Analysis of different
primers used in the PCR method:
diag nosis of tuberculosis in the state of Amazonas, Brazil
MAURICIO MORISHI OGUSKU, JULIA IGNEZ SALEM
Backg round: Various primers are being tested for the detection of Mycobacterium tuberculosis DNA. The accuracy of
the polymerase chain reaction (PCR) depends on the target sequence used and whether the test will be performed in culture or in clinical specimens.
Objectives: To identify DNA sequences, specifically those commonly reported as targets for diagnosis of tuberculosis
(TB), in clinical samples of M. tuberculosis strains.
Method: Eighty- one clinical samples from suspected TB patients were initially processed and submitted to bacilloscopy
(smear) and culture, and PCR was performed with specific primers for the following targets: IS6110, 65 kDa, 38 kDa and MPB64.
Results: Smear and culture results were negative in 24 samples, as was the PCR. The 19 samples testing smear positive,
as well as the isolated strains, were 100% positive on PCR, with the exception of the 89.4% result from PCR with MPB64 primers. In the 38 smear negative and culture positive samples, PCR results were inconsistent. The primers specific for amplifying the 123 bp IS6110 fragment gave the highest positivity (92.1%), diagnostic agreement (0.9143), co- positivity (94.7%) and co- negativity (100%).
Conclusion: The IS6110, 38 kDa, MPB64 and 65 kDa sequences were found in thegenome of all M. tuberculosis strains
isolated in patients from the state of Amazonas. The protocol for processing the clinical samples prior to PCR analysis and the specific primers used to amplify the 123bp IS6110 fragment showed a greater efficiency in diagnosing pulmonary (paucibacillary) tuberculosis in comparison to published data.
Key words: Primers/PCR. Diagnosis/Tuberculosis. Mycobacterium tuberculosis.
*St u d y ca rried o u t in t h e La b o ra t o ry o f Myco b a ct erio lo g y a t t h e Co o rd en a çã o d e Pesq u isa s em Ciên cia s d a Sa ú d e (Scien ce a n d Hea lt h Resea rch Co o rd in a t io n Cen t er) o f t h e In st it u t o Na cio n a l d e Pesq u isa s d a Am a zô n ia (INPA, Na t io n a l Resea rch In st it u t e o f Am a zô n ia ), Ma n a u s, Am a zo n a s. Fin an cial su p p o rt p ro vid ed b y: t h e Min ist ry o f Healt h , CAPES/ RENOR. Red e Brasileira d e Pesq u isa em TB (Red e- TB, Brazilian Tu b ercu lo sis Research Net wo rk)/ Gra n t n o . 6 2 .0 0 5 5 / 01 - 4 - PACDT- Milen io .
Co rresp o n d en ce t o : Ma u ricio Mo rish i Og u sku . In st it u t o Na cio n a l d e Pesq u isa s d a Am a zô n ia – INPA. Av. An d ré Ara ú jo , 2 9 3 6 – Ba irro d o Aleixo . CEP 6 9 0 6 0 - 0 01 - Ma n a u s, AM – Bra z il - Ph o n e : 5 5 9 2 6 4 3 3 0 5 8 . E- m a il: m m o g u sku @ in p a .g ov.b r
Abbre viatio n s u s e d in this pape r:
AFB – Acid - fa st b a cilli b p – Ba se p a irs
IS611 0 – In sert io n seq u en ce 6110 M t b – Myco b a ct e riu m t u b e rcu lo sis P CR – Po lym era se ch a in rea ct io n TB – Tu b ercu lo sis
INTRODUCTION
