Real-time molecular epidemiology of tuberculosis by direct genotyping. of smear-positive clinical specimens

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1 JCM Accepts, published online ahead of print on 29 February 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 Real-time molecular epidemiology of tuberculosis by direct genotyping of smear-positive clinical specimens María Alonso 1,2,3, Marta Herranz 1,2,3, Miguel Martínez Lirola 4,, Milagros González-Rivera 2,5, Emilio Bouza 1,2,3, Darío García de Viedma* 1,2,3 1 Servicio de Microbiología y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain 2 Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain 3 CIBER Enfermedades Respiratorias-CIBERES, Spain 4 Complejo Hospitalario Torrecárdenas, Almería, Spain. On behalf of INDAL-TB group 5 Línea Instrumental Secuenciación, Hospital Gregorio Marañón, Madrid, Spain Running title: Real-time molecular epidemiology in tuberculosis *Corresponding author: Darío García de Viedma Servicio de Microbiología y Enfermedades Infecciosas Hospital General Universitario Gregorio Marañón C/ Dr Esquerdo, 46, Madrid, Spain Fax: dgviedma2@gmail.com 1

2 Abstract We applied MIRU-VNTR to directly analyze the bacilli present in 61 stainpositive specimens from tuberculosis patients. A complete MIRU-type (24 loci) was obtained from all but one (no amplification in one locus) of the specimens (98.4%), and 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 can be integrated into control programs. Downloaded from on December 18, 2018 by guest 2

3 The application of fingerprinting tools has proven crucial in the identification of cases infected by the same Mycobacterium tuberculosis strain (clustered cases), which are considered to result from recent transmission events and constitute a key indicator when evaluating the efficiency of tuberculosis control programs (1). The time to identify clusters from cultured samples has been greatly reduced by a new, faster PCRbased technique, MIRU-VNTR (7). However, one challenge remains unresolved, namely, how to obtain the MTB genotype sufficiently quickly for it to be integrated in 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 analyzing directly the bacilli present in respiratory specimens and this is the aim of this study. We selected all cases (January 2005-December 2010) of culture-confirmed TB with a stain-positive sputum identified in a population-based molecular epidemiology survey on transmission of tuberculosis performed in Almeria (3). We established the following criteria of inclusion: a) to have frozen stain-positive sputum available; b) to have the complete MIRU-VNTR genotype from their corresponding cultured MTB isolate available. Among them, we selected representatives of clustered cases (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: expected proportion of complete agreement (absolute concordance) =.90; width of the confidence interval: +/- 10% (.10); alpha two-sided error = 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) who fulfilled the inclusion criteria mentioned above. 3

4 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 to perform a concordance analysis with the data previously obtained from the cultured MTB isolates. DNA was extracted from 1 ml of decontaminated sputum using a column-based purification method (QIAamp DNA Mini Kit 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 of each unlabeled and labeled oligonucleotide for mixes 1-3, 5, 6 and 8. Mixes 4 and 7 were analyzed with the PuReTaq Ready-To-Go PCR Beads System (GE Healthcare, Buckinghamshire, UK), and the final reaction mixtures (25 μl) included 0.25 μm of 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 the High Pure PCR product Purification Kit (Roche Diagnostics, Mannheim, Germany), and their concentration was measured and diluted, to rule out signal saturation, at 2.5 ng/μl for all the mixes except 4 and 7, in which were fixed at 10 ng/μl. PCR products were analyzed by capillary electrophoresis (3130xl GA- POP-7 polymer LIZ ISS - Applied Biosystems, Foster City, California, USA). The PCR 4

5 fragments were sized using the GeneMapper v 4.0 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), b/f (3+; N=15), 1-9 b/f (2+; N=11) and 1-9 b/10f (1+, N=5). In 7 specimens (two 4+, three 3+ and two 2+), amplification was not detected in one/two of the loci of one of the mixes (three specimens in locus Mtub39, two in locus QUB-4156, one in loci Mtub39/ QUB-4156 and one in loci ETRA/QUB-11b; Table). In these cases the determination was repeated applying a single PCR for each specific locus. The simplex-pcr reaction was run at a final reaction mixture (50 μl) composed of 1 U of HotStart Taq DNA Polymerase (Qiagen), 5 μl of PCR buffer plus 3 μl of MgCl 2 with a final Mg 2+ concentration of 2.25 mm, 10 μl of Q-solution, 0.4 μl of dntp Mix, and 0.1 μm of each unlabeled and labeled oligonucleotide. After applying simplex-pcr, the locus QUB11b from patient 48 still failed to be amplified but all but one of the remaining pendant loci rendered a result, which meant the obtention of a complete genotype for all but one of the 61 specimens (98,36%; CI95%: 91,2-99,9) (Table), even from the specimens with the lowest bacterial load (1-9 b/10 f). When we compared our data with the MIRUtype 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 (Table, C1-C9, 5

