New multilocus sequence typing of MRSA in São Paulo, Brazil

An increased incidence of nosocomial and community-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has been observed worldwide. The molecular characterization of MRSA has played an important role in demonstrating the existence of internationally disseminated clones. The use of molecular biology methods in the surveillance programs has enabled the tracking of MRSA spread within and among hospitals. These data are useful to alert nosocomial infection control programs about the potential introduction of these epidemic clones in their areas. Four MRSA blood culture isolates from patients hospitalized at two hospitals in the city of São Paulo, Brazil, were analyzed; one of them was community acquired. The isolates were characterized as SCCmec, mecA and PVL by PCR, pulsed-field gel electrophoresis (PFGE) profile and molecular sequence typing (MLST) genotyping. The isolates presented type IV SCCmec, and none proved to be positive for PVL. The isolates showed a PFGE profile similar to the pediatric clone. MLST genotyping demonstrated that the isolates belonged to clonal complex 5 (CC5), showing a new yqiL allele gene, resulting in a new sequence typing (ST) (1176). Our results showed that strains of MRSA carrying a new ST are emerging in community and nosocomial infections, including bacteremia, in São Paulo, Brazil.

Evaluation of two detection methods of microorganisms in platelet concentrates.

BACKGROUND: The performance of a bacterial 16S ribosomal DNA real-time polymerase chain reaction (PCR) assay was evaluated and validated with an automated culture system to determine its use for screening of platelet concentrates (PCs). STUDY DESIGN AND METHODS: PCs were spiked with suspensions of Escherichia coli, Serratia marcescens, Staphylococcus epidermidis and St. aureus at 1, 10, and 100 colony-forming units (CFUs) mL and stored for 5 days. DNA amplification was performed using real-time PCR. The BacT/ALERT was used as a reference method and samples were inoculated into an aerobic culture bottle; for the PCR assay, aliquots were drawn from all (spiked) PCs on days 0 to 5 of storage. RESULTS: Real-time PCR detected only the gram-positive bacteria in PCs spiked with low bacterial titres (1 CFU mL) after 48 h; however, it was able to detect all positive samples in PCs spiked with 10 CFU mL of either gram-positive or gram-negative bacteria after 48 h. In addition, real-time PCR detected all positive samples in PCs spiked with high gram-positive bacterial titres (100 CFU mL) after 24 h. On the other hand, the BacT/ALERT system showed positive results in all samples within 24 h. CONCLUSION: The BacT/ALERT method is more sensitive and should continue to be the gold standard for identifying bacterial contaminations in blood samples. The real-time PCR approach can be used for the screening of PCs for microbial detection before they are released from blood centres or shortly before they are used in blood transfusion, and thus allow an extended shelf life of the platelets.

New multilocus sequence typing of MRSA in São Paulo, Brazil.

An increased incidence of nosocomial and community-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has been observed worldwide. The molecular characterization of MRSA has played an important role in demonstrating the existence of internationally disseminated clones. The use of molecular biology methods in the surveillance programs has enabled the tracking of MRSA spread within and among hospitals. These data are useful to alert nosocomial infection control programs about the potential introduction of these epidemic clones in their areas. Four MRSA blood culture isolates from patients hospitalized at two hospitals in the city of São Paulo, Brazil, were analyzed; one of them was community acquired. The isolates were characterized as SCCmec, mecA and PVL by PCR, pulsed-field gel electrophoresis (PFGE) profile and molecular sequence typing (MLST) genotyping. The isolates presented type IV SCCmec, and none proved to be positive for PVL. The isolates showed a PFGE profile similar to the pediatric clone. MLST genotyping demonstrated that the isolates belonged to clonal complex 5 (CC5), showing a new yqiL allele gene, resulting in a new sequence typing (ST) (1176). Our results showed that strains of MRSA carrying a new ST are emerging in community and nosocomial infections, including bacteremia, in São Paulo, Brazil.

An outbreak of catalase-negative methicillin-resistant Staphylococcus aureus.

