Opportunistic and non-opportunistic intestinal parasites in HIV/ AIDS patients in relation to their clinical and epidemiological status in a specialized medical service in Goiás, Brazil.

Patients infected with the Human Immunodeficiency Virus (HIV) often have opportunistic infections, among which strongyloidiasis and coccidiosis are the most common parasitic infections that aggravate their health status. This study examined the prevalence of intestinal parasites, particularly of Strongyloides stercoralis and intestinal coccidia in patients with the Human Immunodeficiency Virus (HIV)/ Acquired Immunodeficiency Syndrome (AIDS) who were treated at the Specialized Assistance Service (SAE) of Jataí, State of Goiás, Brazil, and analyzed its correlation with clinical, laboratory, and socio-epidemiological parameters. A total of 270 stool samples were analyzed by the Lutz technique, Rugai's method, Agar Plate Culture, Ritchie's method and specific staining, Ziehl-Neelsen modified technique, Kinyoun's method and the rapid safranin method. The prevalence of intestinal parasites was 28.88% including 3.8% of S. stercoralis, Cryptosporidium sp. and Cystoisospora belli. There was a significant positive correlation between intestinal parasites and the clinical status and the use of antiretroviral therapy (ART), smoking, CD4+ lymphocyte counts and sexual orientation. In conclusion, the widespread use of antiretroviral therapy and health assistance contributed to the low prevalence of S. stercoralis and coccidiosis in patients with HIV/ AIDS who were followed up at the SAE.

INTRODUCTION

Intestinal parasitic infections are a serious public health problem in developing countries, where poor nutrition is associated with poor hygiene, poor environmental sanitation, inadequate access to potable water and several coinfections , . In Brazil, the tropical climate favors the spread of parasites because it provides ideal conditions for them to complete their life cycle and perpetuate new hosts to contaminate the environment .

Parasitic infections are an important cause of morbidity and mortality, especially with the emergence of immunosuppressive diseases , . After the advent of the human immunodeficiency virus (HIV)/ acquired immunodeficiency syndrome (AIDS) in the world, intestinal parasites that caused only sporadic or zoonotic, benign and asymptomatic infections, have become opportunistic parasites and caused severe episodes of diarrhea in patients with HIV/ AIDS , .

The occurrence of diarrheal stools is considered a marker of poor prognosis, being an evolutionary predictor of AIDS . The most prominent enteroparasitoses in patients with HIV/ AIDS are caused by Strongyloides stercoralis nematodes and intestinal coccidia that are members of the Apicomplexa phylum, which usually aggravate the clinical condition of immunocompromised patients and cause refractory infections , . Because of the chronic and recurrent nature of these infections, the intention of this study was to make the diagnosis of Strongyloides stercoralis and Cryptosporidium sp., Cystoisospora belli and Cyclospora cayetanensis through specific techniques in order to determine the prevalence of these intestinal parasites in HIV-positive/ AIDS patients in order to direct strategies for diagnosis and prevention.

MATERIALS AND METHODS

Characterization of patients and study site

The study participants were adult patients (aged over 18 years old) of both genders, with HIV/ AIDS, who had not used anthelmintics in the previous six months and were followed up at the Specialized Assistance Service (SAE) of Dr. Serafim Carvalho Municipal Health Center, in the city of Jataí, State of Goiás, Brazil, between June 2015 and March 2016. The Research Ethics Committee of the Universidade Federal de Goiás (Jataí, GO, Brazil) approved the study protocol (process Nº 1079116/2015) and all the participants signed an informed consent form.

Study design and delivery of stool samples

Patients who attended medical consultations were invited to meet the researcher, who clarified all the study objectives and the patients’ doubts about them. Individuals who agreed to participate in the study signed the informed consent form and answered a socio-epidemiological questionnaire containing questions related to the variables: gender, age group, type of residence, water treatment, sewerage system, hand washing, number of bowel movements per day, aspects of feces, blood/ mucus in stool, clinical status, CD4+ T-cell count, viral load, taking ART or not and sexual orientation. Patients were instructed on how to collect their stool samples and received vials identified with the initials of their names and the sample number. Fecal samples were stored, further processed and analyzed in the Parasitology Laboratory of the Universidade Federal de Goiás (Jataí, GO, Brazil).

