C.C. Constantine

Efficacy of albendazole against Giardia and hookworm in a remote Aboriginal community in the north of Western Australia.

The parasitological, clinical efficacy and tolerability of albendazole in the treatment for both giardiasis and hookworm infection in a remote Aboriginal population was investigated. Albendazole at a dose rate of 400 mg daily for 5 days was highly effective in reducing hookworm egg numbers and both Giardia antigen and cysts. The 36.6% prevalence of Giardia prior to treatment fell to 12% between days 6 and 9, 15% for days 10-17 and rose to 28% between days 18 and 30. Tolerability and clinical efficacy were excellent. The effect of albendazole on hookworm was longer lasting than that on Giardia, reducing percent infection from over 76-2% on days 6-9 and zero by day 18-30 despite conditions highly conducive to rapid re-infection. We conclude that albendazole is highly efficacious against both parasites when used as described but that long term community benefit may require additional education programmes to avoid re-infection with Giardia although treatment strategies would seem appropriate for hookworm.

PCR-based DNA fingerprinting of Giardia duodenalis isolates using the intergenic rDNA spacer

The potential for the non-coding intergenic rDNA spacer (IGS) to DNA fingerprint Giardia duodenalis isolates was investigated. Conserved PCR primers, specific for the flanking large and small rDNA genes, were used to amplify the IGS from 52 in vitro-cultured Giardia isolates. Four distinct IGS-PCR size groups (1.35-1.6 kb) were observed, which correlated closely with the major genetic assemblages established previously for the same isolates using isoenzyme analysis. IGS-PCR size groups A (1.42 kb) C (1.4 kb) and D (1.35 kb) corresponded to isoenzyme assemblage A, and IGS-PCR group B (1.6 kb) to isoenzyme assemblage B. Amplified products from IGS-PCR size groups A and B, which contained 50/52 isolates, were subsequently digested with 8 different restriction enzymes and their profiles compared. Analysis separated isolates within each IGS-PCR size group into 2 distinct clusters which correlated almost exactly with the same genetic groups established previously using isoenzyme electrophoresis. Within each cluster, both methods exhibited a similar capacity to distinguish between Giardia genotypes although they established different genetic relationships between individual isolates. Much of the variability associated with the IGS was attributed to isolates harbouring multiple IGS-sequence types. Restriction analysis of IGS-PCR products amplified from cloned and parent lines of a human isolate BAH 39, which contains multiple IGS variants, showed that trophozoite populations are homogeneous with respect to the types of IGS-variants they maintain. Furthermore, in vitro culture of the cloned isolate BAH39c9 over a 6-year period also failed to reveal variation in IGS-PCR digestion profiles. These results suggest that IGS-PCR RFLP profiles are inherently stable. IGS-PCR analysis was successfully applied to 11 Giardia cyst samples highlighting the potential for this approach to genotype Giardia isolates without the need for in vitro culture.

RAPD (random amplified polymorphic DNA) analysis of Giardia DNA and correlation with isoenzyme data

Fourteen Giardia duodenalis isolates were examined using the RAPD (random amplified polymorphic deoxyribonucleic acid) technique. Simple reproducible polymorphisms were generated using 3 different RAPD primers. The results generated by each primer were very similar and were significantly correlated with each other. These data were then compared to existing isoenzyme electrophoresis data on the same isolates. The RAPD data divided the isolates into 10 groupings or rapdemes while the isoenzyme data divided them into 10 similar zymodemes. Both methods grouped 4 isolates (BAH42, BAH44c9, BAH12c9 and BAH39c7), which comprised a phenotypically heterogeneous assemblage with respect to growth rate and metabolism, into similar groupings. The 2 methods were significantly correlated (P < 0.001). It will therefore be possible to use RAPD for the characterization of isolates of Giardia, and other parasites such as Cryptosporidium, which are refractory to cultivation in vitro.

Primary infection of dogs with Echinococcus granulosus: systemic and local (Peyer's patches) immune responses

Local and systemic lymphocyte proliferation and antibody production were tested in five dogs 35 days after primary experimental infection with Echinococcus granulosus. A significant cell proliferation was demonstrated by [3H] thymidine incorporation in mesenteric, popliteal and/or Peyers patches (PPs) cells in response to E. granulosus protoscolex or adult worm antigen in three of five infected dogs, but not in five control animals. In contrast, blood mononuclear cells responded very weakly in only two of the infected dogs to parasite antigens. Elevated levels (compared with preinfection status) of protoscolex- and adult worm antigen-specific serum IgG were detected (ELISA) in four of the five dogs 35 days after infection. Furthermore, slightly elevated levels of parasite-specific IgE and IgA were observed in the sera of three and four in four infected dogs, respectively. Specific serum IgM was not significantly higher 35 days after infection than before infection. Local antibody production was studied in vitro using PPs, mesenteric and popliteal cells isolated from three infected and three uninfected dogs by ELISA using adult worm antigen. In two of three cultures of unstimulated PPs cells of infected dogs, parasite-specific IgG was detectable. Parasite-specific IgA and IgM were detected in one of the unstimulated PPs cell culture derived from an infected dog. Following in vitro stimulation with parasite antigen, PPs cells from two infected dogs showed increased parasite-specific IgG and PPs cells of all three infected dogs produced parasite-specific IgA. PPs cells from uninfected dogs did not produce significant quantities of parasite-specific antibodies and cells from mesenteric and popliteal lymph nodes of infected or uninfected dogs neither produced antibodies whilst in in vitro cultures.

Importance and pitfalls of molecular analysis to parasite epidemiology

Molecular tools are increasingly being used to address questions about parasite epidemiology. Parasites represent a diverse group and they might not fit traditional population genetic models. Testing hypotheses depends equally on correct sampling, appropriate tool and/or marker choice, appropriate analysis and careful interpretation. All methods of analysis make assumptions which, if violated, make the results invalid. Some guidelines to avoid common pitfalls are offered here.

The origin of a new focus of infection with Echinococcus granulosus in Tasmania

Hydatid cysts were discovered in cattle on King Island, north of Tasmania, where Echinococcus granulosus was thought to have been eradicated. Using enzyme electrophoresis, isolates from King Island were compared genetically with isolates from Tasmania and the mainland of Australia. The genetic distinctness of the King Island isolates make it unlikely that they originated from a recent introduction from either Tasmania or mainland Australia. Alternative possibilities, that the infection resulted from a recent introduction from another source or from previously undetected persistence of E. granulosus on King Island, could not be distinguished from available data.