Heterogeneity in the sensitivity of stocks and clones of Giardia to metronidazole and ornidazole

The sensitivity in vitro to metronidazole and ornidazole of 7 stocks and of the cloned lines of 5 stocks of Giardia isolated from humans, rodents and monkeys was studied by the growth inhibition test. All 7 stocks of Giardia, irrespective of the host, differed in their sensitivity to these drugs, commonly used in therapy of human giardiasis. The differences were greater with ornidazole than with metronidazole. The 5 Giardia stocks from which clones were prepared were found to consist of populations with significantly (P less than 0.05) differing sensitivities to ornidazole and metronidazole. There was a positive correlation between high resistance in vitro to both drugs of all clones of one parent stock and treatment failures of giardiasis in the patient from which the parasite stock had been isolated. The spectra of sensitivity of Giardia to anti-giardial drugs may have implications concerning the suspected zoonotic character of human giardiasis.     

The viability of Giardia intestinalis and Giardia muris cysts in seawater

The viability of Giardia intestinalis and Giardia muris cysts was determined by excystation at different temperatures (5, 10 and 15C) after exposure to seawater. This was compared with viability in freshwater (milli-Q water) at the same temperatures as controls. There was a significant reduction in viability after exposure to seawater. The significance of this to public health is discussed.     

Cryopreservation of viable Giardia intestinalis trophozoites

A technique is described for the cryopreservation of Giardia intestinalis trophozoites. The most satisfactory results were obtained when organisms were preserved with either 7·5 or 10% dimethyl sulphoxide (Me₂SO) and cooled using a liquid nitrogen controlled freezer. Under these conditions more than 70% of organisms were motile after thawing. Lower recovery rates were obtained using glycerol as the cryopreservant or when samples were placed directly into a ?70 °C refrigerator to cool. Cultures were successfully re-established from material cooled under controlled conditions using either 7·5% Me₂SO or glycerol as the cryopreservant. However, the former had an initial generation time of 12·0 hours compared to 24·5 hours for the latter.     

Giardia intestinalis in Thailand: Identification of Genotypes

This study was undertaken to determine the genetic diversities of Giardia intestinalis isolated in Thailand. G. intestinalis cysts were collected from stool samples of 61 subjects residing in Bangkok or in rural communities of Thailand with and without gastrointestinal symptoms. All the cyst samples gave positive tpi amplicons (100% sensitivity), either of the 148- or the 81-bp tpi segments. Cyst assemblage identification of the 148- and 81-bp tpi gene segments by polymerase chain reaction showed that 8% of the cysts were assemblage A, 41% assemblage A and B combined, and 51% assemblage B. The prevalence of assemblage A was significantly lower than that of assemblage B and the mixed types. Restriction fragment length polymorphism (RFLP) of the 384-bp β-giardin gene segment revealed that 12% and 88% of the assemblage A cysts were AI and AII respectively. RFLP, based on the 432-bp gdh gene segment, showed 45.5% of the assemblage B cysts to be BIII and 54.5% to be BIV. The AI sub-assemblage was less prevalent than the others. All subjects with AI and 50% of the subjects with BIII sub-assemblage cysts were symptomatic; 80% of symptomatic Bangkok residents were adults/elderly while 85% of the rural cases were children.Key words: β-giardin, Genotyping, Giardia duodenalis, Giardia intestinalis, Giardiasis, Glutamate dehydrogenase, Triose phosphate isomerase, Thailand     

M olecular (PCR) differentiation of Giardia muris and Giardia intestinalis

Using random amplification of polymorphic DNA (RAPD) a band present with G. muris but not G. intestinalis DNA was detected. This band was cloned and sequenced and this facilitated the selection of a pair of primers specific for G. muris. Since G. muris does not infect humans, the use of these primers in conjunction with primers specific for G. intestinalis should enable water to be determined as safe or unsafe for human consumption with respect to the species of Giardia.     

Enterococcus faecium SF68 enhances the immune response to Giardia intestinalis in mice

We studied the ability of the probiotic organism Enterococcus faecium SF68 to antagonize Giardia intestinalis infection in mice. Oral feeding of E. faecium strain SF68 starting 7 d before inoculation with Giardia trophozoites significantly increased the production of specific anti-Giardia intestinal IgA and blood IgG. This humoral response was mirrored at the cellular level by an increased percentage of CD4(+) T cells in the Peyer's patches and in the spleens of SF68-fed mice. The improvement of specific immune responses in probiotic-fed mice was associated with a diminution in the number of active trophozoites in the small intestine as well as decreased shedding of fecal Giardia antigens (GSA65 protein). The ability of SF68 to stimulate the immune system at both mucosal and systemic levels highlights mechanisms by which this probiotic might antagonize pathogens in vivo. Taken together, the data demonstrate the strong potential of strain SF68 to prevent protozoa from causing intestinal infections.     