Techniques for parasitological diagnosis of fecal samples

From each participant, three fecal samples were collected on alternate days at the patient’s home and delivered to the researcher. Each fresh stool specimen was processed using the following methods: the method described by Lutz , also known as Hoffman, Pons and Janer’s method or yet the spontaneous sedimentation, which allows detection of helminth eggs and larvae, as well as protozoan cysts ; the Rugai’s method , which is suitable for detection of S. stercoralis and hookworm larvae; the agar plate culture method , ; and the Ritchie technique to concentrate samples to prepare fecal smears. The Ziehl-Neelsen method, the Kinyoun’s method and the safranin technique , were used to identify Cryptosporidium sp, C. belli, and C. cayetanensis. Three slides of each sample were prepared and examined by two analysts, i.e., six slides per sample were analyzed. The slides from samples processed through the Lutz, Rugai and agar plate culture techniques were analyzed by light microscopy under 100- and 400-fold magnification. To identify intestinal coccidia, the slides were analyzed under 1,000-fold magnification using immersion oil. When no parasites and/ or commensals were found in the sample, two additional slides were prepared to confirm the result.

Statistical analysis

Statistical analysis was performed using the GraphPad Prism version 5.0 software (GraphPad Software, Inc., San Diego, USA). Data were analyzed by the t-test (student), chi-square, one-way ANOVA and the Fisher’s exact test when appropriate. The magnitude of association between variables was estimated by the odds ratio (OR) with a 95% confidence interval (95% CI). The level of significance was set at p < 0.05.

RESULTS

Three fecal samples were collected from each one of the 90 patients with HIV/ AIDS. Forty-nine (54.44%) participants were female and 41 (45.55%) were male. Their age ranged from 23 to 77 years old, with mean ± SD of 42 ± 11.61, and predominance of women aged 31 to 40 years (16 or 17.7%) and men aged 41 to 50 years (15 or 16.6%). There was no significant difference between the variables gender and age of all the patients analyzed (p = 0.7132, p = 0.7132; χ2 = 2,914, 5). HIV-infected patients of both genders were positive for intestinal parasites: 13/41 infected men and 13/49 infected women (Table 1). There was no significant difference in positivity regarding the genders.

Table 1

- Prevalence of intestinal parasites according to age and gender in patients with HIV/ AIDS patients, Jataí, State of Goiás, Brazil (N=90)

Age range (years)	Patients HIV/AIDS		Infected with Protozoans/S. stercoralis
	Male	Female	Male	Female
21 a 40	19 (46,3%)	25(51%)	7 (53,8%)	7 (53,8%)
> 41	22 (53,6%)	24(50%)	6 (46,1%)	6 (46,1%)
Total	41	49	13	13

The risk factors associated with transmission of intestinal parasitic infection in patients with HIV/ AIDS are presented in Table 2. The incidence of parasitic infections was strongly associated with individuals living in recovery houses (OR: 7.588; 95 % CI: 0.2 to 192.6). Presence of blood in patients’ stools increased the probability of intestinal parasitic infection (OR: 2.251; 95% CI: 0.7 to 6.8); however, there was no statistical significant difference. The univariate analysis evidenced a significant association between the patients’ clinical status and the risk of parasitic infection (OR: 11.42; p = 0.002). The use of ART and the incidence of parasitic infection were also significantly different (p = 0.041) (Table 2).

Table 2

Demographic, clinical and laboratory characteristics of patients with HIV/ AIDS in Jataí, State of Goiás, Brazil (N=90)

