Tuberculosis in Immigrants to Cartagena de Indias, Colombia

Tuberculosis (TB) is among the top ten causes of death in the world and is the leading cause of death by a single infectious agent. A 2019 report from the World Health Organization (WHO) stated that there were 10 million (range of 9-11.1 million) people with TB in the world in 2018, of which 1.2 million died (range 1.1-1.3 million).1 Two-thirds of the world’s population is infected by the causative agent of TB, Mycobacterium tuberculosis. One in ten people infected with M. tuberculosis develop an active infection; the remaining ninety percent have the latent form of the disease, which can progress to reactivation TB.2 TB affects vulnerable populations such as those with low economic status, persons with comorbidities such as diabetes mellitus or coinfection with HIV, and homeless individuals. Worldwide, immigrants have contributed to the increased incidence of TB. Human migration refers to the movement of persons or groups of people from one geographical region to another across an administrative or political border with the intention of establishing themselves indefinitely or temporarily in a place other than their place of origin.3,4 A 2018 report from the International Organization for Migration (IOM) stated that the estimated number of international migrants has increased in the last 4 and a half decades. The estimated total number of international immigrants in 2015 was 243 700 236 people, equivalent to 3.3% of the world population.5,6 Colombia is experiencing a new surge of immigration from Venezuela.7 This study was carried out in Cartagena de Indias, a tourist city located in the Caribbean region of Colombia with an estimated population of 1 089 683 http://ijtmgh.com Int J Travel Med Glob Health. 2020 Aug;8(3):100-106 doi 10.34172/ijtmgh.2020.18 TMGH IInternational Journal of Travel Medicine and Global Health J


Introduction
Tuberculosis (TB) is among the top ten causes of death in the world and is the leading cause of death by a single infectious agent. A 2019 report from the World Health Organization (WHO) stated that there were 10 million (range of 9-11.1 million) people with TB in the world in 2018, of which 1.2 million died (range 1.1-1.3 million). 1 Two-thirds of the world's population is infected by the causative agent of TB, Mycobacterium tuberculosis. One in ten people infected with M. tuberculosis develop an active infection; the remaining ninety percent have the latent form of the disease, which can progress to reactivation TB. 2 TB affects vulnerable populations such as those with low economic status, persons with comorbidities such as diabetes mellitus or coinfection with HIV, and homeless individuals.
Worldwide, immigrants have contributed to the increased incidence of TB. Human migration refers to the movement of persons or groups of people from one geographical region to another across an administrative or political border with the intention of establishing themselves indefinitely or temporarily in a place other than their place of origin. 3,4 A 2018 report from the International Organization for Migration (IOM) stated that the estimated number of international migrants has increased in the last 4 and a half decades. The estimated total number of international immigrants in 2015 was 243 700 236 people, equivalent to 3.3% of the world population. 5,6 Colombia is experiencing a new surge of immigration from Venezuela. 7 This study was carried out in Cartagena de Indias, a tourist city located in the Caribbean region of Colombia with an estimated population of 1 089 683 inhabitants; 5% were immigrants from Venezuela during the study period, according to the National Administrative Department of Statistics (the Spanish acronym is DANE). 8 Cartagena de Indias has experienced an increase in TB cases; in 2017, the District Administrative Department of Health (DADIS) reported that there were 356 TB cases (2.4% of which were reported in Colombia). Of these cases, 35 (9.98%) had coinfection with HIV, and 25 (7.02%) occurred in patients with Venezuelan nationality. Our objective is to describe the clinical and microbiological characteristics of M. tuberculosis in the immigrant population in Cartagena de Indias, Colombia.

Methods
A descriptive study of 101 patients with a clinical and microbiological diagnosis of TB was carried out in the city of Cartagena de Indias, Colombia during the period from December 2017 to December 2018. The patients came from 3 geographic areas, denominated as localities 1, 2, and 3. The selection criteria included clinical criteria, epidemiological contact, imaging findings, Ziehl Nelsen staining, and histopathology. Participants provided a medical history, and biological samples were taken according to the type of TB (sputum, cerebrospinal fluid, pleural fluid, bone tissue) and seeded in solid culture medium. The samples were subjected to anti-TB drug susceptibility testing by automated susceptibility testing, and the markers of resistance to isoniazid and rifampicin (katG, inhA, rpoB genes) were determined by molecular biology.
Culture method for Isolation of Mycobacterium tuberculosis Biological samples were decontaminated with 4% sodium hydroxide and seeded for culturing in solid Ogawa Kudoh medium in accordance with the protocol of the Pan American Health Organization (PAHO). 9 They were incubated for 8 weeks at 36°C and evaluated for growth every week.

