Reemergence of yellow fever: detection of transmission in the State of São Paulo, Brazil, 2008

INTRODUCTION: Following yellow fever virus (YFV) isolation in monkeys from the São José do Rio Preto region and two fatal human autochthonous cases from the Ribeirão Preto region, State of São Paulo, Brazil, two expeditions for entomological research and eco-epidemiological evaluation were conducted. METHODS: A total of 577 samples from humans, 108 from monkeys and 3,049 mosquitoes were analyzed by one or more methods: virus isolation, ELISA-IgM, RT-PCR, histopathology and immunohistochemical. RESULTS: Of the 577 human samples, 531 were tested by ELISA-IgM, with 3 positives, and 235 were inoculated into mice and 199 in cell culture, resulting in one virus isolation. One sample was positive by histopathology and immunohistochemical. Using RT-PCR, 25 samples were processed with 4 positive reactions. A total of 108 specimens of monkeys were examined, 108 were inoculated into mice and 45 in cell culture. Four virus strains were isolated from Alouattacaraya. A total of 931 mosquitoes were captured in Sao Jose do Rio Preto and 2,118 in Ribeirão Preto and separated into batches. A single isolation of YFV was derived from a batch of 9 mosquitoes Psorophoraferox, collected in Urupês, Ribeirão Preto region. A serological survey was conducted with 128 samples from the municipalities of São Carlos, Rincão and Ribeirão Preto and 10 samples from contacts of patients from Ribeirão Preto. All samples were negative by ELISA-IgM for YFV. CONCLUSIONS: The results confirm the circulation of yellow fever, even though sporadic, in the Sao Paulo State and reinforce the importance of vaccination against yellow fever in areas considered at risk.

Improvement of a PCR test to diagnose infection by Mansonella ozzardi

INTRODUCTION: Mansonelliasis is caused by Mansonella ozzardi. It is widespread in the Amazon region, with a high prevalence. The common exam of thick blood smears stained with Giemsa shows low efficacy levels and has been an obstacle to diagnosing individuals with low blood parasitemia. METHODS: In order to increase diagnosis efficacy, the PCR technique was improved. RESULTS AND CONCLUSIONS: PCR demonstrated the best performance, with sensitivity and negative predictive values (NPV) of 100%, followed by blood filtration through membrane filters, which showed a sensitivity of 88.9% and a NPV of 84.6%, when compared to thick blood smears.

Molecular detection of Treponema pallidum sp. pallidum in blood samples of VDRL-seroreactive women with lethal pregnancy outcomes: a retrospective observational study in northern Brazil

INTRODUCTION: Although control measures of maternal and congenital syphilis are available in Brazil, difficulties exist within the healthcare network in providing a laboratory diagnosis of the infection during the prenatal period. The objective of this study was to confirm the presence of Treponema pallidum by PCR in women with positive VDRL serology and lethal pregnancy outcomes, i.e., abortion, stillbirth and neonatal death. METHODS: A retrospective study was conducted on VDRLseroreactive women with lethal pregnancy outcomes admitted to the Fundação Santa Casa de Misericórdia do Pará (FSCM-PA) between January and July 2004. Serum samples and DNA from whole blood were obtained at the time of screening by the VDRL test. These samples were analyzed by IgG ELISA, IgM FTA-Abs and simple PCR (polA). RESULTS: During the study period, 0.7% (36/4,912) of women with lethal pregnancy outcomes presented a positive VDRL test. The polAgene was amplified in 72.7% (24/33) of these women, with 55.6% (20/36) and 94.4% (34/36) presenting IgM and IgG antibodies against T. pallidum, respectively. Comparison of these results showed a significant difference, with agreement between the PCR and IgM FTA-Abs results, suggesting that maternal syphilis was an active infection. No basic cause of death of the conceptus was reported in 97.2% (35/36) of cases. Among women who were submitted to the VDRL test during the prenatal period, only four of the nine seroreactive patients underwent treatment. CONCLUSIONS: The high frequency of syphilis in the group studied indicates the fragility of the service of infection diagnosis, treatment and monitoring, compromising epidemiological control.

