Prevalence of isosporiasis in relation to CD4 cell counts among HIV-infected patients with diarrhea in Odisha, India

Abstract

To determine the prevalence of Isospora belli and its correlation with CD4+ cell counts in HIV-positive patients with diarrhea in this region.
Stool samples from 250 HIV-positive patients, including 200 with diarrhea and 50 without diarrhea included in the study were examined for the presence of enteric parasites under microscopy. Prevalence of the enteric parasites with special reference to I. belli in HIV-positive patients with and without diarrhea were calculated and correlated with their CD4+ cell counts.
Enteric parasites were detected in 39% of the HIV patients with diarrhea compared to 30% without diarrhea. I. belli was detected in 22% of the patients with diarrhea and in 4% without diarrhea (P = 0.0019). I. belli was the most common parasite, followed by Entamoeba histolytica/dispar (8%) and Cryptosporidium parvum (5%) in HIV-positive patients with diarrhea. In HIV-positive patients without diarrhea, the most common parasite detected was E. histolytica/dispar (12%) followed by C. parvum (6%) and I. belli (4%). The mean CD4 cell count of HIV-positive patients with diarrhea suffering from isosporiasis was 138.35 ± 70.71. In patients with CD4 cell counts <200/μl, I. belli was seen in 36/123 stool samples and 2/27 stool samples which was statistically significant (P = 0.0157).
I. belli was the predominant parasite with a prevalence of 22% among HIV-positive patients with diarrhea, majority having CD4 cell count <200/μl. This study highlights the importance of routine screening for coccidian parasites in HIV-positive patients with and without diarrhea especially in those with low CD4 cell counts.

Full text

Advanced
Biomedical
Research
2013

Advanced Biomedical Research | July - September 2013 | Vol 2 | Issue 3 1
Background: To determine the prevalence of Isospora belli and its correlation with CD4+ cell counts in
HIV-positive patients with diarrhea in this region.
Materials and Methods: Stool samples from 250 HIV-positive patients, including 200 with diarrhea and
50 without diarrhea included in the study were examined for the presence of enteric parasites under
microscopy. Prevalence of the enteric parasites with special reference to I. belli in HIV-positive patients with
and without diarrhea were calculated and correlated with their CD4+ cell counts.
Results: Enteric parasites were detected in 39% of the HIV patients with diarrhea compared to 30% without
diarrhea. I. belli was detected in 22% of the patients with diarrhea and in 4% without diarrhea (P = 0.0019).
I. belli was the most common parasite, followed by Entamoeba histolytica/dispar (8%) and Cryptosporidium
parvum (5%) in HIV-positive patients with diarrhea. In HIV-positive patients without diarrhea, the most
common parasite detected was E. histolytica/dispar (12%) followed by C. parvum (6%) and I. belli (4%). The
mean CD4 cell count of HIV-positive patients with diarrhea suffering from isosporiasis was 138.35 ± 70.71.
In patients with CD4 cell counts <200/µl, I. belli was seen in 36/123 stool samples and 2/27 stool samples
which was statistically significant (P = 0.0157).
Conclusion: I. belli was the predominant parasite with a prevalence of 22% among HIV-positive patients
with diarrhea, majority having CD4 cell count <200/µl. This study highlights the importance of routine
screening for coccidian parasites in HIV-positive patients with and without diarrhea especially in those
with low CD4 cell counts.
Key Words: CD4 cell count, diarrhea, HIV, Isospora belli, isosporiasis
Address for correspondence:
Dr. Indrani Mohanty, Department of Microbiology, M.K.C.G. Medical College, Berhampur ‑ 760 004, Orissa, India. E‑mail: indranimohanty@yahoo.co.in
Received: 22.07.2012, Accepted: 25.09.2012
Abstract
Prevalence of isosporiasis in relation to CD4 cell counts
among HIV‑infected patients with diarrhea in Odisha, India
Indrani Mohanty, Pritilata Panda, Susmita Sahu, Mutikesh Dash, Moningi Venkat Narasimham,
Sanghamitra Padhi, Banojini Parida
Department of Microbiology, ART Centre, M.K.C.G. Medical College, Berhampur, Odisha, India
Original Article
INTRODUCTION
Isosporiasis is an intestinal disease of humans caused
by the coccidian parasite Isospora belli. Isosporiasis
mainly affects children and causes self‑limiting
diarrheal illness in healthy individuals. It is also
recognized as a cause of persistent diarrhea in children
and severe, prolonged diarrhea in persons with acquired
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DOI:
10.4103/2277-9175.115810
How to cite this article: Mohanty I, Panda P, Sahu S, Dash M, Narasimham MV, Padhi S, et al. Prevalence of isosporiasis in relation to CD4 cell counts among
HIV-infected patients with diarrhea in Odisha, India. Adv Biomed Res 2013;2:61.
Copyright: © 2013 Mohanty. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction
in any medium, provided the original author and source are credited.

