Carmen Maria Saraiva Giampaglia

Molecular characterization of Mycobacterium kansasii isolates in the State of São Paulo between 1995-1998

Mycobacterium kansasii is the most common cause of pulmonary nontuberculous mycobacteria infection and classical identification of this pathogen needs a time consuming phenotypic tests. Polymerase chain reaction-restriction fragment length polymorphism analysis (PRA) of the gene enconding for the 65 kDa heat shock (hsp65) protein offers an easy, rapid, and inexpensive procedure to identify and subtype M. kansasii isolates. In the present study, we performed a retrospective analysis of patients who had mycobacteria identified on the basis of phenotypic tests by means of a review of database at Mycobacteria Laboratory of the Instituto Adolfo Lutz in the period 1995-1998. A total of 9381 clinical isolates were analyzed of which 7777 (82.9%) were identified as M. tuberculosis complex and 1604 (17.1%) as nontuberculous mycobacteria. Of the 296 M. kansasii isolates, 189 (63.8%) isolates obtained from 119 patients were viable and were analyzed by PRA-hsp65. Hundred eight two (98.9%) were classified as M. kansasii type I. Two isolates were classified as type II and III and five isolates were characterized as other Mycobacterium species. Clinical isolates of M. kansasii in the state of Sao Paulo was almost exclusively subtype I regardless of HIV status.

Cord formation and colony morphology for the presumptive identification of Mycobacterium tuberculosis complex

The identification of Mycobacterium tuberculosis complex (MT), using non-molecular methods, is time-consuming. The objective of this study was to evaluate a screening test for the presumptive identification of MT, which could potentially decrease laboratory turn-around time for reporting preliminary results. From January 1998 to December 1999, 3056 cultures were analysed at the Mycobacterial Laboratory, Instituto Adolfo Lutz, Sao Paulo, Brasil. The screening test consisted of observation of colony morphology on Lowenstein Jensen medium and evaluation of cord formation on smear microscopy from those positive cultures. After the screening test, the cultures identified as non-tuberculous mycobacteria were identified to species by conventional methods (growth on culture and biochemical tests). Those identified as MT were submitted to drug susceptibility tests. The presumptive identification of MT using the proposed screening test, when compared with conventional tests, presented 98.9, 86.9, 97.8 and 93.0% of sensitivity, specificity, positive and negative predictive values, respectively. The conclusion is that it is possible to make a presumptive identification of MT using visual analysis of colony morphology and cord formation on microscopy examination. This method could be used to report the presumptive identification of MT and to guide laboratory decisions regarding susceptibility and identification tests with little cost and in a very practical way.

Rapid detection of resistant tuberculosis by nitrate reductase assay performed in three settings in Brazil

OBJECTIVES To evaluate nitrate reductase assay (NRA) efficacy for streptomycin, isoniazid, rifampicin and ethambutol susceptibility testing of Mycobacterium tuberculosis strains. METHODS Results were generated by three laboratories: the Instituto Adolfo Lutz (IAL) Mycobacteria Reference Laboratory and two IAL Regional Laboratories in Santo André and Sorocaba, São Paulo State, Brazil. One hundred and twenty M. tuberculosis strains were simultaneously tested using NRA and the proportion method (PM), while 117 strains were tested using both NRA and BACTEC MGIT 960 (M960). RESULTS Repeatability analysis of NRA results showed rates of 100% for isoniazid and ethambutol and 97% for streptomycin and rifampicin susceptibility detection, representing substantial agreement. McNemar testing of the data also indicates that NRA and PM, as well as NRA and M960, do not differ significantly. On average, NRA results were available after 10 days. CONCLUSIONS The data demonstrate that NRA is reliable for susceptibility testing of isoniazid and rifampicin, the two most important drugs for the treatment of tuberculosis. In addition, the reduction in the time necessary to obtain susceptibility results is of fundamental importance.