The wide dissemination of a major epidemic methicillin-resistant Staphylococcus aureus (MRSA) clone in Brazilian hospitals (Brazilian clone) limits the value of molecular typing techniques such as pulsed-field gel electrophoresis (PFGE) for outbreak investigation. We report the first outbreak of a catalase-negative strain of MRSA, which was initially detected by the unusual result of this phenotypical test. The outbreak occurred in the Hospital Sanatorinhos de Carapicuíba, a 237-bed secondary hospital located in São Paulo, Brazil. From May to August 2002, a total of 11 MRSA isolates were recovered from four patients in the intensive care unit. All the isolates were catalase negative and susceptible only to vancomycin and linezolid. Three of the four patients eventually died. Molecular typing demonstrated an indistinguishable PFGE pattern among the 11 isolates, with similarities to the Brazilian clone and the hospital's usual MRSA strain. This report emphasizes the importance of an uncommon phenotypical result as a marker for initiating an outbreak investigation and should encourage clinical laboratories to recognize and report such isolates.

Activity of mupirocin and 14 additional antibiotics against staphylococci isolated from Latin American hospitals: report from the SENTRY antimicrobial surveillance program.

A total of 1,346 Staphylococcus aureus (SA) and 498 coagulase-negative staphylococcal (CoNS) strains isolated from 11 Latin American medical centers between 2000 and 2001 were tested against mupirocin and other antimicrobial agents by reference broth microdilution method as part of the SENTRY Antimicrobial Surveillance Program. Oxacillin resistance (OR) was detected in 38.6% of S. aureus and in 78.1% of CoNS. The overall resistance rate to mupirocin was low among S. aureus (3.1%; MIC > or =8 microd/ml) but significantly higher among ORSA compared to oxacillin-susceptible SA (5.4% versus 1.7%; p <0.001). Mupirocin-resistant S. aureus strains were detected in 9 of 11 centers, with individual center rates varying between 1.8 and 15.7%. Mupirocin resistance rates were high among CoNS (27.5%) and varied widely (10.0 to 48.9%) among the monitored Latin American medical centers. Mupirocin resistance rates appear to be increasing and routine monitoring for potential resistance seems prudent.

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols. Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection. Reticulocyte counting by microscopic examination was employed as the end-point using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry. Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared. Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision. Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 12.1-28.4 percent for the brilliant cresyl blue method, 14.1-30.8 percent for the selective hemolysis method and 8.5-19.7 percent for the flow cytometry method. Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay. Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 5.9-14.2 percent), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products.

Comparative evaluation of the human whole blood and human peripheral blood monocyte tests for pyrogens.

Two different in vitro tests for pyrogens, using human peripheral blood monocytes (PBMNC) and diluted whole blood (WBC), respectively, were applied to different classes of parenteral medicinal products. Many of these products did not have a specified endotoxin limit concentration that was established as the maximum valid dilution to comply with the test. The results of the in vitro tests for pyrogens were compared with the results from the Limulus amoebocyte lysate (LAL) and rabbit pyrogen tests. The Second International Standard for endotoxin was used to calibrate all of the assays and the International Standard for IL-6 was used to calibrate the IL-6 ELISA which provided the readout for the in vitro tests for pyrogens. Preparatory tests were conducted to ensure that the "criteria for validity and precision of the standard curve" were satisfied and that the drugs being tested did not interfere in the tests. The PBMNC/IL-6 test had a detection limit of 0.06 EU/ml and spike recoveries were 62-165%. The whole blood/IL-6 test also had a detection limit of 0.06 EU/ml and spike recoveries were 58-132%. The application to the detection of non-endotoxin pyrogens needs to be evaluated in more detail, but the two in vitro tests for pyrogens showed good agreement overall, both with each other and with the LAL test and the rabbit pyrogen test for the detection of endotoxins.

Biological evaluation of recombinant human erythropoietin in pharmaceutical products.

The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols. Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection. Reticulocyte counting by microscopic examination was employed as the end-point using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry. Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared. Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision. Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 12.1-28.4% for the brilliant cresyl blue method, 14.1-30.8% for the selective hemolysis method and 8.5-19.7% for the flow cytometry method. Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay. Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 5.9-14.2%), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products.

Early electrocardiographic abnormalities in Trypanosoma cruzi-seropositive children.

