Elenice Messias do Nascimento Gonçalves

Evaluation of real-time PCR assay to detect Schistosoma mansoni infections in a low endemic setting

BackgroundSchistosomiasis constitutes a major public health problem, and 200 million people are estimated to be infected with schistosomiasis worldwide. In Brazil, schistosomiasis has been reported in 19 states, showing areas of high and medium endemicity and a wide range of areas of low endemicity (ALE). Barra Mansa in Rio de Janeiro state has an estimated prevalence of 1%. ALE represent a new challenge for the helminth control because about 75% of infected individuals are asymptomatic and infections occur with a low parasite load (<100 eggs per gram of feces), causing a decrease in sensitivity of stool parasitological techniques, which are a reference for the laboratory diagnosis of this helminth. The objective of this study was to evaluate the performance of a TaqMan quantitative polymerase chain reaction (qPCR) technique in serum and feces DNA samples using the techniques of Kato-Katz (KK), Hoffman, Pons and Janer (HH) as references, during an epidemiological survey using fecal samples and sera from randomized residents from an ALE.MethodsA cross-sectional study conducted from April to December 2011 using a probabilistic sampling that collected 572 fecal and serum samples. The laboratory diagnostic techniques used were: KK, HH and qPCR (feces and serum).ResultsWe obtained the following results using the different diagnostic techniques: KK and HH, 0.9% (n =5); qPCR-feces, 9.6% (n =55); and qPCR-serum, 1.4% (n =8). The qPCR-feces presented the highest positivity, whereas the techniques of HH and KK were the least sensitive to detect infections (0.8%). Compared to HH and KK, qPCR-feces showed a statistically significant difference in positivity (p <0.05), although with poor agreement.ConclusionThe positivity rate presented by the qPCR approach was far higher than that obtained by parasitological techniques. The lack of adequate surveillance in ALE of schistosomiasis indicates a high possibility of these areas being actually of medium and high endemicity. This study presents a control perspective, pointing to the possibility of using combined laboratory tools in the diagnosis of schistosomiasis in ALE.

Protocol for DNA extraction of Cryptosporidium spp. oocysts in fecal samples

Molecular characterization of Cryptosporidium spp.oocysts in clinical samples is useful for public health since it allows the study of sources of contamination as well as the transmission in different geographical regions. Although widely used in developed countries, in Brazil it is restricted to academic studies, mostly using commercial kits for the extraction of genomic DNA, or in collaboration with external reference centers, rendering the method expensive and limited. The study proposes the application of the modifications recently introduced in the method improving feasibility with lower cost. This method was efficient for clinical samples preserved at -20 degrees C for up to six years and the low number of oocysts may be overcome by repetitions of extraction.

Retrospective study of the occurrence of Cyclospora cayetanensis at Clinical Hospital of the University of São Paulo Medical School, SP.

Cyclospora cayetanensis causes watery diarrhea in tropical countries, among travelers and after ingestion of contaminated water and food. Very little is known about its epidemiology, pathogenic aspects and reservoirs. In Brazil, its prevalence is unknown and to date there have been reports of three outbreaks. We report here a retrospective study of 5,015 stool samples from 4,869 patients attended at Clinical Hospital of the University of São Paulo Medical School, SP, Brazil between April 1996 and January 2002, with 14 cases of Cyclospora cayetanensis being detected there was a prevalence of 0.3%. Of the 14 infected patients, the mean age was 38 years and 71.4% were female. Ten patients presented symptoms; six presented levels of immunological markers and five patients were immunodeficient.

Comparative Study of the Accuracy of Different Techniques for the Laboratory Diagnosis of Schistosomiasis Mansoni in Areas of Low Endemicity in Barra Mansa City, Rio de Janeiro State, Brazil

