A semi-nested PCR assay for molecular detection of Paracoccidioides brasiliensis in tissue samples

INTRODUCTION: Paracoccidioidomycosis is a systemic infection caused by Paracoccidioides brasiliensis. METHODS: In this study, a semi-nested PCR for paracoccidioidomycosis diagnosis was developed. The primers ITS1 and ITS4 were used in the first reaction, while the primers MJ03 and ITS1 primer were used in the second reaction. The semi-nested PCR was used to investigate biopsies of five patients with oral lesions that resembled paracoccidioidomycosis. RESULTS: The semi-nested PCR was positive for four samples and negative for a sample from a patient later diagnosed with leishmaniasis. CONCLUSIONS: The new semi-nested PCR describe is useful for paracoccidioidomycosis diagnosis.

Improving tuberculosis control through the partnership between university and the health system

INTRODUCTION: Tuberculosis (TB) control is linked to the availability of qualified methods for microbiological diagnostics; however, microscopy with limited sensitivity is the only method available in many locations. The objective of this study was to evaluate the introduction of culture, drug susceptibility testing (DST), and genotyping in the routine of a Municipal Program of Tuberculosis Control. METHODS: Direct microscopy of sputum and culture in Ogawa-Kudoh were performed on 1,636 samples from 787 patients. DST of positive cultures was performed by resazurin microtiter assay and genotyping by mycobacterial interspersed repetitive units-variable number tandem repeat. RESULTS: A total 91 patients with TB were identified. The culture increased case detection by 32% compared with the microscopy; acquired resistance was 3.3% and the genotyping showed high genetic diversity. CONCLUSIONS: Ogawa-Kudoh contributed significantly to the increase in case detection and is suitable for implementation in poor-resource locations. The acquired resistance rate was lower than that reported in a recent Brazilian survey. The high genetic diversity is possibly related to the high TB prevalence in the population, as well as to early detection and suitable treatment of patients. The interaction between research and health care is important for reorienting the practice, transferring technology, and improving TB control.

cagE as a biomarker of the pathogenicity of Helicobacter pylori

IntroductionHelicobacter pylori infection is associated with gastro-duodenal diseases. Genes related to pathogenicity have been described for H. pylori and some of them appear to be associated with more severe clinical outcomes of the infection. The present study investigates the role of cagE as a pathogenicity biomarker of H. pylori compare it to cagA, vacA, iceA and babA2 genes and correlate with endoscopic diagnoses.MethodsWere collected biopsy samples of 144 dyspeptic patients at the Hospital of the Federal University of Rio Grande, Rio Grande do Sul, Brazil. After collection, the samples were sent for histological examination, DNA extraction and detection of all putative pathogenicity genes by PCR.ResultsOf the 144 patients undergoing endoscopy, 57 (39.6%) presented H. pylori by histological examination and PCR by detection of the ureA gene. Based on the endoscopic diagnoses, 45.6% (26/57) of the patients had erosive gastritis, while 54.4% (31/57) had enanthematous gastritis. The genes cagA, cagE, vacAs1/m1, vacAs1/m2 and iceA1 were related to erosive gastritis, while the genes vacAs2/m2, iceA2 and babA2 were associated to enanthematous gastritis. We found a statistically significant association between the presence of cagE and the endoscopic diagnosis. However, we detect no statistically significant association between the endoscopic diagnosis and the presence of cagA, vacA, iceA and babA2, although a biological association has been suggested.ConclusionsThus, cagE could be a risk biomarker for gastric lesions and may contribute to a better evaluation of the H. pylori pathogenic potential and to the prognosis of infection evolution in the gastric mucosa.

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.

Schistosomiasis mansoni in urban Northeast Brazil: influence of rainfall regime on the population dynamics of Biomphalaria sp.

Introduction Our objective was to evaluate the influence of rainfall regime on the population dynamics of Biomphalaria in a potential urban focus of schistosomiasis in Aracaju, Brazil, during 2009-2010. Methods Snails were collected monthly and were counted, measured and identified; the level of infection and fecal contamination at the sampling sites was determined; rainfall data were obtained. Results High levels of fecal contamination were observed, and the abundance of Biomphalaria glabrata increased during the rainy and post-rainy seasons. The snails' size was variable, and infected snails were identified independently of rainfall. Conclusions These results provide evidence of anthropogenic and climate interference in an urban focus of schistosomiasis in the Aracaju metropolitan area.

