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Investigación original / Original research
Multidrug-resistant tuberculosis in
Port-au-Prince, Haiti
Oksana Ocheretina,1 Willy Morose,2 Marie Gauthier,3 Patrice Joseph,2 Richard D’Meza,4 Vincent E. Escuyer,5 Nalin Rastogi,6 Guy Vernet,3 Jean W. Pape,2 and Daniel W. Fitzgerald 1 Suggested citation
Ocheretina O, Morose W, Gauthier M, Joseph P, D’Meza R, Escuyer VE, et al. Multidrug-resistant tuberculosis in Port-au-Prince, Haiti. Rev Panam Salud Publica. 2012;31(3):221–4.
abstract
Objective. To determine the prevalence of multidrug-resistant tuberculosis (MDR-TB)
among patients with new smear-positive pulmonary TB in Port-au-Prince, Haiti. Methods. Sputum samples were cultured from 1 006 patients newly diagnosed with TB
in 2008. The core region of the rpoB gene that is associated with resistance to rifampin was sequenced. All isolates with rpoB mutations were sent to the New York State reference laboratory for conventional drug susceptibility testing (DST). All isolates were also tested with the GenoType MTBDRplus line-probe assay. Results. Mycobacterium tuberculosis was isolated from 906 patients. Twenty-six (2.9%)
of the isolates had missense mutations or deletions in rpoB and were resistant to rifampin by DST. All 26 were also resistant to isoniazid and classified as MDR-TB. Forty-six control isolates without rpoB mutations were found to be rifampin sensitive by DST. The GenoType MTBDRplus line-probe assay correctly identified 26 MDR-TB strains. It misclassified one pansusceptible isolate as rifampin resistant. Conclusions. This study shows an MDR-TB prevalence of 2.9% in newly diagnosed
TB patients in Haiti and suggests that rpoB sequencing and hybridization assays are good screening tools for early detection of MDR-TB. Key words
Tuberculosis, multidrug resistant; cross-sectional studies; Haiti.
with an estimated prevalence of 331 TB cases, and the fifth most frequent losis (TB) in the Western Hemisphere, per 100 000 population (1). Because of in New York, with 5.0% of all TB cases Mycobacterium tuberculosis strains circu- Center for Global Health, Division of Infectious Diseases, Department of Medicine, Weill Cornell tians emigrate to countries around the lating in Haiti, especially drug-resistant Medical College, New York, New York, United States of America. Send correspondence to: O. that approximately 1 million Haitians, global health significance (5, 6).
2 Les Centres GHESKIO, Port-au-Prince, Haiti.
or nearly 10% of Haiti’s resident popula- Findings from the three M. tuberculosis tion, were living abroad in countries that 4 Programme Nationale de Lutte contre la Tubercu- lose, Ministère de la Santé Publique et de la Popu- include the Dominican Republic, the ducted over the past 20 years suggest 5 Laboratory of Clinical Mycobacteriology, Wads- increasing in Haiti. In a 1990 study, the worth Center, New York State Department of Health, Albany, New York, United States of francophone world (2). Haiti was the rate of isoniazid resistance was 19% but most frequent country of birth among multidrug-resistant tuberculosis (MDR- 6 World Health Organization Supranational Tuber- foreign-born TB patients in Montreal, TB), defined as resistance to both isonia- culosis Reference Laboratory, Institut Pasteur de la Guadeloupe, Pointe-à-Pitre, Guadeloupe, France.
Rev Panam Salud Publica 31(3), 2012 Ocheretina et al. • Prevalence of multidrug-resistant tuberculosis in Haiti patient newly diagnosed with TB (7). susceptibility testing (DST). A subset of isolates negative for rifampin resistance by rpoB gene analysis was also sent to defined as isolates that were resistant a QIAquick PCR purification kit to rifampin by rpoB gene analysis with Haiti, with new onset tuberculosis found confirmation of resistance to rifampin with the primers used for PCR ampli- Study setting and population
were compared with the wild-type rpoB sequence of H37Rv for M. tuberculosis au-Prince and its environs included complex identification and for detection Grace Children’s Hospital, the Haitian of mutations. Isolates were provision- State Sanatorium, GHESKIO Centers, ally classified as rifampin resistant if 6.6% in the Dominican Republic (10, 11). nonite Mission of Croix des Bouquets. described as conferring resistance (12).
