Abstract
We applied MIRU-VNTR (mycobacterial interspersed repetitive-unit–variable-number tandem-repeat typing) to directly analyze the bacilli present in 61 stain-positive specimens from tuberculosis patients. A complete MIRU type (24 loci) was obtained for all but one (no amplification in one locus) of the specimens (98.4%), and the allelic values fully correlated with those obtained from the corresponding cultures. Our study is the first to demonstrate that real-time genotyping of Mycobacterium tuberculosis can be achieved, fully transforming the way in which molecular epidemiology techniques can be integrated into control programs.
TEXT
The application of fingerprinting tools has proven crucial in the identification of cases of infection by the same Mycobacterium tuberculosis strain (clustered cases), which are considered to result from recent transmission events and constitute a key indicator when the efficiency of tuberculosis control programs is being evaluated (1). The time required to identify clusters from cultured samples has been greatly reduced by a new, faster PCR-based technique, MIRU-VNTR (mycobacterial interspersed repetitive-unit–variable-number tandem-repeat typing) (7). However, one challenge remains unresolved, namely, how to obtain the M. tuberculosis genotype sufficiently quickly for it to be integrated into the survey of tuberculosis transmission, while contact tracing is still ongoing. To get this target, the only option would be to switch from a culture-based genotyping approach to one based on direct analysis of the bacilli present in respiratory specimens, and this was the aim of this study.
We selected all cases (January 2005 to December 2010) of culture-confirmed TB with a stain-positive sputum sample identified in a population-based molecular epidemiology survey of transmission of tuberculosis performed in Almeria, Spain (3). We established the following criteria for inclusion: (i) frozen stain-positive sputum must be available, and (ii) the complete MIRU-VNTR genotype from the corresponding cultured M. tuberculosis isolate must be available. From the samples that met these criteria, we selected representatives of clustered cases (where different individuals were infected by the same strain) and orphan cases, attempting to maintain a proportion between clustered and orphan cases similar to that found during the survey in Almeria (around 35:65). The estimated sample size was based on the following assumptions: an expected proportion of complete agreement (absolute concordance) of 0.90, a width of the confidence interval of ±10% (0.10), and an alpha two-sided error of 0.05. With these assumptions, the estimated sample size (estimation of one proportion) was 35. In order to increase the precision of our estimation, we decided to extend the recruitment to all the cases (n = 61) that met the inclusion criteria mentioned above.
Specimens were retrieved from the Complejo Hospitalario Torrecardenas in Almeria and sent blind to Hospital Gregorio Marañon in Madrid for analysis. The final genotyping data obtained from the analysis on specimens were sent back to Almeria for a concordance analysis with the data previously obtained from the cultured M. tuberculosis isolates.
DNA was extracted from 1 ml of decontaminated sputum using a column-based purification method (QIAamp DNA minikit protocol; Qiagen, Courtaboeuf, France) and eluted in 70 μl of buffer AE.
The MIRU-VNTR protocol followed was a modified version of the original one (eight triplex PCRs) developed to analyze cultured isolates (6). The final reaction mixture (50 μl) included 25 μl of PCR Master Mix (Qiagen multiplex PCR kit; Qiagen, Courtaboeuf, France), 5 μl of Q solution, and 0.25 μM each unlabeled and labeled oligonucleotide for mixes 1 to 3, 5, 6, and 8. Mixes 4 and 7 were analyzed with the PuReTaq Ready-To-Go PCR bead system (GE Healthcare, Buckinghamshire, United Kingdom), and the final reaction mixtures (25 μl) included 0.25 μM each unlabeled and labeled oligonucleotide (0.5 μM for loci QUB-4156 and MIRU20) and 1.5 μl of dimethyl sulfoxide.
The primers used for PCR amplification were described by Supply et al. (6), and amplification profiles were as described elsewhere (5), except for the number of cycles in multiplex PCR assays (35 cycles). PCR products were purified with a High Pure PCR product purification kit (Roche Diagnostics, Mannheim, Germany), their concentration was measured, and the products were then diluted, to rule out signal saturation, to 2.5 ng/μl for all the mixes except 4 and 7, in which concentrations were fixed at 10 ng/μl.
