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Objective: To investigate the prevalence of non-tuberculosis mycobacteria (NTM) among the samples deposited from the National Tuberculosis Reference Laboratory of Iran between 2011 and 2018. Methods: The study evaluated the prevalence of NTM among specimens from patients with pulmonary tuberculosis symptoms (n=15 771) deposited at the National Tuberculosis Reference Laboratory of Iran from 2011 to 2018. Detection of Mycobacterium (M.) tuberculosis was based on presence of a 190-bp amplicon from IS6110 insertion sequence using Tb1 and Tb2 primers, and amplicon-negative specimens were tested for NTM and M. tuberculosis (refractory to IS6110 amplification) using restriction fragment length polymorphism PCR of hsp65 amplicon fragment. Results: A total of 7 307 (46.33%) M. tuberculosis and 658 (4.17%) NTM specimens were found, the latter mainly comprising M. abscessus (10.18%), M. avium (2.28%), M. chelonae (8.97%), M. intracellulare (10.49%), M. kansasii (4.71%), and M. simiae (56.08%). Conclusions: As treatment for NTM differs from that for M. tuberculosis, accurate detection of Mycobacterium sp. is of public health significance.
ABSTRACT
Mycobacterium tuberculosis has infected more than two billion individuals worldwide, of whom 5%–10% have clinically active disease and 90%–95% remain in the latent stage with a reservoir of viable bacteria in the macrophages for extended periods of time. The tubercle bacilli at this stage are usually called dormant, non-viable, and/or non-culturable microorganisms. The patients with latent bacilli will not have clinical pictures and are not infectious. The infections in about 2%–23% of the patients with latent status become reactivated for various reasons such as cancer, human immunodeficiency virus infection, diabetes, and/or aging. Many studies have examined the mechanisms involved in the latent state of Mycobacterium and showed that latency modified the expression of many genes. Therefore, several mechanisms will change in this bacterium. Hence, this study aimed to briefly examine the genes involved in the latent state as well as the changes that are caused by Mycobacterium tuberculosis. The study also evaluated the relationship between the functions of these genes.
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Objective/background: Detection of mutations in the quinolone resistance-determining region [QRDR] of the gyrA gene could determine resistance to fluoroquinolone antitubercu-losis drugs. The aim of this study was to detect mutations in QRDRs
Methods: From 184 clinical isolates of Mycobacterium tuberculosis, ofloxacin resistance was proven in 42 isolates using the proportion method
The molecular basis of resistance to ofloxacin were investigated by the determination of mutations in the QRDR region of the gyrA gene. Extracted DNA fragments of 194 bp from the gyrA gene were amplified and an automatic DNA sequencer was used for the sequencing process
Results: Molecular genetic analysis of 42 resistant M. tuberculosis strains demonstrated that they belong to Principal Genetic Group [PGG] 1 in 19 cases [45.2 +/- 10.9%], to PGG2 in 15 cases [35.7 +/- 10.5%], and to PGG3 in eight cases [19.0 +/- 8.4%]
Isolates from PGG1 were dominant among resistant isolates [P < .05]. It was found that 24 [57%] resistant isolates carried mutations at codon 94 with five different amino acid changes: D94A [n = 11], D94G [n = 3], D94T [n = 4], D94A [n = 4], and D94Y [n = 2]. The remaining 18 [43%] isolates had mutations in codon A90V [GCG -> GTG] and S91P [TCG -> CCG]
Five isolates had two mutations in codons 90 and 94. There was no difference between mutations at these two codons in resistant isolates of the two countries [P < .001]. There was no polymorphii observed in codon 95 in any of the ofloxacin-susceptible isolates
Conclusion: It was concluded that the determination of nucleotide sequences of QRE can be used as a molecular test for the rapid detection of ofloxacin resistance. Furthermc frequencies in gyrA codons in Belarus and Iran were similar, therefore it is not of geograj ical concern for the two countries
ABSTRACT