Mohanty, et al.: Isosporiasis in Odisha
2 Advanced Biomedical Research | July - September 2013 | Vol 2 | Issue 3
immunodeficiency syndrome (AIDS). Progressive
decline in immunologic response makes HIV‑infected
patients vulnerable to opportunistic infections (OI).
Diarrhea is one of the major complications, occurring
in 90% of patients living with HIV/AIDS (PLHA) in
developing countries.[1] Many studies have outlined
the emergence of opportunistic intestinal coccidian
parasites notably Cryptosporidium species and I. belli
in HIV‑infected individuals.[2] As isosporiasis can be
effectively treated with trimethoprim‑sulfamethoxazole
combination, investigation of the parasitic etiology of
diarrhea can help in decreasing the morbidity in such
cases.[3] Keeping the above facts in view, this study
was conducted to know the prevalence of isosporiasis
and its correlation with CD4 count.
MATERIALS AND METHODS
This study was conducted in the Department of
Microbiology, M.K.C.G. Medical College, Berhampur
University, from April 2010 to March 2012. A total of
250 stool samples from HIV sero‑positive individuals
attending the antiretroviral treatment (ART) center
were included in the study, comprising 200 HIV
patients with diarrhea and 50 HIV patients without
diarrhea. The age group of the patients ranged
from 21 to 55 years. The HIV sero‑positive patients
were dened as those who had been tested positive
for HIV antibodies by the three Rapid tests as per
recommendations given by the WHO. A verbal
informed consent was obtained from all the patients
prior to specimen collection. Fresh stool samples
were collected in a clean, dry, and wide‑mouthed
universal container. All the patients were advised
to give three consecutive stool samples. Diarrhea
was dened as the passage of two or more liquid or
three or more soft or unformed stools per day. The
stool samples were observed macroscopically for
the presence of blood, mucus, and adult helminthes
worms. The samples were rst subjected to wet‑mount
examination (saline and iodine) under low power
(×10) and high power (×40) magnication for the
presence of ova, cysts, and trophozoites of intestinal
parasites. After formal‑ether concentration, a
smear was made in a clean and grease‑free slide,
xed with methanol and stained using the modied
Ziehl‑Nelseen technique for the detection of coccidian
parasites. The CD4 cell counts of the patients
were analyzed using flow cytometry (Becton and
Dickinson, USA, FACS caliber). Three antibody
panels were used, i.e., BD Tri TEST™ CD3 uorescein
isothiocyanate (FITC)/CD4 phycoerythrin (PE)/CD45
peridinin‑chlorophyll‑protein (PerCP), a three‑color
direct immunouorescence reagent to identify and
determine the percentages and absolute counts
of mature T‑lymphocytes (CD3+) and helper
T‑lymphocyte (CD3+CD4+) subsets in erythrocyte‑lysed
whole‑blood, by using Tru Count™ tubes. The ndings
were noted and tabulated.
Statistical analysis
Graph Pad software was used for calculation of mean,
median, standard deviation, range, P value using
Fisher’s extract test.
RESULTS
A total of 250 stool samples from HIV‑infected
patients were studied which included a study group
of 200 patients with diarrhea and a control group of
50 patients without diarrhea. The mean age of the
patients with diarrhea was 34.93 (SD 6.89; median 35)
and those without diarrhea was 32.50 (SD 7.55;
median 32). The ages ranged from 21 to 55 years. Out of
the 200 HIV‑positive patients with diarrhea, 118 (59%)
were male and 82 (41%) were female. Even in the
control group, there was male (66%) preponderance.
Enteric parasites were seen in 78 (39%) stool samples
in the study group and in 15 (30%) stool samples
in the control group. In the study group, the most
common parasite seen was I. belli (22%), [Figure 1],
followed by Entamoeba histolytica/dispar (8%),
Cryptosporidium spp. (5%), Microsporidium spp. (2%),
Giardia lamblia (1%), and Ascaris lumbricoides and
Strongyloides stercoralis (0.5% each). In the control
group (HIV‑positive without diarrhea), the most
common entero‑parasite detected was E. histolytica/
dispar (12%), followed by Cryptosporidium spp. (6%),
I. belli (4%) and A. lumbricoides, A. duodenale,
Cyclospora cayetenensis and Microsporidium
spp. (2% each) [Table 1]. The association of I. belli
infection among HIV‑positive individuals with
diarrhea was signicant (P = 0.0019, Fisher’s extract
test).
Figure 1: Microphotograph showing acid fast Isospora belli in stool
sample

