Evaluation of PCR-based methods for the diagnosis of infection in bancroftian filariasis

TRANSACTIONSOFTHEROYALSOCIETYOFTROPICALMEDICINEANDHYGIENE(2000)94,526-530

Evaluation of PCR-based methods for the diagnosis of infection in bancroftian filariasis

Senarath Dissanayake’ * , Abraham Rocha* , Joaquim Noroes3, Z&a Medeiros’ , Gerusa Dreyeti and Willy F. Piessens4 ‘Department of Microbiology, Faculty of Medicine, UAE University, PO Box 17666, Al Ain, United Arab Emirates; ‘Depanamento de Parasitologia, Centro de Pesquisas Aggeu Magales (FIOCRUZ), Recife, Brazil; 3Servico de Urologia, Hospital das Clinicas, Universidade Federal de Pernambuco, Recife, Brazil; 4Depamnent of Immunology and Infectious Diseases, Haruard School of Public Health, 665 Huntington Avenue, Boston, ML4 02115, USA

Abstract The value of the polymerase chain reaction (PCR) in the diagnosis of Wucheretia bancrofti infection was evaluated in comparison to microscopical examination of night blood smears, Nucleporea filtration, serology and ultrasonography. No correlation was found between PCR-based deoxyribonucleic acid (DNA) probing and serology. We did not find any evidence of free filarial DNA in either blood plasma or chylocoele fluid. We conclude that the 2 PCR-based techniques evaluated are not more sensitive than Nuclepore@ filtration for detection of lV. bancrofti microfilaraemia, need at least 1 intact microfilaria in the volume of blood used for DNA extraction, and were much inferior to ultrasonography for detection of amicrofilaraemic adult worm carriers.

Keywords: filariasis, Wuchereria bancrofci, diagnosis, chylocoele, ultrasonography, polymerase chain reaction, Sri Lanka

Introduction Lymphatic filariasis due to Wuchereria bancrofii infec-

tions continues to be a public health problem in many countries with an estimated -120 million people infected and 900 million at risk of infection (WHO, 1994). Of the many different approaches to controlling transmission, chemotherapy of microfilaraemic individuals (mostly asymptomatic) remains the most practical and commonlv used, and is within the resources available in many endemic areas.

For the diaanosis of individuals infected with W. bancrofii, the &vmerase chain reaction (PCR) has been proposed as- mbre sensitive than mi&osco$cal and serolotical tests (ABBASI et al.. 1996. 1999; SIRIDEWA etal., 7996; ZHO~G etal., 1996). Pri&stud>es on PCR and bancrbftian filariasis indicated that the method nermits detection of N 1 DP of W. bancrofti DNA added io 100 @of human blood,& the equivalint of about 1% of the deoxyribonucleic acid (DNA) present in 1 microfilaria (DISSANAYAKE etal., 1991;Sm~~~~ etal., 1996; ZHONG et al., 1996). Because none ofthe filarial species- specific DNA probes developed to date are also stage- specific, the inherent sensitivity of PCR-based assays might even be an undesirable characteristic of a diagnostic test in areas of intense transmission. A positive result in a highly sensitive PCR reaction with human blood, for example, might result from the presence of DNA from infective larvae, microfilariae or adult worms and thus signify exposure to the parasite, productive infection. or occult filariasis. On the other hand. if the unit of filarial DNA is 1 parasite rather than a certain amount of ‘free’ DNA, the sensitivity of PCR-based diagnostic tests will be governed by the volume or size of the specimen to be analysed and not by the assay performance characteristics per se.

To resolve some of these uncertainties, and to assess further the usefulness of PCR as a diagnostic test, we evaluated 2 PCR-based methods with samples obtained during a population survey for bancroftian filariasis in Sri Lanka and &om patients attending a specialized clinic in Brazil. Results of DNA probing were compared with diagnostic information provided by traditional microscopical examination of blood films and filters, serodiag- nostic methods, and ultrasonography.

