Assessment of the rapid test based on an immunochromatography technique for detecting anti-Treponema pallidum antibodies

A rapid test based on an immunochromatography assay - Determine™ Syphilis TP (Abbott Lab.) for detecting specific antibodies to Treponema pallidum was evaluated against serum samples from patients with clinical, epidemiological and serological diagnosis of syphilis, patients with sexually transmitted disease other than syphilis, and individuals with negative serology for syphilis. The Determine™ test presented the sensitivity of 93.6%, specificity of 92.5%, and positive predictive value and negative predictive value of 95.2% and 93.7%, respectively. One serum sample from patient with recent latent syphilis showed a prozone reaction. Determine™ is a rapid assay, highly specific and easy to perform. This technique obviates the need of equipment and its diagnostic features demonstrate that it may be applicable as an alternative assay for syphilis screening under some emergency conditions or for patients living in remote localities.

Occurrence of Giardia cysts and Cryptosporidium oocysts in activated sludge samples in Campinas, SP, Brazil

Giardia and Cryptosporidium have caused several outbreaks of gastroenteritis in humans associated with drinking water. Contaminated sewage effluents are recognized as a potential source of waterborne protozoa. Due to the lack of studies about the occurrence of these parasites in sewage samples in Brazil, we compared the efficiency of two procedures for concentrating cysts and oocysts in activated sludge samples of one sewage treatment plant. For this, the samples were submitted to i) concentration by the ether clarification procedure (ECP) and to ii) purification by sucrose flotation method (SFM) and aliquots of the pellets were examined by immunofluorescence. Giardia cysts were present in all samples (100.0%; n = 8) when using ECP and kit 1 reagents, while kit 2 resulted in six positive samples (85.7%; n = 7). As for SFM, cysts were detected in 75.0% and 100.0% of these samples (for kit 1 and 2, respectively). Regarding Cryptosporidium, two samples (25.0%; kit 1 and 28.5% for kit 2) were detected positive by using ECP, while for SFM, only one sample (examined by kit 1) was positive (12.5%). The results of the control trial revealed Giardia and Cryptosporidium recovery efficiency rates for ECP of 54.5% and 9.6%, while SFM was 10.5% and 3.2%, respectively. Considering the high concentration detected, a previous evaluation of the activated sludge before its application in agriculture is recommended and with some improvement, ECP would be an appropriate simple technique for protozoa detection in sewage samples.

Optimization of random amplified polymorphic DNA techniques for use in genetic studies of Cuban triatominae

Random amplified polymorphic DNA (RAPD) technique is a simple and reliable method to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of assay. In this study, we analyzed the effect of changing the concentration of primer, magnesium chloride, template DNA and Taq DNA polymerase with the objective of determining their optimum concentration for the standardization of RAPD technique for genetic studies of Cuban Triatominae. Reproducible amplification patterns were obtained using 5 pmoL of primer, 2.5 mM of MgCl2, 25 ng of template DNA and 2 U of Taq DNA polymerase in 25 µL of the reaction. A panel of five random primers was used to evaluate the genetic variability of T. flavida. Three of these (OPA-1, OPA-2 and OPA-4) generated reproducible and distinguishable fingerprinting patterns of Triatominae. Numerical analysis of 52 RAPD amplified bands generated for all five primers was carried out with unweighted pair group method analysis (UPGMA). Jaccard's Similarity Coefficient data were used to construct a dendrogram. Two groups could be distinguished by RAPD data and these groups coincided with geographic origin, i.e. the populations captured in areas from east and west of Guanahacabibes, Pinar del Río. T. flavida present low interpopulation variability that could result in greater susceptibility to pesticides in control programs. The RAPD protocol and the selected primers are useful for molecular characterization of Cuban Triatominae.

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.

Histoplasma capsulatum fungemia in patients with acquired immunodeficiency syndrome: detection by lysis-centrifugation blood-culturing technique

Progressive disseminated histoplasmosis (PDH) is an increasingly common cause of infection in patients with acquired immune deficiency syndrome (AIDS). We report 21 cases of PDH associated with AIDS diagnosed by lysis-centrifugation blood culture method. The most prevalent clinical findings were fever, weight loss, respiratory symptoms, and mucocutaneous lesions. Chest roentgenogram showed diffuse pulmonary infiltrates in 13 of 21 patients (62%). Brochoalveolar fluid has yelded positive culture in four patients only in medium with cycloheximide.