Schistosomiasis constitutes a major public health problem, with an estimated 200 million people infected worldwide. Many areas of Brazil show low endemicity of schistosomiasis, and the current standard parasitological techniques are not sufficiently sensitive to detect the low-level helminth infections common in areas of low endemicity (ALEs). This study compared the Kato-Katz (KK); Hoffman, Pons, and Janer (HH); enzyme-linked immunosorbent assay- (ELISA-) IgG and ELISA-IgM; indirect immunofluorescence technique (IFT-IgM); and qPCR techniques for schistosomiasis detection in serum and fecal samples, using the circumoval precipitin test (COPT) as reference. An epidemiological survey was conducted in a randomized sample of residents from five neighborhoods of Barra Mansa, RJ, with 610 fecal and 612 serum samples. ELISA-IgM (21.4%) showed the highest positivity and HH and KK techniques were the least sensitive (0.8%). All techniques except qPCR-serum showed high accuracy (82–95.5%), differed significantly from COPT in positivity (P < 0.05), and showed poor agreement with COPT. Medium agreement was seen with ELISA-IgG (Kappa = 0.377) and IFA (Kappa = 0.347). Parasitological techniques showed much lower positivity rates than those by other techniques. We suggest the possibility of using a combination of laboratory tools for the diagnosis of schistosomiasis in ALEs.

Microsporidiosis in a Brazilian University Hospital: case report

This is the report on a patient with chronic diarrhea caused by microsporidia. He is married, infected with HIV and has low CD4 cell count. The diagnosis was established through stool parasite search using concentration methods and Gram-chromotrope staining technique. Ileum biopsy was also performed in this case. The etiological diagnosis may be established in a clinical laboratory, by chromotrope staining technique in routine microscopic examination of stool specimens.

DIAGNOSIS OF Strongyloides stercoralis INFECTION IN IMMUNOCOMPROMISED PATIENTS BY SEROLOGICAL AND MOLECULAR METHODS

SUMMARY Strongyloidiasis is a potentially serious infection in immunocompromised patients. Thus, the availability of sensitive and specific diagnostic methods is desirable, especially in the context of immunosuppressed patients in whom the diagnosis and treatment of strongyloidiasis is of utmost importance. In this study, serological and molecular tools were used to diagnose Strongyloides stercoralis infections in immunosuppressed patients. Serum and stool samples were obtained from 52 patients. Stool samples were first analyzed by Lutz, Rugai, and Agar plate culture methods, and then by a quantitative real time polymerase chain reaction (qPCR). Serum samples were evaluated by an enzyme-linked immunosorbent assay (ELISA) using a soluble (AS) or a membrane fractions antigen (AM) obtained from alkaline solutions of the filariform larvae of Strongyloides venezuelensis. Of the 52 immunosuppressed patients, three (5.8%) were positive for S. stercoralis by parasitological methods, compared to two patients (3.8%) and one patient (1.9%) who were detected by ELISA using the AS and the AM antigens, respectively. S. stercoralis DNA was amplified in seven (13.5%) stool samples by qPCR. These results suggest the utility of qPCR as an alternative diagnostic tool for the diagnosis of S. stercoralis infection in immunocompromised patients, considering the possible severity of this helminthiasis in this group of patients.

IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS: USE OF SIX DIFFERENT ANTIGENIC FRACTIONS FROM Strongyloides venezuelensis PARASITIC FEMALES.

SUMMARY The aim of this study was to evaluate six different antigenic fractions from Strongyloides venezuelensis parasitic females for the immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from S. venezuelensis parasitic females were prepared in phosphate-buffered saline (SSF and SMF, respectively), Tris-HCl (TSF and TMF, respectively), and an alkaline buffer (ASF and AMF, respectively). Serum samples obtained from patients with strongyloidiasis or, other parasitic diseases, and healthy individuals were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions SSF, TSF, and ASF showed 85.0%, 75.0%, and 80.0% sensitivity and 93.1%, 93.1%, and 87.5% specificity, respectively. Membrane fractions SMF, TMF, and AMF showed 80.0%, 75.0%, and 85.0% sensitivity, and 95.8%, 90.3%, and 91.7% specificity, respectively. In conclusion, the present results suggest that the fractions obtained from parasitic females, especially the SSF and SMF, could be used as alternative antigen sources in the serodiagnosis of human strongyloidiasis.

