Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units–variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications.

Polyclonal endemicity of Pseudomonas aeruginosa in a teaching hospital from Brazil: molecular typing of decade-old strains

Pseudomonas aeruginosa infections cause significant mortality and morbidity in health care settings. Strategies to prevent and control the emergence and spread of P. aeruginosa within hospitals involve implementation of barrier methods and antimicrobial stewardship programs. However, there is still much debate over which of these measures holds the utmost importance. Molecular strain typing may help elucidate this issue. In our study, 71 nosocomial isolates from 41 patients and 23 community-acquired isolates from 21 patients were genotyped. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) was performed. Band patterns were compared using similarity coefficients of Dice, Jaccard and simple matching. Strain similarity for nosocomial strains varied from 0.14 to 1.00 (Dice); 0.08 to 1.00 (Jaccard) and 0.58 to 1.00 (simple matching). Forty patterns were identified. In most units, several clones coexisted. However, there was evidence of clonal dissemination in the high risk nursery, neurology and two surgical units. Each and every community-acquired strain produced a unique distinct pattern. Results suggest that cross transmission of P. aeruginosa was an uncommon event in our hospital. This points out to a minor role for barrier methods in the control of P. aeruginosa spread.

Characteristics of Molecular Strain Typing of Mycobacterium tuberculosis Isolated from Korea / 대한임상미생물학회지

Molecular strain typing of Mycobacterium tuberculosis is important for the detection of outbreaks of tuberculosis and laboratory cross contamination, as well as the differentiation between re-infection and reactivation of tuberculosis. In the present review, the authors investigated the currently available typing methods for M. tuberculosis and the current status of strain distribution in Korea. IS6110-restriction fragment length polymorphism (RFLP), which is considered a standard method, is based on numbers and positions of the insertion sequence, IS6110. The method has an excellent discriminatory power with a considerable amount of worldwide data, although it is time-consuming and labor-intensive. Spoligotyping is based on the presence or absence of spacer sequences between direct repeat (DR) regions. PCR amplification allows for the possibility of application in the early suspicious stage. The data can be easily digitized; however, it shows identical profiles in Beijing family strains. Mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) is another PCR-based genotyping method with a good discrimination power whose data can also be easily digitized. In Korea, the prevalence of Beijing family strains have been as high as 80 to 87%.

Strain Typing of Mycobacterium tuberculosis Isolates from Korea by Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats / 대한진단검사의학회지

BACKGROUND: Mycobacterium tuberculosis is one of the most clinically significant infectious agents. Especially during mass outbreaks, accurate identification and monitoring are required. The proportion of Beijing family members is very high among infecting strains, and spoligotyping is not suitable for strain typing. Therefore, we studied the homogeneity of isolates using the mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) method and identified its utility for carrying out molecular epidemiologic analysis. METHODS: Eighty-one clinical M. tuberculosis isolates that had previously been analyzed by spoligotyping were used in this study. We used the 12 standard MIRU loci and further four exact tandem repeat (ETR) loci (ETR-A, -B, -C, and -F). Four strains each of randomly selected Beijing and Beijing-like families were subjected to IS6110- restriction fragment length polymorphism analysis. RESULTS: All 81 samples showed amplification products of all VNTR loci, and all of them showed differences in at least one locus. The calculation of the Hunter-Gaston diversity index (HGDI) for MIRU-VNTR gave the value of 0.965. Discriminatory index in the six loci (MIRU-10, -16, -26, -31, -39, and ETR-F) were found to be highly discriminated (HGDI >0.6). Beijing and Beijing-like family isolates were discriminated into different MIRU-VNTR types. CONCLUSIONS: MIRU-VNTR analysis by using well-selected loci can be useful in discriminating the clinical M. tuberculosis isolates in areas where the Beijing family is predominant.

