Intestinal parasitic infections in patients with Diabetes Mellitus : A case-control study

© 2015 The Authors; Tabriz University of Medical Sciences This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Intestinal parasitic infections in patients with Diabetes Mellitus: A case-control study

Patients with diabetes mellitus (DM) are at increased risk of certain infections; however, little is known about the prevalence of intestinal parasitic infections in them.The aim of this study was to assess the risk of intestinal parasitic infections in patients with DM in comparison with a healthy control group.
This case-control study was conducted on 118 patients with DM and 118 healthy people as control group from April to September 2014.Oral glucose tolerance test (OGTT) and hemoglobin A1c level were checked, and checklists including risk factors for parasitic infections were filed for all participants.Three stool samples and one scotch tape were obtained.Samples were examined by direct wet smear, formol-ether concentration, Kinyoun acid-fast staining, and modified trichrome stain.Data were analyzed using chi-square and logistic regression tests.
Patients with DM might be at an increased risk of infection with intestinal parasites specifically B. hominis as an opportunistic infection, and routine stool examination should be considered for them.
immune system such as chronic internal diseases and metabolic disorders can put the patients at higher risks of infectious diseases.
Diabetes mellitus (DM) is a group of chronic diseases characterized by hyperglycemia that is caused by insufficient insulin secretion, impaired insulin action, or both. 5Chronic hyperglycemia leads to vascular and neurologic complications, often accompanied by end-organ damages and susceptibility to certain infections in patients with DM such as urinary tract infections, lower extremity infections, tuberculosis reactivation, surgical wound infection, candidiasis, and pneumonia. 6Probably local and systemic immune defects are responsible for this higher susceptibility. 7ecently, it is demonstrated that both innate and acquired immunities are impaired in DM. 8 In mice with DM with urinary tract infection, chemokine expression, neutrophil infiltration, and bacterial clearance are decreased. 9Functions of neutrophil such as phagocytosis and chemotaxis are impaired in the mice with DM. 8 Because of general immunosuppressive condition, a high prevalence of various infections is expected in DM, but surprisingly, epidemiologic data in this regard are scarce 7 and there are few studies addressing the prevalence of intestinal parasites in patients with DM. 10,11 The purpose of this study was to assess the rate of parasitic infections in patients with DM and a control group to estimate the risk of intestinal parasitic infection in patients with DM in comparison with healthy people.Written informed consent was taken from all participants and the project was approved by Isfahan University of Medical Sciences Ethical Committee (Project number: 293052).
A 75 g oral glucose tolerance test (OGTT) was performed for all participants and hemoglobin A1c level was checked.Diagnosis or exclusion of DM was based on the criteria from the American Diabetes Association: 5 fasting plasma glucose ≥ 126 mg/dl, or 2 hours plasma glucose ≥ 200 mg/dl during OGTT, or A1c ≥ 6.5%.
A checklist including demographic data and risk factors for parasitic infections was filled for each participant.Three stool samples and one scotch tape were obtained from individuals after the full explanation of the process to them.The samples were transported immediately to the Department of Parasitology, School of Medicine, Isfahan University of Medical Sciences, where lab assessments were done.
Stool samples were examined by direct wet smear and formol-ether concentration method for the routine screening of ova and parasites.Each sample was examined separately.Smears were prepared from sediments of formol-ether concentration for specific staining: Kinyoun acid-fast staining was used to detect Cryptosporidium, and modified trichrome stain (Ryan-Blue) was employed for detection of Microsporidia as described elsewhere. 12,13lides were examined by light microscopy at × 400 magnification.Rate of parasitic infection was calculated as the ratio of the number of participants with at least one positive parasitological test to the number of total participants in each group.
Data were analyzed by SPSS software (version 16, SPSS Inc., Chicago, IL, USA).Rate of parasitic infection and the risk factors were compared between the two groups by chi-square or Fisher's exact test when appropriate.Logistic regression analysis determined the degree of relationship between the rate of parasitic infection and the identified risk factors.Odds ratios (OR) and 95% confidence intervals (CI = 95%) for OR were calculated.P < 0.050 was considered as statistically significant.
Three stool samples were obtained from 236 persons (118 controls and 118 patients with DM) with an age range of 4-73 (male/female: 82/154).The rate of parasitic infection was significantly more in patients with DM (26.3%) than in controls (6.8%) (P < 0.001).In this study, the most detected infection was Blastocystis hominis (14 cases), followed by Endolimax nana (10 cases) and Giardia lamblia (5 cases) (Table 1).Only the rate of B. hominis infection significantly different between the two groups (2.5 and 9.3% for control and DM patients, respectively, P < 0.050).Infection with two or more parasites was seen in three patients with DM and two controls, from those four were co-infected with B. hominis and E. nana.
Fisher's exact test showed significantly more parasitic infection in females (20.1%) than males (9.8%) (P < 0.050).Also the rate of infection was more in patients with symptoms such as diarrhea, abdominal Pain, and abdominal discomfort (70.6 vs. 12.3%) (P < 0.001), in those who kept animal at home (53.8 vs. 14.3%)(P < 0.050), and in undereducated persons (less than Diploma: 42.9%, Diploma and more: 14.9%) (P < 0.050).Although parasitic infection was more in persons under 10 years old (20.0%) and over 50 (28.0%) in comparison with ages 10-50 (14.9%), the difference was not statistically significant.The number of households was not related to the rate of parasitic infection.
The distribution of risk factors in the two groups was not different except for the presence of symptoms and keeping animals at home (Table 2).In logistic regression analysis, independent risk factors for parasitic infection were identified as DM (OR = 3.6, 95% CI: 1.5-8.8),female gender (OR = 3.0, 95% CI: 1.1-8.3),and the presence of symptoms (OR = 9.9, 95% CI: 2.5-39.1)(Table 3).
