A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping

Introduction Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5′ untranslated region (5′UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Methods Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. Results The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. Conclusions A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping.

Occurrence of potentially pathogenic Vibrio in oysters (Crassostrea gigas) and waters from bivalve mollusk cultivations in the South Bay of Santa Catarina

Introduction This research aimed to identify and quantify potentially pathogenic Vibrio from different cultivations of bivalve shellfish in the State of Santa Catarina, Brazil, and water regions in the South Bay, as well as correlate the incidence of these microorganisms with the physicochemical parameters of marine waters. Methods Between October 2008 and March 2009, 60 oyster and seawater samples were collected from six regions of bivalve mollusk cultivation, and these samples were submitted for Vibrio counts. Results Twenty-nine (48.3%) oyster samples were revealed to be contaminated with one or more Vibrio species. The Vibrio parahaemolyticus and Vibrio vulnificus counts in the samples ranged from < 0.5 log10 Most Probable Number (MPN) g–1 to 2.3 log10 MPN g–1 oyster and from < 0.5 log10 MPN g–1 to 2.1 log10 MPN g–1 oyster, respectively. Of the 60 seawater samples analyzed, 44 (73.3%) showed signs of contamination with one or more vibrio species. The counts of V. parahaemolyticus and V. vulnificus in the samples ranged from < 0.3 log10 MPN·100mL–1 to 1.7 log10MPN·100mL–1 seawater and from < 0.3 log10 MPN·100mL–1 to 2.0 log10 MPN·100mL–1 seawater, respectively. A positive correlation between V. vulnificus counts and the seawater temperature as well as a negative correlation between the V. parahaemolyticus counts and salinity were observed. Conclusions The results suggest the need to implement strategies to prevent vibrio diseases from being transmitted by the consumption of contaminated bivalve shellfish.

Osteoarticular tuberculosis in an HIV-positive patient: a case report

The authors report a case of a 38-year-old HIV-positive woman, with subcutaneous nodules on the thoracic region with 3 months of evolution. Clinical, laboratory, and epidemiological features were evaluated and associated with apparent damage to the T11-T12 vertebrae, identification by imaging tests, positivity in a polymerase chain reaction-based test, and reactivity to the Mantoux tuberculin skin test (PPD-RT 23). The patient was diagnosed with osteoarticular tuberculosis and received treatment for a year, and clinical cure was achieved.

The occurrence and dissemination of methicillin and vancomycin-resistant Staphylococcus in samples from patients and health professionals of a university hospital in Recife, State of Pernambuco, Brazil

Introduction Methicillin-resistant Staphylococcus aureus (MRSA) strains have been responsible for many nosocomial outbreaks. Within hospitals, colonized employees often act as reservoirs for the spread of this organism. This study collected clinical samples of 91 patients admitted to the intensive care unit (ICU), hemodialysis/nephrology service and surgical clinic, and biological samples from the nasal cavities of 120 professionals working in those environments, of a University Hospital in Recife, in the State of Pernambuco, Brazil. The main objective of this study was to determine the occurrence and dissemination of methicillin- and vancomycin-resistant Staphylococcus spp. Methods The isolates obtained were tested for susceptibility to oxacillin and vancomycin and detection of the mecA gene. In addition, the isolates were evaluated for the presence of clones by ribotyping-polymerase chain reaction (PCR). Results MRSA occurrence, as detected by the presence of the mecA gene, was more prevalent among nursing technicians; 48.1% (13/27) and 40.7% (11/27) of the isolates were from health professionals of the surgical clinic. In patients, the most frequent occurrence of mecA-positive isolates was among the samples from catheter tips (33.3%; 3/9), obtained mostly from the hemodialysis/nephrology service. Eight vancomycin-resistant strains were found among the MRSA isolates through vancomycin screening. Based on the amplification patterns, 17 ribotypes were identified, with some distributed between patients and professionals. Conclusions Despite the great diversity of clones, which makes it difficult to trace the source of the infection, knowledge of the molecular and phenotypic profiles of Staphylococcus samples can contribute towards guiding therapeutic approaches in the treatment and control of nosocomial infections.

Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum

IntroductionKala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2) and N-acetylglucosamine-1-phosphotransferase (NAGT), which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar.MethodsTo determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR), the fragments were sequenced, followed by polymorphism identification.ResultsThe nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival.ConclusionsNAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis.