Materials and Methods Patients, blood and chylocoele$uid samples

Night blood samples were obtained during several years before 1998 from volunteers living in Kurunegala,

*Author for correspondence: fax +971 3 671966, e-mail [email protected]

Sri Lanka (DISSANAYAKE, 1989) or in Recife, Brazil (MEDEIROS et al., 1992, 1999), 2 areas where noctur- nally periodic bancroftian filariasis is endemic, and from residents of an area in north-eastern Brazil (Paraiba state) that is devoid of human filariasis (ANOtiMOUS, 1986; ~JARCHETTI etal., 1998). The SriLankansamnles were*pre-selected fro& a larger batch of previoisly characterized samples for which repeat examinations and confirmatorv parasitological data were available (DISSANAYAKE, -1689; DIS&NAYAKE et al., 1994). Whole blood for PCR amnlification was stored at - 70°C in 20 mM ethylenediaiinetetraacetic acid (EDTA) until processed. Plasma samples were prepared by centrifugation of anticoagulated whole blood for 10 min at 4°C. Live adult worms were detected by ultrasonography as described earlier to define amicrofilaraemic adult worm carriers (DREYER et al., 1996a). Chylocoele fluid was obtained aseptically from a life-long resident of Recife during surgical chylocoele repair, who had a confirmed W. bancrofti infection; i.e., microfilaraemia and live adult worms demonstrated by Nucle- pore@ filtration and ultrasonography respectively (AMARAL et al., 1994; NOR~)ES et al., 1996). The ruptured dilated lymphatic vessel contained live adult W. bancrofti and microfilariae were uresent in the chvlocoele fluid (AMARAL et al., 199i). For PCR/D-NA probing, the fluid was divided into 2 aliquots; 1 was centrifuged to remove the microfilariae and the super- nate was used for DNA probing. The other aliquot was used untreated. The laboratorv investigations were done in 1997-99.

Microfilaraemia levels were determined by microsco- Dical examination of films Drenared from -20 & Cat+ iary blood for Sri Lanka samples or by ekamin-ing Nuclepore” membranes (DENNIS & KEAN, 1971) after filtration of 1 mL of venous blood for Brazilian samples. An individual was considered amicrofilaraemic only if no microfilaria was detected in 16 mL of blood (DREYER et al., 1996a). Because the very large cells that are present in chylocoele fluids rapidly clog Nuclepore@ membranes, 3 mL of fluid were centrifuged and 20 p.L of the sediment were examined for microfilariae. Sera were also screened for the nresence of filarial Oa4C3 antigen (MORE & COPEM&, 1990). Ultrasonogiaphy wasised to detect live adult worms in the scrotal area (AMARAL et al, 1994; NOR~ES et al., 1996). Immunoglobulin (Ig) G4 antibody to mixed adult Brugia malayi worm extracts and IgG antibodv to the recombinant filarial antigen SXPl were measured as described by KWAN-LIM e;al. (1990) and DISSANAYAKE et al. (1992, 1994), remectivelv. Resi- dents of Recife were considered & be hninfecfed ‘endemic controls’ if they met all the following criteria: no

PCR-BASED DIAGNOSIS OF BANCROFTIAN F’ILARIASIS 527

history or physical finding compatible with bancroftian filariasis, no Og4C3 antigen, no microfilaria in 16 mL of blood, no adult worm or lymphangiectasia detected by ultrasonography (NOR~ES et al. 1996; DRE~ER et al., 1999) and no lymphatic nodule formation observed after diethylcarbamazine (DEC) treatment (DRBYER et al., 1994). The Brazilian study was approved by the ethical committee, Hospital das Clinicas, Federal University of Pemambuco.

Extraction offilarial DNA from blood and chylocoele fluid Blood or chylocoele fluid samples (200 ~.IL) were

diluted 1:3 in TE buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA) containing 0.1% sodium dodecylsulphate (SDS) and 100 ug/mL of proteinase K, and incubated for 6 h at 50°C to release DNA from the blood cells and microfilariae (if present). After centrifugation to pellet debris, the digestion mixture was extracted serially with equal volumes ofphenol, phenol/chloroform (5050) and chloroform by standard procedures, and the DNA was precipitated with an equal volume of isopropyl alcohol at room temperature. The precipitate was then washed twice with ice-cold 80% ethyl alcohol and dissolved in 200 l.tL of distilled water.

