Rifampicin and isoniazid resistance mutations in Mycobacterium tuberculosis strains isolated from patients in Kazakhstan.

OBJECTIVE: To analyse possible associations of specific mutations conferring rifampicin (RMP) and isoniazid (INH) resistance with Beijing and non Beijing genotype strains of Mycobacterium tuberculosis from Kazakhstan. METHOD: Genotypic analysis of 92 multidrug-resistant (MDR), 50 INH but not RMP-resistant (INHr/RMPs) and 10 fully susceptible strains of M. tuberculosis from Kazakhstan was performed. In the MDR group, 59 strains (64.1%), and within the INHr/RMPs group, 32 strains (64.0%) were classified as Beijing genotype. RESULTS: Analysis of the rpoB gene of the MDR strains revealed 10 different mutations in five codons, with rpoB codons 531 (65.2%), 526 (23.9%) and 516 (7.6%) most frequently affected. A significantly higher proportion of the rpoB S531L mutation was found among Beijing genotype strains compared with non Beijing strains (71.2% vs. 46.2%, P = 0.027). All 92 MDR isolates (100%), irrespective of their genotype, carried a mutation in codon 315 of the katG gene (S315T). However, in the INHr/RMPS control group, the S315T mutation was significantly more prevalent in the Beijing than in the non Beijing group (96.9% vs. 71.4%, P = 0.012). CONCLUSION: The high similarity of mutations supports the assumption that transmission of resistant strains is a major reason for the emergence of drug resistance in this region.

The Beijing genotype is a major cause of drug-resistant tuberculosis in Kazakhstan.

SETTING: Nine Kazakhstan oblasts, 2001. OBJECTIVE: To analyse the genetic relationship of drug-resistant Mycobacterium tuberculosis strains from Kazakhstan and to determine the frequency of the Beijing genotype. DESIGN: All drug-resistant smear-positive cases identified in nine oblasts during the 2001 nationwide drug resistance survey were analysed by IS6110 fingerprinting and spoligotyping. Isolates were obtained from 150 patients (64 new and 86 retreated cases). RESULTS: Eight cases (5.3%) of dual infection were identified. Of the remaining 142 strains, 91 (64.1%) were grouped in 18 clusters, indicating a high rate of recent transmission of resistant tuberculosis (TB). This was further confirmed by the origin of the patients as well as by the similar drug resistance patterns of the clustered strains. Accordingly, more than one third of all clustered strains were new cases. About 70% of the resistant strains belonged to the Beijing genotype, compared to only 37.5% in a control group of 40 susceptible isolates. CONCLUSIONS: Transmission of drug-resistant strains seems to contribute to the spread of resistant TB in this high incidence region. The Beijing genotype should be seen as a major cause of drug-resistant TB in Kazakhstan and was found to be associated with drug resistance.

The Beijing genotype is emerging among multidrug-resistant Mycobacterium tuberculosis strains from Germany.

SETTING: Germany, 1995 to 2001. OBJECTIVE: To determine the genetic relationship of 451 multidrug-resistant (MDR) Mycobacterium tuberculosis strains from Germany and to identify strains of the Beijing genotype. DESIGN: All strains were analysed using IS6110 fingerprinting and a cluster analysis was performed. Clustering of isolates was used as a measure for recent transmission. RESULTS: Two hundred and fourteen of 433 strains (49.4%) with more than four IS6110 copies formed 46 fingerprint clusters comprising two to 32 patients. Transmission links based on classical epidemiological data could be established for 39 cases (18.2%) and in 14 clusters (30.4%), and included three cases of exogenous reinfection with MDR strains. One hundred and seventy-five strains (38.8%) were of the Beijing genotype with an increasing annual proportion from 19.2% in 1995 to 58.3% in 2001. About 70% of these patients had an indication of foreign birth, mainly the former Soviet Union. CONCLUSION: Transmission of MDR strains seems to be contributing to the spread of MDR-TB in Germany, and exogenous reinfection with MDR strains must be considered as a possible cause of treatment failure. A high proportion of these MDR strains is probably carried over from the former Soviet Union, and strains of the Beijing genotype represent an increasing cause of MDR-TB in Germany.

The species Mycobacterium africanum in the light of new molecular markers.

