TRANSACTIONS OFTHE ROYAL SOCIETY OFTROI’ICAL MEDICINE AND HYGIENE (1998) 92,290-293 Diagnosis of Wuchereria bancroffi infection by the polymerase chain reaction using urine and day blood samples from amicrofilaraemic patients Wagner A. Lucenal*, Rafael Dhalia’, Frederic0 G. C. Abath2, Luc Nicolas3, Leda N. Regisl and Andre F. Furtadol lDepartamento de Entomologia and 2Departamento de Immunologia, Centro de Pesquisas Aggeu Magalhdes- FIOCRUZ, Av. Pro& Moraes Rego sin, Cidade UniversitcEria, 50670-420 Recif, PE, Brasil; 3Unitg d’Immunophysiologie Cellulaire, Institut Pasteur, 25 Rue du Dr Roux, 75724, Paris, France Abstract A sensitive and specific polymerase chain reaction @‘CR) based on a highly repeated deoxyribonucleic acid (DNA) sequence (188 bp; SspI repeat) was tested for the detection of Wuchereria bancrofti DNA in blood and urine samples collected during the day from individuals in Coque, Recife, Brazil, an endemic area for Wbancrofti. All microfilaraemic individuals were also positive by PCR, irrespective of the samples used.The PCR system was capable of detecting Wbancrofti DNA in amicrofilaraemic individuals: c. 93% were positive by PCR when day blood samples were used and 59.7% when urine samples collected at 07:OO were used. Thus, nocturnally periodic IK bancroft infection can be detected in blood samples col- lected during the day, which is convenient for large-scale screening. In addition, non-invasive urine collection provided suitable samples for PCR, which is clearly advantageous for preliminary mass diagnosis. Keywords: filariasis, Wuchereria bancrofti, diagnosis, polymerase chain reaction, blood, urine Introduction Bancroftian filariasis affects over 90 million people in 76 nations, and more than 905 millions live in endemic areas (WHO, 1992). The mean Wucheretia bancrofiimi- crofilaraemia prevalence rate has shown a steady in- crease in the last 10 years in Recife, a city in north- easternBrazil (1.5% in 1983,3.5% in 1991 and 5.1% in 1992). A recent survey estimated that in some urban ar- eas prevalence reached 14% (MACIEL et al., 1996). Evaluation of infection in communities is necessary before initiating mass treatment for control of lymphatic filariasis and also for monitoring control programmes (OTTESEN & RAMACHANDRAN, 1995). Night-time bleeding for detecting the nocturnally periodic micro- tilaraemia has several problems and therefore methods using daytime blood are preferred. Species-specific de- oxyribonucleic acid (DNA) probes for diagnosis of W. bancrofti infection have been reported by several authors (DISSANAYARR et al., 1991; THEODORE et al., 1993; SIRIDEWA et al., 1994). A very sensitive and specific polymerase chain reaction (PCR), based on a highly re- peated DNA sequence (SspI repeat), has been devel- oped (ZHONG et al., 1996). This assay is capable of detecting W bancrofti genomic DNA in blood samples (WILLIAMS et al., 1996), mosquitoes (CHANTEAU et al., 1994; NICOLAS et al., 1996; FURTADO et al., 1997) and paraffin-embedded tissues (MCCARTHY et al., 1996). In addition, it could detect W bancrofti DNA in day blood samples (MCCARTHY et al., 1996; FURTADO et al., 1997). However, blood collection is often rejected by part of the community. It requires a certain degree of expertise and proper precautions, and may therefore not be suitable for mass campaigns (ABBASI et al., 1996). Non-invasive urine collection would be clearly advanta- geous for mass diagnosis. In the present paper we describe a PCR-based tech- nique to detect filarial DNA in urine samples, collected during the day, and compare the results with those ob- tained using blood samples. Material and Methods Human blood and urine samples Nocturnal blood samnles (NBS) were collected from 22:00 to 02:00, and diuinal blood’samples (DBS) from 11:OO to 14:00, by venepuncture into 5 mL Vacutain- ers@ containing ethylenediaminetetraacetic acid (ED- *Address for correspondence: W. Lucena, Departamento de Entomologia, CPqAM-FIOCRUZ, Av. Prof. Moraes Rego s/n, Cidade Universithia, CEP 52020-200, Recife, FE, Brazil; fax +5581 453 3046, e-mail Wagner@cpqam.fiocruz.br TA). First urine samples (FUS) were collected at 07:OO and second urine samples (SUS) at ll:OO, in 50 mL Falcon@ tubes. All PCRs were performed once with each sample; there was no duplicate assay. A total of 54 NBS, 53 DBS, 72 FUS and 43 SUS were collected from individuals living in an endemic area of Brazil (Coque, Recife). Thirty-nine of these in- dividuals, who had been found to be microfilaraemic by microscopical examination of finger-prick blood during a previous survey, had simultaneous blood and urine samples collected. The other 15 NBS, 14 DBS, 33 FUS and 14 SUS were collected separately. All individuals provided nocturnal blood films for microscopical exam- ination; informed consent was obtained from all blood donors. Blood and urine samples from healthy individuals liv- ing in non-endemic areas (7 from USA and France and 20 from Manaus in Brazil) and 10 individuals from an endemic area (Recife, Brazil), who were found to be amicrofilaraemic by blood filtration, were used as nega- tive controls for PCR assays. Extraction of DNA from blood and urine samples Five hundred & of plasma, separated from the hu- man blood samples, were thoroughly mixed with 500 l.rL of phenol:chloroform (1: 1) and centrifuged at 5000 rev/ min for 10 min. The supernatant was transferred to a fresh tube containing 100 uL of 8 M potassium acetate and 1000 l.tL of ice-cold absolute ethanol and then cen- trifuged at 14000 rev/min for 10 min. The pellet was washed with 70% ethanol, dried at room temperature in a vacuum centrifuge, resuspended in 25 & of MilliQ@ autoclaved water and kept at -20°C until used for the PCR. Ten mL of each urine sample were precipitated by ad- dition of 20 mL of ice-cold absolute ethanol and 1 mL of 4 M sodium acetate and centrifuged at 5000 revimin for 10 min. The supernatant was discarded and 1 mL of 4.5 M guanidine thiocyanate, 1.2% Triton X100@, 50 mM Tris-HCI (pH 6.4), 20 mu EDTA solution was added. The total volume was transferred to a 1.5 mL Eppendorf@ tube. The DNA was purified by addition of 40 Ilr, of silica suspension and incubation for 10 min at room temperature. Following centrifugation for 10 s at 5000 rev/min, pellets were washed once with ice-cold absolute ethanol and dried by vacuum centrifugation. DNA was eluted bv resusnension of the nellet in 25 UL of MilliQ@ autocla;ed wafer and incubation at 42°C for 15 min. After brief centrifugation for 10 s at 14 000 rev/ min, the supernatant was transferred to a fresh 0.5 mL microcentrifuge tube and kept at -20°C until used for the PCR.