PCR products were analyzed by capillary electrophoresis (3130xl Genetic Analyzer, POP-7 polymer, 1200 LIZ ISS standard; Applied Biosystems, Foster City, CA). The PCR fragments were sized using the GeneMapper v4.0 software package. Throughout the study, technical performance was evaluated by monitoring a blank and two fixed PCR samples as quality controls. The retention time data from the ISS peaks and the amplified product peaks of the two quality controls were used to rule out deviations in the correct allelic assignment of the unknown samples.
We performed MIRU-VNTR analysis on 61 stain-positive specimens from 61 patients. Based on the number of acid-fast bacilli observed (4), clinical specimens were classified as having >90 bacilli/field (4+; n = 30), 10 to 90 bacilli/field (3+; n = 15), 1 to 9 bacilli/field (2+; n = 11), and 1 to 9 bacilli/10 fields (1+; n = 5).
In 7 specimens (two 4+, three 3+, and two 2+ specimens), amplification was not detected in one or two loci in one of the mixes (Mtub39 in three specimens, QUB-4156 in two specimens, Mtub39 and QUB-4156 in one specimen, and ETRA and QUB-11b in one specimen) (Table 1). In these cases, the determination was repeated by performing a single PCR for each specific locus. The simplex PCR was run with a final reaction mixture (50 μl) composed of 1 U of HotStart Taq DNA polymerase (Qiagen) and 5 μl of PCR buffer plus 3 μl of MgCl2 with a final Mg2+ concentration of 2.25 mM, 10 μl of Q solution, 0.4 μl of a deoxynucleoside triphosphate mix, and 0.1 μM each unlabeled and labeled oligonucleotide. After simplex PCR, the locus QUB11b from patient 48 still failed to be amplified, but all but one of the remaining loci rendered a result, which meant that a complete genotype was obtained for all but one of the 61 specimens (98.36%; 95% confidence interval [CI], 91.2 to 99.9) (Table 1), even from the specimens with the lowest bacterial load (1 to 9 bacilli/10 fields).
Table 1.
MIRU-VNTR data from analysis of respiratory specimens
| Cluster status | Strain | Patient | Bacterial load | MIRU-VNTR locus aliasa |
Full agreement with cultured isolates | |||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIRU2 | MIRU20 | MIRU23 | MIRU24 | MIRU27 | MIRU39 | MIRU4 | MIRU26 | MIRU40 | MIRU10 | MIRU16 | MIRU31 | Mtub04 | ETR C | ETR A | Mtub30 | Mtub39 | QUB-4156 | QUB-11b | Mtub21 | QUB-26 | Mtub29 | ETR B | Mtub34 | |||||
| Clustered | C1 | 1 | +++ | 2 | 2 | 6 | 1 | 3 | 2 | 2 | 3 | 4 | 4 | 3 | 3 | 3 | 4 | 2 | 2 | 1 | 2 | 4 | 2 | 5 | 4 | 2 | 1 | + |
| C1 | 2 | +++ | 2 | 2 | 6 | 1 | 3 | 2 | 2 | 3 | 4 | 4 | 3 | 3 | 3 | 4 | 2 | 2 | 1 | 2 | 4 | 2 | 5 | 4 | 2 | 1 | + | |
| C2 | 3 | ++ | 2 | 2 | 6 | 1 | 3 | 2 | 2 | 4 | 1 | 4 | 2 | 3 | 2 | 4 | 2 | 1 | 2 | 2 | 2 | 3 | 4 | 4 | 1 | 5 | + | |
