Journal of Infection (2006) 53, 331e336 www.elsevierhealth.com/journals/jinf Prevalence of Haarlem I and Beijing types of Mycobacterium tuberculosis strains in Iranian and Afghan MDR-TB patients Parissa Farnia a, *, Mohamad Reza Masjedi a, Mehdi Mirsaeidi a, Foroozan Mohammadi a, Jallaledin-Ghanavi a, Veronique Vincent b, Moslem Bahadori a, Ali Akbar Velayati a a National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shaheed Bahonar Avenue, Darabad, P.O. Box 19575/154, Tehran 19556, Iran b Centre National de Reference des Mycobacteries, Institute Pasteur, 25 Rue du Docteur Roux, Paris 75724, France Accepted 9 December 2005 Available online 14 February 2006 KEYWORDS Spoligotyping; Beijing; MDR-TB Summary Objectives: This survey identified the spoligopatterns of Mycobacterium tuberculosis strains with an international designation responsible for transmission and prevalence of Multi-Drug Resistance Tuberculosis (MDR-TB) among native and immigrant population of Tehran (2000e2005). Methods: The spacer oligonucleotides typing was performed on 263 M. tuberculosis strains isolated from verified cases of MDR-TB. Clinical and demographical data of patients were collected using traditional methods. Results: Classical epidemiological investigation revealed that out of 263 MDR-TB cases, 175, 66.5% were isolated from Afghan immigrants. In both communities, majority of MDR-TB cases had either previous history of TB (107, 40.6%) or had a close contact (84, 31.9%). By spoligotyping, 27 distinct patterns were observed, 253 clinical isolates were grouped in 17 clusters (62.9%) and 10 isolates displayed an orphan pattern (37%). Based on an international spoligotype database, Haarlem I (85, 33.5%), Beijing (52, 20.5%), Central Asia (32, 12.1%), and EAI (21, 8.3%) were the major identified super families. Although, 76.9% of the Beijing genotypes and 100% of ST 253 strains (that was prevalent through former Soviet Union) were isolated * Corresponding author. Iranian National Reference TB Laboratory, National Research Institute of Tuberculosis and Lung Disease (NRITLD), WHO Collaborating Centre of Tuberculosis, Shaheed Bahonar Ave, Darabad, P.O. Box 19575/154, Tehran 19556, Iran. Tel.: þ982 18408275; fax: þ982 12285777. E-mail address: pfarnia@hotmail.com (P. Farnia). 0163-4453/$30 ª 2005 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2005.12.020
332 P. Farnia et al. from Afghan patients only. The linkage patterns between 30 Iranian and Afghan patients were observed. Conclusion: The study highlighted the epidemic potential of Haarlem I and Beijing genotypes among MDR-TB cases in Tehran territory. ª 2005 The British Infection Society. Published by Elsevier Ltd. All rights reserved. Introduction Non-adherence to therapy and inappropriate prescribing of anti-tuberculosis drugs have been associated with the development of multidrug-resistant tuberculosis (MDR-TB). 1 However, in several instances, MDR-TB outbreaks were reported to be the results of the spread and transmission of particular strain of M. tuberculosis. 2 Using spoligotyping, strains and their variants have been found to belong to particular families. 3,4 The technique is based on the analysis of polymorphism in direct repeat (DR) chromosomal region consisting of identical 36-bp DRs alternating with 35e41 bp unique spacers. 5,6 Recently, by systematically analyzing spoligopatterns of M. tuberculosis strains essentially from Europe and U.S.A. (http://www.pasteurgualdeloupe.fr/tb/spldb4) the polymorphism data base mining methods were constructed. 7,8 A total of nine potential super families or clades of M. tuberculosis complex have been identified (Mycobacterium africanum, Beijing, Mycobacterium bovis, EAI, CAS, T, Haarlem I, X and LAM families), 9 among which, the Beijing family accounted for the most reported MDR-TB outbreaks. Member of this family have an IS6110 insertion in the origin of replication (A1 insertion) and display the S00034 spoligopattern, characterized by the absence of spacers 1e34 and positive hybridization to spacers 35e43. 3,8 Although, no one knows exactly how many cases of MDR-TB are due to the Beijing family, but in many Asian countries this genotype is found to be frequently associated with multidrug resistance. 3,4,10,11 In Iran, the TB surveillance is based on conventional methods that have limited practical value. The method cannot identify the source and the route of infection and cannot discriminate between new and old infection. Thereby, no reliable data regarding the transmission pattern of MDR-TB cases are available. For this reasons, we seek to determine the predominant spoligopatterns with international designation responsible for MDR-TB transmission in Tehran. Tehran is a densely populated city and therefore, it is interesting to study the spoligopattern in an effort to understand the extent of polymorphism among MDR-TB isolates. To our knowledge, this is the first documentation of the introduction and spread of Haarlem I and Beijing strains in the Tehran territory. Materials and methods Data collection The study was conducted from January 2000 to January 2005. Case data were collected by trained technicians using standard questionnaires. Information was obtained on sex, age, country of birth, close contact (>8 h contact), previous TB history, present address and associated medical data such as HIV infection, tuberculin skin test and chest radiography finding. Bacterial strains Primary isolation and culturing of Mycobacterial isolates were performed in accordance to procedures manual. 