The use of qualitative and quantitative polymer ase chain r eactions
for diagnosis of cytomegalovir us infections in bone
mar r ow and kidney tr ansplant r ecipients
Desenvolvimento e aplicação de PCRs quali-quantitativas para diagnóstico
de citomegalovirose em transplantados de rim e medula óssea
La ur o Julia no Ma r in1
, Aldo Albuque r que Cunha1
, Victo r Hugo Aquino1 and Luiz Tadeu Mo r aes Figueir edo1
ABSTRACT
The pu rpo se o f thi s wo rk wa s to te st a c yto m e ga lo vi ru s q u a li ta ti ve PCR a n d a se m i - q u a n ti ta ti ve PCR o n the de te rm i n a ti o n o f CMV lo a d i n le u k o c yte s o f b o n e m a rro w a n d k i d n e y tra n sp la n te d ( RT) p a ti e n ts. Th i rty th re e BMT a n d 3 5 RT p a ti e n ts p a rti c i p a te d o f th e stu d y. Th e DNA wa s su b je c te d to a q u a li ta ti ve PCR u si n g p ri m e rs th a t a m p li f y p a rt o f CMV gB ge n e . CMV lo a d o f po si ti ve sa m ple s wa s de te rm i n e d b y a se m i - q u a n ti ta ti ve PCR u si n g q u a n ti f i e d pla sm i ds i n se rte d wi th pa rt o f the gB ge n e o f CMV a s c o n tro ls. Th e se n si ti vi ty o f th e te st wa s d e te rm i n e d to b e 8 6 7 p la sm i d c o p i e s/
µ
g DNA. CMV lo a d s b e twe e n 2 ,1 1 8 a n d 7 2 ,4 4 3 c o p i e s/µ
g DNA we re o b se rve d i n 1 2 .1 % BMT re c i p i e n ts a n d b e twe e n 1 ,2 4 6 a n d 5 8 ,6 1 3 c o p i e s/µ
g DNA i n 2 2 .9 % RT re c i p i e n ts. Fu rth e r stu d i e s a re n e c e ssa ry to c o n f i rm th e u se f u ln e ss o f th i s CMV se m i - q u a n ti ta ti ve PCR i n tra n sp la n te d p a ti e n ts.Ke y-wor ds: CMV i n f e c ti o n . Qu a li ta ti ve PCR. Qu a n ti ta ti ve PCR. Bo n e m a rro w tra n sp la n t. Ki d n e y tra n sp la n t.
RESUMO
O o b je ti vo de ste tra b a lho f o i te sta r u m a PCR q u a li ta ti va e u m a PCR se m i q u a n ti ta ti va pa ra CMV pa ra de te rm i n a r a c a rga de CMV n o s le u c ó c i to s de pa c i e n te s tra n spla n ta do s de m e du la ó sse a e tra n spla n ta do s de ri m . Tri n ta e trê s pa c i e n te s TMO e 35 TR p a rti c i p a ra m d e ste e stu d o . O DNA f o i te sta d o p e la PCR q u a li ta ti va u ti li za n d o pr ime r s q u e a m p li f i c a m p a rte d o ge n e gB de CMV. As c a rga s de CMV da s a m o stra s po sitiva s fo ra m de te rm in a da s pe la PCR se m i- q u a n tita tiva u tiliza n do c o m o c o n tro le p la sm í d i o s q u a n ti f i c á ve i s i n se ri d o s c o m p a rte d o ge n e gB d e CMV. A se n si b i li d a d e d o te ste f o i d e 8 6 7 p la m í d i o s/
µ
g DNA. Ca rga s de CMV e n tre 2.118 e 72.443 c o pia s/µ
g DNA fo ra m o b se rva da s e m 12,1% do s TMO e n tre 1,246 e 58,613 c ó pia s/µ
g DNA f o ra m o b se rva da s e m 22,9% do s TR. Fu tu ro s e stu do s, c o m m a i o re s c a su í sti c a s sã o n e c e ssá ri o s pa ra c o n f i rm a r a u ti li da de d e sta PCR se m i q u a n ti ta ti va p a ra CMV e m p a c i e n te s tra n sp la n ta d o s.Palavr as-chave s: In f e c ç ã o po r CMV. PCR q u a li ta ti va . PCR q u a n ti ta ti va . Tra n spla n ta do s de m e du la ó sse a . Tra n spla n ta do s d e ri m .
1 . Unidade Multide par tame ntal de Pe sq uisa e m Vir o lo gia, Fac uldade de Me dic ina de Rib e ir ão Pr e to , Unive r sidade de São Paulo , Rib e ir ão Pr e to , SP, B r asil. Re se ar c h Suppo r te d b y FAPESP ( 8 2 6 /9 9 )
Addr e ss to:Pr o f. Luiz Tade u Mo r ae s Figue ir e do . Unidade Multide par tame ntal de Pe sq uisa e m Vir o lo gia/FMRP/USP. Av. B ande ir ante s 3 9 0 0 , 1 4 0 4 9 - 9 0 0 Rib e ir ão Pr e to , SP, B r asil.