| C2 | 4 | +++ | 2 | 2 | 6 | 1 | 3 | 2 | 2 | 4 | 1 | 4 | 2 | 3 | 2 | 4 | 2 | 1 | 2 | 2 | 2 | 3 | 4 | 4 | 1 | 5 | + | |
| C2 | 5 | +++ | 2 | 2 | 6 | 1 | 3 | 2 | 2 | 4 | 1 | 4 | 2 | 3 | 2 | 4 | 2 | 1 | 2 | 2 | 2 | 3 | 4 | 4 | 1 | 5 | + | |
| C3 | 6 | ++++ | 1 | 2 | 6 | 1 | 3 | 2 | 2 | 5 | 2 | 5 | 3 | 2 | 3 | 2 | 2 | 1 | 2 | 2 | 2 | 3 | 4 | 4 | 2 | 3 | + | |
| C3 | 7 | ++++ | 1 | 2 | 6 | 1 | 3 | 2 | 2 | 5 | 2 | 5 | 3 | 2 | 3 | 2 | 2 | 1 | 2 | 2 | 2 | 3 | 4 | 4 | 2 | 3 | + | |
| C4 | 8 | ++++ | 2 | 1 | 3 | 1 | 3 | 2 | 2 | 5 | 1 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 3 | 6 | 3 | 4 | 2 | 2 | 3 | + | |
| C4 | 9 | +++ | 2 | 1 | 3 | 1 | 3 | 2 | 2 | 5 | 1 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 3 | 6 | 3 | 4 | 2 | 2 | 3 | + | |
| C4 | 10 | ++++ | 2 | 1 | 3 | 1 | 3 | 2 | 2 | 5 | 1 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 3 | 6 | 3 | 4 | 2 | 2 | 3 | + | |
| C4 | 11 | + | 2 | 1 | 3 | 1 | 3 | 2 | 2 | 5 | 1 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 3 | 6 | 3 | 4 | 2 | 2 | 3 | + | |
| C5 | 12 | +++ | 2 | 2 | 3 | 1 | 4 | 2 | 3 | 6 | 5 | 4 | 3 | 3 | 3 | 4 | 2 | 2 | 3 | 2 | 4 | 3 | 5 | 5 | 2 | 3 | + | |
| C5 | 13 | ++++ | 2 | 2 | 3 | 1 | 4 | 2 | 3 | 6 | 5 | 4 | 3 | 3 | 3 | 4 | 2 | 2 | 3 | 2 | 4 | 3 | 5 | 5 | 2 | 3 | + | |
| C6 | 14 | ++++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 4 | 1 | 3 | 3 | 3 | 2 | 4 | 2 | 2 | 5 | 2 | 4 | 2 | 4 | 4 | 2 | 3 | + | |
| C6 | 15 | ++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 4 | 1 | 3 | 3 | 3 | 2 | 4 | 2 | 2 | 5 | 2 | 4 | 2 | 4 | 4 | 2 | 3 | + | |
| C7 | 16 | ++++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 2 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 1 | 5 | 3 | 5 | 4 | 2 | 3 | + | |
| C7 | 17 | ++++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 2 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 1 | 5 | 3 | 5 | 4 | 2 | 3 | + | |
| C8 | 18 | ++++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 3 | 6 | 3 | 3 | 2 | 3 | 3 | 4 | 2 | 3 | 6 | 2 | 6 | 4 | 2 | 3 | + | |
| C8 | 19 | ++++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 3 | 6 | 3 | 3 | 2 | 3 | 3 | 4 | 2 | 3 | 6 | 2 | 6 | 4 | 2 | 3 | + | |
| C9 | 20 | + | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 4 | 3 | 2 | 3 | 2 | 4 | 3 | 2 | 3 | 2 | 4 | 2 | 5 | 4 | 2 | 3 | + | |
| C9 | 21 | ++++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 4 | 3 | 2 | 3 | 2 | 4 | 3 | 2 | 3 | 2 | 4 | 2 | 5 | 4 | 2 | 3 | + | |
| C9 | 22 | ++++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 4 | 3 | 2 | 3 | 2 | 4 | 3 | 2 | 3 | 2 | 4 | 2 | 5 | 4 | 2 | 3 | + | |
| C9 | 23 | +++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 4 | 3 | 2 | 3 | 2 | 4 | 3 | 2 | 3 | 2 | 4 | 2 | 5 | 4 | 2 | 3 | + | |
