1 3 1
ARTIGO/ARTICLE
Revista da So ciedade Br asileir a de Medicina Tr o pical 3 7 ( 2 ) :1 3 1 - 1 3 4 , mar- abr, 2 0 0 4
Recommendations for the detection of
Leptospira
in ur ine by PCR
Recomendações para detecção de
Le pto spira
em urina pela PCR
Paula M.A. Lucchesi1, Guiller mo H. Ar r o yo1, Analía I. Etchever r ía1,
Alber to E. Par ma1
and Alfr edo C. Seijo2
ABSTRACT
In the pre se nt study PCR wa s a pplie d to de te ct le pto spire s in hum a n urine . Se ve ra l a ppro a che s fo r sa m ple pro ce ssing we re eva lua ted to o ptim ize the detectio n o f lepto spires in urine m ixed with this ba cterium . Furtherm o re, so m e cha nges in the co m po sitio n o f the rea ctio n m ix were studied. No a m plifica tio n wa s o bserved in a cidic urine, therefo re neutra liza tio n o f the sa m ple im m edia tely a fte r co lle ctio n is stro ngly re co m m e nde d. PBS ga ve b e tte r re sults tha n Tris o r Na OH a s ne utra lizing re a ge nts. Fre e zing a nd tha wing o f sa m ple s b e fo re pro ce ssing yie lde d ne ga tive re sults. Elim ina tio n o f e pithe lia l ce lls, le uk o cyte s a nd crysta ls b y ce ntrifuga tio n a t 3,000 rpm a t ro o m tem pera ture increa sed sensitivity. In a dditio n, bo th the wa shing step a fter co llecting lepto spires by centrifuga tio n a nd the inclusio n o f 0.1% b o vine se rum a lb um in in the re a ctio n m ix m inim ize d the inte rfe re nce o f o the r inhib ito ry co m po unds. The se m o difica tio ns we re use ful to im pro ve the de te ctio n o f Leptospira in urine b y PCR.
Ke y-wor ds: Le pto spir a. PCR. Uri n e . PCR i n h i b i to rs. Hu m a n .
RESUMO
No pre se nte e studo , a PCR fo i utiliza da pa ra de te c ta r le pto spira s e m urina hum a na . Dive rsa s a b o rda ge ns pa ra pro c e ssa m e nto d e a m o stra f o ra m a va li a d a s p a ra o ti m i za r a d e te c ç ã o d e le p to sp i ra s e m u ri n a m i stu ra d a c o m e sta b a c té ri a . Alé m d i sso , a lgu m a s m u da n ç a s n a c o m po si ç ã o da m i stu ra de re a ç ã o f o ra m a n a li sa da s. Nã o se o b se rvo u a m pli f i c a ç ã o e m u ri n a á c i da , c o n se q ü e n te m e n te , a n e u tra li za ç ã o da a m o stra i m e di a ta m e n te a pó s a c o le ta é f o rte m e n te re c o m e n da da . PBS a pre se n to u m e lh o re s re su lta d o s q u e Tri s o u Na OH c o m o re a ge n te s n e u tra li za d o re s. Co n ge la m e n to e d e sc o n ge la m e n to d e a m o stra s a n te s d o p ro c e ssa m e n to p ro d u zi ra m re su lta d o s n e ga ti vo s. Eli m i n a ç ã o d e c é lu la s e p i te li a i s, le u c ó c i to s e c ri sta i s p o r c e n tri f u ga ç ã o a 3.000rpm , à te m pe ra tu ra a m b i e n te , a u m e n to u a se n si b i li da de . Ade m a i s, a m b a s, a e ta pa de la va ge m a pó s a c o le ta de le pto spira s po r c e n trifu ga ç ã o e a in c lu sã o de a lb u m in a de so ro b o vin o a 0,1% n a m istu ra de re a ç ã o m in im iza ra m a i n te rf e rê n c i a d e o u tro s c o m p o sto s i n i b i d o re s. Essa s m o d i f i c a ç õ e s c o n tri b u í ra m p a ra m e lh o ra r a d e te c ç ã o d e Le pto spir a e m u ri n a a tra vé s d a PCR.
