Genetic Diversity and Drug Resistance of Mycobacterium tuberculosis Complex Isolates and Nontuberculous Mycobacteria Identification from Presumptive Tuberculosis Cases in Oaxaca, Mexico

Tuberculosis (TB) is a re-emerging health problem worldwide. In Mexico, information about genetic diversity and drug resistance of Mycobacterium Tuberculosis complex (MTBc) is limited. Samples from 463 Presumptive TB cases were tested for TB by smear, culture and PCR, from which 19.2% were identify as MTBc and 16% as nontuberculous mycobacteria. MTBc isolates were characterized by Large Sequence Polymorphisms (LSPs), spoligotyping and MIRU-VNTR 24 loci typing methods. Clade designations showed 10 sub-lineages: Haarlem (35.7%), EAI (26.2%), LAM (9.5%), Ghana (4.8%), X (2.4%), New-1 (2.4%), H37Rv-like (7.1%), M. bovis (2.4%), S (7.1%), Uganda I (2.4%). The finding of EAI as one of the principal genotypes may be associated with high migration rates. Drug resistance was found in 35.71% of the isolates: 14.2% were multidrug-resistant (MDR-TB), 14.2% mono-resistant and 7.14% poly-resistant. This study provides the first description of genetic diversity and drug resistance profile of MTBc in Oaxaca, Mexico.


Introduction
Tuberculosis (TB) is considered since 1993 by World Health Organization as an emergent public health worldwide disease. In 2014, 9.6 million people developed TB and 1.5 million died by this disease (WHO, 2015). Mexico ranks third in TB cases in the Americas (OPS/OMS, 2014) and the state of Oaxaca has incidence and mortality rates above the national average (PUDI, 2014). According to ethnic and geographical criteria, Oaxaca is divided in eight geocultural regions with a high level of indigenous population and, in addition, immigration and emigration rates (nationally and internationally) are the highest among many other states of the south of Mexico. In Oaxaca, about 3.9 million people demand healthcare attention (PUDI, 2014) and 500-700 new TB cases were reported each year in the last decade. 8 Genotyping of Mycobacterium tuberculosis complex (MTBc) is a powerful tool that helps, at individual and population level to the disease management (Coll et al., 2014), however there is no information about the TB genotypic characteristics from Oaxaca. The inclusion of a few number of clinical isolates from this region in previous studies suggest that genotypic composition of circulating strains from the Southwest States (Guerrero, Oaxaca and Chiapas) differ from the rest of the country (Nava-Aguilera et al., 2011), but not conclusive data was previously available about Oaxaca. At the same time, the prevalence of drug resistance or MDR strains in Oaxaca was under represented in recent works (Macias Parra et al., 2011;Martinez-Guarneros et al., 2013). The genotyping methodologies more widely used due to its traceability, robustness and uniformity in the interpretation of results are spoligotyping, MIRU-VNTR and LSP (Allix et al., 2008;Reed et al., 2009;Supply et al., 2006).
In this work, we used the methodologies mentioned above to determine the first insight into the genetic diversity, drug resistance and comorbidities of MTBc isolates circulating in a southwest state of Mexico.

Bioinformatic Analysis
Spoligotype patterns as binary format, 24-loci MIRU patterns and presence/absence of RDs were analyze using the MIRU-VNTR plus database (http://www.miruvntrplus.org) to determine the genotypes and the matrix of distances. MEGA6 software (MEGA software) was used to build the phylogenetic tree using the neighborjoining algorithm.

Mycobacterial Isolation and Drug Susceptibility Testing
From January 2014 to March 2015, 463 individuals with presumptive Mycobacterium tuberculosis infection of the eight regions of Oaxaca were tested. Some 21% samples were positive by PCR/XPERT and 16% were positive to Nontuberculous Mycobacteria (NTM). The samples from "Cañada" region were negative for MTBc nevertheless positive to NTM.
We recovered 42 clinical isolates of MTBc, all from 2014. Two isolates (4.7%) were recovered from extrapulmonar location (lymph node and cerebrospinal) and the rest were from respiratory samples. According to gender, 67% TB patients were men and 33% were women, all of them were diagnosed/treated as new TB cases. The most frequent comorbidities found were type 2 Diabetes Mellitus (DM) (33.3%), malnutrition (21.4%) and HIV coinfection (11.9%).

