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Comparative Study on the Use of Solid Media: Lowenstein–Jensen and Ogawa in the Determination of Anti-Tuberculosis Drug Suscep

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Comparative Study on the Use of Solid Media: Lowenstein–Jensen and Ogawa in the Determination of Anti-Tuberculosis Drug Suscep Tuberculosis (2002) 82(2/3), 63 -- 67 & 2002 Elsevier Science Ltd. All rights reserved doi:10.1054/tube.323, available online at http://www.idealibrary.com on Comparative study on the use of solid media: Lo¨wenstein–Jensen and Ogawa in the determination of anti- tuberculosis drug susceptibility S.Tanoue, S. Mitarai, H. Shishido Department of Respiratory Diseases,National Tokyo Hospital, 3-1-1Takeoka,Kiyose,Tokyo 204-5858, Japan Summary A total of 53 Mycobacterium tuberculosis strains were examined to evaluate the consistency of susceptibility tests using two solid egg-based culture media,Ogawa and L˛wenstein--Jensen (L--J), in order to assessthe inter-media differencesin susceptibility tests foranti-tuberculosis drugs.Fifty-three M. tuberculosis strainswere tested for resistance against isoniazid, rifampicin, ethambutol, streptomycin and other alternative drugs on L--J and Ogawa media.Data from isoniazid (INH) revealed 31 strainswere susceptibleand11strainswereresistant in L--J medium.Onthe other hand, one strain showedresistanceagainst INH onlyon Ogawamedium.Thisgaveaconsistencyratio of 97.7% for INHbetweenthesetwomedia.Similarly,themethodshad100% compatibility with rifampicin, 86.0% with ethambutol and 88.4% with streptomycin.When 41of these strainswere tested against ethionamide, enviomycin, para-aminosalicylate, sparfloxacin, andlevofloxacin, the same numbers of strains were shown to be resistant or susceptible to L--J and Ogawa media. Similarly, the consistency ratio was 75.6% for kanamycin and 61.0% for capreomycin.There are inconsistencies between results obtained with L--J and Ogawa media,Ogawa medium had a tendency to produce results that indicated more resistant strains.However, the differenceswere statistically negligible. & 2002 Elsevier Science Ltd. Allrights reserved INTRODUCTION for agar plates is increasingly becoming the standard for determining M. tuberculosis resistance in western coun- Mycobacterium tuberculosis infection is re-emerging in tries. However, this technology has not been introduced most parts of the world and recently the World Health in most of the developing countries in Asia and sub- Organization (WHO) has sent a stern warning to its Saharan Africa because it is relatively easy to use the members on the problems associated with the treatment conventional egg-based media for initial isolation and of tuberculosis.1 Specifically, WHO warns of the impact of consecutive susceptibility testing. Although susceptibility drug-resistant M. tuberculosis. In this situation, even the testing using egg-based media is popular in many diagnosis of tuberculosis with smear-positive specimens countries, the methods used vary widely. Because of and subsequent directly observed treatment with short- increasing international communication, the comparabil- course chemotherapy (DOTS) are not enough to control ity of susceptibility results from different methods will the disease. Thus, drug susceptibility testing for resistant become essential and will be crucial in understanding the M. tuberculosis is important in designing subsequent real trend of resistant M. tuberculosis in the world. treatment options for such patients. However, there are The present study was conducted to evaluate the currently several methods for susceptibility testing, i.e. compatibility of M. tuberculosis resistance results the absolute method, resistant ratio method and propor- obtained using Lo¨ wenstein–Jensen and Ogawa media. tion method with a variety of culture media.2 Recently, the proportion method using artificial culture media like Middlebrook 7H9 for the BACTEC/MGIT system or 7H10 MATERIALS AND METHODS Isolation of M. tuberculosis Correspondence to: Satoshi Mitarai,The Research Institute of Tuberculosis, M. tuberculosis strains were obtained from pulmonary Japan Anti-Tuberculosis Association, 3-1-24 Kiyose,Tokyo 203-8533, Japan. Tel.:+81-424-93-3090;Fax:+81-424-92-8258;E-mail:[email protected] tuberculosis patients admitted to the National Tokyo Hospital and National Hiroshima Hospital (51 and 2 Accepted: 14 February 2002 strains, respectively) between July 1996 and January 1997. 