| Orphan | O1 | 24 | ++++ | 3 | 2 | 4 | 2 | 3 | 2 | 2 | 4 | 1 | 5 | 2 | 5 | 2 | 5 | 4 | 4 | 4 | 3 | 3 | 4 | 6 | 3 | 4 | 3 | + |
| O2 | 25 | ++++ | 2 | 2 | 5 | 1 | 3 | 2 | 3 | 5 | 4 | 3 | 2 | 3 | 2 | 4 | 3 | 2 | 3 | 2 | 5 | 2 | 5 | 4 | 2 | 3 | + | |
| O3 | 26 | ++++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 3 | 3 | 3 | 3 | 2 | 3 | 3 | 4 | 4 | 2 | 4 | 3 | 7 | 4 | 2 | 3 | + | |
| O4 | 27 | +++ | 2 | 2 | 3 | 1 | 3 | 2 | 2 | 5 | 3 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 3* | 3* | 5 | 3 | 5 | 2 | 2 | 3 | + | |
| O5 | 28 | +++ | 2 | 2 | 5 | 1 | 4 | 2 | 2 | 5 | 3 | 5 | 3 | 3 | 3 | 3 | 3 | 4 | 3 | 3 | 6 | 2 | 4 | 4 | 2 | 2 | + | |
| O6 | 29 | ++++ | 1 | 2 | 6 | 1 | 3 | 2 | 2 | 5 | 1 | 3 | 3 | 3 | 2 | 4 | 3 | 2 | 6* | 2 | 5 | 2 | 5 | 4 | 2 | 3 | + | |
| O7 | 30 | ++++ | 2 | 2 | 4 | 2 | 3 | 2 | 2 | 4 | 1 | 6 | 2 | 5 | 2 | 5 | 7 | 4 | 4 | 3 | 3 | 4 | 6 | 3 | 5 | 3 | + | |
| O8 | 31 | ++++ | 1 | 2 | 3 | 1 | 3 | 2 | 2 | 5 | 5 | 4 | 3 | 2 | 3 | 2 | 2 | 1 | 2 | 2 | 2 | 3 | 6 | 4 | 2 | 3 | + | |
| O9 | 32 | ++++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 3 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 3 | 5 | 3 | 5 | 4 | 2 | 3 | + | |
| O10 | 33 | ++++ | 2 | 2 | 6 | 1 | 3 | 2 | 2 | 4 | 1 | 3 | 2 | 3 | 4 | 4 | 2 | 1 | 2 | 2 | 4 | 3 | 8 | 4 | 1 | 5 | + | |
| O11 | 34 | +++ | 2 | 1 | 5 | 1 | 3 | 3 | 1 | 5 | 3 | 3 | 1 | 4 | 2 | 4 | 3 | 2 | 3 | 2 | 3 | 2 | 5 | 4 | 3 | 3 | + | |
| O12 | 35 | ++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 4 | 0 | 3 | 4 | 3 | 2 | 4 | 2 | 2 | 3 | 2* | 5 | 2 | 5 | 4 | 2 | 3 | + | |
| O13 | 36 | ++ | 2 | 2 | 5 | 1 | 3 | 3 | 2 | 5 | 3 | 3 | 3 | 5 | 4 | 4 | 3 | 4 | 3 | 2 | 6 | 5 | 8 | 4 | 2 | 3 | + | |
| O14 | 37 | ++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 3 | 4 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 3 | 6 | 3 | 7 | 4 | 2 | 3 | + | |
| O15 | 38 | ++++ | 2 | 2 | 3 | 1 | 3 | 2 | 2 | 6 | 3 | 5 | 3 | 3 | 2 | 3 | 3 | 3 | 3 | 3 | 4 | 2 | 7 | 2 | 2 | 3 | + | |
| O16 | 39 | ++ | 1 | 2 | 6 | 1 | 3 | 2 | 1 | 5 | 5 | 2 | 3 | 3 | 5 | 4 | 2 | 1 | 2 | 3* | 2 | 3 | 8 | 4 | 2 | 3 | + | |
| O17 | 40 | +++ | 2 | 2 | 5 | 1 | 1 | 2 | 2 | 5 | 2 | 3 | 3 | 3 | 2 | 4 | 2 | 2 | 3 | 2 | 4 | 2 | 5 | 4 | 2 | 3 | + | |
| O18 | 41 | ++++ | 2 | 2 | 3 | 1 | 3 | 2 | 2 | 5 | 3 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 3 | 4 | 3 | 5 | 2 | 2 | 3 | + | |
| O19 | 42 | ++++ | 2 | 2 | 6 | 1 | 1 | 1 | 2 | 5 | 1 | 3 | 3 | 3 | 2 | 4 | 3 | 2 | 6 | 2 | 4 | 2 | 5 | 4 | 2 | 3 | + | |
| O20 | 43 | + | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 3 | 4 | 3 | 3 | 2 | 3 | 3 | 4 | 4 | 3 | 5 | 3 | 7 | 4 | 2 | 3 | + | |
| O21 | 44 | ++++ | 2 | 2 | 3 | 1 | 3 | 2 | 2 | 5 | 4 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 3 | 6 | 3 | 7 | 2 | 2 | 3 | + | |