Fax: 1 6 6 3 3 - 6 6 9 5 , Te l: 1 6 6 0 2 - 3 0 6 7 e -mail: ltmfigue @ fmr p.usp.b r
Re c e b ido par a pub lic aç ão e m 1 5 /1 /2 0 0 3 Ac e ito e m 1 6 /2 /2 0 0 4
Hum a n c yto m e ga lo vir us e s ( CMVs ) a r e m e m b e r s o f th e
b e tahe r pe svir us sub fam ily o f the He rp e svi ri d a e fam ily. The
vir io n , m e a s ur in g b e twe e n 1 5 0 a n d 2 0 0 n m in dia m e te r, h a s a l i p i d b i l a ye r e n ve l o p e , a n i c o s a h e d r a l c a p s i d
s ur r o un de d b y a pr o te ic m a tr ix , a n d a ge n o m e c o m po s e d
o f a do ub le s tr a n de d lin e a r DNA. Cito me galo vir us ge n o m e
o f a p p r o x im a te ly 2 4 0 k ilo b a s e s , in c lude s m o r e th a n 2 0 0
ge n e s , e n c o din g a t le a s t 3 5 s tr uc tur a l pr o te in s a n d a s till
un de fin e d n um b e r o f n o n s tr uc tur a l p r o te in s1 8. Th e vir a l
e n ve lo pe is c o m po s e d o f a t le a s t e igh t glyc o pr o te in s ; m o s t
o f t h e n e u t r a l i zi n g a n t i b o d i e s a r e d i r e c t e d a g a i n s t
Or gan and b o ne mar r o w tr ansplant r e c ipie nts may b e
infe c te d with CMV b y pe r so nal c o ntac t with the me dic al c ar e staff, b y me dic al pr o c e dur e s suc h as he mo dialysis, b lo o d
tr ansfusio ns, o r b y the tr ansplante d o r gans1 7.
After the infec tion, CMV stays latent inside several c ell types
o r, eventually, may pro duc e disease. Reac tivatio n of latent CMV
infec tio n may o c c ur in per io ds o f dec r easing c ellular immune r e spo nse as o b se r ve d in dise ase s suc h as AIDS, o r unde r
immuno suppr e ssive the r apie s suc h as tho se use d afte r so lid
o r gan and b o ne mar r o w tr ansplantatio n1 2 1 9.
Signs and symptoms of CMV disease inc lude fever, interstitial
p n e u m o n i a , ga s tr o i n te s ti n a l m a n i fe s ta ti o n s , r e ti n i ti s , p a n c yto p e n i a , l e u k o p e n i a , th r o m b o c yto p e n i a , a typ i c a l
lympho c yto sis, myalgia, sple no me galy and ar thr algia8. CMV
disease in so lid o rgans and bo ne marro w transplant rec ipients
usually o c c ur s o ne to fo ur mo nths after tr ansplantatio n9. CMV
c an also pr o duc e im m uno de pr e ssio n that c o uld augm e nt
opportunistic infec tions or episodes of graft rejec tion1 6.
Super-infec tio ns o c c ur when the patients bec o me Super-infec ted with a new
vir us ge no type . A study do ne in Rib e ir ão Pr e to , SP, B r azil, showed that 7 0 .6 % of the kidney transplant rec ipients presented
super-infec tio n by CMV1.
Ac tive CMV infec tion c an be detec ted by virus isolation using human fibroblast c ell c ultures, and the virus identific ation c an be
done with spec ific monoc lonal antibodies in immunofluoresc ent
or immunoperoxidase tests2 0 2 3. During the last dec ade, CMV
DNA detec tion by PCR and virus genome nuc leotide sequenc ing have been used for diagnosis and virus strain analysis in transplant
patients. Several studies using the PCR for CMV diagnosis in kidney transplant rec ipients have been reported in Brazil. Aquino
& Figueiredo1 observed that 1 0 0 % of the patients were infec ted
with CMV before renal transplantation. Costa et al1 0, detec ting
CMV genome in the urine, observed that 4 8 % of the renal transplant
rec ipients presented CMV infec tion and some of these presented
c linic al manifestations. Caballero et al6, found a low inc idenc e of
c linic al manifestations ( 8 .8 % ) in CMV infec ted patients. These studies show that CMV infec tions are highly prevalent among
Brazilian transplant recipients, and that a fraction of them present c linic al manifestatio ns related to CMV. Therefo re, a pro per
diagno sis o f CMV dise ase is impo r tant fo r an appr o pr iate management of transplant rec ipients.
This wo r k aimed to evaluate the usefulness o f a qualitative PCR and a simple and lo w c o st se mi-quantitative PCR fo r CMV
diagno sis in kidney and b o ne mar r o w tr ansplant patients.