Po lym erase ch ain react io n (PCR) is a m o lecu lar t ech n iq u e wh o se a ccu ra cy is d et erm in ed b y t h e ch o ice o f t h e t arg et DNA an d t h e d efin it io n o f t h e primers wit hin t he DNA sequ en ce. The PCR met hod h as b een u sed as an alt ern at ive t h at p resen t s h ig h sen sit ivit y a n d sp ecificit y fo r t h e ra p id d ia g n o sis o f in fect io us d iseases. Ho wever, t h e u se o f PCR in t he det ect ion of Mycobact eriu m t u bercu losis (Mt b) has produ ced varyin g resu lt s, especially in relat ion t o t h e sen sit ivit y o f t h e t est(1 ,2 ,3 ). Va rio u s t a rg et se q u e n c e s, su c h a s in se rt io n se q u e n c e 611 0 (IS6110 ), 6 5 kDa (Gro EL), 3 8 kDa (Ph o S, CIE Ag 7 8 o r Pa b ) a n d MPB6 4 (2 3 kDa ), h a ve t h erefo re b een used. Am on g t hese, IS6110 is more common ly used b eca u se it is a rep et it ive seq u en ce in t h e Mt b g en o m e. Th is ch a ra ct erist ic h elp s in crea se t h e se n sit ivit y o f P CR o ve r t h a t o b t a in e d in t h e am plificat ion of sin gle DNA sequ en ces(4 ). However, t h e IS6110 seq u en ce was rep o rt ed t o b e ab sen t f ro m a n Mt b st ra in iso la t e d in In d ia(5 ), a n d h o m o lo g o u s seq u en ces h a ve b een d et ect ed in pot en t ially pat hogen ic mycobact eriu m st rain s su ch as M. in t racellu lare, M. fo rt u it u m , M. kan sasii, M. xen o p i, M. m a lm o en se a n d M. ch elo n a e(6 ,7 ). In Brazil, t h e exist en ce o f Mt b st rain s lackin g IS6110 h a s n ever b een rep o rt ed , a n d so m e o f t h e sp ecies m en t io n ed a b o ve a re freq u en t ly iso la t ed in t h e st a t e o f Am a zo n a s(8 ,9 ,1 0 ).
These fact s led u s t o ask t he followin g qu est ion s reg a rd in g t h e u se o f PCR fo r t h e d ia g n o sis o f t u bercu losis (TB) in t he st at e of Am azon as: Wou ld t h e seq u en ces rep o rt ed in t h e lit era t u re a lso b e fo u n d in t h e Mt b st ra in s iso la t ed in t h e st a t e o f Am a z o n a s ? Wh ic h p r im e r s w o u ld b e m o s t recommen ded for PCR of clin ical samples collect ed in t h e st a t e o f Am a zo n a s?
In a n a t t em p t t o a d d ress t h ese q u est io n s, we in ve st ig a t e d t h e p re se n ce o f t h e DNA t a rg e t s e q u e n c e s m o s t c o m m o n ly r e p o r t e d in t h e lit erat u re. A set of Mt b st rain s, as well as t he clin ical sam p les fro m wh ich t h e st rain s h ad b een iso lat ed , were an alyzed .
METHOD
Clin ic a l sa m p le s w e r e c o lle c t e d f ro m 8 1 su sp e ct e d TB p a t ie n t s livin g in t h e st a t e o f Am a zo n a s a n d were sen t t o t h e La b o ra t o ry o f Myco b a ct erio lo g y o f t h e In st it u t o Na cio n a l d e Pesq u isas d a Am azô n ia (INPA, Nat io n al Research
Inst it u t e of Am azôn ia) for st u dy. The sam ples were s u b m it t e d t o a c id - f a s t b a c illi (AFB) s m e a r m icro sco p y an d Mt b cu lt u re. Sm ear m icro sco p y a n d cu lt u re resu lt s were n eg a t ive in 2 4 sa m p les an d posit ive in 19 samples (mu lt ibacillary samples). In 3 8 sam p les, t h e sm ear m icro sco p y was n eg at ive a n d t h e cu lt u re w a s p o sit ive (p a u cib a cilla ry sam p les).