Phenotypic Sensitivity Test
All samples were subjected to detection of M. tuberculosis and anti-TB drug susceptibility testing by the BD Bactec MGIT (Mycobacterial Growth Indicator Tube) 960 Mycobacteria Culture System (Becton, Dickinson and Company 7 Loveton Circle Sparks, MD 21152-0999 USA; 800-638-8663), 10 in collaboration with the National Reference Laboratory of the Colombian National Institute of Health (INS). The critical concentrations of isoniazid were 0.1 and 0.4 µg/mL, and that of rifampin was 1.0 µg/mL. 10

Molecular Tests
DNA was extracted from the colonies of the cultures in solid medium using the Wizard technique (Promega®) 11 for grampositive bacteria, which was modified for mycobacteria. Briefly, colonies were taken from the solid culture medium, inactivated at 100°C in a thermoblock, and centrifuged at 13 000 rpm for 3 minutes. Then, the supernatant was removed. The cell button was suspended in 480 µL 50 mM EDTA. Lysis was performed with 50 µL lysozyme (incubation at 37°C) and 600 µL lysis solution (incubation at 80°C). Three microliters of RNAase (incubation at 37°C) was added. Then, 250 µL of the precipitation solution was added (vortex and incubation at -20°C). The supernatant was transferred to a tube with 600 µL 100% ethanol (washed with 600 µL 70% ethanol). DNA button rehydration was performed with rehydration solution, and the samples were stored at 4°C for immediate use or at -80°C for later use.
To identify mutations in the katG, inhA, and rpoB genes, the Genotype MTBDR plus V.2 test was used (Hain LifeScience, Germany, Table S1), following the instructions described in the insert of the commercial kit. 12

Definition of Terms
The following terms are related to the clinical aspects and therapeutic outcomes.
• Clinical behavior: For the purposes of characterizing the patients, symptoms and semiologic findings were predominant in the patients. The symptoms were categorized into condensation syndrome, pleural effusion syndrome, and constitutional type (  Figure S1).

Risk Factors
Among the associated factors, one patient (11.11%) reported consuming illegal addictive substances. Coinfection with HIV in immigrants occurred in 4 patients (95% CI: 44.44%; 13.70-78.80%). Two of the HIV coinfected patients had resistance to first-line anti-TB medications (50%; P = 0.642). In relation to the native population, there was a higher frequency of cases of HIV coinfection in immigrants (6.52% vs. 44.44%; P = 0.005).

Sensitivity Profile
Of the nine immigrants, 5 (55.56%) had sensitive strains of TB, and 4 had drug-resistant strains (44.44%). Similar levels of sensitivity were found with the molecular biology and phenotypic tests (kappa 0.95; 95% CI 0.75-0.97, P<0.05), except for a case of mono-resistance to isoniazid due to a mutation in the inhA gene that was found by the molecular test but not by the phenotypic test.
Three of the resistant TB patients came from the state of Zulia and settled in locality 2 in Cartagena de Indias. Two of these patients had multidrug-resistant (MDR) TB (22.22%; 95% CI 2.81-60.01%) with katGS315T1 (AGC315ACC)/rpoBS531L (TGC/TTG) mutations; they also presented meningeal and pleural EPTB, and both patients died. The other patient from the state of Zulia presented resistance to rifampin (TB-RR) (11.11%; 95% CI 0.28-48.25%) with an rpoBS531L-type mutation (TGC/TTG); the patients presented meningeal-type EPTB and had therapeutic failure. There was also one patient from the state of Miranda who settled in locality 3; the patient presented mono-resistance to H (11.11%; 95% CI: 0.28-48.25%) with a mutation in the promoter region -15C/T of the inhA gene. The patient had therapeutic failure (Figure 1). Figure 2 shows the mutations found in patients with resistant TB. Compared with the native population, immigrants had a higher frequency of resistant cases (15.22 vs. 44.44%; P = 0.051).
Regarding the sensitivity profile in the immigrant patients with sensitive strains, 4 patients (80%) had manifestations of constitutional syndrome, and one (20%) had condensation syndrome. Three (75%) of the patients with resistant strains had manifestations of constitutional syndrome, and one (25%) had pleural effusion syndrome; this difference was statistically significant (P = 0.036; Figure 3).
Two patients were children aged under fifteen years old (22.22%; 95% CI: 2.81-60.01%); one patient was a one-yearold with osteoarticular EPTB, and the other patient was a 2-year-old with BPD. The clinical manifestations in these patients were nonspecific, and the diagnostic impression was based on close contact with adults with active TB. The radiological characteristics in the patient with PTB showed an interstitial pattern. Microbiological isolation was performed by sampling of induced sputum and in the patient with bone EPTB by culturing the inflammatory bone exudate. Sensitivity tests in these patients did not reveal resistance patterns. These patients were managed with the scheme of sensitive PTB and EPTB with an outcome in cure. Compared with the native population, immigrants had a higher prevalence of cases with constitutional manifestations (26.09% vs. 77.78%; P = 0.003) ( Table 2).