The existence of only one haplotype of Leishmania major in the main and potential reservoir hosts of zoonotic cutaneous leishmaniasis using different molecular markers in a focal area in Iran

IntroductionLeishmania major is the causative agent of zoonotic cutaneous leishmaniasis (ZCL), and great gerbils are the main reservoir hosts in Iran. Abarkouh in central Iran is an emerging focal point for which the reservoir hosts of ZCL are unclear. This research project was designed to detect any Leishmania parasites in different wild rodent species.MethodsAll rodents captured in 2011 and 2012 from Abarkouh district were identified based on morphological characteristics and by amplification of the rodent cytochrome b (Cyt b) gene. To detect Leishmania infection in rodents, deoxyribonucleic acid (DNA) of each ear was extracted. Internal transcribed spacer-ribosomal deoxyribonucleic acid (ITS-rDNA), microsatellites, kinetoplast deoxyribonucleic acid (kDNA) and cytochrome b genes of Leishmania parasites were amplified by polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) and sequencing were employed to confirm the Leishmania identification.ResultsOf 68 captured rodents in the region, 55 Rhombomys opimus were identified and nine Leishmaniainfections (9/55) were found. In addition, eight Meriones libycus and two Tatera indicawere sampled, and one of each was confirmed to be infected. Two Meriones persicus and one Mus musculuswere sampled with no infection.ConclusionsThe results showed that all 11 unambiguously positive Leishmania infections were Leishmania major. Only one haplotype of L. major(GenBank access No. EF413075) was found and at least three rodents R. opimus, M. libycus and T. indica—appear to be the main and potential reservoir hosts in this ZCL focus. The reservoir hosts are variable and versatile in small ZCL focal locations.

Awareness of visceral leishmaniasis and its relationship to canine infection in riverside endemic areas in Northeastern Brazil

IntroductionAn awareness of visceral leishmaniasis (VL) is necessary to encourage the population to participate in prevention and control in collaboration with more efficient, centrally organized health programs. The aim of this study was to evaluate the awareness of the riverside population regarding VL and the association between awareness and the prevalence of canine visceral leishmaniasis (CVL).MethodsIn total, 71 people living in riverside areas in the City of Mossoró in State of Rio Grande do Norte participated of the study, and 71 dogs were tested for CVL by polymerase chain reaction (PCR). Association analysis of several variables related to knowledge of the riverside population regarding CVL positivity was performed, yielding odds ratios (OR) and 95% confidence intervals (CI), and significance was determined using chi-square (χ2) and Fisher's exact tests.ResultsAmong individuals whose dogs tested positive for CVL, 60% did not know the cure for CVL, and these subjects were three times more likely to have a dog test positive for CVL than those who were aware the cure for CVL. Knowledge of CVL cure was the only variable that remained in the logistic model after the successive removal of variables, with an adjusted OR of 3.11 (95%CI: 1.1-8,799; p=0.032).ConclusionsInsufficient awareness regarding VL in riverside areas with CVL-positive dogs was associated with increased rates of canine infection, which suggests that changes in habits and the adoption of attitudes and preventive practices may contribute to the control and prevention of this disease. This study reinforces the need to invest in better health education programs regarding VL.

High similarity of Trypanosoma cruzikDNA genetic profiles detected by LSSP-PCR within family groups in an endemic area of Chagas disease in Brazil

IntroductionDetermining the genetic similarities among Trypanosoma cruzi populations isolated from different hosts and vectors is very important to clarify the epidemiology of Chagas disease.MethodsAn epidemiological study was conducted in a Brazilian endemic area for Chagas disease, including 76 chronic chagasic individuals (96.1% with an indeterminate form; 46.1% with positive hemoculture).ResultsT. cruzi I (TcI) was isolated from one child and TcII was found in the remaining (97.1%) subjects. Low-stringency single-specific-primer-polymerase chain reaction (LSSP-PCR) showed high heterogeneity among TcII populations (46% of shared bands); however, high similarities (80-100%) among pairs of mothers/children, siblings, or cousins were detected.ConclusionsLSSP-PCR showed potential for identifying similar parasite populations among individuals with close kinship in epidemiological studies of Chagas disease.