Objective/Background: Mycobacterium tuberculosis [MTB] causes active tuberculosis [TB] in only a small percentage of infected people. In most cases, the infection is clinically latent, where bacilli can persist in human hosts for years without causing disease. Surprisingly, the biology of such persister cells is largely unknown. This study describes the isolation, identification, and whole-genome sequencing [WGS] of latent TB bacilli after 782 days [26 months] of latency [the ability of MTB bacilli to lie persistent]
Methods: The in vitro double-stress model of latency [oxygen and nutrition] was designed for MTB culture. After 26 months of latency, MTB cells that persisted were isolated and investigated under light and atomic force microscopy. Spoligotyping and WGS were performed to verify the identity of the strain
Results: We established a culture medium in which MTB bacilli arrest their growth, reduce their size [0.3-01 microm], lose their acid fastness [85-90%] and change their shape. Spoligopatterns of latent cells were identical to original H[37]R[v], with differences observed at spacers two and 14. WGS revealed only a few genetic changes relative to the already published H[37]R[v] reference genome. Among these was a large 2064-hp insertion [RvD6], which was originally detected in both H[37]R[a] and CDC1551, but not H[37]R[v]
Subject(s)
In Vitro Techniques , Population , Cell Wall , Genotyping Techniques , Genome-Wide Association StudyABSTRACT
The objectives of this study were to determine drug resistance pattern in new and previously treated tuberculosis [TB] patients, to assess function of TB control program, and to characterize multidrug resistant TB [MDR-TB] by molecular fingerprinting methods. Anti-micorbial susceptibility testing [AST] to the first line anti-TB drugs was performed on L?wenstein-Jensen [middlebrook 7H10] medium according to the proportion method. Molecular fingerprinting of all MDR strains was performed by spoligotyping and MIRU-VNTR. Mycobacterium tuberculosis strains were isolated from 53 Iraqi patients with pulmonary TB. Thirty eight patients [71.7%] tested cases, and 15 [28.3%] were previously treated. Four of the 38 new cases [10.5%] had resistant, of which 3 [7.9%] were MDR. Eight [53.3%] of the 15 previously treated patients had resistant strains, of which 7 [46.7%] were MDR. Spoligotyping of MDR strains showed CAS family [40%] as the predominant genotype. Using MIRU-VNTR typing, all isolates had a unique profile. MDR-TB prevalence is higher among previously treated patients than among the new cases. The many drug resistant strains, in absence of evidence of recent transmission and in combination with the many previously treated cases, highlight the need for an improved control program, coupled with a need to improve detection rate and early diagnosis of MDR-TB
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Advancements in molecular technology increased our understanding of genetic mechanism of drug resistance. Nowadays, the chance of rapid detection of resistant Mycobacterium tuberculosis [M. tuberculosis] strains is increased. In the present study, we aimed to investigate the sensitivity and specificity of PCR-SSCP for detecting susceptible and resistant strains of M. tuberculosis compared with DNA sequencing. To calculate the sensitivity and specificity of PCR-SSCP assay to detect drug resistance in M. tuberculosis, respiratory samples were collected from suspected patients referred to Mycobacteriology Research Center [Masieh Daneshvary Hosptial] since 2002. Susceptibility testing against first line drugs was performed on 74 culturepositive specimens. Consequently, PCR-SSCP and DNA sequencing were performed on katG, inhA, ahpC and rpoB genes. Drug-susceptibility testing by the proportional method in selected samples revealed 16 MDR [21.6%], 23 mono-drug resistant [31%] and 35 susceptible strains [47.3%]. In comparison with DNA sequencing as a gold standard for molecular methods, the sensitivity of PCR-SSCP assay for detecting of mutation in 315 codon of katG gene was 94.74% [CI=73.97%-99.87%] with 100% [CI=93.51%-100%] specificity. In contrast, the sensitivity and specificity of this assay in detecting of rpoB gene were 70.8% [CI=48.91%-87.38%] and 88% [CI=75.69%-95.47%], respectively. PCR-SSCP in combination with DNA sequencing can be used as screening method to detect MDR-TB and mono-drug resistant cases
