Echinococcus granulosus sensu lato GENOTYPES IN DOMESTIC LIVESTOCK AND HUMANS IN GOLESTAN PROVINCE, IRAN

Cystic echinococcosis (CE) is a globally parasitic zoonosis caused by larval stages of Echinococcus granulosus. This study investigated E. granulosus genotypes isolated from livestock and humans in the Golestan province, northern Iran, southeast of the Caspian sea, using partial sequencing data of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. Seventy E. granulosus isolates were collected from animals in slaughterhouses: 18 isolates from sheep, 40 from cattle, nine from camels, two from buffaloes and one from a goat, along with four human isolates (formalin-fixed, paraffin-embedded tissues) from CE patients of provincial hospitals. All isolates were successfully analysed by PCR amplification and sequencing. The sequence analysis found four E. granulosus genotypes among the 74 CE isolates: G1 (78.3%), G2 (2.7%), G3 (15%) and G6 (4%). The G1-G3 complex genotype was found in all of the sheep, goat, cattle and buffalo isolates. Among the nine camel isolates, the frequency of G1-G3 and G6 genotypes were 66.7% and 33.3%, respectively. All four human CE isolates belonged to E. granulosus sensu stricto. This study reports the first occurrence of the G2 genotype in cattle from Iran and confirms the previously reported G3 genotype in camels in the same country.

Human parvovirus B19 infection in HIV-positive patients

Parvovirus B19 infects predominantly erythroid cells, leading to transient inhibition of erythropoiesis. Immunocompromised patients may be unable to produce neutralizing antibodies and may develop severe chronic anemia. Epidemiological studies done on Niterói population showed that B19 infection occurs periodically in late spring and summer. We report a study from 55 HIV infected patients attending an infectious diseases outpatient clinic in this city during a 5-month period in which B19 circulation was well documented. All patients were under anti-retroviral therapy. No anti-B19 IgM was found, but a high prevalence of IgG anti-B19 (91%) was observed. In six patients, B19 DNA was found by dot-blot hybridization techniques, but this was not confirmed by PCR. None of these 6 patients manifested anemia and only one had CD4 cell count below 200 x 10(7)/L. We conclude that persistent infection causing anemia is an infrequent finding in our HIV positive patients under drug therapy.

Identification of Leishmania chagasi from skin in Leishmania/HIV co-infection: a case report

A case of HIV/Leishmania co-infection presenting both visceral and cutaneous manifestations is reported. Leishmania infection was confirmed by conventional methods (parasitological approach and serology) and by PCR. Leishmania chagasi isolated from the skin lesion was characterized by enzyme electrophoresis and by restriction fragment length polymorphism of the internal transcribed spacer of the ribosomal gene.

Sensitivity of polymerase chain reaction for detection of known aliquots of Trypanosoma cruzi in the blood of mice: an in vitro study

To evaluate the sensitivity of polymerase chain reaction (PCR) to reveal known number of trypomastigote in the blood of mice, three separate experiments were done. First: To eight samples of 500mul of normal mice blood, one aliquot of 1, 2, 3, 4, 5, 10, and 50 trypomastigotes respectively, were added. Second and third: 10 aliquots with 1 and 10 with 2 trypomastigotes were added to samples of 500mul of normal mice blood. Positive control: 500mul of blood containing 100,000 trypomastigotes. For kDNA minicircles amplification by PCR the primers:S35 and S36 were used. PCR revealed products of 330 b.p in the positive controls. When only one sample with the aliquots of 1 or 2 trypomastigotes was examined, results were negative; results were positive with aliquots of 3 to 50 trypomastigotes. In the 2nd and 3rd experiments, 9/10 aliquots with one parasite and 9/10 with 2 trypomastigotes were positive revealing a high sensitivity of this reaction. In conclusion, the presence of one single parasite in 500mul of blood, is enough for a positive PCR. This method could be used as a complement to the various parasitological cure tests in treated mice, when low volumes of blood are individually examined.