MATERIAL AND METHODS
Pa tie nts. Thir ty-thr e e b o ne mar r o w tr ansplant ( B MT) rec ipients attended at the B o ne Marro w Transplant Unit o f the
Ge ne r al Ho spital o f the Me dic al Sc ho o l o f Rib e ir ão Pr e to ,
Univer sity o f São Paulo , b etween 1 9 9 9 and 2 0 0 0 , par tic ipated in the study. These were 1 8 males and 1 5 females, aged between
3 and 4 0 year s. B lo o d samples o f the B MT patients pr esenting
more than 1 0 0 0 leukoc ytes/mm3 were c ollec ted twic e a month,
fo r 4 mo nths afte r the tr ansplantatio n. A to tal o f 1 4 4 b lo o d
sample s we r e c o lle c te d fr o m B MT gr o up patie nts. So me B MT
patie nts die d imme diate ly afte r tr ansplantatio n and it was no t po ssible to c o llec t mo re than o ne sample.
Thir ty-five r e nal tr ansplant patie nts ( RT) manage d in the
Re nal Tr ansplant Unit o f the Ge ne r al Ho spital o f the Me dic al
Sc hool of Ribeirão Preto, University of São Paulo, between 1 9 9 6
and 1 9 9 7 , par tic ipate d in the study. The se we r e 2 2 male s and 1 3 females, aged b etween 2 1 and 7 3 year s o ld. B lo o d samples
fr o m RT gr o up patie nts we r e c o lle c te d b e fo r e tr ansplantatio n
and twic e a mo nth dur ing 4 mo nths afte r tr ansplantatio n. A
to tal o f 2 6 5 b lo o d sample s we r e o b taine d fr o m RT patie nts. So me RT patients died immediately after transplantatio n and it
was no t po ssib le to c o lle c t mo r e than o ne o r two sample s.
Personal and c linic al data were c ollec ted from the transplant
patient files. Fever asso c iated to pneumo nia was c o nsider ed a
c linic al manifestatio n po tentially asso c iated to CMV infec tio n.
However, though present in the BMT group of patients, diagnosis
c o uld no t b e c o nfir me d b y the de te c tio n o f CMV antige ns in
b lo o d le uk o c yte s1 1. Fo r the RT gr o up, fe ve r asso c iate d to
he pato sple no me galy o r pne umo nia was o b se r ve d in so me
partic ipants. These c linic al manifestations were also c onsidered
as po te ntially asso c iate d to CMV infe c tio n de spite ab se nc e o f
c o nfir m atio n b y CMV antige ns de te c tio n in b lo o d1 1. Gr aft
rejec tion in the RT patients was also analyzed regarding results
o f qualitative and semi-quantitative PCRs fo r CMV.
De te c tio n o f CMV antige ns in b lo o d le uk o c yte s fr o m B MT
patients was c arried out in the laboratory of Dr. Claudio Pannuti
at the Institute fo r Tr o pic al Me dic ine o f the Unive r sity o f São
Paulo , in São Paulo . De te c tio n o f CMV antige ns in b lo o d
le uk o c yte s was no t do ne in the RT patie nts b e c ause sample s
we r e o r iginally c o lle c te d j ust fo r a CMV ge no typing study and
o nly thr e e ye ar s late r we r e se le c te d fo r the pr e se nt study.
This r e se ar c h pr o j e c t was appr o ve d b y the Ethic B o ar d o f
the Gener al Ho spital o f the Medic al Sc ho o l o f Rib eir ão Pr eto ,
Univer sity o f São Paulo ( Pr o c ess 8 2 6 /9 9 ) .
CMV DNA pur ification. Five ml of blood was c ollec ted in tubes containing EDTA as anticoagulant. Peripheral blood leukocytes
( PBL) were separated by centrifugation in a dextran solution ( Sigma,
Germany) . The pellet containing the PBLs was suspended in 2 0 0 µl
of PBS and the DNA was extracted using the QIAamp DNA Blood Kit
( Qiagen, Germany) , following the manufacturer’s recommendation.
CMV DNA was also obtained from the supernatant of fibroblast cell
c ultures infec ted with CMV AD1 6 9 strain using the QIAamp DNA
Blood Kit ( Qiagen, Germany) .
Qu a lita tive P CR a n d n e s te d - P CR fo r CMV. Th e r e ac tio n mixtur e o f the qualitative PCR c o ntaine d, in a to tal
vo lume o f 5 0 µl, 7 5 mM o f Tr is-HCl ( pH 9 ) , 2 mM o f MgCl
2,
5 0 mM o f KCl, 2 0 mM o f ( NH
4) 2 SO4, 5 0 µM o f e ac h o ne o f the
deoxynuc leoside triphosphates, 0 .3 µM of primers gB 1 and gB 2
( as shown in Table 1 ) , and 1 µg of DNA obtained from PB Ls. The
r e ac tio n mixtur e was fir st inc ub ate d at 9 4oC fo r 3 min, the
te mpe r atur e was the n r e duc e d to 8 0oC, and 2 U o f Ta q DNA
po lymer ase wer e added. The PCR mixtur e was subj ec ted to 1 5
and to 3 0 c yc le s o f 6 0 se c at 9 4oC, 9 0 se c at 5 5oC, 1 2 0 se c at
7 2oC, and finally to 3 min at 7 2oC. Two mic roliters of this reac tion
were used in a nested-PCR c o ntaining the same c o mpo nents as
me ntio ne d ab o ve , e xc e pt fo r the inte r nal pr ime r s gB n1 and
gB n2 ( as sho wn in Tab le 1 )2. The r e ac tio n mixtur e was fir st
inc ubated at 9 4oC for 3 min, the temperature was then reduc ed
to 8 0oC, and 1 U o f Taq DNA po lyme r ase was adde d. The PCR
mixtur e was sub j ec ted to 3 0 c yc les o f 6 0 sec at 9 4oC, 6 0 sec at
5 5oC, and 6 0 sec at 7 2oC, and finally to 3 min at 7 2oC. PCR and
nested-PCR pr o duc ts wer e sub j ec ted to elec tr o pho r esis in 2 % agarose gel and the amplic on bands were visualized by UV after
ethidium bromide staining.
