Background: Direct sensitivity test either by sputum concentrate (DS) or swab method (DSM) set up along with the

primary culture would avoid the delay of four or more weeks required for the indirect test. A comparison of these two

methods against the standard indirect sensitivity method under routine laboratory conditions is necessary to prove their

merit.

Method: Smear positive sputum samples were aliquoted and sensitivity tests were set up by both the direct methods as also

an indirect test set up from the primary culture of the same sample.

Results: The agreement with the indirect test results for isoniazid (INH) ranged from 97-98% for the DS method and 93-

97% for the DSM method. The corresponding figures were 96-98% by the DS and 94-99% by the DSM method for

rifampicin (R). The agreement was less satisfactory for ethambutol (Emb).

Conclusion: This study showed that direct sensitivity tests such as DS and DSM methods can detect most of the cultures

resistant to INH and R (MDR) from the time growth appears on the primary culture , even as early as the second week of

setting up the tests

Dastageer, Aszhgar

Mathew, Sarah

Nalini, S M

Paramasivan, C N

Rahman, Fathima

Sundaram, V

NIRT Institutional Repository

Indian Journal of TuberculosisSIMPLE DIRECT DRUG SUSCEPTIBILITY TESTS ON SPUTUM SAMPLES FOREARLY DETECTION OF RESISTANCE IN TUBERCLE BACILLIOriginal ArticleSarah Mathew, S. M. Nalini, Fathima Rahman, Aszhgar Dastageer, V. Sundaram and C. N. Paramasivan*(Original article received on 6.3.2006;  Revised on 6.3.2007; Accepted on 27.7.2007)SummaryBackground: Direct sensitivity test either by sputum concentrate (DS) or swab method (DSM) set up along with theprimary culture would avoid the delay of four or more weeks required for the indirect test.  A comparison of these twomethods against the standard indirect sensitivity method under routine laboratory conditions is necessary to prove theirmerit.Method: Smear positive sputum samples were aliquoted and sensitivity tests were set up by both the direct methods as alsoan indirect test set up from the primary culture of the same sample.Results: The agreement with the indirect test results for isoniazid (INH) ranged from 97-98% for the DS method and 93-97% for the DSM method. The corresponding figures were 96-98% by the DS and 94-99% by the DSM method forrifampicin (R). The agreement was less satisfactory for ethambutol (Emb).Conclusion: This study showed that direct sensitivity tests such as DS and DSM methods can detect most of the culturesresistant to INH and R (MDR)  from the time growth appears on the primary culture , even as early as the  second week ofsetting up the tests.Key words:   M. tuberculosis, Direct tests, Measuring resistance[Indian J Tuberc 2007; 54:184-189]Tuberculosis Research Centre, Indian Council of Medical Research, Chetput, Chennai-600 031*Correspondence: Dr.C.N.Paramasivan, Foundation for Innovative New Diagnostics, 71.av. Louis Casai, P.O. Box 93, CH-1216 Cointrin/Geneva(Switzerland); E-mail: <cn.paramasivan@finddiagnostics.org>INTRODUCTIONAs early as 1969 and 1970, results based ondirect sensitivity tests using a concentrated depositof sputum decontaminated by Petroff’s method(DS), as also by sputum swabs, decontaminated usingcetrimide (DSM) were published 1,2. However, thesemethods had only academic interest during thatperiod, since treatment for TB was not based ondrug susceptibility results. Simple and time savingmethods are needed today, particularly for previouslytreated, smear-positive patients whose continuedtreatment should ideally be based on their drugsusceptibility pattern.Though acceptable definitions emerged forresistance to isoniazid (INH), streptomycin (S) andrifampicin (R) using the two direct sensitivity testsmethods, swab direct sensitivity tests for R andethambutol (Emb) were yet to be standardized 3.In the present study, direct sensitivity testingfor INH, R and Emb by swab (DSM) andconcentration (DS) culture methods were comparedagainst the indirect test, which is the gold standard.MATERIAL AND METHODSSpecimens:  Smear positive sputum samplesobtained from 151 patients attending the Centre’sclinic irrespective of their treatment status formedthe study material. To each of the sputum samples,few sterile glass beads were added to homogenize itand the contents were divided into two aliquots of3-5 ml each for culture by Petroff’s method andswab culture method respectively 4, 5. Smear examination: Sputum smears wereexamined by fluorescent microscopy and graded as3+, 2+ or 1+; the least grade consisted of 4 or morebut less than 100 bacilli in the entire field examinedat high power magnification6. Medium:  Lowenstein-Jensen (L-J) medium wasIndian Journal of Tuberculosisused for culture and drug susceptibility testing 7.  