|Year : 2014 | Volume
| Issue : 1 | Page : 68-74
Efficacy of combination therapy (metronidazole and/or artemether) in experimental giardiasis and its impact on nonenzymatic oxidative stress biomarkers
Eman M Aly, Hoda Y Sabry, Zeinab H Fahmy, Rabab S Zalat
Department of Parasitology, Theodor Bilharz Research Institute, Imbaba, Egypt
|Date of Submission||03-Sep-2013|
|Date of Acceptance||15-Mar-2014|
|Date of Web Publication||25-Sep-2014|
Zeinab H Fahmy
PhD, Department of Parasitology, Theodor Bilharz Research Institute, Imbaba, PO Box 30
Source of Support: None, Conflict of Interest: None
Giardia lamblia trophozoites colonize in the upper small intestine resulting in diarrhea and various clinical manifestations, including abdominal pain, anorexia, and signs of malabsorption. A decrease in the level of trace elements might occur because of this absorption deficiency resulting from giardiasis. Experimentally, the excretory secretory product of G. lamblia trophozoites increased the level of reactive oxygen species in mice enterocytes. The levels of bilirubin, uric acid, and albumin are often used as major nonenzymatic oxidative biomarkers.
This study was designed to determine the effect of therapy by metronidazole (MTZ) and artemether (ART) on trophozoite and cyst forms in experimentally Giardia spp.-infected hamsters and to reveal the changes in iron (Fe), manganese (Mn), copper (Cu), and chromium (Cr) serum levels pretreatment and post-treatment. Another objective was to evaluate the impact of this therapy on serum levels of bilirubin, uric acid, and albumin as nonenzymatic oxidative stress biomarkers.
Materials and methods
Hamsters were divided into four groups: the control group I included two subgroups, Ia (noninfected, nontreated) and Ib (infected, nontreated); group II (infected and treated with MTZ); group III (infected and treated with ART); and group IV (infected and treated with combined therapy of MTZ+ART). Hamsters of all four groups were killed 5 weeks postinfection (PI) - that is, 2 weeks after treatment - to evaluate drug efficacy. Stool samples and duodenal contents were examined to count the number of G. lamblia cysts and trophozoites, respectively. Blood samples were also collected to estimate trace elements (Fe, Mn, Cu, and Cr) as well as nonenzymatic oxidative stress biomarkers (bilirubin, uric acid, and albumin).
There was a significant reduction in trophozoite and cyst counts following treatment with ART alone (88 and 82.5%, respectively) as compared with the infected control group Ib. Treatment with MTZ alone and in combination with ART also yielded a very high percentage of reduction in both trophozoites (94.2 and 98.3%, respectively) and cysts counts (93.9 and 95.5%, respectively). The trace elements in serum of infected controls (Ib) displayed nonsignificant decrease in Fe and significant decrease in Mn levels as compared with their levels in noninfected hamsters of group Ia. Cu levels increased in the infected group and were still increased after treatment with either MTZ or ART but decreased to normal with the combined therapy. Cr levels showed no significant change in all groups. Uric acid increased in infected controls as compared with normal controls. Treatment with MTZ or ART alone decreased uric acid levels lower than normal, and the combination of both drugs normalized its levels. Evaluation of serum bilirubin levels in the infected group and in those treated by MTZ and ART alone did not show any statistically significant differences compared with the normal noninfected group. Treatment with the combined therapy yielded even slightly lower insignificant level. Albumin level also did not differ significantly except in the combined regimen where it was lower than the normal range.
The effect of giardiasis on the changes in the level of trace elements and nonenzymatic oxidative stress biomarkers is relevant in this study. The combined therapy produced significant parasite eradication and normalized the studied parameters with the exception of Mn and albumin levels, which were adversely affected and remained lower than normal. Further studies are needed to evaluate these data in undernourished and chronically infected hamsters.
