Selection of IS6110 conserved regions for the detection of Mycobacterium tuberculosis using qPCR and LAMP.

IS6110 insertion sequence is a frequently used target for Mycobacterium tuberculosis detection. However, its sequence variability is studied insufficiently. We aimed to identify the most conservative and variable regions in IS6110 sequences and develop qPCR and LAMP oligonucleotide sets for the conservative regions. Using in-house Python scripts, 3609 M. tuberculosis genome sequences from the NCBI database were aligned; conservative regions were identified to design oligonucleotide sets. IS6110 fragments located within the 31-231 bp region were the most conservative and represented in genomes and were used to design qPCR and LAMP oligonucleotides. The in silico sensitivity of the qPCR oligonucleotides on the whole genome set was 99.1% and 98.4%. For the LAMP primers developed, the sensitivity was 96.9%. For qPCR, the limit of detection with 95% confidence (LoD95%) was four IS6110 copies per reaction, with LoD90% being 200 BCG cells per ml of artificial sputum. For LAMP, LoD95% was 16 copies per reaction, with LoD90% being 400 Mycobacterium bovis Bacille Calmette-Guerin (BCG) cells per ml of artificial sputum. We have demonstrated the IS6110 sequence variability and designed highly sensitive qPCR and LAMP oligonucleotides to detect M. tuberculosis.

Urogenital tuberculosis, the cause of ineffective antibacterial therapy for urinary tract infections.

BACKGROUND: Urogenital tuberculosis (UGTB) is one of the great imitators; it is commonly masked by urinary tract infections (UTIs). We aimed to estimate how many UGTB patients were among patients with a long history of UTIs. MATERIAL AND METHODS: A total of 244 patients with recurrent UTIs and suspected UGTB were enrolled in an open, noncomparative prospective study. Their urine and expressed prostate secretion or ejaculate were cultured (a total of 1446 samples), and 421 isolates with growth of ⩾104 colony-forming units (CFU)/ml were investigated for drug resistance. Typically, UGTB diagnosis is made by individual case. RESULTS: All 244 patients had a long history of recurrent UTIs (on average, 7.9 ± 3.4 years); all received at least five courses of antibacterial therapy without good result. UGTB was diagnosed in 63 (25.8%), and in 41 of these (65.1%), there was comorbidity of UTI and UGTB. Of 1446 samples investigated, 421 (29.1%) were positive, and 1025 were negative. Escherichia coli was found in 57.3% of gram-negative microflora and in 29.0% only among all uropathogens. E. coli was resistant to amoxicillin/clavulanate in 51.5-57.1%, to cefotaxime in 50.0-52.0%, to gentamycin in 33.3-59.5%, to ciprofloxacin in 63.2-66.7%, to levofloxacin in 54.8-45.2%, and to nitrofurantoin in 23.5-20.8% in 2015 and 2016, respectively. If, in 2015, all isolates of E. coli were susceptible to imipenem, in 2016, 7.1% of strains were resistant to this antibiotic. Level of drug-resistance was higher in 2016, excluding only levofloxacin and nitrofurantoin. CONCLUSIONS: Total prevalence of UGTB among UTI patients with poor results of antibacterial therapy was 25.8%. Comorbidity of UTI and UGTB was diagnosed in 65.1%.

BCG infection in mice is promoted by naïve mesenchymal stromal cells (MSC) and suppressed by poly(A:U)-conditioned MSC.

Mesenchymal stromal cells (MSC) transplantation is an actively studied therapeutic approach used in regenerative medicine and in the field of control of immunoinflammatory response. Conditioning of MSC in culture can form their predominantly pro- or anti-inflammatory phenotypes. We demonstrated that poly(A:U)-conditioning of bone marrow-derived mouse MSC induced predominantly pro-inflammatory phenotype. The effects of administration of naïve MSC (nMSC) or conditioned MSC (cMSC) on the course of mycobacterial infection were studied. BALB/c mice infected i.p. with 5 × 106 M. bovis BCG were successively injected i.v. with 0.75 × 106 of nMSC or cMSC in 11 and 12.5 weeks after infection and sacrificed at the week 14. Histological and bacteriological examination of BCG-infected animals revealed low bacterial loads in liver, lungs and spleen; the bacterial load in spleen was higher than in other organs. Treatment with nMSC induced 3-fold increase of the number of bacteria in spleen granulomas, while cMSC decreased significantly the number of bacteria in BCG-positive granulomas. Analysis of preparations of organ homogenates by luminescent microscopy, MGIT cultures and CFU count on Lowenstein-Jensen medium revealed that nMSC promoted mycobacterial growth whereas cMSC suppressed mycobacterial growth significantly. We concluded that MSC therapy can be effective in mycobacterial infection, but only in a case of appropriate conditioning of the cells.