63 64 Tanoue et al. All the strains were recovered from sputum specimens Ta ble 1 Critical concentrations of anti-tuberculosis drugsin the proportion method collected after informed consent. The initial isolates were identified as M. tuberculosis complex using Ziehl–Neelsen Drug Concentration Drug Concentration (Z–N) staining, niacin accumulation tests and the DNA– (mg/ml) (mg/ml) DNA hybridization (DDH) method (Mycobacterium, Isoniazid 0.2 Enviomycin 20 Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Rifampicin 40 p-Aminosalicylate 0.5 Streptomycin 4 Capreomycin 20 Japan). A total of 53 strains of M. tuberculosis showing Kanamycin 20 Cycloserin 30 complete drug resistance to at least one of the four first Ethambutol 2 Levofloxacin 1 line drugs, i.e. isoniazid (INH), rifampicin (RFP), etham- Ethionamide 20 Sparfloxacin 1 butol (EB), and streptomycin (SM), was obtained. The absolute concentration method on 1% Ogawa media was Ta ble 2 Composition of L˛wenstein-Jensen and Ogawa media used at each laboratory for initial susceptibility testing. Thereafter, the drug susceptibilities were confirmed with Amount the proportion method in the present study. Ingredient 1% Ogawa medium L--J medium Potassium phosphate 1.0 g 1.2 g Drug susceptibility test Sodium glutamate 1.0 g F Magnesium sulphate F 0.12 g F The susceptibilities of all 53 resistant isolates of M. Magnesium citrate 0.3 g L-Asparagine F 1.8 g tuberculosis were examined against several anti-tubercu- Distilled water 100 ml 300 ml losis drugs for data confirmation and comparison. These Egghomogenate 200 ml 500 ml drugs were INH, RFP, EB, SM, kanamycin (KM), ethiona- Glycerol 6.0 ml 6.0 ml 2%Malachitegreensolution 6.0ml 10ml mide (TH), enviomycin (EVM), cycloserin (CS), capreomy- Inspissation temperature 901C/60 min 901C/45 min cin (CPM), para-aminosalicylate (PAS), levofloxacin Expected pH 6.5 6.9 (LVFX) and sparfloxacin (SPFX). The critical concentration of each drug used in the resistance assays is shown in Table 1. They were determined using the standard growth of tuberculosis bacilli was inspected in the MIC concentrations in use on the L–J medium in western tubes. For quality control, a standard susceptible M. countries, clinical experience and treatment effects.2,3 tuberculosis strain was tested in the same experiments. INH, RFP, EB, and SM were stipulated as first-line drugs for the treatment of tuberculosis. The proportion method Consistency of two different methods was used to evaluate resistances against anti-tuberculosis drugs. The drug susceptibility results of 53 strains examined The cultured M. tuberculosis strain was harvested and using L–J and Ogawa media were compared and grouped suspended in distilled water at McFarland turbidity No. 1. into two different categories, i.e. consistent and incon- The original bacterial suspension was further diluted sistent, according to the results. The consistency and the 100 Â and 10,000 Â . One hundred microlitres of each of MIC of each drug with these media were evaluated. the two suspensions were inoculated onto each drug- Conventionally, the results with the L–J medium were containing medium. If the number of colonies on the stipulated as the standard. drug-containing medium was more than that on another plain medium with a 100 Â diluted suspension of the inoculum, the strain was judged to be 1% or more Statistical analysis resistant to the drug. L–J and 1% Ogawa media were used Statistical analysis was done by chi-square or Fisher’s for the tests. A summary of the ingredients in each exact test for consistency results. Parametric (two-way medium is shown in Table 2. analysis of variance) and non-parametric (Wilcoxon) The minimum inhibitory concentration (MIC) of each analytic methods were used for MIC results. A p-value drug was determined using commercially prepared drug- of o0.05 was considered significant. containing medium (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan). MIC is the minimum drug concentration that inhibits the growth of the tested RESULTS strain. Approximately 100,000 cfu of resistant tuberculo- Confirmation of drug resistance sis bacilli were inoculated onto the control medium and the medium of a two-fold dilution series of each drug The 53 strains that were judged as having no resistance in ranging from 0.08 to 100 mg/ml. When the control tubes initial testing were confirmed with the proportion method showed sufficient growth (basically 3–4 weeks), the in newly designated concentrations as shown in Table 1. Tuberculosis (2002) 82(2/3),63--67 & 2002 Elsevier Science Ltd. All rights reserved Solid media for anti-tuberculosis susceptibility tests 65 Ta bl e 3 M. tuberculosis susceptibility comparisons using L--J and Ogawa media Consistent Inconsistent Susceptible Resistant Ratio (%) Resistant Resistant on Ogawa on L--J INH 31 11 97.7 1 0 RFP 26 17 100 0 0 EB 31 6 86.0 6 0 SM 10 28 88.4 5 0 KM 24 7 75.6 10 0 TH 29 12 100 0 0 EVM 32 9 100 0 0 PAS 2 9 12 10 0 0 0 CPM 12 13 61.0 16 0 CS 35 4 95.1 0 2 SPFX 40 1 100 0 0 LVFX 38 3 100 0 0 Note:Forty-three and 41strains were examined for first-line and other drugs, respectively. 100 100 Isoniazid (n=25) Rifampicin (n=28) 80 80 Ogawa 60 L-J 60 L-J d 40 40 Ogawa 20 20 0 0 0.08 0.16 0.32 0.64 1.25 2.5 5 10≤ (µg/ml) 0.78 1.56 3.13 6.25 12.5 25 50 100≤ (µg/ml) t of strains inhibite 100 100 Ethambutol (n=17) Streptomycin (n=27) 80 80 60 L-J 60 L-J Cumlative percen 40 40 Ogawa Ogawa 20 20 0 0 0.08 0.16 0.32 0.64 1.25 2.5 5 10≤ (µg/ml) 0.78 1.56 3.13 6.25 12.5 25 50 100≤ (µg/ml) Fig. 1 MICs of first-line drugs against M.
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