Smear microscopy was performed in accordan ce wit h t h e g u id elin es o f t h e Min ist ério d a Saú d e (Min ist ry o f Healt h )(11 ). Cu lt u res were p erfo rm ed as follows: samples were t ran sferred t o gradu at ed 15-m L co n ical cen t rifu g e t u b es an d an eq u al vo lu 15-m e of 4% NaOH solu t ion was added. The samples were a g it a t ed a n d left t o set t le fo r 1 5 m in u t es. St erile dist illed wat er was added u p t o a combin ed volu me o f 1 2 m L wit h . Sa m p les were t h en cen t rifu g ed a t 3 0 0 0 x g a n d t h e su p ern a t a n t wa s set a sid e. Th e sedim en t was su spen ded in 2 m L of st erile dist illed wat er, n eu t ralized wit h 4 % HCl so lu t io n , an d 0 .2 -m L a liq u o t s w e re t ra n sf e rre d f o r c u lt u re in Lö wen st ein - J en sen cu lt u re m ed iu m (t h ree t u b es p er sam p le). Th e rem ain d er o f t h e su sp en sio n was st ored for lat er PCR an alysis. The cu lt u re t u bes were in cu bat ed at 37ºC for u p t o 60 days. Iden t ificat ion o f t h e iso la t ed m yco b a ct eriu m st ra in s a s Mt b wa s p erfo rm ed a s reco m m en d ed b y Da vid et a l. (1 2 )
a m p lified a fra g m en t o f 1 5 5 b p . Wh en t h e t a rg et wa s 3 8 kDa(1 5 ), a m p lified p ro d u ct s fro m st a n d a rd PCR an d n est ed PCR p ro d u ced frag m en t s o f 41 9 bp an d 322 bp, respect ively. Primers for t he MPB64 reg io n a m p lified a fra g m en t o f 2 4 0 b p(1 6 ).
DNA e xt ra ct io n f ro m sa m p le se d im e n t wa s p e rf o rm e d in a cco rd a n ce wit h t h e Og u sku e t a l. p r o t o c o l(1 7 ). Th e DNA w a s p u r if ie d w it h p h e n o l- c h lo r o f o r m e xt r a c t io n a n d a b s o lu t e e t h a n o l p re cip it a t io n , in a cco rd a n ce wit h t h e Da vis e t a l. m e t h o d(1 8 ). Fo r DNA e xt ra ct io n fro m t h e 5 7 Mt b st ra in s iso la t e d f ro m t h e cu lt u re sa m p le s, we u se d a n a d a p t a t io n o f t h e P e ro sio e t a l.(1 9 ) p ro t o co l, in wh ich a p p ro xim a t e ly 1 m g o f e a ch st ra in su sp e n sio n wa s t ra n sf e rre d in t o a scre w ca p t u b e co n t a in in g g la ss b e a d s. Th e s a m p le s in t h e s e t u b e s w e r e a g it a t e d o n a vo rt e x m ixe r f o r ce ll se p a ra t io n , a n d st e rile d ist illed wa t er wa s a d d ed t o a d ju st t h e t u rb id it y o f t h e su sp e n sio n t o a 1 .0 McFa rla n d st a n d a rd . Fro m ea ch su sp en sio n , a 5 0 -
µ
L a liq u o t wa s u sed fo r DNA ext ra ct io n . To ea ch a liq u o t , 5 0µ
L lysis b u f f e r so lu t io n (2 0 0 m M Tris- HCl p H 8 ,0 , a n d 8 0 0µ
g / m L P ro t e in a se K) wa s a d d e d . Aliq u o t s w e r e i n c u b a t e d a t 3 7 ° C o v e r n i g h t a n d su b se q u e n t ly a t 1 0 0 °C f o r 1 0 m in u t e s, t h e n ce n t rif u g e d a t 1 4 .0 0 0 x g f o r 1 5 se co n d s. We u se d 5µ