Mohanty, et al.: Isosporiasis in Odisha
Advanced Biomedical Research | July - September 2013 | Vol 2 | Issue 3 3
The study and the control groups were categorized into
three groups based on their CD4 cell counts, i.e., <200,
200‑499 and >500 cells/µl. In HIV‑positive patients
with CD4+ cell counts <200 cells/µl, I. belli was seen
in 36/123 stool samples with diarrhea and 2/27 stool
samples without diarrhea, which was statistically
signicant (P = 0.0157, Fisher’s extract test). Eight
cases of I. belli were seen in HIV‑positive patients
with diarrhea having CD4+ cell counts in the range
of 200‑499 cell/µl. No cases of I. belli were seen in
the CD4 cell count range >500 cells/µl [Table 2]. The
mean CD4+ count of the patients who harbored I. belli
in was 138.35 (SD 70.71; minimum 55; median 106;
maximum 316).
DISCUSSION
Intestinal parasitic infections are the commonest
among the OI’s and are the major cause of morbidity
and mortality in HIV‑positive patients.[4] The
coccidian parasites like Cryptosporidium spp.,
Isospora spp., Cyclospora spp. and Microsporidium
spp. are the foremost among the enteric parasites in
HIV‑positive patients with diarrhea. These organisms
cause self‑limiting illness in immunocompetent
individuals, but in the case of immunocompromised
patients, they can cause life threatening, profuse,
watery diarrhea.[5] The line of treatment being
different for the diverse parasites, necessitates a
denitive diagnosis of the etiological agents causing
diarrhea, especially when the outcome can be fatal
in this group of individuals.
In the present study, enteric parasites were detected
in 39% of the HIV‑positive patients with diarrhea.
Various studies from India and other countries have
reported a high prevalence of intestinal parasites
ranging from 18.4% to 70%.[4,6‑11] Like many studies
we found that coccidian parasites were the leading
cause of diarrhea in HIV‑positive patients. Among the
non‑opportunistic pathogens, E. histolytica/dispar
was the commonest in HIV‑positive patients with and
without diarrhea.
I. belli (22%) was identied as the most prevalent
coccidian parasite among the HIV‑positive patients with
diarrhea in the present study. Vignesh et al. showed
similar detection rates of Isospora spp. in HIV‑positive
patients with diarrhea but it was slightly higher than
our study.[2] Similarly, higher prevalence of isosporiasis
has also been reported from southern India ranging
from 18% to 26.1%[1,2,12‑14] [Table 3]. The prevalence
of isosporiasis in the western part of India revealed
a lower prevalence of I. belli (4.7‑8%) in comparison
to our study.[9,10,15] But in the northern part of India,
studies have shown a wide variation in the prevalence
of isosporiasis ranging from 2.5% to 50%.[4,11,16,17]
Studies from countries like Nigeria, France, and Spain
have shown the prevalence of isosporiasis as 9.8%,
0.44%, and 5.0%, respectively[7,8,18‑21] [Table 3]. The
frequency of isosporiasis is often underestimated due
to asymptomatic shedding of oocysts and treatment of
Table 1: Parasites detected from the stool samples of
HIV‑positive individuals with and without diarrhea
Parasites HIV patients Total
With diarrhea
(n=200) (%)
Without diarrhea
(n=50) (%)
Ascaris lumbricoides 1 (0.5) 1 (2) 2
Ankylostoma duodenale 0 (0) 1 (2) 1
Strongyloides stercoralis 1 (0.5) 0 (0) 1
Entamoeba
histolytica/dispar
16 (8) 6 (12) 14
Giardia lamblia 2 (1) 0 (0) 2
Isospora belli 44 (22) 2 (4) 46
Cyclospora cayetenensis 0 (0) 1 (2) 1
Cryptosporidium parvum 10 (5) 3 (6) 13
Microsporidium spp. 4 (2) 1 (2) 5
Total 78 (39) 15 (30) 85
Table 2: Correlation of CD4+ cell counts with isosporiasis
CD4 cell counts HIV‑positive with
diarrhea (n=200)
HIV‑positive
without
diarrhea (n=50)
No. of
cases
Isospora
positive
No. of
cases
Isospora
positive
<200/µl123 36* 27 2
200‑499/µl49 8 14 0
<500/µl28 0 9 0
*P=0.0157, Fisher’s extract test. Mean CD4 count in HIV‑positive with
isosporiasis 138.35±70.71
Table 3: Prevalence of isosporiasis in HIV‑positive patients with
diarrhea in different studies within India and abroad
Place Prevalence of
isosporiasis (%)
Reference
number
Studies outside India
Nigeria, 2010, 2012 7.8; 9.8 [8,7]
Ethiopia, 2009 5; 12 [19,20]
Spain, 1987 5 [21]
France, 2008 0.44 [22]
Studies within India
Vellore, 1999, 2007 18; 20 [ 1,13 ]
Lucknow, 2000 31 [11]
Chennai, 2002, 2007 18; 26.1 [14,2]
Chandigarh, 2002 2.5 [17]
Madurai, 2007 1.2 [12]
New Delhi, 2008 50 [4]
Pune, 2009 8 [9]
Wardha, 2010 4.7 [10]
Ahmednagar, 2011 5.72 [15]
Jaipur, 2012 10.9 [16]
Our study 22