Pal avr as-chave s: Le pto spir a. PCR. Uri n a . In i b i d o re s PCR. Hu m a n a .
1 . Lab o r ato r io de Inmuno químic a y B io tec no lo gía, Fac ultad de Cienc ias Veter inar ias, Univer sidad Nac io nal del Centr o de la Pr o vinc ia de B ueno s Air es, Pinto 3 9 9 , 7 0 0 0 Tandil, Ar gentina. 2 . Ser vic io de Zo o no sis, Ho spital de Enfer medades Infec c io sas “Dr. Fr anc isc o Javier Muñiz”, Uspallata 2 2 7 2 , 1 2 8 2 B ueno s Air es, Ar gentina. Financ ial suppo r t: FONCYT, CIC and SECYT- UNCPB A. AEP and AIE ar e me mb e r s o f CIC and GHA is a me mb e r o f the CONICET.
Addr e ss to: Dr a. Paula Luc c he si. Lab o r ato r io de Inmuno q uímic a y B io te c no lo gía, Fac ultad de Cie nc ias Ve te r inar ias, Unive r sidad Nac io nal de l Ce ntr o de la Pr o vinc ia de B ue no s Air e s, Pinto 3 9 9 , 7 0 0 0 Tandil, Ar ge ntina.
Te le fax: 5 4 2 2 9 3 - 4 2 - 6 6 6 7 /4 2 - 2 3 5 7 . e -mail: paulaluc @ ve t.unic e n.e du.ar Re c e b ido par a pub lic aç ão e m 2 8 /0 2 /2 0 0 3 Ac e ito e m 2 2 /1 2 /2 0 0 3
Leptospirosis is a widespread disease that affec ts wild and domestic animals as well as humans1 2. There are several reports on the frequency of leptospirosis among livestock, wild animals and human beings in South America mainly in Argentina and Brazil4 1 9 2 0. Humans may be infec ted indirec tly from animals by c ontac t with c ontaminated water, soil or mud in a moist environment, or by direc t infec tion from urine, fresh c arc asses or organs6.
There are many possible c linic al presentations and c ourses of human leptospirosis. In the past two dec ades, an inc reasing
number of c ases of leptospiral pulmonary hemorrhages have been reported8 1 9 2 1. As a c onsequenc e of the broad spec trum of nonspec ific symptoms, leptospirosis c an be misdiagnosed and c linic al differential diagnosis is required between leptospirosis and severe influenza, viral meningitis, hepatitis, hemorrhagic fevers or nephritis, among other conditions6. A conclusive diagnosis cannot be made without laboratory confirmation.
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Luc c he s i PMA e t al
although it is a c omplex test to c ontrol, perform and interpret1 2. Live c ultures of all serovars required for use as antigens must be maintained. Another drawback is that antibodies are only detectable in blood approximately 5 to 7 days after the onset of symptoms.
Efforts for early diagnosis of leptospirosis are directed towards the detection of leptospires or their DNA or antigens in blood, cerebrospinal fluid, urine, and tissues. The techniques available are direct examination for leptospires, culture, detection of leptospiral antigens with antibodies, and detection of leptospiral DNA with homologous nuc leic ac id sequenc e probes, with or without amplification by polymerase chain reaction ( PCR)6 1 6.
The PCR assay can be applied to selectively amplify specific DNA sequences by more than 1 06 fold1 7. Therefore, PCR is very useful for the rapid detection of organisms involved in acute infections. In fact, oligonucleotide primers have been developed by several research teams and applied for the specific detection of certain serovars of
Lepto spira1 8 2 2 2 3. Gravekamp e t a l9 designed two pairs of primers
for the specific amplification of DNA of pathogenic leptospires. With some modifications, this method was applied in the analysis of several strains isolated in Argentina, from water, soil and human patients1 5.
