1 . Núc le o de Do e nç as Infe c c io sas do Ce ntr o B io mé dic o da Unive r sidade Fe de r al do Espir ito Santo , Vitó r ia, ES, B r azil. Suppo r te d b y TB RU.
Addr e ss to: Dr. Mo isé s Palac i. NDI/CB /UFES. Av. Mar e c hal Campo s 1 4 6 8 , Mar uípe , 2 9 0 4 0 - 0 9 1 Vitó r ia, ES, B r azil. e - mail: mpalac i@ ndi. ufe s. b r
Te l: 5 5 2 7 3 3 3 5 - 7 2 0 9 , Fax: 5 5 2 7 3 3 3 5 - 7 2 0 6 Re c e b ido par a pub lic aç ão e m 2 3 /5 /2 0 0 3 Ac e ito e m 2 6 /7 /2 0 0 4
The r e e me r ge nc e o f tub e r c ulo sis ( TB ) thr o ugho ut the wo rld and o utbreaks o f multi-drug resistant ( MDR) TB during
the last de c ade de mo nstr ate the ne c e ssity fo r an e ar ly and
ac c ur ate diagno sis o f this infe c tio us dise ase1 6 1 8. I n m o st
r o utine c linic al lab o r ato r ie s the de te c tio n o f Myc o b a c te ri u m tu b e rc u lo si s is still b ase d o n the mic r o sc o pic e xaminatio n o f ac id-fast staine d sme ar s and c ultur e . Ho we ve r, staining
fo r ac id-fast b ac illi ( AFB ) lac k s se nsitivity, var ying fr o m 5 0 %
to 6 0 % , and does not distinguish M. tub e rculo sis from other myc obac teria1 1 2 3. Cultures in solid media are more sensitive but
c an take 3 to 6 weeks until the results are available5. The BACTEC
radiometric ( Bec ton-Dic kinson Diagnostic s, Towson, MD, USA)
system, the MGIT ( Bec ton Dic kinson Diagnostic s, Towson, MD, USA) c ulture system and nuc leic ac id probes have improved the
speed of isolation and identific ation but still require several days
before a definitive diagnosis c an be made1 1 9.
Evaluation of a commer cial test based on ligase chain r eaction for dir ect
detection of
Mycobacteriu m tu bercu losis
in r espir ator y specimens
Avaliação do método comercial baseado na reação em cadeia da ligase para detecção direta
do
Myco ba cte rium tube rculo sis
em espécimes pulmonares
Fabíola Kar la Cor r êa Ribeir o
1, Valdér io do Valle Dettoni
1, Renata Lyr io Per es
1, Solange Alves
Vinhas
1, Tatiana Resende Có
1, Reynaldo Dietze
1and Moisés Palaci
1ABSTRACT
A li ga se c h a i n re a c ti o n DNA a m p li f i c a ti o n m e th o d f o r d i re c t d e te c ti o n o f Myc o b ac te r ium tub e r c ulo sis ( Ab b o tt LCx MTB) i n re sp i ra to ry sp e c i m e n s wa s e va lu a te d . Re su lts f ro m LCx MTBAssa y we re c o m p a re d wi th th o se f ro m a c i d f a st b a c i lli sm e a r, c u ltu re , a n d f i n a l c li n i c a l d i a gn o si s f o r e a c h p a ti e n t. A to ta l o f 2 9 7 re sp i ra to ry sp e c i m e n s ( sp u tu m a n d b ro n c h i a l la va ge ) f ro m 1 9 3 p a ti e n ts we re te ste d . Th e se n si ti vi ty, sp e c i f i c i ty, p o si ti ve p re d i c ti ve va lu e a n d n e ga ti ve p re d i c ti ve va lu e o f LCx vs c u ltu re we re 9 2 .7 %, 9 3 %, 6 7 .8 % a n d 9 8 .7 %, re sp e c ti ve ly. Wh e n c o m p a re d to th e c li n i c a l f i n a l d i a gn o si s, th e se n si ti vi ty, sp e c i f i c i ty, PPV a n d NPV f o r LCx we re 8 8 .9 %, 9 6 .8 %, 8 6 .5 % a n d 9 7 .4 %, re sp e c ti ve ly. Th e se n si ti vi ty o f LCx MTB a ssa y wa s 7 5 % f o r sm e a r- n e ga ti ve , c u ltu re p o si ti ve sa m p le s. Th e re su lts i n d i c a te th a t LCx MTB a ssa y i s a ra p i d , si m p le a n d va lu a b le te c h n i q u e a s a c o m p le m e n ta ry to o l f o r th e d i a gn o si s o f tu b e rc u lo si s.