Eac h PCR assay inc luded a positive c ontrol with CMV AD1 6 9
DNA and a negative c o ntr o l c o ntaining distilled water. PCR fo r
β
-globin gene detec tion was performed in order to c onfirm theinte gr ity o f the DNA e xtr ac ts.
Cloning a fragment of the gB gene of CMV into a pCR2 .1 pla smid. The amplic o n o f 2 9 6 b p o b taine d fr o m the CMV AD1 6 9 DNA in the PCR using the primers gB 1 and gB 2 was fused
into the plasmid vec tor pCR2 .1 ( Invitrogen, USA) . The plasmids
were amplified in bac teria (Esc he ric hia c o li) and purified with
the Plasmid Mini Kit ( Qiagen, Germany) . The plasmids were
sequenc ed with the Thermo Sequenase CY5 .5 terminator kit
( Amersham, England) , using the M1 3 /Forward-Reverse primers. The sequencing reaction was resolved in an automated sequencer
( Seq 4 x 4 , Pharmacia, USA) . The obtained sequence was compared
with that of the CMV AD1 6 9 strain ( Genbank, seqüenc e M6 0 9 3 1 )
using the DNAsis software ( Hitac hi, Japan) .
Pla smid qua ntifica tio n. The mo le c ular we ight o f the pla s m id c o n ta in in g th e in s e r t o f 2 9 6 b p wa s c a lc ula te d c onsidering as 6 6 0 the average molec ular weight of a base pair
and was fo und to b e 1 4 0 0 1 9 0 . The r e fo r e , 1 4 0 0 1 9 0 g o f the
plasmid c o ntain 6 .0 2 x 1 02 3 plasmid mo le c ule s ( Avo gr ado ’s
numb e r ) . Thus, the numb e r o f plasmid par tic le s in o ur sto c k s o l u ti o n wa s d e te r m i n e d b a s e d o n th e p l a s m i d D NA c o nc e ntr a tio n o b ta ine d b y spe c tr o pho to m e tr y a t 2 6 0 nm .
Aliquo ts o f the quantifie d plasmid we r e sto r e d at -2 0oC until
use fo r the CMV semi-quantitative PCR tests.
Se nsitivity o f the CMV se mi- qua ntita tive PCR. The sensitivity o f the CMV semi-quantitative PCR using primers gB 1 and gB 2 was deter mined b y testing, in quadr uplic ate, dec imal dilutio ns o f the c lo ne d plasmid so lutio n. Spe c ific amplic o ns
with 2 9 6 b p we r e de te c te d until the 1 0-8-fo ld dilutio n, whic h
c o r r e spo nds to 8 6 7 plasmids, as sho wn in Figur e 1 .
CMV se mi- qua ntita tive PCR. CMV lo ad was deter mined in all c linic al sample s with a po sitive r e sult in the qualitative PCR. The pr o to c o l use d in the se mi-quantitative PCR was the
same as that used in the qualitative PCR, exc ept for the addition
of 2 .5 U of Taq DNA polymerase and 1
µ
g of the clinical sample DNA.For the semi-quantitative PCR, samples c ontaining 8 0 0 0 0 , 8 0 0 0 ,
8 0 0 and 8 0 plasmid partic les were tested simultaneously with the c lin ic a l s a m p le s . Th e PCR p r o duc ts we r e s ub j e c te d to
electrophoresis in 2% agarose gel and the amplicons were visualized
by UV light and photographed with the Sc ienc e 1 D digital c ame
( Kodak, USA) . The amplicon band densities were determined using the DC1 2 0 Digital Ac c ess software ( Kodak, USA) .
The CMV lo ad o f the c linic al sample s was de te r mine d b y
plo tting the amplic o n band density value o f eac h sample into a
graphic inc luding samples c ontaining 8 0 0 0 0 , 8 0 0 0 , 8 0 0 and 8 0
plasmid par tic le s, as sho wn in Figur e 2 .
Ta b le 1 - Prim e rs use d fo r the PCR a nd fo r the ne ste d- PCR de te c ting CMV in c lin ic a l sa m ple s.
