182 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 112(3): 182-187, March 2017 online | memorias.ioc.fiocruz.br Tuberculosis associated factors caused by Mycobacterium tuberculosis of the RDRio genotype Eloise Brasil Moraes1,2/+, Letícia Slompo1, Amanda Juliane Finardi1,2, Heloisa da Silveira Paro Pedro3, Luciana Ruiz4, Harrison Magdinier Gomes5, Virginia Bodelão Richini4, Philip Suffys5, Carlos Magno Castelo Branco Fortaleza2, Ricardo Cavalcanti2, Ida Maria Foschiani Dias Baptista1,2 1Instituto Lauro de Souza Lima, Divisão de Pesquisa e Ensino, Equipe Técnica de Microbiologia, Bauru, SP, Brasil 2Universidade Estadual Paulista Júlio de Mesquita Filho, Faculdade de Medicina de Botucatu, Doenças Tropicais, Botucatu, SP, Brasil 3Instituto Adolfo Lutz, Centro de Laboratório Regional de São José do Rio Preto, Núcleo de Ciências Biomédicas, São José do Rio Preto, SP, Brasil 4Instituto Adolfo Lutz, Centro de Laboratório Regional de Bauru, Núcleo de Ciências Biomédicas, Bauru, SP, Brasil 5Fundação Oswaldo Cruz-Fiocruz, Laboratório de Biologia Molecular aplicada a Micobactérias, Rio de Janeiro, RJ, Brasil BACKGROUND Tuberculosis (TB) continues to be a disease that affects many countries around the world, including Brazil. Recently, a subtype of Latin American-Mediterranean family strain was identified and characterised by RDRio. The strain has been associated with different characteristics of the disease. OBJECTIVES In the present study we investigated the association of epidemiological, clinical, radiological and bacteriological variables with pulmonary tuberculosis caused by RDRio Mycobacterium tuberculosis strain in large regions of São Paulo. METHODS We conducted a cross-sectional study in 530 patients with pulmonary tuberculosis, diagnosed using sputum culture, from two regions of the São Paulo state in Brazil. The samples were brought to São Paulo reference laboratories for epidemiological, clinical, radiological and bacteriological analyses, and the data were obtained from a TB notification system. RDRio genotyping and Spoligotyping of the samples were performed. For the analysis of the categorical variables we used the chi- square test or the Fisher’s exact test, and for the continuous variables, the Mann-Whitney test. In addition, a logistic regression was used for multivariate analysis. Differences with p < 0.05 were considered significant. FINDINGS The RDRio deletion was identified in 152 (28.7%) samples. In the univariate analysis, both the age groups above 25 years and alcohol consumption were associated with the RDRio deletion. The multivariate analysis confirmed the association of the RDRio deletion with the age groups: 25-35 years old [OR: 2.28 (1.02-5.07; p = 0.04)] and 36-60 years old (OR: 2.36 (1.11-5.05); p = 0.03], and also with alcohol consumption [OR: 1.63 (1.05-2.54); p = 0,03]. MAIN CONCLUSIONS In this study, we identified new factors associated with the M. tuberculosis of the RDRio deletion strains infection. Key words: Mycobacterium tuberculosis - RDRio - tuberculosis - genotyping - epidemiology doi: 10.1590/0074-02760160347 Financial support: FAPESP (nº 2013/09538-3) + Corresponding author: eloise.moraes@hotmail.com Received 30 July 2016 Accepted 7 November 2016 Tuberculosis (TB) is an infectious disease that kills nearly 2 million people each year. The disease emerged about 70,000 years ago, with the migration of anatomically modern humans from Africa, and expanded as a result of the increase in human population density during the Neo- lithic (Comas et al. 2013). TB continues to be a global epi- demic, and if left untreated, has a mortality rate of ~70% in people with positive sputum smears (Fogel 2015). Although the incidence of tuberculosis has decreased in recent years, 9.6 million new cases and 1.5 million deaths were reported worldwide in 2014 (WHO 2014). Brazil is among the 22 countries with the highest TB incidence worldwide. The majority of the cases reported