HIV Infection and Geographically Bound Transmission of Drug-Resistant Tuberculosis, Argentina

Disease trends are driven by HIV co-infection and transmission of a few strains within narrow geographic niches.

D uring the early 1990s, HIV-associated multidrug-resistant tuberculosis (MDR TB) emerged in Argentina (1). In Buenos Aires, the country's most heavily populated city, certain multidrug-resistant Mycobacterium tuberculosis strains spread quickly among patients with AIDS (2,3). Specifi cally, the so-called M strain caused a major MDR TB outbreak at the Hospital Muñiz, a referral treatment center for infectious diseases (4). HIV-infected patients repeatedly seeking assistance at different health centers introduced the M strain into hospitals in nearby districts, where secondary transmission occurred (5). This strain was later responsible for the emergence of MDR TB in HIV-negative patients who had not previously undergone TB treatment (6). In 2002, the M strain was isolated from 2 patients with extensively drug-resistant TB (XDR TB). Two other MDR TB outbreak strains, Ra and Rb, emerged in Rosario, the third largest city in Argentina, simultaneously with the M strain (7).
MDR TB emergence highlighted the need for a MDR/ XDR TB surveillance system focused on incidence and transmission. In 2003, the National TB Laboratory Network launched a systematic registry of all incident MDR/ XDR TB cases diagnosed throughout the country. The registry includes a genotype database for all MDR/XDR TB patients going back to the initial outbreaks and population studies. We present the fi ndings of a 7-year follow-up study of MDR and XDR TB in Argentina, with emphasis on potential transmission events involving strains responsible for previous outbreaks.

Study Group
Isolates from all patients with newly diagnosed MDR or XDR TB from January 2003 through December 2009 were included in the study (1 isolate per patient, collected at time of diagnosis). MDR TB was defi ned as disease caused by M. tuberculosis resistant to at least isoniazid and rifampin and XDR TB as disease caused by MDR M. tuberculosis showing further resistance to any fl uoroquino-lone and any second-line injectable anti-TB drug. A patient with newly diagnosed MDR or XDR TB was defi ned as a patient with disease fi rst confi rmed by drug susceptibility testing (DST) during the study period, regardless of previous treatment history. A hotspot was defi ned as an area where a MDR TB outbreak had been documented before the study period. Two or more patients were considered to be epidemiologically related when they were in the same place and time or shared similar behavioral risk factors.
Available demographic and clinical data were collected through the national TB laboratory network. A special effort was made to retrieve data from clinical records in special groups, i.e., XDR TB, patients in hotspot areas, and those in clusters with <6 bands in the IS6110 restriction fragment length polymorphism (RFLP). This research was approved by the research review board of the Instituto Nacional de Enfermedades Infecciosas, Administración Nacional de Laboratorios e Institutos de Salud (INEI ANLIS) "Carlos G. Malbran."

Bacteriologic Studies
On the basis of programmatic guidelines, DST was performed on isolates from patients at risk for drug resistance: patients with TB treatment failure and retreatment, HIV or other concomitant conditions, or exposure to drugresistant TB in household, prison, or hospital. In Buenos Aires and Rosario, culture and DST are available to test virtually all persons with suspected TB who seek assistance at large referral treatment centers. In the rest of the country, persons not included in the high-risk group are highly unlikely to contract MDR TB. In all, ≈10,000 TB cases are reported annually in Argentina, of which »4,500 are diagnosed on the basis of a positive culture. Among cases that are culture-positive, ≈3,000 have isolates submitted for DST; MDR TB is diagnosed for 4% of these patients.
M. tuberculosis DST to fi rst-line drugs (isoniazid, rifampin, streptomycin, ethambutol, and pyrazinamide) was performed in 19 TB network laboratories under regular profi ciency testing, according to World Health Organization standards (8). The supranational reference laboratory at Instituto Nacional de Enfermedades Infecciosas ANLIS conducted external quality control, confi rmed multidrug resistance, and tested susceptibility to second-line drugs (kanamycin, amikacin, capreomycin, and ofl oxacin), according to World Health Organization recommendations (9).