The usefulness of Umelosa hepatitis C vírus qualitative kit as supplemental test for confirmation of hepatitis C virus infection

Forty voluntary blood donors from two different blood banks in Havana, Cuba, who were repeatedly reactive on the routine screening of antibodies to hepatitis C virus, by Umelisa HCV test, were analyzed for the presence of HCV RNA using a nested PCR assay of the HCV 5' untranslated region, Umelosa HCV qualitative. Sera from 45 patients of a specialized gastroenterology consultation, positive to Umelisa HCV, were also assayed with the Umelosa HCV qualitative, to establish their condition related to the presence of HCV RNA previously to the indication of a treatment or after three, six or twelve months of antiviral therapy. Serum HCV-RNA was detected in 21/40 (52.5%) donors who had repeatedly positive ELISA results, confirming the HCV infection for them. In specialized consultation HCV-RNA was detected by PCR analysis in 30/45 (66%) analyzed sera.

Establishment of HTLV-I-infected cell lines from peripheral blood mononuclear cells of Brazilian patients

To investigate epidemiological and pathogenetic features of HTLV-I infection, a cohort of carriers has been followed at the USP Teaching Hospital since 1991. This study describes the establishment of cell lines from peripheral blood mononuclear cells (PBMC) of infected subjects. Ex vivo PBMC were cultured with those from a seronegative donor and morphologic evidence of cell transformation was obtained after 90 days with detection of multinucleated cells exhibiting cerebriform nuclei. Integration of HTLV-I proviral DNA and expression of viral antigens was demonstrated in culture by PCR and immunofluorescence. Cell lines were maintained for 240 days, gradually weaned from exogenous IL-2. Immunophenotyping of cell lines on flow cytometry yielded evidence of cell activation. Establishment of HTLV-I-infected cell lines from ex vivo PBMC is feasible and may be useful for studies on lymphocyte phenotypic changes and on mechanisms of HTLV-induced cell proliferation. Moreover they may be used with diagnostic purposes in immunofluorescence tests.

Polymerase chain reaction and restriction fragment length polymorphism analysis of the ITS2 region for differatiation of Brazilian Biomphalaria intermediate hosts of the Schistosoma mansoni

We sequenced the internal transcribed spacer 2 of the ribosomal DNA (ITS2-DNAr) from the three Schistosoma mansoni intermediate hosts in Brazil: Biomphalaria glabrata, Biomphalaria tenagophila and Biomphalaria straminea. Analysis of a restriction map from those sequences allowed us to select putative restriction enzymes able to identify the snail species under study. Four restriction enzymes were used and HpaII provided simple species-specific profiles easily visualized in polyacrylamide gels. The use of ITS2 is advantageous as it provides a small fragment of 460 bp which may be easily amplified by PCR. In the current work, we showed that the amplification of ITS2-DNAr together with HpaII enzyme restriction is an auxiliary molecular tool for the morphological identification of such snails as well as for taxonomic and phylogenetic studies of neotropical planorbids.

Immunoblotting analyses using two-dimensional gel electrophoresis of Trypanosoma cruzi excreted-secreted antigens

Trypanosoma cruzi trypomastigotes excrete-secrete a complex mixture of antigenic molecules. This antigenic mixture denominated trypomastigote excreted-secreted antigens contains a 150-160 kDa band that shows excellent performance in Chagas' disease diagnosis by immunoblotting. The present study partially characterized by two-dimensional gel electrophoresis the immunoreactivity against the 150-160kDa protein using sera samples from chagasic patients in different phases of the disease. Trypomastigote excreted-secreted antigen preparations were subjected to high-resolution two-dimensional (2D) gel electrophoresis followed by immunoblotting with sera from chagasic and non-chagasic patients. The 150-160kDa protein presented four isoforms with isoelectric focusing ranging from 6.2 to 6.7. The four isoforms were recognized by IgM from acute phase and IgG from chronic phase sera of chagasic patients. The 150-160kDa isoform with IF of approximately 6.4 became the immunodominant spot with the progression of the disease. No cross-reactivity was observed with non-chagasic or patients infected with Leishmania sp. In this study we provide basic knowledge that supports the validation of trypomastigote excreted-secreted antigens for serological diagnosis of Chagas' disease.