Isolation and identification of freshwater bacteria antagonistic to Giardia intestinalis cysts

We have isolated three freshwater bacterial strains that demonstrate the ability to degrade Giardia intestinalis cysts. These strains have been identified by 16S rRNA sequencing and phylogenetic analysis as belonging to the Flavobacterium columnare clade of the Cytophaga-Flavobacterium group. While the cyst degradation mechanism is unclear, two different effects on the cysts were observed: non-viability and lysis. Cysts exposed to bacterial strains BR1 and SC1 were generally non-viable, but remained structurally intact. In contrast, cysts exposed to strain SR1 were clearly lysed. Increases in bacterial densities with a concomitant decrease in cyst viability suggest that these bacterial strains are capable of using the cysts to enhance their growth. We propose that the presence of bacterial strains such as SR1, BR1 and SC1 may play a role in reducing the viability of G. intestinalis cysts in natural waters.     

Improvement in isolation and axenic growth of Giardia intestinalis strains

Twenty fresh Giardia intestinalis strains were isolated by inoculation of trophozoites obtained by in vitro excystation procedure or by duodenal aspiration. The strains were continuously cultivated in TP-S-1 or bile-supplemented BI-S-33 media without antibiotics. No correlation was found between the ease of isolation and axenization of Giardia strains and the source of material for inoculum preparation (faeces v. duodenal fluid and symptomatic patients v. asymptomatic cyst passers). Panmede and Trypticase used in TP-S-1 and TYI-S-33 media vary in their ability to support the growth of particular strains. Of the five sera used in TP-S-1 medium no variations were associated with bovine and calf serum. The bile-supplemented BI-S-33 medium supported the growth more consistently than did the TP-S-1 and TYI-S-33 media; depending on the strain, the mean number of trophozoites at 120 hours was 1.1 X 10(6) to 1.8 X 10(6), and the generation times were 8.1 to 16.6 hours.     

Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin.

A genetic analysis of Giardia intestinalis, a parasitic protozoan species that is ubiquitous in mammals worldwide, was undertaken using organisms derived from a variety of mammalian hosts in different geographical locations. The test panel of 53 Giardia isolates comprised 48 samples of G. intestinalis, including representatives of all known genetic subgroups, plus an isolate of G. ardeae and four isolates of G. muris. The isolates were compared by allozymic analysis of electrophoretic data obtained for 21 cytosolic enzymes, representing 23 gene loci. Neighbour Joining analysis of the allelic profiles supported the monophyly of G. intestinalis but showed that the species encompasses a rich population substructure. Seven major clusters were evident within G. intestinalis, corresponding to lineages designated previously as genetic assemblages A-G. Some genotypes, e.g. those defining assemblage A, are found in divergent host species and may be zoonotic. However other genotypes, e.g. those defining assemblages C-G, appear to be confined to particular hosts or host groups. The findings reinforce other evidence that G. intestinalis, which was defined on the basis of morphological criteria only, is a species complex.     

Inhibition of adherence of Giardia intestinalis by human neutrophils and monocytes

Human neutrophils and monocytes were found to be capable of interfering with the in vitro adherence of Giardia intestinalis. Significantly greater inhibition of adherence was shown by stimulated phagocytic cells than by unstimulated cells. Both azurophil and specific granules extracted from the cytoplasm of neutrophils were equally potent in their adherence inhibitory activity. Dose-dependent effects on adherence were observed with granular enzymes and reactive oxygen species. Lower concentrations of reactive oxygen species were required for adherence inhibition that for growth inhibition. These results suggest that the adherence mechanism of G. intestinalis may be a feasible target for immunological attack by phagocytic cells.     

Giardia intestinalis is unlikely to be a major cause of the poor growth of rural Gambian infants

Parasite-specific plasma immunoglobulins have been used to indicate the presence of Giardia intestinalis infection in 60 infants living in a rural area of The Gambia. Infants were studied longitudinally between 2 and 8 mo of age. The median age for first exposure to G. intestinalis was between 3 and 4 mo, and by 8 mo all but 3 infants (95%) showed a positive titer on at least one occasion. Raised Giardia-specific IgM titers were associated with reduced weight gain in the 2 wk preceding a positive titer, but catch-up growth occurred in the following 2 wk. IgM antibody titers were also positively associated with intestinal permeability (lactulose/mannitol ratio), urinary lactose excretion, plasma concentrations of alpha1-antichymotrypsin and total IgM, IgA and IgG immunoglobulins. However, infant growth over the whole 6-mo period (i.e., between 2 and 8 mo of age) was not related to mean Giardia-specific antibody titers, nor the time of first exposure to the parasite. The data suggest that giardiasis in these very young breast-fed children occurs as a mild, acute disease, and its presence could not explain the marked, long-term growth faltering observed in many of the subjects.     