Hepatitis B and C virus infection among Brazilian Amazon riparians

INTRODUCTION: Viral hepatitis is a major public health concern in Brazil. There are few past studies on this issue, especially among riparian communities. This study aims at determining the seroprevalence of viral hepatitis B and C in the riparian community of Pacuí Island, within the Cametá municipality of Pará State, Brazil. Moreover, this study aims to investigate the principal risk factors that this community is exposed to. METHODS: The current study has accessed blood samples from 181 volunteers who have answered an epidemiological questionnaire. Analyses on serological markers have been tested with commercial ELISA kits for detecting HBsAg, total anti-HBc, anti-HBs, and anti-HCV. Within seroreactive patients for HCV, RT-PCR and line probe assay have been performed to identify the viral genotype. RESULTS: In the serological marker analysis for hepatitis B, no reactivity for HBsAg, rate of 1.1% for total anti-HBc, and rate of 19.3% for anti-HBs have been observed. On hepatitis C, 8.8% seroprevalence has been found, in which 62.5% have gotten viral RNA. Among the risk factors studied, the following have been highlighted: non-use of condoms, sharing of cutting instruments, use of illicit drugs, and reports of family disease with HBV or HCV. CONCLUSIONS: The vaccination coverage against HBV is low, and the high prevalence of HCV within this community has been observed.

Epidemiological factors related to the transmission risk of Trypanosoma cruzi in a Quilombola community, State of Mato Grosso do Sul, Brazil

INTRODUCTION: This work was an epidemiological investigation of the risk of Trypanosoma cruzi transmission in the rural Quilombola community of Furnas do Dionízio, State of Mato Grosso do Sul, Brazil. METHODS: Of the 71 animals examined, seven were captured (two opossums, Didelphis albiventris; four rats, Rattus rattus; and one nine-banded armadillo, Dasypus novemcinctus) and 64 were domestic (one canine, Canis familiaris; five pigs, Sus scrofa; two bovines, Bos taurus; five caprines, Capra sp.; and 51 ovines, Ovis aries). Parasitological tests were performed to detect parasites in the blood and to identify the morphology of flagellates. These methods included fresh examinations, buffy coat tests and blood cultures. Molecular analysis of DNA for identification of trypanosomatids was performed by polymerase chain reaction (PCR) with primers S35 and S36. RESULTS: The parasitological tests showed flagellates in an opossum and two cattle. The molecular tests showed DNA from T. cruzi in an opossum and a pig. Triatoma sordida was the only triatomine species found in the community, and it colonized households (four specimens) and the surrounding areas (124 specimens). Twenty-three specimens tested positive for flagellates, which were subsequently identified as T. cruzi by PCR. CONCLUSIONS: Data analysis demonstrated that T. cruzi has a peridomestic life cycle that involves both domestic and wild mammals.

Rapid detection of Van genes in rectal swabs by real time PCR in Southern Brazil

INTRODUCTION: Laboratory-based surveillance is an important component in the control of vancomycin resistant enterococci (VRE). METHODS: The study aimed to evaluate real-time polymerase chain reaction (RT-PCR) (genes vanA-vanB) for VRE detection on 115 swabs from patients included in a surveillance program. RESULTS: Sensitivity of RT-PCR was similar to primary culture (75% and 79.5%, respectively) when compared to broth enriched culture, whereas specificity was 83.1%. CONCLUSIONS: RT-PCR provides same day results, however it showed low sensitivity for VRE detection.

In-house ELISA method to analyze anti-Trypanosoma cruzi IgG reactivity for differential diagnosis and evaluation of Chagas disease morbidity

INTRODUCTION: The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS: With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS: The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100% sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS: These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.

High prevalence of HTLV-1 and 2 viruses in pregnant women in São Luis, state of Maranhão, Brazil

INTRODUCTION: Human T cell lymphotropic virus type 1 (HTLV-1) is endemic in the Caribbean, Japan, South America and regions of Africa. HTLV-2 is present in Native American populations and associated with IV drug use in Europe and North America. In Brazil, it is estimated that 1.5 million people are infected with HTLV-1/2. The study objective was to determine HTLV-1/2 prevalence in pregnant women in the prenatal care from three public services in São Luis, State of Maranhão, Brazil, and to counsel seropositive women to reduce viral transmission. METHODS: A cross-sectional study was conducted from February to December 2008; women with age of 18 to 45 years, with low risk for sexually transmitted disease (STD) were invited to participate. Blood samples were collected in filter paper, and HTLV-1/2 immunoenzymatic test (ELISA) was performed as a screening test. Women with reactive results were submitted to peripheral venous blood collection for ELISA repetition, followed by Western blot (WB) and real-time PCR to confirm and discriminate the infection between virus types 1 and 2. RESULTS: Of the 2,044 women tested, seven (0.3%) were ELISA reactive and confirmed positive (four were HTLV-1, and three were HTLV-2). All positive women were oriented not to breastfeed their newborns. CONCLUSIONS: This study showed that the virus is present in high prevalence in that population. Further studies covering other segments of the population are necessary to better characterize the presence of HTLV-1/2 in Maranhão and to elicit measures to prevent its spread.

