Leila de Souza Fonseca

Comparison of Flow Cytometric and Alamar Blue Tests with the Proportional Method for Testing Susceptibility of Mycobacterium tuberculosis to Rifampin and Isoniazid

ABSTRACT The performance of flow cytometry and the microplate Alamar Blue assay in determining susceptibility of Mycobacterium tuberculosis was assessed by testing 150 Brazilian isolates. The overall agreement was 97.3 and 98% for isoniazid and 94.7 and 100% for rifampin by flow cytometry and MABA, respectively. This study was entirely done in a developing country.

Correlations of mutations in katG, oxyR-ahpC and inhA genes and in vitro susceptibility in Mycobacterium tuberculosis clinical strains segregated by spoligotype families from tuberculosis prevalent countries in South America.

BackgroundMutations associated with resistance to rifampin or streptomycin have been reported for W/Beijing and Latin American Mediterranean (LAM) strain families of Mycobacterium tuberculosis. A few studies with limited sample sizes have separately evaluated mutations in katG, ahpC and inhA genes that are associated with isoniazid (INH) resistance. Increasing prevalence of INH resistance, especially in high tuberculosis (TB) prevalent countries is worsening the burden of TB control programs, since similar transmission rates are noted for INH susceptible and resistant M. tuberculosis strains.ResultsWe, therefore, conducted a comprehensive evaluation of INH resistant M. tuberculosis strains (n = 224) from three South American countries with high burden of drug resistant TB to characterize mutations in katG, ahpC and inhA gene loci and correlate with minimal inhibitory concentrations (MIC) levels and spoligotype strain family. Mutations in katG were observed in 181 (80.8%) of the isolates of which 178 (98.3%) was contributed by the katG S315T mutation. Additional mutations seen included oxyR-ahpC; inhA regulatory region and inhA structural gene. The S315T katG mutation was significantly more likely to be associated with MIC for INH ≥2 μg/mL. The S315T katG mutation was also more frequent in Haarlem family strains than LAM (n = 81) and T strain families.ConclusionOur data suggests that genetic screening for the S315T katG mutation may provide rapid information for anti-TB regimen selection, epidemiological monitoring of INH resistance and, possibly, to track transmission of INH resistant strains.

Emergence of High-Level Mupirocin Resistance in Methicillin-Resistant Staphylococcus aureus Isolated From Brazilian University Hospitals

Surveillance for methicillin-resistant Staphylococcus aureus (MRSA) was implemented in Rio de Janeiro and Uberlândia University Hospitals, which had different policies on use of mupirocin. One hundred fourteen multiresistant MRSA strains were isolated from 62 patients. Mupirocin resistance was observed in 63% of strains in Rio de Janeiro, where there was extensive use of topical mupirocin, and 6.1% in Uberlândia, where its use was rare.

Clinical Evaluation of the Microscopic-Observation Drug-Susceptibility Assay for Detection of Tuberculosis

BACKGROUND There is an urgent need for low-cost methods for rapid, accurate detection of Mycobacterium tuberculosis in clinical specimens. The microscopic-observation drug-susceptibility (MODS) assay is a relatively low-cost and simple liquid culture method that has been proposed for use in resource-limited environments. METHODS This prospective study evaluated the performance of the MODS assay for detection of M. tuberculosis in persons undergoing evaluation for pulmonary tuberculosis in Brazil and Honduras. Respiratory specimens were evaluated using smear microscopy, culture on Lowenstein-Jensen medium, and culture using the MODS assay. A subset of specimens was also cultured using the Mycobacterial Growth Indicator Tube (MGIT) 960 automated system (Becton Dickinson). A study subject was considered to have tuberculosis if at least 1 culture on Lowenstein-Jensen medium was positive for M. tuberculosis. FINDINGS A total of 1639 respiratory specimens obtained from 854 study subjects were analyzed. On a per-subject basis, MODS sensitivity was 97.5% (95% confidence interval [CI], 95.7-98.6), and specificity was 94.4% (95% CI, 93.1-95.2). Median times to detection were 21 days (interquartile range [IQR], 17-25 days) and 7 days (IQR, 5-10) for culture on Lowenstein-Jensen medium and for the MODS assay, respectively (P<.01). For 64 specimens cultured using the MGIT 960 automated system, median time to growth was similar for the MODS assay (7 days; IQR, 7-10 days) and the MGIT 960 automated system (8 days; IQR, 6-11.5 days; P=.16). The percentage of contaminated cultures was lower for the MODS assay than for culture on Lowenstein-Jensen medium (3.8% vs. 5.8%; P<.01). CONCLUSIONS The MODS assay is a relatively simple test whose good performance characteristics for detection of pulmonary tuberculosis may make it suitable for resource-limited environments.