Bec ause endogenous substanc es present in urine c an inhibit PCR, we r epo r t sever al appr o ac hes fo r sample pr o c essing and some c hanges in the c omposition of the reac tion mix to optimize detec tio n o f this bac terium.
MATERIALS AND METHODS
Ba c te r ia l s tr a in a nd c ultur e me dium. Le p to sp i ra
i n te rro ga n s ser o var po m o n a, do nated b y Dr. Gleyr e Do r ta de
Mazzo ne lli ( DILACOT-SENASA, B ue no s Air e s) , was gr o wn in EMJH medium5 1 1 at 2 7 ºC and perio dic ally subc ultured in fresh medium. This strain had been typed by the c ro ss-agglutinatio n ab so r ptio n test2 4.
Ur ine mixed with lepto spir es. Aliquots were taken from a L. i n te rro ga n s ser o var po m o n a c ultur e in EMJH. Cells wer e washed twic e in sterile distilled water by c entrifugation at 1 2 ,0 0 0 r pm ( m ic r o c e n tr ifuge So r va ll RMC 1 4 ) fo r 2 0 m in a n d r e suspe nde d in distille d wate r. Afte r e stimating the b ac te r ial c o nc entr atio n by nephelo metr y, differ ent aliquo ts wer e added to ur ine fr o m a he althy human to ac hie ve c o nc e ntr atio ns o f 1 ,0 0 0 , 1 0 ,0 0 0 , 1 0 0 ,0 0 0 and 1 millio n bac teria per ml.
Sa mple pr e pa r a tio n fo r PCR. Sample s o f ar tific ially inoc ulated urine were inc ubated at 4 0 ºC for 1 0 min, to eliminate amorphous sediment, and then c entrifuged at 3 ,0 0 0 rpm for 1 0 min at room temperature to eliminate epithelial c ells, leukoc ytes and c r ystals c o mmo nly pr e se nt in ur ine . Le pto spir e s we r e c o nc e ntr ate d b y c e ntr ifugatio n at 1 2 ,0 0 0 r pm fo r 2 0 min, resuspended in 1 0 0
µ
l distilled water and boiled for 1 0 min. Two different volumes ( 1 0 and 3 5µ
l) of sample were tested by PCR in a total volume of 5 0µ
l.To this general proc edure the following modific ations were made in o r de r to inc r e ase the se nsitivity o f the PCR.
Ne u tra li za ti o n o f u ri n e :ur ine was ne utr alize d to pH 7 .6
befo re adding the lepto spires. Different so lutio ns were used to ac hieve this pH: 0 .1 M Tris, 0 .2 M NaOH and phosphate-buffered saline ( PB S) .
Fr e e z i n g th e u r i n e : s a m p l e s o f u r i n e s e e de d wi th le pto s pir e s we r e s ub j e c te d to fr e e zin g a t - 2 0 º C, b e fo r e c entr ifugatio n. This mo dific atio n was analyzed to deter mine if ur ine samples c o uld b e fr o zen b efo r e pr o c essing.
Wa sh i n g th e p e lle t o f le p to sp i re s: afte r c e ntr ifuging to c onc entrate leptospires and before boiling, a washing step with distilled water was added.
Additio n o f b o vin e se ru m a lb u m in ( BSA) to the re a c tio n m ix: different c onc entrations of B SA, from 0 .0 1 3 to 0 .1 % ( w/v) , were added to the PCR reac tio n7 instead o f gelatin.
PCR. Th e r e a c tio n m ix wa s c o n s titute d b y 5 0 m M KCl, 1 0 mM Tris-HCl pH 9 .0 , 0 .1 % ( v/v) Triton X-1 0 0 , 2 .5 mM MgCl2, 0 .0 1 % ( w/v) ge latin, 2 5 0 µM e ac h dNTP, 0 .5 µM e ac h pr ime r ( G1 , G2 , B 6 4 -I, B 6 4 -II) , 1 U Ta q DNA polymerase. Amplific ation was pe r fo r me d with pr ime r s whic h o nly de te c t patho ge nic le pto spir e s9 1 5.