Genotyping Profiles of MTBc Strains
Using MIRU-VNTR 24 loci, spoligotyping and LSP, 42 MTBc isolates were analyzed. Considering data of the three markers, a distance matrix was calculated and a phylogenetic tree was constructed using the neighbor-joining algorithm. The isolates were classified into three major lineages: Indo-Oceanic, Euro-American and M. bovis (Fig. 1).
As shown in Table 1, the Euro-American was the principal genetic lineage represented with thirty isolates, including 13 Haarlem (30.9%), 4 LAM (9.5%), 5 H37Rv-like (11.9%) sub-lineages, accounting for 52% of all isolated strains. The second principal genetic lineage identified was the Indo-Oceanic with eleven (26.1%) M. tuberculosis isolates of EAI sublineage, which was also found highly associated to first-line drug resistance with five (45%) isolates being resistant to at least one drug (Table 1).
The frequency of other minor genotypes and their association with drug resistance is indicated in Table 1. It is noteworthy the high burden of EAI sub-lineage in Oaxaca and the high genetic diversity of the strains belonging to the Euro-American lineage.

Discussion
The genotypic diversity of clinical isolates in the state of Oaxaca differs from other national reports (Macias Parra et al., 2011;Martinez-Guarneros et al., 2013;Zenteno-Cuevas et al., 2013), specifically those from the northern and central region of the country (Lopez-Alvarez et al., 2010;Molina-Torres et al., 2010), where the genotypes T, LAM and X are the most frequently reported. The T lineage was not detected in the oaxacan population, besides LAM and X which occurred in low frequency, 12% and 10% respectively. Unexpectedly we detected a high occurrence of EAI sub-lineage (26.1%), belonging to the Indo-Oceanic lineage, which has been reported at low frequencies throughout the country (Macias Parra et al., 2011;Molina-Torres et al., 2010), except for Acapulco City, in the state of Guerrero (Nava-Aguilera et al., 2011), where it was reported as the main genotype (44.6%). It has been proposed that high occurrence of Indo-Oceanic strains could be due to their arrival directly from Philippines between XVI and XIX centuries when both countries were Spanish colonies and kept frequent maritime communication through the Pacific Ocean, where Oaxaca and Guerrero are located (Nava-Aguilera et al., 2011).
Similar to previous studies that report type 2 Diabetes Melltitus (DM) as one of the most frequent comorbidities in TB patients (Workneh et al., 2016), in this study it was present in 30.9% of the cases. Besides, TB-HIV coinfection has been reported between 28.8% and 64% (Wejse et al., 2015;Middelkoop et al., 2015), here we found this binomial in just 11.9% of the patients. Haarlem sub-lineage predominates among DM patients (46%). Similar results were found in another Mexican population study, where 53% of patients infected by Haarlem strains were diabetic (Pérez-Navarro, 2014). The EAI sub-lineage was the most frequent among the HIV co-infected patients (60%), in concordance with another report in which this sublinage was present in 46.2% of TB-HIV coinfected patients (Kibiki et al., 2007).
In our study we observed a high percentage of primary resistance to at least one first line drug (35.71%): MDR-TB (14.2%), mono-resistant (14.2%) and poly-resistant (7.14%); being 1.5 times higher than in a previous drug resistance report from Oaxaca (Granich et al., 2000).
H37Rv-like sub-lineage was more associated with drug resistance (60%) than the other lineages with two mono-resistant isolates to STR and RIF and one MDR-TB isolate. To our knowledge there is only one report about drug resistance in this sub-lineage (Niemann et al., 2010), where no MDR-TB isolates were found.