Coagulase-Negative Staphylococci: Comparison of Phenotypic and Genotypic Oxacillin Susceptibility Tests and Evaluation of the Agar Screening Test by Using Different Concentrations of Oxacillin

ABSTRACT This study evaluated the oxacillin susceptibilities of 152 coagulase-negative staphylococcal (CoNS) strains of 12 species by disk diffusion; agar dilution; E-test; the slide latex agglutination test (Slidex MRSA Detection test; bioMérieux S/A, Paris, France); the agar screening test with 1, 2, 4, or 6 μg of oxacillin per ml and incubation for 24 or 48 h; and detection of the mecA gene by PCR. The results revealed that the agar screening test with 4 μg of oxacillin per ml and incubation for 48 h was superior to any single phenotype-based susceptibility assay, presenting a sensitivity and a specificity of 100% each. For the different methods evaluated, the sensitivities and specificities were as follows: for disk diffusion, 94.2 and 91.8%, respectively; for the agar dilution test 100 and 73.5%, respectively; for E-test, 100 and 71.4%, respectively; and for the slide latex agglutination test, 97.1 and 98%, respectively. A good correlation was observed between oxacillin susceptibility testing results and PCR results for Staphylococcus epidermidis, S. haemolyticus, S. hominis subsp. hominis, and all mecA-positive strains. However, at least 60% of the mecA-negative isolates of the species S. saprophyticus, S. cohnii subsp. urealyticum, S. lugdunensis, and S. sciuri were erroneously classified as oxacillin resistant by the agar dilution test. Conversely, the slide latex agglutination test presented a high sensitivity (97.1%) and a high specificity (98%) for all CoNS species. Our results demonstrated the accuracy of the agar screening test with 4 μg of oxacillin per ml and incubation for 48 h and the slide latex agglutination test for the appropriate detection of the oxacillin susceptibilities of CoNS isolates. Both assays are technically simple and can be easier to perform in routine laboratories than PCR.

Identification of specific proteins and peptides in Mycobacterium leprae suitable for the selective diagnosis of leprosy

Diagnosis of leprosy is a major obstacle to disease control and has been compromised in the past due to the lack of specific reagents. We have used comparative genome analysis to identify genes that are specific to Mycobacterium leprae and tested both recombinant proteins and synthetic peptides from a subset of these for immunological reactivity. Four unique recombinant proteins (ML0008, ML0126, ML1057, and ML2567) and a panel of 58 peptides (15 and 9 mer) were tested for IFN-γ responses in PBMC from leprosy patients and contacts, tuberculosis patients, and endemic and nonendemic controls. The responses to the four recombinant proteins gave higher levels of IFN-γ production, but less specificity, than the peptides. Thirty-five peptides showed IFN-γ responses only in the paucibacillary leprosy and household contact groups, with no responses in the tuberculosis or endemic control groups. High frequencies of IFN-γ-producing CD4+ and CD8+ T cells specific for the 15- and 9-mer peptides were observed in the blood of a paucibacillary leprosy patient. 9-mer peptides preferentially activated CD8+ T cells, while the 15-mer peptides were efficient in inducing responses in both the CD4+ and CD8+ T cell subsets. Four of the six 9-mer peptides tested showed promising specificity, indicating that CD8+ T cell epitopes may also have diagnostic potential. Those peptides that provide specific responses in leprosy patients from an endemic setting could potentially be developed into a rapid diagnostic test for the early detection of M. leprae infection and epidemiological surveys of the incidence of leprosy, of which little is known.

DNA typing of methicillin-resistant Staphylococcus aureus : isolates and factors associated with nosocomial acquisition in two Brazilian university hospitals

Control and prevention of methicillin-resistant Staphylococcus aureus (MRSA) infections should include early identification of patients at higher risk of MRSA acquisition and analysis of isolates by discriminatory bacterial DNA typing methods. One hundred and three MRSA isolates cultured between Sept. 1994 and Sept. 1995 from 62 patients in two teaching hospitals (hospital 1, in Rio de Janeiro; hospital 2, in Minas Gerais) were tested for antimicrobial resistance and genomic DNA was analysed by pulsed-field gel electrophoresis (PFGE). Ten profiles were identified: A, B, C, I and J in hospital 1 and A, B, D, E, F, G and H in hospital 2. PFGE patterns A and B were isolated at both hospitals. The majority (80%) of isolates had similar PFGE patterns (type A). Subtype A1 was isolated at both hospitals, but was more frequent in hospital 2 (54%), while subtype A2 predominated in hospital 1 (63%). MRSA isolates were resistant to the majority of antimicrobial agents tested. However, susceptibility to vancomycin alone was found in 32% of the isolates at hospital 1, whereas 48% of isolates from hospital 2 were susceptible to both vancomycin and mupirocin, and 34% demonstrated susceptibility to vancomycin, mupirocin and chloramphenicol. Thirty-nine percent of all isolates were mupirocin-resistant, with 90% of these belonging to PFGE pattern A. Four main risk factors were associated with MRSA infection or colonisation which may be useful in the early identification of patients at risk: >7 days hospitalisation (95%), very dependent patients (84%), invasive procedures (79%) and recent antimicrobial therapy (79%). The data demonstrate that PFGE pattern A is disseminated in both hospitals. However, at both hospitals subtypes of pattern A and the other PFGE types were associated with different antibiotic resistance patterns.

