Quantitative toxoplasma gondii oocyst detection by a modified Kato Katz test using Kinyoun staining (KKK) in ME49 strain experimentally infected cats

We detected Toxoplasma gondii oocysts in feces of experimentally infected cats, using a Kato Katz approach with subsequent Kinyoun staining. Animals serologically negative to T. gondii were infected orally with 5x10² mice brain cysts of ME49 strain. Feces were collected daily from the 3rd to the 30th day after challenge. Oocysts were detected by qualitative sugar flotation and the quantitative modified Kato Katz stained by Kinyoun (KKK). In the experimentally infected cats, oocysts were detected from the 7th to 15th day through sugar flotation technique, but oocysts were found in KKK from the 6th to 16th day, being sensitive for a larger period, with permanent documentation. The peak of oocysts excretion occurred between the 8th to 11th days after challenge, before any serological positive result. KKK could be used in the screening and quantification of oocysts excretion in feces of suspected animals, with reduced handling of infective material, decreasing the possibility of environmental and operator contamination.

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples

Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

Excretion-secretion products and proteases from live Sporothrix schenckii yeast phase: immunological detection and cleavage of human IgG

Antigenic preparations from Sporothrix schenckii usually involve materials from mixed cultures of yeast and mycelia presenting cross-reactions with other deep mycoses. We have standardized pure yeast phase with high viability of the cells suitable to obtain specific excretion-secretion products without somatic contaminations. These excretion-secretion products were highly immunogenic and did not produce noticeable cross-reactions in either double immunodiffusion or Western blot. The antigenic preparation consists mainly of proteins with molecular weights between 40 and 70 kDa, some of them with proteolytic activity in mild acidic conditions. We also observed cathepsin-like activity at two days of culture and chymotrypsin-like activity at four days of culture consistent with the change in concentration of different secreted proteins. The proteases were able to cleave different subclasses of human IgG suggesting a sequential production of antigens and molecules that could interact and interfere with the immune response of the host.

Rapid HIV diagnostic test in undocumented pregnant women applied at an inner-city teaching hospital

A significant number of Brazilian gestational-age women are still not tested for HIV, representing a high risk of transmission to their newborns. The current study sought to identify the number of pregnant women with no previous testing or undocumented for HIV referred to the Gynecology and Obstetrics Department of a Regional Teaching Hospital and included diagnosis of HIV infection determined by a rapid test and perinatal transmission in pregnancy. Medical records of all pregnant women admitted to hospital from January 2001 to December 2005 were reviewed. Pregnant women without HIV results were submitted to a rapid HIV test. Those who tested positive were further tested by ELISA and confirmed by indirect immunofluorescence assay (IIA) or Western blot (WB). The viral load from babies born to HIV-infected mothers was assessed by bDNA. Of the 16,424 pregnant women analyzed (6.6%), 1,089 were undocumented for HIV. Eleven women were positive in rapid testing and 10 were confirmed by ELISA, IIA or WB, with 0.9% seropositivity. Mother/infant pairs received zidovudine monotherapy prophylaxis and infant viral load was lower than 50 copies/mL. A higher number of pregnant women previously tested for HIV during antenatal care was verified, compared to that obtained nationwide.

Validation of a rapid stool antigen test for diagnosis of Helicobacter pylori infection

The aim of this study was to validate the rapid lateral flow Helicobacter pylori stool antigen test (One step H. pylori antigen test, ACON laboratories, San Diego, USA; Prime diagnostics, São Paulo), using 13C-Urea Breath Test as the gold standard for H. pylori infection diagnosis. A total of 98 consecutive patients, asymptomatic or dyspeptic, entered the study. Sixty-nine were women, with a mean age of 45.76 ± 14.59 years (14 to 79 years). In the H. pylori-positive group, the rapid stool antigen test detected H. pylori antigen in 44 of the 50 positive patients (sensitivity 88%; 95% CI: 75.7-95.5%), and six false-negative; and in the H. pylori-negative group 42 presented negative results (specificity 87.5%; 95% CI: 74.7-95.3%), and six false-positive, showing a substantial agreement (Kappa Index = 0.75; p < 0.0001; 95% CI: 0.6-0.9). Forty four of fifty patients that had positive stool antigen were H. pylori-positive, the PPV of the stool antigen test was 88% (95% CI: 75.7-95.5%), and 42 patients with negative stool antigen test were H. pylori-negative, the NPV of the stool antigen test was 87.5% (95% CI: 74.7-95.3%). We conclude that the lateral flow stool antigen test can be used as an alternative to breath test for H. pylori infection diagnosis especially in developing countries.

