Jornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
Evaluation of rapid microplate assays using
cellular-viability indicators to determine patterns of susceptibility
to isoniazid and rifampin in
Mycobacterium tuberculosis
strains*
MARTA OSÓRIO RIBEIRO, MARLEI DA SILVA GOMES, SIMONE GONÇALVES SENNA, MARIA LUCIA ROSA ROSSETTI, LEILA DE SOUZA FONSECA.
Background: Knowledge of the rates of drug resistance is one of the pillars of tuberculosis control program evaluation. Data from low- resource countries are scarce and results are delayed due to the techniques employed. There is therefore an urgent need for evaluation of faster and less onerous testing methods.
Objective: To compare the performance of rapid colorimetric assays for phenotyping that employ oxidation- reduction indicators to determine the susceptibility profile of Mycobacterium tuberculosis with the gold- standard proportion method on Lowenstein-Jensen Medium.
Method: We analyzed 166 M. tuberculosis strains of known susceptibility. Minimal inhibition concentrations for isoniazid and rifampicin were determined in microplates, using a liquid medium and Alamar Blue and tetrazolium bromide indicators. To measure agreement the Kappa value was used. Cutoff values between sensitive and resistant strains were defined as 0.2µg/mL and 1.0µg/mL for isoniazid and rifampicin, respectively.
Results: There was 100% concordance between Alamar Blue and tetrazolium bromide methods in the determination of minimal inhibition concentrations. Agreement between the colorimetric method and the Lowenstein- Jensen was 95% for isoniazid and rifampicin. Using the colorimetric method, results were obtained within 7 days, in contrast to the 28 days required for the conventional method.
Conclusions: Assays to determine minimal inhibition concentrations in liquid medium and employing oxidation- reduction indicators proved to be rapid and inexpensive. This method has the potential to become a faster, alternative method for determining susceptibility of M. tuberculosis strains in developing countries.
Key words: Mycobacterium tubercolis. Disease susceptibility. Isoniazid/therapeutic use. Rifampin/therapeutic use.
*St u dy carried ou t at t he In st it u t o de Microbiologia (Microbiology In st it u t e) of t he Un iversidade Federal do Rio de Jan eiro (Rio de Jan eiro Federal Un iversit y), Rio de J an eiro, RJ , Brazil.
Correspon den ce t o: Mart a Osório Ribeiro. Av. Ipiran ga, 5400, Bairro J ardim Bot ân ico, CEP: 90610- 000 Port o Alegre, RS, Brasil. Tel: 55 51 3288 4 0 3 4 . E- m a il: m a rt a o so @ t erra .co m .b r
Fin a n cia l Su p p o rt : Red e Bra sileira d e Pesq u isa em TB (Red e- TB, Bra zilia n Tu b ercu lo sis Resea rch Net wo rk)/ Gra n t n o . 6 2 .0 0 5 5 / 01 - 4 - PACDT-Milen io .
ABM – Alamar Blu e met hod MTTM – MTT met hod
emergen cy, requ irin g in t ensified effort s in order t o co m b a t t h is d ise a se(2 ). Alt h o u g h co n t ro l
measu res an d t herapeu t ic regimes have been well established, there are difficulties that are inherent t o t h e d is e a s e , c h ie f a m o n g w h ic h is t h e appearance of drug- resistant strains(3,4).
Ep id e m io lo g ica l st u d ie s o f a n t i- TB d ru g re sist a n c e p ro vid e in d ic a t o rs, su c h a s t h e prevalen ce of primary an d acqu ired resist an ce, which are u sefu l for t he evalu at ion of t he qu alit y of treatment and of TB control programs. For such studies, the classically recommended method (gold standard) is the proportion method, performed on solid mediu m or on au t omat ed (MB/ BacT an d BACTEC 960) an d semi- au t omat ed (BACTEC 460) syst ems. These met hods are eit her prohibit ively expen sive or difficu lt t o execu t e in t he laborat ory ro u t in e. Th ere is t h erefo re lit t le in fo rm a t io n available regardin g t ran smission an d resist an ce p a t t e rn s, w h e t h e r f o r p rim a ry o r a c q u ire d resist an ce(5). In t his st u dy, we aimed t o compare
t h e su sce p t ib ilit y p ro file o f Myco b a ct e riu m tuberculosis (Mt b) st rain s in relat ion t o ison iazid (INH) and rifampin (RIF). This was done through a n ew met hod t hat u ses cell- viabilit y in dicat ors t o det ermin e t he min imal in hibit ory con cen t rat ion s (MIC) for INH an d RIF.