Non-tuberculous mycobacteria I: one year clinical isolates identification in Tertiary Hospital Aids Reference Center, Rio de Janeiro, Brazil, in pre highly active antiretroviral therapy era

The aim of this study was to determine the prevalence of non-tuberculous mycobacteria (NTM) isolates at University Hospital, Reference Center for Aids in Rio de Janeiro, Brazil, during one year. We used standard biochemical tests for species identification and IS1245 PCR amplification was applied as a Mycobacterium avium specific identification marker. Four hundred and four specimens from 233 patients yielded acid-fast bacilli growth. M. tuberculosis was identified in 85% of the patients and NTM in 15%. NTM disseminated infection was a common event correlated with human immunodeficiency virus (HIV) infected patients and only in HIV negative patients the source of NTM was non sterile site. M. avium complex (MAC) was biochemically identified in 57.8% (49/83) of NTM isolates, most of them from sterile sites (75.5%), and in 94% (46/49) the IS 1245 marker specific for M. avium was present. Twenty NTM strains showed a MAC biochemical pattern with the exception of a urease-positive (99% of MAC are urease-negative), however IS1245 was detected in 96% of the strains leading to their identification as M. avium. In this group differences in NTM source was not significant. The second most frequently isolated NTM was identified as M. scrofulaceum (7.2%), followed by M. terrae (3.6%), M. gordonae (2.4%), M. chelonae (1.2%), M. fortuitum (1.2%) and one strain which could not be identified. All were IS1245 negative except for one strain identified as M. scrofulaceum. It is interesting to note that non-sterile sites were the major source of these isolates (92.8%). Our finding indicated that M. avium is still the major atypical species among in the MAC isolates recovered from Brazilian Aids patients without highty active antiretroviral therapy schema. Some discrepancies were seen between the identification methods and further investigations must be done to better characterize NTM isolates using other phenotypic and genotypic methods.

RFLP patterns and risk factors for recent tuberculosis transmission among hospitalized tuberculosis patients in Rio de Janeiro, Brazil

Isolates of Mycobacterium tuberculosis from 120 tuberculosis patients seen in the 12 months ending September 1994 at 2 tertiary-care centres in Rio de Janeiro were characterized by IS6110 restriction fragment length polymorphism (RFLP) analysis. Ninety-seven patients (81%) had isolates with unique RFLP patterns, while 23 patients (19%) had isolates that belonged to 11 different RFLP cluster patterns. The strains from the latter patients were distributed among 1 group of 3 patients and 10 groups of 2 patients each. The cluster-pattern strains were not associated with gender, age, HIV infection, type of residence, living in shelter, homelessness or previous history of tuberculosis. However, clustering was strongly associated with multidrug resistance (P = 0.006). These data suggest that recent exogenous transmission may be important for the development of new cases of multidrug-resistant disease in patients attending tertiary-care centres in Rio de Janeiro, Brazil.

Surgical site infection: Rates, etiology and resistance patterns to antimicrobials among strains isolated at rio de janeiro university hospital

SummaryA 6-month prospective surveillance was conducted in the Department of General Surgery of the Rio de Janeiro University Hospital. Postoperative infections were classified according to CDC criteria. This study reports a significant rate (16.9%) in surgical infections detected by surveillance in a series where 45% of surgical interventions were classified as clean. The majority (52.7%) was apparent only after patient discharge from the hospital. Bacterial cultures were obtained from 42 out of 55 infected wounds.Staphylococcus aureus was the most frequently found pathogen (33.9%), followed byEscherichia coli (20.3%). With the exception ofS. aureus isolates, multiresistance was found in 66% of coagulase-negative staphylococci and 60% of gram-negative bacteria. This study showed that community surveillance associated with hospital surveillance is necessary in order to determine accurate rates of surgical site infections, and also showed that the emergence of multiresistant bacterial strains was common among isolates of surgical infections.A 6-month prospective surveillance was conducted in the Department of General Surgery of the Rio de Janeiro University Hospital. Postoperative infections were classified according to CDC criteria. This study reports a significant rate (16.9%) in surgical infections detected by surveillance in a series where 45% of surgical interventions were classified as clean. The majority (52.7%) was apparent only after patient discharge from the hospital. Bacterial cultures were obtained from 42 out of 55 infected wounds.Staphylococcus aureus was the most frequently found pathogen (33.9%), followed byEscherichia coli (20.3%). With the exception ofS. aureus isolates, multiresistance was found in 66% of coagulase-negative staphylococci and 60% of gram-negative bacteria. This study showed that community surveillance associated with hospital surveillance is necessary in order to determine accurate rates of surgical site infections, and also showed that the emergence of multiresistant bacterial strains was common among isolates of surgical infections.