cases treated in the Port-au-Prince area. nosed with an acid-fast bacillus-positive It is hoped that with recent advances smear in 2008. They did not report a according to the manufacturer’s instruc-in molecular techniques, more drug- prior diagnosis of TB.
the WHO will be able to determine 5-mL aliquot of sputum from each patient into a 50-mL plastic tube. The tube was la- to the results of rpoB sequence analysis.
beled with the patient’s age, gender, and All isolates with rpoB mutations, HIV status (positive, negative, unknown). five largest TB treatment centers in Port- out rpoB mutations, were sent to the au-Prince was conducted to determine transported within 2 days to a central Laboratory of Clinical Mycobacteriol-the prevalence rates of primary MDR-TB Laboratory studies
York, United States). All specimens were jective of this study was to validate the confirmed as M. tuberculosis complex use of molecular tests for the diagnosis of MDR-TB in Haiti before their clinical York, drug sensitivity testing to first-line on Lowenstein–Jensen slants (Becton antituberculosis drugs was performed Dickenson, Franklin Lakes, New Jersey, Study design
instructions. For isolates resistant to at tion at 95°C, and disrupted by three least one first-line drug, DST to isonia- study of M. tuberculosis drug resistance cycles of freezing at –70°C and heating zid, rifampin, ethambutol, streptomycin, among patients presenting with new at 95°C. DNA was separated from de- acid-fast bacillus smear-positive active bris using Spin-X centrifuge filter tubes, kanamycin, p-aminosalicylc acid, amika- 0.22-µm pore size (COSTAR, Corning cin, and ofloxacin was performed with est TB treatment centers in the metropol- Inc., Lowell, Massachusetts, United the proportion method on 7H10 agar as itan Port-au-Prince area in 2008. Sputum States). The 329-bp rpoB fragment was recommended by the Clinical and Labo- samples were cultured from these five amplified in 35 cycles of polymerase ratory Standards Institute (14).
centers for M. tuberculosis. Isolates were were 30 s of denaturation at 95°C, 1 min Analysis
the rpoB gene, which is known to be of annealing at 55°C, and 1 min of exten-associated with > 95% of rifampin resis- tance (12). M. tuberculosis isolates resis- tant to rifampin by rpoB DNA sequence 0.2 µM each primers rpoB-F (5’-CCA- riology Laboratory of the New York CAC-3’) and rpoB-R (5’-CGT-TTC-GAT- State Department of Health for drug GAA-CCC-GAA-CGG-GTT-GAC-3’), Rev Panam Salud Publica 31(3), 2012 Ocheretina et al. • Prevalence of multidrug-resistant tuberculosis in Haiti rpoB gene and the one isolate that had a Review Board at GHESKIO and Weill New York, and one isolate was nonvi- able upon subculture. All 26 isolates phism in rpoB codon T508 (ACC → ACT). The remaining isolates were sensitive to as M. tuberculosis. All 26 isolates tested Study population
DISCUSSION
The five sites sent sputum samples and hence were classified as MDR-TB. from 1 006 patients to the central labora- The complete drug sensitivity profile of the 26 MDR isolates is shown in Table 2.
909 grew Mycobacteria on Lowenstein– In total, 26 of 906 (2.9%) M. tuberculosis au-Prince had MDR M. tuberculosis. This value is an increase from two studies in statistically significant associations were isolates, 906 belonged to the M. tubercu- found between patient age, gender, or tected in any new TB cases in Haiti. This losis complex. The characteristics of the HIV status and multidrug resistance.
study also suggests that rpoB sequencing 906 patients from whom M. tuberculosis was isolated are presented in Table 1.
tide polymorphism in rpoB codon T508 female, and 77.9% of patients were HIV-1 in the reference laboratory and found to proximately the same number of samples.
