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Journal of Clinical Microbiology, May 2009, p. 1581-1584, Vol. 47, No. 5
0095-1137/09/$08.00+0     doi:10.1128/JCM.02284-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

First Insight into Genetic Diversity of the Mycobacterium tuberculosis Complex in Albania Obtained by Multilocus Variable-Number Tandem-Repeat Analysis and Spoligotyping Reveals the Presence of Beijing Multidrug-Resistant Isolates {triangledown}

Silva Tafaj,1,{dagger} Jian Zhang,2,{dagger} Yolande Hauck,3 Christine Pourcel,3 Hasan Hafizi,1 Grigor Zoraqi,4 and Christophe Sola2,5*

National TB Reference Laboratory, University Hospital of Lung Diseases Shefqet Ndroqi, Tirana, Albania,1 Institute of Genetics and Microbiology, UMR8621, IGEPE Team, Universud, CNRS Université Paris-Sud 11, Campus d'Orsay, F-91405 Orsay-Cedex, France,2 Institute of Genetics and Microbiology UMR8621, GPMS Team, Universud, CNRS Université Paris-Sud 11, Campus d'Orsay, F-91405 Orsay-Cedex, France,3 Center of Molecular Diagnosis and Genetic Research Mbreteresha Geraldine, University Hospital of Obstetrics and Gynecology, Tirana, Albania,4 Unité de Génétique Mycobactérienne, Institut Pasteur, Paris, France5

Received 28 November 2008/ Returned for modification 17 February 2009/ Accepted 3 March 2009


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ABSTRACT
 
We characterized a set of 100 Mycobacterium tuberculosis complex clinical isolates from tuberculosis (TB) patients in Albania, typing them with a 24-locus variable-number tandem-repeat-spoligotyping scheme. Depending on the cluster definition, 43 to 49 patients were distributed into 15 to 16 clusters which were likely to be epidemiologically linked, indicative of a recent transmission rate of 28 to 34%. This result suggests that TB is under control in Albania. However, two multidrug-resistant (MDR) Beijing genotypes harboring the same S531A mutation on the rpoB gene were also found, suggesting a potential recent transmission of MDR TB. Three brand new genotypes, Albania-1 to Albania-3, are also described.


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TEXT
 
Albania is a country in political transition and one of the poorest nations in Europe (Fig. 1) (9). In 2006, the official incidence of tuberculosis (TB) was estimated to be 15.8/100,000 inhabitants (www.eurotb.org). This number varies from 4/100,000 to 50/100,000 among districts in the northern regions of the country (Fig. 1). The number of young adults affected with TB indicates active transmission. Underreporting is also suspected because of the serious clinical condition of new cases, and epidemiological data on TB are scarce for this country. In 2006, 502 cases were reported by the National Tuberculosis Program. Emigration from Albania to Italy and Greece decreased the number of TB-affected people in Albania; however, most of these people immigrated illegally to Italy or Greece and have limited access, if any, to health services.


Figure 1
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  		FIG. 1. Map of Albania with main geographic, demographic, and administrative information about the country. The regional distribution of TB is shown. Dark-gray-shaded areas have an incidence of >40/100,000 inhabitants; gray-shaded areas, >20/100,000 inhabitants; and white areas, 10/100,000 inhabitants. The numbers of recruited cases in this study are indicated by numbers on the districts. (Adapted from a TB prevalence map extracted from the grant proposal document "Albania: Scaling Up the National Response to Tuberculosis, Tirana, June 8, 2005," submitted to the Global Fund for TB.)

A TB laboratory network was established in 2000 in Albania. It is made up of 16 peripheral first-level laboratories that perform direct smear staining, 2 intermediate-level laboratories that perform culture (Shkodra and Korca), and the National Reference Laboratory (NRL), which performs culture and first-line drug susceptibility testing. The NRL processes an average of 20 samples per day. Since 1999, the Albanian NRL has had reference to the Italian Supranational Reference Laboratory. Ninety percent agreement between the NRL and the Supranational Reference Laboratory was achieved for each drug in 2002, and 100% agreement was achieved in 2005 and 2008 (7).

Molecular epidemiology is a complementary support to conventional epidemiology (2). Large, worldwide, representative spoligotype databases have been released, allowing local genetic specificities and the microevolution of the Mycobacterium tuberculosis complex to be studied (3). The use of both variable-number tandem-repeat (VNTR) and spoligotyping is progressively replacing the traditional IS6110-restriction fragment length polymorphism technique in molecular epidemiology (20, 26). The aim of this work was to study the genetic diversity of M. tuberculosis clinical isolates in Albania as a step to prevent multidrug-resistant (MDR) TB strains from spreading in the southeastern part of Europe.

