Rev Cubana Med Trop 1996;48(2)

Departament of Micobacteriology, Pasteur Institute, Tehran - Iran

Reporte de la resistencia a las drogas en cepas de Mycobacterium tuberculosis aisladas de pacientes en Irán

Dr. A.R. BAHRMAND, Dr. A. SIADATI, Dr. G SAMAR y Dr. A. SANAMI

RESUMEN

Se estudiaron 6 472 muestras clínicas de pacientes con sospecha de tuberculosis entre marzo de 1993 a marzo de 1994. Se obtuvieron resultados positivos en 443 pacientes; 238 correspondieron al sexo femenino (53,7 %) y 205 (46,3 %) al masculino, predominó el grupo de edad entre 30 y 39 años (31,5 %). La prueba cutánea de sensibilidad al derivado proteico purificado (PPD) fue positiva en 178 pacientes con un rango de 10-14 mm. Se encontraron imágenes radiológicas anormales en 222 pacientes (50,1 %). Se detectó mayor frecuencia de resistencia en las cepas de Mycobacterium tuberculosis en casos con tuberculosis pulmonar. Cuarenta y dos cepas (9,5 %) fueron resistentes a la isoniacida y 31 (7,0 %) a la estreptomicina. Se registró resistencia a 1 droga en 25 aislamientos (5,4 %). Pocas cepas (1,3 %) resultaron resistentes a 3 drogas y 1 de ellas a 5 drogas. Los datos clínicos y epidemiológicos sugieren que la resistencia a las drogas en la tuberculosis comienza a ser un problema importante en la región. El diagnóstico rápido de esta infección y el uso de antibióticos con un espectro reducido puede facilitar el control de esta forma de tuberculosis.

Palabras clave: MYCOBACTERIUM TUBERCULOSIS/aislamiento & purificación; RESISTENCIA MICROBIANA A LAS DROGAS; ISONIACIDA; ESTREPTOMICINA; TUBERCULOSIS PULMONAR/radiología; IRAN.

INTRODUCTION

Tuberculosis (TB) is considered as one of the most serious infectious disease, especially in developing countries. It depends on socio-economical situation, inappropriate funding TB control programmes, etc.^1,2 The pandemic of AIDS has seriously endangered the infectious potential of mycobacteria in United States and Africa.^1-3 A high incidence of disease is observed in Asia. Up to five million people acquiring this disease annually or 2/3 of all cases in the world are residing in the countries of this continent.⁴

In spite of the significant efforts in the control of infection TB remains to be an important problem in Iran and other countries of the region.^5,6 The disease is not distributed uniformly throughout the country and incidence rates are strongly associated with geographical origin. According to the survey of 1984-85 and annual investigations of Pasteur Institute of Iran, pulmonary TB is predominantly spreaded in Tehran, Sistan, Hamedan, and Khorasan provinces (Report on Communicable Disease Control).

At present, chemotherapy is the most effective method for the treatment and control of TB worldwide. However, the growing emergence and spreading of drug-resistant form of disease have caused a great concern. Correct diagnosis of these infections is the only way to prevent ineffective use of narrow-spectrum antibiotics. Such issue have also increased the importance of epidemiological survey on drug-resistant Mycobacterium tuberculosis (MTB) infections.

The aim of this report is to present a cumulative data on TB spreading in the regional population and its drug susceptibility in order to strengthen the control of resistant forms of disease.

MATERIALS AND METHODS

PATIENTS AND SAMPLES

Between March 1993 and March 1994, 6 472 patients suspected of primary tuberculosis were referred to Mycobacteriology Department of Pasteur Institute of Iran with their clinical and radiological data and PPD skin tests. The majority of cases were from Tehran region. Samples were collected from sputum, body tissues, blood, bronchoalveolar lavage, urine and other sources.

SPECIMEN PROCESSING

Smears were made and examined by fluorescence stain for preliminary screening and confirmed by Kinyon staining technique.⁷ The samples were decontaminated in accord with laurylsulphate method, the concentrated and cultured on the two slopes of Lowenstein-Jensen medium (LJ) containing pyruvate or glycerol in an atmosphere of 5 % CO₂ at 35 °C for 8 weeks. Positive cultures were identified in accord with CDC recommended protocols⁷ using standard biochemical tests including production of niacin, catalase activity, nitrate reduction, arylsulfatase activity, pigment production, growth rate. Tween 80 hydrolysis (Kilborn et al., 1973; Wayne, 1964), and sodium chloride test.