Features	Protozoans and Helminths		Total(%)	OR	IC-95%	p value
	Infected	No Infected
Sex
Male	13 (50)	28 (43,75)	41 (45,55)	1,286	0.5- 3.2	0,644
Female	13 (50)	36 (56,25)	49 (54,44)
Age group (years)
21 a 30	6 (23,07)	10 (15,62)	16 (17,77)	1,620	0.5- 5.0	0,543
31 a 40	7 (26,92)	21 (31,25)	27 (30)	0,810	0.2- 2.2	0,802
41 a 50	6 (23,07)	21 (32,81)	27 (30)	0,614	0.2- 1.7	0,451
51 a 60	5 (19,23)	9 (14,06)	14 (15,55)	1,455	0.4 - 4.8	0,535
> 60	2 (7,69)	3 (4,6)	5 (5,55)	1,694	0.2-10.7	0,624
Type of residence
Own home	11 (42,30)	31 (48,43)	42 (46,66)	0,780	0.31- 1.9	0,646
Rented house	8 (30,76)	14 (21,87)	22 (24,44)	1,587	0.57- 4.4	0,421
Earned home	3 (11,53)	7 (10,93)	10 (11,11)	1,062	0.25- 4.4	1,000
Home courtesy	3 (11,53)	11 (17,18)	14 (15,55)	0,628	0.16- 2.4	0,749
Recovery House	1 (3,84)	-	1 (1,11)	7,588	0.29-192	0,288
Did not	-	1 (1,56)	1 (1,11)	0,798	0.03- 20	1,000
Water treatment
Mineral water	1 (3,84)	1 (1,56)	2 (1,11)	2,520	0.1- 41.9	0,496
Filtered water	11 (42,30)	31(48,43)	42 (46,66)	0,780	0.3- 1.9	0,646
No treatment	14 (53,84)	32 (50)	46 (51,11)	1,167	0.4- 2.9	0,818
Sewerage system
Yes	15 (57,69)	34 (53,12)	49 (54,44)	1,203	0.4- 3.0	0,816
No/ do not know	11(42,30)	30 (46,87)	41 (45,55)
Wash hands
Yes	25 (27,77)	58 (90,62)	83 (92,22)	2,586	0.2- 22.6	0,668
No	1 (3,84)	6 (9,37)	7 (7,77)
Number of bowel movements per day
1 a 2	21 (80,76)	47 (73,43)	68 (75,55)	1,519	0.4-4.6	0,592
3 a 4	-	8 (12,5)	8 (8,88)	0,125	0.06- 2.2	0,099
5 a 6	-	1 (1,56)	1 (1,11)	0,798	0.03- 20	1,000
Do not know	5 (19,23)	8 (12,5)	13 (14,44)	1,667	0.4-5.6	0,509
Aspects of faeces
Diarrheal	10 (38,46)	23 (35,93)	33 (36,66)	1,114	0.4- 2.85	0.814
No diarrheal	16 (61,53)	41 (64,06)	57 (63,33)
Blood/mucus in stool
Yes	4 (15.4)	3 (4.6)	7 (7.80)	3.697	0.7-17.8	0.186
No	22 (84.6)	61 (95.4)	83 (92.2)
Clinical status
AIDS	7 (26,92)	2 (3,12)	9 (10)	11,42	2.1-59.6	0,002
HIV	19 (73,07)	62(96,87)	81 (90)
CD4+T (cels/mm 3 )
< 200	3 (11,53)	6 (9,37)	9 (10)	1,261	0.29-5.4	0,714
200 a 500	5 (19,23)	7(10,93)	12 (13,33)	1,939	0.5- 6.7	0,317
> 500	18 (69,23)	51 (79,68)	69 (76,66)	0,573	0.20 -1.6	0,288
Viral charge (copies/mL)
< minimun limit	14 (15,55)	39 (60,93)	53 (58,88)	0,747	0.29-1.8	0,637
< 1.000	4 (15,38)	13 (20,31)	17 (18,88)	0,713	0.20- 2.4	0,768
1.001-10.000	4 (15,38)	5 (7,81)	9 (10)	2,145	0.5- 8.7	0,275
10.001-100.000	3 (11,53)	4 (6,25)	7 (7,77)	1,957	0.40- 9.4	0,407
>100.000	2 (7,69)	2 (3,12)	4 (4,44)	1,240	0.1-14.3	1,000
Did not	-	1 (1,56)	1 (1,11)	0,798	0.03-20	1,000
TARV
Yes	21 (80,76)	61 (95,31)	82 (91,11)	0,206	0.04-0.9	0,041
No	5 (19,23)	3 (4,68)	8 (8,88)
Sexual orientation
Heterossex	20 (76,92)	54 (84,37)	74 (82,22)	0,617	0.19-1.9	0,543
Homossex	1 (3,84)	5 (7,81)	6 (6,66)	0,472	0.05-4.2	0,668
Bisex	4 (15,38)	2 (3,12)	6 (6,66)	5,636	0.9-32.9	0,055
Unknown	1 (3,84)	3 (4,68)	4 (4,44)	0,813	0.08-8.2	1,000
Total	26 (28,88)	64 (71,11)	90 (100)

No – number, OR – Odd ratio, IC 95% - Confidence interval, p value – significant when p< 0.05.

