A Finer Snapshot of Circulating Mycobacterium tuberculosis Genotypes in Guadeloupe, Martinique, and French Guiana

Transcription

1 JCM Accepts, published online ahead of print on May J. Clin. Microbiol. doi:.8/jcm.78- Copyright, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved NOTE - JCM78-v, A Finer Snapshot of Circulating Mycobacterium tuberculosis Genotypes in Guadeloupe, Martinique, and French Guiana Julie Millet, William Laurent, Thierry Zozio, and Nalin Rastogi* WHO Supranational TB Reference Laboratory, Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, Abymes, Guadeloupe, France Running title: TB in ultra-peripheral Europe Key words: Tuberculosis, Mycobacterium, Spoligotyping, MIRU, Genotyping * Corresponding author. Mailing address: WHO Supranational TB Reference Laboratory, Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, 978-Abymes, Cedex, Guadeloupe, France Phone: Fax: nrastogi@pasteur-guadeloupe.fr Downloaded from on June 7, 8 by guest

2 This study shows the benefit of spoligotyping coupled to MIRU typing to pinpoint circulating Mycobacterium tuberculosis genotypes in Guadeloupe, Martinique, and French Guiana. We hereby propose reduced -loci and 6-loci subsets for LAM and Haarlem lineage strains that predominate in South America and the Caribbean, retaining 99.% and 99.6% of the total discriminatory power of the -loci scheme, respectively. The present investigation provides an improved phylogenetic characterization of existing Mycobacterium tuberculosis transmission chains in Guadeloupe, Martinique and French Guiana. It also aimed to estimate the benefit of -loci MIRU typing coupled to spoligotyping, and to select a reduced MIRU-loci typing scheme for an efficient subtyping of clusters belonging to LAM and Haarlem lineages that predominate in South America and the Caribbean (, -, ). Under a longitudinal universal genotyping program covering a -year period (January to December ), all culture-positive TB cases (n=76) from Guadeloupe (n=), Martinique (n=) and French Guiana (n=9) were studied. Mycobacterial identification, drug susceptibility testing, spoligotyping and -loci MIRU typing was performed as reported earlier (,,, 6). Additional typing schemes were used retrospectively to compare a subset of strains using -loci ETRs (9) and additional MIRU loci (). The genotyping data was compared to the international SITVIT database of the Pasteur Institute of Guadeloupe as described (). In this database, SIT (Spoligotype International Type) and MIT (MIRU International Type) designate spoligotyping and MIRU patterns shared by or more patient isolates, respectively, whereas orphan designates patterns reported for a single isolate. Major phylogenetic clades were assigned according to signatures provided in SpolDB, which defines 6 genetic lineages/sub-lineages (). Discriminatory power of a typing method (or a combination of methods) was calculated using the Hunter and Gaston Discriminatory Index (HGDI; ), and significant difference between percentages was estimated using a Chi-square test. Using the Perl programming language ( we also developed and implemented a dynamic programming algorithm named MIRU-Selector. For a given set of strains, this program calculates the discriminatory ability of each possible combination of MIRUs by subtracting loci at a time (-loci, then -loci and so on), until a single loci is left. Spoligotyping allowed a first-line screening of the clinical isolates and lineage assignments (Figure A to C, and Table ; refer to Supplemental Tables S to S for demographic characteristics of patients, drug-resistance of M. tuberculosis clinical isolates, and detailed typing results). A total of 76 patterns obtained corresponded to clusters (n= strains or 7.9%, to strains/cluster), and 6 (6.%) unique patterns with 8 orphans. The bulk of genotypes in our study (n=9/76 or 7.%) concerned families: evolutionary recent LAM (%), Haarlem (.9%) and ill-defined T clades (.%). MIRU typing was performed on all the 76 strains, nonetheless strains with incomplete profiles were excluded from the analysis (Supplemental Table S); 97 patterns obtained corresponded to clusters (n=8 strains or.6%, to strains/cluster), and 7 (7.%) unique patterns with 8 orphans. Combined spoligotyping and MIRU analysis grouped 8/ (7.7%) strains in clusters ( to strains per cluster), while 96 strains were unclustered. Downloaded from on June 7, 8 by guest