As part of a major epidemiologic study on Chagas' disease, we compared the prevalence of electrocardiographic (ECG) abnormalities among 141 school children 7-12 years of age and seropositive for Trypanosoma cruzi, and 282 age-, sex-, and school-matched seronegative children in an endemic area in Brazil. The prevalence of ECG abnormalities was 11.3% among seropositive children and 3.5% among seronegative children (odds ratio = 3.5, 95% confidence interval [CI] = 1.5-8.4). The prevalence rate of ECG alterations was 10.7% for seropositive males versus 8.9% for seropositive females. Complete right bundle branch block (CRBBB), which is highly suggestive of Chagas' disease cardiopathy, was diagnosed in nine (6.4%) seropositive children and in only one (0.3%) seronegative child (odds ratio = 18.5, 95% CI = 2.3-146.5, attributable fraction = 58.3%). Five incident new cases of CRBBB were diagnosed after a 36-month follow-up of seropositive children who were enrolled in an independent clinical field trial. No case of frequent and/or multifocal ventricular premature beats was found in the cohort of children. The surprisingly high frequency of early ECG abnormalities, which indicates a rapid evolution from infection to disease, suggests the existence of endemic areas with a particular accelerated disease progression that was not described before. Under such conditions, a public health chemotherapy program focusing on the treatment of young seropositive children would be recommended.

Randomised trial of efficacy of benznidazole in treatment of early Trypanosoma cruzi infection.

BACKGROUND: Benznidazole, a nitroimidazole derivative, has been recommended for the treatment of acute and congenital Trypanosoma cruzi infection (Chagas' disease). We have examined the safety and efficacy of this drug in the treatment of the early chronic phase of T cruzi infection. METHODS: Between 1991 and 1995, we carried out a randomised, double-blind, placebo-controlled trial in a rural area of Brazil with endemic Chagas' disease. 82% of 2434 schoolchildren (aged 7-12 years) identified in a census were screened for antibodies to T cruzi by indirect immunofluorescence, indirect haemagglutination, and ELISA. 130 were positive in all tests and were randomly assigned benznidazole (7.5 mg/kg daily for 60 days by mouth) or placebo. The primary endpoint for efficacy was the disappearance of specific antibodies (negative seroconversion) by the end of 3-year follow-up. The secondary endpoint was the reduction of antibody titres on repeated serological tests. One child moved away from the area just after randomisation and was excluded from the analyses. Insecticidal measures were taken throughout the trial to reduce the risk of reinfection. FINDINGS: Minor side-effects requiring no specific medication were recorded in a small proportion of individuals. On a chemiluminescent ELISA with purified trypomastigote glycoconjugate, serum from all participants was positive at the beginning of the trial. At the end of follow-up, 37 (58%) of the 64 benznidazole-treated participants and 3 (5%) of those who received placebo were negative for T cruzi antibodies. The efficacy of benznidazole treatment estimated by intention to treat was 55.8% (95% CI 40.8-67.0). At the end of follow-up, children who received benznidazole had five-fold lower geometric mean titres by indirect immunofluorescence than placebo-treated children (196[147-256] vs 1068[809-1408], p < 0.00001). INTERPRETATION: The trial showed that a 60-day course of benznidazole treatment of early chronic T cruzi infection was safe and 55.8% effective in producing negative seroconversion of specific antibodies. The results are very encouraging and justify the recommendation of treatment for seropositive children as public health policy.

Evaluation of risk factors for house infestation by Triatoma infestans in Brazil.

An active entomologic survey was conducted by a team of trained health workers in a rural area endemic for Chagas' disease in central Brazil. They used pyrethrum as a flushing agent and 4,232 houses were inspected for triatomine bugs both inside and in the immediate environs. Houses with Triatoma infestans or evidence of an established colony were identified and defined as infested houses (cases). The building and environmental characteristics of 161 randomly selected infested houses were compared with 161 matched, noninfested houses (controls) that were the shortest distance from the infested house. Domestic and peridomestic potential risk factors associated with house infestation by Triatoma infestans were assessed by logistic regression analysis. Incomplete house construction (odds ratio [OR] = 2.5, 95% confidence interval [CI] = 1.5-4.1) was confirmed as a risk factor related to the presence or evidence of Triatoma infestans in the dwellings. The study also disclosed a statistically significant association between the presence of rats (OR = 1.6, 95% CI = 1.1-2.6) and indoor crop storage (OR = 2.3, 95% CI = 1.1-5.2) and house infestation. Further experimental field studies using tagged rodents should be conducted to assess their epidemiologic role in the domestic chain of Trypanosoma cruzi transmission.