METHODS
A total of 166 Mtbstrains were evaluated. These st rain s were from t he collect ion s of t he followin g laboratories: Laboratório Central de Saúde Pública do Rio Grande do Sul (Rio Gran de do Su l Cen t ral Laboratory of Public Health); Hospital Clementino Fraga Filho (Clemen t in o Fraga Filho Hospit al) of t he Universidade Federal do Rio de Janeiro (Rio de Janeiro Federal University); Centro de Referência Pro fesso r Hélio Fra g a Pro fesso r Hélio Fra g a Referen ce Cen t er) in Rio d e J an eiro ; an d t h e Instituto Adolfo Lutz (Adolfo Lutz Institute) in São Paulo. The standard Mtb H37Rv ATCC 27294 strain and Mycobacterium fortuitum ATCC 6841 (the latter as a st an dard for bot h INH an d RIF resist an ce),
both of which were provided by the Professor Hélio Fraga Referen ce Cen t er, were also used.
All st rain s were iden t ified as belon gin g t o t he Mtbcomplex through standard colorimetric assays for phenotyping. The susceptibility test, using the Lowenstein- Jensen proportion method (LJPM), was carried ou t in accordan ce wit h t he Man u al de Ba ct erio lo g ia d a Tu b ercu lo se (Gu id eb o o k fo r Tuberculosis Bacteriology)(6). When the Mtb began
t o mu lt iply in t he Lowen st ein - Jen sen mediu m, su sp e n sio n s wit h t u rb id it y e q u iva le n t t o a McFarlan d t u rbidit y st an dard of 3 were prepared in phosphat e bu ffer at pH 7.0 an d t ran sferred t o 2- mL cryotubes, where they were stored at –20°C. The t echn iqu es u sed in t he det ermin at ion of MICs were performed accordin g t o t he prot ocols devised by various authors(7- 9). The test was carried
out in 96- well flat- bottom microplates, with sterile covers (Corn in g Cost ar, Cambridge, MA, USA). We u sed Mid d leb ro o k 7 H9 b ro t h m ed iu m (Difco , Detroit, MI, USA), containing glycerol and enriched wit h 10% oleic acid- albu min - dext rose- cat alase (OADC), as well as with Bacto Casitone (Difco). The INH an d RIF (Sigma, St . Lou is, MO, USA) were prepared as st ock solu t ion s at con cen t rat ion s of 10,000
µ
g/mL in sterile distilled water and ethylene glycol (Difco), respect ively. The con cen t rat ions of INH an d RIF were chosen based on t he cu t off va lu es, st a n d a rd ized fo r t h e BACTEC 4 6 0 TB Syst em: 0.2µ
g/ mL for INH an d 1.0µ
g/ mL for RIF(1 0 ). Aft er t he serialized dilu t ion s, t he fin alJornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
suspension was prepared in tubes containing glass beads and sterile distilled water. After agitation and subsequent settling, the suspension was transferred by eyedropper into another tube containing sterile d ist illed wa t er, u n t il t u rb id it y sim ila r t o t h e McFarlan d t u rbidit y st an dard of 1 was achieved. The in ocu lat e was dilu t ed at 1:25 in Middlebrook 7H9 medium.