3 The combined numerical analysis of isolates clustered by spoligotyping for which MIRU data was available (n=9) is summarized in Supplemental Figure S. It additionally shows all single vs. double locus variants (SLV vs. DLV) representing strains differing in or copies of a given minisatellite for enhanced cluster analysis. As can be seen, MIRU loci 6 and were most important contributors to the locus variations observed. Four clusters were specific for Guadeloupe: with identical patterns (SIT-MIT, and SIT99-MIT) and being SLVs (SIT9-MIT /MIT 78 and SIT-MIT/MIT78). We also detected introduction in Martinique of the Beijing genotype (SIT) in young and epi-linked patients (brother and sister aged <8 years) for the first time since 99 (); however the strains differed by -copy changes upon -loci MIRU typing (MIT 7 and MIT 9). Finally, 9 clusters were specific to French Guiana among which concerned patients originating in Brazil (SIT76-MIT8, and DLVs: SIT9-MIT898/MIT). Lastly, none of the clusters were made up of only multiple drug-resistant (MDR) isolates, however clusters of isolates each (SIT-MIT, and SIT7-MIT) found respectively in Guadeloupe and French Guiana, contained single MDR strains as a result of acquired drug resistance. As summarized in Table, the discriminatory power of typing methods used alone or in combination increased in the order: -loci ETR < spoligotyping < -loci MIRU; achieving an HGDI of.99 for spoligotyping + -loci MIRU (subset B with isolates). In subset C (n=97 isolates) with additional data on -loci ETRs, the highest HGDI was.99 for the combination of all markers pooled together. These results argue in favor of a two-step approach for large-scale, prospective genotyping of M. tuberculosis based on spoligotyping and MIRU-VNTRs (6, 7). We further decided to evaluate the potential of additional MIRU- VNTRs () by screening a subset containing SIT-MIT clusters (n=6, to 7 strains per cluster). As summarized in Supplemental Table S, only out of clusters did not subdivide. Thus use of additional loci reduced the clustering by.8% to as little as 6 clusters (n=7 strains, strains per cluster). Last but not least, we attempted to define a minimal subset of MIRU loci allowing satisfactory discrimination of strains of the largest clades in the present study, i.e. LAM and Haarlem. Figure D to F summarize the performance (in percentage) of each of the individual MIRU loci as compared to the discriminatory power of the -loci typing scheme for the Haarlem (Fig. D, n=7, HGDI=.86) and LAM (Fig. E, n=, HGDI=.98) lineage strains. The whole sample is shown as reference in Fig. F (n=, HGDI=.98). Using MIRU-Selector, we were able to define reduced 6-loci (MIRU loci,,,,, and ), and -loci (MIRU loci, 6, and ) typing schemes for studying specifically the Haarlem and LAM lineage strains. In our sample, these schemes allowed to achieve 99.6% (HGDI=.88) and 99.% (HGDI=.9) of the total discriminatory power of the full -loci scheme respectively. As previously (), Haarlem (.9%), LAM (%) and T Family (.%) constituted the major clades in our setting. Strains of the EAI lineage were mainly found in patients living in French Guiana (9/ patients) with the predominance of SIT (EAI6-BGD sublineage, /9 strains). According to the SITVIT database, strains were reported to present SIT among which.9% (8/) originated in Suriname, where it represents as high as 6% of all M. tuberculosis isolates (our unpublished observations). Hence, the SIT strains may represent emerging trans-border M. tuberculosis clone in French Guiana. The presence of the X strains, of presumed Anglo-Saxon origin (), in all the French departments of America, and the Caribbean territories, probably reflects the past history of this geographical area. Lastly, despite a Downloaded from on June 7, 8 by guest