Falso diagnóstico de tuberculosis por cultivo / False diagnosis of tuberculosis by culture

Las herramientas de genotipificación intra-especie de Mycobacterium tuberculosis desarrolladas durante los años 90 no sólo dieron un impulso notable a la epidemiología de la tuberculosis, también pusieron de manifiesto un fenómeno hasta entonces soslayado en los laboratorios de tuberculosis: la contaminación cruzada de muestras. Este error consiste en la transferencia accidental de bacilos de una muestra con alta carga bacilar a la o las procesadas subsecuentemente. La consiguiente aparición de falsos cultivos positivos puede inducir al diagnóstico erróneo de tuberculosis y la instauración de tratamientos prolongados con drogas potencialmente tóxicas. Esa secuencia de errores conduce al mal manejo de los pacientes involucrados, la distracción de los recursos del sistema de salud y la distorsión de los resultados de análisis epidemiológicos. Se detectó contaminación cruzada en todos los laboratorios donde fue investigada sistemáticamente, con tasas de alrededor del 3% de los cultivos positivos. La confirmación requiere confrontar resultados bacteriológicos, clínicos, epidemiológicos y de genotipificación. Realizamos aquí una revisión de la información nacional e internacional sobre el tema y describimos las medidas recomendadas para minimizar el riesgo, vigilar la ocurrencia y evitar las consecuencias clínicas de este error de laboratorio que vulnera la certeza de un cultivo positivo.

A remarkable input to the epidemiology of tuberculosis was not the only benefit of the molecular tools developed in the early nineties for Mycobacterium tuberculosis intra-species differentiation. These genotyping methods served also to unveil specimen crosscontamination, which was until then overlooked in laboratories culturing mycobacteria. This error consists in the accidental carry-over of bacilli from a specimen with high bacterial load to that, or those, processed subsequently. The ensuing detection of falsely positive cultures can result in a wrong diagnosis of tuberculosis and the initiation of a long-lasting treatment with potentially toxic drugs. This series of errors implies the mismanagement of patients, the distraction of public health system resources, and the distortion of epidemiological data. M. tuberculosis laboratory cross-contamination was detected wherever investigated systematically, with a median rate of 3% of all positive cultures. The confirmation of this error requires a critical appraisal of bacteriological, clinical, epidemiological and genotyping results. We present here a review of national and international information on laboratory crosscontamination and describe measures recommended for minimizing the risk, surveying the occurrence, and avoiding clinical consequences of this laboratory error that raises a question on the reliability of a positive culture.

Molecular Epidemiologic Analysis of Legionella pneumophila by Infrequent-Restriction-Site Polymerase Chain Reaction / 대한임상미생물학회지

BACKGROUND: The frequent outbreak of legionellosis makes it critical to identify infection sources for the prevention and blockade of transmission of the disease. METHODS: Thirty-one strains of Legionella pneumophila isolated from the cooling towers of big buildings in Busan and Gyungsangnamdo Province areas, 12 strains of L. pneumophila from patients in Japan, and one type strain (L. pneumophila ATCC 33152) were used for molecular strain typing by using an infrequent-restriction-site polymerase chain reaction (IRS-PCR). RESULTS: Each strain revealed to have 7-16 bands of 200-1000 bp size. All 44 strains showed band patterns different from each other, except two strains sharing 90% homology. CONCLUSION: The molecular typing of Legionellaby IRS-PCR is an excellent and rapid method for discriminating strains; therefore, it should be useful in demonstrating the identity of possible outbreak strains.

Molecular Typing of Trichophyton rubrum Isolated from Multiple Skin Lesions / 中华皮肤科杂志

Objective To determine whether Trichophyton rubrum isolated from different lesions in the same patient is of different strains. Methods DNA was extracted from the isolates, then subjected to a polymerase chain reaction-based typing which analyzed the number of repetitive elements in the non-transcribed spacer region of the ribosomal DNA gene repeats. Results Thirty-six strains of T. rubrum were isolated from 17 patients with fungal infection on multiple sites. All strains could be classified into 10 genotypes. The genotype distribution was unrelated to sites of infection. It happened to 8 of the 17 patients that multiple genotypes were involved in T. rubrum infection on different sites in the same body. Conclusion The study shows that multiple genotypes are involved in T. rubrum infection on different sites in the same patient, suggesting external sources of infection rather than infection from a different site in the same individual.