Analysis of the prevalence of intestinal parasites stratified by HAART (highly active antiretroviral therapy) and non-HAART revealed the strongest correlations with infections caused by the commensal Endolimax nana (OR: 2.33; IC95 0.21 to 24.9) and Entamoeba coli (OR: 2.20; IC95: 0.10 to 46.4), with significant differences (p = 0.0094) (Table 3). Analysis of the clinical and laboratory variables of patients with diarrhea evidenced that CD4+ T counts (< 200 cells/ mm3) and viral load (between 10,001 and 100,000) increased the probability of diarrhea (OR: 1.434; CI 95%: 0.35 to 5.7; OR: 4.911; CI 95%: 0.8 to 26.9, respectively), but not in a significant manner (p > 0.05) (Table 4).

Table 3

Relationship between HAART and Non-HAART and infection with intestinal parasites in patients HIV/ AIDS in Jataí, State of Goiás, Brazil (N=90)

Prevalence of intestinal parasites	HAART (N = 81)	NON-HAART ( N = 9)	OR	CI 95%	p value
E. nana	14	1	2.33	0.21–24.9	0.6257
E. hartmanni	5	2	0.18	0.02–1.63	0.1626
E. coli	6	0	2.20	0.10–46.4	1.0000
B. hominis	2	0	0.73	0.03–18.0	1.0000
G. lamblia	1	0	0.42	0.01–12.2	1.0000
I. butschlii	1	0	0.42	0.01–12.2	1.0000
E. histolytica/dispar	1	0	0.42	0.01–12.2	1.0000
C. belli	0	1	0.035	0.001–1.0	0.1111
Cryptosporidium sp.	1	0	0.42	0.01–12.2	1.0000
S. stercoralis	1	0	0.42	0.01–12.2	1,0000
Total	32	4

OR – odds ratio, CI 95% – confidence interval, p value of Mann-Whitney = 0,0094; (p < 0.05**).

Table 4

- Analysis of clinical and laboratory parameters of the 90 patients with HIV/ AIDS stratified by the presence of diarrhea in Jataí, State of Goiás, Brazil

Variables		Diarrhea		OR	IC 95%	p value
		YES (%)	NO (%)
CD4+T (cells/mm 3 )	< 200	4 (12,12)	5 (8,77)	1,434	0.3568-5.767	0,7197
	200-500	4 (12,12)	8 (14,03)	0,8448	0.2336-3.055	1,0000
	>500	25 (75,75)	44 (77,19)	0,9233	0.3368-2.531	1,0000
Viral charge (copies/mL)	< minimum	20 (60,60)	33 (57,89)	1,119	0.4667-2.682	0,8278
	<1000	6 (18,18)	11 (19,29)	0,9293	0.3085-2.799	1,0000
	1.001-10.000	1 (3,03)	8 (14,03)	0,1914	0.02282-1.605	0,1467
	10.001-100.000	5 (15,15)	2 (3,50)	4,911	0.8951-26.94	0,0951
	>100.000	1 (3,03)	2 (3,50)	0,8594	0.07488-9.863	1,0000
	Did not	-	1 (1,75)	0,5622	0.02224-14.21	1,0000
TARV	Yes	31 (93,93)	51 (89,47)	1,824	0.3461-9.608	0,7053
	No	2 (6,06)	6 (10,52)
Total		33 (36,66)	57 (63,33)

HAART –highly active antiretroviral therapy, OR – Odd ratio, IC 95% - Confidence interval, p value (significant when p<0.05).

Table 5 summarizes the significant associations between the incidence of different intestinal parasitic species and CD4+ T-cell counts. Cell counts > 500 cells/ mm3 were positively associated with monoparasitism (p = 0.0132). The type of sexual relationship significantly influenced (p = 0.0001) the parasitic associations with monoparasitism.