Pr im e r s Nuc leotide sequenc e Annealing site in CMV genome
GB 1 5’GAAACGCGCGGCAATCGG 3’ 8 1 8 7 4 - 8 1 8 9 1
GB 2 5’ TGGAACTGGAACGTTTGGC3’ 8 2 1 5 8 - 8 2 1 7 6
gB n1 5 ’GCGCCGTTGATCCACACACC 3 ’ 8 1 9 6 0
gB n2 5 ’TACGCTGCAGTTCACCCCAG 3 ’ 8 2 0 5 5
Fi gu re 1 - Aga ro se ge l sta i n e d w i th e th i d i u m b ro m i d e sh o w i n g, u n d e r UV li gh t, a m p li c o n s o b ta i n e d f ro m d e c i m a l d i lu ti o n s o f th e gB CMV c lo n e d p la sm i d b y u si n g th e CMV se m i - q u a n ti ta ti ve PCR: c o lu m n 1 sh o w s th e 1 0 0 b p DNA la d d e r; c o lu m n 2 sh o w s a 2 9 6 b p a m p li c o n b a n d f ro m a 8 6 7 0 0 0 p la sm i d so lu ti o n ; c o lu m n 3 sh o w s a m p li c o n b a n d f ro m a 8 6 7 0 0 p la sm i d so lu ti o n , c o lu m n 4 , sh o w s a m p li c o n b a n d f ro m a 8 6 7 0 p la sm i d so lu ti o n ; c o lu m n 5 , sh o w s a w e a k a m p li c o n b a n d f ro m a 8 6 7 p la sm i d so lu ti o n ( li m i t d e te c ti o n o f th e te st) ; a n d c o lu m n 6 f ro m a 8 6 p la sm i d so lu ti o n , d o e s n o t sh o w a n y a m p li c o n b a n d .
0 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000
Plasm id num ber
A
m
pl
ic
on
de
ns
it
y
v
a
lue
1000 10000 100000 1000000 10000000
Sta ti sti c a l a n a lysi s. Pe r so nal and c linic al data as we ll as lab o r ato r y te st r e sults we r e c o nfr o nte d to r e sults o f the CMV qualitative amplific ation and with the semi-quantitative PCR tests
b y using the Chi-squar e and the Fisher ’s exac t test ( p < 0 .0 5 ) .
RESULTS
CMV qua lita tive PCR. One hundr e d fo r ty fo ur b lo o d samples from 3 3 BMT patients were tested by the CMV qualitative
PCR, and the CMV genome was detec ted in 1 6 ( 1 1 .1 % ) samples belonging to nine ( 2 7 .3 %) patients. Likewise, 2 6 5 blood samples
fr o m 3 5 RT patie nts we r e te ste d b y the CMV qualitative PCR,
and the CMV ge no me was de te c te d in 2 3 ( 8 .7 % ) sample s
b e lo nging to 1 3 ( 3 7 .1 % ) patie nts. All the se po sitive r e sults wer e c o nfir med by nested-PCR.
CMV s e m i- q u a n tita tive P CR. CMV DNA lo a d wa s de te r mine d in tho se sample s having CMV ge no me de te c te d b y
qualitative PCR. Four ( 2 5 % ) samples belonging to four ( 4 4 .4 % )
B MT patients showed CMV loads between 2 1 1 8 and 7 2 4 4 3 virus
par tic les/
µ
g DNA. Thir teen ( 2 5 % ) samples b elo nging to eight( 6 1 .5 %) RT patients showed CMV loads between 1 2 4 6 and 5 8 6 1 3
vir us par tic le s/
µ
g DNA.Asso cia tio n a na lysis o f pe r so na l a nd clinica l da ta fr o m BMT a n d RT p a t i e n t s wi t h r e s u l t s o f CMV qualitative and semi- quantitative PCRs. Personal, c linic al data and CMV lo ads fr o m B MT and RT patie nts having CMV
geno me detec ted by qualitative PCR ar e sho wn in Tables 2 and
3 , respec tively. Personal and c linic al data of these patients were c ompared with those of the remaining 2 4 B MT patients and the
2 2 RT patie nts who did no t have CMV ge no me de te c te d. No
assoc iation of positive qualitative PCR with personal and c linic al
Ta b le 2 - Pe rso na l a nd c linic a l da ta a lso inc luding CMV lo a ds ( b o ld type ) fro m the 9 BMT pa tie nts ha ving CMV ge no m e de te c te d b y q ua lita tive PCR.