Keywords: artemether, biomarkers, combination therapy, giardiasis, metronidazole, trace elements
|How to cite this article:|
Aly EM, Sabry HY, Fahmy ZH, Zalat RS. Efficacy of combination therapy (metronidazole and/or artemether) in experimental giardiasis and its impact on nonenzymatic oxidative stress biomarkers. Parasitol United J 2014;7:68-74
|How to cite this URL:|
Aly EM, Sabry HY, Fahmy ZH, Zalat RS. Efficacy of combination therapy (metronidazole and/or artemether) in experimental giardiasis and its impact on nonenzymatic oxidative stress biomarkers. Parasitol United J [serial online] 2014 [cited 2017 Aug 17];7:68-74. Available from: http://www.new.puj.eg.net/text.asp?2014/7/1/68/139692
| Introduction|| |
Giardia lamblia is a flagellated protozoan parasite that frequently coexists with Entamoeba histolytica and is transmitted in the same way. It occurs worldwide, particularly where sanitation is poor, and it is a common cause of both acute and persistent diarrhea among children in developing countries. In addition, several large waterborne epidemics have occurred almost all over the world, including northern regions of the former USSR and also Canada and USA, where beavers may provide a reservoir of infection  . Ingested cysts release trophozoites that attach firmly to the jejunal mucosa. These multiply and eventually form another generation of cysts, which are excreted intermittently in the feces. Many carriers are symptomless, but others lose weight and complain of diarrhea or gastrointestinal discomfort  . Diagnosis requires skilled microscopy, and false-negative tests are common because cysts are excreted in stools irregularly. Confirmatory examination of jejunal aspirates may be necessary. Extensive infections result in intestinal malabsorption and impairment of growth. Severe symptoms are more likely to occur in patients who are malnourished, hypochlorhydric, or immunocompromised  .
This is significant as the prevalence of vitamin and mineral deficiencies is high in developing countries  . These micronutrients act as essential cofactors of enzymes and as organizers of molecular structures of the cell  . Children and pregnant and lactating women are the most vulnerable groups. In preschool age children, micronutrient deficiencies increase the risk for acute diarrhea and pneumonia. These deficiencies are responsible for a great number of deaths and a large proportion of the disease burden worldwide  , and also impinge on economic and human capital development  .
Iron (Fe) deficiency impairs immune function and limits cognitive development in children, causes low productivity in adults, and increases the risk of perinatal death , . Zinc deficiency affects children's physical growth and increases the risk for and severity of diarrhea, pneumonia, and other infections , . Manganese (Mn) is a cofactor of more than 300 enzymes. It participates in the synthesis of proteins and nucleic acids, neuromuscular transmission, and in cardiac contraction  . In addition, the low bioavailability of Mn in giardiasis can be caused by binding to free fatty acids in the gut lumen as a result of fat malabsorption  . Copper (Cu) participates in the synthesis of connective tissue and the myelin sheath of nerves, as well as in energy and iron metabolism; it is the reductive agent of iron oxidase enzymes and is a constituent of the ceruloplasmins involved in iron transport and absorption  . Cu deficiency manifests as hypochromic anemia, and in children it has been associated with osteoporosis and stunting , . Yet, elevated Cu levels could be attributed to changes in the concentration of specific tissue proteins controlled by cytokines  .
A decrease may occur in the levels of trace elements because of absorption deficiency resulting from giardiasis  . In experimental giardiasis, an enterotoxin purified from the excretory secretory product of Giardia spp. trophozoites increased the level of reactive oxygen species (ROS) in mice enterocytes, and this correlated well with decline in the activity of superoxide dismutase and catalase  . Intestinal homogenates of G. lamblia-infected rats displayed high degree of cell injury and lipid peroxidation as indicated by the statistically significant higher lactate dehydrogenase activity and higher malondialdehyde level, respectively, compared with the noninfected rats  . Estimation of levels of uric acid, albumin, and bilirubin is often used as major nonenzymatic antioxidant biomarkers , .