4 decrease in the proportion of drug resistant strains and MDR as compared to previous study (), the detection of the Beijing genotype in Martinique and French Guiana must be taken with utmost care since it is often associated with development of drug resistance (, 8). Acknowledgements We thank colleagues at various university hospitals, local clinics, dispensaries, health services, and research institutions for their precious collaboration. We are grateful to colleagues at Institut Pasteur de la Guadeloupe for their precious help regarding identification and drug susceptibility testing of the isolates (M. Berchel, K.S. Goh, and F. Prudenté), genotyping and database comparison (C. Sola, B. Liens, I. Filliol, S. Ferdinand, K. Brudey), for retrospective genotyping of clustered isolates using additional MIRU loci (J. Vanhomwegen), and for developing the MIRU-Selector application (C. Demay). REFERENCES. Abadia, E., M. Sequera, D. Ortega, M.V. Mendez, A. Escalona, O. Da Mata, E. Izarra, Y. Rojas, R. Jaspe, A.S. Motiwala, D. Alland, J. de Waard, H.E. Takiff. 9. Mycobacterium tuberculosis ecology in Venezuela: epidemiologic correlates of common spoligotypes and a large clonal cluster defined by MIRU-VNTR-. BMC Infect. Dis. 9:.. Bifani, P.J., B. Mathema, N.E. Kurepina, B.N. Kreiswirth.. Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. Trends Microbiol. :-.. Brudey, K., J.R. Driscoll, L. Rigouts, W.M. Prodinger, A. Gori, S.A. Al-Hajoj, C. Allix, L. Aristimuno, J. Arora, V. Baumanis, L. Binder, P. Cafrune, A. Cataldi, S. Cheong, R. Diel, C. Ellermeier, J.T. Evans, M. Fauville- Dufaux, S. Ferdinand, D. Garcia de Viedma, C. Garzelli, L. Gazzola, H.M. Gomes, M.C. Gutierrez, P.M. Hawkey, P.D. van Helden, G.V. Kadival, B.N. Kreiswirth, K. Kremer, M. Kubin, S.P. Kulkarni, B. Liens, T. Lillebaek, H. M. Ly, C. Martin, I. Mokrousov, O. Narvskaia, Y.F. Ngeow, L. Naumann, S. Niemann, I. Parwati, M.Z. Rahim, V. Rasolofo-Razanamparany, T. Rasolonavalona, M.L. Rossetti, S. Rusch-Gerdes, A. Sajduda, S. Samper, I. Shemyakin, U.B. Singh, A. Somoskovi, R. Skuce, D. van Soolingen, E.M. Streicher, P. N. Suffys, E. Tortoli, T. Tracevska, V. Vincent, T.C. Victor, R. Warren, S.F. Yap, K. Zaman, F. Portaels, N. Rastogi, and C. Sola. 6a. Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB) for classification, population genetics and epidemiology. BMC Microbiol. 6:.. Brudey, K., I. Filliol, S. Ferdinand, V. Guernier, P. Duval, B. Maubert, C. Sola, and N. Rastogi. 6b. Longterm population-based genotyping study of Mycobacterium tuberculosis complex isolates in the French departments of the Americas. J. Clin. Microbiol. :8-9.. Candia, N., B. Lopez, T. Zozio, M. Carrivale, C. Diaz, G. Russomando, N.J. de Romero, J.C. Jara, L. Barrera, N. Rastogi, and V. Ritacco. 7. First insight into Mycobacterium tuberculosis genetic diversity in Paraguay. BMC Microbiol. 7:7. 6. Cowan, L. S., L. Diem, T. Monson, P. Wand, D. Temporado, T. V. Oemig, and J. T. Crawford.. Evaluation of a two-step approach for large-scale, prospective genotyping of Mycobacterium tuberculosis isolates in the United States. J. Clin. Microbiol. : García de Viedma, D., I. Mokrousov, and N. Rastogi.. Innovations in the molecular epidemiology of tuberculosis. Enferm. Infecc. Microbiol. Clin. 9 (Suppl ):8-. Downloaded from on June 7, 8 by guest

