Jul 13, 2011 - Pulmonary Tuberculosis in Honduras. Isolated From Patients With Suspected and Atypical Mycobacteria tuberculosis. Mycobacterium. Drug- ...
Drug-Resistant Mycobacterium tuberculosis and Atypical Mycobacteria Isolated From Patients With Suspected Pulmonary Tuberculosis in Honduras Lelany Pineda-Garcia, Annabelle Ferrera, Carlos Alvarado Galvez and Sven E. Hoffner Chest 1997;111;148-153 DOI 10.1378/chest.111.1.148 The online version of this article, along with updated information and services can be found online on the World Wide Web at: http://chestjournal.chestpubs.org/content/111/1/148
Chest is the official journal of the American College of Chest Physicians. It has been published monthly since 1935. Copyright1997by the American College of Chest Physicians, 3300 Dundee Road, Northbrook, IL 60062. All rights reserved. No part of this article or PDF may be reproduced or distributed without the prior written permission of the copyright holder. (http://chestjournal.chestpubs.org/site/misc/reprints.xhtml) ISSN:0012-3692
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Drug-Resistant Mycobacterium tuberculosis and Atypical Mycobacteria Isolated From Patients With Suspected Pulmonary Tuberculosis in Honduras* Pineda-Garcia, BSc; Annabelle Ferrera, MSc; Lelany Carlos Alvarado Galvez, MD; and Sven E. Hojfner, PhD
Background: Tuberculosis is a major health problem in Central America. In Honduras, with an incidence rate of 81/100,000, it is an increasingly common cause of morbidity and hospitalization. This study was conducted to examine drug-resistant tuberculosis and prevalence of infection with atypical mycobacteria in Honduran patients with suspected pulmonary tuberculosis. Methods: Pulmonary specimens from 235 Honduran patients with suspected tuberculosis were examined by acid-fast smears and culture. The 95 mycobacterial strains isolated were identified to species level and drug susceptibility tests were carried out. Resistant Mycobacterium tuber¬ culosis strains were tested for susceptibility to six additional drugs. Their possible relationship was studied by DNA restriction fragment length polymorphism. Results: Drug-resistant strains were found in 13 of 85 culture-verified tuberculosis patients, including 10 with isolates of multidrug-resistant bacteria. Seven of the patients with multidrugresistant tuberculosis had smear-positive disease. Nine of them had a history of specific therapy. Two patients with drug-resistant disease were shown to be infected by identical strains. Only one of 11 HIV-positive patients had drug-resistant tuberculosis. Most resistant strains were suscep¬ tible to ciprofloxacin, amikacin, kanamycin, and pyrazinamide. Atypical mycobacteria were isolated from 10 patients with suspected tuberculosis. Seven of them were receiving antituberculosis chemotherapy and five had smear-positive samples. Conclusions: These results illustrate the importance of mycobacterial culture and subsequent species identification and in vitro susceptibility testing for identification of patients with drug-susceptible or drug-resistant tuberculosis and those infected or colonized with other mycobacteria. (CHEST 1997; 111:148-53) Keywords: atypical mycobacteria; drug resistance; Honduras; multidrug-resistant tuberculosis (MDR-TB); tuberculosis Abbreviations: INT=Instituto National del Torax; MDR-TB=multidrug-resistant tuberculosis; RFLP=restriction fragment length polymorphism; TB=tuberculosis
increasing incidence of tuberculosis (TB) is today reported from many parts of the world. Deterioration of public health-care facilities, poverty and crowded living conditions, increasing numbers A
n
^*-
of homeless persons, and the AIDS *From the
Department of Microbiology,
epidemic have
Universidad National
Autonoma de Honduras, Tegucigalpa, Honduras (Drs. PinedaGarcia and Ferrera); the Instituto National Del Torax and
Medicine, UNAH, Teg¬ PhysiologyHonduras Department, School of the Swedish Institute for (Dr. Alvarado); ucigalpa, Infectious Disease Control (Drs. Garcia and Hoffner); and the Microbiology and Tumorbiology Center, Karolinska Institute (Dr. Hoffner), Stockholm, Sweden. Supported by the Karolinska International Research and Train¬ ing Programme. Manuscript received December 12, 1995; revision accepted July 26, 1996. Reprint requests: Sven Hojfner, Swedish Institute for Infectious S-105 21 Disease Sweden Control,
148
Stockholm,
contributed to the emergence of the disease. In¬ creasing numbers of patients carrying drug-resistant tuberculosis strains make the situa¬ Mycobacterium tion even more severe, since the effectiveness of chemotherapy is significantly reduced in these cases.1-4 In Honduras, Central America, TB is the sixth cause of morbidity and ninth cause of hospitalization,
with
an
annual incidence
rate of 81/100,000, al¬ reaches 250/100,000 (Al¬
though regions varado, unpublished). TB was diagnosed in 4,290 patients in 1994, 95% of whom had pulmonary in
some
it
disease. In the adult population, more than 15 years of age, an incidence rate of 127/100,000 was seen. The laboratory diagnosis was in most cases based on sputum microscopy. Mycobacterial culture and drug susceptibility tests were normally not carried out.