Therapeutic strategy in giardiasis has included diverse pharmaceutical agents of traditional use, such as MTZ, quinacrine, furazolidone, and paromomycin , . Other drugs of more recent introduction, such as albendazole and nitazoxanide, have also been applied in clinical practice  . Of these, MTZ (a 5-nitroimidazole) and albendazole (a benzimidazole) may be considered the most representative antigiardial agents of traditional and recent use, respectively. However, reports pointed to an increasing frequency of cases refractory to treatment with both drugs , , the causes of which include treatment noncompliance and emergence of drug-resistant Giardia spp. strains . An in vitro study evaluated the effect of natural plant extracts (Myrtus communis and olibanum) as alternative antigiardial agent to overcome the side effects and the emerging drug resistance against imidazole derivatives. Both plants inhibited G. lamblia multiplication in vivo and resulted in ventral disc and axoneme fragmentations after 48 h in vitro incubation  .
The generally accepted mechanism of action of the antimalarial drug ART involves the interaction of a peroxide-containing drug with heme, a hemoglobin degradation byproduct derived from proteolysis of hemoglobin. This interaction is believed to result in the formation of a range of potentially toxic oxygen and carbon-centered radicals. The WHO has been promoting an artemisinin-based combination therapy for treating malaria , . ART and its related compounds enhance the efficacy of antimalarial combination therapy and have the potential of lowering the rate at which resistance emerges and spreads. Artemisinin-based combination therapies are particularly known to be the most effective because of their high killing rates, and they have become the mainstay chemotherapeutic agents for malaria  . Few studies have reported the ART effect against other protozoa including Giardia spp. , . Numerous studies have investigated the type of damage that may be induced by oxygen radicals [19-21,31].
This study was designed to reveal the changes in iron, manganese, copper, and chromium levels pretreatment and post-treatment with ART and/or MTZ in infected animals. In addition, evaluation of the changes in bilirubin, uric acid, and albumin levels in hamsters experimentally infected with Giardia spp. was also performed, as they are considered as nonenzymatic oxidative stress biomarkers.
| Materials and methods|| |
Type of the study
This is a case control study conducted at the animal house and Parasitology Department, Theodore Bilharz Research Institute (TBRI), Egypt.
Male golden hamsters, weighing 100-110 g each, were provided by Schistosome Biological Supply Center (SBSP) at TBRI.
G. lamblia was collected from stool of patients attending outpatient clinic at TBRI. Positive stools were pooled, filtered, and then concentrated by a series of centrifugations and filtrations. Cysts were counted in 0.1 ml of sediment and the infection dose was calculated in 1 ml to reach about 10 +3 cysts/ml. Hamsters were infected orally by esophageal tube  .
Drugs and dose
ART was obtained in tablet form (Kunming Pharmaceutical Cooperation, Yunnan province, China) with a documented purity of 99.6%. A dose of 400 mg/kg body weight was given for 3 consecutive days  . MTZ suspension (Rhone Poulenc Rorer, Sanofi Aventis, Cairo, Egypt) was given at a dose of 120 mg/kg body weight orally for 2 successive days  .
Each group comprised 10 hamsters. The control group included two subgroups: Ia (noninfected, nontreated) and Ib (infected, nontreated). Group II received MTZ. Group III received ART. Group IV was infected and treated with a combination of 1/3 the dose of MTZ and 2/3 the dose of ART  .
Study design and methods
Three weeks PI, animals were administered medication orally using stainless steel esophageal tube. Two weeks after treatment, stool analysis was performed by the merthiolate iodine formaldehyde concentration technique, and G. lamblia cysts were counted in a weighed aliquot then calculated in 1 g of stool  . Animals of all four groups were killed 5 weeks PI - that is, 2 weeks after treatment - to evaluate drug efficacy. The small bowel was removed and the number of trophozoites was counted in 1 cm of duodenal tissue  . Blood samples were collected and sera were separated and stored at −20°C for analysis. Levels of Mn, Fe, Cu, and Cr were determined by a direct method using an atomic absorption spectrophotometer  . Serum total bilirubin, uric acid, and albumin were also determined ,, .
Statistical analysis of the results was carried out using one-way analysis of variance  . Comparison between the two groups was made by the Student test. The data were considered significant if P value was less than 0.05.