12 All isolates were identified as M. tuberculosis complex by using biochemical tests, including production of niacin, catalase activity, nitrate reduction, pigment production and growth rate. Drug susceptibility testing against isoniazid (INH), rifampicin (RIF), streptomycin (STM) and ethambutol (ETB) was performed by proportional method on LowensteineJensen media at concentrations of 0.2, 40, 4.0 and 2.0 mg/ml, respectively. Susceptibility to pyrazinamide (PZA) was tested using a two-phase medium and two different concentrations of the inoculums. Multi-drug resistance was defined as resistance to at least isoniazid and rifampicin. Spoligotyping Spoligotyping was performed as described by Kamerbeek et al. 5 with a commercially available kit, according to the instructions supplied by the manufacturer (Isogen Bioscience B.V., Maarsen, The Netherlands). In brief, DR region was amplified by PCR using primers [DRa (5 0 -GGT TTT GGG TCT GAC GAC-3 0 biotinylated at 5 0 end ] and [ DRb (5 0 - CCG AGA GGG GAC GGA AAC-3 0 ] derived from the DR sequence. The amplification was carried out in a Master Cycler5330 (Eppendorf, Germany) by
Haarlem I and Beijing types of Mycobacterium tuberculosis strains 333 using 53 cycles of denaturation for 1 min at 95 C, annealing for 1 min at 55 C and extension for 30 s at 72 C. The first denaturation and final extension steps were held for 10 min. The amplified DNA was hybridized to a set of 43 immobilized oligonucleotides derived from the spacer sequences of M. tuberculosis H37Rv and M. bovis BCG P3 by reverse line blotting. Hybridized DNA was revealed with the ECL detection kit (Amersham, Buckingham shire, United Kingdom) and by exposing ECL-Hyper film (Amersham) to the membrane for 12 min. 5 Statistical analysis The results obtained were entered in a binary format as a Excel spreadsheet (Microsoft) and compared with published data. 6e9 The strains with spoligotype similar to any pattern of an M. tuberculosis strain already found in the database were automatically labeled with an already defined shared type number, whereas a spoligotype exhibiting a profile not yet found anywhere in the SpolDB4 database was termed an orphan strain. Categorical data in each groups (Iranian and Afghan TB cases) were compared by Chi-square test or Fisher s exact test. Results Study population From December 2000 to January 2005, 3812 patients with pulmonary TB were referred to Iranian National Reference TB Laboratory (INRTL) Tehran, Iran. Of 3812 TB patients, 1582; 41% were excluded because they were either culture negative or had smear examination only. Additional patients (n ¼ 394; 10.3%) were excluded because their culture were contaminated. Ninety three patients (93, 2.4%) were infected with diseases other than tuberculosis. The study, therefore, involved 1074; 28.1% Iranian and 668; 17.5% Afghan cases. Nine hundred and eighty six patients (986, 56.6%) had in ratio susceptible to all four drugs tested. Two hundred and sixty three patients (263, 15%) had a multi-drug resistance in ratio. Notably, 175; 66.5% of MDR-TB cases were isolated from Afghan immigrants. These 263 MDR-TB patients were selected for further analysis. Demographical characteristic of MDR-TB patients The demographic characteristics of the MDR-TB patients are reported in Table 1. One hundred and Table 1 Detailed demographic data of the studied population: PGG 1 and 2 Total number of patients 263 Iranian 88 (33.4%) eighty four (184, 69.9%) were men and seventy nine (79, 30%) were women. The mean age for Iranian cases was 44 18 (SD) and for Afghanis was 35.2 16.3 (SD). Among 88 well documented Iranian cases, 9; 10.2% were HIV-positive and 20; 22.7% were intravenous drug abusers. The HIV and intravenous drug abuse was not available for Afghan cases. Reviewing the patients questionnaires revealed that the majority of patients (Iranian and Afghani) had either a previous history of TB (107, 40.6%) or had a close contact (83, 31%). The crowded and poor living condition (196, 74.5%), low-salary (230, 87.4%) and poor access to health services (177, 67.3%) were common factors increasing the risk of TB among native and immigrant population. In this respect, the unemployment in Afghanis were almost two fold (91, 52%) of Iranian (21, 23%). Distribution of the 263 MDR-TB spoligotypes Afghanis 175 (66.5%) Mean