The anti-microbial drug susceptibility tests (AMST) of the isolated organisms were performed with conventional anti-tubercle drugs such as rifampin (RMP) - 40 mg/L, isoniazid (INH) -0,2 mg/L, ethambutol (EMB) - 2 mg/L, ethionamide (ETH) - 20 mg/L, streptomycin (SM) -4 mg/L, and kanamycin (K) - 20 mg/L using proportional method.⁸

RESULTS

MTB isolates were recovered from 443 (6,4 %) samples Simultaneously, 82 isolates (1,2 %) of atypical non-tuberculous mycobacteria were identified (results will be published else-where). Of the 443 patients, 238 (53,7 %) were female and 205 (46,3 %) were male. The predominant age group of infection was 30-39 years old (138 cases - 31,2 %). However, signi-ficant number of cases has been registered for the age from 20 to 59 years old for both sexes: 20-29 years old group (74 cases) and 50-59 years old (91 cases).

Houseviwes (123), clerks (41), workers (34) and farmers (24) have covered in sum the majority of cases, when patients were grouped in accordance with their occupation. However, the rate of infection was highest in housewives (27,8 %): students (1,1 %) and servicemen (1,1 %) were among the groups with lowest incidence of disease. Patients were mostly from Tehran region, that explained the predominancy among the positive cases (287 - 64,8 %).

243 positive cultures were isolated from sputum (54,8 %), 59 - from gastric washing (13,3 %), 45 - from bronchial lavage (10,1 %), 26 - from lymph nodes (5,9 %), and rest - from other sources (figure 2). Lowest number of positive samples were found in sterile body fluids, i.e., cerebrospinal fluid (11 cases - 2,5 %), pericardial fluid (4 cases - 0,9 %), pleural fluid (9 cases - 2,0 %) and bone marrow aspiration (2 cases - 0,45 %). Pulmonary TB was diagnosed in 314 (70,9 %) and extra-pulmonary - in 129 (29,1 %) cases.

PPD skin tests with indurations of 10-14 mm or more were common for majority of TB infected patients (271 results - 61,2 %). However, 145 (32,7 %) of the patients have demonstrated negative tuberculin reactivity (figure 3). Tuberculin test was positive in 215 (68,5 %) patients with pulmonary and 71 (55,0 %) with extrapulmonary disease.

The chest roentgenograms were abnormal in 222 (50,1 %) patients and radiological data revealed infiltration in 62 (14,0 %), turbidity in 53 (12,0 %), nodules in 19 (4,3 %), cavitation in 17 (3,8 %), calcification in 20 (4,5 %), caverns in 11 (2,5 %) cases, and other pulmonary abnormalities.

The results of AMST for isolated strains are shown in table. The drug susceptibility tests demonstrated substantial resistance of 42 to INH (9,5 %) followed by SM - 31 isolates (7,0 %), RMP - 15 isolates (3,4 %) and EMB - 10 isolates (2,3 %). The resistance was greatly correlated with the origin of organisms. Of the strains isolated from patients with pulmonary TB 22 (7,0 %) were resistant at least to one drug, while strains from extra-pulmonary form of disease had a limited resistant range. Only one strain (0,8 %) resistant to RMP was isolated from non-pulmonary patients compared to 14 (4,5 %) isolates from pulmonary TB.

TABLE. Drug resistance of MTB isolated from patients with pulmonary and non-pulmonary TB

	Pulmonary (314 cases)		Non-pulmonary (129 cases)		Total (443 cases)
Drug	No.	%	No.	%	No.	%
INH	36	11,5	6	4,6	42	9,5
SM	26	8,3	5	3,9	31	7,0
RMP	14	4,4	1	0,8	15	3,4
EMB	10	3,2	0	0,0	10	2,3

The majority of the drug-resistant strains-25 from 42 (59,5 %) were resistant to one drug. However, significant part (16 strains) of the isolates were shown to be resistant to 2-4 drugs used. It was also found that one MTB isolate demonstrated resistance to five drugs tested, including kanamycin.

DISCUSSION

TB is caused by several mycobacterial species, such as MTB, Mycobacterium bovis or Mycobacterium africanum. It is a disease of world-wide importance and major health problem, particularly in developing countries.^1,2,9 According to WHO report of June 1994, mortality caused by TB is currently increasing in countries of Eastern Europe and the former Soviet Union. This increase follows a steady decline in death rate over nearly 40 years and is most marked in large cities.¹⁰ TB is also responsible for more than 80 % of all cases of communicable diseases notified in the South Africa and can be regarded as one of the most serious health problems affecting this country.¹¹ Over 3,9 million new cases are expected annually in South East Asia, 2,3 million in West Pacific region, and 2,1 million in Sub-Saharan Africa by 2000.^12,13

Demographic factor has greatly contributed to the rise in death rate due to TB. The age- -specific incidence for the years 1990-2000 is predicted as follows: 70 % of new cases are aged between 15 and 59 years old, 20 % are prognosed for group of 60 and more years old and 10 % - under 15 years old.¹² According to the results of our study of the referred patients 443 (mostly in the age range from 20 to 59 years old) were positive for MTB. We have also found that housewives, clerks, workers, and farmers belong to occupational groups in which highest rate of disease was detected.