One dr o p o f miner al o il was added to eac h tub e to pr event e vapo r atio n. PCR amplific atio ns we r e pe r fo r me d as fo llo ws: o ne initial c yc le o f 9 4 ºC fo r 1 8 0 " , o ne final c yc le o f 7 2 ºC fo r 2 4 0 " and 3 8 c yc le s o f 9 0 " at 9 4 ºC ( de natur ing) , 9 0 " at 5 5 ºC ( annealing) and 1 5 0 " at 7 2 ºC ( extension) .
A po sitive c o ntr o l with le pto spir e s in the ab se nc e o f ur ine was always r un, and r e sults we r e r e fe r r e d to this.
Agar o se gel electr o pho r esis. Ten µl aliquo ts fro m eac h DNA amplific atio n we r e analyze d b y ho r izo ntal agar o se ge l e le c tr o pho r e sis and UV tr ansilluminatio n ( 3 0 0 nm) . Ge l was c o nstitute d b y 1 .5 % ( m/v) agar o se and 1 .2 g/ml-1 e thidium b r o m ide in r un n in g b uffe r ( 8 9 m M Tr is , 8 9 m M b o r ic a c id, 1 mM EDTA, pH 8 .0 ) .
RESULTS AND DISCUSSION
Sample pro c essing fo r PCR is c ritic al and must be adj usted to the tissue, fluid, and spec ies being tested. Several substanc es fo und in the var io us type s o f c linic al mate r ial inhib it PCR, the r e fo r e po sitive spe c ime ns may go unde te c te d b e c ause o f false-negative results.
There are many referenc es about inhibition of Ta q DNA polymerase by several factors such us chelation of free magnesium ions, hemoglobin, bile salts, acidic polysaccharides from glycoproteins and extreme pH variations3 1 0 1 3 1 4. Phenol and chloroform, often used for DNA extraction and purification, are also considered to be inhibitors1 0.
As a consequence of the presence of inhibitors, some DNA purification steps are necessary before performing PCR amplification. These purification steps increase the cost of the test as well as the time required for diagnosis. Therefore, we tested procedures and conditions for sample preparation which are quick and inexpensive, and do not require the use of expensive kits to purify DNA.
1 3 3 Re vis ta da So c ie da de Br a s ile ir a de Me dic ina Tr o pic a l 3 7 : 1 3 1 - 1 3 4 , m a r- a br, 2 0 0 4
by PCR. This emphasizes the need to neutralize the urine sample imme diate ly afte r c o lle c tio n, to avo id lo sing b ac te r ial DNA in the washing step. PB S buffer rendered better results than either 1 M Tris or 0 ,2 M NaOH as neutralizing reagents ( Figure 1 , lanes 1 to 3 ) .
Wh e n th e u r i n e m i x e d wi th l e p to s p i r e s wa s fr o ze n a t - 2 0 ºC and thawe d, b e fo r e c e ntr ifugatio n ste ps, no le pto spir e s c o uld b e de te c te d b y PCR ( Figur e 1 , lane s 4 and 5 ) . This sugge sts that whe n ur ine sam ple s ar e go ing to b e te ste d b y PCR to de te c t le pto spir e s, the y have to b e ne utr alize d, and washe d b e fo r e sto r age at -2 0 º C. I n the c ase o f sam ple s that ar e no t pr o c e sse d o n the sam e day, the y c an b e sto r e d at 5 º C until the fo llo wing day, afte r ne utr alizatio n.
The steps of sample preparation to improve the detec tion of le pto spir e s in ur ine ar e summar ize d in Figur e 2 .
The optimum c onc entration of B SA used in the reac tion mix fo r PCR was 0 .1 % . ( Figur e 3 , lane s 1 3 to 1 5 ) . This pr o te in
to note that an internal c ontrol for the amplific ation reac tion is ne c e ssar y b e c ause inhib ito r y sub stanc e s c an var y am o ng patients o r between samples fr o m the same patient.