Forthe standard indirect test, the concentrations of drugsused were 0.2, 1.0 and 5.0 mg/l of INH; 32, 64 and128 mg/l of rifampicin; and 2, 4 and 8 mg/l of Emb.For both the direct sensitivity tests, 0.2 mg/l of INH;40 and 64 mg/l of R (R40, R64) and 4 mg/l of Emb(E) were used.  In addition, L-J medium containing500 mg/l of para nitro benzoic acid (PNB) was usedwith all three drug susceptibility methods as anidentification test for M. tuberculosis.8Indirect sensitivity tests:  Deposit from the aliquotsof sputum samples treated by Petroff’s method wasinoculated onto a pair of L-J media. As and whensufficient growth was observed, an indirectsensitivity test was set up for INH, R and Emb, andalso for the control strain M.tuberculosis H37RVusing the standard method4,8. The minimal inhibitoryconcentration (MIC) was determined at the end offour weeks.Direct Sensitivity Test (DS):  The sputum depositsobtained by Petroff’s method were inoculated witha 5 mm loop (27 SWG) onto a pair of plain L-J anddrug containing L-J media (INH 0.2; R-40, R-64and Emb4) and one slope of PNB.  The slopes wereincubated and examined weekly for 8 weeks and thegrowth was recorded each week.Direct Swab Sensitivity Test (DSM):  Swab culturewas set up from the remaining aliquot of sputum 2.In brief, 11 sterile moistened swabs were dipped inthe sputum and gently rotated and transferred to asmany test tubes half filled with 1% sterile cetrimidesolution.  After one hour, each swab was carefullydrained of excess fluid and smeared over the surfaceof 2 slopes of plain L-J and drug containing L-J Media(INH 0.2; R40, R64 and Emb4) and one slope ofPNB.  The slopes were incubated and examinedweekly for 8 weeks and the level of growth wasrecorded.Thus, the results for the direct tests DS andDSM were available from the time growth was firstrecorded on the plain L-J slopes.  For all tests, growthon L-J was graded as 3+ if confluent, as 2+ if therewere more than 100 discrete colonies, as 1+ for 20-99 colonies, and the actual count if there were lessthan 20 colonies.  The two direct tests were identifiedby different series of numbers and read by twoindependent readers to avoid bias. The concentrationculture and indirect tests were read by a third readerwho had no access to the results of the direct tests,thereby avoiding giving subjective results.Interpretation of testsIndirect tests: Resistance was defined as MIC(based on a 20 colony end point) of 1 mg/l or morefor INH, 128 mg/l or more for R, and 8 mg/l ormore for Emb.Direct tests (DS and DSM):  The definition ofresistance was based on the amount of growth seenon the drug-free medium as already reported.1,2,3Thus, when the growth on the drug-free mediumwas 2+ or more, growth of 1+ or more on the drug-containing medium was defined as resistance to thedrug.  When growth on the drug-free medium was1+ or less (i.e. < 100 colonies), any growth on thedrug-containing medium was considered to be anindication of resistance to that particular drug. Forthis purpose, the higher growth observed on thepaired slopes was considered for interpretation.Cultures were classified as sensitive orresistant to the drugs INH, R and Emb by the indirectand direct tests (DS and DSM), depending on theirrespective definitions.Analysis: The disagreements, if any, were lookedinto to determine their statistical significance usingthe McNemar test and to decide the optimum weekfor correct interpretation of the results.  The twodirect tests were compared together as well as withthe indirect test to assess their relative merit.RESULTSThe DS test was set for 118 smear positivesamples among which 10 were negative on culture(all 1+ smear grades) and 8 were contaminated onthe drug-free L-J medium.  Thus, 100 samplesremained in the analysis.  