The TBRI approved the experimental study, which was conducted according to the husbandry guidelines.
| Results|| |
[Table 1] shows that, as compared with infected control (Ib), a significant reduction in trophozoite count occurred in intestinal tissue following treatment with ART (88%), and a higher reduction (94.2%) was obtained following MTZ treatment. Group IV, given both drugs at reduced dose, also yielded a very high percentage of reduction (98.3%) [Table 2], revealing the changes in G. lamblia cyst counts in 1 g stool. The infected group treated with ART showed significant reduction (82.5%), but a higher significant reduction was encountered with MTZ (93.9%). Group IV (combined regimen) showed the highest reduction percentage (95.5%).
|Table 1: Effect of treatment by ART and/or MTZ on Giardia lamblia trophozoite forms in the small intestine, 5 weeks PI and 2 weeks after treatment|
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|Table 2: Effect of treatment by ART and/or MTZ on the number of Giardia lamblia cysts excreted in stool, 5 weeks PI and 2 weeks after treatment|
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Evaluation of serum bilirubin [Table 3] levels in groups Ib, II, and III did not show any statistically significant differences compared with the normal noninfected group (Ia). Treatment with the combined therapy (group IV) yielded even slightly lower insignificant level. Albumin levels also did not differ significantly except in the combined regimen where it was lower than the normal range. Uric acid, in contrast, increased in infected control (Ib) as compared with normal, but treatment with MTZ or ART alone (groups II and III) decreased the level of uric acid even lower than normal. The combination of both drugs normalized uric acid levels. Considering the trace elements in serum of infected hamsters [Table 4], Fe level displayed nonsignificant changes in all groups as compared with their levels in the normal noninfected animal group (Ia). Level of Mn decreased in the infected control group (Ib). Treatment with either therapy alone (groups II and III) did not improve the Mn level. With combined therapy (group IV), the level improved but remained significantly lower than normal. In all groups, the Cr levels were not different compared with normal control. In contrast, Cu levels increased in infected group (Ib) and were still increased after treatment except after the combined therapy in group IV.
|Table 3: Nonenzymatic biochemical parameters in serum, pretreatment and post-treatment with ART and/or MTZ in experimental giardiasis|
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|Table 4: Concentration (mg/ml) of Cr, Cu, Mn, and Fe in serum samples, pretreatment and post-treatment with ART and/or MTZ in experimental giardiasis|
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| Discussion|| |
Chronic giardiasis is one of the most common causes of severe malabsorption and weight loss especially in children and pregnant women, and it contributes to increased mortality in the immunocompromised patients  . Adhesion of trophozoites to the intestinal epithelium is crucial for both the initial colonization and the maintenance of infection, as parasites that do not attach or cannot move in the flow of intestinal fluid are expelled  . The majority of patients with chronic giardiasis were successfully treated with MTZ and only 10-20% of patients were reported to fail the response to MTZ therapy [25, 27, 41]. Nash et al.  proposed six potential causes for treatment failures in giardiasis: reinfection, inadequate drug levels, immunosuppression, drug-resistant strains, sequestration of G. lamblia trophozoites in the gallbladder or pancreatic ducts, and unknown reasons. Drug-resistant strains and trophozoites sequestration are reasons that could not be determined in the clinical patients with failed response to MTZ.
Epidemiological studies showed variable results regarding response of protozoa to MTZ therapy. Up to 30% of Helicobacter pylori and Trichomonas vaginalis patients showed relevant clinical resistance to MTZ , . In contrast, clinical MTZ resistance was not observed in E. histolytica, and laboratory-acquired resistance could only be induced at low parasite levels  , whereas clinical G. lamblia patients showed an intermediate position in MTZ-resistant strains  with strong evidence for marked laboratory-acquired MTZ resistance , .