Prevalence of MDR-TB
tations in the core region of rpoB, were The 81-bp core region of the rpoB gene tory for control DST. All 46 isolates were was sequenced for all isolates. Of the 906 M. tuberculosis isolates, 27 (2.9%) con- Screening with GenoType
in the rpoB gene that are known to be MTBDRplus line-probe assay
rate of 10.2% in all cases and 6.6% in new associated with rifampin resistance. One TB cases (11). Given the large amount of additional isolate had a silent mutation DNA extracts from the 906 M. tuberculosis morphism in rpoB codon T508 (ACC → of these studies are of particular concern fied 28 (3.1%) samples as resistant to ri- The 27 M. tuberculosis isolates with fampin. This number included all 27 iso- missense mutations or deletions in rpoB lates identified by sequencing as having were sent to the New York State TB a missense mutation or deletion in the even more relevant since the 12 January 2010 earthquake, when the headquar-ters of the National TB Program and TABLE 1. Characteristics of 906 patients with
TABLE 2. Drug resistance of Mycobacterium
a diagnosis of new onset sputum smear-
tuberculosis isolates with mutations in rpoB
positive tuberculosis, Port-au-Prince, Haiti, gene from 26 patients with new diagnosis
of active pulmonary tuberculosis, Port-au-
Prince, Haiti, 2008
now living in crowded tent cities. Rates samples that tested positive for rpoB isolates that were negative for the rpoB ever, all 46 isolates without rpoB muta- No strains were resistant to p-aminosalicylic acid, ofloxa- cin, capreomycin, cycloserine, kanamycin, or amikacin.
b Mutations include missense mutations or deletions in the 81-bp core region of rpoB but do not include isolates with silent mutations or synonymous single nucleotide and other studies suggest a > 95.0% cor- Note: NA: not applicable.
a May not sum to 100 due to rounding.
relation between rpoB sequence analysis Rev Panam Salud Publica 31(3), 2012 Ocheretina et al. • Prevalence of multidrug-resistant tuberculosis in Haiti and phenotypic drug sensitivity testing. Acknowledgments. This project was
Therefore, it is unlikely that the actual MDR-TB. Sequencing or hybridization supported by Fondation Merieux and rates of MDR-TB are significantly higher assays of the rpoB gene are good screen- ing tools in this population for early TW00018 from the Fogarty Interna- tional Center of the National Institutes nosed with smear-positive pulmonary MDR-TB.
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Objetivo. Determinar la prevalencia de tuberculosis (TB) multirresistente en
pacientes con TB pulmonar nueva con baciloscopia positiva en Puerto Príncipe, Haití. Métodos. Se cultivaron muestras de esputo de 1 006 pacientes con diagnóstico
Tuberculosis multirresistente reciente de tuberculosis efectuado durante el 2008. Se secuenció la región nuclear
en Puerto Príncipe, Haití
del gen rpoB, que se asocia con la resistencia a la rifampicina. Todos los aislados con mutaciones de rpoB se enviaron al laboratorio de referencia del estado de Nueva York para llevar a cabo un antibiograma convencional. Todos los aislados se estudiaron también con el ensayo de sonda lineal GenoType MTBDRplus. Resultados. Se aisló Mycobacterium tuberculosis de 906 pacientes. Veintiséis (2,9%) de
los aislados presentaban mutaciones de sentido erróneo o deleciones en rpoB y fueron resistentes a la rifampicina en el antibiograma. Los 26 aislados fueron resistentes también a la isoniacida y se clasificaron como TB multirresistente. Cuarenta y seis aislados de control sin mutaciones de rpoB resultaron sensibles a la rifampicina en el antibiograma. El ensayo de sonda lineal GenoType MTBDRplus identificó correctamente a las 26 cepas de TB multirresistente y clasificó de manera errónea un aislado sensible a múltiples fármacos como resistente a la rifampicina. Conclusiones. Este estudio revela una prevalencia de TB multirresistente de 2,9%
en los pacientes con TB recién diagnosticada en Haití e indica que los ensayos de secuenciación e hibridación de rpoB son estudios de detección sistemática adecuados para la detección temprana de la TB multirresistente. Palabras clave
Tuberculosis resistente a múltiples medicamentos; estudios transversales; Haití.
Rev Panam Salud Publica 31(3), 2012

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