age 44 18 35.2 16.3 Female 21 (23%) 58 (33.1%) Male 67 (76.1%) 117 (66.8%) Pulmonary 56 (63.6%) 145 (82.8%) Extra-pulmonary 5 (5.6%) 18 (10.2%) Both 27 (30.6%) 21 (12%) AIDS 9 (10.2%) NA Intravenous 20 (22.7%) NA drug abuse Previous 43 (48.8%) 64 (36.5%) history of TB History of family 21 (23.8%) 63 (36%) or contact tracing New-smear positive 24 (27.2%) 48 (27.4%) Crowded and poor 69 (78.4%) 127 (72.5%) living condition Low-salary 71 (80.1%) 159 (90.8%) Poor access to 65 (73.8%) 112 (64%) health services Unemployment 21 (23%) 91 (52.0%) NA, the data were not available. A total of 27 distinct patterns were observed. Ten unique patterns (orphan patterns) were seen, and the remaining 253 strains were contained within 17 clusters. The diversity of observed clustered was 0.01 (diversity ¼ the number of shared types divided by the total number of found isolates, 27
334 P. Farnia et al. of 263; 0.01). Among clustered isolates, two broad categories of clusters (shared types) could be identified; the clusters represented by more than 10 isolates i.e., 7 clusters containing 225 isolates and those represented by less than 10 isolates, i.e., 10 clusters containing 28 isolates (five shared types contained only two patients each, 3 shared types contained only three patients, one shared type contained four patients, and one shared type contained five patients). Epidemiological analysis of clustered MDR-TB cases Afghan-TB cases in clusters Out of 175 isolates, 167; 95.4% were clustered in 14 clusters. Seven clusters contained 148 isolates (more than 10 isolates per cluster) and the remaining 19 isolates fell under 8 clusters (less than 10 isolates per clusters). Based on the published worldwide spoligopatterns, the identified super families were Haarlem I by 41 isolates; 24.5%, Beijing by 40 isolates; 23.9%, CAS by 21; 12.5%, ST253 by 12 isolates; 7.1%, X by 12 isolates; 7.1%, EAI and T by 11 isolates each; 6.5% (Fig. 1). Among identified clusters, the Beijing family and ST 253 had a particular distribution. Indeed, of the 44 strains displaying the Beijing genotype, 31 patients; 70% were below the age of 30 and 18 patients in this cluster were family or close contact; 40.9%. In contrast, 11 out of 12 of patients belonging to the ST 253 cluster were older than 45 years of age and all (100%) had a previous history of TB. Overall, a direct epidemiological link could be established for 92 Afghani patients; 55% in different clusters, 75 were family or close contact. Figure 1 The predominant spoligotypes which are prevalent among Iranian and Afghan-born immigrants.
Haarlem I and Beijing types of Mycobacterium tuberculosis strains 335 Iranian cases in clusters Of 88 isolates, 86; 97% were grouped into 8 clusters. Five clusters contained 77 isolates (more than 10 isolates per cluster) and 2 clusters involved nine isolates (less than 10 isolates per clusters). The five dominant super families were: Haarlem I by 44 isolates; 57.1%, Beijing by 12 isolates; 15.5%, EAI by 10 isolates; 12.9%, and CAS by 11 isolates; 14.2% (Fig. 1). A direct epidemiological link could be established for 15 patients; 17.4% in different clusters, 11; 12.7% were family or close contact. Iranian and Afghan-born immigrants in clusters The direct transmission link could be established for 30 patients, 11.4%. Twenty-two of these isolates were of the Haarlem I type and eight belonged to Beijing family. The patients in these clusters were either living in the same locality (n ÿ 14; 46%), had a job connection (n ÿ 7; 23%), were using similar shelter for night (n ÿ 5; 16.6%) and or had intra-family connection (n ÿ 4; 13.3%). Conclusion This study describes the molecular epidemiology of MDR-TB in Tehran. Tehran is a highly populated city where not only people from other parts of Iran have flooded into, but also has considerable number of immigrants from other countries. 13 During the study period, 32% of the initial TB patients (referred to our unit) were from Afghan-born immigrants. As shown in Table 1, the rate of resistance to any drug or drug combination (including MDR-TB), was almost two fold as compared to Iranian. These findings highlighted the need to reinforce the TB policy measures with regards to screening immigrants from neighbouring countries. The result of this study shows that the most frequently occurring super families or clades among MDR-TB cases are Haarlem I (85, 32.3%), Beijing (53, 20%), CAS (32, 12.1%) and EAI (21, 7.9%). The Haarlem I family of M. tuberculosis strains was first isolated from a patient living in Haarlem, The Netherlands. 6 Today, its widespread distribution in different geographical regions of the world such as Asia, Europe and Africa has been documented. 4,6 In addition, its ability to cause outbreaks has been reported in Argentina, 14 in Czech Republic 15 and in Tunisia. 