| O22 | 45 | ++++ | 1 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 5 | 4 | 3 | 2 | 3 | 2 | 2 | 1 | 2 | 2 | 2 | 3 | 6 | 4 | 2 | 3 | + | |
| O23 | 46 | ++++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 6 | 4 | 4 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 3 | 3 | 2 | 7 | 4 | 2 | 3 | + | |
| O24 | 47 | ++ | 2 | 1 | 3 | 1 | 3 | 2 | 2 | 5 | 3 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 3 | 6 | 3 | 7 | 2 | 2 | 3 | + | |
| O25 | 48 | ++++ | 2 | 2 | 5 | 1 | 3 | 3 | 2 | 7 | 3 | 5 | 4 | 5 | 5 | 2 | TA | 2 | 3 | 4 | 4 | 8 | 4 | 2 | 3 | NA | ||
| O26 | 49 | +++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 4 | 5 | 2 | 3 | 2 | 3 | 3 | 4 | 3 | 3 | 4 | 3 | 5 | 4 | 2 | 3 | + | |
| O27 | 50 | + | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 6 | 3 | 4 | 1 | 3 | 2 | 3 | 3 | 4 | 3 | 1 | 3 | 2 | 6 | 4 | 2 | 2 | + | |
| O28 | 51 | ++++ | 2 | 2 | 3 | 1 | 3 | 2 | 2 | 5 | 4 | 5 | 2 | 3 | 2 | 3 | 3 | 3 | 4 | 3 | 3 | 3 | 7 | 2 | 2 | 3 | + | |
| O29 | 52 | ++ | 2 | 2 | 5 | 1 | 3 | 3 | 2 | 7 | 3 | 3 | 3 | 5 | 4 | 4 | 4 | 4 | 3 | 2 | 5 | 5 | 8 | 4 | 2 | 3 | + | |
| O30 | 53 | + | 2 | 1 | 5 | 1 | 3 | 2 | 2 | 5 | 4 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 3 | 3 | 6 | 3 | 7 | 4 | 2 | 3 | + | |
| O31 | 54 | ++++ | 2 | 2 | 5 | 1 | 3 | 2 | 2 | 5 | 3 | 4 | 3 | 3 | 2 | 3 | 4 | 4 | 3 | 3 | 5 | 3 | 7 | 4 | 2 | 2 | + | |
| O32 | 55 | ++ | 1 | 2 | 7 | 1 | 1 | 2 | 2 | 5 | 6 | 4 | 3 | 3 | 3 | 4 | 2 | 1 | 2 | 3 | 2 | 3 | 7 | 4 | 2 | 3 | + | |
| O33 | 56 | ++ | 1 | 2 | 6 | 1 | 3 | 2 | 2 | 5 | 4 | 4 | 2 | 4 | 3 | 4 | 2 | 1 | 2 | 3 | 2 | 3 | 6 | 4 | 2 | 3 | + | |
| O34 | 57 | +++ | 2 | 1 | 5 | 1 | 3 | 2 | 2 | 5 | 3 | 3 | 3 | 3 | 2 | 4 | 4 | 2 | 6* | 2 | 6 | 3 | 5 | 4 | 2 | 3 | + | |
| O35 | 58 | +++ | 2 | 1 | 3 | 1 | 3 | 2 | 2 | 5 | 3 | 5 | 3 | 3 | 2 | 3 | 3 | 4 | 5* | 3 | 5 | 3 | 7 | 2 | 2 | 3 | + | |
| O36 | 59 | +++ | 1 | 2 | 6 | 1 | 3 | 2 | 2 | 6 | 4 | 4 | 3 | 3 | 3 | 4 | 2 | 1 | 2 | 3 | 2 | 3 | 5 | 4 | 2 | 3 | + | |
| O37 | 60 | ++ | 2 | 2 | 6 | 1 | 3 | 2 | 2 | 3 | 4 | 5 | 2 | 3 | 3 | 4 | TA | 2 | 1 | 2 | 2 | 2 | 7 | 4 | 2 | 1 | + | |
| O38 | 61 | ++++ | 2 | 2 | 6 | 1 | 3 | 2 | 2 | 4 | 1 | 4 | 2 | 3 | 2 | 4 | 2 | 1 | 2 | 2 | 3 | 3 | 3 | 4 | 1 | 5 | + | |
Locus order is according to the MIRU-VNTRplus database (http://www.miru-vntrplus.org). TA, target absence; NA, no amplification of allele 2 in locus QUB-11b;
, locus amplification required the simplex PCR format.
When we compared our data with the MIRU type obtained by analyzing the corresponding cultured isolates, the results were identical for all of them. The analysis of all patterns enabled us to identify 23 patients grouped in nine clusters (C1 to C9, including 2 to 4 cases each) and 38 orphan cases (cases 24 to 61) (Table 1), which coincided with the distribution of clustered and orphan cases obtained from the analysis of the cultured isolates.