Incidence of enteroparasites with molecular characterization of Cryptosporidium spp. in different Brazilian communities

The study was developed with the purpose to detect and characterize Cryptosporidium spp. in patients of different Brazilian communities attended at the HC-FMUSP. Fecal samples from 2,410 individuals came from the Central Laboratory Division of the University of Sao Paulo Medical School Hospital (DLC-HC-FMUSP) searching for enteroparasites, in the period from 2000 to 2006. Most samples (96.82%) were from Sao Paulo State (DIR 1 to DIR 4) of which 58.18% were from Sao Paulo city (DIR 1). Their relationship with other enteroparasites, clinical data and geographical localization was also assessed. In the search for enteroparasites, all fecal samples were submitted to concentration methods with a 4.6% and 27.8% positive result for helminths and protozoans, respectively. Cryptosporidium spp. oocysts were detected, semi-quantified and measured in 233 fecal samples (9.7%), using light microscopy after staining by Kinyoun’s method. Most samples presented few oocysts. In the biologic isolates genomic DNA extraction was performed using 223 fecal samples stored at -20 C, incubated with lysing solution (Tris-HCl + EDTA + 10% SDS + β mercaptoethanol + PVP) and proteinase K followed by extraction with phenol-chloroform-isoamylic alcohol in a Phase Lock Gel Light tube. Amplification of target DNA was performed with double PCR, with 18 SSUrRNA gene as genetic marker. The double PCR amplification products (amplicons) were digested by TaqI and AseI restriction enzymes. Double PCR confirmed Cryptosporidium in 37.2% (83/223) of the analyzed fecal samples with characterization in 62.7% (52/83) after digestion of its products. Characteristic profiles of C. hominis (88.5%), C. parvum (3.8%), Cryptosporidium non-parvum nonhominis (34.9%) and mixed infection with C. hominis (27.2%) were observed. Those not characterized were considered to be Cryptosporidium spp. Cryptopsoridium hominis presented significant associations with all evaluated risk groups and diarrhea. A statistically significant correlation between size of the oocysts detected by microscopy and the Cryptosporidium hominis and Cryptosporidium non-hominis non-parvum genotypes was observed. It is concluded that different Cryptosporidium genotypes circulate in the same geographical region, infecting both immunocompetent and immunodepressed individuals. The higher frequency of Cryptosporidium hominis indicates the fecal-oral pathway as the most important in the transmission of this infection. Molecular genotyping methods are essential for epidemiological studies on this parasite.

Molecular diagnosis of Strongyloides stercoralis among transplant candidates

Strongyloidiasis can occur without any symptoms or as a potentially fatal hyperinfection or disseminated infection, principally in immunosuppressed patients. Our study aimed to evaluate the application of conventional polymerase chain reaction (cPCR) and real‐time PCR (qPCR). Polymerase chain reaction (PCR) and real‐time PCR (qPCR) targeting the 18S rRNA gene for detection of Strongyloides stercoralis infection among transplant candidates were applied in stool samples obtained from 150 transplant candidates, preliminarily analyzed by parasitological methods. S. stercoralis larvae were visualized in 15/150 (10.0%) transplant candidates by parasitological methods. DNA from S. stercoralis was amplified in 26/150 (17.3%) and 49/150 (32.7%) stool samples of transplant candidates, using cPCR and qPCR, respectively. The results suggest that molecular methods, especially qPCR, should be used as an additional tool for diagnostic of S. stercoralis infection among transplant candidates.

HEMORRAGIA DIGESTIVA OCASIONAL EM CRIANÇA POR ESTRONGILOIDÍASE: IMPORTÂNCIA DO EXAME PARASITOLÓGICO

ABSTRACT Objective: To describe an uncommon case of infection by Strongyloides stercoralis (S. stercoralis) in a 4-month-old child and to highlight the importance of early diagnosis. Case description: The patient was a male child from the city of Videira, State of Santa Catarina, Southern Brazil, who was born preterm by Cesarean-section, weighing 1,655 g, and stayed in the neonatal intensive care unit for 20 days. At four months of age, the child started presenting blood in stools and the possibility of cow’s milk protein allergy was considered, given the symptoms and the use of infant formula in his 1st semester of life, which was then replaced by infant formula with hydrolyzed protein. White blood cell count and a parasitological stool sample were requested. Both tested positive and the stool ova and parasite examination showed a rhabditoid larva of S. stercoralis. The clinician maintained the initial hypothesis and diet, but requested three new stool samples, which tested positive for rhabditoid larvae of S. stercoralis. Since the child presented abdominal pain and vomiting, and there was still blood in stools, treatment with thiabendazole was initiated twice a day for two days. Treatment was repeated after seven days along with a new parasitological examination, which was then negative. Comments: Although strongyloidiasis is usually a mild parasitic infection, it may be severe and disseminated in immunocompromised patients. This agent must be considered in patients who live in endemic areas, and the diagnosis should be established by searching S. stercoralis larvae in tracheal secretions and in stools.