Intestinal parasitic infections and associated risk factors in diabetic patients: a case-control study

Abstract

Diabetes mellitus (DM) is a metabolic disease that puts the individual at immune-suppression state. The present study aims to detect the burden of intestinal parasites and associated risk factors among diabetic patients in a case-control study. Stool samples from 100 diabetic patients, and 100 non-diabetic controls attending Beni-Suef University Hospital were collected and processed by direct smear examination, concentration technique, permanent staining by modified Ziehl-Neelsen and modified trichrome stains, and culture on nutrient agar plates. The overall prevalence of intestinal parasites was 38%; higher in diabetic patients (44%) than control group (32%) with non-statistical significance. The most predominant intestinal parasites detected among diabetics were Blastocystis hominis (29%), followed by Cryptosporidium sp. (12%), Giardia lamblia (7%), Microsporidia sp. (5%), Entamoeba histolytica/dispar, Hymenolepis nana, and Capillaria philippinensis (each representing 2%). No statistical difference was detected between both groups in all parasites except for Microsporidia sp. (P = 0.008). In diabetic patients; age ≥ 41 years, living in rural areas, and patients having uncontrolled and complicated DM were significantly associated with intestinal parasitosis by univariate analysis (P = 0.016, 0.035, 0.014, 0.043) respectively. By multivariate analysis, age and rural residence were the only statistically significant risk factors (OR = 6.192, and 2.614) respectively. Intestinal parasites were highly associated with diarrhea (P < 0.001), and flatulence (P = 0.042) in the diabetic patients. Diabetic patients should be screened routinely for intestinal parasites, especially the opportunistic ones, and treated for their overall well-being.

Introduction

Intestinal parasitic infections constitute a substantial estimate of morbidity and mortality in low-income developing countries, and even in developed countries. More than three billion persons are infected with one or more intestinal parasites worldwide (WHO 2014).

Although intestinal parasites generally create non-aggressive diseases, some of them gained increased interest as important opportunistic pathogens responsible for hostile up to life threatening sequences in immunocompromised individuals (Bora et al. 2016). Worthily noted, the possibility of establishing the infection, the chronicity of carriage state, the complications, and the mortality rate of the intestinal parasites is increased in immune suppression conditions such as AIDs, cancer, use of immunosuppressive drugs, and chronic diseases including diabetes mellitus (DM) (Tangi et al. 2016).

DM is a one of the most frequent metabolic diseases where an individual has chronic hyperglycemia, either due to inadequate insulin secretion, impaired insulin sensitivity, or both (David et al. 2011). This overt hyperglycemia found in both type 1 and type 2 DM is usually accompanied by the classical triad of polyuria, polydipsia, and polyphagia (Aknibo et al. 2013).

The number of diabetic patients, in particular type 2 DM, is incredibly increasing worldwide mainly due to changes in diet habits associated with sedentary life styles (Bafghi et al. 2015). According to the latest report of International Diabetes Federation (IDF), DM affects more than 425 million individuals worldwide. Egypt is listed on the top 10 countries of the world in the number of diabetics with estimated 8.2 million in 2017, and the number is expected to rise to 16.7 million by 2045 (IDF 2017).

Diabetics have been described to be immunocompromised individuals. In general, both innate and acquired immunity are suppressed in DM through various modalities. Diabetes impairs the physiological and barrier functions of the intestine, decreases T cell function, as well as causes changes in humoral immunity (Chen et al. 2012). Hyperglycemia decreases mobilization, chemotaxis, phagocytosis, and intracellular killing of polymorphonuclear leukocytes (Vardakas et al. 2007). Also, DM decreases the complement C4 component and some interleukins (ILs), particularly IL-1, IL-6, IL-10, interferon gamma, and tumor necrosis factor (Klekotka et al. 2015).

Because of general immunosuppressive condition, diabetic patients are predisposed to various opportunistic helminthic and protozoan infections, in particular the gastrointestinal ones, including Strongyloides stercoralis, Cryptosporidium spp., Cyclospora cayetanensis, Cystoisospora belli, and Microsporidia spp. (Garcia 2007). Regarding Blastocystis, it is still indistinct whether it is a commensal, a pathogen, or an opportunistic parasite (Scanlan and Stensvold 2013).

Considering the higher percentage of intestinal parasites, the increasing number of diabetics, and the need for their continuous medical care to decrease the risk of complications, it is meaningful to reveal the rate of enteric parasites in DM and draw adequate attention to it in clinical practice. This study aimed to clarify the frequency of intestinal parasites in a group of diabetic patients and cross matched non-diabetic controls. Furthermore, the risk factors associated with acquiring intestinal parasites were estimated.

