Tuberculosis under the Influence of COVID-19 Lockdowns: Lessons from Tehran, Iran.

This study investigates the short-term effects of the coronavirus disease 2019 (COVID-19) pandemic lockdown on tracing and detection of tuberculosis (TB) patients in Tehran, Iran. Results of this study have demonstrated that due to the significant decrease in the identification of patients with suspected TB during the COVID-19 outbreak in Tehran, it is imperative that patients with suspected TB be tracked and diagnosed more quickly to make up for some of the decline in TB diagnosis in recent months and to recover lost cases.

Prediction of the hidden genotype of mixed infection strains in Iranian tuberculosis patients.

BACKGROUND: Patients with mixed-strain Mycobacterium tuberculosis infections may be at a high risk of poor treatment outcomes. However, the mechanisms through which mixed infections affect the clinical manifestations are not well recognized. Evidence suggests that failure to detect the pathogen diversity within the host can influence the clinical results. We aimed to investigate the effects of different genotypes in mixed infections and determine their relationship with heteroresistance in the treatment of Iranian tuberculosis patients. METHODS: One of the genotypes was identified in the culture and another genotype pattern in the mixed infection was predicted by comparing the pattern of MIRU-VNTR between the clinical specimens and their respective cultures in each patient. For all patients, the drug susceptibility testing was carried out on three single colonies from each clinical sample. The follow-up of patients was carried out during six months of treatment. RESULTS: Based on MIRU-VNTR profiles of clinical samples, we showed that 55.6% (25/45) of the Iranian patients included in the study had mixed infections. Patients with mixed infections had a higher rate of treatment failure, compared to others (P=0.03). By comparing clinical sample profiles to profiles obtained after culture, we were able to distinguish between major and hidden genotypes. Among hidden genotypes, Haarlem (L4.1.2) and Beijing (L2) were associated to treatment failure (6/8 patients). CONCLUSIONS: To conclude, we propose a procedure using the MIRU-VNTR method to identify the different genotypes in mixed infections. The present findings suggest that genotypes with potentially higher pathogenicity may not be detected when performing experimental culture in patients with mixed infections.

Application of MIRU-VNTR on smear slides: a shortcut for detection of polyclonal infections in tuberculosis patients.

Mixed (polyclonal) infections are one of the main problems in tuberculosis (TB) management. The best available method for detecting polyclonal infections in TB is mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). According to multiple studies, MIRU-VNTR method can be applied to detect TB-related polyclonal infections in sputum samples or cultures. Setup of MIRU-VNTR on smear slides can be an efficient approach, regardless of the limitations of cultures and sputum samples in many laboratories. The present study aimed at investigating the diagnostic potential of MIRU-VNTR on smear slides in detecting mixed infections. Ziehl-Neelsen-stained microscopic slides were prepared from 14 clinical specimens. For amplifying 24 MIRU-VNTR loci, PCR assay was performed on the smear slides, clinical specimens, and cultures. Based on the 24-locus MIRU-VNTR analysis, polyclonal infections were reported in 42.85% of smear slides, while the corresponding rate was estimated at 57.1% (8/14) in the clinical samples. In the corresponding cultures, the rate of mixed infection was 7.14% (1/14). Use of smear slides can be a safe option for transferring clinical specimens between environmental and reference laboratories. Considering their significant impact on TB treatment, it is essential to diagnose mixed infections in low-resource countries with a high prevalence of mixed infections. The present findings show that direct MIRU-VNTR on smear slides can be conveniently used for the detection of mixed infections.

Comparison of MIRU-VNTR genotyping between old and fresh clinical samples in tuberculosis.

