Use of laboratory methodologies to confirm an outbreak of acute diarrheal disease caused by multiple pathogens / Uso de metodologias laboratoriais para confirmação de surto de doença diarreica aguda causada por múltiplos patógenos

The aim of this study was to investigate an outbreak caused by protozoa, which occurred in a municipality in the Brazil southern region. The investigations were carried out analyzing 47 fresh stool samples and 26 water samples by parasitological and molecular methods, as well as, direct immunofluorescence. After the filtrations of water samples and purification of stool samples, the concentrates were evaluated microscopically for presence of parasites. Molecular analyses were performed by polymerase chain reaction (PCR) for DNA detection of Giardia spp., Cryptosporidium parvum, C. hominis and Cyclospora cayetanensis. Out of 26 water samples, 30.8% (8/26) had waterborne protozoa and C. cayetanensis was the most prevalent (15.5%). Out of the 47 stool samples, 23.4% (11/47) were infected with C. cayetanensis and Giardia spp. The results showed that backwash water samples from filters of the Water Treatment Station were contaminated with C. cayetanensis, C. hominis and Giardia spp., suggesting the contamination of water sources with human waste brought by sewage. These results show the importance of protozoa investigation in water and stool samples by laboratory methodologies principally in outbreaks causing acute diarrheal disease (AU).

O objetivo do presente estudo foi investigar um surto causado por protozoários, ocorrido em um município da região sul do Brasil. As investigações foram realizadas analisando 47 amostras de fezes frescas e 26 amostras de água por métodos parasitológicos, moleculares e de imunofluorscência direta. Após as filtrações das amostras de água e purificação das amostras de fezes, os concentrados foram avaliados microscopicamente a procura de parasitas. A seguir, foram analisadas, pela reação em cadeia da polimerase (PCR), a detecção de DNA de Giardia spp., Cryptosporidium parvum, C. hominis e Cyclospora cayetanensis. Das 26 amostras de água, 30,8% (8/26) apresentaram protozoários de veiculação hídrica, sendo que, C. cayetanensis foi o mais prevalente (15,5%). Das 47 amostras de fezes, 23,4% (11/47) estavam infectadas por C. cayetanensis e Giardia spp. Os resultados mostraram que as águas de retrolavagem dos filtros da Estação de Tratamento de Água estavam contaminadas com C. cayetanensis, C. hominis e Giardia spp. sugerindo a contaminação dos mananciais com dejetos humanos trazidos pelo esgoto. Estes resultados mostram a importância da investigação de protozoários em água e fezes por metodologias laboratoriais, principalmente em surtos que causam doença diarreica aguda (AU).

Gene expression profile of cytokines produced in biopsies from patients with American cutaneous leishmaniasis

American cutaneous leishmaniasis (ACL) causes a local inflammatory process, inducing expression of several cytokine genes. Particularly, IFN-γ can predict to disease susceptibility. Based in these data, this study was aimed to investigate the gene expression profile of IFN-γ, IL-10, IL-27, TNF-γ, TGF-ß and IL-6 produced in biopsies from ACL patients; and whether the gene expression profile of IFN-γ could determine the disease evolution. Gene expression of 6 cytokines was investigated in 40 formalin-fixed paraffin embedded (FFPE) biopsies from patients with cutaneous leishmaniosis (CL); and 10 FFPE biopsies from patients with mucosal leishmaniasis (ML) (control). All 50 patients were infected with Leishmania (Viannia) braziliensis. Gene expression was determined by qPCR; and a normal control group was used for calculations (5 normal biopsies). Values were expressed as Relative Quantification (RQ). The 40 CL patients were classified into 2 groups. CLlowIFN-γ, 35 patients with RQ for IFN-γ below 100; and CLhighIFN-γ, 5 (12.5%) patients with RQ above 100. Significant increase of mRNA levels of IFN-γ, IL-10 and IL-27 was shown in CLhighIFN-γ group when compared with CLlowIFN-γ and ML groups. TNF-α levels in CLlowIFN-γ group were higher than CLhighIFN-γ and ML groups. TGF-ß and IL-6 were similar in 3 groups. Comparison of cytokine expression/group showed that CLlowIFN-γ group had an equilibrium between the cytokines analyzed. In ML group, IFN-γ was over-expressed; but in CLhighIFN-γ group, besides IFN-γ, IL-27 was also over-expressed. The immune response to Leishmania induces to identification of some markers, which can be determined by analysis by gene expression of cytokines produced in biopsies

Performance of cryptococcal antigen lateral flow assay in serum, cerebrospinal fluid, whole blood, and urine in HIV-infected patients with culture-proven cryptococcal meningitis admitted at a Brazilian referral center