4 This family itself accounts for more than half of all clustered strains among Iranian MDR-TB patients. The epidemiological link could be established for 39 out of 58 patients with a Haarlem spoligopattern; 27 (79.1%) were family or close contact, four were HIV and drug abuser (4.7%), three had a job connection (3.5%) and five were staying in same locality (5.8%). Therefore, our result demonstrated that Haarlem genotypes can cause epidemic and from epidemiological point it is necessary to conduct extensive surveillance of MDR strains because they might cause serious outbreaks. Another identified super family was Beijing type of M. tuberculosis strains. This family was originally described by Van Soolingen et al. 16 in China and is reported to be highly prevalent throughout Asia and in the countries of the former Soviet Union. The Beijing genotype is thought to possess selective advantages compared with strains of other M. tuberculosis genotypes and is sometimes associated with multi-drug resistance with specific pathogenic properties. In our study also, 20% of MDR-TB patients had Beijing characteristics. It has been claimed that alteration within DNA repair (mutator genes) might favor the emergence of MDR strains with an increased adaptability. 11 Association of Beijing strains with drug resistance pattern as demonstrated in this study might be related to accumulation of mutations within their putative mutator genes that has to be further investigated. Doroudhchi et al. 17 has demonstrated that 40% of Iranian isolates in the Beijing family were recovered from Afghan refugees living in Iran. In present data, 55% of Afghan patient with Beijing characteristic were below the age of 30 and had stayed in Iran for more than 10 years without any previous history of TB. At present, the nature of the driving force(s) that contribute to the selection and dissemination of Beijing strains is unknown. However, it is clear, that the majority of Afghan immigrants were living and mixing among themselves and that, 55% Beijing-related cases develop diseases because they were family or close contact. Therefore, on-going transmission of Beijing family in Afghan immigrants might be related to their socio-economical state such as low income, psychological stress, low education and poor access to health services. Our results also showed that the East-African Indian (EAI) and Central Asian I (CAS) were the other most frequent super families in both communities. The EAI super family is characterized by presence of spacer 33 and absence of spacer 34. Members of the CAS family are devoid of spacers 4e7 and 23e34. Until now, these strain families have been reported in different countries of the Middle East (Iran, Pakistan, and India), Oceanic (Australia), the United States and Europe. 8,9 In Europe and Australia, they were regularly found to be linked with immigrants from Central Asia and Middle East. These two genotypes were analyzed and no significance regarding the age, sex and geographical origin of the patients was found. We also
336 P. Farnia et al. found another conserved genotype of M. tuberculosis strains that was prevalent through the former Soviet Union. 7,8 The presence of ST 253 in Afghanis could be linked to the former Soviet Union history in that region. However, further investigations are in progress to estimate the prevalence of these genotypes in Afghanistan itself. Previously, Sreevatsan s et al. 18 assigned M. tuberculosis isolates into three principal genetic groups (PGG). PGG I has the allele combination KatG codon 463 CTG (leu) and gyra codon 95 ACC (Thr); PGG 2 has KatG 463 CGG (Arg) and gyra codon 95 ACC (Thr) and PGG 3 organisms have KatG 463 CGG (Arg) and gyra codon 95 AGC (Ser). Later on, they demonstrated a relation between spoligotyping pattern and the major genetic groups of M. tuberculosis isolates. PGG 2 and 3 failed to hybridize with spacers 33e36, suggesting that these spacer and DRs have been deleted from the genome of all PGG 2 and 3 organisms. In the present investigation about 65% of M. tuberculosis strains belonged to the PGG 1 group, while the remaining were PGG 2. Hence, the current epidemiological picture of MDR-TB reflects on the persistence of ancestral clones of M. tuberculosis as well as those emerging recently (PGG 1 and 2). In conclusion, by using spoligotyping the well-defined genotype families of MDR-TB strains were identified. The obtained data would help to guide the TB control program to act accordingly. Acknowledgements First part of this work was supported by a grant from UNESCO-IUMS Mircens-SGM short-term fellowship and its second part was sponsored by grant from NRITLD-WHO/12/004. We gratefully thank Centre of National de Reference des Mycobacteries, Institute Pasteur, Paris (France) for technical instruction and training. Finally, we thank all the TB patients and their family who have patiently helped us to complete the required information. References 1. Drobniewski F, Balabanova Y, Coker R. 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