Table 5

- Parasitic association stratified by CD4+ T-cell count and sexuality of the 26 patients with HIV/ AIDS who were positive for intestinal parasites in Jataí, State of Goiás, Brazil

Parasitism	N	%	CD4+T (cells/mm3)			p	Sexual Orientation (%)			p
			< 200	200-500	>500		Homo	Hetero	Bi
Monoparasitism	20	22,22
E. nana	10	11,11	1 (1,11)	3 (3,33)	6 (6,66)	0,0132	-	8 (8,88)	2 (2,22)	0,0001
E. hartmanni	4	4,44	-	-	4 (4,44)		-	2 (2,22)	2 (2,22)
E. coli	3	3,33	-	1 (1,11)	2 (2,22)		-	3 (3,33)	-
B. hominis	1	1,11	-	-	1 (1,11)		-	1 (1,11)	-
S. stercoralis	1	1,11	-	-	1 (1,11)		-	1 (1,11)	-
C. belli	1	1,11	1 (1,11)				-	1 (1,11)	-
Biparasitism	3	3,33
E. coli + E. nana	1	1,11	-	-	1 (1,11)		-	1 (1,11)	-
E. coli + hartmanni	1	1,11	-	-	1 (1,11)		1 (1,11)	-	-
E. nana + B. hominis	1	1,11	1 (1,11)	-	-		-	1 (1,11)	-
Polyparasitism	3	3,33
E. nana + E. coli + I. butschlii	1	1,11	-	-	1 (1,11)		-	1 (1,11)	-
G. lamblia + E. nana + E. hartmanni	1	1,11	-	-	1 (1,11)		-	1 (1,11)	-
Cryptosporidium sp. + E. nana + E. histolytica/dispar + E. hartmanni	1	1,11	-	1 (1,11)	-			1 (1,11)
Total	26	28,88	3	5	18		1	21	4

DISCUSSION

Opportunistic infections are common in patients with HIV/ AIDS and are related to high morbidity in this population , . The HIV virus induces an immunodeficient state that favors infection by enteroparasites , which, in turn, contributes to worsen the patients’ clinical condition by causing malnutrition, weight loss, and chronic diarrhea - . The Epidemiological Bulletin of the State of Goiás, Brazil, reported 8,785 cases of AIDS in individuals aged over 13 years in the period between 2002 and 2015 . In this context, the present study examined the occurrence of intestinal parasites in patients with HIV/ AIDS who were treated at the SAE.

The socio-epidemiological profile of the studied patients, whose ages ranged from 23 to 77 years, with predominance of the age groups 31-40 years and 41-50 years old, corroborates the national data reported by Bertoni et al. . As the 31-40 age group corresponds to active individuals with respect to their capacity to work, procreate, and have sexual intercourses, the prevalence of HIV-infected people in this age group can result in significant damage to the population and the economic development of the country .

Detection of S. stercoralis, Cryptosporidium sp. and C. belli in 3.8% of the fecal samples of patients with HIV/ AIDS was lower than the prevalence reported by Soúza-Junior and Garcia-Zapata , in a similar study conducted in Goiânia, Goiás. It is important to note that there are few studies on the prevalence of intestinal parasites, including S. stercoralis and intestinal coccidia, in patients with HIV/ AIDS in the State of Goiás - this is the first study carried out in the Southwestern region of this State.

The frequency of commensal protozoa is 35,8% in patients with HIV/ AIDS from Rio de Janeiro . The high positivity rate for commensals observed in the present study may be considered an indicator of poor hygiene and sanitation, as well as of consumption of contaminated water and food by these patients . Good hygiene habits are the best way to avoid contamination and reinfection by intestinal parasites and health security is necessary for the prevention of diseases transmitted by water and food . Food handlers are unaware of the safety procedures for the handling, disposal and storage of food , and patients with HIV/ AIDS do not adopt food safety practices, making them more susceptible to this source of parasitic infection .

The poor management of water and sewage services, associated with the lack of regulation and public policies represent a serious problem in Brazil. Unfortunately, most of these services dump garbage and sewage directly into the soil, rivers and river springs, causing environmental degradation and proliferation of diseases , . The waterborne diseases, especially those caused by intestinal protozoa, are among the most relevant pathogens of drinking water-associated disease outbreaks , .