Treatment and therapeutic outcome
Patients initially received the standard regimen of rifampicin, isoniazid, pyrazinamide, and ethambutol (RHPE).
Patients who were diagnosed with resistance to anti-TB drugs underwent a change in their therapeutic regimen. One patient with mono-resistance to isoniazid due to the inhA-15 C/T mutation was managed as sensitive TB due to not showing resistance in the phenotypic test.
The patient with meningeal EPTB with rifampicinresistant strain type rpoBS531L was treated with levofloxacin, kanamycin, pyrazinamide, ethionamide, and ethambutol. The other patient with meningeal EPTB and the patient with pleural TB with MDR strains (katGS315T-rpoBS531L) received treatment with pyrazinamide, ethambutol, ethionamide, levofloxacin, kanamycin, and cycloserine.
Two of the patients who died had sensitive PTB (50%), and 2 had katGS315T-rpoBS531L type MDR-TB (50%) with extrapulmonary manifestations. One of the deceased patients with sensitive PTB came from the state of Miranda, and the other came from Sucre; both patients were coinfected with HIV, presented symptoms of constitutional syndrome, and had a cavernous pattern image. The patients with MDR TB who died came from the state of Zulia; they had settled in locality 2 in Cartagena de Indias and presented pleural and meningeal EPTB.