L o f t h e su p e rn a t a n t t o p e rf o rm P CR wit h t h e va rio u s p rim e rs.We verified t he in t rin sic sen sit ivit y of st rain s in a cco rd a n ce wit h t h e p ro t o co l d evised b y Bo llela e t a l.(2 0 ) in o rd e r t o e s t a b lis h t h e m in im u m co n cen t ra t io n o f b a cilli t h a t wo u ld b e d et ect a b le b y PCR wit h t h e va rio u s p rim ers. Th erefo re, a su sp e n sio n o f Mt b H3 7 Rv wa s p re p a re d a n d adju st ed t o a 1.0 McFarlan d st an dard t u rbidit y level (eq u iva len t t o 3 x 1 08 b a cilli/ m L). Th is su sp en sio n was su bject ed t o su ccessive 1:10 dilu t ion s in st erile d ist illed wa t er – u p t o a co n cen t ra t io n o f 3 x 1 0- 1 b a cilli/ m L. Fo r DNA ext ra ct io n , ea ch d ilu t io n wa s h ea t ed a t 1 0 0 ºC fo r 1 0 m in u t es a n d cen t rifu g ed at 1 3 ,5 0 0 rp m fo r 1 0 m in u t es. We t h en u sed 5
µ
L o f t h e su p ern at an t fo r PCR. In t rin sic sp ecificit y o f p rim ers wa s verified in rela t io n t o t h e DNA o f t h e f o llo w in g m ic r o o r g a n is m s : M yc o b a c t e r iu m fo rt u it u m , Myco b a ct eriu m a viu m - in t ra cellu la re, Shigella son n ei, Salmon ella parat yphi A, Escherichia co li a n d St a p h ylo co ccu s a u re u s. Th e a b o ve -m en t io n ed p ro t o co l wa s u sed fo r DNA ext ra ct io n fro m t h ese sp ecies.Th e PCR fo r ea ch t a rg et seq u en ce in t h e DNA e xt ra ct e d fro m clin ica l sa m p le s a n d fro m t h e iso la t ed st ra in s wa s p erfo rm ed u sin g a so lu t io n co n t a in in g 1 0 m M o f Tris- HCl a t p H 8 .3 , 5 0 m M o f KCl, 0 .01 % g elat in ; 2 m M o f Mg Cl2, 200
µ
M of d NTP (Sig m a, St . Lo u is, MO, USA), 0 .1µ
M o f each p rim er, 2 U o f Taq DNA Po lym erase (In vit ro g en , Melb o u rn e, Au st ralia) an d 5µ
L o f t h e ext ra ct ed DNA (fro m t h e iso la t ed st ra in , clin ica l sa m p le o r PCR produ ct ) – in a fin al volu m e of 50µ
L. Prim er se q u e n ce s fo r t h e re sp e ct ive t a rg e t DNA a n d am p lificat io n p aram et ers are d escrib ed in Tab le 1 . Prim ers were syn t h esized b y In vit ro g en , a n d we u sed a Gen eAm p PCR Syst em 2 4 0 0 t h erm a l cycler (Applied Biosystems, Foster City, CA, USA). A positive con t rol con t ain in g Mt b H37Rv DNA an d a n egat ive co n t ro l (react io n wit h o u t DNA) were in co rp o rat ed in t o each DNA am p lificat io n sessio n .Am p lified p ro d u ct s were elect ro p h o resed o n a 1 . 5 % a g a r o s e g e l . Th e a g a r o s e g e l w a s su b seq u en t ly st a in ed wit h et h id iu m b ro m id e a n d PCR p ro d u ct s were d isp la yed u sin g a n Ea g le Eye II u lt raviolet t ran sillu m in at or (St rat agen e, La J olla, CA, USA).
Resu lt s were a n a lyzed u sin g t h e Ka p p a in d ex t o d e t e rm in e c o n c o rd a n c e b e t w e e n t h e t w o d iag n o st ic m et h o d s (cu lt u re an d PCR) an d am o n g t he variou s primers. Co- posit ivit y an d co- n egat ivit y were an alyzed u sin g 2 X 2 t ab les.