ABSTRACT
Over several decades, morphological variation of Mycobacterium tuberculosis [M. tuberculosis] has engaged the attention of numerous investigators. The single point on which all investigators have agreed is that tubercle bacillus does not always manifest itself in the classical rod shape. While most commonly the organism appears as a granular rod, the other forms i.e., coccid, filament and club shapes are also present. Aside from the more purely academic aspect of the subject, the possible significance of variant forms in the etiology, prognosis, and control of tuberculosis infection were objects of heated controversies, even before 1900. These differences have never been resolved, and have been ignored by most recent workers. The main questions were centered on the following points: [1] Dose the tubercle bacillus produce endospore? [2] Does it normally undergo a complicated life cycle? [3] What is the importance of the non-acid-fast forms? [4] And what happens to the bacteria during latent infection? Today, based on various in-vitro and in-vivo models, the researchers agreed to consider M. tuberculosis as a two-phase microorganism which can appear either in its metabolically active acid-fast or in its inactive forms. It is the purpose of this chapter to review and discuss morphological variation and its challenges in M. tuberculosis. Furthermore, the cell shape and cell division were illustrated using atomic force microscopy. The present information will discuss the adaptation mechanism in M. tuberculosis and may help scientists to identify targets for novel therapies
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Molecular epidemiology analyses are frequently used in determining epidemiology of tuberculosis. Recently, Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat [MIRU-VNTR] and Spoligotyping has become an important method, as it allows high-through put, discriminatory and reproducible analysis of clinical isolate. The purpose of this study is to compare techniques of "MIRU-VNTR" versus "MIRU-VNTR and Spoligotyping" together for study of genetic pattern of Mycobacterium tuberculosis [M. tuberculosis] strains. Sixty M. tuberculosis [MTB] isolates were selected [30 susceptible, 30 multi-drug resistant] for this study. Thereafter, the "MIRU-VNTR and spoligotyping" were performed to identify their genetic patterns. The frequency of unknown genetic pattern of MTB was compared using technique of "MIRU-VNTR" alone versus "MIRU-VNTR and Spoligotyping" together. The MIRU-VNTR allelic diversity at each of the loci was calculated by Hunter - Gaston Discriminatory Index [HGDI]. Based on differentiation index of all strains 10, 16, 26, 31 and 40 loci were identified as the most distinctive [HGI >/= 0.6] and 2, 4, 20 and 24 as the weakest distinctive locus [HGI
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Tuberculosis/epidemiology , Polymorphism, Restriction Fragment Length , Molecular Epidemiology , AllelesABSTRACT
Nowadays the molecular methods widely use for rapid identification of Mycobacterium other than tuberculosis [MOTT]. The Mycobacterium simiae isolates are cause of majority of human pulmonary diseases compared with other atypical mycobacteria. As sensitivity of primers and digestion patterns for diversified fragments is different, this survey evaluated the three various fragments using the PCR- restriction fragment length polymorphism analysis [PRA] for rapid diagnostic of M simiae isolates. Strains that were identified as M. simiae [1.7 isolates] by phenotypic [photochromogen and positive niacin] methods were selected for this study. The fragments of the 16S-23S rRNA gene spacer and hsp65 gene were amplified by PCR. Subsequently the amplicons were digested with three restriction enzyme namely Avail, Hphl and Hpall for a 644bp region of hsp65 DNAs, BstEll and Haelll endonucleases for 439bp region of hsp65 gene [TB11 and TB12 fragment] and Haelll digestion for 225bp region of 16S-23S rRNA gene spacer. Of 962 culture positive specimens, 17 [1.7%] were identified as M. simiae species; majority of them were multidrug-resistance [12; 70.5%]. The overall detection rate by Tbll, Tbl2 and SP primers were 82.3% whereas hsp65 primer was 100% [p>0.005]. We also found out that the Hpall and Hphl enzymes were more specific to distinguish M simiae species than other restriction enzyme used in this study. The high discriminative power of hsp65 pattern particularly Hpall digestion, provide an exact and cost-effective method for rapid identification of M. simiae strains among registered pulmonary cases
