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The aim of this study was to investigate the significance of multiple mutations in the rpoB gene as well as predominant nucleotide changes and their correlation with high levels of resistance to rifampin (rifampicin) in Mycobacterium tuberculosis isolates that were randomly collected from the sputa of 46 patients with primary and secondary cases of active pulmonary tuberculosis from the southern region (Afghanistan border) of Iran where tuberculosis is endemic. Drug susceptibility testing was performed using the CDC standard conventional proportional method. DNA extraction, rpoB gene amplification, and DNA sequencing analysis were performed. Thirty-five (76.09%) isolates were found to have multiple mutations (two to four) in the rpoB (β-subunit) gene. Furthermore, we demonstrate that the combination of mutations with more prevalent nucleotide changes were observed in codons 523, 526, and 531, indicating higher frequencies of mutations among patients with secondary infection. In this study, 76.08% ( n = 35) of all isolates found to have mutation combinations involving nucleotide changes in codons 523 (GGG→GCG), 531 (TCG→TTG or TTC), and 526 (CAC→CGC, TTC, AAC, or CAA) demonstrated an association with higher levels of resistance to rifampin (MIC, ≥100 μg/ml).

One hundred two Corynebacterium diphtheriae strains (93 of the gravis biotype and nine of the mitis biotype) isolated from clinical cases during the Belarus diphtheria epidemic were characterized by biotyping, toxigenicity testing by the Elek test and an indirect hemagglutination assay, phage typing, and ribotyping. The gravis biotype strains were characterized as high and medium toxin producers, and strains of biotype mitis were characterized as low and medium toxin producers. Most strains (82 of 102) were distributed among five phage types. Seventy-two strains (64 of the gravis biotype and 8 of the mitis biotype) belonged to phage type VI ls5,34add. Hybridization of genomic DNA digested with Bst EII and Pvu II revealed five ribotype patterns, namely, D1, D4, D6, D7, and D13. The majority of gravis biotype strains belonged to ribotypes D1 (49 of 93) and D4 (33 of 93) and included one clonal group of C . diphtheriae . This clone predominated in all regions in Belarus. There was a statistical association between ribotypes and phage types but not between ribotypes and levels of toxin production.

An immunochromatographic strip (ICS) test was developed for the detection of diphtheria toxin by using an equine polyclonal antibody as the capture antibody and colloidal gold-labeled monoclonal antibodies specific for fragment A of the diphtheria toxin molecule as the detection antibody. The ICS test has been fully optimized for the detection of toxin from bacterial cultures; the limit of detection was approximately 0.5 ng of diphtheria toxin per ml within 10 min. In a comparative study with 915 pure clinical isolates of Corynebacterium spp., the results of the ICS test were in complete agreement with those of the conventional Elek test. The ICS test was also evaluated for its ability to detect toxigenicity from clinical specimens (throat swabs) in two field studies conducted within areas of the former USSR where diphtheria is epidemic. Eight hundred fifty throat swabs were examined by conventional culture and by use of directly inoculated broth cultures for the ICS test. The results showed 99% concordance (848 of 850 specimens), and the sensitivity and specificity of the ICS test were 98% (95% confidence interval, 91 to 99%) and 99% (95% confidence interval, 99 to 100%), respectively.

Toxigenic Clostridium difficile is the most common etiologic agent of hospital-acquired diarrhea in developed countries. The role of this pathogen in nosocomial diarrhea in Eastern Europe has not been clearly established. The goal of this study was to determine the prevalence of C. difficile in patients and the hospital environment in Belarus and to characterize these isolates as to the presence of toxin genes and their molecular type. C. difficile was isolated from 9 of 509 (1.8%) patients analyzed and recovered from 28 of 1,300 (2.1%) environmental sites cultured. A multiplex PCR assay was used to analyze the pathogenicity locus (PaLoc) of all isolates, and strain identity was determined by an arbitrarily primed PCR (AP-PCR). The targeted sequences for all the genes in the PaLoc were amplified in all C. difficile strains examined. A predominantly homogenous group of strains was found among these isolates, with five major AP-PCR groups being identified. Eighty-three percent of environmental isolates were classified into two groups, while patient isolates grouped into three AP-PCR types, two of which were also found in the hospital environment. Although no data on the role of C. difficile infection or epidemiology of C. difficile -associated diarrhea (CDAD) in this country exist, the isolation of toxigenic C. difficile from the hospital environment suggests that this pathogen may be responsible for cases of diarrhea of undiagnosed origin and validates our effort to further investigate the significance of CDAD in Eastern Europe.