In vitro susceptibility testing of dermatophytes isolated in Goiania, Brazil, against five antifungal agents by broth microdilution method

The antifungal activities of fluconazole, itraconazole, ketoconazole, terbinafine and griseofulvin were tested by broth microdilution technique, against 60 dermatophytes isolated from nail or skin specimens from Goiania city patients, Brazil. In this study, the microtiter plates were incubated at 28 ºC allowing a reading of the minimal inhibitory concentration (MIC) after four days of incubation for Trichophyton mentagrophytes and five days for T. rubrum and Microsporum canis. Most of the dermatophytes had uniform patterns of susceptibility to the antifungal agents tested. Low MIC values as 0.03 µg/mL were found for 33.3%, 31.6% and 15% of isolates for itraconazole, ketoconazole and terbinafine, respectively.

Immunoperoxidase technique using an anti-Leishmania (L.) chagasi hyperimmune serum in the diagnosis of culture-confirmed American tegumentary leishmaniasis

The present study reports the production of the rabbit anti-Leishmania (L.) chagasi hyperimmune serum, the standardization of the immunohistochemistry (IHC) technique and the evaluation of its employment in cutaneous leishmaniasis (CL) lesions diagnosed by Leishmania sp. culture isolation. Thirty fragments of active CL lesions were examined as well as 10 fragments of cutaneous mycosis lesions as control group. IHC proved more sensitive in detecting amastigotes than conventional hematoxylin-eosin (HE) stained slides: the former was positive in 24 (80%) biopsies whereas the latter, in 16 (53%) (p = 0.028). The reaction stained different fungus species causing cutaneous mycosis. Besides, positive reaction was noticed in mononuclear and endothelial cells. Nevertheless, this finding was present in the control group biopsies. It is concluded that IHC showed good sensitivity in detecting amastigotes.

Influence of variables on centrifuge-flotation technique for recovery of Toxocara canis eggs from soil

The purpose of this study was to evaluate the influence of variables in a flotation technique for the recovery of Toxocara canis eggs from soil. The trials were done under standardized conditions on one gram of previously sterilized soil samples contaminated with 200 eggs of T. canis. The following variables were evaluated in serial steps: sieving; type of wash; time of stirring; resuspension of sediment; solution flotation. Centrifuge-flotation in sodium nitrate (d = 1.20 g/cm³) was adopted as an initial technique, using Tween 80 (0.2%) and decinormal sodium hydroxide as solutions for washing the samples. Ten tests were done to compare the variables, using counting in triplicate. The sieving of the material reduced significantly the recovery of eggs (p < 0.001) and the number of eggs recovered was higher when the sediment was resuspended (p < 0.05). After standardization, flotation solutions sodium chloride, zinc sulfate, sodium dichromate, magnesium sulfate, and sodium nitrate (d = 1.20g/cm³) were compared. The best results were obtained by using zinc sulfate solution. In conclusion, the chances of recovering T. canis eggs from samples using flotation solutions can be increased by washing of soil twice using distilled water, and resuspension of sediment. On the other hand, the sieving procedure can drastically reduce the number of eggs.

Genetic variability of Triatoma flavida and Triatoma bruneri (Hemiptera: Reduviidae) by RAPD-PCR technique

The Triatominae (Hemiptera:Reduviidae) contains the principal and potential Chagas disease vectors present in Mexico, Central America and South America. Triatoma flavida and T. bruneri are Cuban species. These species are closely related according to morphology and were considered synonyms until 1981, when they were separated on the grounds of external characters of the body and the morphology of male genitalia. The present study seeks to analyze genetic polymorphism of T. flavida and T. bruneri populations using RAPD techniques, and to assess the genetic relationship between these species. Ten random primers were used to evaluate the genetic variability among species using RAPD-PCR. The genetic flow among them was calculated. The dendrogram based on calculated Jaccard distances showed two clearly distinguishable clusters which coincided with the studied species. Within each species, moderate genetic differentiation (Fst 0.05-0.15) and migration rates (N > 1) were found among populations, that reveal gene flow and genetic homogeneity. Between species, the Fst value showed a high genetic differentiation and the migration rate was insufficient to maintain genetic homogeneity, and confirmed the absence of gene flow between them. Our results confirm the genetic variability among T. flavida and T. bruneri species.