Th e so lu t io n u sed fo r t h e in d ica t o rs wa s prepared on e day prior t o t he t est . Usin g Alam ar Blu e® dye (Fisher Scientific, Pittsburgh, PA, USA)
as a b ase, a m ixt u re o f 1 0 % Tween 8 0 so lu t io n in wa t er wa s p rep a red in vo lu m e t o vo lu m e p ro p o rt io n s(7 ). A vo lu m e o f 2 5
µ
L was u sed ineach well o f t h e m icro p lat e. Alam ar Blu e® is a
flu o rescen t / co lo rim et ric in d ica t o r wit h red o x propert ies. The oxidized form (n on - flu orescen t / n o n - viab le cell) is b lu e, an d t h e red u ced fo rm (flu o re sce n t / via b le ce ll) is ro se - co lo re d . To p r e p a r e t h e s o l u t i o n f o r u s e , 3 (4 , 5 -d im et h ylt h ia zo l- 2 - yl)- 2 ,5 - -d ip h en ylt et ra zo liu m b ro m id e (MTT, Sig m a), wh ich is a t et razo liu m salt t h at is red u ced b y d eh yd ro g en ase en zym es, forming formazan crystals soluble in ethanol, was u se d . Th e MTT wa s d ilu t e d in 1 m g / m L o f ab so lu t e et h an o l an d eq u ivalen t am o u n t s (v/ v) were a d d ed t o 1 0 % Tween 8 0 so lu t io n . Th e volu me u sed in each microplat e well was 50
µ
L. Th e oxid ized fo rm o f MTT is yello w, an d t h e red u ced fo rm is p u rp le.St an dard st rain s were t est ed for each bat t ery of tests. The peripheral microplate wells were filled wit h 200
µ
L of st erile dist illed wat er t o avoid evaporation of the medium in the incubator. Wells d esig n at ed as co n t ro ls fo r d ru g s an d fo r t h e culture medium were included. With the exception of t he peripheral wells, all wells received 100µ
L of 7H9 liqu id mediu m. To t he in it ial wells in each row, 100µ
L of each dru g was added. Usin g t hese wells an d a mu lt ichan n el pipet t e, serial dilu t ion s were m ad e. No d ru g s were ad d ed t o t h e last colu mn , wit h t he in t en t ion of preservin g it as a con t rol for bact erial growt h. Therefore, all wells had a volu me of 100µ
L. We t hen added 100µ
L of su spen sion of each st rain , for a fin al volu me of 200µ
L.Each microplat e was sealed wit h plast ic film and remained in the incubator at 37°C for five days. On t he fift h day, t he respect ive in dicat ors were added t o t he bact erial con t rol wells as well as t o
t h e d ru g a n d m ed iu m co n t ro l wells. On t h e followin g day, if t he in dicat ors in t he bact erial g ro wt h co n t ro l wells h a d ch a n g ed co lo r, a n in dicat or was added t o all remain in g wells. The microplat es were placed in t he in cu bat or for an additional 24 hours. The MIC was then determined, defin ed as t he lowest dru g con cen t rat ion capable of impedin g t he chan ge in color, i.e. of in hibit in g cell growt h.
RESULTS
The su scept ibilit y profile t o INH an d RIF was det ermin ed u sin g t he LJPM. Of t he 166 st rain s studied, 34 were sensitive and 91 were resistant to both drugs. There were 38 strains resistant to INH only and 3 strains resistant to RIF only. Considering each drug separately, there were 129 INH- resistant strains and 37 INH- sensitive strains versus 94 RIF-resist an t st rain s an d 72 RIF- sen sit ive st rain s.