The findings of recent studies addressing the molecular characteristics of Mycobacterium tuberculosis complex isolates have initiated a discussion on the classification of M. africanum, especially of those isolates originating from East Africa (cluster F, subtype II) and displaying phenotypic and biochemical characteristics more similar to those of M. tuberculosis. To further address this question, we analyzed a representative collection of 63 M. tuberculosis complex strains comprising 30 M. africanum subtype I strains, 20 M. africanum subtype II strains, 10 randomly chosen M. tuberculosis isolates, and type strains of M. tuberculosis, M. bovis, and M. africanum for the following biochemical and molecular characteristics: single-nucleotide polymorphisms (SNPs) in gyrB and narGHJI and the presence or absence of RD1, RD9, and RD12. For all molecular markers analyzed, subtype II strains were identical to the M. tuberculosis strains tested. In contrast, the subtype I strains as well as the M. africanum type strain showed unique combinations of SNPs in gyrB and genomic deletions (the absence of RD9 and the presence of RD12), which proves their independence from M. tuberculosis and M. bovis. Accordingly, all subtype I strains displayed main biochemical characteristics included in the original species description of M. africanum. We conclude that the isolates from West Africa were proved to be M. africanum with respect to the phenotypic and genetic markers analyzed, while the isolates from East Africa must be regarded as phenotypic variants of M. tuberculosis (genotype Uganda). We propose the addition of the molecular characteristics defined here to the species description of M. africanum, which will allow clearer species differentiation in the future.

HOPE-fixation enables improved PCR-based detection and differentiation of Mycobacterium tuberculosis complex in paraffin-embedded tissues.

Standard PCR-based detection of mycobacterial DNA in paraffin-embedded specimens may lack sufficient sensitivity because of the degradation of nucleic acids caused by routinely used formalin fixation. Therefore, we set up an approach that aimed at improving the results by applying the novel HOPE-fixative in PCR-detection of mycobacteria in paraffin-embedded tissues. Comparison of PCR-results using DNA extracted from either HOPE- or formalin-fixed specimens in BCG-infected SCID-mice revealed a more than 100fold enhanced sensitivity for the HOPE-fixed material. Owing to the preservation of DNA from degradation in HOPE-fixed tissues, even differentiation within the M. tuberculosis complex was possible by spoligotyping. We therefore conclude that the HOPE-fixative is a useful tool for molecular pathology that enhances the sensitivity of PCR-based methods for the detection of pathogens in paraffin-embedded tissues compared to formalin-fixation. Owing to the better preserved DNA, improved differentiation of mycobacteria from archived materials is possible. These results promise new and a substantially wider range of possibilities in the field of molecular pathology.

Analysis of renal function in onchocerciasis patients before and after therapy.

The occurrence of renal abnormalities was investigated in patients with onchocerciasis in comparison to individuals without onchocerciasis in Guinea. Serum creatinine levels, excretion of urinary marker proteins, and kidney size by ultrasound were determined. A high prevalence of glomerular as well as tubular dysfunctions was observed; however, no association with onchocerciasis could be detected. We also hypothesized that patients with hyperreactive onchocerciasis might be prone to develop immune-mediated glomerular disorders; however, this could not be verified. Following treatment with ivermectin, a slight but significant increase in the excretion of urinary albumin and alpha1-microglobulin was seen five days after treatment in all treated patients, whereas levels of proteinuria were significantly higher five days after treatment only in patients with high microfilarial densities. Our results indicate that ivermectin can cause glomerular and tubular disturbances in patients with onchocerciasis; however, these are minor and do not seem to be clinically relevant.

Eosinophils, eosinophil cationic protein and eosinophil-derived neurotoxin in serum and urine of patients with onchocerciasis coinfected with intestinal nematodes and in urinary schistosomiasis.

Eosinophils, eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN/EPX), myeloperoxidase (MPO) and IgE were measured in blood, serum and/or urine in Schistosoma haematobium- and Onchocerca volvulus-infected Guineans and O. volvulus- and S. haematobium-negative Guineans coinfected or infected with intestinal nematodes. The number of eosinophils and levels of eosinophil granule proteins but not of MPO were found to be strongly elevated in all Africans as compared to European controls. The highest serum ECP and serum and urinary EDN/EPX levels were observed in the hyperreactive form of onchocerciasis (sowda). Onchocerciasis patients and O. volvulus-negative Africans coinfected or infected with intestinal nematodes (hookworm and/or Ascaris lumbricoides) revealed higher serum granule protein concentrations and/or absolute eosinophil counts and urinary ECP than those without nematode infections. Statistical differences between both sections were found for the absolute eosinophil counts and for serum EDN/EPX and IgE in generalized onchocerciasis, and for urinary ECP in sowda, indicating stimulation of the eosinophil potential of O. volvulus-positive patients by coexistent hookworm infection. This worm species, in contrast to A. lumbricoides, causes especially high eosinophil counts and EDN/EPX and IgE levels. From these results it is concluded that in nematode diseases, ECP and EDN/EPX levels reflect the degree of antigenic stimulation, eosinophil activation and eosinophil turnover rates. Serum ECP and serum and urinary EDN/EPX may, therefore, serve as parameters to monitor helminth infection. Urinary ECP may be a marker of eosinophiluria secondary to urogenital manifestation of S. haematobium. It is elevated in hyperreactive onchocerciasis activated by intestinal nematodes.