Patient Sample Age Sex Disease Do no r Date of Date CMV load Antig Clinic al aspec t Outc ome
transplant of sample ( vp/µg DNA)
3 ; 2 3 1 M N H L auto lo go us 2 6 - 0 4 - 9 9 + 3 2 < 8 6 7 neg fever death
3 ; 3 + 4 3 < 8 6 7 neg
4 ; 2 3 9 F AML auto lo go us 2 4 - 0 6 - 9 9 + 3 4 < 8 6 7 neg fever death
4 ; 3 + 4 5 2 1 1 8 neg
6 ; 1 4 0 M CML allogenic 0 8 - 0 6 - 9 9 + 3 0 7 2 2 4 neg fever r e c o ve r y
6 ; 2 + 4 9 < 8 6 7 neg
7 ; 3 3 F SAA allogenic 1 5 - 0 6 - 9 9 + 5 2 < 8 6 7 neg fever r e c o ve r y
7 ; 6 + 1 0 1 < 8 6 7 neg
8 ; 5 1 9 M Hodgkin’s auto lo go us 0 1 - 0 7 - 9 9 + 5 9 6 3 0 9 5 neg fever, PN r e c o ve r y
disease
1 2 ; 1 2 2 F Hypo-plastic s allogenic 1 9 - 0 8 - 9 9 + 2 1 < 8 6 7 neg fever, PN death
yndr o m e
2 1 ; 2 2 4 F + 4 5 < 8 6 7 1 c ell
2 1 ; 3 B LA allogenic 1 8 - 0 2 - 0 0 + 6 0 7 2 4 4 3 neg fever, PN r e c o ve r y
2 1 ; 4 + 7 4 < 8 6 7 neg
2 1 ; 5 + 1 0 2 < 8 6 7 neg
2 7 ; 5 2 9 M CML allogenic 1 4 - 0 7 - 0 0 + 9 7 < 8 6 7 neg fever, PN death
2 8 ; 3 2 6 F Hodgkin’s disease auto lo go us 1 0 - 0 7 - 0 0 + 5 7 < 8 6 7 neg fever r e c o ve r y
Antig = CMV antigen detec tion in blood leukoc ytes, NHL = Non Hodgkin’s lymphoma B LA = biphenotypic ac ute leukemia, AML = ac ute myeloid leukemia, CML = c hronic myeloid leukemia, SAA = severe aplastic anemia, PN = pneumonia, M = male, F = female, Neg = negative test.
data o f the B MT and RT patie nts was o b se r ve d. Ho we ve r, B MT
patients having CMV loads higher than 1 0 0 0 0 CMV partic les/
µ
gDNA sho we d 1 0 0 % spe c ific ity and 1 0 0 % po sitive pr e dic tive
va lue r e ga r ding fe ve r a nd pne um o nia , a s we ll a s 7 0 . 8 %
se nsitivity. The ne gative pr e dic tive value was lo w, 2 2 . 2 % .
Corroborating this assoc iation, B MT patients having the highest
CMV lo ads ( 6 3 0 9 5 and 7 2 4 4 3 par tic le s/
µ
g DNA) pr e se nte dfever and pneumo nia. Asso c iatio n between CMV lo ad and fever
was no t o b se r ve d in RT patie nts, inc luding tho se with mo r e
than 1 0 0 0 0 CMV par tic le s/
µ
g DNA. Lik e wise , asso c iatio n o fgraft rejec tion with CMV genome detec tion was not observed in
the RT gr o up.
DISCUSSION
PCR h a s b e e n c o m m o n ly us e d fo r dia gn o s is o f CMV
infec tio ns sho wing a higher sensitivity when c o mpared to virus
isolation in tissue c ulture and to antigenemia detec tion. CMV is
also detec ted ear lier b y PCR than b y o ther tec hniques2 6. Other
a dva nta ge s o f the PCR inc lude sim plic ity, r e q uir e s sm a ll
quantities o f c linic al samples, and the use o f sto r ed samples.
The qualitative PCR fo r CMV geno me detec tio n fo llo wed by c onfirmatory nested-PCR with gB primers has been used in our
lab o r ato r y fo r pr o c essing ur ine, b lo o d, saliva, milk and o ther
se c r e tio n sample s, sho wing high se nsitivity and spe c ific ity.
The se se nsitivity and spe c ific ity r ate s ar e highe r than tho se
repo rted fo r CMV sero lo gic tests2 5. The CMV geno me detec tio n
b y qualitative PCR fo llo we d b y c o nfir mato r y ne ste d-PCR with
gB pr ime r s, pr o c e ssing ur ine and b lo o d sample s o f k idne y
tr ansplant patie nts, was ab le to de te c t 9 4 .3 % o f ac tive CMV
pr e s e n t s tudy, CMV q ua lita tive a n d s e m i- q ua n tita tive PCRs we r e performed using DNA extrac ts from PB L. These c ells were
se le c te d b e c ause dur ing CMV infe c tio n, b lo o d le uk o c yte s
par tic ipate in vir us spr e ading into diffe r e nt o r gans and the se
c e lls ar e k no wn as pr e fe r e ntial site s fo r vir al r e plic atio n
spec ially in immuno c o mpro mised ho sts and latenc y2 2.
A proportion of 2 7 .3 % of the B MT patients had CMV genome
detec ted by qualitative PCR. This result is similar to that reported
by Woo et al2 4, who detec ted CMV by PCR in 3 4 .1 % of bone
marrow transplant patients. The positivity rate of CMV qualitative
PCR was higher than that of antigenemia. Only one of the B MT
patients having CMV genome detec ted by qualitative PCR had CMV
pp6 5 detec ted in PBLs. This low positive rate of CMV antigenemia
suggests that only a small number of the B MT patients had ac tive
CMV infec tion or disease. Our results, as reported by other
authors, show that genome detec tion by PCR is more sensitive
than antigen detec tion in PB Ls for CMV diagnosis3. Considering
that some patients having fever, pneumonia, and virus genome
detec ted by PCR were negative for antigen detec tion in PB Ls,
highly suggestive evidenc es of CMV disease, this low sensitivity
antigenemia should not be c onsidered a suitable method for
diagnosis of CMV ac tive infec tion or disease. Likewise it should
not be c onsidered a suitable method for use as the gold standard
in the present study.