The patient cohort consisted of a total of 71 men and 29 women. The mean age was 47.4 years. Ninety-eight patients were Albanian citizens, and two were Kosovars. Ninety-two patients had pulmonary TB, and eight had extrapulmonary TB (pleuritis cases); in these cases, the bacteria were isolated from pleural fluids. Culture was done on both solid (Löwenstein-Jensen) and liquid (Bactec MGIT 960; Becton Dickinson, NJ) media. Drug susceptibility testing was performed by the proportion method with isoniazid (0.2 µg/ml), rifampin (40.0 µg/ml), streptomycin (4.0 µg/ml), and ethambutol (2.0 µg/ml) (5). Identification at the species level was performed by phenotypic methods and verified by a genotypic method on isolates with rare spoligotypes and/or unusual exact tandem repeat D (ETR-D) sequences (6, 23). Two Beijing clinical isolates were MDR; four isolates were monoresistant to streptomycin (n = 1), rifampin (n = 1), or isoniazid (n = 2), and one was resistant to both streptomycin and isoniazid (data not shown).

One hundred M. tuberculosis clinical isolates from 2006-2007, representing as many patients, were randomly selected and subcultured for DNA preparation. The geographic origin of the patients is shown in Fig. 1. DNA was extracted following three cycles of hot-freeze cell disruption (thermolyzates). Supernatants were collected and kept at –20°C until further use. Spoligotyping was performed using a commercial membrane (Isogen, The Netherlands) (11). VNTR analysis was performed as previously described (12, 20). The rpoB and ETR-D loci were sequenced as described previously (6, 22). Genotyping results were entered into two databases, the MIRU-VNTRplus database (1) and the SITVIT2 database (13, 14), and spoligo-international type (SIT) labels were assigned (Fig. 2). A combined spoligotype-VNTR dendrogram was computed and drawn, using the unweighted paired-group method with mathematical averages and BioNumerics version 5.1 software (Applied Maths, Sint-Marten-Latem, Belgium) (Fig. 1) (19). A recent transmission index (RTI) was computed using the n–1 method (18).


Figure 2
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  		FIG. 2. Genotyping results and dendrogram for 100 M. tuberculosis DNA sequences extracted from clinical isolates representative of 100 TB patients from Albania. (A) Dendrogram (unweighted paired-group method using mathematical averages; distance matrix average of spoligotyping-based and VNTR results) built with BioNumerics version 5.1. (B) Spoligotyping results in binary format, spacers 1 to 43. (C) Twenty-four-locus VNTR results with their original designations and VNTR aliases, from left to right: ETR-A (VNTR0154), ETR-B (VNTR2461), ETR-C (VNTR0577), MIRU02 (VNTR0154), MIRU04 (VNTR0580), MIRU10 (VNTR0960), MIRU16 (VNTR1644), MIRU20 (VNTR2059), MIRU23 (VNTR2531), MIRU24 (VNTR2687), MIRU26 (VNTR2996), MIRU27 (VNTR3007), MIRU31 (VNTR3192), MIRU39 (VNTR4348), MIRU40 (VNTR0802), Mtub04 (VNTR0424), Mtub21 (VNTR1955), Mtub29 (VNTR2347), Mtub30 (VNTR2401), Mtub34 (VNTR3171), Mtub39 (VNTR3960), QuB11b (VNTR2163), QuB26 (VNTR4052), and QuB4156 (VNTR4156). (D) Strain identification. (E) Main spoligotyping-based defined clades and associated spoligotyping international type. ALB, Albania; H1, Haarlem-1; TUR, Turkish genotype.

Spoligotyping results showed 32 different types, among which 16 clusters containing between 2 and 22 isolates totaling 84 isolates and 16 unique patterns were found (Fig. 2). Four new clusters (SIT2900, 2936, 2937, and 2938) were detected. The presence of three MDR Beijing genotypes (SIT1), three Haarlem-1 genotypes (SIT47), and six Turkish genotypes (SIT41) was observed. One specific spoligotyping-based cluster (SIT613) showed a striking intermediate-sized ETR-D allele, which was sequenced and showed the presence of a partial extra copy of the repeat (results not shown). Sequencing of the rpoB locus of the Beijing isolates confirmed the presence of a S531L mutation in all isolates (16). A likely case of intrafamilial transmission of Beijing MDR TB clinical isolates was found.