DNA amplajkation and detection W. bancrojki DNA was amplified with 2 distinct sets of

primers based on the sequence of 2 different species- specific DNA probes, pWb35 (DISSANAYAKE & PIES- SENS, 1990; DISSANAYAKE et al., 1991) and pWb12 (SIRIDEWA et al., 1996). For detection with pWb35, the PCR primers used were S’AACCACTTGCGA CGTCACTI’lTG (F2) and 5’CCGTCCACAAA GCCAAACTGCTG (R5); for detection with pWbl2, the mimers used were S’CTGAGTGAAATC AATGAACTGC (F3) and S’GTCCATCCGATGAA GTTCCACC (R2). The expected sizes of the amplified nroducts were 380 bn c~‘&‘b35) and 490 bn (~Wb12). Forty-cycle PCR amp&cations were perfokgd as previously described (DISSANAYAKE et al., 1991). Each amplification reaction was done in a final volume of 100 & with 10 u.L of DNA-containing extract from blood or chylocoele fluid. All PCR amplification experiments also included a positive control consisting of pooled normal human serum from a filariasis non- endemic area in Sri Lanka, ‘spiked’ with W. bancrofi genomic DNA (1 ng/mL), and a negative control consisting of 10 l.tL distilled water.

PCR products (10 @.+ aliquots of the 100 uL reaction mixture) were separated on agarose gels, transferred to nylon membranes by Southern blotting and fured by exposure to ultraviolet light (using StratalinkerTM, Stra- tagene Inc., La Jolla, California, USA). The blots were then nrobed with the F2-R5 fraament of ~wb35 (DIS- SANAi’AKE et al., 199 1) or the F3&2 fragment of pvirb 12 (SIRIDEWA et al., 1996), both labeled with [32P]d- adenosine triphosphate by primer extension with the Klenov fragment of Escherichia coli DNA polymerase (New England Biolabs, Beverly, Massachusetts, USA) using the same primers as in the PCR amplification. Both probes were used at N 1 O6 cpm per mL of hybridization solution [5 X SSC (1 X SSC is 0.15 M NaCl, O-015 M sodium citrate, pH 7*0), 5 X Denhardt’s solution (1 X Denhardt’s is 0.02% Ficoll, 0.02% polyvinylpirrolidone, 0.02% bovine serum albumin), 1% SDS and 50% formamide] and hybridized at 42°C for 8-12 h. The membranes were washed extensively at 50°C in 0.1 X SSC with 0.5% SDS and exposed to X-ray film for 3 h without intensifying screens. The signal strength of the 380 bp or 490 bp bands was quantified on an arbitrary scale of O-5 (0 being no hybridization and 5 being approximately equal to that generated by the positive control of 1 ng/mL W. bancrofn’ DNA amplified in parallel).

Results Comparison of FCR-based DNA probing of blood with other diagnostic tests for bancroftan filariasis

A batch of 40 blood samples consisting of 8 micro- tilaraemic individuals (I-20 microfilariae in a ~20 & smear) and 32 smear negative persons from Sri Lanka were screened by the pWb35 and pWb12 PCR-based assays. ThepWb35basedPCRassayon 200 &ofblood detected all 8 blood smear positive persons and 9 of 32 blood smear negative persons. The pWbl2-based PCR assay detected only 6 of the 8 microfilaraemic subjects, but 21 of the 32 smear negative persons were also positive. Because of this observed discrepancy between the 2 DNA nrobes. a second batch of 39 blood samnles from Sri La&an donors, comprising 3 1 amicrofilaraemic (smear negative) and 8 microfilaraemic (smear positive) individuals, was examined by both methods under identical conditions. Thirteen samples gave positive results with each probe including all smear-positive nersons, and 9 were negative bv both. Four smear- negative samples that w&e positive by pWb35 were negative by pWb12, but 13 of the smear-negative and nWb35-negative samples were nositive bv uWbl2. Over- all, the pWb 12-based assay defected as positive a higher number of smear-negative persons (43/63, Table 1) than the pWb35-based assay (18163, Table 1). When the microtilaraemia diagnostic SXP-1 enzyme-linked im- munosorbent assay (DISSANAYAKE et al., 1992, 1994) was used as the base criterion, among 28 SXP-l-positive samples, pWb35 detected 14 while pWb 12 detected 18. As was the case with smear-negative blood samples, the pWb12 assay detected a higher number (9/l 1) of SXP-I- negative and smear-negative persons than pWb35 (21 11). Although the significance of this remains to be determined, for the purposes of this investigation a higher sensitivity for smear and/or antibody-negative samples was considered more suitable. For this reason, pWb12 was used to examine all Brazilian samples.