RESULTS
In t rin sic se n sit ivit y o f t h e p rim e rs t e st e d ext en d ed t o a d ilu t io n o f 3 x 1 00, t h at is, 3 b acilli/ mL, except for MPB64 primers, which showed lower sen sit ivit y sin ce posit ive resu lt s were on ly obt ain ed a t a d ilu t io n o f 3 x 1 02 b acilli/ m L. Sp ecificit y o f t h e vario u s p rim ers was co n sid ered h ig h sin ce n o a m p lifica t io n o ccu rred in DNA ext ra ct ed fro m M. fo rt u it u m , M. aviu m - in t racellu lare, S. so n n ei, S. p arat yp h i A, E. co li an d S. au reu s.
TABLE 1
Se qu e n ce s o f prim e rs an d po ly m e ras e chain re actio n param e te rs
Target s Seq u en ces o f p rim ers Am p lified p ro d u ct Cycles
IS6110 5 ´- CTC GTC CAG CGC CGC TTC GG - 3 ´ 1 2 3 p b 9 4 ºC – 1 m in
5 ´- CCT GCG AGC GTA GGC GTC GG - 3 ´ 6 6 ºC – 1 m in 35
7 2 ºC – 1 m in
IS6110 5 ´- GTG CGG ATG GTC GCA GAG AT - 3 ´ 5 41 p b 9 4 ºC – 1 m in
5 ´- CTC GAT GCC CTC ACG GTT CA - 3 ´ 6 5 ºC – 1 m in 35
7 2 ºC – 1 m in
Ext ern a l: 41 9 p b 9 4 ºC – 1 m in
38 5 ´- ACC ACC GAG CGG TTC GCC TGA - 3 ´ 6 3 ºC – 1 m in 35
k Da 5 ´- GAT CTG CGG GTC GTC CCA GGT - 3 7 2 ºC – 1 m in
In t ern a l: 3 2 2 p b 9 4 ºC – 1 m in
5 ´- TGA CGT TGG CGG AGA CCG - 3 ´ 6 3 ºC – 1 m in 35
5 ´- ATG GTG CCC TGG TAC ATG - 3 ´ 7 2 ºC – 1 m in
Ext ern a l: 3 8 3 p b 9 4 ºC – 1 m in
65 5 ´- GAG ATC GAG CTG GAG GAT CC - 3 ´ 6 0 ºC – 1 m in 35
k Da 5 ´- AGC TGC AGC CCA AAA GGT GTT - 3 ´ 7 2 ºC – 1 m in
In t ern a l: 1 5 5 p b 9 4 ºC – 1 m in
5 ´- CCA TCG ATC CGA GAC CCT GCT CAA GGG C - 3 ´ 6 0 ºC – 1 m in 35
5 ´- TGC TCT AGA CTC CTC GAC GGT GAT GAC G - 3 ´ 7 2 ºC – 1 m in
MPB64 5 ´- TCC GCT GCC AGT CGT CTT CC - 3 ´ 2 4 0 p b 9 4 ºC – 1 m in
5 ´- GTC CTC GCG AGT CTA GGC CA - 3 ´ 5 0 ºC – 1 m in 35
7 2 ºC – 1 m in
Fig u re 1. Percen t a g es o f PCR p o sit ivit y in rela t io n t o t h e p rim ers u sed in p a u cib a cilla ry clin ica l sa m p les a n d in Myco b a ct eriu m t u b ercu lo sis st ra in s Percen t a g es o f p o sit ivit y fo r t h ese sa m p les a re
sh o wn in Fig u re 1 .