Of these, 11 werecontaminated on the INH medium, one each on theR40 and R64 medium and 9 on the Emb medium.SARAH  MATHEW  ET  AL 185Indian Journal of TuberculosisThe comparison with the indirect test was thereforeavailable for 89 tests on INH, 99 on R and 91 onEmb.Sensitivity tests for INH by DS wereavailable for 64 (72%) samples at 2 weeks, 76 (85%)at 3 weeks and 84 (94%) at 4 weeks.  Thecomparison of the results by DS with that obtainedwith the indirect test is tabulated based on readingstaken at 2, 3 and 4 weeks (Table 1).  When INH wasanalyzed, the extent of agreement was 97% at 2weeks, 99% at 3 weeks and 98% at 4 weeks. A totalof 29 out of 30 resistant samples were detected byDS.  The little disagreement there showed nostatistical significance to suggest a trend in any onedirection. Thus, results available by DS were highlyreliable as early as the second week of growth.Since R40 and R64 gave similar results forR, R64 alone is tabulated, since it is the criticalconcentration for determining resistance by theindirect test method.  The agreement between thetwo tests was 96% at 2 weeks and 98% at 3 and 4weeks. In the following weeks, the disagreementswere fewer.  By week 6, 25 out of 26 resistant strainswere detected by DS (not tabulated). At 2 weeks, 3resistant cultures were read as sensitive but nonethe other way.The DS test for Emb showed 94%agreement with the indirect test at 2 weeks, 88% at3 weeks and 92% at 4 weeks.  At 2 weeks, 4 resistantcultures were classified as sensitive.  At 3 weeks,the disagreements involved six resistant and threesensitive strains.  From week 4, the disagreementswere greater both in sensitive and resistant strains.Direct sensitivity test by the swab method (DSM):Swab cultures and sensitivity tests were set up fromall 151 smear-positive samples.  Among them, 33were excluded from the analyses, since they did nothave results either by one or both of the methods.Of the remaining sample, 24 showed no result byDSM (17 negative and 7 contaminated), 23 showedno result by DS (10 negative and 13 contaminated)and 14 among them were common to both methods.Among the remaining 118 tests, Emb medium wascontaminated for one sample.  The comparison ofthe classification by DSM and the indirect tests arepresented in Table 2. Identical classification obtained with DS test INH n = 89 R 64 n =99 Emb n= 91 Classification based on indirect test 2W 3W 4W 2W 3W 4W 2W 3W 4W Sensitive  48 50 55 57 63 70 58 62 71 Resistant  14 25 27 11 19 22 2 6 8 Per cent Agreement  97 99 98 96 98 98 94 88 92 Results available  64 76 84 71 84 94 64 77 86  Identical classification obtained with DSM test INH n = 118 R 64 n =118 Emb n= 117 Classification based on indirect test 2W 3W 4W 2W 3W 4W 2W 3W 4W Sensitive 47 66 71 53 75 81 58 82 90 Resistant 15 33 37 9 22 28 3 10 14 Per cent Agreement 93 97 96 91 95 97 91 91 94 Results available 67 102 112 68 102 112 67 101 111 Table 1:  Comparison of Direct Concentration Method (DS) with indirect sensitivity test resultsTable 2:  Comparison of Direct Swab Method (DSM) with indirect sensitivity test resultsDIRECT  DRUG  SUSCEPTIBILITY  TESTS  FOR  M. TUBERCULOSIS186Indian Journal of TuberculosisThe number of samples with results for INHwas 67 (56%), 102 (86%) and 112 (95%) at 2, 3and 4 weeks respectively. The extent of agreementseen for INH was 93% at 2 weeks, 97% at 3 weeksand 96% at 4 weeks.  At 2 weeks, 4 resistant cultureswere classified as sensitive by DSM and one sensitiveas resistant by DSM.  These differences, however,were not statistically significant from the third weekonwards, when the disagreements were fewer anda total of 41 out of 43 INH-resistant strains weredetected using this method.The rifampicin test based on R64 showed91% agreement at 2 weeks, 95% at 3 weeks and97% at 4 weeks.  At 2 and 3 weeks, 5 resistantstrains were classified as sensitive by DSM but nosensitive culture appeared to be resistant.  Thisdifference was statistically significant (P<0.02).  Inthe following weeks the disagreements were fewerand not significant.  At sixth weeks, 33 out of 34 R-resistant strains were detected by DSM.Agreement seen for Emb with the indirecttest was 91% at 2 and 3 weeks and 94% at 4 weeks.Misclassification by the DSM method occurred with6 resistant strains at 2 weeks, 8 at 3 weeks and 6 at4 weeks, with only one sensitive culture beingclassified as resistant at 3 and 4 weeks.  