Pseudo-outbreak of Clostridium difficile associated diarrhea (CDAD) in a tertiary-care hospital

The objective of this study was to describe a pseudo-outbreak of C. difficile in a hospital, following a change in the method used to detect the toxin. In February 2002, there were two cases of CDAD and in March 7 occurred, coinciding with a change of the test (from detection of toxin A to toxin A/B). An outbreak was suspected. Active surveillance and education of staff were started. A CDAD case was defined as a patient with acute onset of diarrhea (³ three episodes of liquid stools) and a positive stool test. They were classified as hospital or community-acquired. Stool samples were also collected for C. difficile culture and isolates were typed using AP-PCR. From March 2002 through December 2003 there were 138 cases of CDAD: 70% were hospital-acquired and among the 30% with CDAD present on admission, most (81%) came directly from the community (50% had no history of hospitalization). Fifty-two percent of hospital-acquired CDAD and 94% of cases on admission had already used antibiotics. The incidence of CDAD in hospitalized patients during surveillance was 3.3 per 1000 patient-admissions. The incidence of CDAD present on admission was 6.1/1000 patients. Sixteen isolates were typed and presented 13 different profiles. In conclusion, the CDAD increase in our study occurred due to change in diagnostic methods and not due to an outbreak, as suspected initially. The incidence in hospitalized patients was much lower than in reported outbreaks. There were 13 molecular types suggesting that an outbreak did not occur. CDAD was largely community-acquired.

Botulism in Brazil, 2000-2008: epidemiology, clinical findings and laboratorial diagnosis

Botulism is a rare and potentially lethal illness caused by Clostridium botulinum neurotoxin. We describe the findings of a laboratorial investigation of 117 suspected cases of botulism reported to the surveillance system in Brazil from January 2000 to October 2008. Data on the number and type of samples analyzed, type of toxins identified, reporting of the number of botulism cases and transmission sources are discussed. A total of 193 clinical samples and 81 food samples were analyzed for detection and identification of the botulism neurotoxin. Among the clinical samples, 22 (11.4%) presented the toxin (nine type A, five type AB and eight with an unidentified type); in food samples, eight (9.9%) were positive for the toxin (five type A, one type AB and two with an unidentified type). Of the 38 cases of suspected botulism in Brazil, 27 were confirmed by a mouse bioassay. Laboratorial botulism diagnosis is an important procedure to elucidate cases, especially food-borne botulism, to confirm clinical diagnosis and to identify toxins in food, helping sanitary control measures.

Canine visceral leishmaniasis: study of methods for the detection of IgG in serum and eluate samples

The Brazilian Ministry of Health recommends the culling and euthanasia of dogs with a positive serological test for canine visceral leishmaniasis (CVL). In the Municipality of Rio de Janeiro, the technique used for the diagnosis of CVL is the indirect fluorescent antibody test (IFAT), using blood samples eluted on filter paper (eluate). A dog survey was conducted over a period of one year in the region of Carapiá, in order to evaluate the diagnosis of CVL in this region. All animals underwent clinical examination, and blood samples (serum and eluate) were collected for analysis by enzyme immunoassay (ELISA) and IFAT. A skin biopsy was obtained for parasitological examination (culture). A total of 305 animals were studied and Leishmania chagasi was isolated from nine animals. Sensitivity and specificity were 100% and 96.6% for ELISA, respectively, 100% and 65.5% for IFAT (cut-off at a 1:40 dilution), 100% and 83.4% for IFAT (cut-off at a 1:80 dilution), and 22.2% and 97.0% for eluate IFAT. In conclusion, ELISA was the best tool for the diagnosis of CVL among the serological techniques tested. The present results suggest the need for a better evaluation of filter paper IFAT as the only diagnostic method for CVL in the Municipality of Rio de Janeiro.