Hepatitis B virus genotypes and mutations in the basal core promoter and pre-core/core in chronically infected patients in southern Brazil: a cross-sectional study of HBV genotypes and mutations in chronic carriers

Introduction In Brazil, little data exist regarding the distribution of genotypes in relation to basal core promoter (BCP) and precore/core mutations among chronic hepatitis B virus (HBV) carriers from different regions of the country. The aim of this study was to identify HBV genotypes and the frequency of mutations at the BCP and precore/core region among the prevalent genotypes in chronic carriers from southern Brazil. Methods Nested-polymerase chain reaction (nested-PCR) products amplified from the S-polymerase gene, BCP and precore/core region from 54 samples were sequenced and analyzed. Results Phylogenetic analysis of the S-polymerase gene sequences showed that 66.7% (36/54) of the patients were infected with genotype D (D1, D2, D3), 25.9% (14/54) with genotype A (A1, A2), 5.6% (3/54) with subgenotype C2, and 2% (1/54) with genotype E. A comparison of virological characteristics showed significant differences between genotypes A, C and D. The comparison between HBeAg status and the G1896A stop codon mutation in patients with genotype D revealed a relationship between HBV G1896A precore mutants and genotype D and hepatitis B e antigen (HBeAg) seroconversion. Genotype D had a higher prevalence of the G1896A mutation and the presence of a thymine at position 1858. Genotype A was associated with a higher prevalence of the G1862T mutation and the presence of a cytosine at position 1858. Conclusions HBV genotype D (D3) is predominant in HBV chronic carriers from southern Brazil. The presence of mutations in the BCP and precore/core region was correlated with the HBV genotype and HBeAg negative status.

Histological and endoscopic features of the stomachs of patients with Chagas disease in the era of Helicobacter pylori

Introduction Most studies that have evaluated the stomachs of patients with Chagas disease were performed before the discovery of Helicobacter pylori and used no control groups. This study compared the gastric features of chagasic and non-chagasic patients and assessed whether gastritis could be associated with Chagas disease. Methods Gastric biopsy samples were taken from patients who underwent endoscopy for histological analysis according to the Updated Sydney System. H. pylori infection was assessed by histology, 16S ribosomal ribonucleic acid (rRNA) polymerase chain reaction (PCR), serology and the 13C-urea breath test. Patients were considered H. pylori-negative when all of these diagnostic tests were negative. Clinical and socio-demographic data were obtained by reviewing medical records and using a questionnaire. Results The prevalence of H. pylori infection (70.3% versus 71.7%) and chronic gastritis (92.2% versus 85%) was similar in the chagasic and non-chagasic groups, respectively; such as peptic ulcer, atrophy and intestinal metaplasia. Gastritis was associated with H. pylori infection independent of Chagas disease in a log-binomial regression model. However, the chagasic H. pylori-negative patients showed a significantly higher grade of mononuclear (in the corpus) and polymorphonuclear (PMN) (in the antrum) cell infiltration. Additionally, the patients with the digestive form of Chagas disease showed a significantly lower prevalence of corpus atrophy than those with other clinical forms. Conclusions The prevalence of H. pylori infection and of gastric histological and endoscopic features was similar among the chagasic and non-chagasic patients. Additionally, this is the first controlled study to demonstrate that H. pylori is the major cause of gastritis in patients with Chagas disease.

Geographical distribution of Trypanosoma cruzi in triatomine vectors in the State of Mato Grosso do Sul, Brazil

Introduction This work presents the initial findings of a molecular epidemiological investigation of Trypanosoma cruzi in triatomine insects in State of Mato Grosso do Sul. Methods A total of 511 triatomines from different regions of the state were examined. Deoxyribonucleic acid (DNA) was extracted from the intestinal contents of the insects using phenol-chloroform-isoamyl alcohol (25:24:1). Polymerase chain reaction (PCR) using primers 121/122 targeting DNA kinetoplast (kDNA) was then performed to identify T. cruzi, and positive samples were subjected to PCR using the primer pair TcSC5D-F/R followed by restriction fragment length polymorphism (RFLP) with the restriction enzymes SphI and HpaI (1 U/reaction), cloning and sequencing. Results One hundred samples were positive for T. cruzi, and three discrete typing units (DTUs) were identified (TcI, TcII, and TcBat). Triatoma sordida had the highest T. cruzi occurrence (83.3%), and DTUs were found in three samples: 58.3% of the samples were TcI, 33.3% were TcII and 8.3% were TcBat. There was a clear geographical distribution of the DTUs throughout the state, with TcI, TcII and TcBat located in the center, TcI located in the east, and TcII located in the west. Conclusions This study showed the occurrence of overlapping DTUs in State of Mato Grosso do Sul. The distributions of the DTUs were different, with TcI, TcII and TcBat in the center of the state, TcI predominantly in the east, and TcII in the west. Further studies may reveal a more defined mosaic distribution of DTUs in MS.