The Haarlem sub-lineage has been linked with drugresistance and clonal expansion (Ramazanzadeh et al., 2015). We found that in "Papaloapan" region two of the three Haarlem isolates characterized were resistant to all drugs tested, in contrast with the isolates belonging to "Mixteca" region that were pan-susceptible. "Papaloapan" region is characterized by trade routes of agricultural products, is part of one of the migration routes from Central America to the USA and is better communicated with Veracruz than with Oaxaca. Reports about TB in Veracruz indicate that, together with Baja California, is the state with the highest prevalence of MDR-TB in the country (Juarez-Eusebio et al., 2017). So drug-resistant strains found in "Papaloapan" region may be associated with its geographical proximity to Veracruz. Furthermore, in the "Mixteca" region people prefer to use traditional medicine to treat diseases, in consequence they aren't exposed to drugs, which may not favour the development of drug-resistant strains.
There are some reports in which MDR-TB showed significant association with the EAI sub-lineage (Chen et al., 2017;Phyu et al., 2009). We observed that 45.5% of these strains were resistant to at least one first line drug: 9.1% monoresistant to RIF, 9.1% monoresistant to STR, 18.2% MDR-TB and 9.1% polyresistant to INH/STR.
It has been demonstrated that Mexican diabetic patients present 4.7-fold and 3.5-fold higher risk to develop drug-resistance and MDR-TB, respectively (Pérez-Navarro et al., 2015). We found that among DM patients, 35.71% showed resistance to at least one drug and 7.14% was MDR-TB. In fact, in our study the most frequent comorbidities associated with drug-resistance were malnutrition (40%) and HIV (40%). Regarding to HIV, there are reports of resistance to at least one drug that ranged from 10.6% in Tanzania (Kibiki et al., 2007), to 33.3% in Mexico City (Lopez-Alvarez et al., 2010), the latter more consistent with our results.
It is noteworthy that 16% of the analyzed isolates corresponded to NTM. This is an important percentage although lower than those previously reported for Mexico City (30.8%) (Hernández-Solís et al., 2017) and Northern India (27.4%) (Maurya et al., 2015). As in the above mentioned reports, we found M. avium, M. fortuitum and M. intracellulare as the most frequent infecting species. Now a days, AFB smear is still the preferred diagnosis method for TB, however it does not 17 provide accurate information about the infecting specie, consequently, patients receive TB drug treatment which is not effective in NTM infections. It is extremely important that public health official policies include molecular techniques as part of the initial diagnosis in TB suspicious cases, in order to differentiate between MTBc and NTM. This methods may also provide drug resistance information and give faster results than mycobacterial isolation by culture.
This is a first approach to the description of the lineages circulating in Oaxaca and its association with drug resistance and co-morbidities. It is necessary to increase the number of isolates to draw wider conclusions. Although by molecular methods the detection of members belonging to MTBc was significant, it was not possible to recover all AFB (+) strains in culture; this probably due to the grueling journey to reach some communities, climate conditions and time of delivery to the laboratory. All of this circumstances may have affected the viability of the bacillus. Improving sample transport conditions should be considered in future studies.

Conclusion
According to this study, ten different MTBc sublineages were identified in Oaxaca, being Haarlem and EAI the most prevalent and related to patients with DM, HIV and malnutrition.
Drug resistance, including MDR-TB cases, was observed in isolates belonging to these two lineages, while most isolates form the rest of the identified sublineages were mainly pan-sensitive to all first line drugs.
The high diversity of sub-lineages found in "Valles Centrales" could be associated to the location of the Capital City in this region which implies migration, tourism and regional trading.
The identification of highly pathogenic MTBc genotypes and NTM in Oaxaca reveals the significance of implementing strategic surveillance molecular systems in order to identify possible outbreaks which may impact public health management in the state of Oaxaca.