Impact of Giardia intestinalis on vitamin a status in schoolchildren from northwest Mexico

We conducted a cross-sectional study in northwest Mexico in order to investigate the association between giardiasis and serum vitamin A in 40 Giardia-infected and 70 Giardia-free schoolchildren who were covered by a regional school breakfast program. There were no significant differences in age, Z-scores for nutritional indices of height for age, weight for age, or weight for height, socioeconomic conditions (employment and education of the parents, household conditions, sanitation facilities, type of drinking water, and family income), and mean daily intakes of vitamin A in the Giardia-free (899 +/- 887 microg) and the Giardia-infected (711 +/- 433 microg) groups. A higher concentration of serum retinol was found in the Giardia-free group than in the Giardia-infected group (0.75 micromol/L versus 0.61 micromol/L, respectively; p < 0.0001). Giardia-infected children were more likely to be vitamin A-deficient than the Giardia-free children (OR = 3.2; 95% CI = 1.2-8.5). Although 95% of the children met the daily-recommended intakes of vitamin A, half of them showed subclinical vitamin A deficiency. It is recognized that vitamin A deficiency is multifactorial and giardiasis was a factor significantly associated with this deficiency in this study. Mexican program developers and policymakers should be aware about the distinction between dietary deficiencies and deficiency diseases when current national program strategies for parasitic control and vitamin A supplementation are redesigned.     

Age and sex distribution of infections with Entamoeba histolytica and Giardia intestinalis in the Lagos population

Stool samples were collected from 2,099 persons and examined for parasites: 9.5 percent were infected with Entamoeba histolytica, 7.9 percent were infected with Giardia intestinalis and 15.5 percent were infected with Iodamoeba butschlii. The infections with E. histolytica and I. butschlii occurred more frequently in females than in males and adults were also more frequently infected than children. Infection with G. intestinalis, on the other hand, was more prevalent in males than in females and children were more frequently infected than adults with the highest incidence of infection (13.8 percent) in the age-group 1-5 years.     

Lack of an adverse effect of Giardia intestinalis infection on the health of Peruvian children

Giardia intestinalis is a common gastrointestinal protozoan worldwide, but its effects on childhood growth in developing countries are not clearly understood. The authors aimed to describe its effects on child growth. They followed 220 Peruvian children daily for diarrhea, weekly for stool samples, and monthly for anthropometry. The authors modeled the effect of nutritional status on the risk of Giardia infection and the risk of diarrhea attributable to Giardia using negative binomial regression. They modeled the effects of Giardia infection on growth using linear regression, with 85% of children becoming infected with Giardia and 87% of these becoming reinfected. In multivariable analysis, the risk of Giardia infection did not vary with weight for age (relative risk = 1.00, 95% confidence interval: 0.89, 1.12) or height for age (relative risk = 0.92, 95% confidence interval: 0.82, 1.04). Giardiasis did not affect growth at 1 or 2 months following the first infection at any age interval. The longitudinal prevalence of Giardia between 6 and 24 months of age was not associated with height gain in that interval (p = 0.981). Giardia was not associated with an increased risk of diarrhea at any age interval. Study results question the importance of Giardia as a childhood pathogen in developing countries where giardiasis is hyperendemic.     

Altered uptake of metronidazole in vitro by stocks of Giardia intestinalis with different drug sensitivities

Growth of Giardia intestinalis in TYI-S-33 medium containing a sub-lethal concentration of metronidazole over a period of 66 weeks selected a line of organisms that was over 8 times less sensitive to the drug. This resistance was unstable and the organisms reverted to their original sensitivity within 22 weeks of growth in the absence of drug. A comparison of the uptake of [14C]metronidazole by the original and selected lines showed a highly significant reduction in drug uptake by the resistant line, indicating either a defective transport mechanism across the cell membrane or decreased reduction of metronidazole to the active metabolite within the cell. There was no significant difference in the uptake of metronidazole by 9 recent isolates of G. intestinalis maintained in axenic culture.     

Sensitivity in vitro of Giardia intestinalis to dyadic combinations of azithromycin, doxycycline, mefloquine, tinidazole and furazolidone

The new macrolide antibiotic, azithromycin, produced significant growth inhibition of Giardia intestinalis at 100 micrograms/ml, but adherence inhibition was significant at concentrations as low as 1 microgram/ml for the two strains used in these experiments. The dyadic combinations of azithromycin-furazolidone, doxycycline-mefloquine, doxycycline-tinidazole and mefloquine-tinidazole were synergistic for inhibition of adherence. These results suggest that these dyadic combinations may be worthy of consideration for chemotherapy of recalcitrant giardiasis.