in Brazil were concentrated in the Southeast, where the state of São Paulo (SP) accounted for 20% of the disease incidence in the country (Wysochi et al. 2016). Molecular typing, based on genetic markers, allows the rapid identification of Mycobacterium tuberculosis com- plex strains (MTC) and provides useful tools for examining the transmission and the development of the mycobacteria (Sola et al. 2001, Brosch et al. 2002, Gagneux et al. 2006). A polymerase chain reaction (PCR) identification based on the amplification of multiple deleted loci can distinguish between the MTC species (Huard et al. 2003). Lazzarini et al. (2007) reported that MTC isolates, designated as RDRio, were found to contain a chromo- somal deletion of more than 26 kb. The authors reported that further analysis of the RDRio strains with a spoli- gotyping molecular technique confirmed that all iso- lates belong to Latin American-Mediterranean family (LAM). The presence of RDRio M. tuberculosis isolates have been reported in at least 18 other countries in Eu- rope, Africa, and the Americas, in which all the isolates belonged to the LAM genotype of the Euro-American lineage (Gibson et al. 2008, Weisenberg et al. 2012). The RDRio M. tuberculosis is the most common cause of TB in Rio de Janeiro and other regions of Brazil (Lazzarini et al. 2008, Oelemann et al. 2011). Tuberculosis factors associated RDRio strain • Eloise Brasil Moraes et al. 183 Recent studies suggest that M. tuberculosis strains are well adapted to human populations and that they are older and have a greater genetic diversity than previously thought (Alix et al. 2006). This diversity may have an important effect in different clinical and epidemiological aspects of tuberculosis. However, the current knowledge about the influence of TB strain diversity on the development of the disease is still scarce (Barbosa et al. 2012). Considering this, in the present study we investigated the association of epidemiological, clinical, radiological and bacteriological variables with pulmonary tuberculosis caused by RDRio M. tuberculosis strains in large regions of São Paulo. PATIENTS AND METHODS A cross-sectional population-based study was con- ducted on pulmonary tuberculosis cases reported in the period 2012-2014. Two regions of the state of São Paulo were considered for this study: 69 municipalities belong- ing to the Regional Health Department (DRS) Section VI (Bauru) and 102 municipalities of the DRS Section XV (São José do Rio Preto). A total of 530 tuberculosis patients with positive spu- tum cultures for M. tuberculosis were recruited. Since the detection of the RDRio deletion is only possible us- ing the isolated culture, patients without sputum culture were not included in the study. The patients’ data were obtained through the Notification System and Monitor- ing of Tuberculosis Cases (TB-WEB) of the State De- partment of Health from the state of São Paulo - Brazil (SES/CCD/CVE). The epidemiological, clinical, radio- logical and bacteriological data for each patient were filled in the TB-WEB system for the epidemiological surveillance in each municipality. M. tuberculosis culture samples - The M. tuberculo- sis culture samples were collected in the laboratories of the Adolfo Lutz Institute from both DRS Sections (VI and XV) and brought to the Instituto Lauro de Souza Lima for analysis. Biochemical characterisation and multidrug-resistance tests (INH, RMP, SM, and EMB) were performed as recommended by the Ministry of Health of Brazil. The susceptibility of the samples to PZA was tested separately by means of the pyrazinami- dase assay (MS/SVS/DVE 2008). Multiplex PCR and Spoligotyping of RDRio strains - The identification of RDRio deletions was performed as described by Lazzarini et al. (2007) and Gibson et al. (2008). The RDRio and wild type (WT) genotypes were identified by the detection of the 1175 and 530 bp stan- dard bands, respectively. Sample