Transmission dynamics and molecular characterization of Mycobacterium tuberculosis isolates with low copy numbers of IS6110.

Population-based analysis of Mycobacterium tuberculosis transmission in Houston, Tex., over 5 years identified 377 patients infected with an isolate containing one to four copies of IS6110. The isolates were analyzed by spoligotyping and assigned to one of three major genetic groups based on nucleotide polymorphisms in codons katG 463 and gyrA 95. Prospectively obtained patient interviews were reviewed to assess epidemiologic links between apparently clustered patients. A total of 13 groups of isolates with the same IS6110 profile were identified, representing 326 of the 377 patients (86.5%; range 2 to 113 patients). In contrast, 28 groups of isolates containing 334 patients (88.6%) had the same spoligotype (range, 2 to 143 patients). Combination of IS6110 profile and spoligotype data identified 31 clusters with 300 patients (79.6%; range, 2 to 82 patients). All 377 isolates belonged to major genetic group 1 (77 patients) or genetic group 2 (300 patients); no major genetic group 3 isolates were identified. Among the 228 patients interviewed, 33 patients (14.5%) were directly linked to another patient in the same cluster. Possible epidemiologic links were also found among 11 patients. Moreover, many clusters consisted of individuals with the same ethnicity. In conclusion, we confirmed that IS6110 profiling and spoligotyping together provide enhanced molecular discrimination of M. tuberculosis isolates with low copy numbers of IS6110. Identification of epidemiologic links among some of the patients verified that the combination of these two methods reliably indexes tuberculosis transmission.

Extensive cross-contamination of specimens with Mycobacterium tuberculosis in a reference laboratory.

A striking increase in the numbers of cultures positive for Mycobacterium tuberculosis was noticed in a mycobacterial reference laboratory in Campinas, Sao Paulo State, Brazil, in May 1995. A contaminated bronchoscope was the suspected cause of the increase. All 91 M. tuberculosis isolates grown from samples from patients between 8 May and 18 July 1995 were characterized by spoligotyping and IS6110 fingerprinting. Sixty-one of the 91 isolates had identical spoligotype patterns, and the pattern was arbitrarily designated S36. The 61 specimens containing these isolates had been processed and cultured in a 21-day period ending on 1 June 1995, but only 1 sample was smear positive for acid-fast bacilli. The patient from whom this sample was obtained was considered to be the index case patient and had a 4+ smear-positive lymph node aspirate that had been sent to the laboratory on 10 May. Virtually all organisms with spoligotype S36 had the same IS6110 fingerprint pattern. Extensive review of the patients' charts and investigation of laboratory procedures revealed that cross-contamination of specimens had occurred. Because the same strain was grown from all types of specimens, the bronchoscope was ruled out as the outbreak source. The most likely source of contamination was a multiple-use reagent used for specimen processing. The organism was cultured from two of the solutions 3 weeks after mock contamination. This investigation strongly supports the idea that M. tuberculosis grown from smear-negative specimens should be analyzed by rapid and reliable strain differentiation techniques, such as spoligotyping, to help rule out laboratory contamination.

Genotypic characterization of drug-resistant Mycobacterium tuberculosis isolates from Peru.

SETTING: Twenty-nine epidemiological unrelated and mostly multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strains from Peruvian patients. OBJECTIVE: To investigate the molecular genetics of MDR-TB strains recovered in a Latin American country. DESIGN: Antimicrobial agent susceptibility testing, major genetic group designation, IS6110 fingerprinting, spoligotyping, and automated deoxyribonucleic acid sequencing of regions of the katG, rpoB, embB, gyrA, and pncA genes with mutations commonly associated with drug resistance. RESULTS: Nineteen isolates were found to be multidrug-resistant by susceptibility testing. IS6110 typing showed that virtually all isolates were unique and therefore had independently acquired drug resistance. Seventy-nine percent of isoniazid-resistant strains had a Ser315Thr amino acid change in KatG. Ninety-five percent of rifampin-resistant isolates had amino acid replacements in the rifampin-resistance determining region of RpoB. Six of 11 ethambutol-resistant strains had EmbB alterations. Eleven pyrazinamide-resistant strains had distinct mutations in pncA. CONCLUSION: Virtually all organisms evolved drug resistance independently. The types of drug resistance-associated mutations identified were very similar to changes occurring in isolates from other areas of the world. Nucleotide sequence-based strategies for rapid detection of drug resistance-conferring mutants will be applicable to organisms recovered in Peru, and potentially other areas of Latin America.