Several studies proposed a switch to other antiprotozoal agents , or to antimalarial agents  . Artemisinin was evaluated in several experimental studies as antihelminthic  as well as antiprotozoal agent , . In the present study, administration of combination therapy (MTZ+ART) produced the highest significant decrease in Giardia spp. cysts count in stool, as well as remarkable reduction in the number of trophozoites in the intestinal mucosa. The effect of treatment on infection intensity in all groups was highly significant. In a study conducted in Mexico, ART was shown to ameliorate the deleterious effect of G. lamblia on the intestinal mucosa of infected animals. The investigators claimed that there was complete villus regeneration compared with the infected control group  . Another study conducted in Egypt showed that artesunate was active in vitro against both E. histolytica and G. lamblia. The investigators attributed this activity to a reduction in the production of interleukin-12, a cytokine that is important in the regulation of cell-mediated immune responses  . The association between giardiasis and Whipple's disease led to raising the question of whether alteration in the immune system facilitates infections or whether the development of infection leads to immunosuppression  .
On the basis that an adequate supply of trace elements is required in gastro intestinal tract for the structure and function of metalloproteinases that participate in energy production and protection against ROS  , and that diarrhea may be exacerbated by micronutrients deficiency , , we measured serum levels of Fe, Mn, Cu, and Cr before and after treatment in hamsters of the study groups. Fe levels were not affected and displayed no significant decrease in all groups as compared with the normal noninfected animal group Ia. However, Mn levels decreased significantly in the infected nontreated control group Ib and treatment in groups II, III, and IV did not improve Mn levels, which were still lower, although the combined MTZ and ART therapy in group IV ameliorated its level. This could be due to the reduced doses of these drugs when used in combination, pointing to their possible potential role as stress agents. Drug exposure could induce free radical formation and lipid peroxidation initiated by free radicals, which is considered deleterious for cell membranes and has been implicated in a number of pathological situations  .
In addition, combined therapy in the present study also normalized Cu levels, which increased significantly, whereas Cr levels were not altered. The study of trace elements in giardiasis has gained much attention especially in children living under poor socioeconomic conditions. In other reports, chronic giardiasis was found to lower the serum Fe and zinc levels, whereas Cu levels did not change  . Recently, another study conducted in Egypt also recorded significantly decreased zinc and Fe levels in giardiasis-infected children (1-5 years) with lower weights  .
In the present study, nonenzymatic markers such as bilirubin, uric acid, and albumin were measured to evaluate their levels as oxidative stress markers in experimental giardiasis. Despite a number of studies pointing to bilirubin antioxidant capacity , , it was reported that its role as a scavenger of ROS is still controversial  . This seems to account for its dual nature, acting as an antioxidant at low physiologic levels, whereas beyond a given threshold it is no longer beneficial  . In our study, bilirubin level did not show significant change, whereas albumin level was lower than the normal range in the combined regimen.
In contrast, uric acid revealed significant increase after infection. This was confirmed by experimental and clinical evidence showing that uric acid has an important role as an oxidative stress marker and a potential therapeutic role as an antioxidant  . Our results showed that uric acid levels increased in the infected nontreated control group (Ib) as compared with the normal group (1a), but treatment with MTZ or ART alone (groups II and III) decreased its level even lower than the normal level, and combination therapy (group IV) normalized its levels. Uric acid is known to provide a significant antioxidant defense against nitration by peroxynitrite  . In experimental giardiasis, uric acid probably counterbalances the excessive production of free radicals and acts as a compensatory host response to the deficiency of antioxidant micronutrients  .
| Conclusion|| |
The relevant effect of giardiasis on changes in levels of trace elements and nonenzymatic oxidative stress biomarkers was determined. Combined MTZ/ART therapy resulted in significant parasite eradication, significantly normalized Cu levels but Mn levels were still decreased, and normalized bilirubin and uric acid but decreased albumin levels. The animals in this study were kept on a standard diet, which could be considered a factor in the minimal variation in serum trace elements. Further studies are needed to evaluate these parameters in undernourished and chronically infected hamsters. Meanwhile, further studies are recommended to evaluate combined therapies in clinical randomized studies.
| Author contribution|| |
E.M. Aly, Z.H. Fahmy, and R.S. Zalat shared equally in the practical work, including parasite isolation and conduction of the experimental studies. H.Y. Sabry planned the study and wrote the manuscript.
| Acknowledgements|| |
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[Table 1], [Table 2], [Table 3], [Table 4]