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Clinical
Investigations
This study was conducted to examine drug resistance of M tuberculosis strains and prevalence of atypical mycobacteria isolated from patients in Honduras with suspected pulmonary TB. Materials
and
Methods
During the period September 1994 to February 1995, pulmo¬ nary specimens (mainly sputum) were collected from 235 pa¬ tients with suspected or known pulmonary TB at the Instituto National del Torax (INT) reference center in Tegucigalpa. This center receives 15 to 20% of all new Honduran cases of TB and patients apparently not responding to anti-TB therapy referred from local health centers. An epidemiologic questionnaire, fol¬ lowing World Health Organization guidelines,5 including clinical and chest radiographic data as well as previous drug therapy, was filled in for each patient. The HIV status was tested by a serologic
(Serodia-HIV; Fujirebio Inc; Tokyo, Japan); positive samples were confirmed by western blot (Diagnostic Biotechnology Ltd; Singapore). Statistical analysis was done with a computer pro¬ gram (Epi Info version 5.0; CDC; Atlanta, Ga).6
Sweden), rifampicin (Ciba-Geigy AG; Basel, Switzerland), strep¬
tomycin (Sigma Chemicals Company; St. Louis;) and ethambutol (Lederle; Wayne, NJ). For all strains, resistant to one or more of these drugs, susceptibility was also tested to pyrazinamide (Merck Sharp & Dohme International; Rahway, NJ), amikacin (Bristol-Myers Squibb Co; New York), kanamycin (Sigma Chem¬ icals Company), clarithromycin (Abbott Labs; Abbott Park, 111), ciprofloxacin (Bayer AG; Leverkusen, Germany) and para-amin-
osalicylic acid (Ferrosan AB; Malmo, Sweden). Stock solutions of each drug were prepared and diluted in BACTEC broth as described earlier,1314 to obtain the following test concentrations: isoniazid (0.2 mg/L); rifampicin (2 mg/L); streptomycin (4 mg/L); ethambutol (5 mg/L); pyrazinamide (100 mg/L); amikacin (4 mg/L); kanamycin (4 mg/L); clarithromycin (4 mg/L); ciprofloxacin (2 mg/L); and para-aminosalicylic acid (4 mg/L). Results
test
nitrate reductase8 the Swedish Institute for Infectious Disease Control, the strains were examined by a M tuberculosis complex specific hybridization probe (Accuprobe; Gen Probe Inc; San Diego, Calif). Atypical mycobacteria were characterized by growth rate, pigmentation, standard biochemical tests, and species-specific innucleic hybridization probes (Accuprobe). Strains negative hybridization probes and with an inconclusive biotyping were finally identified by sequencing of the 16S rRNA gene.9 Epidemiologic typing was done by restriction fragment
A total of 100 mycobacterial isolates were obtained from 235 specimens (43%) from the same number of Honduran patients, aged 15 to 82 years, with sus¬ or known pulmonary TB. M tuberculosis was pected isolated in 90 cases and atypical mycobacteria in 10 cases. Mycobacterium bovis was not detected. Of the 90 M tuberculosis isolates, 85 could be studied further, one was contaminated, and four failed to grow on subculture. Of these 85 strains, 24 (28%) were derived from patients with previous of TB. Around 50% of the patients chemotherapy came from the Tegucigalpa area; 72% were men and 70% were 15 to 42 years old. Most of the patients (62%) had a low income (less than $53 a month) and were alcoholics and smokers (61% and 58%, respec¬ tively); many (34%) were living in crowded condi¬ tions or were household contacts with known TB cases (24%). The HIV seropositivity was 11 of 77 (14%), nine men and two women with a mean age of 32 years. No significant differences were seen in socioeconomic conditions between the HIV-positive and HIV-negative TB patients. Most clinical findings were similar for the two groups, although a higher incidence of diarrhea and adenopathy was seen in the HIV-positive patients (p16 mg/L) and were resis¬ tory tant also to isoniazid. Thus, multidrug-resistant tuber¬ culosis (MDR-TB), defined as resistance to at least and isoniazid, was seen in 10 cases (12%). rifampicin Nine of these 10 patients had a history of previous chemotherapy (Table 1). Resistance to streptomycin