Among the Brazilian samples, pWbl2 correctly identified all 13 individuals who were microfilaraemic as determined by filtration of 1 mL of venous blood obtained at night (Table 2). No correlation was observed between microfilarial density (range 9-1477 per mL) and intensity of the hybridization signal. However, the pWbl2 DNA probe did not identify any of the 10 adult worm carriers without microfilaraemia (i.e., with <l microfilaria in UD to 16 mL of blood) (Table 2). Subse- quent reanalysis-of these samples with ihe pWb35 probe failed to identify any positive sample in this group (data not shown). All endemic and non-endemic controls were negative by PCR-DNA hybridization (Table 2),

As expected, Og4C3 antigenaemia and IgG antibodies to recombinant SXPl antigen were uresent in the majority of microfilaraemic donors whose blood samples hybridized to pWb 12 (Table 3). All 3 tests were negative in 3 untreated amicrofilaraemic adult worm carriers (i.e.,

Table 1. Comparison of the species-specific DNA probes pWb35 and pWbl2 for detection of Wucher- ia bancrofh’ DNA in blood fkom Sri La&an donors

Subject group”

Smear pwb35 pwb12 Batch 1 Batch 2

+ + + - - - - Total

+ + 6 + - - + t + + 9 + - 0

+ 12 - - 11

40

8 0 0 5 4

13

3;

a+, positive; -, negative.

528 SENARATH DISSANAYAKE ETAL.

been due to a technical failure of the PCR method per se but due to a true absence of free DNA from blood.-

Chvlocoele fluids containing microfilariae also hvbri-

Table 2. Comparison of adult worms and microfilariae as source of Wuchereria bancrofti DNA in blood samples from Brazilian donors

Parasite stage detecteda No. in No. pWb12

Adult worms Microfilariae group positive

Yes No Yes No No

Yes Yes No No No

13 13 2b 2

0 ;;I 0 12’ 0

“Adult worms detected by ultrasonography of the scrotal area and microfilariae by filtration of 1 mL of venous blood; microfilaraemia 9-1477 per mL. bIvIicrofilaraemic individuals in whom dead adult worms (not induced by DEC) were detected by ultrasonography. ‘All subjects were amicrofilaraemic by examination of 16 mL of blood at the time of PCR-DNA probing; 8 had been microfilaraemic previously but had become amicrofilaraemic after ivermectin treatment. ‘%habitanta of endemic area but with no evidence of infection. ‘Inhabitants of an area not endemic for filariasis.

those with < 1 microfilaria in 16 mL of blood) identified by ultrasonography. In contrast, the serological tests remained positive in some of the patients who became amicrofilaraemic following treatment with ivermectin but who remained live adult worm carriers as determined by ultrasonography (DREYER et al., 1995, 1996b), but pWbl2 PCR-based DNA probing was negative in all such persons (Table 3).

