Transmission dynamics of tuberculosis in a high-incidence country: prospective analysis by PCR DNA fingerprinting.

We have prospectively analyzed the DNA fingerprints of Mycobacterium tuberculosis strains from a random sample of patients with newly diagnosed tuberculosis in Windhoek, Namibia. Strains from 263 smear-positive patients in whom tuberculosis was diagnosed during 1 year were evaluated, and the results were correlated with selected epidemiological and clinical data. A total of 163 different IS6110 fingerprint patterns were observed among the 263 isolates. Isolates from a high percentage of patients (47%) were found in 29 separate clusters, with a cluster defined as isolates with 100% matching patterns. The largest cluster included isolates from 39 patients. One predominant strain of M. tuberculosis caused 15% of cases of smear-positive pulmonary tuberculosis in Windhoek. That strain was also prevalent in the north of the country, suggesting that in contrast to other African countries with isolates with high levels of diversity in their DNA fingerprint patterns, only a restricted number of different strains significantly contribute to the tuberculosis problem in Namibia.

Universal pattern of RpoB gene mutations among multidrug-resistant isolates of Mycobacterium tuberculosis complex from Africa.

SETTING: Multidrug-resistant tuberculosis (MDR-TB) presents an increasing burden in Southern Africa. Rapid diagnostic tests for drug resistance to rifampicin have been developed based on mutation analysis of the rpoB gene. However, geographic differences of underlying mutations have recently been suggested. OBJECTIVE: Drug-resistant strains of Mycobacterium tuberculosis complex from Africa were analysed for geographic differences in frequency and location of rpoB mutations. DESIGN: A random sample of rifampicin-resistant strains was collected from 87 patients with pulmonary MDR-TB treated in 12 hospitals from six different regions of South Africa. In addition, 18 isolates of M. tuberculosis complex from Namibia, Sierra Leone, and Uganda, including 13 isolates of M. africanum, were analyzed. Point mutations were detected by direct sequence analysis of the rpoB gene. RESULTS: Missense mutations were identified for 91 isolates (87%). Double mutations were present in eight (8%) MDR-TB isolates, two of which carried one mutation outside a previously described diagnostic region. We found no geographic differences regarding the frequency and pattern of single rpoB gene mutations. CONCLUSION: Our results confirm that molecular genetic analysis of rifampicin resistance based on a core region within the rpoB gene is universally applicable to strains of M. tuberculosis complex from different geographic regions.

Preoperative diagnosis of Mycobacterium avium lymphadenitis in two immunocompetent children by polymerase chain reaction of gastric aspirates.

BACKGROUND: Analysis of gastric aspirates is a routine procedure for detection of Mycobacterium tuberculosis in pediatric pulmonary tuberculosis. However, identification of nontuberculous mycobacteria in gastric aspirates of immunocompetent children is not thought to be clinically significant. METHODS: A PCR method was devised for the detection of M. avium in clinical specimens. The method is based on the amplification of a M. avium-specific DNA fragment present in the 3'-end of the repetitive element IS1245. Surgically removed lymphatic tissue was analyzed prospectively by microscopy, culture and PCR in 13 children admitted to our hospital with suspected mycobacterial lymphadenitis. In 4 of these children 1 to 4 gastric aspirates were obtained before surgical treatment and submitted to the same analysis. RESULTS: We report the detection of M. avium in the gastric aspirates of two children with cervical lymphadenitis before surgical intervention by a novel PCR method. The subsequently surgically removed lymph nodes were also positive by PCR and culture. In one child cultures of both sources grew M. avium. The isolates could be identified as the same strain by DNA fingerprinting. The PCR assay was almost twice as sensitive as culture in detecting M. avium. CONCLUSIONS: Our findings suggest the possibility for noninvasive diagnosis of cervical lymphadenitis caused by nontuberculous mycobacteria before surgery. In addition detection of M. avium in gastric aspirates without evidence of fistula formation provides new insights into the pathogenesis of mycobacterial infection and disease in immunocompetent children.

DNA fingerprinting of Mycobacterium tuberculosis complex culture isolates collected in Brazil and spotted onto filter paper.

The usefulness of filter paper for preservation of bacterial cells was shown by mixed-linker DNA fingerprint analysis of Mycobacterium tuberculosis isolates from 77 Brazilian patients. DNA fingerprints of samples spotted onto filter paper and conventional culture material were identical. Thus, filter paper specimens analyzed by an amplification-based typing method provide a new resource for epidemiological studies of infectious diseases.

A new agent of mycobacterial lymphadenitis in children: Mycobacterium heidelbergense sp. nov.