Seroprevalence of hepatitis B virus infection and seroconvertion to anti-HBsAg in laboratory staff in Goiânia, Goiás

Were analyzed 648 serum samples from laboratory staff in Goiânia, Goiás aiming detection of three serological markers of HBV: HBsAg, anti-HBsAg and anti-HBcAg. The HBsAg and anti-HBcAg positive samples were also analyzed for HBeAg, anti-HBeAg and anti-HBcAgIgM markers. HBV infection rate of 24.1% was observed and, from them, 0.7% were positive for HBsAg. Viral DNA was detected by PCR in two HBsAg positive samples. A vaccination index of 74.5% and a global index of 89.9% of serological response to vaccination were observed. The direct work with biological fluids as well as cleaning workers represented significant risks for acquisition of HBV infection. The data from the present study showed an increase of the vaccination index among laboratory staff but the rates of HBV infection did not change through the years in the region.

Extended-spectrum β-lactamase-producing Salmonella enterica serovar Oranienburg (CTX-M-2 group) in a pediatric hospital in Tucumán, Argentina

INTRODUCTION: Salmonella sp infections have been reported over recent years in hospitals in Argentina and other countries due to multiresistant strains. The aim of this study was to characterize the extended-spectrum β-lactamases in third-generation cephalosporin-resistant strains of Salmonella enterica serovar Oranienburg. METHODS: We studied 60 strains isolated from children with gastroenteritis and/or extraintestinal complications. The antibiotic susceptibility patterns of the isolates were analyzed and the β-lactamases were characterized using phenotyping and genotyping methods. RESULTS: All the strains were resistant to ampicillin, cefotaxime, cefepime and aztreonam and partially susceptible to ceftazidime, thus corresponding well with the resistance phenotype conferred by CTX-M-type β-lactamases. An isoelectric point enzyme (pI = 7.9) was detected in all of the strains, and this was confirmed by PCR as a member of the CTX-M-2 group. CONCLUSIONS: This is the first report of Salmonella enterica serovar Oranienburg producing β-lactamases of the CTX-M-2 group in a pediatric hospital in Tucumán, Argentina.

Intrahospital spread of carbapenem-resistant Pseudomonas aeruginosa in a University Hospital in Florianópolis, Santa Catarina, Brazil

INTRODUCTION: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) has been isolated with increasing frequency in Brazilian hospitals. Since June 2003, its detection in a teaching hospital in the city of Florianópolis, Brazil, has increased. This study aimed to investigate the minimal inhibitory concentration (MIC), presence of Metallo-β-lactamase (MβL) and a possible clonal relationship among the isolates. METHODS: The study included 29 CRPA and seven isolates with reduced susceptibility. The MIC was determined by agar-dilution. Detection of MβL was performed by Double Disk Sinergism (DDS) and Combined Disk (CD). The MβL gene was verified by PCR and nucleotide sequence analysis. Epidemiological typing was performed by pulsed-field gel electrophoresis. RESULTS: Among the 29 carbapenem-resistant isolates, polymyxin B presented 100% susceptibility and piperacillin/tazobactam 96.7%. Seventeen (62%) strains were verified as clonal (A clone) and among these, six isolates indicated phenotypically positive tests for MβL and harbored the blaSPM-1 gene. The first CRPA isolates were unrelated to clone A, harbored blaIMP-16 and were phenotypically positive only by CD. CONCLUSIONS: The spread of a high-level of resistance clone suggests cross transmission as an important dissemination mechanism and has contributed to the increased rate of resistance to carbapenems. This study emphasizes the need for continuous surveillance and improved strategies.

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.