All 1 6 6 Mt b st rain s were t est ed u sin g t h e Alamar Blu e® met hod (ABM) an d t he MTT met hod
Sen sit ive t o INH (< 0.2) 6 (4%) 36 (97.4%) -
-Resist an t t o RIF (> 1.0) - - 86 (93.5%)
-Sen sit ive t o RIF (< 1.0) - - 8 (8.7%) 72 (100%)
TOTAL 129 (100%) 37 (100%) 94 (100%) 72 (100%)
ABM: Alam ar Blu e m et h o d ; MTTM: 3 - (4 ,5 - d im et h ylt h iazo l- 2 - yl)- 2 ,5 - d ip h en ylt et razo liu m b ro m id e m et h o d ; INH: ison iazid; RIF: rifampin
DISCUSSION
Du e t o con cern abou t emergen cy con t rol an d transmission of resistant strains, it is essential that new phenotyping methods used to determine strain susceptibility to anti- TB drugs, especially INH and RIF, be t est ed. These met hods shou ld be pract ical and effective and should allow a faster result than t h a t p ro vid e d b y t h e co n ve n t io n a l m e t h o d . Therefore, with the objective of decreasing the cost of these tests and the time spent waiting for results, fast , reliable an d easily performed phen ot ypin g methods have been developed. Susceptibility tests co n d u ct ed in m icro p la t es rep resen t o n e su ch alternative. The use of indicators, such as resazurin and tetrazolium salts, to determine cell viability is an alternative for better viewing during the reading of t hese t est s in microplat es. These compou n ds f u n c t io n a s c h r o m o g e n ic s u b s t r a t e s o f d eh yd ro g en ase en zym es, act in g as o xid at io n -redu ct ion in dicat ors, an d are -redu ced (t hrou gh gain in g hydrogen molecu les) by flavin s bou n d t o t ran sport syst em en zymes presen t du rin g t he cell met abolism process(11).
Since 1995, studies have been conducted using Alamar Blue® (resazurin salt) as an indicator of cell
growt h. The au t hors of on e su ch st u dy(12) du bbed
the method MABA (Microplate Alamar Blue Assay) an d compares favorably wit h t he agar proport ion m et h o d , o b t a in in g a co n co rd a n ce o f 9 7 % in determining the MIC of Mtb strains. Other studies applying changes in the way the test is read of the t est , u sin g flu orescen ce, also showed posit ive resu lt s(13,14). When compared t o t he BACTEC 460(7)
syst em an d t o t he proport ion met hod on solid mediu m met hod, MABA presen t ed con cordan ces
of 93.6% an d 97.1%, respect ively, an d has t he added advantage of the fact that the results remain available for a period of 8 t o 10 days(8).
The MTT dye has been u sed as a cell growt h indicator to determine MIC for the Mycobacterium aviu m complex(15). Gu idelin es for det ect ion of RIF
resist a n ce in Mt b st ra in s a n d fo r t h e u se o f microplat es were est ablished in 1998, in a st u dy t hat det ermin ed t he appropriat e cu t off valu es for defin in g resist an ce an d sen sit ivit y t o RIF(16). The
technique of performing this method in tubes was standardized through comparison with the BACTEC 460 syst em, used t o det ect resist an ce t o RIF, wit h con cordan t resu lt s(17).
In t his st u dy, 166 Mt b st rains were evalu at ed wit h t he object ive of an alyzin g t he performan ce of n ew MIC- det ermin in g met hodologies, which were evalu at ed wit h regard t o sen sit ivit y an d con cordan ce in comparison wit h t he st an dard met hod on solid mediu m. In order t o evalu at e t he degree of con cordan ce bet ween t he n ew met hods a n d t h e c o n ve n t io n a l m e t h o d s , w e u s e d predetermined cutoff values of 0.2
µ
g/mL for INH and 1.0µ
g/ mL for RIF. The new methods showed 100% con cordan ce when compared wit h each ot her. When t he met hods ABM an d MTT were compared t o PMLJ, we observed a con cordan ce o f 9 5 % fo r b o t h d ru g s. Th e sen sit ivit y a n d specificit y valu es for INH were 95.3% an d 97.3%, respectively, whereas those for RIF were 91.5% and 100%, respect ively.In t wo previou sly pu blished st u dies(9 ,1 8 ), t he
Jornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
resazurin microtiter assay plate method, which uses resazurin as an indicator of cell viability, was used for det ermin in g MIC. The LJPM met hod was u sed as the gold standard. Sensitivity and specificity for INH were 96.2% an d 100%, respect ively. For RIF, all 80 resu lt s showed a 100% con cordan ce. In t he secon d st u dy, t he au t hors compared t he MABA met hod wit h t he t et razoliu m microplat e assay (MTT) method in determin in g the MICs of 35 Mtb strains for INH, RIF, streptomycin and ethambutol. The authors found a strong concordance between the two methods, although it was unclear whether t h ere wa s a g o ld - st a n d a rd m et h o d u sed fo r co m p a riso n o r t h e n ew m et h o d s were sim p ly compared wit h each ot her.