The pulmonary TB was diagnosed to be the predominant form of TB in patients. We have worked out in detail the situation for Tehran region when we demonstrated that 70,9 % of all positive cases were related to pulmonary form of infection. PPD tuberculin test was positive for more than 2/3 of all patients. However, about 1/3 of them had negative PPD reactivity. These findings have emphasized the fact that a negative tuberculin skin reaction and X-ray evidence do not exclude active disease.

At present TB is one of the most cost- -effective disease. However, increasing drug resistance threatens to make treatment of TB an expensive and difficult for future generations.^1,2 Current therapy for TB is based on multidrug treatment, predominantly with INH, SM, RMP, and EMB for period of several months. However, a number of reports about multidrug- -resistant TB (MDR-TB) arising in recent years have alerted physicians in the different regions of the world.^11,14 Furthermore, large outbreaks of MDR-TB occured during last years gave evidences that TB may come to uncontrollable state using currently available therapy.

The prevalence of drug resistant MTB in patients with pulmonary forms of disease in the United States has steadily increased from appr. 2 % to 9 % in the past three decades and similar finding have occured in many other countries.¹⁴ Rate of the resistance was found to be higher in developing countries.¹⁵ In the 20-years period from 1970 to 1990, 5 TB serious outbreaks involving strains of MTB resistant to two or more drugs (predominantly INH, SM) were reported.¹⁶ In our study primary resistance was more frequent to INH (9,5 % of isolates) followed by SM (7,0 %), RMP (3,4 %) and EMB (2,3 %). These data are very close to results published recently for Istanbul, except for the highest resistance to SM (21,9 %) found by the authors.¹⁷

Until recently the resistance to RMP has been relatively infrequent. During the period of 1982-1986 resistance to this drug was found in 0,6 % of strains from previously untreated patients and in 3,3 % of strains from treated patients in the United States. Our data on the substantial level of primary resistance of MTB strains to RMP (3,4 %) suggest an increase of TB resistance to this agent in the region.

In a survey of new TB cases reported to CDC during the first quarter of 1991, 13,3 % of them were resistant to at least one anti-TB drug and 3 % - to both INH and RMP, the two, most effective drugs presently available for TB treatment.^11,18 Our data are different in part showing that one drug resistant strains encompassed 5-6 % of all isolates. However, 3,6 % of the organisms were resistant to 2-4 drugs used, that is in agreement with the above demonstrations. The important differences were noted between drug susceptibility of our isolates from pulmonary site of disease and organisms from other sites. Resistance of strains to RMP and EMB was predominantly due to isolates from patients with pulmonary TB.

This epidemiologic study presented cumulative information on the incidence of TB, including its drug-resistant form, in the Tehran province. It can provide proper recommendation for the treatment of patients with drug-susceptible TB in order to prevent development of drug- -resistant disease. Effective initial antituberculosis therapy regimens may include 5-drug combination of INH, SM, EMB, RMP, and K. Screening and preventive therapy among persons at risk for MDR-TB should be implemented.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. V. Bakaev for his constructive review of the manuscript, Mr. N. Nouroozi, Mr. M. Yaghli, Mrs. Argang, Mrs. Khardoosh, and Mrs. Azad for their excellent technical assitance. The help of Ms. F. Farahtaj and Ms. Khalilzadeh in preparation of the manuscript is highly appreciated.

SUMMARY

6 472 clinical samples of patients with tuberculosis suspicion between March, 1993 and March, 1994, were studied. Positive results were obtained in 443 patients; 238 females (53,7 %) and 205 males (46,3 %). The predominant age group was that between 30 and 39 years (31,5 %). The cutaneous test of sensitivity to the purified protein derivate (PPD) was positive in 178 patients with a range of 10-14 mm. Abnormal radiological images were found in 222 patients (50,1 %). Higher resistance frequency was detected in Mycobaterium tuberculosis strains among cases suffering from pulmonary tuberculosis. 42 (9,5 %) strains were resistant to isoniazid and 31 (7,0 %) to streptomycin. Resitance to one drug was observed in 25 isolations (5,4 %). A few strains (1,3 %) were resistant to 3 drugs, and 1 of them to 5 drugs. Clinical and epidemiological data suggest that resistance to drugs in tuberculosis is becoming an important problem in the region. The fast diagnosis of this infection and the use of antibiotics with a reduced spectrum may enable the control of this form of tuberculosis.

Key words: MYCOBACTERIUM TUBERCULOSIS/isolation & purification; DRUG RESISTANCE; MICROBIAL; ISONIAZID; STREPTOMYCIN; TUBERCULOSIS; PULMONARY/radiology; IRAN.

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Recibido: 10 de agosto de 1995. Aprobado: 11 de noviembre de 1995.

Dr. A. R. Bahrmand. Micobacteriology Dept, Pasteur Institute of Iran. Pasteur Avenue, Tehran 13164, Islamic Republic of Iran.