Rifampin resistance among individuals with extrapulmonary tuberculosis: 4 years of experience from a reference laboratory.

Information is limited about the drug resistance patterns in extrapulmonary tuberculosis (EPTB) in Iran. This study aimed to determine the prevalence of EPTB and to investigate the drug-resistance pattern in Mycobacterium tuberculosis strains collected from extrapulmonary samples at the Tehran regional TB reference laboratory. Extrapulmonary specimens from individuals with suspected TB referred to the TB reference laboratories in five cities of Iran were collected. Both standard conventional methods (culture and direct smear microscopy) and Xpert MTB/RIF assay were used for the identification of mycobacteria. Drug susceptibility testing was done using Xpert MTB/RIF. The proportion method on Lowenstein-Jensen medium was performed for confirmation. Between 2016 and 2020, a total of 12 050 clinical specimens from individuals with suspected TB were collected, of which 10 380 (86%) were pulmonary specimens and 1670 (14%) were extrapulmonary. Of the extrapulmonary specimens, 85 (5.0%) were positive for M. tuberculosis, and the remaining 1585 (95.0%) samples were negative by standard methods. Of 85 M. tuberculosis isolates, drug susceptibility testing was performed for 32 isolates, of which 1 (3.1%, 95% CI 0.0%-9.4%) was rifampin resistant and 31 (96.9%, 95% CI 90.1%-100%) were pan-susceptible. The rifampin-resistant isolate was also resistant to isoniazid, so was assigned as a multidrug-resistant TB. Our study indicated the frequency of drug-resistance among EPTB in Iran. Establishing rapid diagnostic methods for detection of drug-resistance in EPTB, performing drug susceptibility testing for all EPTB cases to provide effective treatment, and continuous monitoring of drug resistance, are suggested for prevention and control of drug resistance in EPTB in Iran.

Genotyping and drug susceptibility testing of Mycobacterium tuberculosis in Iran: a multi-centre study.

Tuberculosis (TB) is a deadly infection and caused 1.4 million deaths in 2018. Assessing the geographic distribution of major lineages of Mycobacterium tuberculosis can contribute greatly to TB control. Mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) typing is commonly used to differentiate various lineages of M. tuberculosis. A total of 2747 clinical specimens were collected consecutively from October 2018 through June 2019. Clinical isolates were identified as M. tuberculosis using standard biochemical tests. The standard 15-locus MIRU-VNTR typing was used for the genotyping of clinical isolates. Drug susceptibility testing was performed using the conventional proportion method. From the collected specimens, 100 were culture positive for M. tuberculosis. Using MIRU-VNTR, 99 different patterns were detected among the 100 isolates. They were distributed in one cluster comprising two strains and 98 unique patterns. Most of our isolates were similar to New-1 and Delhi/CAS strains. Of the M. tuberculosis isolates, 83 (83.0%) were pan-susceptible and 17 (17.0%) were resistant to at least one drug. Our study showed that MIRU-VNTR is a useful method for studying the genetic diversity of M. tuberculosis isolates in different regional settings and will help the health authorities to construct a preventive programme for TB.

Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation-mediated instability using small molecules.

Regulatory T (Treg ) cell therapy is a promising approach for immune tolerance induction in autoimmunity conditions and cell/organ transplantations. Insufficient isolation yields and impurity during downstream processes and Treg instability after adoptive transfer in inflammatory conditions are major limitations to Treg therapy, and indicate the importance of seeking a valid, reliable method for de-novo generation of Tregs . In this research, we evaluated Treg -like cells obtained from different Treg differentiation protocols in terms of their yield, purity and activity. Differentiation was performed on naive CD4+ cells and a naive CD4+ /Treg co-culture by using three different protocols - ectopic expression of forkhead box protein P3 (E-FoxP3), soluble transforming growth factor ß (S-TGF) and small molecules [N-acetyl puromycin and SR1555 (N-Ac/SR)]. The results showed that a high yield of a homogeneous population of Treg -like cells could be achieved by the N-Ac/SR method under a T helper type 17 (Th17)-polarizing condition, particularly interleukin (IL)-6 and TGF-ß, when compared with the E-FoxP3 and S-TGF methods. Surprisingly, SR completely inhibited the differentiation of IL-17-producing cells and facilitated Treg generation in the inflammatory condition and had highly suppressive activity against T cell proliferation without Treg -specific demethylase region (TSDR) demethylation. For the first time, to our knowledge, we report the generation of efficient, pure Treg -like cells by using small molecules during in-vitro inflammatory conditions. Our results suggested that the N-Ac/SR method has several advantages for Treg generation when compared with the other methods, including a higher purity of Tregs , easier procedure, superior suppressive activity during the inflammatory condition and decreased cost.