Discussion
Migratory waves have contributed to an increased number of TB cases, especially in countries that have medium and low incidence rates. TB patients from other countries import    13 Many immigrants have to work in unclassified activities. 14 A total of 66.67% (95% CI: 29.93-92.51% ) of the immigrant participants in this study stated that they were working in informal jobs. The most common activities were those that involved contact with the public, such as minority commerce and street vending; these situations favor the spread of TB.
The difficult economic situation that some immigrants face in both their country of origin and their host country precludes access to a balanced diet; that fact is reflected in their musculoskeletal state. All immigrant patients studied herein were in a state of malnutrition (100%; 95% CI: 66.37-100%;  BMI: 15.49 (95% CI: 13.92-17.06)). Malnutrition (MNT) was not related with any other factor, including coinfection with HIV (P = 0.688). The relationship between MNT and TB is interactive because prolonged MNT makes people more susceptible to infectious-contagious pathologies, and, in turn, TB leads to or worsens MNT. 15 Nutritional deficits can lead to impaired immune homeostasis, which greatly increases an individual's susceptibility to infection progression. 16,17 TB in the immigrant population has some common characteristics that are globally recognized. These include a younger average age, which is explained by the search for job offers. [18][19][20][21] Casals et al 22 carried out a literature review on TB and immigration in Spain between 1998 and 2012. They found 219 publications that included differential characteristics between immigrants and native populations. The average age was lower in immigrants; 93.5% were aged under 51, while in the local population, 64.9% were aged under 51. Furthermore, they found that the prevalence of resistance to isoniazid was 3.8% in natives and 7% in immigrants. 22 Another feature of TB in immigrants is an increased presentation of EPTB. The frequency of EPTB in the group of immigrant patients was higher than that of the autochthonous population (44.44% vs. 4.35%; P = 0.002). The 2 cases of meningeal TB were related to the nonapplication of the Calmette Guerin (BCG) vaccine, which is applied at the neonatal stage to protect against serious forms of TB, such as meningeal and miliary TB. 23 The tendency for extrapulmonary dissemination in the strains that infect migrant patients confers characteristics of clinical behavior. The presence of cavitation with hypervirulent strains that produce cord factor or trehalose dimycolate has been described. 24 Some authors have described the increased presence of lung cavitation in immigrants. 18 In the immigrant patients in this study, cavitation was the most frequent finding on radiography thorax (33.33%; 95% CI: 7.49-70.07%) and of extrapulmonary images such as pleural effusion (11.11% vs. 6.52%; P = 0.065) and bone destruction.
Pierluigi et al 25 carried out a comparative study in 236 patients (44% were natives; 56% were immigrants) in northern Italy. They found that immigrants were younger than natives on average (30.5 vs. 62.2%; P = 0.001), had more radiological signs of cavitary lesions (54.5 vs. 33.8%; P = 0.004), and had a higher prevalence of coexisting chronic noncommunicable diseases (17.4% vs. 8.0%). The prevalence of resistance to any medication was 25.7% among immigrants, and the prevalence of MDR was 3.9%. In addition, they found no significant difference in resistance between native and immigrant patients (29.2% vs. 19.6%; P = 0.138). 25 In the current study, a higher proportion of resistant cases was found among immigrants than among native immigrants (44.44% vs. 15.22%; P = 0.050). The tendency to spread and resist foreign strains raises an alert for the possible presence of hypervirulent strains. 26 One patient in this study who was from the state of Miranda had a mutation in the promoter region -15 C/T of the inhA gene; however, no resistance was detected in the phenotypic test, although the patient had therapeutic failure. Another patient from the same state who had sensitive PTB and was coinfected with HIV died. Both patients had settled in the same locality in Cartagena de Indias. In this study, strains were found with katGS315T/ rpoBS531L mutations, which confer MDR resistance; these strains were present in patients from the state of Zulia who had settled in locality 2, which is an economically depressed area in Cartagena de Indias. These strains exhibited hypervirulent Immigrant cases had a higher frequency of presentation of constitutional syndrome than the native population (P = 0.003). behavior, manifesting with a clinical picture of constitutional type, extrapulmonary spread, and a fatal outcome. In a sensitivity identification study on 59 M. tuberculosis strains in Venezuela (Sucre state), Mendoza et al 27 found prevalence rates of 6.3% and 14.3% for primary resistance and acquired resistance, respectively. They found one MDR strain and one strain with RR. They identified 2 other mutation points in the rpoB gene, different than the one found in our group of patients. The positions with changes were 516, 526, and 531. 27 Among our immigrant participants, one came from the state of Sucre (Venezuela). This patient had a grade II malnutrition status and was found to be very smear-positive (positive +++). Sensitivity tests did not show drug resistance, but the patient died during treatment with the RHPE scheme.
In another study carried out with data from the National Reference Laboratory for Tuberculosis on 193 patients from different areas of Venezuela, researchers detected the presence of mutations in the katG gene in 19 isolates (9.8%), of which 17 had resistance to other anti-TB drugs (6.2%), including 11 patients with resistance to rifampicin (64.7%). Six of the multiresistant clinical isolates presented resistance to 4 antimicrobials. 28 The clinical and microbiological characteristics of the immigrant patients were different than those of the local population with respect to undesired outcomes, such as therapeutic failure (8.70% vs. 33.33%; P = 0.001) and death (4.35% vs. 44.44%; P = 0.001).
This study was descriptive, and therefore, an association between the type of sensitivity of the strains and the clinical characteristics in the study participants was not examined; however, statistically significant data was found between immigrants and constitutional-type clinical behavior, resistance to anti-TB, and undesired outcomes.
TB, along with measles, diphtheria, Chagas disease, HIV, and malaria, are among the infectious pathologies that have resurfaced in the countries bordering Venezuela as a result of the migration of its inhabitants. Colombia is among the countries that have been most affected by these pathologies within the problem of migratory waves. 29

Conclusion
Immigrants have contributed to the increase in incidence of TB in the city of Cartagena de Indias, representing 8.91% of the patients studied herein. They came from geographical areas where the presence of virulent strains has been demonstrated, and they had conditions such as coinfection with HIV and malnutrition; thus, these patients presented particular clinical characteristics such as constitutional manifestations, extrapulmonary spread, the presence of caverns, and undesired outcomes such as therapeutic failure and death. Disease control programs should take these characteristics into account in the immigrant population to prevent the spread of potentially hypervirulent strains.