Mohanty, et al.: Isosporiasis in Odisha
4 Advanced Biomedical Research | July - September 2013 | Vol 2 | Issue 3
other OI’s with trimethoprim‑sulfamethoxazole which
may confer some protection against this parasite. In
most studies, Cryptosporidium spp. was the most
common coccidian parasite responsible for diarrhea
in HIV‑positive patients.[9,10,15] There has been no
documented report on the prevalence of coccidian
parasite from this region prior to this study. We report
a high detection rate of I. belli in comparison to other
coccidian parasites among HIV‑positive patients
with diarrhea. E. histolytica/dispar (8%) was the
next common parasite followed by Cryptosporidium
spp. (5%) in the present study. A comparison listed
in Table 3, on the studies conducted throughout the
world showed a marked geographical variation in the
prevalence of isosporiasis.
In the present study, I. belli was the predominant
pathogen among patients having CD4+ cell counts
<200/µl which was similar to the study done by Gupta
et al.[4] There are very few studies on the correlation
of isosporiasis with CD4+ cell counts. Opportunistic
parasitic infections may be acquired any time during
the course of HIV infection and cause self‑limiting
illness in individuals with normal CD4+ cell counts,
but as the immune status decreases, life‑threatening
diarrhea results. Spontaneous clearing of these
opportunistic parasites generally occurs as the CD4+
counts increases. All the patients included in this study
were attending ART center and were on Highly active
anti retroviral therapy (HAART) and other antibiotics
for OI’s including trimethoprim‑sulfamethoxazole
prophylactically for these infections. In spite of
the prophylactic treatment with trimethoprim–
sulfamethoxazole, the patients were suffering from
isosporiasis. This may be due to under dosage,
non‑compliance, relapses after discontinuation, or
emergence of drug resistance.
CONCLUSION
Screening of HIV‑patients with diarrhea is not done
routinely in most of the cases even though coccidian
parasites are considered as AIDS‑dening opportunistic
pathogens according to CDC guidelines. The incidence
and prevalence of infection with a particular enteric
parasite in HIV/AIDS patients is likely to depend
upon the endemicity of that particular parasite in
the community.[22] Intestinal parasitic infections in
HIV‑positive individuals would be expected to be
higher in developing countries due to the higher
prevalence of infection in the general population. As
most protozoan infections are treatable, an early and
accurate diagnosis can decrease the morbidity and
mortality in most cases. As the techniques used in the
diagnosis of coccidian parasites are simple, rapid, and
do not require sophisticated or costly equipments, they
can be used as screening tests in all diarrheal cases in
HIV‑positive patients. In resource‑poor settings like
ours, the patients usually present late in the course of
illness with low CD4+ counts due to delay in diagnosis.
It should be stressed that routine screening of stool
samples of HIV‑positive patients with diarrhea should
be done to prevent morbidity and mortality. As most of
the enteric pathogens gain entry through the feco‑oral
route, improvement of sanitation and proper drinking
water and health education can help in decreasing
the prevalence. The health practitioners should also
be made aware about the routine screening methods
for coccidian parasites.
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Source of Support: Nil, Conict of Interest: None declared.