An optimized one-tube, semi-nested PCR assay for Paracoccidioides brasiliensis detection

Introduction Herein, we report a one-tube, semi-nested-polymerase chain reaction (OTsn-PCR) assay for the detection of Paracoccidioides brasiliensis. Methods We developed the OTsn-PCR assay for the detection of P. brasiliensis in clinical specimens and compared it with other PCR methods. Results The OTsn-PCR assay was positive for all clinical samples, and the detection limit was better or equivalent to the other nested or semi-nested PCR methods for P. brasiliensis detection. Conclusions The OTsn-PCR assay described in this paper has a detection limit similar to other reactions for the molecular detection of P. brasiliensis, but this approach is faster and less prone to contamination than other conventional nested or semi-nested PCR assays.

Syphilis seroprevalence estimates of Santa Catarina blood donors in 2010

Introduction Knowledge of blood donor characteristics is essential to better guide clinical and serological screening for hemotherapy. The objective of this study was to determine the syphilis seroprevalence and the associated factors of blood donors in the State of Santa Catarina, Brazil. Methods This population-based study from the State of Santa Catarina used information obtained from blood donation records. We analyzed 83,396 blood donor records generated from donors who were considered eligible to donate between January and August 2010. The aim of the study was to estimate the syphilis seroprevalence and its relationship with educational level, age, gender, geographical region and having donated blood in the past 12 months. We used descriptive analyses and a Poisson regression to calculate the prevalence ratios for the variables of interest. Results We found a 0.14% overall seroprevalence and significant differences among the following: first-time blood donors (0.19%) versus repeat donors (0.03% to 0.08%); low educational levels (0.30%) versus medium and high educational levels (0.08% to 0.19%); and donors who did not report their residence (0.88%) or age (6.94%) versus those who did. Increased syphilis seroprevalence was also significantly associated with increased age. Conclusion High syphilis seroprevalence was associated with lower educational level, age, first-time donation and the failure to provide age or residence information.

Molecular identification of the agent of Q fever – Coxiella burnetii – in domestic animals in State of Rio de Janeiro, Brazil

Introduction Over the last recent years, the number of Q fever cases have has increased throughout the world. An epidemiological investigation was performed in the area in which the first molecular documentation of Q fever in Brazil was previously reported. Methods Indirect immunofluorescence assay (IFA) and PCR of Coxiella burnetii targeting the htpAB gene were performed in samples from 14 dogs (blood); 1 cat (blood); 10 goats (blood, milk, vaginal swab and anal swab); 3 sheep (blood); and 2 horses (blood). Results Two dogs, two sheep and five goats were seroreactive. DNA was amplified from 6 milk and 2 blood samples from goats and from dogs, respectively. The sequence of the amplicons exhibited 99% sequence similarity with the homologous sequence of the htpAB gene of C. burnetii RSA 331 (GenBank - CP000890). Conclusions The results confirm C. burnetii infection in animals in Rio de Janeiro and reinforce the need for the surveillance of Q fever in Brazil.

The first canine visceral leishmaniasis outbreak in Campinas, State of São Paulo Southeastern Brazil

Introduction Early detection of American visceral leishmaniasis (AVL) outbreak in animals is crucial for controlling this disease in non-endemic areas. Methods Epidemiological surveillance (2009-2012) was performed in Campinas, State of São Paulo, Brazil. Results In 2009, Leishmania chagasi was positively identified in four dogs. Entomological research and three serological studies (2010-2012) were undertaken as monitoring measures; these approaches revealed a moderate prevalence of Leishmania present in 4% of the canine population. Nyssomyia whitmani and Lutzomyia longipalpis were the predominant species identified. Conclusions Detection of an AVL outbreak in dogs in an area with an evolving natural landscape containing sand flies is crucial for control programs.