Physical form of W. bancrofti DNA in blood One possible explanation for the inability of PCR-

based assays to detect adult worm carriers with ultra-low levels of microfilaraemia (or no circulating microfilaria) is that no ‘free’ worm DNA is present in blood and that intact microfilariae are the major source of parasite DNA detected by this method. This possibility was formally examined by processing and probing in parallel extracts prepared from blood cells and microfilariae precipitated by low speed centrifugation and from the corresponding plasma samples. A total of 23 whole blood samples, comprising of 11 samples PCR-positive by both probes and 12 which were PCR-negative by both probes, were used. Filarial DNA was detected with both probes in extracts from the precipitated pellets of the 11 positive samples, but in none of the corresponding plasma samples (data not shown). In control experiments, plasma samnles ‘sniked’ with W. bancrofn’ DNA imme- hiately before extraction and PCR ampl&ication consis- tentlv vielded nositive results with nrobe ~wb35. Thus,

our inability io detect worm D&IA in’ plasma from patients with bancroftian filariasis is not likely to have

dized-with the DNA probes, but fluids without r&ro- filariae failed to do so. Chylocoele fluid from 1 donor was divided into 2 aliauots before DNA nrobina. One aliauot was centrifuged to remove intact microfiariae, which were known to be present in the uncentrifuged replicate sample. Both DNA probes hybridized to the uncentrifuged sample, but neither reacted with the centrifuged replicate.

Discussion In this investigation, we attempted to answer the

question whether PCR is more sensitive than the available night blood smear, filtration, serological and ultrasonomanhic techniaues in the diagnosis of asvmn- tomatic @ bancrojii infections. Our dita indicate that PCR-based DNA orobine of venous blood is not more sensitive than Nuclepore@ filtration for the detection of microfilaraemia and is much inferior to ultrasonography as a method to detect amicrofilaraemic or ultra-low microfilaraemic adult worm carriers (DREYER et al., 1996a). We conclude that free DNA is absent from blood and lvmvh fluid and. for detection bv PCR-based assays, at leas; 1 microfilaria must be present in the volume of blood used for DNA extraction. During chvlocoele collection, adult worms were seen in the &nity of the ruptured lymphatic vessel. The negative PCRwith centrifuged chvlocoele fluid clearlv established the absence of free DNA from the fluid tested. Others have shown that a different DNA probe also yielded negative results with blood samples from a variable proportion of individuals with low level microfilaraemia andfrom most antigenaemic individuals without microfilaraemia WILLIAMS et al.. 1996). Some of the latter harbour viable adult worms and thus are actively infected (DREYER et al., 1996a; ROCI-IA et al., 1996). These findings by other investigators, together with our inability to detect parasite DNA in blood from amicrofilaraemic adult worm carriers, strongly indicate that microfilariae, not adult worms, are the major source of W. bancrofn’ DNA in the systemic circulation. PCR- based probing of blood also does not appear to detect pre-adult stages, at least under the conditions of filarial transmission that prevail in the study sites in Sri Lanka and Brazil. This conclusion is not entirely surprising, given the relative numbers of different developmental worm stages that may be present in an infected individual.

Worm DNA in blood is evidently associated with microfilariae and is not present ‘free’ in solution, at least not in sufficient amounts to be detectable by PCR-based DNA probing. Further, we found Og4C3 antigen [re- lated to excretory-secretory products from adult worms (CHANTEAU et al., 1994a)], but no filarial DNA in blood samples from several patients who became amicrofilar-

Table 3. Comparison of PCR-based and serological diagnostic methods for Wuchereria bancrofti infection

Parasitological stat& No. positive

Adult worms Microfilariae No. Og4C3b SXPI’ pWbl2d

+ + 13 13 11 13 + -e 5 4 0 + - ; 0 Endemic controls 15 0 : : Non-endemic controls 12 0 0 0

“+, adult worms detected by ulnasonography or microfilatiae detected by filtration of 1 mL of venous blood; -, no adult worm detected or no microfilaria detected by filtration of 16 mL of venous blood. “Antigen Og4C3 detected in serum. ‘IgG antibodies to antigen SXPl present. dDNA extracted from whole blood hybridized to probe pWb 12 after PCR amplification. ‘Previously microfilaraemic individuals treated with ivermectin.

PCR-BASED DIAGNOSIS OF BANCROFTIAN FILARIASIS 529

aemic after ivermectin treatment, suggesting that re- leased worm DNA is degraded and/or cleared rapidly from the circulation. This-is consistent with the fact that WILLIAMS et al. (1996) detected no increase in blood levels of parasite l?)NA &mediately following treatment with diethylcarbamazine and/or ivermectin.