Ke y-words: Tu b e rc u lo si s. Di a gn o si s. Mo le c u la r b i o lo gy.
RESUMO
O m é to d o d e a m p li f i c a ç ã o d e DNA b a se a d o n a re a ç ã o e m c a d e i a d a li ga se ( Ab b o tt LCx MTB) f o i a va li a d o p a ra d e te c ç ã o do Myc o b ac te r ium tub e r c ulo sis e m e sp é c i m e s p u lm o n a re s. Os re su lta d o s d o LCx MTBf o ra m c o m p a ra d o s a o s re su lta d o s d e b a c i lo sc o p i a , c u ltu ra e d i a gn ó sti c o c lí n i c o p a ra c a d a p a c i e n te . Um to ta l d e 2 9 7 e sp é c i m e s ( e sc a rro e la va d o b ro n c o a lve o la r) d e 1 8 9 p a c i e n te s f o ra m te sta d a s.Os va lo re s d e se n si b i li d a d e , e sp e c i f i c i d a d e , va lo r p re d i ti vo p o si ti vo e va lo r p re d i ti vo n e ga ti vo d o LCX vs c u ltu ra f o ra m 9 2 ,7 %, 9 3 %, 6 7 ,8 % e 9 8 ,7 %, re sp e c ti va m e n te . Qu a n d o c o m p a ra d o s a o d i a gn ó sti c o c lí n i c o , o s va lo re s d e se n si b i li d a d e , e sp e c i f i c i d a d e , VPP e VPN p a ra o LCx f o ra m 8 8 ,9 %, 9 6 ,8 %, 8 6 ,5 % e 9 7 ,4 %, re sp e c ti va m e n te . A se n si b i li d a d e d o LCx MTB f o i d e 7 5 % p a ra a s a m o stra s c o m b a c i lo sc o p i a n e ga ti va e c u ltu ra p o si ti va . Os re su lta d o s i n d i c a m q u e o te ste LCx MTB é si m p le s, rá p i d o , e f i c i e n te e p o d e se r u ti li za d o c o m o u m re c u rso c o m p le m e n ta r p a ra o d i a gn ó sti c o d a tu b e rc u lo se .
Diagnostic tec hniques based on molec ular biology methods
are able to dramatic ally reduc e the time of detec tion ( within ho ur s) as we ll as inc r e ase the se nsitivity fo r de te c ting M. tub e rculo sis. The sensitivity of non-c ommerc ial PCR assays has varied widely, from 5 5 % to 1 0 0 % in various studies3 9 1 3 1 7. The
use of c ommerc ial kits with spec ific proc edures and c ontrols, suc h as the Amplified M. tub e rculo sis Direc t Test ( Gen-Probe Inc ., San Diego, CA, USA) and the Amplic or M. tub e rculo sis test ( Roc he Diagnostic s, Basel, Switzerland) , may reduc e this
inter-laboratory variation6.
In the last years, ligase c hain reac tion ( LCR) tec hnology has bec ome c ommerc ially available for the direc t detec tion of M. tub e rculo sis in c linic al samples. The LCxTM M. tub e rculo sis Assay
( Ab b o tt Lab o r ato r ie s, No r th Chic ago , IL, USA) is the fir st
c o mmer c ial semi-auto mated nuc leic ac id amplific atio n test developed for use with respiratory spec imens1 0. However, the
c linic al utility of this method, mainly c onc erning its sensitivity
for smear-negative samples, in a population with high tuberculosis
prevalenc e, suc h as in Brazil, has not been widely studied.
In this study, we evaluated the LCxTM M. tub e rculo sis assay in
a c linic al laboratory using pulmonary samples. A c linic al c ase
definition of tuberc ulosis c onsistent with c ase reporting c riteria
was used as the referenc e-standard for evaluating the utility of
all diagnostic tests.