ACKNOWLEDGEMENTS
The autho r s thank M.R. Or tiz fo r her tec hnic al assistanc e.
REFERENCES
1 . Ale xande r AD. Le p to sp i ra. In: Le nne tte EH, Spaulding E, Tr uant J P ( e ds) Ma n u a l o f c l i n i c a l m i c r o b i o l o gy, 2n d e di ti o n , Am e r i c a n S o c i e ty fo r
Mic r o b io lo gy, Washingto n, DC, p. 3 4 7 - 3 5 4 , 1 9 7 4 .
2 . Alsto n J M, B r o o m J C. Le pto spir o sis in Man and Animals. Livingsto ne Ltd, Lo ndo n, p. 2 7 7 - 3 0 0 , 1 9 5 8 .
3 . B e j AK, Mahb ub ani MH. The r mo stab le DNA po lyme r ase fo r in vitr o DNA a m plific a tio n . In : Gr iffin H, Gr iffin A ( e ds ) PCR te c h n o lo gy: c ur r e n t inno vatio ns, CRC Pr e ss, Lo ndo n, p. 2 1 9 - 2 3 7 , 1 9 9 4 .
4 . Co sta E, Co sta YA, Lo pe s AA, Sac r ame nto E, B ina J C. Fo r mas gr ave s de le pto spir o se : aspe c to s c línic o s, de m o gr áfic o s e am b ie ntais. Re vista da So c ie dade B r asile ir a de Me dic ina Tr o pic al 3 4 : 2 6 1 - 2 6 7 , 2 0 0 1 .
5 . Ellinghause n J r HC, Mc Cullo ugh WC. Nutr itio n o f Le p to sp i ra p o m o n a and gro wth o f 1 3 o ther sero types: a serum-free medium emplo ying o leic albumin c o mple x. Ame r ic an Jo ur nal o f Ve te r inar y Re se ar c h 2 6 : 3 9 -4 4 , 1 9 6 5 .
6 . Faine S. Le pto spi ra and Le pto spir o sis. CRC Pr e ss, Inc , B o c a Rato n, Flo r ida, 1 9 9 4 .
7 . Fo r b e s B A, Hi c k s KE. S u b s ta n c e s I n te r fe r i n g wi th Di r e c t De te c ti o n o f Myc o b a c te r i u m tu b e r c u lo s i s i n Cl i n i c a l S p e c i m e n s b y P CR : Effe c ts o f B o vi n e S e r u m Al b u m i n . J o u r n a l o f Cl i n i c a l Mi c r o b i o l o gy 3 4 : 2 1 2 5 -2 1 -2 8 , 1 9 9 6 .
TREATMENT FOR URINE SAMPLES
1) NEUTRALIZE
IMMEDIATELY
PH 7.6 PBS
2) ELIMINATE
AMORPHOUS
SEDIMENT
40 ºC 10MIN
3) ELIMINATE
EPITHELIAL CELLS,
LEUKOCYTES AND
CRYSTALS
3000RPM10MIN AT
ROOM TEMP.
4) CONCENTRATE
LEPTOSPIRES
12000RPM 20MIN
AT 4ºC
5) ELIMINATE OTHER
INHIBITORS IN THE
PELLET
WASH BY
CENTRIFUGATION
Fi gu re 2 - Re c o m m e n d e d ste p s f o r u ri n e sa m p le p re p a ra ti o n .
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6
Fi gu re 3 - Ef f e c t o f b o vi n e se ru m a lb u m i n ( BSA) o n the de te c ti o n li m i t o f le pto spi re s i n u ri n e b y PCR. La n e s 1, 2 a n d 3 c o rre spo n d to u ri n e c o n ta i n i n g 104, 105 a nd 106 le pto spire s/m l, re spe c tive ly, with 0.013% BSA a dde d to the
re a c tio n m ix; la n e s 4, 5 a n d 6: with 0.025% BSA; la n e s 7, 8 a n d 9: with 0.05% BSA; la n e s 10, 11 a n d 12: wi th 0.078% BSA; la n e s 13, 14 a n d 15: wi th 0.1% BSA; la n e 16: po si ti ve c o n tro l ( a m pli f i c a ti o n o f u ri n e - f re e le pto spi ra l DNA) .