Cryptococcal meningitis is the most common cause of opportunistic meningitis in HIV-infected patients in Brazil and causes unacceptable high mortality rates. In this study, HIV-infected patients with a first episode of culture-proven cryptococcal meningitis in cerebrospinal fluid (CSF) were prospectively included in order to evaluate sensitivity of cryptococcal antigen (CrAg) lateral flow assay (LFA) in serum, CSF, whole blood (fingerstick), and fresh urine. In addition, HIV-infected patients with other neurological confirmed diseases were included in order to evaluate the specificity of CrAg LFA in serum. Twenty patients with cryptococcal meningitis were included and in 19 of them, CrAg LFA in CSF, serum, and whole blood were positive (95% sensitivity). In 18 patients, India ink test was positive in CSF (90% sensitivity), and in 16 cases, CrAg LFA was positive in urine (80% sensitivity). Thirty-six HIV-infected patients with other neurological diseases had negative results of CrAg LFA in serum (100% specificity). In conclusion, CrAg LFA in serum, CSF, and whole blood showed high sensitivity and specificity. Whole blood CrAg LFA seems to be a good and reliable strategy to improve AIDS-related cryptococcal meningitis diagnosis in Brazil

Asymptomatic cryptococcal antigen prevalence detected by lateral flow assay in hospitalised HIV-infected patients in São Paulo, Brazil

OBJECTIVE: To determine the prevalence of asymptomatic cryptococcal antigen (CRAG) using lateral flow assay (LFA) in hospitalised HIV-infected patients with CD4 counts <200 cells/ll. METHODS: Hospitalised HIV-infected patients were prospectively recruited at Instituto de Infectologia Emilio Ribas, a tertiary referral hospital to HIV-infected patients serving the S~ao Paulo State, Brazil. All patients were >18 years old without prior cryptococcal meningitis, without clinical suspicion of cryptococcal meningitis, regardless of antiretroviral (ART) status, and with CD4 counts <200 cells/ll. Serum CRAG was tested by LFA in all patients, and whole blood CRAG was tested by LFA in positive cases. RESULTS: We enrolled 163 participants of whom 61% were men. The duration of HIV diagnosis was a median of 8 (range, 1­29) years. 26% were antiretroviral (ART)-na€ive, and 74% were ARTexperienced. The median CD4 cell count was 25 (range, 1­192) cells/ll. Five patients (3.1%; 95%CI, 1.0­7.0%) were asymptomatic CRAG-positive. Positive results cases were cross-verified by performing LFA in whole blood. CONCLUSIONS: 3.1% of HIV-infected inpatients with CD4 <200 cells/ll without symptomatic meningitis had cryptococcal antigenemia in São Paulo, suggesting that routine CRAG screening may be beneficial in similar settings in South America. Our study reveals another targeted population for CRAG screening: hospitalised HIV-infected patients with CD4 <200 cells/ll, regardless of ART status. Whole blood CRAG LFA screening seems to be a simple strategy to prevention of symptomatic meningitis

Genotyping of Toxoplasma gondii: DNA extraction from formalin-fixed paraffin-embedded autopsy tissues from AIDS patients who died by severe disseminated toxoplasmosis

This study investigated the genetic features of Toxoplasma gondii isolated directly in autopsies of HIV-infected patients who died with severe disseminated toxoplasmosis. This retrospective analysis was conducted in a cohort of 15 HIV-infected patients with clinical and laboratory data. They had previous cerebral toxoplasmosis at least 6 months before the disseminated toxoplasmosis episode. The hypothesis was that they were infected with highly virulent parasites due to the condition in which they died. T. gondii genotyping was done directly in DNA extracted from 30 autopsy brain and lung samples (2 per patient) and mutilocus PCR-RFLP genotyping was done using 12 molecular markers. The 30 clinical samples were genotyped successfully in 8 or more loci and six suggestive genotypes were identified. One of them was Toxo DB #11, previously identified in different domestic animals and virulent in experimental animals. The other five suggestive genotypes identified in 14 patients were not described. TgHuDis1 was the most frequent and was determined in 8 patients. TgHuDis3 and TgHuDis5 were identified in two patients each. TgHuDis2 and TgHuDis4 have been identified in one patient each. These suggestive genotypes could be considered as virulent, since they caused severe tissue damage and had similar characteristics as Toxo # DB 11

Genotyping of toxoplasma gondii: DNA extraction from formalin-fixed paraffin-embedded autopsy tissues from AIDS patients who died by severe disseminated toxoplasmosis

This study investigated the genetic features of T. gondii isolated directly in autopsies of HIV-infected patients who died with severe disseminated toxoplasmosis. This retrospective analysis was conducted in a cohort of 15 HIV-infected patients with clinical and laboratory data. They had previous cerebral toxoplasmosis at least 6 months before the disseminated toxoplasmosis episode. The hypothesis was that they were infected with highly virulent parasites due to the condition in which they died. T. gondii genotyping was done directly in DNA extracted from 30 autopsy brain and lung samples (2 per patient) and mutilocus PCR-RFLP genotyping was done using 12 molecular markers. The 30 clinical samples were genotyped successfully in 8 or more loci and six suggestive genotypes were identified. One of them was Toxo DB #11, previously identified in different domestic animals and virulent in experimental animals. The other five suggestive genotypes...(AU)