MATERIAL AND METHODS
Clinical specimens. A to tal o f 2 9 7 respirato ry spec imens ( sputum a nd b r o nc hia l la va ge ) fr o m 1 9 3 pa tie nts b e ing
s c r e e n e d o r u n d e r tr e a tm e n t fo r tu b e r c u l o s i s a t th e
Pneumo lo gy Clinic o f Ho spital Univer sitár io Cassiano Antô nio
Mo r ae s ( Espír ito Santo , B r azil) we r e inc lude d in the study.
Sample pr ocessing. Respiratory spec imens were liquefied and dec ontaminated with an equal volume of N-ac etyl-c ysteine-NaOH to a final c onc entration of 2 % and inc ubated for 1 5 min at
room temperature. After dec ontamination, PBS was added to all
the spec imens for a final volume of 5 0 ml. The mixture was c entrifuged at 3 0 0 0 x g for 1 5 min at 4 ºC and the sediment was resuspended in PBS.
Micr o sco py. The sediment was subjec ted to mic rosc opic examination for ac id-fast bac illi ( AFB) by standard proc edures
with fluoresc ent and/or Ziehl Neelsen stains1 2.
Cultur e . The sediment was inoc ulated in Lowenstein-Jensen medium, inc ubated at 3 7 ºC for a maximum of 6 weeks and
inspec ted weekly for growth, and c ultivated in B ACTEC 4 6 0
radiometric method ( Bec ton-Dic kinson Diagnostic s) for 6 weeks
with the growth index c hec ked 3 times a week. The myc obac teria
isolates were identified using standard methods1 2.
LCx MTB a s s a y. Th e tr e a te d s a m ple wa s pr o c e s s e d ac c o r ding to the manufac tur e r ’s r e c o mme ndatio ns. B r ie fly,
5 0 0
µ
l o f e a c h tr e a te d s a m ple we r e put in a s c r e w- c a pmic r o c e ntr ifuge tub e , c e ntr ifuge d twic e and r e suspe nde d in o r d e r to m i n i m i ze th e p o te n ti a l i m p a c t o f i n h i b i to r s ,
inac tivate d fo r 2 0 min at 9 5 ºC in the LCx c o ve r e d dr y b ath
( Abbott Laboratories) and lysed for 1 0 min in the LCx lysor
( Abbott Laboratories) . For the amplific ation reac tion, 1 0 0
µ
l of th e s upe r n a ta n t we r e tr a n s fe r r e d to a r e a dy- to - us e tub ec ontaining 1 0 0
µ
l of the LCR mixture. The specimens and controlswere plac ed in the LCx thermal c yc ler and amplified for 3 7 c yc les
of inc ubation for 1 s at 9 4 ºC, 1 s at 6 4 ºC and 4 0 s at 6 9 ºC. Amplified tubes were transferred unopened to the c arousel of the LCx
analyzer, whic h direc tly detec ts the amplific ation produc ts by a
mic ropartic le enzyme immunoassay. Results were expressed as
fluoresc enc e rates and were c ompared to the c alibrator rate. A sample rate/c utoff value ratio of > 1 .0 indic ates an LCx MTB
assay positive result.
Pa tie nts’ clinica l da ta . After spec imen c ollec tion, the me dic al and e pide mio lo gic al r e c o r ds o f all patie nts we r e
reviewed. In c ases in whic h disc repant results for the LCx MTB assay and the c ulture were obtained, the responsible physic ians
we r e c o ntac te d and c linic al data we r e e valuate d. Clinic al
assessment inc luded patient history, signs, symptoms, c hest
X-rays, laboratory results, and follow-up observations as well as results obtained from additional specimens previously taken from
the patient.
Da ta a na lys is . The mic robiologic al data of all patients we r e r ec o r ded using TB No tes So ftwar e ( NDI-UFES & Gaia
Informátic a) . Sensitivity, spec ific ity, and positive and negative predic tive values were c alc ulated for LCx MTB as c ompared with
standard c ulture of spec imens and, later, with c linic al data.