Changes in the prevalence, incidence and residual risk for HIV and hepatitis C virus in Southern Brazilian blood donors since the implementation of NAT screening

Introduction Previous studies have shown high residual risk of transfusing a blood donation contaminated by human immunodeficiency virus (HIV) or hepatitis C virus (HCV) in Brazil and motivated the development of a Brazilian platform for simultaneous detection of both viruses by nucleic acid amplification test (NAT) denominated HIV/HCV Bio-Manguinhos/Fundação Oswaldo Cruz (FIOCRUZ). The objective of this study was to verify seroprevalence, incidence and residual risk for both viruses before and after the implementation of NAT. Methods Over 700,000 blood samples from all blood banks in the southern Brazilian State of Santa Catarina were analyzed during the period between January 2007 and July 2013. Results Compared with the period preceding the NAT screening, HIV prevalence increased from 1.38 to 1.58 per 1,000 donors, HIV incidence rate increased from 1.22 to 1.35 per 1,000 donor-years, and HIV residual risk dropped almost 2.5 times during the NAT period. For HCV, seroprevalence increased from 1.22 to 1.35 per 1,000 donors, incidence dropped from 0.12 to 0.06 per 1,000 donor-years, and residual risk decreased more than 3 times after the NAT implementation. Conclusions NAT reduced the duration of the immunologic window for HIV and HCV, thus corresponding to approximately 2.5- and 3-fold respective residual risk reductions.

Invasive aspergillosis in a user of inhaled cocaine: rhinosinusitis with bone and cartilage destruction

Aspergillosis is an infection caused by saprophytic fungi of the genus Aspergillus, which typically occurs in immunosuppressed individuals, but has also been reported in immunocompetent patients. The main routes of entry are the respiratory tract, skin, cornea, and ear, and the infection may be localized or disseminated by contiguity or vascular invasion. We report a severe case of rhinosinusitis with cutaneous involvement, caused by invasive aspergillosis, in an immunocompetent user of inhaled cocaine. Invasive aspergillosis related to cocaine abuse has not yet been reported in the literature. After itraconazole treatment and surgical debridement, complete clinical remission was achieved. Nasal reconstruction with a skin graft over a silicone prosthesis resulted in a satisfactory esthetic outcome.

Opportunistic infections among individuals with HIV-1/AIDS in the highly active antiretroviral therapy era at a Quaternary Level Care Teaching Hospital

INTRODUCTION : In this study, clinical-laboratory and epidemiological characteristics are described for a group of 700 individuals with HIV (human immunodeficiency virus)/AIDS (acquired immunodeficiency syndrome) in the ART (antiretroviral therapy) era at a teaching hospital that provides a quaternary level of care, with an emphasis on opportunistic infections (OIs), co-infections and immune profile. METHODS : A retrospective cross-sectional study of AIDS cases was conducted from 1998 to 2008 by reviewing medical records from the Base Hospital/FUNFARME (Fundação Faculdade Regional de Medicina), São José do Rio Preto, São Paulo, Brazil. RESULTS: The individuals were 14 to 75 years of age, and 458 were males. Heterosexuals accounted for 31.1% of all patients. Eighty-three percent were on ART, and 33.8% of those presented difficulties with treatment adherence. OIs were analyzed from medical records, and Pneumocystis jiroveci pneumonia was the most prevalent, regardless of the LTCD4+ (TCD4+ Lymphocytes) levels. Individuals whose viral loads were ≥10,000 showed a 90% greater chance of neurotoxoplasmosis. For P. jiroveci pneumonia, neurotoxoplasmosis, esophageal candidiasis, pulmonary tuberculosis and neurocryptococcosis, the chances of infection were higher among patients with LTCD4+ levels below 200 cells/mm3. HIV/hepatitis C virus (HCV) and HIV/hepatitis B virus (HBV) co-infections were significantly associated with death. CONCLUSIONS : OIs remain frequent in the ART era even in populations where the access to medical care is considered satisfactory.

The BACTEC MGIT(tm) 320 system as a laboratory tool to diagnose tuberculosis in a Brazilian hospital with a high prevalence of HIV infection

Abstract: INTRODUCTION: The World Health Organization endorses the BACTEC Mycobacterial Growth Indicator Tube (MGIT)(tm) system as a rapid, sensitive, and specific method to diagnostic of tuberculosis. Here, we compared the performance of this system against Ogawa-Kudoh cultures and microscopy. METHODS: A total of 927 samples were obtained between December 2011 and December 2013 from 652 cases of suspected tuberculosis at the School Hospital of the Federal University of Rio Grande in Brazil. RESULTS: The MGIT system confirmed tuberculosis in more cases in less time. CONCLUSIONS: The MGIT system is an effective tool for early diagnosis of tuberculosis, especially in patients with HIV/AIDS.