Materials and methods

Study design and populations

A case-control study was employed from November 2017 to September 2018 on stool samples collected from a total of 200 subjects attending the outpatient clinics and internal departments of Beni-Suef University Hospital. The study populations were divided into two groups; G1: 100 diabetic patients complaining of variable gastrointestinal manifestations, and G2: 100 matched non-diabetic individuals as controls.

Inclusion criteria for diabetic participants based on the presence of fasting glucose level ≥ 26 mg/dl, or two hours glucose level ≥200 mg/dl, or HbA1c ≥ 6.5% according to the American Diabetes Association, or patients on anti-diabetic treatment. Exclusion criteria include patients having cancer, chronic diseases other than DM, or other immunodeficiency illness, and patients currently on chemotherapy, radiotherapy, or corticosteroids, and those taking antibiotics and anti-parasitic drugs in the last 2 weeks before sample collection. A checklist was designed to record the subjects’ socio-demographic features, gastrointestinal complaints, and risk factors for parasitic infections. Full diabetic history including onset, duration, manifestations, type of treatment, and presence of complications such as retinopathy, neuropathy, nephropathy, cardiovascular complications, increased susceptibility to common infections, etc. was taken from each diabetic patient.

Sampling and procedures

Fresh stool specimens were collected from the studied population, and processed using direct saline and iodine wet smears, and formol-etheyl acetate concentration technique. Smears prepared from the sediments were stained by modified Ziehl Neelsen (MZN) stain (cold method) (Garcia 2007) for detecting acid fast protozoa and modified trichrome stain (MT) stain (Ryan Blue modification) (Ryan et al. 1993) for detecting Microsporidia spores, then microscopically examined at ×40 and ×1000 magnification. An ocular micrometer was used to measure the size of any suspected stained oocysts or spores. Culture on nutrient agar plates was done for detection of Strongyloides stercoralis or hookworm larvae (Arakaki et al. 1990). Agar plates were incubated at room temperature for 3 to 5 days and microscopically examined on the 3rd and 5th days for the presence of tracks from motile larvae or free-living adults.

Statistical methods

Data were coded, entered and analyzed using Statistical Package for Social Sciences (SPSS) version 23 (IBM, Somers, NY, USA) software. Statistical analysis in the form of mean and standard deviation was used to describe quantitative data, while frequency and percentage for qualitative data. To detect the association between different predictors and intestinal parasites in diabetic patients, univariate analysis was performed using the chi square test where p was considered significant if ≤ 0.05, while binary logistic regression analysis was used to do the multivariate analysis.

Results

Age of the studied population ranged from 7 to 78 years with mean age of 42.7 ± 17.7 years. Number of females were higher than males among the diabetic cases (male/female: 39/61), and in the control group (male/female: 44/56). All participants suffered from various gastrointestinal manifestations in the form of diarrhea (61%), abdominal pain (51.5%), nausea (43%), flatulence (38%), anorexia (37.5%), vomiting (25.5%) and the least symptom was constipation (16.5%).

In the present study, examination of stool samples showed overall intestinal parasites in 76 (38%) out of 200 participants; higher in diabetic group than non-diabetic group (44% vs. 32%), but this difference was statistically insignificant (P = 0.08).

The most predominant intestinal parasite detected was B. hominis where it was totally detected in 50 (25%) of the studied population followed by Cryptosporidium sp. (8.5%), G. lamblia (8%), H. nana (3%), Microsporidia sp. (2.5%), E. histolytica/dispar (2%), and the least prevalent was C. philippinensis (1.5%) (Fig. 1). Strongyloides stercoralis, Cyclospora and Cystoisospora were neither detected in cases nor in controls. No statistical significance was detected in any of the intestinal parasites between both groups except for Microsporidia sp. infection which was significantly different (5% in DM patients and 0% in controls, P = 0.008). Distribution of intestinal parasites in both groups is shown in Table 1.

Fig. 1.

Some detected intestinal parasites in the studied population. Blastocystis vacuolar form ×100 (a), G. lamblia cyst (b) and trophozoite ×100 (c), E. histolytica cyst x100 (d), H. nana egg ×40 (e), C. Philippinensis egg x40 (f), Cryptosporidium sp. oocyst stained with MZN stain (g) and MT stain x100 (h) and Microsporidia sp. stained by MT stain ×100

Table 1.