RESULTS
A to ta l o f 2 9 7 s pe c im e n s fr o m 1 9 3 pa tie n ts ( a ve r a ge
o f 1 . 5 s a m p l e s p e r p a ti e n t) b e i n g s c r e e n e d o r u n de r
tr e a tm e n t fo r tub e r c ulo s is we r e e xa m in e d b y m ic r o s c o py, c ultur e and the LCx assay. Of the se , 4 2 ( 1 4 % ) we r e po sitive
fo r m yc o b a c te r ia e ith e r b y s o lid c ultur e o r b y B ACTEC 4 6 0
T B . T h e LCx MT B As s a y ga ve a p o s i ti ve r e s u l t o n 5 6
( 1 8 . 8 % ) s a m ple s , o f wh ic h 3 8 ( 1 2 . 8 % ) we r e po s itive a ls o
b y c ultur e a n d 1 8 ( 6 % ) o n ly b y LCx MTB ; 2 3 8 ( 8 0 . 1 % ) s a m p l e s we r e n e g a t i ve b y a l l m e t h o d s . Al l p o s i t i ve
s p e c i m e n s we r e i d e n t i fi e d a s M. t u b e r c u l o s i s. T h e s e n s itivity, s pe c ific ity, po s itive pr e dic tive va lue ( PPV) a n d n e g a t i ve p r e d i c t i ve va l u e ( NP V) o f t h e LCx t e s t , i n
c o m pa r is o n with c ultur e r e s ults , we r e 9 2 . 7 % , 9 3 % , 6 7 . 8 %
a n d 9 8 . 7 % , r e s pe c tive ly ( Ta b le 1 ) .
Ta ble 1 - Co m pa riso n o f LCx MTB a nd AFB results with culture results fo r detectio n o f M. tuberculosis in 297 pulm o na ry sa m ples.
Culture Sens. Spec. PPV NPV
Test result positive negative % % % %
AFB 6 2 .0 9 9 .6 9 6 .3 9 4 .0
positive 26 1
negative 16 2 5 4
LCx-MTB 9 2 .7 9 3 .0 6 7 .8 9 8 .7
positive 38 18
negative 3 2 3 8
Ta ble 2 - Perfo rm a nce o f LCx-MTB in AFB sm ea po sitive versus sm ea r-nega tive sa m ples
LCx-MTB
Test result positive negative Sensitivity %
AFB pos/culture pos 26 0 1 0 0 .0
AFB neg/culture pos 12 4 7 5 .0
Ta ble 3 - Co m pa riso n o f LCx-MTB a nd culture results fo r 193 pa tients with a nd witho ut tuberculo sis.
Culture Sens. Spec. PPV NPV
Test result positive negative % % % %
AFB
positive 29 0 8 0 .5 1 0 0 .0 1 0 0 .0 9 5 .7
negative 7 1 5 7
LCx-MTB 8 8 .9 9 6 .8 8 6 .5 9 7 .4
positive 32 5
negative 4 1 5 2
Sens. = Sensitivity; Spec. = Specificity; PPV = Positive Predictive Value; NPV = Negative Predictive Value
Ta ble 4 - Ana lysis o f discrepa nt results between LCx-MTB a nd clinica l dia gno sis.
Patient’s
Patient SR/CVRa LCx/result Culture Ageb sexc Clinical diagnosisd Comments Final
inter-no
result pretation of LCxf
1 1 .6 7 + - 60 M transient cough FP
2 1 .5 4 + - 22 M bronchitis FP
3 1 .6 8 + - 67 M COPDe FP
4 4 .2 1 + - 36 F transient cough FP
5 2 .4 6 + - 45 F bacterial pneumonia FP
6 0 .1 2 - + 76 M active TB FN
7 0 .0 5 - - 64 F active TB FN
8 0 .0 5 - - 40 M active TB FN
9 0 .0 4 - - 28 M active TB HIV positive FN
aSample rate/cutoff value ratio of > 1 .0 . bAge in years. cF. female; M. male . dAssessment based on signs, symptoms, routine laboratory results, chest X rays, results of TB culture and follow up. eChronic obstructive pulmonary disease.. fFP False positive; FN False negative.