Again, thesedifferences were statistically significant (P<0.05 at2 and 3 weeks), showing that the DSM method underread resistance in the initial weeks.  Results nottabulated showed that the trend was reversed in thelater weeks probably as a result of deterioration ofthe drug in the medium with prolonged incubation.Though 19 out of 23 Emb-resistant strains weredetected at 7 weeks by DSM, the method was notextremely reliable at any time during the 8 weeks.Further analyses were undertaken tocompare the two direct tests, DS and DSM, witheach other as well as against the indirect test, basedon readings at four weeks for all three drugs (Table3).  The number of cultures with results by all threemethods was 78 for INH, 85 for R and 77 for Emb.The extent of agreement among the three tests interms of classifying cultures as sensitive or resistantwas as high as 95% for INH, 94% for R and 90%for Emb.  One culture which was sensitive to INHwas classified as resistant by both the two directtests, though both were based on 3+ growths on thedrug-free medium. One sensitive and one resistantculture to Emb were misclassified by both the directtests.  Rifampicin showed complete agreement byall three methods.Analyses not tabulated have shown that themean time for culture positivity at 2-3 weeks wassimilar when processed by the concentration or bythe swab method.  However, the grades of positivitywere considerably higher with the concentrationmethod; growth of 2+ or more was seen in 87% ofcultures by the concentration method compared to55% by the swab method.  The number of cultureswith less than 20 colonies was 3 by the concentrationmethod and 22 by the swab method.  However, thequantitative difference in growth did not affect theagreement in classification of strains when comparedto the indirect sensitivity test.Table 3: Correlation of identical results obtained by direct tests (DS & DSM) with results of theindirect testSARAH  MATHEW  ET  AL 187Indian Journal of TuberculosisAll cultures in the analysis were identifiedas strains of M. tuberculosis based on theirsusceptibility to PNB and positive Niacin production.DISCUSSIONSimple, inexpensive speedier drugsusceptibility test results in resource poor settings,are urgently needed to control the spread of MDR-TB. In this study, two methods of direct drugsusceptibility tests (DS and DSM) yielded 85% ofthe sensitivity results from the third week. The levelof agreement with the indirect sensitivity test was99% and 97% for INH, 98% and 95% for R and88% and 91% for Emb by the DS and DSM methodsrespectively. For INH and R, the agreement was 98.4and 99.4 respectively. There was no false report ofresistance at two weeks for any drug by eithermethod. Analyses of disagreement seen beyond 2weeks in INH and R were statistically non-significant.With incubation beyond 4 weeks, a few false resistantresults occurred with Emb. This could perhaps beattributed to the deterioration of the drug.Rifampicin susceptibility was assessed basedon two concentrations, namely, 40 mg/l, theconcentration used internationally in proportionsensitivity method and 64 mg/l the criticalconcentration differentiating the resistant from thesensitive strains by the MIC method. The twoconcentrations gave 96% and 97% agreement whencompared with the indirect method12.Ethambutol gave more discrepant resultswhen compared to the indirect test throughout the 8weeks of incubation, under reading resistance up to4 weeks and over reading in excess of 4 weeks, afinding suggestive of the deterioration of the drug inthe medium with incubation.Lack of standardization of the inoculum isthe main criticism raised against the reliability of thedirect tests. This study showed that the growth wassignificantly more on the plain medium of theconcentration direct test then of the swab direct test,as could be expected. Yet it did not alter the resultsas the two direct tests were equally accurate inclassifying the cultures from the second week forINH and third week for R.This was possible because the definition forresistance was based on the quantity of growth onthe drug containing medium relative to that on theplain