Distribution of positive intestinal parasites among diabetic cases (n. = 100) and controls ( n. = 100)

Detected parasites	Total		Cases		Control		P
	n.	%	n.	%	n.	%
Opportunistic parasites
Cryptosporidium sp.	17	8.5	12	12	5	5	0.076
Microsporidia sp.	5	2.5	5	5	0	0	0.008*
Other parasites
B. hominis	50	25	29	29	21	21	0.191
E. histolytica/dispar	4	2	2	2	2	2	1
G. lamblia	16	8	7	7	9	9	0.602
H. nana	6	3	2	2	4	4	0.407
C. philippinensis	3	1.5	2	2	1	1	0.56

*Significant P

Out of 76 positive cases for intestinal parasitic infections, 52 (68.4%) cases had mono-parasitic infection, while 24 (31.6%) cases had poly-parasitism; 14 in diabetic group, and 10 in the control group. However, this difference was statistically insignificant (P = 0.197). The highest number of parasites present concomitantly was three; Microporidia sp., Cryptosporidium sp. and B. hominis. This combination was found in one diabetic patient. However, the commonest co-infections were between Cryptosporidium sp. & B. hominis (7 cases) followed by G. lamblia & B. hominis (5 cases), Microporidia sp. & B. hominis (3 cases), H. nana & B. hominis (3 cases), G. lamblia & Cryptosporidium sp. (2 cases), and one case for each combination of H. nana & C. philippinensis, Microporidia sp. & Cryptosporidium sp., and G. lamblia & E. histolytica/dispar.

Peak values of intestinal parasitic infection were in diabetic cases aged ≥ 41 years old (61.4%). In comparison with other ages, the difference was statistically significant (P = 0.016). Although intestinal parasites were higher in diabetic females (63.6%) than males (36.4%), gender was not a risk factor for acquiring intestinal parasites in DM patients. The occurrence of parasitic infections in diabetics was significantly higher among rural inhabitances (77.3%) compared to urban inhabitances (22.7%) with statistical significant value (P = 0.035). Other variables such as occupation, educational level, source of water supply and sewage system were not significantly linked to intestinal parasitic infections in diabetic patients (Table 2).

Table 2.

Association of different risk factors of acquiring intestinal parasites in diabetic patients using univariate analysis

Variables		Intestinal parasites				P
		Positive (n. = 44)		Negative (n. = 56)
Age range	<20	0	0%	8	14.3%	0.016*
	20–40	17	38.6%	13	23.2%
	≥41	27	61.4%	35	62.5%
Gender	Male	16	36.4%	23	41.1%	0.632
	Female	28	63.6%	33	58.9%
Residence	Urban	10	22.7%	24	42.9%	0.035*
	Rural	34	77.3%	32	57.1%
Education	Illiterate	13	29.5%	14	25%	0.407
	Primary	8	18.2%	17	30.4%
	High school	0	0%	1	1.8%
	Tertiary	23	52.3%	24	42.9%
Occupation	House wife	16	36.4%	17	30.4%	0.099
	Retired	6	13.6%	5	8.9%
	Worker	6	13.6%	10	17.9%
	Student	0	0%	8	14.3%
	Employee	16	36.4%	16	28.6%
Water supply	Tap water	36	81.8%	50	89.3%	0.285
	Filtered water	8	18.2%	6	10.7%
Sewage system	Yes	35	79.5%	42	75%	0.592
	No	9	20.5%	14	25%
Treatment	Insulin	20	45.5%	23	41.1%	0.660
	Oral hypoglycemic	24	54.5%	33	58.9%
Diabetic control	Uncontrolled	34	77.3%	30	53.6%	0.014*
	Controlled	10	22.7%	26	46.4%
Complications	Uncomplicated	9	20.5%	22	39.3%	0.043*
	Complicated	35	79.5%	34	60.7%

*Significant P

Intestinal parasites were statistically higher in uncontrolled diabetics than controlled ones (77.3% vs. 22.7%, P = 0.014), and in complicated DM than uncomplicated cases (79.5% vs. 20.5%, P = 0.043). Although the prevalence of intestinal parasites was higher in the patients treated with oral hypoglycemic than those treated with insulin, type of treatment was not a risk factor for acquiring intestinal parasites (Table 2).

Binary logistic regression (Table 3) established that the age (OR = 6.192, 95% CI: 1.478–25.941) and rural residence (OR = 2.614, 95% CI: 0.970–7.043) could statistically significantly predict the parasitic infection acquisition in diabetic patients when the rest of predictors are constant.

Table 3.