In t e st s d e sig n e d t o d e t e rm in e MIC a n d co n d u ct e d in liq u id m e d iu m , t e ch n ica l a n d biological variables, su ch as size of t he in ocu lat e, phase of bact erial growt h, charact erist ics of t he medium and drug stability, hinder reproducibility(19).
In t his st u dy, we t ried t o con t rol some of t hese variables, u sin g t he same in ocu lat e, t he same cu lt u re mediu m an d t he same dru g preparat ion s. The t echn ical man ipu lat ion of t he serial dilu t ion s an d t he charact erist ics of each in dicat or were identified as the possible causes for the differences in t he MICs observed. We fou n d t hat t he MTT method facilitates the visualization of color change, sin ce yellow chan ges t o pu rple, wit h n o possible c o n f u s io n c a u s e d b y n u a n c e s o f c o lo r s intermediate between the two.
In conclusion, susceptibility tests in microplates, using oxidation- reduction indicators, are rapid, are ea sily p erfo rm ed , a n d p resen t a h ig h ra t e o f co n co rd a n ce wit h t h e g o ld - st a n d a rd m et h o d (LJPM) used in t he presen t st u dy. These t est s may rep resen t a t im esa vin g a lt ern a t ive fo r b a sic laboratories, since most possess the infrastructure n ecessary for cu lt u rin gMtb.
ACKNOWLEDGMENTS
Th e a u t h o rs a re g ra t efu l fo r t h e fin a n cia l su pport provided by t he Con selho Nacion al de Desenvolvimento Científico e Tecnológico (CNPq, Nat ion al Cou n cil for Scien t ific an d Techn ological Develo p m en t ; Milên io / Pro n ex), t h e Fu n d a çã o Carlos Chagas Filho de Amparo à Pesqu isa do Estado do Rio de Janeiro (FAPERJ, Carlos Chagas Filho Fou n dat ion for t he Su pport of Research in t he St at e of Rio de Jan eiro, Brazil), an d John s
Hopkins University (Process NIH: n° 1U19AI45432-01). We wou ld also like t o t han k An n a Grazia Marsico of t he Hospital Universitário Clementino Fraga Filho an d Ân gela Maria Wern eck Barret o of t he Centro de Referência Professor Hélio Fraga, as well as Maria Alice da Silva Telles of t he Instituto Adolfo Lutz for kin dly su pplyin g t he Mt b st rain s.
REFERENCES
1 . Dye C, Schelle S, Dolin P, Pat han a V, Raviglion e MC. The WHO Global Su rveillan ce an d Mon it orin g Project . Glo b a l b u rd en o f t u b ercu lo sis. Est im a t ed in cid en ce, p r e va le n c e , a n d m o r t a lit y b y c o u n t r y. J AM A. 1 9 9 9 ; 2 8 2 : 6 7 7 - 8 6 .
2 . World Healt h Organ izat ion . TB. Global emergen cy. WHO rep o rt o n t h e t u b ercu lo sis ep id em ic. Gen eva ; 1 9 9 4 . (WHO/ TB/ 9 4 .1 7 7 ).
3 . Kaufmann SHE, Van Embden JD. Tuberculosis: a neglected disease strikes back. Trends Microbiol. 1993; 1:2- 5. 4 . Bra sil. Min ist é rio d a Sa ú d e . Fu n d a çã o Na cio n a l d e
Sa ú d e . Co n t ro le d a t u b e rcu lo se : u m a p ro p o st a d e in t egração en sin o–serviço. Cen t ro de Referên cia Prof. Hélio Fra g a . So cied a d e Bra sileira d e Pn eu m o lo g ia e Tisiologia. 5ª ed. Rio de Jan eiro: FUNASA/ CRPHF/ SNPT; 2 0 0 2 .