Amo ng RT patie nts, 3 7 .1 % had CMV ge no me de te c te d b y
qualitative PCR. Likewise, 2 3 % of these patients presented fever,
a c linic al manifestation that c ould be assoc iated to CMV disease. A highe r po sitive r ate o f CMV ge no me de te c tio n ( 6 3 .1 % ) was
reported by Mas et al1 5, studying PB Ls of 6 5 renal transplant
patie nts, altho ugh o nly 1 9 . 5 % o f the studie d patie nts had
symptoms suggestive of CMV disease. Costa et al1 0, studying urine
of 3 7 kidney transplant patients by PCR, observed CMV genome
detec tion in 6 4 .9 % of the patients, 4 3 .2 % of these with symptoms
suggestive of CMV disease. However, only three patients were c onfirmed as having CMV disease based on the presenc e of virus
in bronc hoalveolar sec retions or by lung histopathology. In the
study by Costa et al1 0, most of these reported symptoms were
probably c aused by other etiologic agents.
The detection of CMV genome in about 1 /3 of both BMT and RT
patients, shows that the qualitative PCR test with gB primers is a suitable technique for screening CMV infections in transplanted
patients. However, CMV qualitative PCRs were not suitable to
disc riminate latent or ac tive CMV infec tions. Other studies of
immunocompromised patients show a positive correlation between
CMV load and active infection or disease3. Some of these studies have
demonstrated an association between high CMV viremias and clinical
manifestations or increased risk of CMV disease4. In the last five
years, several CMV semi-quantitative and quantitative diagnostic
techniques were reported3. However, in spite of their high sensitivity,
these techniques are expensive, which impedes its routine use in third world countries, such as Brazil. In the present work, we evaluated
the usefulness of a low cost and easily handled CMV semi-quantitative
PCR technique for CMV diagnosis of active infections and disease.
This CMV semi-quantitative PCR technique uses gB CMV cloned
plasmids obtained in our laboratory as positive controls.
The ab se nc e o f a suitab le go ld standar d te c hniq ue fo r
c o mpariso n with PCR fo r the diagno sis o f CMV ac tive infec tio n
and CMV disease is a limitatio n o f the present study3. Likewise,
Ta b le 3 - Pe rso na l a nd c linic a l da ta a lso inc luding CMV lo a ds ( b o ld) fro m the 13 RT pa tie nts ha ving CMV ge no m e de te c te d b y q ua lita tive PCR.
Patient Age Sex Do no r Date of Date of CMV load Clinic al c risis of Outc ome
Sample transplant sample ( pv/µg DNA) aspec t Rejec tion
9 ; 4 2 3 M c adave r 3 0 - 1 1 - 9 6 + 3 4 5 8 2 1 0 fever, PN ye s nephrec to my
9 ; 6 + 4 8 1 9 3 1
9 ; 7 + 5 5 4 9 2 0
1 1 ; 9 7 0 M c adave r 2 0 - 1 1 - 9 6 + 2 9 6 < 8 6 7 n o n o R KG
1 3 ; 6 2 7 F c adave r 1 1 - 1 2 - 9 6 + 3 7 < 8 6 7 fever, PN ye s R KG
1 5 ; 3 + 1 5 4 7 8 6
1 5 ; 4 + 2 2 3 4 3 5
1 5 ; 5 4 8 M c adave r 2 6 - 0 3 - 9 7 + 2 9 2 2 8 0 3 n o n o R KG
1 5 ; 6 + 3 5 < 8 6 7
1 5 ; 7 + 1 1 1 < 8 6 7
1 5 ; 8 + 1 5 4 < 8 6 7
1 6 ; 4 5 1 F c adave r 2 7 - 3 - 9 7 + 2 8 < 8 6 7 no ye s R KG
1 6 ; 5 + 4 1 < 8 6 7
1 9 ; 3 6 2 F c adave r 0 6 - 0 6 - 9 7 + 4 0 < 8 6 7 n o n o R KG
2 0 ; 3 4 3 F c adave r 2 8 - 0 6 - 9 7 + 2 6 < 8 6 7 n o ye s nephrec to my
2 0 ; 4 + 4 0 2 6 1 2 1
2 1 ; 5 3 3 M c adave r 3 0 - 0 6 - 9 7 + 3 8 1 2 4 6 n o ye s R KG
2 4 ; 8 4 1 M c adave r 2 0 - 0 6 - 9 7 + 1 7 3 < 8 6 7 n o n o R KG
2 5 ; 4 4 1 M c adave r 2 0 - 0 6 - 9 7 + 6 2 5 5 7 1 n o n o R KG
2 7 ; 8 3 5 M c adave r 1 3 - 0 8 - 9 7 + 1 0 5 4 9 3 1 7 fever ye s nephrec to my
2 9 ; 4 1 8 F c adave r 0 5 - 0 9 - 9 7 + 3 3 5 8 6 1 3 n o n o R KG
2 9 ; 5 + 5 4 3 9 8 1 0
3 1 ; 5 5 2 M c adave r 2 8 - 0 9 - 9 7 + 4 5 3 7 4 1 n o n o R KG
despite of many reported CMV quantitative PCR tec hniques, the
c omparison of these tec hniques with ours is diffic ult bec ause they were used for proc essing different kinds of c linic al samples suc h
as plasma, whole blood, or urine1 3 1 4. Furthermore, some authors
who used leukocyte samples for quantification of CMV also showed
unsuitable results for comparison due to very different sensitivities.