The absence of free worm DNA from blood has profound practical implications. If the lower limit of detection is 1 parasite rather than a certain portion of the DNA present in 1 worm, then the sensitivity of DNA probing for routine diagnosis of bancroftian filariasis is whollv deoendent on the volume of blood firorn which DNA- is extracted. If the minimum requirement is 200 ILL of blood for PCR (SIRIDEWA et al.. 1996). and if at least 1 microfilaria mu& be present in &is vol&e to be detected by PCR (this paper), then the same objective can be achieved by examination of several thick blood smears by microscopy or by filtration of 1 mL of blood. The like&ood that % least-1 worm is present in the test samnle for PCR (200 uL) can be increased bv concen- tratiig microfilariie bkfoie DNA extraction, b;t even so the theoretical sensitivity of DNA probing cannot exceed the sensitivity of microscopical examination of compar- able blood volumes concentrated, e.g.., by filtration or Knott’s method. Clearly, some serologuzal tests are more sensitive than DNA probing to detect adult worm carriers with low microfilaraemia levels, but even serology fails to identify a proportion of individuals who can be shown to harbour live adult worms by ultrasonography (ROCHA et al., 1996). The relatively low sensitivity of Knott’s concentration method when only part of the sediment is examined may explain why SIRIDEWA et al. (1996) reported that more hydrocoele fluids were positive bv PCR than bv microscouv. That fi+ee filarial DNA may ge present in some tissues;; suggested by the finding of W. bancroti DNA in oatients’ SDU~LUII (ABBASI et al., 1996, 1999)” and urine Samples (~UCEN~ et al., 1998): However, microfilariae can be detected by microscopy in sputum and urine samples from patients with bancroftian filariasis and the physical form of parasite DNA in these biological samples remains to be determined.

A second issue that arose from this investigation was the lack of absolute agreement between the 2 PCR-DNA probe-based methods used. Although the observed discrepancy for amicrofilaraemic individuals can be attrib- uted to differences in the sensitivities of the 2 methods, 2 microfilaraemic individuals who were consistently positive by the pWb35-based assay were consistently negative by the pWb12 assay. Whether different parasite isolates are different in their reuetitive element composi- tion, upon which the sensiti&y and specificity ot the PCR-DNA probe depend, is not known. If the latter is true, that will be a serious limitation on DNA probe- based diagnosis of W. bancmfi microfilaraemia.

Overall. PCR-based assavs aooear to have much less practical value as diagnosticiesti for bancroftian filariasis than is the case in onchocerciasis, where the sensitivity of the technique exceeds that of traditional skin snips (ZIMMERMAN et al., 1994). In contrast to its poor performance in the human host, DNA probing remains a powerful tool to identify vectors of lymphatic filariae and to monitor transmission (DISSANAYAKE et al., 199 1; CHANTEAU et al., 1994b).

Acknowledgements Most of the experiments described were conducted in the

laboratory of Dr W. F. Piessens, Harvard School of Public Health, Boston, MA 02 115, USA, during 1997-99.

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Received 12 May 1999; revised 28 Februa y 2000; accepted for publication 28 Februa y 2000

1 Announcements 1

3rd MOH-AMM Scientific Meeting and International Congress of Medicine in the Tropics

Kuala Lumpur, Malaysia l-4 November 2000

This meeting is organized by the Ministry of Health/Academy of Medicine of Malaysia. For further information please contact the Congress Secretariat, 3rd MOH-AMM Scientific Meeting, Academy

ofMedicine ofMalaysia, 19 Jalan Folly Barat, 50480 &ala Lumpur, Malaysia; phone +60 3 2530100 or 2530200, fax +60 3 2530900, e-mail [email protected]

14th Seminar on Amebiasis

Mexico City, Mexico 27-30 November 2000

For further details please contact Dra Martha Espinosa, Cantellano, CINVESTAV, Aptdo Postal 14-740,070OO Mexico, D. F., fax +52 5 747 7107, e-mail [email protected]

Documents

Evaluation of PCR-based methods for the diagnosis of infection in bancroftian filariasis