5 European Concerted Action on New Generation Genetic Markers and Techniques for the Epidemiology and Control of Tuberculosis. 6. Beijing/W genotype Mycobacterium tuberculosis and drug resistance. : Frothingham, R., and W.A. Meeker-O Connell Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats. Microbiology : Hunter, P.R., and M.A. Gaston Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J. Clin. Microbiol. 6: Kamerbeek, J., L. Schouls, A. Kolk, M. van Agterveld, D. van Soolingen, S. Kuijper, A. Bunschoten, H. Molhuizen, R. Shaw, M. Goyal, and J. van Embden Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. : Millet, J., S. Baboolal, P.E. Akpaka, D. Ramoutar, N. Rastogi. 9. Phylogeographical and molecular characterization of an emerging Mycobacterium tuberculosis clone in Trinidad and Tobago. Infect Genet Evol. 9:6-.. Molina-Torres, C.A., E. Moreno-Torres, J. Ocampo-Candiani, A. Rendon, K. Blackwood, K. Kremer, N. Rastogi, O. Welsh, and L. Vera-Cabrera.. Mycobacterium tuberculosis spoligotypes in Monterrey, Mexico. J. Clin. Microbiol. 8:8-.. Sola, C., I. Filliol, E. Legrand, S. Lesjean, C. Locht, P. Supply, and N. Rastogi.. Genotyping of the Mycobacterium tuberculosis complex using MIRUs: Association with VNTR and spoligotyping for molecular epidemiology and evolutionary genetics. Infect. Genet. Evol. :.. 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. 6. Proposal for standardization of optimized Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat typing of Mycobacterium tuberculosis. J. Clin. Microbiol. : 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 mycobacterial interspersed repetitive units. J. Clin. Microbiol. :6-7. LEGENDS TO FIGURES Figure. Distribution of the SITs found in this study and corresponding lineage attribution in various settings (A C), and relative discriminatory power of individual MIRU loci among major genotypic lineages (D F). Insets: A, Guadeloupe; B, Martinique; C, French Guiana; D, Haarlem lineage; E, LAM lineage; F, Total sample. Note that various lineages in Insets A C are shown by different colors. Refer to Supplemental Table S for detailed results. Downloaded from on June 7, 8 by guest

6 Table : Discriminatory power of various typing methods used alone and in combination on different subsets of M. tuberculosis clinical isolates. Subset (No. of isolates) Typing methods No. of distinct profiles No. of unique profiles No. (size) of clusters No (%) clustered isolates Recent transmission rate A (76) Spoligotyping 76 6 (-) (7.9) HGDI B () Spoligotyping 7 6 (-) 9 (7.8).9.96 B () -loci MIRU 97 7 (-) 8 (.6) B () Spoligotyping + -loci MIRU 6 96 (-) 8 (7.7).7.99 C (97) Spoligotyping 7 (-) 6 (6.9) C (97) -loci ETR a 9 7 (-6) 8 (8.) C (97) -loci MIRU 7 6 (-) 6 (7.) C (97) Spoligotyping + -loci ETR 7 7 (-8) (.) C (97) Spoligotyping + -loci MIRU (-8) (.9).6.99 C (97) Spoligotyping + -loci MIRU + -loci ETR 8 7 (-6) 7 (7.8) a -loci ETRs corresponded to Exact tandem repeats A, B, C, D, E according to Frothingham et al. (9) Downloaded from on June 7, 8 by guest

7 Number of strains A 8 9.% 8.%.% 6.% 8.% 7.%.9%.% 6.%.% O r p h a n O r p h a n SITs O r p h a n O r p h a n D E F 6.7%.%.6%.6% 9.%.6%.9%.6% 6 6.7% 6.6% 6.9%.9%.%.7% 6.% 6.% % 7.% 6 6 B.% 8.8% 8.% 6 C.%.6% O rp h a n O rp h a n %.6%.%.% O rp h a n 7 O rp h a n 8 79% 7% 8% % 8% % % 6 % % % 6% 67% 6 Downloaded from on June 7, 8 by guest Figure