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Prospective genotyping of Mycobacterium tuberculosis from fresh clinical samples.

Bidovec-Stojkovič U, Seme K, Žolnir-Dovč M, Supply P - PLoS ONE (2014)

Bottom Line: Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases.Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%).For 20 of the 26 patients for whom complete or even incomplete M. tuberculosis isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Mycobacteria, University Clinic of Respiratory and Allergic Diseases, Golnik, Slovenia.

ABSTRACT
Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases. We performed a prospective study to evaluate the use of standardized MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) typing of Mycobacterium tuberculosis directly on 79 fresh clinical samples from 26 TB patients consecutively enrolled over a 17-month period. Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%). The degree of completion of the genotypes obtained significantly correlated with smear microscopy grade both for 26 first samples (p = 0.0003) and for 53 follow-up samples (p = 0.002). For 20 of the 26 patients for whom complete or even incomplete M. tuberculosis isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates. Standard 24 locus MIRU-VNTR typing of M. tuberculosis can be applied directly to fresh clinical samples, with typeability depending on the bacterial load in the sample.

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Related in: MedlinePlus

The number of amplified loci in correlation with smear microscopy.The number of amplified loci in correlation with smear microscopy grades is shown by dots for first (A) and follow-up (B) samples. The calculated curves and coefficients (R) of linear correlation are also indicated for both sample sets. Smear microscopy grades are coded as follows: 3+ (>10 bacilli/field); 2+ (1–10 bacilli/field); 1+ (10–99 bacilli/100 fields); 2AFB (7–9 bacilli/100 fields), and 1 AFB (3–6 bacilli/100 fields).
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pone-0109547-g002: The number of amplified loci in correlation with smear microscopy.The number of amplified loci in correlation with smear microscopy grades is shown by dots for first (A) and follow-up (B) samples. The calculated curves and coefficients (R) of linear correlation are also indicated for both sample sets. Smear microscopy grades are coded as follows: 3+ (>10 bacilli/field); 2+ (1–10 bacilli/field); 1+ (10–99 bacilli/100 fields); 2AFB (7–9 bacilli/100 fields), and 1 AFB (3–6 bacilli/100 fields).

Mentions: There was a significant positive correlation between the number of loci successfully amplified and the bacterial load, both for the first (p = 0.0003) and the follow-up samples (p = 0.002). That is, the number of alleles obtained was higher when the bacterial load was higher (Table 1, Figure 2, Table S2). The same sensitivity to bacterial load was also noticed at the individual locus level because the most frequently non-amplified locus overall, 4156, was missing in 16% of 3+, 25% of 2+, 62.5% of 1+, 62.5% of 2AFB, and 80% of 1AFB samples. This locus was also the most frequent single locus missing when the 23 other loci were successfully amplified (three out of four cases; in the remaining case, locus 2163b was missing). In terms of non-amplification frequencies, this locus was followed by loci 1644, 960, and 3192. As could be expected, we generally observed lower proportions of complete M. tuberculosis types among samples that were collected only after the first 3 weeks of treatment, probably reflecting their often lower bacterial loads (Table 1).


Prospective genotyping of Mycobacterium tuberculosis from fresh clinical samples.

Bidovec-Stojkovič U, Seme K, Žolnir-Dovč M, Supply P - PLoS ONE (2014)

The number of amplified loci in correlation with smear microscopy.The number of amplified loci in correlation with smear microscopy grades is shown by dots for first (A) and follow-up (B) samples. The calculated curves and coefficients (R) of linear correlation are also indicated for both sample sets. Smear microscopy grades are coded as follows: 3+ (>10 bacilli/field); 2+ (1–10 bacilli/field); 1+ (10–99 bacilli/100 fields); 2AFB (7–9 bacilli/100 fields), and 1 AFB (3–6 bacilli/100 fields).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196917&req=5

pone-0109547-g002: The number of amplified loci in correlation with smear microscopy.The number of amplified loci in correlation with smear microscopy grades is shown by dots for first (A) and follow-up (B) samples. The calculated curves and coefficients (R) of linear correlation are also indicated for both sample sets. Smear microscopy grades are coded as follows: 3+ (>10 bacilli/field); 2+ (1–10 bacilli/field); 1+ (10–99 bacilli/100 fields); 2AFB (7–9 bacilli/100 fields), and 1 AFB (3–6 bacilli/100 fields).
Mentions: There was a significant positive correlation between the number of loci successfully amplified and the bacterial load, both for the first (p = 0.0003) and the follow-up samples (p = 0.002). That is, the number of alleles obtained was higher when the bacterial load was higher (Table 1, Figure 2, Table S2). The same sensitivity to bacterial load was also noticed at the individual locus level because the most frequently non-amplified locus overall, 4156, was missing in 16% of 3+, 25% of 2+, 62.5% of 1+, 62.5% of 2AFB, and 80% of 1AFB samples. This locus was also the most frequent single locus missing when the 23 other loci were successfully amplified (three out of four cases; in the remaining case, locus 2163b was missing). In terms of non-amplification frequencies, this locus was followed by loci 1644, 960, and 3192. As could be expected, we generally observed lower proportions of complete M. tuberculosis types among samples that were collected only after the first 3 weeks of treatment, probably reflecting their often lower bacterial loads (Table 1).

Bottom Line: Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases.Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%).For 20 of the 26 patients for whom complete or even incomplete M. tuberculosis isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Mycobacteria, University Clinic of Respiratory and Allergic Diseases, Golnik, Slovenia.

ABSTRACT
Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases. We performed a prospective study to evaluate the use of standardized MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) typing of Mycobacterium tuberculosis directly on 79 fresh clinical samples from 26 TB patients consecutively enrolled over a 17-month period. Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%). The degree of completion of the genotypes obtained significantly correlated with smear microscopy grade both for 26 first samples (p = 0.0003) and for 53 follow-up samples (p = 0.002). For 20 of the 26 patients for whom complete or even incomplete M. tuberculosis isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates. Standard 24 locus MIRU-VNTR typing of M. tuberculosis can be applied directly to fresh clinical samples, with typeability depending on the bacterial load in the sample.

Show MeSH
Related in: MedlinePlus