DISCUSSION
When the performance of a new diagnostic method is evaluated,
the performance of a standard test is a critical parameter. For detection of M. tube rculo sis, culture using either conventional medium such as Lowenstein-Jensen or BACTEC 1 2 B medium has
been c onsidered to be the gold standard. The sensitivity and
specificity of these methods are often reported to be higher than 9 0 %1 1 1 5. However, when nucleic acid amplification procedures
are used, clinical diagnosis should also be taken into account,
since the DNA detected by these methods can indicate the presence
of non-viable bacilli1 5. For that reason, the calculated values in
our study were based not only on the performance of the culture
method but also on clinical and historical laboratory data. Nucleic
acid amplification test evaluations may also be biased as a result
of inclusion of multiple specimens from individual patients4. The
present study avoided these biases by collecting an average of only
1 .5 specimens per patient.
Our r e sults sho w that unde r r o utine c linic al c o nditio ns
b o th c ultur e and LCx-MTB te sts pr e se nte d go o d analytic al
performanc es. When c ompared to c ulture, the sensitivity value
( 9 2 .7 % ) of the LCx-MTB assay was shown to be as high as those
reported by other investigators for respiratory spec imens with
several amplific atio n tests7 2 0 2 2 2 3. The three negative results
obtained by LCx amplific ation assay for c ulture-positive samples
may be explained by the presenc e of inhibitors of enzymatic
amplific ation. We did not searc h for the presenc e of inhibitory
substanc es; however, these samples were retested and negative
results were obtained upon repeat assay. Disc repanc y between
positive predic tive values may be explained by the fac t that several
samples c ame from tuberc ulosis patients undergoing treatment.
The long lasting ability to detec t DNA after c ultures bec ome
negative is well doc umented for DNA amplific ations systems and
makes LCx-MTB unsuitable for the monitoring of therapeutic
effic ac y1 5 2 1.
The ab ility o f a te st to de te c t M. tu b e rc u lo si s r apidly in AFB sme ar-ne gative sample s fr o m patie nts sympto matic fo r pulmo nar y TB is o f o b vio us impo r tanc e . Fr o m an o pe r ative
standpo int, mic r o sc o pic e xaminatio n o f staine d sme ar s is the The data in Tab le 2 de mo nstr ate the c o r r e latio n b e twe e n
LCx and sme ar /c ultur e r e sults. Of the 4 2 po sitive spe c ime ns, 2 6 we r e po sitive fo r AFB . The se nsitivity o f the LCx me tho d
was 1 0 0 % fo r the sme ar-po sitive sample s and 7 5 % fo r the
sme ar-ne gative sample s.
A c linic al c ase de finitio n o f tub e r c ulo sis was use d as the
r e fe r e nc e -standar d to de te r mine the utility o f all diagno stic te sts ( Tab le 3 ) . In o ur study a to tal o f 3 6 ( 1 8 .6 % ) patie nts
we r e c o nfir me d as ne w pulmo nar y tub e r c ulo sis c ase s. An
ave r age o f 1 .8 spe c ime ns was c o lle c te d fo r the se patie nts and
th e y h a d s im ila r c ultur e a n d LCx r e s ults . Th e r e we r e 9 disc r e pant r e sults b e twe e n LCx and final c linic al diagno sis
( Tab le 4 ) . Five patie nts with o the r r e spir ato r y pr o b le ms at
the time o f e xaminatio n had the ir spe c ime ns c o nside r e d false
po sitive b e c ause b o th fo llo w up o b se r vatio n and c ultur e r e sults did no t c o nfir m the pr e se nc e o f ac tive TB . Spe c ime ns
fr o m the r emaining 4 patients wer e c o nsider ed false-negative.