medium. This definition evolved in studies in1969 and 1970, have stood the test of time and canbe considered highly reliable.Unpublished data from this centre hasshown that the agreement of indirect sensitivity testsdone on the same culture was highly reproduciblefor INH, R and Emb. The advantage of the swabdirect test the concentrate direct test is that it requiresno special equipment such as the centrifuge, is lesshazardous to the worker since the procedure doesnot create much aerosol contamination of the area,and requires only one relatively inexpensive chemicalreagent, i.e. the commercial cetrimide in 1% solution.The advantage of the direct tests overthe indirect test is that in most samples it givessensitivity results at the same time as theprimary culture. This process not only reducesthe turn around time by four weeks, but alsocontamination by eliminating the step of makinga subculture. Most importantly, the results ofthe direct tests are more closely representativeof the bacterial population in the given sputumsample, unlike in the indirect test, which cansuffer from errors of selection when drugsusceptibility test was set up from a primaryculture. In the direct tests, resistance can bereported if adequate growth is seen on the drugslopes even when the plain medium iscontaminated. Resistance could be detected withgrowth as low as 10 colonies and with totalagreement with the result of the indirect test;however, it is recommended that such resultsbe accepted provisionally and confirmed withindirect test on the subculture. The direct testsdescribed here would serve the purpose well incountries with limited resources.REFERENCES1. Devaki V, Mohan K, Gangadharam PRJ. Direct sensitivitytest for isoniazid.  Indian J Tuberc 1969, 57: 1006-10.DIRECT  DRUG  SUSCEPTIBILITY  TESTS  FOR  M. TUBERCULOSIS188Indian Journal of Tuberculosis2. Mathew S , Nair N G K , Radhakrishna S ,  GangadharamP R J Direct drug susceptibility test for tubercle bacilli bythe sputum swab culture method.  Int  J Tuberc  Lung Dis2000; 4: 168-173.3. Mathew S.  Paramasivan C. N, Fathima Rehman, BalambalR.  K. Rajaram K, Prabhakar R.  A direct rifampicinsensitivity test for tubercle bacilli.  Indian J Med Res1995; 102: 99-103.4. Petroff S. A.  A new method for isolation and cultivationof tubercle bacilli from sputum and faeces.  J Exp Med1915; 21: 38.5. Paramasivan, C. N.; Daniel Herbert; Narayana, A. S. L.;Prabhakar, R.; Somasundaram, P. R. Evaluation ofhomogenizing substances in estimating the viability oftubercule bacilli. Indian Journal of Medical Microbiology1986; 4: 177-180.6. Andrews R. A, Radhakrishna S. TuberculosisChemotherapy Center, Madras. A comparison of twomethods of sputum collection in the diagnosis ofpulmonary tuberculosis. Tubercle 1959: 40: 155 – 162.7. Jensen K A.  Towards a standardization of laboratorymethod.  Second report of the subcommittee oflaboratory methods of International Union againsttuberculosis.  Bull.  Int Union against Tub 1955; 25(1-2):89-1048. Allen B W, Baker F J.  Mycobacteria: Isolation,identification and sensitivity testing.  London: Butterworth & Co. (Publishers) 1968 P. 24-6.9. Tuberculosis Chemotherapy Centre, Madras.  A controlledcomparison of a twice-weekly and three once-weeklyregimens in the initial treatment of pulmonarytuberculosis.  Bull WHO 1970; 43: 143-206.10. Vareldzis  B. P., Grosset J. de Kantor I., Crofton J., LaszloA., Felten M., et al.  Drug-resistant tuberculosis:Laboratory issues.  World Health Organizationrecommendations.  Tubercle Lung Dis 1994; 75: 1-7.11. Subbammal S., Nair N. G. K, Radhakrishna S.  Tripathy S.P.  Comparison of various measures of sensitivity of M.tuberculosis ethambutol.  Tubercle 1978; 59:155-191.12. Paramasivan C. N, Rahman F, Nalini S, Dakshayani.Gand Venkataraman.P Comparison of differentmethods of assessing in vitro resistance of M.tuberculosis to rifampicin. Indian J Med Res. 2001,114: 187- 191SARAH  MATHEW  ET  AL 189

Simple Direct Drug Susceptibility Tests on Sputum Samples for Early Detection of Resistance in Tubercle Bacilli

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Simple Direct Drug Susceptibility Tests on Sputum Samples for Early Detection of Resistance in Tubercle Bacilli

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