Our study is the first evaluation, with molecular-epidemiology purposes, of the feasibility of a tuberculosis genotyping scheme applied directly to clinical specimens. If MIRU-VNTR had been performed directly on specimens, we would have succeeded in classifying isolates as clustered or orphan around 30 days sooner, without waiting for the M. tuberculosis culture results. However, we must admit that there could be a gap between our study and the routine performance of applying MIRU-VNTR to clinical samples. The size of this gap should be measured after final evaluation of the true potential of our proposal in forthcoming studies which evaluate the feasibility to introduce this scheme into a prospective routine practice.
Genotyping M. tuberculosis directly from uncultured clinical specimens would also make it possible to extend the applicability of molecular epidemiology approaches to developing areas where M. tuberculosis culture is not systematically performed and standard genotyping cannot be performed and where, consequently, precise information on tuberculosis transmission dynamics is lacking.
Traditional retrospective descriptive epidemiology will soon be replaced by interventionist molecular epidemiology (2), a new format which attempts to rapidly integrate available molecular cluster data to optimize tuberculosis control programs by offering first-line information on transmission routes and dynamics. Our data show that a revolution in the way we understand the molecular epidemiology of tuberculosis is now possible.
ACKNOWLEDGMENTS
María Alonso is supported by a contract (REF CA09/00054) from the Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias) and provides technical support in the Unidad Central de Análisis Molecular of Hospital General Universitario Gregorio Marañón. LightCycler 2.0 was acquired with a grant (REF IF08/36173) from Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias). The 3130xl Genetic Analyzer was partially financed by grants from Fondo de Investigaciones Sanitarias (IF01-3624, IF08-36173). The study was partially financed by Fondo de Investigaciones Sanitarias (S09/02205), Junta de Andalucía (PI-0444/2008 and PI-0306-2009), and SEPAR (763-09).
We are grateful to Ainhoa Simón Zárate, the sequencer technician, who holds a contract from the Fondo de Investigaciones Sanitarias (CA08/00160). We thank Thomas O'Boyle for proofreading the manuscript.
Footnotes
Published ahead of print 29 February 2012
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