Elimination of Giardia duodenalis BIV in vivo using natural extracts in microbiome and dietary supplements.

In this study, a combination therapy of several natural products was evaluated in vivo in the Giardia duodenalis infection model. G. duodenalis infected mice were treated as follows: distilled water (infected control C+), BIOintestil® (BIO; natural products of Cymbopogon martinii and Zingiber officinale), MicrobiomeX® (MBX; extract of Citrus sinensis and Citrus paradisi), MBX + BIO, Camellia sinensis tea (CPR; black tea). These natural compounds were administered in a dose of 100 mg/day and were compared to G. duodenalis-infected mice treated with albendazole (ALB; 50 mg/Kg/day) and metronidazole (MET; 500 mg/Kg/day), the conventional therapies used to this day. One group remained un-infected and untreated as our control group (C-). Treatment started 8 days after infection, and after 5 days of treatment (7 days for MET), all animals were followed for 15 days. We continuously checked for the presence of G. duodenalis by Faust method, in association with detection of the parasite by PCR from feces, as well for the presence of trophozoites in the intestinal mucosa after sacrifice. Animals treated with MBX, BIO and MBX + BIO presented an undetectable parasitic load until the 15th day of monitoring, while animals treated with CPR, MET and ALB continued to release cysts. Animals in the MBX, MBX + BIO, ALB groups consumed lower feed, MBX, CPR, MET had greater weight and MBX, MBX + BIO, BIO, CPR, C- consumed more water when compared to infected-group control. MBX and BIO alone or associated eliminated G. duodenalis without apparent adverse effects and animals of these groups showed better clinical performance in relation to those with high parasitic load. MET, ALB and CPR only decreased the number of cysts, indicating limitations and therapeutic failure.

Comparative analysis of routine parasitological methods for recovery of cysts, molecular detection, and genotyping of Giardia duodenalis.

In order to improve the diagnosis of giardiasis, fecal samples (high/medium/low concentration of cysts) were processed by the parasitological methods used in the routine: Faust, Lutz e Ritchie modified (replacement of formaldehyde by distilled water). The cysts were quantified; the DNA was extracted and amplified by semi-nested PCR (GDH gene). Fifteen clinical samples were analyzed to validate the study by PCR-RFLP. The results showed that the parasite was only detected and genotyped correctly when samples from children with high, medium, and low parasitic load, belonging to genotype AII, were processed by the modified Ritchie method, different from what was observed for the other methods used in laboratory routine (Faust and Lutz). The modified Ritchie method proved to be more suitable, recovering a greater number of cysts from samples, regardless of parasitic load, which reduces the chance of false negative results and has epidemiological repercussions since individuals with low parasite load are usually asymptomatic and the main disseminators of this infection.

Improvement in cyst recovery and molecular detection of Giardia duodenalis from stool samples.

Molecular detection of Giardia duodenalis by polymerase chain reaction (PCR) is difficult in faecal samples due to inhibitors that contaminate DNA preparations, or due to low cyst concentrations. In order to eliminate inhibitors, improve cyst recovery and molecular detection of G. duodenalis, different types of water, distillates (MDs), deionized (MDz), injection (MI) or Milli-Q® (MM) were used instead of formaldehyde (F) in the laboratory routine method (Ritchie). Cysts were isolated from faecal samples with low cyst concentrations (< 1 cyst/field), medium (1-2 cysts/field) or high (> 2 cysts/field). Cyst recovery was improved using all water types (MDs, MDz, MI, MM) compared to formaldehyde. At all cyst concentrations, the use of MM consistently showed the greatest recovery of G. duodenalis cysts . DNA samples from recovered cysts were tested for the glutamate dehydrogenase (GDH) and ß-giardin (ßg) genes. The use of Milli-Q® water allowed to detect both genes in all cyst concentrations, including low. The method processed with the other types of water amplified these genes at high and medium cyst concentrations. GDH and ßg genes were not detected when the sample was processed with formaldehyde. These experimental results were confirmed in clinical samples. The results suggest that Milli-Q® water provides the highest cyst recovery from stool samples and, correspondingly, the highest sensitivity for detecting G. duodenalis by microscopy or PCR for GDH and ßg genes, even at low concentration of cysts.

Synergistic effects of fenbendazole and metronidazole against Giardia muris in Swiss mice naturally infected.

In this study were proposed different protocols for the treatment of mice naturally infected with Giardia muris. Male Swiss mice were divided into seven groups, with five animals each, in a blind, controlled, randomized by drawing lots and once-repeated experiment. Parasite detection and cure control were performed using the Faust method and search by trophozoites in the intestinal mucosa. Clinical parameters (weight, water and feed consumption, elimination of excreta, aspect of the fur and feces) were also evaluated. All animals were treated with metronidazole (M), fenbendazole (F), and probiotics (P), administered intragastrically, during 7 days. M1, FM1, and F1 groups were treated 1×/day; M3, FM3, and PM3 groups 3×/day; and ST (control group) received only water. After the 5th and 7th days of treatment, the animals in FM1/FM3 and PM3/M3 groups presented, respectively, negative results and remained negative in the following 10 days. Animals in F1 group consumed less water (p = 0.00010) compared with FM1/FM3/PM3. The animals in M1 group compared with FM3/M3, F1 compared with M3, and ST compared with FM1/FM3/M3/PM3 consumed a larger amount of feed (p = 0.00001). The animals in F1 group compared with FM3/M1/M3/PM3, FM1 compared with FM3, and ST compared with FM3/M1/M3/PM3 eliminated lower volume of excreta (p = 0.00001). The results show that the association between F and M potentiates the effects, indicating a synergistic action of these two drugs, and FM1 is the best protocol due to early negativity in the animals, lower concentrations of the drugs, lower risk of toxicity and stress, and less alterations in clinical parameters.