Fungal agents in different anatomical sites in Public Health Services in Cuiabá, state of Mato Grosso, Brazil

INTRODUCTION: A contribution to the regional epidemiological profile of the most common fungal agents in Public Health Services in Cuiabá, state of Mato Grosso, including university hospitals and polyclinics. METHODS: Clinical specimens (n = 1,496) from 1,078 patients were collected, submitted to direct mycological exam (potash or stick tape method) and cultured in specific mediums. Dermatophytic and non-dermatophytic agents were identified according to micromorphology (Ridell technique). RESULTS: The majority of the 1,496 specimens were skin (n = 985) and nail exams (n = 472). Of the 800 positive cultures, 246 (30.8%) corresponded to dermatophytes and 336 (42%) to yeasts of the genus Candida, 190 (23.7%) to other yeasts, 27 (3.4%) to non-dermatophytic filamentous fungi and one (0.1%) the agent of subcutaneous mycosis. Lesions considered primary occurred in greater numbers (59.5%) than recurrent lesions (37.4%), with a greater concentration of positivity occurring on the arms and legs. CONCLUSIONS: Comorbidities, allergies and diabetes mellitus were conditions associated with greater positivity in direct mycological exams and cultures. Positive culture was considered a definitive diagnosis of fungal infection and confirmed 47.8% of diagnostic hypotheses.

PREVALENCE OF INTESTINAL PARASITISM AND ASSOCIATED SYMPTOMATOLOGY AMONG HEMODIALYSIS PATIENTS

Intestinal parasites are an important cause of morbidity and mortality. Immunocompromised individuals may develop more severe forms of these infections. Taking into account the immunity impairment in patients suffering from chronic renal failure (CRF), we will determine the prevalence and associated symptoms of intestinal parasites in these patients. Controls without CRF were used for comparison. Stool samples were collected and processed for microscopic identification of parasites using the Formalin-ether concentration method. For Cryptosporidium diagnosis, the ELISA technique was used. One hundred and ten fecal samples from hemodialysis patients were analyzed, as well as 86 from a community group used as control group. A result of 51.6% of intestinal parasites was observed in hemodialysis patients and 61.6% in the control group. Cryptosporidium and Blastocystis were the most common infections in patients with CRF (26.4% and 24.5%, respectively). Blastocystis was the most common infection in the control group (41.9%), however no individual was found positive for Cryptosporidium. Among the CRF patients, 73.6% were symptomatic, 54.3% of these tested positive for at least one parasite, in contrast to 44.8% in asymptomatic patients (p = 0.38). The most common symptoms in this group were flatulence (36.4%), asthenia (30.0%) and weight loss (30.0%). In the control group, 91.9% were symptomatic, 60.8% of these tested positive for at least one parasite, in contrast to 71.4% in asymptomatic patients (p = 0.703). A significant difference between the two groups was observed with regard to symptoms, with bloating, postprandial fullness, and abdominal pain being more frequent in the control group than in the hemodialysis group (all p < 0.05). Comparing symptomatic with asymptomatic, there was no association in either group between symptoms or the prevalence of parasitic infection, nor with the type of parasite or with multiple parasitic infections. Patients with chronic renal failure are frequent targets for renal transplantation, which as well as the inherent immunological impairment of the disease itself, results in immunosuppression by medication. For this reason, carriers of intestinal parasites with pathogenic potential can develop serious clinical complications influencing the success of transplantation. This fact, coupled with the high prevalence of intestinal parasites and the dissociation between symptoms and infection in CRF patients, suggests that the stool test should be incorporated in routine propedeutics. Furthermore, preventive measures for the acquisition of parasites through the fecal-oral contamination route should be introduced.

FREQUENCY, URINALYSIS AND SUSCEPTIBILITY PROFILE OF PATHOGENS CAUSING URINARY TRACT INFECTIONS IN ENUGU STATE, SOUTHEAST NIGERIA