Characterization of extensively drug-resistant Mycobacterium tuberculosis isolates circulating in Siberia.

BACKGROUND: The spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis compromises effective control of tuberculosis (TB) in Siberia. Early identification of drug-resistant isolates is, therefore, crucial for effective treatment of this disease. The aim of this study was to conduct drug susceptibility testing and identify mutations in drug resistance genes in clinical isolates of M. tuberculosis from some TB patients presenting for treatment in Siberia. METHODS: Thirty randomly selected clinical isolates of M. tuberculosis were obtained from the Novosibirsk Research Institute of Tuberculosis, Russia. Isolates were screened for drug resistance and characterized by variable number of tandem repeats (VNTR)-typing using 15 standard and four additional loci. Deligotyping on multiple large sequences was performed using 10 loci. RESULTS: Twenty-nine of the isolates were assigned XDR status. Twenty-eight isolates belonged to the M. tuberculosis Beijing family, from which 11 isolates were considered the M11 type (39%), two the M2 type (7%), and one the M33 type (3%). Seventeen isolates (60.7%) from this family exhibited unique genetic patterns. The remaining two isolates belonged to the Latino-American Mediterranean family. Gene sequences (rpoB, katG, rrs, rpsL, tlyA, gidB, gyrA, gyrB) were analyzed to identify mutations that confer resistance to rifampicin, isoniazid, amikacin, kanamycin, capreomycin, and ofloxacin. The most common mutations among the XDR isolates were S531L in RpoB, S315T in KatG, various codon 94 mutations in gyrA, A90V in GyrA, K43R in RpsL, and 1401 A → G in rrs; these confer resistance to rifampicin, isoniazid, ofloxacin, streptomycin and kanamycin/capreomycin, respectively. There was high congruence between the two typing methods (VNTR typing and deligotyping) and RD105, RD149, RD152, RD181, and RD207 regions of difference were absent from the 28 Beijing family isolates. CONCLUSIONS: Deligotyping can be used for rapid and reliable screening of M. tuberculosis isolates, followed by more in-depth genotyping. Identification of Beijing family isolates with extensive drug resistance confirms that such strains have epidemiological importance in Siberia. Rapid detection of mutations that lead to drug resistance should facilitate selection of effective drug therapies, and the development of early prevention strategies to combat this infection.

Molecular characteristics of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates from the Russian Federation.

OBJECTIVES: Three Mycobacterium tuberculosis genetic loci--rpoB and katG genes and the fabG1(mabA)-inhA operon promoter region--were studied to reveal the mutations associated with rifampicin and isoniazid resistance. METHODS: Four hundred and twelve isolates of M. tuberculosis from different regions of the Russian Federation were collected during 1997-2005. A matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based minisequencing method was used for the detection of mutations. RESULTS: Thirteen different variants of single mutations in codons 533, 531, 526, 516, 513 and 511 of the rifampicin resistance-determining region of the rpoB gene as well as the TTG insertion in the 514a position were found among the rifampicin-resistant isolates. Single nucleotide substitutions in codons 531, 526 and 516 (64.8%, 10.3% and 7.7%, respectively) were the most prevalent mutations. Codon 526 was shown to be the most variable of all. No mutations were detected in rpoB genes for 29 (10.7%) of the rifampicin-resistant isolates. 76.9% of the isoniazid-resistant isolates carried single mutations in codon 315 of the katG gene. For another 12.9% of them, double mutations in the katG gene and the fabG1(mabA)-inhA promoter region were revealed. No mutations were detected in 8.2% of the isoniazid-resistant isolates. CONCLUSIONS: Molecular analysis of the loci of rpoB and katG genes and the inhA promoter region of 412 M. tuberculosis clinical isolates from various parts of the Russian Federation was carried out. The new MALDI-TOF MS-based method may be used for rapid and accurate monitoring of the spread of drug resistance.