spoligotyping was per- formed according to the method reported by Kamerbeek et al. (1997). The spoligotype patterns were recorded in an octagonal code and in a 43-digit binary format, representing 43 spacers (Filliol et al. 2002). The M. tu- berculosis patterns were compared with the SpolDB4 database (Brudey et al. 2006) of the Pasteur Institute in Guadeloupe (http://www.pasteur-guadeloupe.fr:8081/ SITVITDemo) for type classification. Statistical analysis - For the bivariate evaluation of categorical variables we used the chi-square test or the Fisher’s exact test, whereas for continuous variables anal- yses, the Mann-Whitney test was performed using the Epi Info version 3.5.4. These methods were applied to com- pare the results of the regions of Bauru (DRS-VI) and São José do Rio Preto (DRS-XV). In addition, a multivariate analysis with a logistic regression function was used to investigate the association between the presence of the RDRio marker deletion and the epidemiological variables. Variable selection for the multivariate analysis was performed following a hierarchical procedure. Initially, the demographics were considered as distal variables. The variables that were significant (p < 0.05) were maintained and analysed in conjunction with the comor- bidities and lifestyle variables, which were considered as intermediate variables. The variables which in this analysis resulted in a p < 0.05 were retained and further analysed together with the proximal variables (clinical, bacteriological and radiological). For the logistic regres- sion, the selected variables were presented in terms of the odds ratios and their 95% confidence interval. This analysis was performed using the SPSS software version 20 considering significant variables for p < 0.05. RESULTS In the period covered by this study, 530 patients with positive sputum cultures for tuberculosis were reported: 176 from Bauru and 354 from São José do Rio Preto. Be- cause this study used secondary data, it was not possible to obtain all the information from all the patients. The epide- miological, clinical, radiological and bacteriological data of the patients of both regions are described in Table I. The analysis resulted in a higher prevalence of pa- tients of African descent (16.1 vs. 6.7%; p = 0.003) with pulmonary tuberculosis only (99.4 vs. 94.8%; p = 0.02) and a higher rate of treatment abandonment (18.3 vs. 7.4%; p = 0.001) in the DRS-VI Bauru region. On the other hand, smokers (31.0 vs. 13.3%; p = 0.0001) pre- vailed in the cases detected in the municipalities of the DRS-XV São José do Rio Preto region. The sensitivity to antituberculosis drugs is shown in Table II. The incidence of drug resistance was higher in the DRS-VI Bauru region (20.4 vs. 10.2%; p = 0.08), with significant data for pyrazinamide resistance (50.0 vs. 4.7; p = 0.02). The RDRio deletion was identified in 152 (28.7%) cul- ture samples. The analysis of the factors associated with the presence of this deletion was analysed in 429 patients only. The rest of the patients were not admitted in the analysis for the lack of data. In the univariate analysis, the age groups above 25 years and alcohol-using patients were associated with the presence of the RDRio deletion, while a history of tuberculosis and drug resistance were marginally significant (Table III). The multivariate analysis confirmed the associa- tion between RDRio deletion and the 25-35 years old age group [OR: 2.28 (1.02 to 5.07)] and the 36-60 year old age group [OR: 2.36 (1.11 to 5.05)], as well as with elit- ism [OR: 1.63 (1.05 to 2.54)], Table IV. Spoligotyping results were unsatisfactory due to problems with the membranes used for sample hybridization. Based on the 184 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 112(3), March 2017 TABLE I Epidemiological, clinical, radiological and bacteriological data of 530 tuberculosis patients from two regions of the state of São Paulo, Brazil Male Bauru (DRS-VI) São José do Rio Preto (DRS-XV) p value 130/176 (82,8) 281/354 (80,1) 0,54 Age (years) 35,0 (19,0 - 74,0) 37,0 (14,0 - 80,0) 0,05 Ethnicity white 79/137 (57,7) 195/314 (62,1) 0,43 black 22/137 (16,1) 21/314 (6,7) 0,003 brown 35/137 (25,5) 94/314 (29,9) 0,40 indian 1/137 (0,7) 0/314 (0,0) 0,30 yellow 0/137 (0,0) 4/314 (1,3) 0,23 Schooling unlettered 4/106 (3,8) 12/306 (3,9) 0,60 1 - 3 years 13/106 (12,3) 39/306 (12,7) 0,96 4 - 7 years 47/106 (44,3) 133/306 (43,5) 0,96 8 - 11 years 29/106 (27,4) 109/306 (35,6) 0,15 12 - 14 years 2/106 (1,9) 8/306 (2,6) 0,50 Comorbidities diabetes mellitus 8/128 (6,3) 22/329 (6,7) 0,96 HIV infection 12/125 (9,6) 56/324 (17,3) 0,06 alcoholism 32/129 (24,8) 96/329 (29,2) 0,41 smoking 17/128 (13,3) 102/329 (31,0) 0,0001 drogadiction 42/129 (32,6) 88/324 (27,2) 0,30 mental disease 1/90 (1,1) 8/323 (2,5) 0,38 Prisoner 34/109 (31,2) 94/228 (41,2) 0,09 History of tuberculosis 34/132 (25,8) 69/328 (21,0) 0,32 Tuberculosis forms pulmonary 164/165 (99,4) 311/328 (94,8) 0,02 extrapulmonary 1/165 (0,6) 16/328 (4,9) 0,02 Positive sputum smear 96/134 (71,6) 198/310 (63,9) 0,13 Chest X-ray normal 7/93 (7,5) 24/222 (10,8) 0,49 cavitary 23/93 (24,7) 74/222 (33,3) 0,16 suggestive of tuberculosis 63/93 (67,7) 124/222 (55,9) 0,06 Treatment abandonment 23/126 (18,3) 24/325 (7,4) 0,001 Deaths (all causes) 7/126 (5,6) 17/325 (5,2) 0,92 TABLE II Drug resistance in tuberculosis patients from two regions of the state of São Paulo, Brazil No. of patients with resistance Bauru São José do Rio Preto p value 4/44 (9,1) 22/314 (7,0) 0,39 No. total of identified resistance 9/44 (20,4) 32/314 (10,2) 0,08 No. drug resistance Rifampicin 2/43 (4,7) 6/313 (1,9) 0,24 Isoniazid 3/41 (7,3) 10/314 (3,2) 0,18 Pyrazinamide 2/4 (50,0) 3/64 (4,7) 0,02 Ethambutol 0/32 (0,0) 0/240 (0,0) --- Streptomycin 2/33 (6,1) 13/238 (5,5) 0,56 Tuberculosis factors associated RDRio strain • Eloise Brasil Moraes et al. 185 spoligotype analysis, 21 RDRio isolates belonged to the families LAM 1, LAM 2, LAM 3, LAM 4, LAM 5, and LAM 9, whereas the rest of the isolates were typified within the U H1, T1 and Unknown families. DISCUSSION TB is a global disease that affects not only individual patients, but also the community. Tuberculosis control goes beyond conventional strategies, therefore it is nec- essary to understand the interaction of the disease with the social and economic culture (Eufrásio et al. 2016). M. tuberculosis has a clonal population structure and until recently, it has been regarded to have little genetic varia- tion (Sreevatsan et al. 1997, Hirsh et al. 2004). However, studies examining M. tuberculosis isolates from broader geographical distributions via whole-genome scanning revealed a cladistics phylogeographical distribution with significant variation between the main lines, each asso- ciated with a specific geographical region (Gagneux & Small 2007, Caws et al. 2008). TABLE III Univariate analysis of the epidemiological, clinical, microbiological and radiological factors tassociated with the presence of RDRio deletion in 429 patients with tuberculosis Male Deletion RDRio Odds ratio (CI 95%) p value Presence nº patients (%) Absence nº patients (%) 127 (83,6) 302 (79,9) 1,27 (0,76 - 2,19) 0,19 Age group under 24 years* 9 (5,9) 53 (14,0) --- --- 25 - 35 years 43 (28,3) 101 (26,7) 2,50 (1,13 - 5,53) 0,03 36 - 60 years 89 (58,6) 202 (53,4) 2,59 (1,22 - 5,48) 0,01 over 60 years 11 (7,2) 22 (5,8) 2,94 (1,07 - 8,09) 0,03 Ethnicity white* 79 (52,0) 195 (51,6) --- --- brown 35 (23,0) 94 (24,9) 0,91 (0,57 - 1,46) 0,72 black 10 (6,6) 33 (8,7) 0,74 (0,35 - 1,59) 0,45 others 1 (0,7) 4 (1,1) 0,61 (0,06 - 5,60) 0,66 Schooling 1 - 3 years* 15 (9,9) 37 (9,8) --- --- 4 - 7 years 57 (37,5) 123 (32,5) 1,14 (0,58 - 2,24) 0,69 8 - 11 years 36 (23,7) 102 (27,0) 0,87 (0,42 - 1,77) 0,70 12 - 14 years 1 (0,7) 9 (2,4) 0,24 (0,03 - 2,35) 0,23 over - years 0 (0,0) 5 (1,3) --- 0,97 unlettered 5 (3,3) 11 (2,9) 1,12 (0,33 - 3,78) 