Epstein Barr virus detection in cervical samples of women living with human immunodeficiency virus

Epstein Barr Virus (EBV) is transmitted commonly by saliva, but it has been found in genital secretions, which suggests sexual transmission and led researchers to connect EBV and cervical neoplasia. People living with human immunodeficiency virus (HIV) are reported to be at high risk of acquiring genital infections and cervical lesions. To verify the presence of EBV in the genital tract and/or it could affect cervical changes, we analyzed cervical smears from 85 HIV seropositive women for EBV DNA determination. EBV was only detected in two (2.3%) samples. The present study provides neither evidence for EBV as sexually transmitted infection nor discards this possibility.

Identification of respiratory virus in infants with congenital heart disease by comparison of different methods

Respiratory virus infections are the main cause of infant hospitalization and are potentially severe in children with congenital heart disease (CHD). Rapid and sensitive diagnosis is very important to early introduction of antiviral treatment and implementation of precautions to control transmission, reducing the risk of nosocomial infections. In the present study we compare different techniques in the diagnosis of respiratory viruses in CHD infants. Thirty-nine samples of nasopharyngeal aspirate were obtained from CHD infants with symptoms of respiratory infection. The Multiplex PCR (Seeplex® RV 12 ACE Detection) driven to the detection of 12 respiratory viruses was compared with the direct immunofluorescence assay (DFA) and PCR, both targeting seven respiratory viruses. The positivity found by DFA, Multiplex and PCR was 33.3%, 51.3% and 48.7%, respectively. Kappa index comparing DFA and Multiplex, DFA and PCR and PCR and Multiplex PCR was 0.542, 0.483 and 0.539, respectively. The concordance between techniques was considered moderate. Both Multiplex PCR (p = 0.001) and PCR (p = 0.002) detected significantly more respiratory virus than DFA. As the performance of the tests may vary, the combination of two or more techniques may increase diagnostic sensitivity favoring the diagnosis of co-infections, early introduction of antiviral therapy and implementation of appropriate measures.

Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

Evaluation of dengue NS1 antigen detection for diagnosis in public health laboratories, São Paulo State, 2009

The present work evaluated the diagnostic accuracy of detection of Dengue NS1 antigen employing two NS1 assays, an immunochromatographic assay and ELISA, in the diagnostic routine of Public Health laboratories. The results obtained with NS1 assay were compared with virus isolation and, in a subpopulation of cases, they were compared with the IgM-ELISA results obtained with convalescent samples. A total of 2,321 sera samples were analyzed by one of two NS1 techniques from March to October 2009. The samples were divided into five groups: groups I, II and III included samples tested by NS1 and virus isolation, and groups IV and V included patients with a first sample tested by NS1 and a second sample tested by IgM-ELISA. Sensitivity, specificity, positive and negative predictive values, Kappa Index and Kappa Concordance were calculated. The results showed that NS1 testing in groups I, II and III had high sensitivity (98.0%, 99.5% and 99.3%), and predictive values and Kappa index between 0.9 - 1.0. Groups IV and V only had Kappa Concordance calculated, since the samples were analyzed according to the presence of NS1 antigen or IgM antibody. Concordance of 92.1% was observed when comparing the results of NS1-negative samples with IgM-ELISA. Based on the findings, it is possible to suggest that the tests for NS1 detection may be important tools for monitoring the introduction and spread of Dengue serotypes.

Temporal differences in blood meal detection from the midguts of Triatoma infestans

We used genus/species specific PCRs to determine the temporal persistence of host DNA in Triatoma infestans experimentally fed on blood from six common vertebrate species: humans, domestic dogs, guinea pigs, chickens, mice, and pigs. Twenty third or fourth instar nymphs per animal group were allowed to feed to engorgement, followed by fasting-maintenance in the insectary. At 7, 14, 21, or 28 days post-feeding, the midgut contents from five triatomines per group were tested with the respective PCR assay. DNA from all vertebrate species was detected in at least four of five study nymphs at seven and 14 days post-feeding. DNA of humans, domestic dogs, guinea pigs, pigs, and chickens were more successfully detected (80-100%) through day 21, and less successfully (20-100%) at day 28. Findings demonstrate that species-specific PCRs can consistently identify feeding sources of T. infestans within two weeks, a biologically relevant time interval.