positivities of two methods for the diagnosis of localised cutaneous leishmaniasis (CL) were estimated in 280 patients enrolled in a clinical trial. The trial was conducted in an endemic area of Leishmania (Viannia) braziliensis and trial participants were patients with skin ulcers and positive leishmanin skin tests. Patients underwent aspirative skin punctures of the ulcerated lesions and lymph nodes for in vitro cultures, which were processed under field conditions at the local health centre. Skin lesion biopsies were tested at a reference laboratory using kinetoplastid DNA (kDNA)-PCR to detect DNA. The median time required to obtain a positive culture from the skin samples was seven days and the contamination rate of the samples was 1.8%. The positivities of the cultures from skin lesions, kDNA-PCR and the combination of the two methods were 78.2% (95% CI: 73-82.6%), 89.3% (95% CI: 85.1-92.4%) and 97.1% (95% CI: 94.5-98.5%). We conclude that parasite culture is a feasible method for the detection of Leishmania in field conditions and that the combination of culture and PCR has a potential role for the diagnosis of CL in candidates for clinical trials.

Diagnosis of Chagas disease: what has been achieved? What remains to be done with regard to diagnosis and follow up studies?

In the acute phase and in the chronic forms of Chagas disease, the etiological diagnosis may be performed by detection of the parasite using direct or indirect parasitological methods and by the presence of antibodies in the serum by way of serological tests. Several techniques are easily available, ranging from the simplest wet smear preparation to immuno-enzymatic assays with recombinant antigens that will meet most diagnostic needs. Other tests under evaluation include a molecular test using polymerase chain reaction, which has shown promising results and may be used as a confirmatory test both in the acute and chronic phases of the disease. Better rapid tests are needed for diagnosis, some of which are already under evaluation. Additionally, there is a need for tools that can identify patients cured shortly after specific treatment. Other needs include a marker for prognosis and early diagnosis of congenital transmission.

Therapy, diagnosis and prognosis of chronic Chagas disease: insight gained in Argentina

The purpose of this review is to describe research findings regarding chronic Chagas disease in Argentina that have changed the standards of care for patients with Trypanosoma cruzi infection. Indirect techniques (serological tests) are still the main tools for the primary diagnosis of infection in the chronic phase, but polymerase chain reaction has been shown to be promising. The prognosis of patients with heart failure or advanced stages of chagasic cardiomyopathy is poor, but a timely diagnosis during the initial stages of the disease would allow for prescription of appropriate therapies to offer a better quality of life. Treatment of T. cruzi infection is beneficial as secondary prevention to successfully cure the infection or to delay, reduce or prevent the progression to disease and as primary disease prevention by breaking the chain of transmission. Current recommendations have placed the bulk of the diagnostic and treatment responsibility on the Primary Health Care System. Overall, the current research priorities with respect to Chagas disease should be targeted towards (i) the production of new drugs that would provide a shorter treatment course with fewer side effects; (ii) the development of new tools to confirm cure after a full course of treatment during the chronic phase and (iii) biomarkers to identify patients with a high risk of developing diseases.

Tissue and serum immune response in chronic hepatitis C with mild histological lesions

The immunopathogenesis of chronic hepatitis C virus (HCV) infection is a matter of great controversy and has been suggested to involve a complex balance between cytokines with pro and anti-inflammatory activity. We investigated the expression of inflammatory cells and cytokines in the liver and serum of 51 chronically HCV infected patients and compared them to data from two sets of normal controls: 51 healthy blood donors and 33 liver biopsies of healthy liver donors. We also assessed the relationship between selected cytokines and cell populations in hepatic compartments and the disease stage. Compared with controls, hepatitis C patients had a greater expression of portal TNF-α, TGF-β and CD4+ and acinar IFN-γ, TNF-α, IL-1β and IL-4, as well as a higher serum concentration of IL-2, IL-10 and TGF-β. Significant positive correlations were found between portal CD4+ and TNF-α, portal CD8+ and TGF-β, portal CD45+RO and TNF-α, acinar CD45+RO and IFN-γ and acinar CD57+ and TGF-β. In conclusion, we have shown that (i) in this sample of predominantly mild disease, the immune response was associated with a pro-inflammatory response pattern, (ii) CD4+ T-lymphocytes played a major role in orchestrating the immune response and (iii) these events primarily took place in the portal space.

Evaluation of polymerase chain reaction in the routine diagnosis for tegumentary leishmaniasis in a referral centre

The present study investigated the diagnostic value of polymerase chain reaction (PCR) performed in parallel to conventional methods at an American tegumentary leishmaniasis (ATL) referral centre for diagnosis. Accuracy parameters for PCR were calculated using 130 patients with confirmed ATL (ATL group), 15 patients established with other diseases and 23 patients with a lesion suggestive of ATL, but without parasitological confirmation (NDEF group). PCR showed 92.3% sensitivity, 93.3% specificity, a 99.2% positive predictive value and a 13.84 positive likelihood ratio. In the NDEF group, PCR confirmed ATL in 13 of the 23 patients, seven of whom responded to leishmaniasis treatment and six who presented spontaneous healing of the lesion. PCR should be included in the routine diagnostic procedures for ATL, especially for cases found to be negative by conventional methods.