Population structure and circulating genotypes of drug-sensitive and drug-resistant Mycobacterium tuberculosis clinical isolates in São Paulo state, Brazil

São Paulo is the most populous Brazilian state and reports the largest number of tuberculosis cases in the country annually (over 18,500). This study included 193 isolates obtained during the 2nd Nationwide Survey on Mycobacterium tuberculosis Drug Resistance that was conducted in São Paulo state and 547 isolates from a laboratory based study of drug resistance that were analyzed by the Mycobacteria Reference Laboratory at the Institute Adolfo Lutz. Both studies were conducted from 2006 to 2008 and sought to determine the genetic diversity and pattern of drug resistance of M. tuberculosis isolates (MTC) circulating in São Paulo. The patterns obtained from the spoligotyping analysis demonstrated that 51/740 (6.9%) of the isolates corresponded to orphan patterns and that 689 (93.1%) of the isolates distributed into 144 shared types, including 119 that matched a preexisting shared type in the SITVIT2 database and 25 that were new isolates. A total of 77/144 patterns corresponded to unique isolates, while the remaining 67 corresponded to clustered patterns (n=612 isolates clustered into groups of 2-84 isolates each). The evolutionarily ancient PGG1 lineages (Beijing, CAS1-DEL, EAI3-IND, and PINI2) were rarely detected in São Paulo and comprised only 13/740, or 1.76%, of the total isolates; all of the remaining 727/740, or 98.24%, of the MTC isolates from São Paulo state were from the recent PGG2/3 evolutionary isolates belonging to the LAM, T, S, X, and Haarlem lineages, i.e., the Euro-American group. This study provides the first overview of circulating genotypes of M. tuberculosis in São Paulo state and demonstrates that the clustered shared types containing seven or more M. tuberculosis isolates that are spread in São Paulo state included both resistant and susceptible isolates.

Modified protocol for drug susceptibility testing of MGIT cultures of Mycobacterium tuberculosis by the MGIT 960

A rapid detection of resistance in Mycobacterium tuberculosis is crucial for management and control of tuberculosis. This study evaluated a more rapid and cost-effective drug susceptibility testing (DST) protocol using primary isolates of M. tuberculosis in mycobacteria growth indicator tube (MGIT). Ninety-four M. tuberculosis isolates in MGIT were subjected to DST by the manufacturers method, i.e., primary isolates were subcultured and DST was performed from positive cultures for a maximum of 5days; and by our modified method, i.e., DST was performed directly from primary MGIT cultures positive for more than 5days. Results were concordant for 76 (81%) isolates. Agreement between both methods was 92.0%, 98.9%, 97.7%, and 95.5% for streptomycin, isoniazid, rifampicin, and ethambutol, respectively. Six isolates failed to grow on the recommended method, including 3 resistant isolates. Not performing subculture of primary M. tuberculosis isolates yields reliable results, decreasing the turnaround time and the cost of the test.

Viability of stressed Mycobacterium tuberculosis and association with multidrug resistance.

This study investigated biological characteristics of recovered stressed M. tuberculosis isolates that failed to grow in differential culture media for phenotypic identification and in culture media containing anti-tuberculosis drugs for drug-susceptibility testing, despite of having grown in primary culture. It represents an improvement in the diagnosis of MDR tuberculosis and tuberculosis control.

Monitoramento em cabine de segurança biológica: manipulação de cepas e descontaminação em um laboratório de micobactérias

OBJECTIVES: To verify the evidence of aerosol formation during the manipulation of mycobacteria strains for susceptibility (ST) and identification tests (IT) as well as the decontamination effect of alcohol solution 70% and ultraviolet (UV) radiation in biological safety cabinets (BSC) after laboratory procedures. METHODS: One plate was exposed in a BSC during ST and IT procedures. Afterwards, the BSC was cleaned and decontaminated with alcohol solution 70% and exposed to UV radiation for 15 minutes. After that, another plate was exposed for two hours, only with the BSC ventilation on. Both plates were incubated at 37°C and observed for 30 days. The smears from the isolated colonies were stained with Ziehl Neelsen and Gram techniques, and acid fast bacilli (AFB) were identified by conventional methods. RESULTS: In 38 plates exposed during ST, there was mycobacteria growth in 10 plates (26.3%), fungi in one (2.6%) and bacilli in two (5.3%). Among those plates that presented mycobacteria growth, eight (80%) were identified as M. tuberculosis and two (20%) had inconclusive identification. Even after decontamination with alcohol solution 70% and UV radiation, two plates presented fungi growth (5.3%) and other two presented cocci growth (5.3%). Among 30 plates exposed during IT procedures, there was mycobacteria growth in 10 of them (33.3%), fungi in two (6.6%), cocci in one (3.4%) and one (3.4%) mixed mycobacteria and another bacillus. No growth was observed when alcohol solution 70% and UV radiation were used for decontamination after IT procedures. CONCLUSION: During the procedures there was aerosol formation with mycobacteria, which was proved by mycobacteria growth on the exposed plates. Not only should adequate laboratory techniques be respected to minimize aerosol formation, but professional expertise, the continuity of capacity programs and periodic BSC maintenance should also be observed.