Characterization of group A Streptococcus strains recovered from Mexican children with pharyngitis by automated DNA sequencing of virulence-related genes: unexpectedly large variation in the gene (sic) encoding a complement-inhibiting protein.

Sequence variation was studied in several target genes in 54 strains of group A Streptococcus (GAS) cultured from children with pharyngitis in Mexico City. Although 16 distinct emm alleles were identified, only 4 had not been previously described. Virtually all bacteria (31 of 33 [94%] with the streptococcal pyrogenic exotoxin gene (speA) had emm1-related, emm3, or emm6 alleles. The gene (sic) encoding an extracellular GAS protein that inhibits complement function was unusually variable among isolates with the emm1 family of alleles, with a total of seven variants identified. The data suggest that many GAS strains infecting Mexican children are genetically similar to organisms commonly encountered in the United States and western Europe. Sequence variation in the sic gene is useful for rapid differentiation among GAS isolates with the emm1 family of alleles.

Molecular epidemiology of Haemophilus influenzae within families in Santiago, Chile.

Thirty-six consecutive patients with invasive Haemophilus influenzae type b (Hib) infections at Roberto del Rio Children's Hospital, Santiago, Chile, were enrolled in a prospective study. Throat cultures were obtained from household contacts of each index case, adjacent neighbors, and matched community control households. Colonization rates for H. influenzae were comparable among groups; however, among household contacts 18% of colonizing isolates were Hib, compared with 2% and 3% among neighbor and community controls. When selected isolates were evaluated further by outer membrane protein (OMP) profiles and multilocus enzyme electrophoresis, only one of the four Hib isolates from household members matched the corresponding index case isolate. One serologically nontypeable isolate from a household contact had an OMP profile and electrophoretic type identical to that of the corresponding Hib index case isolate; hybridization studies with a 9-kb capsular gene probe showed a profile consistent with a capsule-deficient mutant. Hib strains were isolated more frequently from household contacts than from control persons living in Santiago, but colonizing Hib strains were often unrelated to the index case strain.

Brazilian purpuric fever: evolutionary genetic relationships of the case clone of Haemophilus influenzae biogroup aegyptius to encapsulated strains of Haemophilus influenzae.

As a first step toward identifying the evolutionary origin of a pathogenic clone of Haemophilus influenzae biogroup aegyptius causing Brazilian purpuric fever, chromosomal variation and genetic relationships were indexed among 17 isolates of biogroup aegyptius and 2209 previously characterized encapsulated H. influenzae strains recovered from 30 countries on six continents. Biogroup aegyptius isolates form three distinct evolutionary lineages of the species H. influenzae and isolates of the case clone are genetically not closely related to other isolates classified as biogroup aegyptius. The Brazilian purpuric fever case clone was found to be genetically allied with H. influenzae isolates producing serotype c polysaccharide capsule. The population genetic evidence suggests that biogroup aegyptius isolates may represent cell lineages occasionally transmitted from nonhuman hosts or spawned from a much larger base population consisting of genetically diverse nonpathogenic precursor clones.

Effect of a novel leukotriene D4 antagonist with 5-lipoxygenase and cyclooxygenase inhibitory activity, Wy-45,911, on leukotriene-D4-and antigen-induced bronchoconstriction in the guinea pig.

Wy-45,911 (4-[hydroxy-[3-(2-quinolinylmethoxy)phenyl] amino]-4-oxabutanoic acid, methyl ester) was found to inhibit competitively leukotriene D4 (LTD4)-induced contractions of the isolated guinea pig trachea but not those of leukotriene C4 (LTC4), even in the presence of a gamma-glutamyltranspeptidase inhibitor, reduced glutathione (GSH). Tracheal contractions induced by histamine or pilocarpine were also not significantly altered by Wy-45,911. The drug inhibited the tracheal contractions induced by antigen, even in the presence of GSH. This latter effect resulted from inhibition of 5-lipoxygenase (5-LO), as the synthesis of 5-LO products by rat polymorphonuclear leukocytes and by mouse macrophages was markedly reduced by Wy-45,911. The drug inhibited both LTD4-induced and antigen-induced bronchoconstriction when injected intraduodenally or intragastrically into intact guinea pigs though it was more potent against LTD4-induced bronchoconstriction. We conclude that Wy-45,911 is a novel, orally active LTD4 antagonist in the guinea pig, with some 5-LO inhibitory activity.