Objective: This study was designed to determine the frequency and causative agent(s) of urinary tract infections (UTIs) in individuals with symptoms of urinary tract infections in Enugu State of Southeast Nigeria, and to determine the antibiotic susceptibility pattern of microbial agents isolated from urine culture.Methods: The study involved 211 individuals (149 females and 62 males) clinically suspected for UTI. Urine samples were collected by the mid-stream ‘clean catch’ method and tested using standard procedures. Antibiotic susceptibility of the isolated pathogens was tested using the Kirby-Bauer technique according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.Results: Microscopy of centrifuged urine samples showed 16 patients had pyuria while 54 had pus cells. Calcium oxalate crystals were found in 14 samples. Urinalysis performed with urine samples showed 17 had protein; seven were nitrite positive and three had moderate to high glucose concentration. Fifty-four urine samples (36.2%) from females and 12 (19.4%) from males showed significant growth upon culture. Gram stain and biochemical tests identified nine different organisms with Escherichia coli as the most common isolated species. Forty three randomly selected strains were further tested for their susceptibility against a panel of antibiotics. Thirty isolates (81.08%) were resistant to four or more antibiotics with the highest resistance shown by E. coli (76.67%). All the Gram- negative isolates were resistant to Ampicilox, Cefuroxime and Amoxicillin.Conclusion: Urinary tract infections were found more in females in the area under study. As found in other studies, E. coli was the most predominant isolate, although other organisms seem to be on the increase.

THE EFFECT OF HOOKWORM INFECTION AND URINARY SCHISTOSOMIASIS ON BLOOD HEMOGLOBIN CONCENTRATION OF SCHOOLCHILDREN LIVING IN NORTHERN MOZAMBIQUE

This study aims to assess the association between schistosomiasis and hookworm infection with hemoglobin levels of schoolchildren in northern Mozambique. Through a cross-sectional survey, 1,015 children from five to 12 years old in the provinces of Nampula, Cabo Delgado and Niassa were studied. Hookworm infection and urinary schistosomiasis were diagnosed, through Ritchie and filtration methods, with a prevalence of 31.3% and 59.1%, respectively. Hemoglobin levels were obtained with a portable photometer (Hemocue®). The average hemoglobin concentration was 10.8 ± 1.42 g/dL, and 62.1% of the children presented levels below 11.5 g/dL, of which 11.8% of the total number of children had hemoglobin levels below 9 g/dL. A multiple linear regression analysis demonstrated negative interactions between hemoglobin levels and ancylostomiasis, this being restricted to the province of Cabo Delgado (β = -0.55; p < 0.001) where an independent interaction between hemoglobin levels and urinary schistosomiasis was also observed (β = -0.35; p = 0.016). The logistical regression model indicated that hookworm infection represents a predictor of mild (OR = 1.87; 95% CI = 1.17-3.00) and moderate/severe anemia (OR = 2.71; 95% CI = 1.50 - 4.89). We concluded that, in the province of Cabo Delgado, hookworm and Schistosoma haematobium infections negatively influence hemoglobin levels in schoolchildren. Periodical deworming should be considered in the region. Health education and improvements in sanitary infrastructure could achieve long-term and sustainable reductions in soil-transmitted helminthiases and schistosomiasis prevalence rates.

EVALUATION OF METHODS IN DETECTING VANCOMYCIN MIC AMONG MRSA ISOLATES AND THE CHANGES IN ACCURACY RELATED TO DIFFERENT MIC VALUES

INTRODUCTION: Methicillin-Resistant Staphylococcus aureus (MRSA) presenting reduced susceptibility to vancomycin has been associated to therapeutic failure. Some methods used by clinical laboratories may not be sufficiently accurate to detect this phenotype, compromising results and the outcome of the patient. OBJECTIVES: To evaluate the performance of methods in the detection of vancomycin MIC values among clinical isolates of MRSA. MATERIAL AND METHODS: The Vancomycin Minimal Inhibitory Concentration was determined for 75 MRSA isolates from inpatients of Mãe de Deus Hospital, Porto Alegre, Brazil. The broth microdilution (BM) was used as the gold-standard technique, as well as the following methods: E-test® strips (BioMérieux), M.I.C.E® strips (Oxoid), PROBAC® commercial panel and the automated system MicroScan® (Siemens). Besides, the agar screening test was carried out with 3 µg/mL of vancomycin. RESULTS: All isolates presented MIC ≤ 2 µg/mL for BM. E-test® had higher concordance (40%) in terms of global agreement with the gold standard, and there was not statistical difference among E-test® and broth microdilution results. PROBAC® panels presented MICs, in general, lower than the gold-standard panels (58.66% major errors), while M.I.C.E.® MICs were higher (67.99% minor errors). CONCLUSIONS: For the population of MRSA in question, E-test® presented the best performance, although with a heterogeneous accuracy, depending on MIC values.