Multivariate analysis of statistically significant risk factors of acquiring intestinal parasites among diabetic group

Independent risk factor	B	P	OR	95% C.I. for EXP (B)
				Lower	Upper
Age	1.823	0.013*	6.192	1.478	25.941
Residence	0.961	0.040*	2.614	0.970	7.043
Diabetic control	− 1.485	0.128	4.414	0.653	29.820
Complications	− 0.794	0.456	0.452	0.056	3.644

*Significant P

From all gastrointestinal manifestations presented in the diabetic patients, rate of intestinal parasites was highly associated with diarrhea (P < 0.001) and flatulence (P = 0.042) only, and not significantly associated with abdominal colic, anorexia, nausea or vomiting (Table 4).

Table 4.

Association of parasitic infections in diabetic patients with different gastrointestinal manifestations using univariate analysis

Risk factor	Intestinal parasites
	Positive (n. = 44)		Negative (n. = 56)		P
Bowel habits
Normal	1	2.3%	26	46.4%	<0.001**
Diarrhea	43	97.7%	22	39.3%
Constipation	0	0%	8	14.3%
Abdominal pain
Yes	17	38.6%	28	50%	0.257
No	27	61.4%	28	50%
Anorexia
Yes	17	38.6%	17	30.4%	0.386
No	27	61.4%	39	69.6%
Nausea
Yes	22	50%	25	44.6%	0.594
No	22	50%	31	55.4%
Vomiting
Yes	10	22.7%	14	25%	0.792
No	34	77.3%	42	75%
Flatulence
Yes	11	25%	25	44.6%	0.042*
No	33	75%	31	55.4%

*Significant P, **Highly significant P

Discussion

Local estimation of the burden of intestinal parasitosis and concomitant risk factors among diabetics ensures good prognosis of DM patients. The present study showed that diabetic patients were more parasitized than the control group (44% vs. 32%) which could be attributed to decrease in the immune response in these patients which favor the establishment of some parasites; in particular the opportunistic ones.

Studies conducted in Egypt showed the same finding that intestinal parasites were higher in diabetic patients (20 and 25%) compared to healthy controls (10 and 7%) (Baiomy et al. 2010; Elnadi et al. 2015) respectively. Similar results were also reported worldwide by Akinbo et al. (2013) and Bafghi et al. (2015) where intestinal parasites were present in 18.7 & 24.4% among diabetic patients, and 0 & 23.2% in controls respectively. In addition, Mohtashamipour et al. (2015) revealed that the risk of intestinal parasitosis was 3.6 times more in diabetics than non-diabetics. However, some researchers have drawn different conclusions where enteric parasites were higher in control group than diabetic patients (Nazligul et al. 2001; Tangi et al. 2016).

The higher prevalence of intestinal parasitosis in our study can be explained by inclusion of only patients suffering from various GIT manifestations which increase the possibility of finding intestinal parasites, adding to higher prevalence of B. hominis, which is endemic in Egyptian population (El Shazly et al. 2006; Abaza et al. 2014).

The prevalence rate of Cryptosporidium sp. in the present study was higher in the diabetic patients (12%). This agrees with Ali et al. (2018) who reported similar percentage, while disagrees with Elnadi et al. (2015), Sabah and Temsah (2015) who reported lower prevalence rates; 5 and 0% in Sohag and Gharbia Governorates. Also, lower rates among diabetics were documented by others (Akhlaghi et al. 2005; Mohtashamipour et al. 2015; Alemu et al. 2018). Such variations in Cryptosporidium prevalence in diabetics could be related to immunologic status, duration of treatment, seasonal disparities as well as the sensitivity of the diagnostic techniques.

In the present study, Microsporidia sp. was detected by MT stain in 5% of diabetic patients, compared to none in the control group which emphasizes the theory of immunodeficiency as a determinant for microsporidial infections. Prior few studies showed that prevalence of Microsporidia in diabetic patients varies from 0 to 3% (Baiomy et al. 2010; Mohtashamipour et al. 2015; Elnadi et al. 2015).

Because of the minute size of microsporidian spores, they may be easily missed even if stained by MT, in particular when present in undetectable levels (Kazemi et al. 2017). So, the molecular techniques are recommended to increase the detection rate of Microsporidia.

No evidence for presence of Cyclospora or Cystoisospora oocysts in all samples which is consistent with others (Bafghi et al. 2015; Bora et al. 2016; Alemu et al. 2018). However, C. cayetanensis was detected in only one case (3.3%) among diabetic patients by Baiomy et al. (2010).

Likewise, all samples were negative for S. Stercoralis which come in agreement with Baiomy et al. (2010) and Bora et al. (2016). However, Ali et al. (2018) detected S. stercoralis in 1.6% of diabetic patients suffering from gastroenteritis.