One o f them had a sputum po sitive c ultur e and the o ther thr ee
patie nts had no b ac te r io lo gic al c o nfir matio n o f TB b ut the ir c linic al pr e se ntatio n and r e spo nse to tr e atme nt, r e sulte d in
mo st r apid way to de te c t myc o b ac te r ia in r e spir ato r y tr ac t
spe c ime ns. Ho we ve r, it is no t a ve r y se nsitive te st, as 4 0 to 5 0 % o f patie nts with pulm o nar y tub e r c ulo sis have sm e ar
ne gative spe c ime ns5 1 1 2 3. The de lay in initiating dr ug the r apy
m a y r e s u l t i n p r o gr e s s i o n o f th e d i s e a s e , a s we l l a s
tr ansmissio n o f Myc o b a c te ri u m tu b e rc u lo si s to o the r s. In these c ases, the r apid detec tio n o f M. tu b e rc u lo si s b y a dir ec t a m p l i fi c a ti o n te s t c o u l d l e a d to e a r l i e r i n i ti a ti o n o f
antitub e r c ulo us tr e atme nt. In o ur study the high se nsitivity
o f the LCx-MTB was espec ially relevant fo r the 1 6 AFB -negative sample s, 1 2 ( 7 5 % ) o f whic h we r e ide ntifie d b y LCx MTB .
Se ve r al studie s have de m o nstr ate d that PCR m e tho ds ar e
signific antly less sensitive ( 4 5 -7 0 % ) o n AFB -negative samples,
and it has b e e n an o b stac le fo r the use o f PCR o n sme ar-ne gative sample s2 1 4 2 1.
A c linic al c ase de finitio n o f tub e r c ulo sis was use d as the
r e fe r e nc e -standar d to de te r mine the utility o f all diagno stic
te sts in o ur study. Thus, whe n c o m par e d to final c linic al
dia gn o s is , th e s e n s itivity wa s h igh e r fo r th e LCR - b a s e d amplific atio n me tho d ( 8 8 .9 % ) than fo r the c ultur e me tho ds
( 8 0 . 5 % ) ; ho we ve r, the y we r e lo we r than the se nsitivitie s
r e po r te d b y Fa dda e t a l, whic h we r e 9 6 . 8 % a nd 9 2 . 7 % , r e s pe c tive ly8. Th is diffe r e n c e c o uld b e e xpla in e d b y th e
he te r o ge ne o us gr o up o f patie nts se le c te d in o ur study, whic h
inc luded patients with HIV infec tio n and / o r AIDS and patients
with no n-c avitar y tub e r c ulo sis.
Studie s have var ie d in the ir manage me nt o f disc r e pant
r esults1 0 2 2 2 3. Our c o mpr ehensive study, enc o mpassing 1 9 3
patients, demonstrates only 9 disc repant results between LCx
and final c linic al diagnosis. Five patients had their spec imens
c onsidered false positive, although the follow-up was too short
to exc lude eventual ac tive tuberc ulosis. The false-negative results for 4 patients may be explained by ( i) the presenc e of possible
amplific atio n inhib ito r s in the sample ; ( ii) a no n-unifo r m
distribution of mic roorganisms in the test suspension; or ( iii) a
low c onc entration of mic roorganisms in the sample.
I n s um m a r y, o ur s tudy de m o n s tr a te d th a t LCR- MTB is
a s e n s i t i ve a n d s p e c i f i c m e t h o d f o r t h e d e t e c t i o n o f
M. t u b e r c u lo s i s i n r e s p i r a to r y s p e c i m e n s . T h e a s s a y pro to c o ls were easy to perfo rm and were suitable fo r the wo rk
flo w o f a r o utine mic r o b io lo gy lab o r ato r y. LCR-MTB pr o vide s the c linic ian and infe c tio n c o ntr o l pr o gr am with valuab le ,
r apid and c linic ally r e le vant info r matio n fo r the diagno sis o f
pulmo nar y tub e r c ulo sis. Ho we ve r, the LCR assay sho uld no t b e c o n s ide r e d a s ub s titute b ut r a th e r a c o m ple m e n t to
tr a d i ti o n a l m i c r o b i o l o gy te c h n i q u e s , wi th th e a i m o f
i n c r e a s i n g th e s e n s i ti vi ty a n d s p e e d o f d i a g n o s i s o f tub e r c ulo sis.
ACKNOWLEDGEMENTS
We thank Ab b o tt Lab o r ató r io s do B r asil fo r supplying the
LCx MTB Assay k its and Ne il Gupta fo r he lp in pr e par atio n o f
th e m a n us c r ipt. We a ls o a c k n o wle dge th e Tub e r c ulo s is Re se ar c h Unit – TB RU fo r te c hnic al suppo r t.
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