6 including 2-4 cases) and 38 orphan cases (cases 24-61; Table), which coincided with the clustered/orphan distribution 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 applied directly on specimens, we would have succeeded in classifying isolates as clustered or orphan, around 30 days in advance, without waiting for the MTB culture results. However we must admit that could there be a gap between our study and the routine performance of applying MIRU-VNTR on 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 MTB directly from uncultured clinical specimens would also make it possible to extend the applicability of molecular epidemiology approaches to developing areas where MTB culture is not systematically performed and standard genotyping cannot be performed, and, 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. 6

7 129 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 (IF , IF ). The study was partially financed by Fondo de Investigaciones Sanitarias (S09/02205), Junta de Andalucía (PI-0444/2008 and PI ), 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 would like to thank Thomas O Boyle for proofreading the manuscript. 7

8 References 1. Cattamanchi, A., P. C. Hopewell, L. C. Gonzalez, D. H. Osmond, L. Masae Kawamura, C. L. Daley, and R. M. Jasmer A 13-year molecular epidemiological analysis of tuberculosis in San Francisco. Int J Tuberc Lung Dis 10: Garcia de Viedma, D., I. Mokrousov, and N. Rastogi Innovations in the molecular epidemiology of tuberculosis. Enferm Infecc Microbiol Clin 29 Suppl 1: Martinez-Lirola, M., N. Alonso-Rodriguez, M. L. Sanchez, M. Herranz, S. Andres, T. Penafiel, M. C. Rogado, T. Cabezas, J. Martinez, M. A. Lucerna, M. Rodriguez, C. Bonillo Mdel, E. Bouza, and D. Garcia de Viedma Advanced survey of tuberculosis transmission in a complex socioepidemiologic scenario with a high proportion of cases in immigrants. Clin Infect Dis 47: Nolte, F Mycobacterium. Manual of Clinical Microbiology 6ed. Washington, DC. 5. Oelemann, M. C., R. Diel, V. Vatin, W. Haas, S. Rusch-Gerdes, C. Locht, S. Niemann, and P. Supply Assessment of an optimized mycobacterial interspersed repetitive- unit-variable-number tandem-repeat typing system combined with spoligotyping for population-based molecular epidemiology studies of tuberculosis. J Clin Microbiol 45: Supply, P., C. Allix, S. Lesjean, M. Cardoso-Oelemann, S. Rusch-Gerdes, E. Willery, E. Savine, P. de Haas, H. van Deutekom, S. Roring, P. Bifani, N. Kurepina, B. Kreiswirth, C. Sola, N. Rastogi, V. Vatin, M. C. Gutierrez, M. Fauville, S. Niemann, R. Skuce, K. Kremer, C. Locht, and D. van Soolingen Proposal for standardization of optimized mycobacterial interspersed 8

9 repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 44: Supply, P., S. Lesjean, E. Savine, K. Kremer, D. van Soolingen, and C. Locht Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on interspersed repetitive units. J Clin Microbiol 39: mycobacterial Downloaded from on December 18, 2018 by guest 9

10 Table. MIRU-VNTR data from analysis of respiratory specimens MIRU-VNTR loci aliases a Clustered strains Cluster status Patient Bacterial load 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 Full agreement with cultured isolates C C C C C C C C C C C C C C C C C C C C C C C O O O O * 3* O O * O O O O O O * O O O O * O O O O O O O O O TA NA O O O O O O O O O * O * O O TA O TA: Target Absence. NA : No amplification of the alelle 2 in locus QUB-11b. *: Locus amplification required simplex-pcr format. a : Loci order according to MIRU-VNTRplus database ( CN: Cluster number, ON: Orphan strain number Orphan strains