Increased detection of schistosomiasis with Kato-Katz and SWAP-IgG-ELISA in a Northeastern Brazil low-intensity transmission area

INTRODUCTION: The laboratory diagnosis of schistosomiasis is based mainly on the detection of parasite eggs in stool samples through the Kato-Katz (KK) technique, reading one slide by test. However, a widely known limitation of parasitological methods is reduced sensitivity, particularly in low endemic areas. METHODS: To increase sensitivity, we conducted further slide readings from the same stool sample using the parasitological method associated with a serological test. We used the KK method (three slides) and the IgG anti-Schistosoma mansoni-enzyme-linked immunosorbent assay (ELISA) technique to diagnose schistosomiasis in low endemic areas in the Brazilian State of Ceará. Fecal samples and sera from 250 individuals were analyzed. RESULTS: Sixteen percent and 47.2% of samples were positive in parasitological tests and serological tests, respectively. Parasitological methods showed that 32 (80%) individuals tested positive on the first slide, 6 (15%) on the second slide, and 2 (5%) on the third. The performance of the ELISA test in the diagnosis, using the KK method as diagnostic reference, showed a negative predictive value of 100%, with specificity and positive predictive values of 62.8% and 33.9%, respectively. CONCLUSIONS: In this study, the increase from one to three slides analyzed per sample using the KK technique was shown to be a useful procedure for increasing the diagnostic sensitivity of this technique.

First description of autochthonous canine visceral leishmaniasis in the metropolitan region of Vitória, State of Espírito Santo, Brazil

INTRODUCTION: We investigated autochthonous canine visceral leishmaniasis (CVL) in the metropolitan region of Vitória (MRV), an area in which a human case was previously reported. METHODS: Serological, parasitological, and molecular tests were performed in 201 dogs. RESULTS: Twenty-six (13%) and 12 (6%) dogs were identified as positive using in-house enzyme-linked immunosorbent assay (ELISA) and rK39 tests, respectively. Two dogs had a positive culture for Leishmania chagasi, and 4 were polymerase chain reaction (PCR)-positive for Leishmania spp. One positive dog belonged to the aforementioned patient. CONCLUSIONS: Although the responsible vector was not found, our results provide evidence of autochthonous CVL in the MRV, a non-endemic area for VL.

Rotavirus G2P[4] and G2P[4]+[6] infections during norovirus gastroenteritis outbreak: summer season 2010, Brazil

IntroductionThis study aimed to monitor the seasonality of rotavirus infection, and gain insight into the variability of Brazilian strains.MethodsA total of 28 stool samples were analyzed from 698 revised cases of gastroenteritis during a norovirus outbreak in the summer of 2010 in Guarujá, Brazil. Diagnosis was performed using enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), and sequencing.ResultsRotavirus infection was detected in 17.9% (5/28) of samples; 4 samples were G2P[4] genotype, and one G2P[4]+P[6] genotype. G2 and P[4] sequences showed a genetic relationship to strains from India and Russia, respectively.ConclusionsThe seasonal pattern of rotavirus may be a consequence of human activity apart from climate factors.

Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

Introduction Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. Methods First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. Results In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. Conclusions The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work.

Monitoring Trypanosoma cruzi infection in triatomines using PCR in Mato Grosso do Sul, Brazil

Introduction The aim of the present study was to assess the polymerase chain reaction (PCR) as a method for detecting Trypanosoma cruzi infection in triatomines that had been previously determined by microscopic examination in the State of Mato Grosso do Sul, Brazil. Methods In total, 515 specimens were collected. Material from the digestive tract of each triatomine was analyzed for the presence of T. cruzi by microscopic examination and PCR using the 121/122 primer set. Results Among the 515 specimens tested, 58 (11.3%) were positive by microscopy and 101 (19.61%) were positive by PCR and there was an association between the results of the techniques (χ2 = 53.354, p = 0.001). The main species of triatomine identified was T. sordida (95.5%) Conclusions The use of PCR in entomological surveillance may contribute to a better assessment of the occurrence of T. cruzi in triatomine populations.

Development of duplex-PCR for identification of Aeromonas species

Introduction The number of reports of intestinal infections caused by Aeromonas spp. has increased significantly in recent years. In most clinical laboratories, identification of these bacteria is carried out by general phenotypic tests that sometimes do not accurately differentiate Aeromonas and Vibrio. Methods A duplex-polymerase chain reaction (PCR) was developed directed to 2 targets identifying Aeromonas spp. pathogenic to humans. Results The duplex-PCR results were reproducible and specific for Aeromonas spp. pathogenic to humans. Conclusions This method will allow differentiation between Vibrio and Aeromonas spp. in patients with in cholera-like symptoms and can also be used in water quality monitoring.

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Introduction The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. Methods The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) Results Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identification of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the final diagnosis from the attending physician. Conclusions Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifiable using conventional biochemical and phenotypic techniques.