Ta b le 2 sh o ws t h e resu lt s o f t h e co n co rd a n ce an alysis bet ween TB diagn osis t hrou gh cu lt u re an d t hat made by PCR in relat ion t o t he variou s primers st u d ied , a s well a s t h e resu lt s o f co - p o sit ivit y a n d co - n eg a t ivit y t est in g , b o t h u sin g t h e iso la t io n o f Mt b a s a d ia g n o st ic st a n d a rd .
Co n co rd a n ce o f TB d ia g n o ses u sin g PCR wit h t h e va rio u s p rim ers st u d ied , a s well a s d ia g n o ses m a d e t h ro u g h co - p o sit ivit y a n d co - n eg a t ivit y t est in g , b o t h usin g IS6110 p rim er as a d iag n o st ic st an d ard d u e t o it s h avin g t h e h ig h est p ercen t ag e o f p o sit ivit y, are sh o wn in Tab le 3 .
DISCUSSION
Ou r resu lt s sh o w t h a t t h e t a rg et seq u en ces IS6110, 65 kDa, 38 kDa an d MPB64 were preserved in all st rain s st u died an d t hat t he respect ive primers st u d ied can b e u sed in t h e d iag n o sis o f TB u sin g
Clinical Samples
PCR in clin ical sam p les. In m u lt ib acillary sam p les, o n ly p rim ers fo r t h e MPB6 4 seq u en ce (2 4 0 b p ) were less t h a n 1 0 0 % effica cio u s, a m p lifyin g o n ly 8 9 .4 % o f su ch sam p les. In p au cib acillary sam p les, p ercen t a g es o f p o sit ivit y va ried d ep en d in g o n t h e p rim ers st u d ied , as sh o wn in Fig u re 1 .
Th e p e r c e n t a g e o f p o s i t i v i t y f o r t h e a m p lifica t io n o f t h e 1 2 3 - b p fra g m en t o f t a rg et IS6110 in t h e p resen t st u d y (9 2 .1 %) was h ig h er t han t hat report ed by Nolt e et al.(2 1 ), Shawar et al.(2 2 ) o r Mo n t en eg ro et a l.(2 ), wh o o b t a in ed 5 7 .0 % , 5 3 .0 %, an d 7 6 .7 %, resp ect ively. Wh en we used primers t hat amplified a 541- bp fragmen t of t arget IS611 0 , w e o b t a in e d 8 6 . 8 % p o s it ivit y. Th e p ro p o rt io n s we n o t ed were h ig h er t h a n t h o se rep o rt ed b y Ab e et al.(1 3 ), wh o o b t ain ed o n ly 5 0 %
wit h t hese primers. Usin g 65- kDa primers, posit ivit y a ft er n est ed - PCR (6 5 kDa – 1 5 5 b p ) wa s 7 6 .3 %, h ig h er t h a n t h a t rep o rt ed b y Pierre et a l.(1 4 ), wh o report ed on ly 40%. As for 38- kDa prim ers, n est ed-PCR p o sit ivit y wa s 8 6 .8 %, h ig h er t h a n t h e 3 8 .4 % rep o rt ed b y Miyazaki et al.(1 5 ). Th e PCR fo r MPB6 4 p r e s e n t e d t h e lo w e s t p o s it ivit y (6 0 . 5 % ) in p a u cib a cilla ry sa m p les. Ou r resu lt wa s lo wer t h a n t h e 7 0 % rep o rt ed b y Ma rt in s et a l.(2 3 ), a ft er t h e in clu sio n o f t h e n est ed - PCR st ep . Ho wever, in t h e p resen t st u d y, t h is st ep wa s n o t p erfo rm ed fo r MPB64.