Genotyping was done successively on 5 ETRs, 12 mycobacterial interspersed repetitive units (MIRUs), and 9 additional VNTR loci (21). Results were obtained for all isolates (Fig. 2). We achieved the best discriminatory power possible, as suggested by the recent VNTR international standardization proposal (20). Some loci could not be typed for some isolates (9 missing data points) for technical reasons. A total of 57 different genotypes were observed (plus 8 incomplete types with one or two missing values), among which were 17 clusters totaling 53 clinical isolates. Depending on the cluster definition (100% identity or inclusion of single- and double-locus variants), we estimated that 15 to 16 epidemiologically linked clusters were found in from 43 to 49 patients (8, 15). Hence, we estimated the RTI, determined by using the n–1 method (18), to be between 28% and 34%.

We also compared the genotypes from Albania to those from two different databases, SITVIT2 (13, 14; http://www.pasteur-guadeloupe.fr:8081/SITVITDemo/) and MIRU-VNTRplus (1). Three brand new spoligotype signatures, designated Albania-1 to Albania-3 (SIT2936 to SIT2938) and representing 4.95%, 2.97%, and 5.94%, respectively, of the studied population, were found in comparisons with SITVIT2 and/or MIRU-VNTRplus. One orphan pattern from a previous study in Greece was found to match SIT2936 (4). Another cluster was created between an orphan pattern found in Rome, Italy, and an isolate from Durres. Comparison of our data with that of former Bulgarian studies showed the presence of Bulgarian and Turkish types in Albania (17, 24, 25). SIT41 (also designated "the Turkish family"; 5.94%) and SIT284 (East-Med-1) were found to reflect various influences (4).

In conclusion, we got a first insight into M. tuberculosis genetic diversity in Albania by characterizing a set of 100 DNAs from TB patients living within Albania. Our results suggest a moderate RTI (28 to 34%) and a high level of genetic diversity. This result should, however, be confirmed in a more exhaustive study (15). Two identical Beijing MDR TB cases were the result of familial transmission in Elbasan, an industrial city. However, for most clusters, investigating from the tightest to the loosest social circles was not done (27). Three new M. tuberculosis complex genotypes (Albania-1 to Albania-3) will require further molecular characterization. One of these was found exclusively in Tirana (SIT2938), suggesting an emergent strain (10). The finding of isolates of the Beijing genotype family is a feature characteristic of Albania, since no Beijing strains could be found in Bulgaria or Romania (17, 24, 25; S. Hoffner, personal communication).


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ACKNOWLEDGMENTS
 
The NRL and the MoH of Albania received a 5-year grant (2006-2011) from the Global Fund (http://www.theglobalfund.org). J.Z. holds a Ph.D. fellowship from the presidency of the University of Paris Sud 11. C.S. holds an Excellency Chair in Microbiology at the University of Paris Sud 11. This work was also made possible thanks to a research grant from the Fondation Mérieux, Lyon, France, to C.S.

Gilles Vergnaud, head of the GPMS research team at IGM, and Brigitte Gicquel, head of the Unité de Génétique Mycobactérienne, Institut Pasteur, are warmly acknowledged for their support. Nalin Rastogi and M. Thierry Zozio (Institut Pasteur of Guadeloupe) are acknowledged for the designation of spoligotype international types within the SITVIT2 database (Institut Pasteur of Guadeloupe test version). The IGEPE research team was created thanks to the University Paris Sud 11 and the director of the UMR8621 (CNRS-University), Institut of Genetics and Microbiology (IGM), Monique Bolotin-Fukuhara.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Genetics and Microbiology, UMR8621, IGEPE Team, Universud, CNRS Université Paris-Sud 11, Campus d'Orsay, F-91405 Orsay-Cedex, France. Phone: 33 (0) 1 69 15 46 48. Fax: 33 (0) 1 69 15 66 78. E-mail: christophe.sola{at}u-psud.fr Back

{triangledown} Published ahead of print on 11 March 2009. Back

{dagger} The first two authors contributed equally to the work. Back


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Journal of Clinical Microbiology, May 2009, p. 1581-1584, Vol. 47, No. 5
0095-1137/09/$08.00+0     doi:10.1128/JCM.02284-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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