DNA probes for the faecal diagnosis of Giardia fumbfia infections in man

Giardia lamblia is the most frequently identified intestinal protozoan parasite in the U.S.A. and U.K., and is one of the more common causes of diarrhoea in children in the developing world. Chronic infections can result in enteropathy and malabsorption, contributing to growth retardation in children (Farthing et al., 1986). Giardia is commonly found in patients with acquired immunodeficiency syndrome and in other immunocompromised individuals. An important practical problem in the management of this infection is that diagnosis is not straightforward. Diagnosis of giardiasis relies at present mainly on the demonstration of Giurdia cysts in stools by microscopy. Cyst excretion is known to be erratic, such that a single faecal specimen may only detect 50% of cases (Burke, 1975), while sequential stool analysis only decreases the false negative rate to 30% (Burke, 1977). Duodenal aspiration and biopsy followed by microscopy for Giardia trophozoites are only reportive in 44% of cases (Madanagopalan et al., 1975). Serodiagnosis by measurement of serum anti-Giardia IgM levels appears to be useful in acute giardiasis but IgG levels do not discriminate current from previous infections (Goka et al., 1986). Clearly there is a need for a simple, rapid, sensitive and specific diagnostic technique that can detect Giardia in faecal specimens. The techniques of gene cloning as applied to infectious diseases now allow the production of gene probes that have the potential to be exquisitely sensitive and specific diagnostic reagents. Increasing numbers of gene probes for bacterial and parasite infections are rapidly being developed and exploited (Tenover, 1988). We report here the generation of cloned genomic DNA probes for Giardia lamblia and their application to the diagnosis of human giardiasis by detection of Giardia DNA in faeces. A fully representative BamHI-digested genomic DNA library of G. lamblia, strain Portland I, was constructed in Escherichia coli XL-1 Blue host cells with the plasmid vector pBluescript (Stratagene L-TD, NBL U.K.) consisting of > lo5 clones. Cloning efficiencies of lo5 clones/pg of Giardia DNA were achieved. Recombinant clones were selected on ampicillin plates by blue/white colour selection in the presence of X-Gal after isopropyl /3-D-thiogalactopyranoside induction of the plasmid LacZ gene /3-galactosidase fusion protein. Initial screening of 2000 white clones was performed with total DNA from three strains of G. lamblia (Portland I. WB and Bart’s 1) by colony hybridization in situ (Grunstein & Hogness, 1975). Clones were also screened with total DNA from closely related protozoa, Entamoeba histolytica, Trichomonas vaginalis and Tritrichomonas foetus, as well as human DNA. Greater than 99% of the clones were specific for Giardia DNA. Twenty white colonies with strong hybridization signals were selected and plasmid isolated (Birnboim & Doly, 1979). Cloned Giardia insert DNA was sized by 1% (w/v) agarose gel electrophoresis and ethidium bromide staining after digestion with the restriction endonuclease BamHI. Southern blotting and hybridization of BamHI-restricted G. lamblia DNA with [33P]dCTP-labelled clones revealed a single band of the same size as the cloned DNA insert. No hybridization was observed with DNA from other protozoal or human DNA. This confirmed the origin and specificity of the selected Giardia clones. A single clone, pGL-2, with a 2 kilobase insert was selected and the insert excised with BumH1, separated by electrophoresis in low melting temperature agarose and labelled with [33PpCTP (Amersham; 3000 Ci/mmol) by random hexanucleotide priming and Klenow extension (Fienberg & Vogelstein, 1983). Specific activities of 4-8 x loR c.p.m./pg of DNA were achieved. This was used as the probe to detect Giardia DNA in faeces by dot-blot hybridization. Faecal samples from 2 1 confirmed cases of giardiasis, containing between lo3 and 10’ cysts/g of stool (Hospital for Tropical Diseases, London), and four negative control samples were studied. Faecal suspensions (1 g/2 ml of water) were made and centrifuged at 10000 g for 10 min. The supernatant containing soluble DNA was removed. The pellet was washed free of bacteria in 10 vol. of water and centrifuged at 1500 g for 10 min. This fraction was the crude washed faecal material containing cysts. Enriched cyst fractions were prepared by sucrose flotation (Bingham et al., 1979). DNA extraction from crude pellets and enriched cyst preparations was by lysis in 1% (v/v) SDS in TEN buffer (50 mM-Tris/HCI, pH 8.0, 100 mM-EDTA, 150 mM-NaCI) and digestion with 500 pg of Proteinase K/ml at 55°C for 3 h. Nucleic acid was recovered by phenol/chloroform extraction and ethanol precipitation and redissolved in TE (10 mM-Tris/ HCl, pH 8.0, 1 mM-EDTA). Soluble fractions and DNA extracts from 0.1 g of faeces (200 p l ) were made 10 x SSC (1 X SSC = 0.1 5 M-NaCI, 0.1 M-sodium citrate, pH 7.0), denatured at 300°C for 5 min, chilled and 500 pl applied to nylon filters (Hybond; Amersham) using a dot-blot apparatus (Biorad). DNA was cross-linked by U.V. irradiation and baked for 1 h at 80°C. Hybridization of dot-blotted faecal fractions with radiolabelled pGL-2 insert DNA was at 65°C for 18 h in 6 X SSC, 5 X Denhardts, 1% (w/v) SDS and 100 pg of denatured salmon sperm DNA/ml, after preincubation for 4 h without probe (Maniatis et al., 1982). Filters were washed in 1 X SSC/O.l% (w/v) SDS at 65°C for 3 x 15 min and autoradiographed. Sensitivity calibration of the detection of Giardia DNA was performed by dot-blotting dilutions of total Giardia DNA containing 0.1 ng to 1 pg of DNA. A miniumum of 1 ng of DNA was detectable, equivalent to about 5 x lo3 Giardia trophozoites based on a genome size of 3.2 x lo7 base pairs (Nash et al., 1985). No Giardia DNA was detected in the soluble faecal fraction or crude washed pellets of any of the samples tested. However, DNA from a minimum of 1 X lo5 enriched cysts was readily detectable. Comparisons based on the strength of hybridization signal of pure Giardia