Genotyping
All available isolates underwent standard IS6110 DNA RFLP fi ngerprinting and spoligotyping (10,11). Patterns were compared by using BioNumerics 5.1 software (Applied Maths, St-Martens-Latem, Belgium), using the Dice coeffi cient with 1% tolerance and the unweighted pairgroup method with arithmetic averages (12). The RFLP pattern of the reference strain M. tuberculosis Mt14323 was used for gel normalization.
For RFLP patterns with >6 bands, a cluster was defi ned as a group of >2 isolates whose RFLP patterns and spoligotypes were 100% identical when compared with all other patterns found within the study period. RFLP patterns with <6 bands were included in a cluster when epidemiologic links were established in addition to identical spoligotypes. Similarly, a proven epidemiologic link was required for including in a cluster a variant of a cluster genotype; a genotype variant was defi ned as a genotype with a 1-band difference in the RFLP or 1-spacer difference in the spoligotype but not both. Because of the unusually large size of some clusters, a major cluster was defi ned as >15 patients and a minor cluster as <15 patients with MDR TB newly diagnosed during the period. Shared International Type (SIT) and genotype family were assigned by consulting the SITVIT database (www.pasteurguadeloupe.fr:8081/SITVIT) (13), except for genotypes H4, T1-Tuscany, and LAM3/S convergent, which were reclassifi ed as Ural, LAM-Tuscany, and S, respectively, according to Abadia et al. (14). Orphan genotypes were those lacking a SIT in the SITVIT database.

Statistical Analysis
We used univariate and multivariate logistic regression analyses to determine factors associated with being in a cluster and being in a major cluster. The explanatory variables were patient age, country of birth, place of diagnosis, HIV status, previous TB treatment, and disease localization. Within the subgroup included in major clusters, we used logistic regression analysis to determine factors associated with being infected by the M strain. In this latter model, the explanatory variables were patient age, country of birth, HIV status, previous TB treatment, hospital exposure, and isolate drug resistance.
We divided patients into 3 age groups: <15, 16-45, and >45 years of age. Gender was removed from the models because it was associated with particular settings in 2 major clusters; unknown categories were removed from all the variables included in the model. Because of the limited numbers per category, the age category <15 years was removed from the multivariate analyses.
We applied 3 tests to assess the performance of the models: overall model fi t, Hosmer & Lemeshow test, and receiver operating characteristic area under the curve. We considered a model to be adequate when values were: overall model fi t p<0.2, Hosmer & Lemeshow test p>0.5, and area under the curve >0.70. We used the χ 2 test for linear trends for assessing changes in the annual number of MDR TB patients in cluster M compared with changes in numbers in other major clusters. Statistical analyses were performed by using MedCalc version 12 software (MedCalc, Mariakerke, Belgium).  (Figure 1). Coverage was lower in 2006 because of a technical mishap that resulted in a loss in the isolate collection.

Clusters
Of 787 patients for whom isolate genotype was available, 438 (55.7%) fi tted into 7 major clusters and 151 (19.2%) into 45 minor clusters; 198 (25.2%) harbored unique genotypes (Table 1). In the multivariate regression analysis, the outcome of being in a cluster was signifi cantly dependent on being 15-44 years of age, born in Argentina, and HIV-infected. Being in a major cluster was signifi cantly dependent on the 2 latter predictors and of having the MDR TB diagnosis occur in a hotspot area ( Table 2).
Characteristics of the 7 major clusters are described in Table 3 and DST profi les in Table 4. Genotype patterns are shown in Figure 2 and geographic distribution in Figure 3. Altogether, the 3 largest clusters (M, Ra, and Rb) accounted for 355 (45.1%) patients; isolate patterns matched 3 genotypes previously associated with MDR TB outbreaks (4,7,15). The other 4 major clusters (Pr, At, Ob, and Os) accounted for 83 (10.5%) patients; these genotypes had been reported only sporadically before the study period.
The predominant cluster, M, was largely confi ned to the city of Buenos Aires and the surrounding area, with only 5/228 patients having MDR TB diagnosed elsewhere. Twenty patients in this cluster were immigrants from neighboring countries (Bolivia 11, Paraguay 6, Peru 2, Uruguay 1). Most patients had >1 commonly acknowledged risk factors for MDR TB (129 patients had 1, 64 had 2, and 16 had 3 risk factors) ( Table 3). The cluster included the 2 previously reported outbreak variants of the M strain (4), Mm in 180 patients and Mn in 35 patients (Figure 2), and 9 sporadic variants, observed in 13 patients who had proven epidemiologic links with other patients in cluster M. An isolate resistant to 5 drugs was strongly associated with disease produced by the M strain ( Table 5). The numbers of patients affected by this strain decreased signifi cantly within the period when compared with the numbers of patients in the other 6 major clusters (p = 0.002). In particular, the proportion of HIV-infected patients affected by the M strain decreased signifi cantly during the study period, from 65% in 2003 to 24% in 2009 (p = 0.02; Figure 4). No similar trend was observed in the HIV-negative group (p = 0.77). The second major cluster, Ra, was mainly limited to the overpopulated area of Rosario City and surroundings, another MDR TB hotspot (7). Only 8 patients in this cluster were found outside that area. Of 38 patients in the third major cluster, Rb, 26 received a diagnosis of MDR TB in Buenos Aires, including 10 transvestite sex workers; 8 patients had the disease diagnosed in Rosario, and 4 elsewhere. Cluster Pr consisted mainly of inmates in several state prisons for men. An association of gender with clustering was only observed in clusters Rb and Pr, in which men predominated (32/38 and 24/26, respectively).