Background: Twenty-four loci mycobacterial interspersed repetitive unit-variable number tandem repeat analysis (MIRU-VNTR) is extensively used for genotyping and detection of polyclonal infections in tuberculosis. The aim of the present study was to compare the direct and indirect MIRU-VNTR genotyping and detection of polyclonal infections between old and fresh clinical samples. Method: Two series of TB samples were collected for comparison. After genomic DNA extraction from clinical samples and their respective cultures, 24 loci MIRU-VNTR was performed. Results: In the 14 old samples, no mixed infections were observed, in clinical samples and their respective cultures. In nine fresh samples, 44.4% of mixed infection was observed in the clinical samples, but no mixed infections were observed in their respective cultures. Surprisingly, in the old samples, 92.86% of samples (13/14) had an allelic change between clinical samples and their respective cultures. On the other hand, in fresh samples, only one sample (1/9) had an allelic change between clinical samples and their respective cultures. Conclusions: We concluded that 24 loci MIRU-VNTR undoubtedly is successful in direct genotyping of clinical samples, especially for the fresh samples. However, selecting starting material, such as clinical sample or respective culture can be controversial for the old samples. Regarding polyclonal infections, the fresh samples gives us a better view to detect these infections, especially in the clinical sample.

Challenge in direct Spoligotyping of Mycobacterium tuberculosis: a problematic issue in the region with high prevalence of polyclonal infections.

OBJECTIVE: Based on our recent studies the prevalence of polyclonal infection in tuberculosis clinical specimens is more than 50% in Tehran, Iran. With this background, Spoligotyping was performed on clinical specimens and their respective cultures, and we examined whether mixed infections interfere with the results or not. RESULTS: Based on the Spoligotyping pattern, among the fourteen patients, 57.1% had different genotypes in clinical samples and their respective cultures. These discrepant patterns were suggestive of polyclonal infections in clinical samples with possible overlapping Spoligotype patterns. We propose that in societies with high mixed infections (e.g. Iran), direct Spoligotyping on clinical samples can be controversial.

Evaluation of the impact of polyclonal infection and heteroresistance on treatment of tuberculosis patients.

Mixed strain infections of Mycobacterium tuberculosis make diagnosis, treatment, and control of tuberculosis (TB) more difficult. This study was aimed to evaluate the relationship between mixed infections, antibiotic resistance patterns and treatment of TB patients. In this study, among 2850 suspected TB clinical samples, a total of ninety-six clinical samples from 66 TB confirmed patients were subjected to the 24-locus variable-number tandem repeat method to evaluate the prevalence of mixed infections. For all studied strains, 288 colonies (three individual clones for each sample) were isolated from different colonies and separately analyzed by the Drug Susceptibility Test (DST). For all patients, follow up was done after 6 months of treatment. Based on direct 24 loci MIRU-VNTR, in the 66 TB patients, 53% (35/66) showed mixed infection. In the mixed samples, 45.71% (16/35) showed different antibiotic resistant patterns. Among the mixed infection patients, eight (22.9%; 8/35) showed treatment failure after six- month therapy. Six of these non-treated patients (75%; 6/8) had different antibiotic resistant patterns. We conclude that mixed infections, have a negative impact on treatment of TB patients especially when co-infecting M. tuberculosis strains display heteroresistance.

Bias in detection of Mycobacterium tuberculosis polyclonal infection: Use clinical samples or cultures?

The application of MIRU-VNTR has unveiled that infection by Mycobacterium tuberculosis can be polyclonal. Our comparative study demonstrated that based on the studied samples (clinical specimen or culture) detection of polyclonal M. tuberculosis infection can be significantly different.

Pros and cons of direct genotyping on tuberculosis clinical samples.

OBJECTIVE: Prompt genotyping of Mycobacterium tuberculosis (M. tuberculosis) is crucial for improving molecular epidemiological investigation of tuberculosis (TB). METHODS: We performed a retrospective study to evaluate the use of 24 loci MIRU-VNTR (mycobacterial interspersed repetitive unit-variable number of tandem-repeat) directly on 135 clinical samples from 84 TB patients. RESULTS: There was a direct correlation between genotyping on clinical samples by MIRU-VNTR and bacterial load (P = 0.001). VNTR loci were amplified successfully for 41.5% of the clinical samples (19-24 loci), 32.6% (13-18 loci), 23.7% (7-12 loci) and 2.2% (1-6 loci). Loci of 2401, 577, 2996 and 154 had the highest power to show the mixed strains infection in clinical samples. CONCLUSIONS: Direct MIRU-VNTR is partially successful in complete genotyping of M. tuberculosis strains. On the other hand, detection of polyclonal infection is undoubtedly reliable based on the direct MIRU-VNTR.