The Coccidia life cycles are suited to waterborne and foodborne transmission and their oocysts are resistant to conventional water treatment protocols . Intestinal parasites are highly prevalent in sludge and sewage, thereby representing potential risks to public health . All the raw water samples from the Atibaia River (Campinas, SP, Brazil) collected in 2001 were positive for Cryptosporidium spp. and Giardia spp . Giardia spp. cysts were found in 90.5% of the raw sewage samples; 96.2% of treated sewage samples not disinfected by UV lightand 94.7% of treated wastewater disinfected by UV lights - in other words, the cysts resist to the wastewater treatment process . These reports stress the importance of the parasitological control of water and sludge from municipal power stations and the urgent need to expand sanitation coverage to improve wastewater treatment in Brazil.

Monoparasitism predominated among the study participants (22.22%), corroborating the findings of Brito et al. and Marchi Blatt and Cantos . Parasitic associations were prevalent in patients with CD4+ cell counts greater than 500 cells/mm3, probably because the majority of study participants were of this group (76.66%). Analysis of the correlation between parasitic association and the type of sexual intercourse resulted in significant differences (p = 0.0001). The sexual behavior of men having homosexual intercourses is considered a risk factor for infections by Cryptosporidium sp and Entamoeba histolytica .

Coinfection of different parasitic species depends on the environmental contamination and increases the probability of polyparasitic contamination . In addition, nutritional and immunological factors of the host are crucial in determining the establishment and the course of parasitic infections . As AIDS causes a progressive immunodeficiency characterized by depletion of CD4+ T-cells, patients with HIV/ AIDS are 11.42 times more likely to develop parasitic infection than healthy individuals (OR: 11.42; 95% CI: 2.18 to 59.6; p = 0.0020) .

The use of antiretroviral therapy provided protection against parasites (OR < 1, p = 0.0419). Patients’ high adherence to antiretroviral therapy markedly improves the immune response, slows down the progression of diseaseand reduces the susceptibility to opportunistic infections , . HIV-infected children who were virgin to antiretroviral therapy had a higher prevalence of intestinal parasites than those undergoing treatment with HAART .

Analysis of medical records showed that most patients who were positive for intestinal parasites showed CD4+ T lymphocyte counts above 500 cells/ mm3 (69.23%), but there was no significant association (p > 0.05). The literature reports that the prevalence of intestinal parasites was significantly higher in subjects with CD4+ cell counts below 200 cells/ mm3, proving that CD4+ T lymphocytes are essential to fight against HIV and slow down the progression of AIDS - . In the present study, patients who were positive for Cryptosporidium sp. and C. belli had CD4+ cell counts of 173 and 282 cells/ mm3, respectively. This finding is in line with the frequent infection by intestinal Coccidia found in patients with low CD4+ cell counts - .

The presence of diarrhea did not significantly correlate with infection by intestinal parasites, corroborating the findings of Bachur et al. and Wanyiri et al. . In contrast, Pavie et al. have reported a positive relationship between diarrhea and intestinal parasites positive samples, while Attili et al. reported that protozoa infections are associated with a higher incidence of diarrhea in HIV-infected individuals than in the general population. Other causes of diarrhea in patients with HIV/ AIDS are infection by other pathogens, such as bacteria, fungi and viruses; entheropathy caused by HIV and side effects of HAART .

Antiretroviral therapy has gradually reduced the incidence of opportunistic infections in patients with HIV/ AIDS. However, poor adherence to treatment negatively affects the effectiveness of antiretroviral therapy and the prognosis of patients. The time between the diagnosis of infection and the onset of AIDS, adverse drug reactions, the patient’s age and education level, and the use of illicit drugs are the main reasons reported for non-compliance to treatment . A recent study revealed that most patients (98.3%) stop taking the medication due to psychological reasons, which can reduce the effectiveness of antiretroviral therapy and increase the susceptibility to opportunistic infections .

In conclusion, the prevalence of intestinal parasites found in this study has attracted the attention of health professionals to the need for parasitological examinations in the routine treatment of patients with HIV/ AIDS, including specific tests, given the clinical importance of these diseases in the evolution to AIDS. In addition, this study demonstrates the risk behaviors for the infection by enteroparasites according to the epidemiological scenario in Jataí, and evidences the need to implement preventive measures to reduce infection rates and prevent the worsening of the patients’ clinical conditions.