Th e d if f e re n c e s in p o sit ivit y p e rc e n t a g e s b e t we e n t h e p re se n t st u d y a n d t h o se in t h e lit era t u re m a y b e rela t ed t o slig h t ch a n g es in t h e adopted protocols, su ch as sample decon tamin ation
PCR
IS6110 IS6110 6 5 kDa 3 8 kDa MPB64
(123 bp) (541 bp)
Pos Neg Pos Neg Pos Neg Pos Neg Pos Neg
Mt b Pos 54 03 52 05 48 09 52 05 32 25
cu lt u re Neg 0 24 0 24 0 24 0 24 0 24
Tot al 81 81 81 81 81
Observed con cordan ce 96.3% 93.8% 88.9% 93.8% 69.1%
Expect ed con cordan ce 56.8% 55.8% 53.7% 55.8% 45.7%
Ka p p a in d ex (K) 0.9143 0.8604 0.7596 0.8604 0.4313
In t erp ret a t io n (K) Excellen t Excellen t Go o d Excellen t No rm a l
Co - p o sit ivit y 94.7% 91.2% 84.2% 91.2% 56.1%
Co - n eg at ivit y 100% 100% 100% 100% 100%
PCR: p o lym era se ch a in rea ct io n ; Po s: Po sit ive; Neg : Neg a t ive; IS6110 : in sert io n seq u en ce 6110 ; Mt b : Myco b a ct eriu m t u b ercu lo sis TABLE2
Co n co rdan ce an d accu racy o f po ly m e ras e chain re actio n w ith vario u s prim e rs in re latio n to cu ltu re in Lö w e n s te in - Je n s e n m e diu m
TABLE 3
Co n co rdan ce an d accu racy o f DNA am plificatio n s w ith vario u s
prim e rs in re latio n to ins e rtio n s e qu e n ce 6110 prim e rs (1 2 3 bp)
Primers IS6110 6 5 kDa 3 8 kDa MPB64
(541 bp)
Pos Neg Pos Neg Pos Neg Pos Neg
IS6110(123 bp) Pos 51 03 47 7 51 3 32 22
Neg 01 26 01 26 01 26 0 27
Tot al 81 81 81 81
Observed con cordan ce 95.0% 90.1% 95.0% 72.8
Expect ed con cordan ce 54.7% 53.1% 54.7% 46.5%
Ka p p a in d ex (K) 0.8909 0.7895 0.8909 0.4923
In t erp ret a t io n (K) Excellen t Go o d Excellen t No rm a l
Co - p o sit ivit y 94.4% 82.4% 94.4% 59.2%
processes an d variat ion s in t he composit ion of lysis b u ffer so lu t io n s. Th ese resu lt s co rro b o ra t e t h e st a t em en t m a d e b y Bu t ch er et a l. (2 4 ) t o t h e effect t h a t PCR p o sit ivit y va ries wid ely in sa m p les t h a t t est n eg a t ive in sm ea r m icro sco p y a n d p o sit ive in cu lt u re fo r Mt b, t o wh ich we ad d t h at variat io n s in PCR p ro t o co ls m a y a lso a ffect p o sit ivit y.
Con cordan ce of diagn osis, posit ivit y an d co-n eg at ivit y am o co-n g p rim ers aco-n d cu lt u res, u sico-n g Mt b iso la t io n a s a st a n d a rd (Ta b le 2 ), sh o ws t h a t t h e p rim ers a m p lifyin g t h e 1 2 3 - b p IS6110 frag m en t a re h ig h ly in d ica t ed fo r t h e d ia g n o sis o f TB fro m clin ica l sa m p le s, in d e p e n d e n t ly o f t h e sm e a r m icro sco p y resu lt s. Alt h o u g h d ia g n o sis o f TB wa s n o t co n firm ed t h ro u g h t h e u se o f t h ese p rim ers in 3 sa m p les p ro ven t o co n t a in Mt b (9 4 .7 % co -p o sit ivit y), n o fa lse- -p o sit ive resu lt s were fo u n d (1 0 0 % co - n eg a t ivit y). It is im p o rt a n t t o h ig h lig h t t h e fa ct t h a t n o fa lse- p o sit ive resu lt s were fo u n d wit h a n y o f t h e p rim ers t est ed .