Circulation of the rabies virus in non-hematophagous bats in the city of Rio de Janeiro, Brazil, during 2001-2010

INTRODUCTION: Rabies is one of the most known lethal zoonosis, responsible for 55,000 human deaths per year. It is transmitted to humans mainly by the bite of domestic or wild animals infected with the virus. This paper shows the circulation of this virus in non-hematophagous bats in the City of Rio de Janeiro, Brazil. METHODS: A survey was performed on the number of bats that had been sent for diagnosis by the Seção de Virologia of the Instituto Municipal de Medicina Veterinária Jorge Vaitsman and were positive for rabies. The positive animals were identified, and the isolated viruses were sent for antigenic typification with indirect immunofluorescence. The results were compared with the antigenic panel of the Centers for Disease Control and Prevention. RESULTS: During 2001-2010, the laboratory received 555 non-hematophagous bats for rabies diagnosis, with 198 (35.7%) from Rio de Janeiro City. A total of 11 (5.5%) animals were positive for this disease. Antigenic typification revealed the predominance of variant 3 in 9 (81.8%) of the isolated viruses; 1 virus was classified as variant 4 and 1 variant was identified that segregated with the viruses in insectivorous bats. CONCLUSIONS: The data obtained in this study showed the presence of the rabies virus in synanthropic populations of non-hematophagous bats in the City of Rio de Janeiro. The circulation of this agent in these animals represents a serious risk to human and animal health and requires attention and control measures by the authorities.

Detection and molecular analysis of Toxoplasma gondii and Neospora caninum from dogs with neurological disorders

INTRODUCTION: Toxoplasma gondii and Neospora caninum are related Apicomplexa parasites responsible for systemic diseases in many species of animals, including dogs. METHODS: This study aimed to determine the occurrence of T. gondii and N. caninum infections in 50 dogs with neurological signs that were admitted to the Veterinary Hospital of Universidade Estadual Paulista, City of Botucatu, Brazil. All animals were screened for antibodies using an immunofluorescent antibody test for both parasites. Tissues of positive animals were bioassayed in mice (T. gondii) and gerbils (N. caninum), and DNA was analyzed using the polymerase chain reaction (PCR). Positive samples for T. gondii by PCR were typed using restriction fragment length polymorphism-PCR for 11 markers: SAG1, SAG2 (5′-3′-SAG2 and alt.SAG2), SAG3, Btub, GRA6, L358, c22-8, c29-6, PK1 and Apico, and CS3 marker for virulence analysis. RESULTS: Specific antibodies were detected in 11/50 (22%; 95% confidence interval (CI95%), 12.8-35.3%) animals for T. gondii and 7/50 (14%; CI95%, 7.02-26.3%) for N. caninum. In the bioassay and PCR, 7/11 (63.6%; CI95%, 34.9-84.8%) samples were positive for T. gondii and 3/7 (42.9%; CI95%I, 15.7-75.5%) samples were positive for N. caninum. Three different genotypes were identified, but only 1 was unique. CONCLUSIONS: These data confirm the presence of T. gondii and N. caninum in dogs from Brazil, indicating the importance of this host as a sentinel of T. gondii for human beings, and the genotypic variation of this parasite in Brazil.

Community-acquired methicillin-resistant Staphylococcus aureus carrying SCCmec type IV in southern Brazil

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen commonly associated with nosocomial infections. However, it has also been associated with community-acquired skin and soft tissue infections (CA-MRSA). There are few data on the identification and prevalence of CA-MRSA infections in Brazil. METHODS: This is a cross-sectional study of 104 patients with community-acquired skin infections attending two health care centers in Porto Alegre, southern Brazil. MRSA isolates were characterized by molecular methods, including detection of the mecA gene by PCR, gene SCCmec typing, Panton-Valentine leukocidin (PVL) detection, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). RESULTS: From the 104 samples, 58 Staphylococcus aureus isolates were obtained, of which five (8.6%) had a CA-MRSA-resistant profile. All five isolates had the mecA gene and amplified to SCCmec type IV. Analysis of chromosomal DNA by PFGE revealed the presence of two clusters related to international clones (OSPC and USA 300), with a Dice similarity coefficient >80%. The study was complemented by MLST, which detected three different strains: ST30, ST8, and ST45, the latter not presenting any relation with the clones compared in PFGE. CONCLUSIONS: The presence of CA-MRSA reveals an important change in the epidemiology of this pathogen and adds new elements to the knowledge of the molecular biology of infections by MRSA with SCCmec type IV in southern Brazil.