XDR TB Patients and Genotypes
XDR TB was newly diagnosed in 53 patients during 2003-2009. Of these patients, 37 fi rst received a diagnosis of MDR TB during the same period, so these patients were included in the MDR and XDR TB groups. The other 16 XDR TB patients received a diagnosis of MDR TB before this period. The male:female ratio for XDR TB patients was 1.25:1; median age was 37.4 years (SD 11.6, range 21-72 years). Fifty-two patients were born in South America (Argentina 30, Bolivia 4, Peru 6, Paraguay 1, Brazil 1, undetermined 11), and 1 was born in Indonesia.  Characteristics of XDR TB patients related to clustering are described in Table 6. Four major clusters (M, Os, At, and Rb) comprised 31 (58%) XDR TB patients; 6/14 HIV-positive patients with XDR TB were in cluster Os, and 5 in cluster M. Five of 11 immigrants from other South American countries to Argentina harbored the M strain, and the patient from Indonesia harbored a Beijing strain. Of 15 patients with no record of previous TB treatment, 12 were included in major clusters. Annual numbers of XDR TB patients by genotype are shown in Table 7.

Discussion
The annual number of newly diagnosed MDR and XDR TB cases decreased slightly, with minor fl uctuations, during the study period. HIV infection was associated with almost one third of MDR TB cases; this proportion is 2-4´ higher than that attributed by different studies to all forms of TB in the country (16,17). As previously observed (18), annual fl uctuations in the numbers of total MDR/XDR TB cases during the study period paralleled closely annual fl uctuations in the numbers of HIV-infected patients.
All 7 major clusters in our study were connected with particular geographic areas, institutional settings, or both. Furthermore, most patients in these clusters underwent TB treatment in health centers that had ongoing MDR TB transmission or had a household or a prison contact with persons who had MDR TB. These fi ndings indicate that these major clusters represent true transmission events.
Many cases in this outbreak were caused by the M strain, an apparently autochthonous outbreak genotype. In a countrywide survey performed in 1998, this strain was found to be responsible for 42% of all MDR TB cases but was confi ned to the metropolitan area of Buenos Aires (National TB Laboratory Network, unpub. data). Since then, the M strain has been the most frequently identifi ed in every MDR TB investigation performed in the country. We found the M strain to be the most prevalent and that its transmission was virtually restricted to the area of the initial outbreak. However, its numbers decreased by more than half within the study period, particularly among HIV-infected patients, which suggests that the epidemic curve of the M strain has entered a declining phase. The other 2 strains associated with previous MDR TB outbreaks, Ra and Rb, were found to persist with lower, fl uctuating frequencies.
The expansion of M strain transmission in Argentina was initially fostered by clinical mismanagement. During the early 1990s, patients with advanced AIDS were hospitalized in large, referral treatment centers, where they shared facilities with patients who had MDR TB. At that time, virtually no respiratory protection policy was in force because it was wrongly assumed that MDR TB patients were barely infectious. After genotyping confi rmed outbreaks of MDR TB among the patients with AIDS (4), hospital infection control interventions were adopted, microbiologic diagnosis and drug-resistance detection were expedited, and second-line TB drugs and highly active antiretroviral therapy became available. As a result of those interventions, hospital transmission was substantially reduced but not completely controlled (18). At the time of our study, however, the M strain had long expanded beyond the hospital environment (6).
The national M. tuberculosis genotype database in Argentina identifi ed very few patients with non-MDR TB harboring H2 genotypes (V. Ritacco, unpub. data). Another study supported this observation (19), and the H2 genotype was found to be infrequent in other South American countries (13,(20)(21)(22). The IS6110 RFLP pattern of the M strain was absent outside Argentina in the Ibero-American MDR  TB genotype database (23) and was not present in the M. tuberculosis genotype database in the Netherlands (D. van Soolingen, pers. comm.). This pattern was registered in other countries, anecdotally, in 2 MDR TB patients with AIDS: a patient from Argentina who died in San Francisco, California, shortly after his arrival in the United States (24); and a patient from Asuncion, Paraguay, who visited the Hospital Muñiz in Buenos Aires each month for antiretroviral therapy (20). Different M. tuberculosis genotypes may have affi nity with certain geographic areas and ethnic groups (25), which could explain why the M strain persists in the Buenos Aires area. In addition, most persons at risk for MDR TB are underprivileged and cannot afford to travel far distances. This mobility limitation might also help to explain why this strain has remained virtually confi ned to the original hotspot area.