1 2 3 4 5
Fi gu r e 1 - I n f lu e n c e o f t h e n e u t r a li z i n g a ge n t o n le pto spi ra l DNA a m pli f i c a ti o n f ro m u ri n e . Sa m ple s wi th: PBS ( la n e 1) , 0.2 M Na OH ( la n e 2) , 1M Tri s ( la n e 3) . Pro du c ts c o rre spo n di n g to a sa m ple f ro ze n a n d tha we d e i the r a f te r ( la n e 4) o r b e f o re ( la n e 5) b e i n g pro c e sse d f o r DNA e xtra c ti o n , we re a lso e le c tro pho re se d .
plays a dual ro le sinc e it c an adso rb residual quantities o f PCR inhib ito r s and also inc r e ase s the stab ility in so lutio n o f Ta q DNA po lymer ase.
A s a m ple vo lum e o f 3 5
µ
l in a 5 0µ
l PCR r e a c tio n m ix r e nde r e d a b and with a gr e ate r inte nsity than the use o f 1 0µ
l o f sample vo lume fo r PCR. Ho we ve r, the r e ar e c ase s in whic h inc r e asing sam ple vo lum e c an r e sult in inc r e ase d inhib itio n o f the e nzym e , b e c ause inhib ito r y sub stanc e s pr e se nt in the sample also inc r e ase in q uantity. The lo we r limit o f de te c tio n wa s 1 04 le pto s pir e s in 1 m l o f ur in e .1 3 4
Luc c he s i PMA e t al
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2 0 . Se ij o A, Dr aghi G, Do r ta de Mazzo ne lli G, Mazzo ne lli J, Stie b e l C, Ar ge nto E, Ca m in o a R , De o da to B y c o la b o r a do r e s de la CCLA. I n fo r m e s o b r e le pto spir o sis e n la Re púb lic a Ar ge ntina. Fundac ió n Mundo Sano , B s As. Se r ie Enfe r me dade s Tr ansmisib le s, pub lic ac ió n mo no gr áfic a 3 , 2 0 0 2 .
2 1 . Tr evej o RT, Rigau-Per ez JG, Ashfo r d DA, Mc Clur e EM, Jar quin-Go nzalez C, Amado r JJ, de lo s Reyes JO, Go nzalez A, Zaki SR, Shieh WJ, Mc Lean RG, Nasc i RS, We yant RS, B o lin CA, B r agg SL, Pe r k ins B A, Spie ge l RA. Epide m ic le pto spir o sis asso c iate d with pulm o nar y he m o r r hage - Nic ar agua, 1 9 9 5 . Jo ur nal o f Infec tio us Diseases 1 7 8 :1 4 5 7 - 1 4 6 3 , 1 9 9 8 .
2 2 . Van Eys GJ J M, Gr ave k amp C, Ge r r itse n MJ , Quint W, Co r ne lisse n MTE, Te r Sc he gge t J , Te r pstr a WJ . De te c tio n o f le pto spir e s in ur ine b y po lyme r ase c hain r e ac tio n. J o ur nal o f Clinic al Mic r o b io lo gy 2 7 : 2 2 5 8 - 2 2 6 2 , 1 9 8 9 . 2 3 . Wo o dwa r d MJ , Sulliva n GJ , Pa lm e r NMA, Wo o lle y J C, R e ds to n e J S .
De ve lo pme nt o f a PCR te st spe c ific fo r Le p to sp i ra h a rd jo ge no type b o vi s. Ve te r inar y Re c o r d 1 2 8 : 2 8 2 - 2 8 3 , 1 9 9 1 .