Toyoda et al2 1 described a highly sensitive technique, able to detect
five virus partic les/
µ
g DNA. Caballero et al5, desc ribed a CMVsemi-quantitative PCR having a similar sensitivity to that of the
present work, 8 0 0 virus partic les/
µ
g DNA. We suppose that thesensitivity of our CMV semi-quantitative PCR tec hnique, 8 6 7 virus
particles/
µ
g DNA, is suitable for the diagnosis of CMV active infectionand disease based on the need of viral replic ation and high viral
loads for the expression of these manifestations3.
Vir al lo ads o f 2 1 1 8 to 7 2 4 4 3 vir us par tic le s/
µ
g DNA we r edetec ted in the B MT patients by using the CMV semi-quantitative
PCR. The se patie nts c o r r e spo nde d to 4 4 .4 % o f tho se having
CMV genome detec ted by the qualitative PCR. The B MT patients
pr e se nting the highe st CMV lo ads ( 6 3 0 9 5 and 7 2 4 4 3 vir us
par tic le s/
µ
g DNA) also pr e se nte d fe ve r and pne umo nia. Anasso c iatio n with presenc e o f fever and pneumo nia, despite no t
b eing c o nfir med as CMV disease, was investigated in two B MT
patients with a CMV lo ad higher than 1 0 0 0 0 partic les/
µ
g DNA,sho wing 1 0 0 % spec ific ity and 1 0 0 % po sitive pr edic tive value.
All the samples of RT that had CMV detec ted by the qualitative
test, were subjec ted to CMV semi-quantitative PCR. CMV loads
above 8 6 7 partic les/
µ
g DNA were observed in 6 1 .5 % of thesamples. CMV loads between 1 2 4 6 and 5 8 6 1 3 virus partic les/
µ
gDNA were observed. No association between CMV loads, including
spec ific ally those higher than 1 0 0 0 0 partic les/
µ
g DNA, withpresence of fever was observed among the RT patients. Our results
are in agreement with those reported by other authors. Toyoda et
al2 1, studying 2 5 kidney transplanted and 9 5 heart transplanted
patients during three years, showed that patients having 5 0 0 CMV
partic les/
µ
g DNA did not present symptoms suggestive of CMVdisease while other patients with 5 0 to 1 0 0 CMV partic les/
µ
gDNA presented these c linic al symptoms.
Gr aft r ej ec tio n c r isis in the RT patients was no t asso c iated
with CMV loads. Nevertheless, kidney graft rejec tion c risis c ould
b e e xplaine d b y many o the r c ause s no t asso c iate d with CMV
infec tions.
In sho r t, qualitative PCR fo llo wed b y c o nfir mato r y nested-PCR with gB primers allowed the sc reening of CMV infec tions in
B MT and RT patie nts. The CMV ge no me was de te c te d in an
e xpr e ssive amo unt o f patie nts. Ho we ve r, qualitative PCR was
not able either to diagnose or to discriminate CMV active infection fr o m dise ase . Our se mi-quantitative PCR was ab le to de te c t
CMV lo ads ab o ve 8 6 7 par tic le s/
µ
g DNA in 4 B MT and 9 RTpatients suggesting that these were c ases of ac tive infec tion; this
is a nec essary c ondition for CMV disease. One hundred perc ent spe c ific ity and 1 0 0 % po sitive pr e dic tive value fo r fe ve r and
pneumonia was observed among B MT patients having high CMV
lo ads ( mo r e than 1 0 0 0 0 vir al par tic le s/
µ
g DNA) . High CMVlo ads we r e also o b se r ve d in five RT patients but a signific ant asso c iatio n o f high CMV lo ad with the pr esenc e o f c linic al
manifestations suggestive of CMV disease among them was not
observed. We suppose that only a small number of the transplant rec ipients that partic ipated in the present study had CMV disease.
However, it is possible that CMV was the etiologic al agent of the
c linic al manifestations in the two BMT patients having high CMV
load. We c onc lude that the studied population did not allow a definitive opinion on the usefulness of our CMV semi-quantitative
PCR for the diagnosis of CMV disease in BMT and in RT patients.
Further studies are necessary in order to confirm the usefulness of
this CMV semi-quantitative PCR in transplanted patients.
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