High-resolution melt curve analysis for rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a single-centre study in Iran.

This study aimed to compare the diagnostic accuracy of high-resolution melting (HRM) analysis in comparison with Xpert MTB/RIF as well as conventional drug susceptibility testing (DST) for the detection of rifampicin (RIF) resistance in Mycobacterium tuberculosis in Iran. A comparative cross-sectional study was carried out from April 2017 to September 2018. A total of 80 culture-positive clinical samples selected during the study period were analysed for detection of RIF-resistant TB by conventional DST, Xpert MTB/RIF, and sequencing. Sensitivity and specificity of the HRM calculated according to DST was our reference standard test in this study. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of HRM assay were found to be 100%, 89.33%, 38.46%, and 100% respectively. The analysis demonstrated that the diagnostic accuracy of HRM tests is insufficient to replace Xpert MTB/RIF and conventional DST. HRM tests have the advantage of time to result and may be used in combination with culture. Further work to improve molecular tests would benefit from standardized reference standards and the methodology.

High rates of nontuberculous mycobacteria isolation from patients with presumptive tuberculosis in Iran.

Nontuberculous mycobacteria (NTM) can cause disease which can be indistinguishable from tuberculosis (TB), posing a diagnostic and therapeutic challenge, particularly in low- and middle-income settings. We aimed to investigate the mycobacterial agents associated with presumptive clinical pulmonary TB in Iran. A total of 410 mycobacterial isolates, obtained between March 2014 and January 2016, from 7600 clinical samples taken from consecutive cases of presumptive diagnosis of TB were identified. Phenotypic and molecular tests were used to identify the isolated organisms to the species level. Single-locus and multilocus sequence analysis based on 16S rRNA, rpoB, hsp65 and ITS locus were used to confirm the results. Of 410 consecutive strains isolated from suspected TB subjects, 62 isolates (15.1%) were identified as NTM. Patients with positive NTM cultures met American Thoracic Society diagnostic criteria for NTM disease. Mycobacterium simiae was the most frequently encountered (38.7%), followed by Mycobacterium fortuitum (19.3%), M. kansasii (17.7%) and M. avium complex (8.0%). Isolation of NTM, including M. simiae, from suspected TB cases is a serious public health problem and merits further attention by health authorities, physicians and microbiologists.

Catalase-negative Staphylococcus aureus isolated from a diabetic foot ulcer.

We report a catalase-negative Staphylococcus aureus isolated from a 56-year-old male diabetic patient with foot ulcer who attended our surgery ward. Species identification was confirmed by Gram staining, standard biochemical tests and PCR amplification of the nuc and fem genes. Antibiotic susceptibility showed that the strain was sensitive to imepenem, chloramphenicol, amoxicillin, vancomycin and resistant to oxacillin, penicillin, ceftriaxone, erythromycin, clindamycin, and amikacin. Clinicians and microbiologists must be encouraged to identify and report these atypical strains and the infections associated with them in order to establish their role in pathogenesis.

Determination of Helicobacter pylori virulence by analysis of the cag pathogenicity island isolated from Iranian patients.

BACKGROUND: The cag pathogenicity island (PAI), which can be divided into two parts, cagI and cagII, is the most well-known virulence factor of Helicobacter pylori. AIMS: We investigated the association between genetic variations within the cag PAI (cagA and cagE in the cagI and cagT in the cagII) and clinical outcomes in Iranian patients. SUBJECTS: A total of 231 patients including 182 patients with gastritis, 41 with peptic ulcer and 8 with gastric cancer. METHODS: The presence of the cagA, cagE and cagT genes were measured by polymerase chain reaction and the results were compared with clinical outcomes and gastric histology. RESULTS: The cagA, cagE and cagT genes were found in 154 (66.7%), 90 (39.0%) and 70 (30.3%) of clinical isolates. At least 144 (62.3%) strains possessed partially deleted cag PAI (e.g., 69 [29.9%] strains were cagA-positive, but cagE and cagT-negative). CONCLUSION: The single genes as well as the combination of genes in the cag PAI appeared not to be useful markers to predict H. pylori-related diseases in Iranian patients. The genomic sequences of the cag PAI in Iranian strains might be considerably different from those in other geographic locations.