In t he PCR an alysis of t he samples st u died, on ly o n e o f t h e p rim ers sp ecific fo r t h e a m p lifica t io n of the 123- bp IS6110 fragment was unable to detect Mt b DNA, wh ich was o n ly d et ect ed b y t h e p rim ers am p lifyin g t h e 5 41 - b p IS6110, t h e 6 5 kDa an d t h e 3 8 kDa t a rg et fra g m en t s, a s sh o wn in Ta b le 3 (a n a l y s i s o f c o n c o r d a n c e b e t w e e n p r i m e r a m p lifica t io n s o f t h e DNA). Th is o ccu rred in a p a u cib a cilla ry sa m p le wit h t h e lo west n u m b er o f iso la t ed co lo n ies p er sa m p le (fo u r co lo n ies in t h e t h ree Lö wen st ein - J en sen cu lt u re t u b es).
Mu lt ip le co p ies o f t h e IS6110 seq u en ce a re frequ en t ly presen t in t he Mt b gen om e. This resu lt s in h ig h e r P CR se n sit ivit y wh e n co m p a re d t o a m p lifica t io n s o f sin g le co p y seq u en ces (3 8 kDa , 6 5 kDa , a n d MP B6 4 ), e ve n w h e n t h e s e a re su b m it t ed t o t wo co n secu t ive PCR react io n s, su ch a s in t h e PCR- n est ed PCR t ech n iq u e. Th e h ig h p ercen t a g e o f p o sit ivit y in t h e sa m p les iso la t ed in t h e st a t e o f Am a zo n a s m a y in d ica t e t h a t t h e Mt b st rain s presen t a higher frequ en cy of copies of t his seq u en ce in t h eir g en o m e, wh ich can b e verified t h r o u g h t h e r e s t r i c t i o n f r a g m e n t l e n g t h p o lym o rp h ism . Th is p re m ise , a s w e ll a s t h e d ifferen ces in PCR p ro t o co ls, m ay exp lain t h e fact t h at p ro p o rt io n s o f p o sit ivit y fo u n d in t h e p resen t st u d y were h ig h er t h a n t h o se rep o rt ed in t h e lit erat u re.
Ou r resu lt s sh o w t h a t t h e p rim er p a irs st u d ied a re u se fu l fo r t h e id e n t ifica t io n o f Mt b g e n o m e
se q u e n ce s in co lo n ie s iso la t e d f ro m cu lt u re s, a st ra t e g y re co m m e n d e d b y Ko n t o s e t a l.(2 5 ). Th e id e n t if ica t io n p ro t o co l u sin g P CR wit h in 2 4 t o 4 8 h o u rs a f t e r cu lt u re b e co m e s m o re e sse n t ia l wh en t h ere is a n in su fficien t n u m b er o f co lo n ies sin ce , in su ch a ca se , id e n t if ica t io n t e st s ca n o n ly b e p e rf o rm e d a f t e r su b cu lt u re , d e la yin g t h e id e n t if ica t io n o f t h e m yco b a ct e riu m b y a t le a st 2 1 d a ys.
D e s p i t e t h e s m a l l n u m b e r o f s a m p l e s st u d ie d , we ca n co n clu d e t h a t , in t h e st a t e o f Am a z o n a s, IS6110 (1 2 3 - b p ) p rim e rs a re t h e m o st h ig h ly re co m m e n d e d f o r P CR in clin ica l sa m p les a n d in Mt b st ra in s o b t a in ed fro m h ea lt h ca re se rvice s t h a t p e rfo rm la b o ra t o ry d ia g n o sis o f TB. In o rd e r t o c o n f irm t h e s e re s u lt s , a g re a t e r n u m b e r o f sa m p le s a n d st ra in s sh o u ld b e a n a lyz e d u sin g t h e p rim e rs st u d ie d a n d t h a t o t h er la b o ra t o ries in t h e st a t e sh o u ld a lso b eg in t o e m p lo y t h is t e ch n iq u e .
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