Polymerase chain reaction of peripheral blood as a tool for the diagnosis of visceral leishmaniasis in children

The diagnosis of visceral leishmaniasis (VL) generally requires the use of invasive tests for the collection of infected tissue (aspirates of bone marrow, spleen, liver or lymph nodes). This difficulty has led to the search for safer and less painful techniques to confirm the occurrence of the disease in children. Polymerase chain reaction (PCR) is a method that is advantageous in that it allows the use of peripheral blood samples for diagnosis. This paper reports the utilisation of PCR on peripheral blood samples to diagnose VL in 45 children in Mato Grosso do Sul, Brazil. This technique is compared with methods carried out using tissue collected by invasive procedures, including direct microscopy, culture and detection of Leishmania DNA by PCR in bone marrow aspirates. The results show that PCR of peripheral blood provides great sensitivity (95.6%) that is similar to that from the PCR of bone marrow aspirates (91.1%) and higher than that achieved with microscopy (80%) or culture (26.7%) methods. PCR of peripheral blood proved to be a suitable tool for the diagnosis of VL in children because it is highly sensitive and safe, with tissue collection being less invasive than in traditional tests.

IgG and IgM western blot assay for diagnosis of congenital toxoplasmosis

The aim of this study was to evaluate the utility of western blot (WB) analysis as a diagnostic tool for congenital toxoplasmosis in 215 newborn infants. The children were submitted to clinical examinations to assess macular, neurological and hearing signals. The WB results obtained were compared to the persistence of IgG antibodies at the end of 12 months, which is regarded as the "gold standard" diagnosis of congenital toxoplasmosis. Association between the WB results and the clinical signs presented by the infants was also assessed. Of the 215 children, 177 had a confirmed congenital toxoplasmosis diagnosis and 38 were uninfected. IgG-WB showed a sensitivity of 73.5% and a specificity of 97.4%. IgM-WB showed a sensitivity of 54.8% and a specificity of 94.7%. The IgG-WB and IgM-WB combination increased the sensitivity to 86.5%. The IgM-WB-positive children had a 1.4-fold greater risk of presenting active macular lesions than did those that were IgM-WB-negative. This study showed that the WB assay is a useful tool to confirm a diagnosis of congenital toxoplasmosis and that the IgM-WB-positive results can indicate active macular lesions in newborn infants.

Serological diagnosis of paracoccidioidomycosis in HIV-coinfected patients

Paracoccidioidomycosis should be differentiated from other opportunistic diseases in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients who live in Latin America. Laboratory investigation can begin with serological tests, which are rapid and efficient. In the present study, double immunodiffusion (DID), counterimmunoelectrophoresis (CIEP) and an enzyme linked immunosorbent assay (ELISA) tests were assessed for the detection of anti-Paracoccidioides brasiliensis antibodies in 40 patients coinfected with HIV. The results were compared to those obtained for 75 non-HIV-infected patients with endemic paracoccidioidomycosis. Anti-P. brasiliensis antibodies were detected in 65% (DID), 79% (CIEP) and 95% (ELISA) of the patients with HIV/AIDS, significantly lower rates than those detected in cases of endemic paracoccidioidomycosis, which were 89%, 99% and 100%, respectively. The reactive sera of HIV-infected patients also showed lower anti-P. brasiliensis antibody titres than those of non-HIV-infected patients. Despite the lower intensity of the specific humoral response, serological tests are useful for the diagnosis of opportunistic paracoccidioidomycosis in the HIV/AIDS population. We suggest optimization of the laboratory diagnosis by combining the ELISA test with CIEP or DID.

Use of heterologous antigens for the immunodiagnosis of abdominal angiostrongyliasis by an enzyme-linked immunosorbent assay

Angiostrongylus costaricensis has a broad geographic distribution spanning from North to South America and the infections of vertebrates with this nematode can result in abdominal complications. Human infections are diagnosed by histological or serological methods because the isolation of larvae from feces is not feasible, as most parasites become trapped in intestinal tissues due to intense eosinophilic inflammation. Because A. costaricensis is difficult to maintain in the laboratory, an immunodiagnostic IgG enzyme-linked immunosorbent assay (ELISA) using antigens from the congeneric Angiostrongylus cantonensis species was evaluated against a panel of serum samples from patients who were histologically diagnosed with A. costaricensis infections. Sera from uninfected individuals and individuals infected with other parasites were used as controls. The sensitivity and specificity of the assay were estimated at 88.4% and 78.7%, respectively. Because the use of purified or cloned antigens has not been established as a reliable diagnostic tool, the use of heterologous antigens may provide a viable alternative for the development of an ELISA-based immunodetection system for the diagnosis of abdominal angiostrongyliasis.

An indirect immunofluorescence antibody test employing whole eggs as the antigen for the diagnosis of abdominal angiostrongyliasis

Abdominal angiostrongyliasis is a potentially fatal zoonotic disease with a broad geographical distribution throughout Central and South America. This study assessed the performance of Angiostrongylus costaricensis eggs as the antigen in an indirect immunofluorescence assay for the determination of parasite-specific IgG and IgG1 antibodies. For prevalence studies, an IgG antibody titre > 16 was identified as the diagnostic threshold with the best performance, providing 93.7% sensitivity and 84.6% specificity. Cross reactivity was evaluated with 65 additional samples from patients with other known parasitic infections. Cross reactivity was observed only in samples from individuals infected with Strongyloides stercoralis. For clinical diagnosis, we recommend the determination of IgG only as a screening test. IgG1 determination may be used to increase the specificity of the results for patients with a positive screening test.

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.