0,85 Prisoner 32 (21,1) 96 (25,4) 0,78 (0,48 - 1,25) 0,17 Health professional 3 (2,0) 2 (0,5) 3,77 (0,42 - 45,60) 0,14 HIV 18 (11,8) 50 (13,2) 0,88 (0,46 - 1,60) 0,39 Diabetes 7 (4,6) 23 (6,1) 0,74 (0,26 - 1,84) 0,33 Alcoholism 46 (30,3) 82 (21,7) 1,56 (1,02 - 2,38) 0,02 Smoking 35 (23,0) 84 (22,2) 1,04 (0,64 - 1,67) 0,46 Mental disease 3 (2,0) 6 (1,6) 1,24 (0,19 - 5,93) 0,50 Previous history of tuberculosis 23 (17,6) 80 (24,3) 0,66 (0,37 - 1,13) 0,07 Year of diagnosis 2012* 23 (19,7) 72 (26,1) --- --- 2013 50 (42,7) 102 (37,0) 1,49 (0,83 - 2,65) 0,17 2014 44 (37,6) 102 (37,0) 1,25 (0,73 - 2,15) 0,40 Pulmonary tuberculosis 146 (96,1) 365 (96,6) 0,86 (0,30 - 2,83) 0,47 Positive sputum smear 94 (61,8) 250 (66,1) 0,83 (0,55 - 1,25) 0,20 Cavitary 24 (15,8) 73 (19,3) 0,78 (0,45 - 1,32) 0,20 Drug resistance 4 (2,6) 22 (5,8) 0,43 (0,10 - 1,32) 0,08 CI 95%: confidence interval of 95%; *: category of reference. 186 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 112(3), March 2017 TABLE IV Multivariate analysis of the epidemiological, clinical, microbiological and radiological factors associated with the presence of RDRio deletion in 429 patients with tuberculosis Age group Odds ratio (CI 95%) p value under 24 years* --- --- 25 - 35 years 2,28 (1,02 - 5,07) 0,04 36 - 60 years 2,36 (1,11 - 5,05) 0,03 over 60 years 2,69 (0,96 - 7,52) 0,06 Alcoholism 1,63 (1,05 - 2,54) 0,03 CI 95%: confidence interval of 95%; *: category of reference. Several studies have used genotyping assays to char- acterise the population structure of M. tuberculosis and its transmission in defined communities. The high prev- alence of TB caused by identical or related strains in a community may be caused by recent introduction of iso- lates, increased of its virulence, or even epidemiological factors that facilitate TB transmission. However, it is still unknown how the genetic makeup of M. tuberculosis iso- lates determines the transmission or the severity of the disease, as only a few studies have reported this type of analysis (Coscolla & Gagneux 2010, Vinhas et. al. 2013). Lazzarini et al. (2007) described an M. tuberculosis strain belonging to the LAM family. The strain (RDRio) had a large 26.3-Kb deletion (Long Sequence Polymorphism - LSP), which included 10 genes, in its DNA sequence. The prevalence of M. tuberculosis samples with an RDRio deletion varied with the geographical location. In this study, we found this behaviour in 28.7% of the samples. These results were similar to those reported by Barbosa et al. (2012) in the city of Rio de Janeiro, in which the strain was detected in 26.5% of the cases (Bar- bosa et al. 2012). The occurrence of the RDRio deletion in the samples was lower than that found in Belo Horizonte (37%) and in Porto Alegre (38%). Patients in both the regions analysed in this study, DRS-VI Bauru and DRS-XV São José do Rio Preto, showed some differences. There was a higher preva- lence of patients of African descent, patients with only pulmonary tuberculosis, and higher treatment-dropout rate in DRS-VI Bauru, while smokers prevailed in the municipalities of DRS-XV São José do Rio Preto. Ap- parently, these differences did not have an impact on the presence or absence of the RDRio deletion, and they may be related to the geographical characteristics of every region. In general, the strains were not drug resistant, with the DRS-VI Bauru samples possessing a greater drug resistant capacity. The resistance of the strains to pyrazinamide was higher in the latter region. However, since the number of samples was very small, this result may not be representative of the local reality. The age groups 25-35 and 36-60 years old were asso- ciated with the presence of RDRio deletion, indicating that these strains are less infectious in young individuals un- der 25 years old and in patients over 60 years old. In pre- vious studies, no positive association was found between age and the presence of RDRio deletion (Lazzarini et al. 2007, Gibson et al. 2008, Barbosa et al. 2012, Weisenberg et al. 2012). The discrepancy on these results may be at- tributed to the fact that the latter studies considered the average or the median values rather than the age. Considering the multivariate analysis, a positive as- sociation was found between alcohol consumption and the presence of RDRio deletion. The former condition in- creased 1.6 times the chance of M. tuberculosis disease by this strain. A couple of studies have assessed the effect of alcohol consumption on the disease and found no positive association (Gibson et al. 2008, Dalla Costa et al. 2013). Although we did not assess the nutritional status of the patients due to a lack of data, it is possible that the associa- tion of the disease with alcohol consumption may reflect the fact that an impaired nutritional status and a poor im- mune system favour M. tuberculosis infection. Lazzarini et al. (2007) found an association between weight loss and the presence of RDRio deletion, which supports the critical effect of malnutrition on the disease. There is evidence that the RDRio deletion strains are associated with an in- creased transmissibility of tuberculosis bacillus (Lazzari- ni et al. 2007, Weisenberg et al. 2012). In general, alcoholic patients live in unhealthy and crowded environments and this could also explain this association. However, we did not evaluate the association of the socioeconomic condi- tions of the patients with alcohol consumption. Previous studies have reported additional positive associations with RDRio strains carrying the deletion. In a prospective study comprising 105 patients, Lazzarini et al. (2008) found 8.9 times more lung cavitation cases in patients infected by RDRio deletion strains. We were not able to prove this association because with the sec- ondary data used in this study we could not confirm the occurrence of cavitation. In this study, no association was found between an- tituberculosis-drug resistance and RDRio deletion. This can be explained by the low resistance prevalence in the studied strains. A Brazilian study conducted in Porto Alegre reported an RDRio association with drug resis- tance (Dalla Costa et al. 2013). In the present study, 99% of the samples with deletions were resistant, while only 63% of the M. tuberculosis wild strains presented mul- tidrug resistance (MDR). The samples were obtained from a referral centre for MDR TB. It is possible that this association is the result of the high rate of resistance in the population, so that it does not represent the pop- ulation with TB in Brazil. A study conducted in New York City reported a 1.6-fold increase in the resistance to isoniazid in the presence of RDRio deletion (Weisenberg et. al. 2012). Further studies are needed to verify the as- sociation of resistance with the RDRio deletion strain. Barbosa et al. (2012) evaluated 272 patients in Rio de Janeiro. Using a univariate analysis, the authors did not find an association of the infection by RDRio deletion strains with epidemiological, clinical and radiological factors. These re- sults demonstrate the need for further studies to assess the role of this strain as the cause of tuberculosis. Tuberculosis factors associated RDRio strain • Eloise Brasil Moraes et al. 187 The major limitation of this study was the use of sec- ondary data. Considering the lack of important patient in- formation regarding the epidemiological, clinical, radiolog- ical and bacteriological results, a bias must be considered in the interpretation of the associations reported in this work. Finally, this study contributes to the knowledge of the association between infection by M. tuberculosis strains with RDRio deletion and the 25-60 years-old age groups and/ or alcohol consumption. Future studies with larger sample sizes are needed to better understand these associations. 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