Subject(s)
Humans , Nontuberculous Mycobacteria/isolation & purification , Nontuberculous Mycobacteria/genetics , Tuberculosis, Pulmonary/microbiology , Polymorphism, Restriction Fragment Length , Sensitivity and Specificity , Mycobacterium/geneticsABSTRACT
Vitamin-D receptor [VDR] and tumor necrosis factor-alpha [TNF-alpha] genes are thought to be important in the intracellular killing of mycobacteria. This study aimed to determine the association of VDR and TNF-alpha variant with development of pulmonary tuberculosis [PTB] among Iranian patients. Selected regions of VDR and TNF-alpha were amplified, and then the PCR products were digested using restriction enzyme [RFLP]. Digested products were run on 8% polyacrylamide gel, and were stained with silver-nitrate. Single nucleotide polymorphisms [SNPs] at restriction sites of BsmI, and FokI of VDR gene and SNPs of TNF-alpha at-238,-308,-244,-857,-863 positions were analyzed by PCR-RFLP among 117 PTB cases and 60 healthy controls. No statistically significant difference was observed in allele frequencies of FokI of VDR and TNF-alpha at-238,-244,-863 and-857 position. Although, the frequency of b allele of BsmI [p=0.001] and-308 A variant in TNF-alpha promoter region [p=0.006] were significantly more in PTB patients than healthy controls. The frequency of extended diplotypes were different in patients and control subjects [p<0.05]. This study confirmed the association of VDR BsmI and TNF-alpha-308A with susceptibility to tuberculosis in Iranian PTB patients. In addition, the results showed the importance of haplotypes and diplotypes analysis in determining the host susceptibility against TB
Subject(s)
Humans , Receptors, Calcitriol , Tumor Necrosis Factor-alpha , Polymorphism, Genetic , Polymorphism, Restriction Fragment Length , Polymerase Chain Reaction , Mycobacterium tuberculosis , Tuberculosis, PulmonaryABSTRACT
Prevention and treatment of drug-resistant clones is important in guiding TB control strategies. The simultaneous rapid detection of the type of mutation conferring resistance and the genotype reflect the extent of drug resistant TB transmission within the communities.Mutations conferring resistance to rifampin in rifampin-resistant clinical Mycobacterium tuberculosis isolates occur mostly in the 81 bp rifampin-resistance-determining region [RRDR] of the rpoB gene.Spoligotyping, IS6110- restriction fragment length polymorphism [RFLP] typing and sequencing of the rpoB gene were performed for 30 rifampin resistant M. tuberculosis isolates from patients referred to "Iranian National TB Laboratory" from 2006 to 2007 Mutations in the RRDR of the rpoB gene were identified in 96.6% of rifampin-resistant isolates. The spoligotyping analysis identified one [3.3%] East African-Indian [EAI] family, 7 [23.3%] Haarlem family, 9 [30.0%] Beijing family and 12 [40.0%] Central Asia [CAS] family isolates. Sixty- six percent of CAS isolates carried a mutation in codon 516, 37% of Beijing isolates carried a mutation in codon 531 and 33% of Haarlem isolates carried a mutation in codon 526 Overall, there appeared to be a correlation between the genotype and specific mutations conferring resistance to rifampin in the Beijing and Haarlem families
Subject(s)
Rifampin , Mutation/genetics , Mycobacterium tuberculosis/genetics , Microbial Sensitivity Tests , Genotype , Polymerase Chain ReactionABSTRACT
The aim of this study was to determine IS6110 banding pattern of Mycobacterium tuberculosis [MTB] isolates for evaluation of tuberculosis [TB] transmission. These isolates were obtained from intermediate laboratories of six major provinces of Iran; East Azarbaijan, West Azarbaijan, Khorasan, Kerman, Kermanshah and Fars. Restriction fragment length polymorphism [RFLP] was performed on 100 suitable isolates, which have been obtained from some laboratories thought Iran. Fingerprinting was done using the oligonucleotide 6110 a' [5