A small number of patients affected by the M strain were immigrants from neighboring countries who had settled in Buenos Aires. Cross-border and domestic migration toward large metropolitan areas is a long-observed demographic and public health concern in Argentina. More than 80% of the patients in this study were assisted in metropolises designated as MDR TB hotspots. Even though immigrants with TB have access to higher quality health care in these areas than in other parts of the country, they are also at higher risk of becoming newly infected with an outbreak MDR TB strain.
The M strain was overrepresented among patients with XDR TB; isolate resistance to >5 anti-TB drugs was found to be a strong predictor of disease caused by the M strain (4,26). The accumulation of drug resistance-conferring mutations would be expected to have reduced the epidemiologic fi tness of this strain, but it has prevailed for 15 years. The epidemiologic fi tness of a strain can be infl uenced by a range of factors, e.g., the genetic backgrounds of host and pathogen, host-pathogen interactions, and the environment (27)(28)(29). Compensatory evolution restoring in vivo fi tness, as well as social and behavioral factors, might   have played a role in the epidemiologic persistence of the M strain (30). These factors might also have preferentially fostered the spread of drug-resistant strains of the H2 genotype in our setting. Further studies are needed to evaluate the most critical risk factors. Drug-resistance profi les were not uniform within the M strain clusters. The variations in susceptibility to individual drugs refl ect the existence of various ongoing chains of transmission, some of which might have started before, or simultaneously with, the fi rst documented outbreak. One limitation of our study is the failure to identify individual chains. Factors that precluded the reliable characterization of subclusters were the long time elapsed since the outbreak onset, the insuffi cient epidemiologic documentation in many cases, and the unavailability of additional molecular markers.
Our study has another major limitation. Incomplete demographic and clinical data on patients were retrieved, and several observations had missing values. If missing values were systematically associated with a given force or factor, results presented here would be biased. We are not aware of any association of missing values with the dependent variables under study and assume that those data were missing at random. Missing values may have affected the analyses by reducing the number of observations, which may have reduced the power of the model to detect signifi cant associations but without necessarily biasing the associations reported. However, the possibility that bias might have resulted from missing data cannot be ruled out. Therefore, statistical signifi cances of our analyses should be interpreted cautiously.
In the MDR TB hotspots in Argentina, the distinction between primary and acquired MDR TB on the basis of a history of previous TB treatment was not decisive because patients could have been exposed to hospital-associated MDR TB infection while being treated for community-acquired TB. This fact could explain why clustering was not more frequent among patients without previous TB treatment in our study.
The national TB network includes all the laboratories performing bacteriological TB diagnosis in the country; therefore, the patients in this study represent all newly diagnosed MDR TB cases in Argentina. The structure, geographic coverage, and personnel of the TB laboratory network are adequate to provide DST for all patients at risk for MDR TB in Argentina. However, a few MDR TB patients might remain undiagnosed because of operational factors, e.g., ineffi cient detection of risk factors, insuffi cient or delayed requests for DST, and disorganized information systems.
The geographically restricted distribution of successful MDR TB genotypes that we found has public health implications. As a result of this study, specifi c interventions are being reinforced, particularly in the MDR TB hotspots: implementing universal culture and strategies to expedite drug