This study demonstrated that the risk of infection with intestinal parasites was 3.6 times greater for patients with DM than healthy people.This is in agreement with a previous study in two cities near Tehran (Karaj and Savojbolagh) Iran, in which the rate of intestinal parasitic infections in patients with DM was more than healthy controls (5.6 vs. 10.0%). 10 In another study in Egypt, patients with DM were examined among other immunocompromised groups, and high risk of parasitic infection was found among them. 11The results indicate that the clearance of parasites and also commensals from intestine might have been impaired in DM; however, the exact mechanisms are not clear.Resolving of intestinal parasitic infections is dependent on both innate and adaptive immune responses, but cell-mediated immunity, specifically T-cells, plays the main role in pathogen clearance from intestine. 1 Although some defects in the function of neutrophil and macrophage are documented in several studies, 6 there are controversies about the defective T-cell function in type II DM.Spatz et al. found that the expression of cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) (involved in down regulation of immune response) on CD4 + T cells is increased in type I DM. 14 Likewise, in response to stimulation, CD4 + T-cells from DM type I patients secreted elevated levels of the regulatory cytokine transforming growth factor-beta 1 and their monocytes produced more inhibitory cytokine interleukin-10 (IL-10) in comparison with cells from healthy people or DM type II patients. 14Thus, it seems that T-cell function is intact in DM type II. 7Local intestinal immune response to parasites is an important factor which can explain high parasitic infection in our patients; however, basic studies in this regard are scarce and actually there is no experiment on the mechanisms of intestinal infections in DM. 7 Also impaired mucosal integrity, due to defective microcirculation in DM, 15 can predispose parasitic infections although it is not evaluated in any study.
The most infection detected in this study was B. hominis followed by E. nana and G. lamblia.We found that the rate of infection with pathogenic and opportunistic agents as well as commensals was not different between the two groups except for B. hominis which was significantly more in with DM.We found only four cases of infection with Cryptosporidium spp.and Microsporidia (each 1.6%) in patients with DM.The rate of Cryptosporidium infection was lower in our setting in comparison with a similar study (2.4%) in which only Cryptosporidium infection was meaningfully more in DM. 10 As the cryptosporidiosis is a zoonotic infection, the reason might be little contact of our patients with reservoir animals.
While there have been many doubts about pathogenic role of Blastocystis in humans, now it is accepted as a potential pathogen which can specifically trouble immunocompromised host. 16Symptoms associated with Blastocystis are more likely to develop in HIV-infected patients and transplant recipients than in healthy hosts. 17,18tudies of intestinal parasitic infections show different parasites as the dominant infectious agents; however, Blastocystis nearly always has been among the most prevalent parasites in immunocompromised patients.For example, in Ethiopia, the prevalence of Cryptosporidium and Blastocystis spp. was significantly associated with lower CD4 + T-cell count in patients with HIV/AIDS (acquired immune deficiency syndrome). 19In Laos, Blastocystis was the most frequent protozoa (26.3%) compared with Cryptosporidium spp.(6.6%). 20n another study in Iran, B. hominis (4.4%) was the most prevalent parasite after G. lamblia (7.3%) in HIV-positive individuals. 21. hominis (16.7%) and Cryptosporidium parvum (8.3%) were the most infections in HIV (+) patients in south of Iran. 22Also in another study in Iran, B. hominis was the most prevalent intestinal parasite in hemodialysis patients. 23It is suggested that the pathogenesis of Blastocystis depends upon subtype; subtypes 1-4 are more common and have a cosmopolitan distribution. 16ucosal invasion and intestinal inflammation have been shown in animal models of subtypes 3 and 4. 24,25 The theory of impaired intestinal mucosal integrity in DM might explain the increased rate of Blastocystis infection in these patients.
In addition to DM, female gender and the presence of symptoms were the risk factors identified for infection with intestinal parasites.The reason for the increased risk of infection in females is not clear for us.Several studies including a national survey of the prevalence of intestinal parasitic infections in Iran showed no sex related significant difference, [26][27][28] however, in some regions, these infections predominated in either males or females. 29,30Sex dependent distribution of intestinal parasitic infections depends on cultural, social, and environmental factors which are different in each area, thus those inconsistencies are expected.

Limitations
We used conventional microscopic methods for diagnosis of intestinal protozoa and helminthes rather than the molecular methods.The combination of both methods would lead to more strict results; however, conventional methods are steel and the most cost-effective methods for detection of intestinal parasites. 12Small sample size was another limitation of this study, but the power was high enough to show the difference in intestinal parasitic infection rate between patients with DM and healthy people.
DM patients are at higher risk of infection with intestinal parasites than normal population.In this regard, B. hominis specifically is an important opportunistic infection which can cause gastrointestinal symptoms; therefore patients with DM should be screened for this parasite routinely.
Authors have no conflict of interest.
Authors gratefully acknowledge personnel of Endocrine and Metabolism Research Centre for their valuable cooperation in this project.

Table 1 .
Different parasitic infections detected in patients with DM (diabetes mellitus) and control group

Table 2 .
Distribution of risk factors for parasitic infection in patients with DM (diabetes mellitus) and control group * By chi-square or Fisher's exact test, DM: Diabetes mellitus

Table 3 .
Risk factors for intestinal parasitic infection * By logistic regression test, OR: Odds ratio; CI: Confidence interval