The commonest parasite, B. hominis, was detected in one quarter of participants: 29% in the diabetic group and 21% in the control group. This is similar to Iranian study which reported B. hominis as the commonest parasite detected in diabetic patients with a significant difference between diabetics and controls (Mohtashamipour et al. 2015). Higher prevalence rate of B. hominis (35.5%) in diabetic patients was reported by Ali et al. (2018). Though there have been many debate about Blastocystis pathogenicity, it has been reported as the most prevalent parasite in immunocompromised patients including HIV-infected patients (Paboriboune et al. 2014), hemodialysis patients (Barazesh et al. 2015), and cancer patients (Wassef et al. 2016). Rates of B. hominis in Egypt reached up to 67.4% in Alexandria Governorate (Eassa et al. 2016) and 82% in Minia Governorate (Gabr et al. 2018). Polymorphism of Blastocystis sp., and tiny size of some isolates make microscopical detection difficult even after staining, especially when present in small numbers, and this may account for this difference (El-Badry et al. 2018; Hamdy et al. 2020).

G. lamblia, E. histolytica/dispar, and H. nana prevalences in diabetic patients in this study was recorded as 7%, 2 and 2% respectively. There are several reports with variable results ranging from 2.8 to 25.8% for Giardia (Baiomy et al. 2010; Alemu et al. 2018), (2–39.4%) for E. histolytica/dispar (Akinbo et al. 2013; Sabah and Temsah 2015; Tangi et al. 2016), and (0.8–5%) for H. nana (Akhlaghi et al. 2005; Elnadi et al. 2015; Ali et al. 2018). Although C. philippinensis was reported to be endemic in Upper Egypt including Beni-Suef Governorate (El-Dib and Doss 2002; El-Dib and Ali 2020), it was only observed in 3 cases, two cases in the diabetic group and one case in the control group. However, C. philippinensis wasn’t detected in other studies among diabetic patients (Bafghi et al. 2015; Bora et al. 2016).

Polyparasitism in the present study was approximately the same in diabetic patients (31.8%) and the control (31.25%). This is consistent with the fact that, 30% of infections may be co-infections (Petney and Andrews 1998). Polyparasitism usually results from presence of common risk factors facilitating parasites establishment and has greater impact on morbidity than single species infection (Mekonnen et al. 2016).

In this study, the highest percentage of intestinal parasites in diabetic group (61.4%) was in patients aged ≥ 41 years old. This finding agrees with others (Akinbo et al. 2013; Ali et al. 2018). Weakened immune system that comes with aging coupled with DM status with increased complications may clarify this finding.

The result of this study that intestinal parasites were higher in female diabetics (63.6%) than males (36.4%) is comparable with the reports of others Nazligul et al. (2001) and Akinbo et al. (2013), but contrary to the result of others (Baiomy et al. 2010; Alemu et al. 2018).

Results showed significantly more parasitic infection in diabetic patients inhabiting rural areas (77.3%) than urban ones (22.7%) by univariate analysis and multivariate analysis. Lower socioeconomic and hygiene standards may explain higher prevalence rates in rural areas than the remainder of the community (Fan et al. 2019).

Level of education, occupation, source of water supply and sewage system were not found to be significant risk factors affecting the prevalence of intestinal parasitic infections in diabetic patients. This is similar to a study conducted by Akinbo et al. (2013) in Nigeria.

Intestinal parasites were higher in uncontrolled and complicated diabetic patients with significant statistics. Diabetic patients in developing countries including Egypt are probably unable to receive adequate treatment, control blood glucose level, or manage diabetic complications well. This in turn leads to deterioration of immune system and facilitate the acquiring intestinal parasitic infections. Studies focused on this point are scarce.

In this study, intestinal parasites were highly statistically associated with diarrhea (P < 0.001) consistent with Mohtashamipour et al. (2015) and Bora et al. (2016), and flatulence (P = 0.042) as recorded by others Monib et al. (2016) and Siddig et al. (2017).

Conclusion

Our study showed that diabetic patients are at higher risk of infection with intestinal parasites than normal population. Also, B. hominis is an important parasitic infection which can cause gastrointestinal symptoms both in diabetic patients and control population.

Author contribution

All authors contributed to the study conception and design. Materials preparation, and samples collection from diabetic patients and controls were performed by [Walaa Ramadan Waly]. Performing the practical work, data analysis, and designing the results were done by [Walaa Ramadan Waly] and [Wegdan Mohamed Abd El Wahab]. All authors contributed to writing, revising the manuscript, and preparing it for publication.

Declarations

Conflict of interest

The authors declare that there is no conflict of interest.

Ethical approval

The study protocol was permitted by Scientific Research Ethical Committee of Beni-Suef University. Subjects were verbally informed about the purpose of the study. All individuals infected with intestinal parasites were personally informed or through their physicians to receive adequate treatment.