Importance of high IgG anti-Toxoplasma gondii titers and PCR detection of T. gondii DNA in peripheral blood samples for the diagnosis of AIDS-related cerebral toxoplasmosis: a case-control study

BACKGROUND: Cerebral toxoplasmosis (CT) continues to cause significant morbidity and mortality in human immunodeficiency virus (HIV)-infected patients in Brazil. In clinical practice, the initial diagnosis is usually presumptive and alternative diagnosis tools are necessary. Our objective was to evaluate whether the detection of high titers of IgG anti-Toxoplasma gondii and T. gondii DNA in blood samples are associated with the diagnosis of CT. METHODS: In this case-control study we included 192 patients with HIV-1 infection: 64 patients with presumptive CT (cases) and 128 patients with other diseases (controls). Blood samples to perform indirect immunofluorescense reaction (IFI) to detect anti-T. gondii IgG antibodies and polymerase chain reaction (PCR) were collected before or within the first three days of anti-Toxoplasma therapy. Two multivariate logistic regression models were performed: one including the variable qualitative serology and another including quantitative serology. RESULTS: In the first model, positive IgG anti-T. gondii (OR 4.7, 95 percent CI 1.2-18.3; p = 0.027) and a positive T. gondii PCR result (OR 132, 95 percent CI 35-505; p < 0.001) were associated with the diagnosis. In the second model, IgG anti-T. gondii titres > 1:1024 (OR 7.6, 95 percent CI 2.3-25.1; p = 0.001) and a positive T. gondii PCR result (OR 147, 95 percent CI 35-613; p < 0.001) were associated with the diagnosis. CONCLUSIONS: Quantitative serology and molecular diagnosis in peripheral blood samples were independently associated with the diagnosis of CT in HIV-infected patients. These diagnostic tools can contribute to a timely diagnosis of CT in settings where Toxoplasma infection is common in the general population.

Fase aguda da infecção por toxoplasma gondii: avaliação do parasitismo sanguíneo e resposta humoral em camundongos isogênicos AS/n / Toxoplasma gondii acute infection: estimation of humoral response andblood parasitism in mice AS/n inbred

Objetivos: analisar experimentalmente a evolução da resposta imune humoral nas fases aguda e crônica recente da infecção por Toxoplasma gondii e sua correlação com o parasitismo sanguíneo. Métodos: foram analisados, por 60 dias, 10 camundongos fêmeas da linhagem AS/n infectados, por via oral, com 10 cistos por animal da cepa ME-49 de Toxoplasma gondii. As coletas de sangue foram feitas a cada 3-4 dias. O parasitismo sanguíneo foi avaliado pela reação em cadeia da polimerase e os títulos de anticorpos por enzimaimunoensaio e imunofluorescência indireta. Resultados: a reação em cadeia da polimerase foi positiva em amostras com intervalos de aproximadamente 7 dias até o 28º dia, e a seguir, negativas até o 60º dia. Os soros avaliados pela imunofluorescência indireta apresentaram anticorpos IgM após o 7º dia, com pico entre o 18º e 27º dia. Após o primeiro mês os títulos foram baixos até o 60º dia. Anticorpos IgG surgiram no 14º dia e persistiram em altos títulos até o 60º dia. A cinética dos anticorpos IgG, bem como a avidez destes, demonstrou que os níveis de anticorpos aumentaram a partir do 14º dia e as porcen-tagens de avidez evoluíram com pico máximo após 28 dias, estabelecendo-se então a fase crônica da infecção. Conclusões: os dados aqui demonstrados enfatizam que taquizoítos podem estar presentes na circulação sanguínea durante toda a fase aguda da toxoplasmose, mesmo que já se tenha instalado a resposta imune protetora.

Aims: To analyze experimentally the humoral immune response in acute and recent chronic infections by Toxoplasma gondii and its correlation with the blood parasitism. Methods: Ten female mice AS/n inbred strain orally infected with 10 cysts per animal from T. gondii ME-49 strain were evaluated during 60 days. Blood collections were made in each 3-4 days. Blood parasitism was evaluated by polymerase chain reaction, and antibody titres by enzyme-linked immunosorbent assay and indirect immunofluorescence. Results: Positive polymerase chain reaction was detected around seven day-intervals until the 28th day and was negative after the 30th day pos-infection. Sera assayed by immunofluorescence presented IgM antibodies after the 7th day of infection and high titers were found between the 18th and 27th day. After 30 days, IgM titers were low until the 60th day. IgG antibodies were produced around the 14th day and were high until the 60th day. The avidity and kinetic exhibited by IgG antibody levels increased from the 14th day and the avidity percents increased with maximum peak after 28 days, establishing the chronic infection. Conclusions: These data emphasize that tachyzoites can be detected in blood during the acute phase of toxoplasmosis, even though the protective immune response was already developed.