Frequency of the toxic shock syndrome toxin-1 gene in methicillin-susceptible and -resistant Staphylococcus aureus isolates from teaching hospitals in Shiraz, Iran

INTRODUCTION: Staphylococcus aureus produces a range of virulence factors such as toxic shock syndrome toxin-1. METHODS: In this cross-sectional study of 345 clinical S. aureus isolates, the presence of the tst gene was assessed by polymerase chain reaction (PCR). RESULTS: The study revealed 53/345 (15.4%) isolates were positive for the tst gene. The tst gene was present in 18.1% of methicillin-susceptible S. aureus (MSSA) isolates and 11.6% of methicillin-resistant S. aureus (MRSA) isolates (p = 0.136). CONCLUSIONS: These results reveal the remarkable risk of S. aureus infections in hospitals, regardless of methicillin-resistance status.

Diagnosing lymphoma in a setting with a high burden of infection: a pediatric case of Epstein-Barr virus-associated aggressive B-cell lymphoma with t(8;14) (q23;q32) and extensive necrosis mimicking tuberculosis

The association of lymphoma with necrotic granuloma can pose diagnostic challenges and delay treatment, especially in settings with a high burden of infection. In these settings, the timely use of cytogenetic and molecular methods is most relevant. Here, we report a case of B-cell lymphoma with t (8;14) in a 5-year-old male child. The lymphoma was associated with necrotic granuloma and was initially misdiagnosed as tuberculosis. Polymerase chain reaction was used to detect clonal lymphoproliferation and to rule out Mycobacterium tuberculosis infection. Tumor cells harbored Epstein-Barr virus and expressed CD20, CD10, BCL6, and Ki67 (30%), leading to the diagnosis of B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma.

Evaluation of arboviruses of public health interest in free-living non-human primates (Alouatta spp., Callithrix spp., Sapajus spp.) in Brazil

INTRODUCTION: The aim of the present study was to evaluate the presence of arboviruses from the Flavivirus genus in asymptomatic free-living non-human primates (NHPs) living in close contact with humans and vectors in the States of Paraná and Mato Grosso do Sul, Brazil. METHODS: NHP sera samples (total n = 80, Alouatta spp. n = 07, Callithrix spp. n = 29 and Sapajus spp. n = 44) were screened for the presence of viral genomes using reverse transcription polymerase chain reaction and 10% polyacrylamide gel electrophoresis techniques. RESULTS: All of the samples were negative for the Flavivirus genome following the 10% polyacrylamide gel electrophoresis analysis. CONCLUSIONS: These negative results indicate that the analyzed animals were not infected with arboviruses from the Flavivirus genus and did not represent a risk for viral transmission through vectors during the period in which the samples were collected.

Distribution of extended-spectrum β-lactamase types in a Brazilian tertiary hospital

INTRODUCTION: Epidemiological data on the prevalence of extended-spectrum β-lactamases (ESBLs) are scarce in Brazil despite the fact that these data are essential for empirical treatment and control measures. The objective of this study was to evaluate the prevalence of different ESBLs by type and distribution in a tertiary hospital in southern Brazil. METHODS: We evaluated 1,827 enterobacterial isolates between August 2003 and March 2008 isolated from patients at a tertiary hospital. Samples were identified using a Vitek automated system and were confirmed by biochemical testing. The identified ESBL strains were characterized by phenotypic methods, polymerase chain reaction (PCR), and sequencing. Genetic similarities were evaluated by pulsed-field gel electrophoresis. RESULTS: It was 390 (21.3%) ESBL-producing strains, which expressed the ESBLs CTX-M (292), SHV (84), CTX and SHV (10), TEM (2), and PER (2). CONCLUSIONS: The prevalence of ESBL-expressing strains was high, especially in Klebsiella pneumoniae and Enterobacter spp. CTX-M was the predominant type of ESBL observed, and its genetic variability indicates a polyclonal distribution.