Nontuberculous mycobacterial lymphadenitis presents an increasing clinical problem in immunocompetent young children. A slowly growing, nonphotochromogenic mycobacterium was recovered twice (isolates 2553/91 and 2554/91) from the lymphatic tissue of a child with recurrent cervical lymphadenitis. It could be differentiated biochemically from described Mycobacterium species, although it most closely resembled Mycobacterium malmoense by thin-layer chromatography and high-performance liquid chromatography of mycolic acids. A striking characteristic of the isolate was its high degree of susceptibility to antituberculous drugs in vitro, including isoniazid. Direct determination of the 16S rRNA gene sequence revealed a unique sequence and positioned the strain phylogenetically on a branch separate from M. malmoense within a group of slowly growing mycobacteria that show a high degree of similarity to M. simiae at the 16S rRNA gene level. Despite 99.6% sequence identity with M. simiae at the 16S rRNA gene level, DNA-DNA hybridization studies (hydroxyapatite method) demonstrated DNA relatedness of less than 40%. We conclude that this organism is a new species for which we propose the name M. heidelbergense. A culture of the type strain, strain 2554/91, has been deposited in the American Type Culture Collection as strain ATCC 51253.

Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis complex from Africa.

A sample of 124 isoniazid (INH)-resistant and 88 susceptible strains of Mycobacterium tuberculosis complex from south, central, and west Africa was analyzed by direct sequence analysis and PCR-restriction fragment length polymorphism analysis of their catalase-peroxidase (katG) genes. Point mutations at codon 315 were found in the genomes of 64% of INH-resistant strains, but no complete deletions were identified. Mutations at codon 463 were independent of INH resistance and were linked to the geographic origins of the strains.

Comparison of DNA fingerprint patterns of isolates of Mycobacterium africanum from east and west Africa.

Mycobacterium africanum is a pathogen found in tuberculosis patients in certain parts of Africa and is a member of the Mycobacterium tuberculosis complex. Biochemically, strains of M. africanum exhibit a high degree of variability, with some tendency to cluster according to their geographical origin. To investigate whether this phenotypic variability is reflected at the genetic level, we performed DNA fingerprint analysis of strains isolated from patients with pulmonary tuberculosis in Uganda and Sierra Leone. IS6110 DNA fingerprinting was carried out by the mixed-linker PCR method. A total of 138 strains of M. africanum were analyzed: 42 isolates from Uganda and 96 isolates from Sierra Leone. With few exceptions, the resulting DNA fingerprint patterns grouped together according to their country of origin. A striking lack of variability of DNA fingerprints was found for strains from Sierra Leone, where 70 of 96 isolates (61.5%) fell into clusters. The two largest clusters accounted for 41.7% of all isolates and differed by only one band, as confirmed by standard DNA fingerprinting. In contrast, only two clusters (7.1%) with two and three isolates, respectively, were found for M. africanum isolates collected in Uganda, and three of the DNA fingerprints contained fewer than seven bands. Strains of M. tuberculosis collected and processed during the same time period were highly variable in both countries. Our results support the concept of geographically defined subtypes of M. africanum. In addition, they demonstrate that natural geographic differences in the variability of IS6110 DNA fingerprints within the M. tuberculosis complex must be considered if this technique is used for epidemiologic studies.

Short antisense oligonucleotide-mediated inhibition is strongly dependent on oligo length and concentration but almost independent of location of the target sequence.

The inhibitory effect of short antisense oligodeoxynucleotides (aODNs) on cRNA expression in Xenopus oocytes was measured using an electrophysiological assay based on subunit-specific block of cloned alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors. The effect of both phosphorothioate-modified (PS) and phosphodiester (PO) aODNs was strongly length dependent with a half-maximal inhibition calculated for an oligo length of 7.6 nucleotides (nt) and 9.9 nt, respectively. More than 95% inhibition was mediated by a PS aODN of 12 nt and by PO aODNs > or = 15 nt. At a given length PS and PO aODNs showed differential dependence of their inhibitory effect on the injected aODN concentration (half-maximal inhibition at 18 ng/microliter for a PO 12-mer and at 0.19 ng/microliter for a PS 12-mer) and differential saturation behavior. The inhibitory effect of aODNs, even as short as 8 nt for PS oligomers, was highly sequence specific, but almost independent of the position of the respective target site on the cRNA (for PS 8-mers, > or = 70% expression inhibition throughout the tested target sites from the translation initiation to the 3'-untranslated region). Thus, short PS aODNs can be reliably used in order to specifically inhibit protein expression in experiments addressing physiological, molecular biological, and perhaps even therapeutical issues.