5 . Wo rld He a lt h Org a n iz a t io n . An t i- t u b e rcu lo sis d ru g resist an ce in t he world: t he WHO/ IUATLD global project on an t i- t u bercu losis dru g resist an ce su rveillan ce, 1994– 1 9 9 7 . Swit zerlan d ; 1 9 9 7 . Gen eva. (WHO/ TB/ 9 7 .2 2 9 ). 6 . Bra sil. Min ist é rio d a Sa ú d e . Fu n d a çã o Na cio n a l d e Sa ú d e. Cen t ro d e Referên cia Pro fesso r Hélio Fra g a . Ma n u a l d e Ba c t e rio lo g ia d a Tu b e rc u lo s e . Rio d e J an eiro: Edit ora Gu an apá, 1994.
7 . Fra n zb la u SG, Wit zig RS, McCla u g h lin J C, To rres P, Madico G, Hern an dez A, et al. Rapid, low- t echn ology CMI d e t e r m in a t io n w it h c lin ic a l Myc o b a c t e r iu m t u b ercu lo sis iso lat es b y u sin g t h e Micro p lat e Alam ar Blu e Assay. J Clin Microbiol. 1998;36:362- 6. 8 . Pa lo m in o J C, Po rt a els F. Sim p le p ro ced u re fo r d ru g
su scep t ib ilit y t est in g o f Myco b a ct eriu m t u b ercu lo sis u sin g a co m m e rcia l co lo rim e t ric a ssa y. Eu r J Clin Micro b io l In fect Dis. 1 9 9 9 ;1 8 :3 8 0 - 3 .
9 . Ca vie d e s L, De lg a d o J , Gilm a n RH. Te t r a z o liu m microplat e assay as a rapid an d in expen sive colorimet ric m et h o d fo r d et erm in at io n o f an t ib io t ic su scep t ib ilit y o f Myco b a ct e riu m t u b e rcu lo sis. J Clin Micro b io l. 2 0 0 2 ; 4 0 (5 ): 1 8 7 3 - 4 .
1 0 . In d erlied CB, Sa lfin g er M. An t im yco b a ct eria l a g en t s an d su scept ibilit y t est s. In : Mu rray PR (Edit or). Man u al o f clin ica l m icro b io lo g y. 7 t h e d . Wa sh in g t o n , DC: Am erican Societ y for Microbiology; 1999.
11 . Ko n em a n EW, St ep h en SD, J a n d a WM, Sch ren b erg er PC, Win n WC, (Edit ors). Diagn ost ic microbiology. Color at las an d t ext book. 5aª ed. New York: Lippin cot t ; 1997.
1 2 . Yajko DM, Madej JJ, Lan cast er MV, San ders CA, Owt hon VL, Gee B, et al. Colorimet ric met hod for det ermin in g MICs o f a n t im ic ro b ia l a g e n t s f o r Myc o b a c t e riu m t u bercu losis. J Clin Micro b io l. 1 9 9 5 ;3 3 :2 3 2 4 - 7 . 1 3 . Collin s LA, Fran zblau SG. Microplat e Alamar Blu e Assay
Det erm in at io n o f MICs fo r Myco b act eriu m aviu m - M. in t r a c e llu la r e c o m p le x in liq u id m e d iu m b y a c o lo r im e t r ic m e t h o d . J Clin M ic r o b io l. 1 9 9 5 ; 3 3 (7 ): 1 8 4 2 - 6 .
1 6 . Mshan a RN, Tadesse G, Abat e G, Miörn er H. Use of 3-(4 ,5 - Dim et h ylt h ia zo l- 2 - yl)- 2 ,5 - Dip h en yl Tet ra zo liu m Bro m id e fo r ra p id d e t e ct io n o f Rifa m p in - re sist a n t M yc o b a c t e r iu m t u b e r c u lo s is . J Clin M ic r o b io l. 1 9 9 8 ; 3 6 : 1 2 1 4 - 9 .
Ag en t s Ch em o t h er. 2 0 0 2 ;4 6 :2 7 2 0 - 2 .
