Evaluation of IS6110 as amplification target for direct tuberculosis diagnosis.

We describe in the present study an evaluation of the IS6110 repetitive element in the rapid diagnosis of pulmonary and extrapulmonary tuberculosis by polymerase chain reaction (PCR). A pair of oligonucleotide primers was designed to amplify a 201-bp DNA fragment of IS6110. The amplified DNA was detected by ethidium bromide stained agarose gel electrophoresis and confirmed by Sal I digestion and Southern blot hybridization with a 32P-labeled probe. To detect the presence of amplification inhibitors, an internal control DNA that used the same primers as for the target sequence was added to each PCR reaction. PCR results were compared with the results of acid fast stained smears, cultures, and clinical data in 102 sputum and 41 extrapulmonary specimens. With the exception of four samples, M. tuberculosis was detected by PCR in all smear- and culture-positive cases and in all smear-negative, culture positive cases. Additionally, PCR was able to detect 6 cases that were smear and culture negative but clinically strongly suspected of tuberculosis. The final PCR sensitivity and specificity were 93.1% and 95.18%, respectively. One M. tuberculosis strain isolated from a sputum was found to lack IS6110. This study shows that (1) PCR diagnosis based on IS6110 reached the best sensitivity and specificity but must be considered carefully since some M. tuberculosis strains lack IS6110; and (2) PCR must be interpreted in conjunction with clinical and radiological data when it is discordant with conventional methods results.

Survey of primary drug resistance of Mycobacterium tuberculosis in Casablanca, Morocco.

SETTING: In 1990, a 6-month short-course regimen (2 SHRZ/4 RH) was introduced for the treatment of tuberculosis in Morocco. OBJECTIVE: To assess the efficacy of the national tuberculosis control programme, a prospective study of primary drug resistance was conducted from April 1992 to July 1994 in Casablanca. DESIGN: A total of 402 strains isolated from 402 patients living in Casablanca with no previous history of tuberculosis was included in the study. RESULTS: The overall rate of primary drug resistance to at least one drug was 23.9%; it was 19.7% to streptomycin, 11.4% to isoniazid, and 8.2% to both streptomycin and isoniazid. The rates of resistance to rifampicin and ethambutol were both less than 1%. The survey was divided into two periods of 14 months each. The rates of primary drug resistance increased from 21.1% to 27.6% during these two periods (Odds Ratio [OR] 1.43; 95% Confidence Interval [CI] 0.88 to 2.32); this increase occurred only for streptomycin (15.9% to 24.7%, OR 1.73; 95% CI 1.02 to 2.93). CONCLUSION: The rate of primary drug resistance of Mycobacterium tuberculosis in Casablanca has risen in recent years to an ominous level. Urgent measures are needed in order to interrupt this trend.

[Molecular typing of Moroccan strains of Mycobacterium tuberculosis]. / Typage moléculaire de souches marocaines de Mycobacterium tuberculosis.

The insertion sequence IS 6110 was used to differentiate clinical Moroccan isolates of Mycobacterium tuberculosis by using two non radioactive probes. Among 16 strains isolated from patients clinically related, 10 had similar IS 6110 restriction fragment length polymorphism (RFLP) patterns, confirming that they were derived from a common source. Two strains were isolated from the same patient (sputum, lymph node) showed identical profiles hybridized with IS 6110 element. Four sequential strains isolated from the same patients before treatment and after one year had identical IS 6110 RFLP patterns suggesting relapse and not reinfection. Twenty-one strains with identical drug susceptibility showed different IS 6110 RFLP profiles confirming no correlation between antibiotic resistance profiles and IS 6110 RFLP patterns. Since RFLP analysis by using IS 6110 element is a useful tool for the epidemiological survey, of tuberculosis.

[PCR detection of Mycobacterium tuberculosis lacking IS 6110]. / Détection par PCR de Mycobacterium tuberculosis sans IS 6110.

We have evaluated the frequency of M. tuberculosis strains which lack IS 6110 among 102 sputa isolated from Moroccan patients. A pair of primers was designed to amplify a 201bp DNA fragment of IS 6110. The amplified DNA was detected by ethidium bromide stained agarose gel electrophoresis and confirmed by southern blot hybridization with a 32P-labelled probe (PMTO2). To detect the presence of amplification inhibitors, an internal control DNA was added in each negative PCR result. Among 102 samples, 6 sputa were negative by PCR-IS 6110 but culture positive. The test of detection of M. tuberculosis for 2/6 sputa by PCR Amplicor amplifying 584 pb of rRNA 16s sequence was positive. RFLP analysis of these 2 strains revealed no bands hybridizing IS 6110 but PCR-Mt 308 was positive. These results confirmed that these M. tuberculosis strains are lacking IS 6110.

Isolation and characterization of temperature sensitive mutants of the F5 deletion mutant of mycobacteriophage D29.

Seven thermosensitive mutants of the F5 deletion mutant of the mycobacteriophage D29 were described. The mutants were obtained using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis, and were characterized using temperature shift assays, complementation and recombination tests, electron microscopy of infected host bacteria at non-permissive temperature, and serum blocking power. Mutants deficient in tail assembly, and mutants deficient in head and tail assembly were described. Mutants deficient in head assembly but capable of assembling tails were not isolated during this study. From the data, 3 provisional linkage map of the phage F5 was proposed.

Conjugative transfer of a shuttle plasmid from Escherichia coli to Mycobacterium smegmatis [corrected].

The chimeric plasmid pMY10 containing the origin of replication of pAL5000, the origin of replication of pBR322, the origin of transfer of pRK2 and a kanamycin resistance gene was constructed and successfully transferred by conjugation from Escherichia coli harbouring the helper plasmid pRK4.24 into Mycobacterium smegmatis. This is the first report of conjugtive transfer of plasmid between E. coli and an acid fast organism.

Restriction map of mycobacteriophage D29 and its deletion mutant F5.

A physical map of mycobacteriophage D29 was constructed, including positions for 25 restriction sites for 9 endunocleasic enzymes. D29 DNA contains about 48 150 bp. Analysis of a deletion mutant (F5) has allowed to determine the location of two non essential regions in the genome, allowing further insertion of foreign genes and construction of cosmids.