of Mycobacterial Strains All samples were examined by microscopy after Ziehl-Neelsen staining at the TB laboratory of INT, and quantified according to the international recommendations.7 After decontamination with the Petroff method,8 culture was performed on standard Lowenstein-Jensen egg medium with glycerol and on a modified Lowenstein-Jensen media with pyruvate as a carbon source. The cultures were incubated at 37°C and examined weekly for 9 weeks. Mycobacterial growth was confirmed by acid-fast staining. The 100 strains isolated were transported by air to the mycobac¬ terial reference laboratory at the Swedish Institute for Infectious Disease Control in Stockholm for further characterization and drug susceptibility testing. Demonstration
of Mycobacterial Isolates production, catalase activity, and
Characterization Niacin
were
tested
at INT. At
length polymorphism (RFLP) analysis, using a 245-bp polymerase chain reaction fragment of the IS-element IS6110, according to internationally standardized guidelines.10 Mycobacterial DNA was extracted, cut with the restriction enzyme PvuII, and sepa¬ rated by electrophoresis on an 0.8% agarose gel. The restriction fragments were transferred by southern blotting onto a nylon membrane, and hybridized with the insertion sequence IS6110. The targets were detected by a chemiluminescence kit (ECL Direct System; Little Chalfont, England).
AIDS
patients.
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149
Table Strain No.
1.Origin and Resistance ofM tuberculosis Strains Isolated From 13 Honduran Patients Smear HIV Resistant tof Sample Therapy* Age, yr/Sex Sputum Sputum Sputum Sputum Sputum Sputum Sputum Sputum Sputum Sputum Sputum Sputum Sputum
H-982/94 H-985/94
H-997/94 H-1005/95 H-1081/94 H-1105/94 H-l 173/94 H-1261/94 H-1263/94 H-1311/94 H-1346/94 H-44/95 H-87/95
25/M 42/M 46/M 23/M 59/M 51/F 29/M 44/M 40/M 59/M 36/M 52/M 67/F
1983 1989 1993 1989 1970 1988 1994 1993 1994 1988 1989 1994 1995
+++ + +++ +++ +++ +++ +++ +++ +++
Neg Neg Neg Neg Neg Neg
INH, RIF, SM INH, RIF INH
INH, RIF, INH, RIF, INH, RIF, INH, RIF, INH, RIF,
Pos
Neg Neg Neg Neg
SM, EMB SM EMB SM, EMB SM
INH
INH, RIF INH, RIF INH, RIF
Not tested
Neg
SM
Ul initiation *Year lCcU of llllllclLlUll.
Drugs: INH=isoniazid (0.2 mg/L); RIF=rifampicin (2 mg/L); SM=streptomycin (4 mg/L); EMB=ethambutol (5 mg/L).
susceptibleandto the other tested aminamikacin oglycosides, kanamycin. RFLP patterns of the 13 drug-resistant strains and five of the susceptible strains are shown in Figure 1. In one case, in strains H-1005/94 and H-1173/94 (lanes 4 and 7), identical restriction patterns were seen. These MDR-TB strains were isolated from two young men from Tegucigalpa. Another MDR-TB strain, H-1311/94, and one of the susceptible strains, H-1218/94 (Fig 1, lanes 10 and 16) were also clearly related. However, most of the patients were infected with unrelated strains, and no specific patterns sug¬ gesting a clonal relationship of, eg, drug-resistant strains were detected. Mycobacteria other than M tuberculosis were iso¬ lated from 10 patients with suspected TB (Table 3). intracellulare was most commonly Mycobacterium found, in three cases including one from an AIDS patient. Five of the patients infected with atypical
shown in six and three cases, respectively. Altogether nine of the 24 patients (38%) with previous treatment were infected by resistant strains. Initial drug resistance was seen in four of 58 and ethambutol
strains
was
patients (7%). Thus, previous anti-TB drug therapy was
strongly associated with drug-resistant disease (p
