How common is intestinal parasitism in HIV-infected patients in Malaysia?

Abstract

Human immunodeficiency virus (HIV)-infected individuals have greater susceptibility to infections by a myriad of microorganisms which can cause significant morbidity and mortality compared to immunocompetent individuals. Of these microbial infections, intestinal parasitic infections (IPIs) however are receiving less attention than bacterial and viral infections, hence, the lack of information of parasitic infections in HIV individuals. Prevalence of IPIs among 346 HIV-infected individuals in Malaysia was determined in this study. The overall prevalence of intestinal parasitic infections (IPIs) was 37.9% (131 of 346) with protozoa infections (18.8%) being more common compared to helminth infections (7.5%). Observed protozoa include Entamoeba histolytica/dispar (16.8%), Cryptosporidium parvum (12.4%), Isospora belli (10.1%), Cyclospora cayetanensis (4.9%) and Giardia duodenalis (intestinalis) (3.2%) whilst helminthes which were detected comprised of Ascaris lumbricoides (13.9%), Trichuris trichiura (6.4%) and hookworms (0.6%). Among those 131 infected, 50.4% had multiple infections and 48.9% had single parasitic infection. The CD4 counts were significantly lower (i.e., 200 cells/mm³) in patients harbouring IPIs. Of those individuals infected with intestinal parasites, 49% were intravenous drug users and 58% were not on any antiretroviral therapy. Most were asymptomatic and had concurrent opportunistic infections (OIs) mainly with Mycobacterium tuberculosis infection. These results confirmed that IPIs are ubiquitous among HIV-infected individuals, especially those presenting with low CD4 T cells counts, and provide useful insights into the epidemiology of these infections among HIV-infected patients in Malaysia. It is therefore recommended, that diagnosis of these intestinal parasitic pathogens should be conducted on a routine basis for better management of gastrointestinal illnesses among HIV individuals.

Full text

Page 1

400
Tropical Biomedicine 28(2): 400–410 (2011)
How common is intestinal parasitism in HIV-infected
patients in Malaysia?
Asma, I.1, Johari, S.1, Benedict, L.H. Sim2 and Yvonne, A.L. Lim1*
1 Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur
2 Infectious Disease Unit, Department of Medicine, Hospital Sungai Buloh, 47000 Sungai Buloh,
Selangor Darul Ehsan
* Corresponding author email: limailian@um.edu.my
Received 25 January 2011; received in revised form 31 March 2011; accepted 4 April 2011
Abstract. Human immunodeficiency virus (HIV)-infected individuals have greater
susceptibility to infections by a myriad of microorganisms which can cause significant morbidity
and mortality compared to immunocompetent individuals. Of these microbial infections,
intestinal parasitic infections (IPIs) however are receiving less attention than bacterial and
viral infections, hence, the lack of information of parasitic infections in HIV individuals.
Prevalence of IPIs among 346 HIV-infected individuals in Malaysia was determined in this
study. The overall prevalence of intestinal parasitic infections (IPIs) was 37.9% (131 of 346)
with protozoa infections (18.8%) being more common compared to helminth infections (7.5%).
Observed protozoa include Entamoeba histolytica/dispar (16.8%), Cryptosporidium parvum
(12.4%), Isospora belli (10.1%), Cyclospora cayetanensis (4.9%) and Giardia duodenalis
(intestinalis) (3.2%) whilst helminthes which were detected comprised of Ascaris lumbricoides
(13.9%), Trichuris trichiura (6.4%) and hookworms (0.6%). Among those 131 infected, 50.4%
had multiple infections and 48.9% had single parasitic infection. The CD4 counts were
significantly lower (i.e., 200 cells/mm3) in patients harbouring IPIs. Of those individuals
infected with intestinal parasites, 49% were intravenous drug users and 58% were not on any
antiretroviral therapy. Most were asymptomatic and had concurrent opportunistic infections
(OIs) mainly with Mycobacterium tuberculosis infection. These results confirmed that IPIs
are ubiquitous among HIV-infected individuals, especially those presenting with low CD4 T
cells counts, and provide useful insights into the epidemiology of these infections among HIV-
infected patients in Malaysia. It is therefore recommended, that diagnosis of these intestinal
parasitic pathogens should be conducted on a routine basis for better management of
gastrointestinal illnesses among HIV individuals.
INTRODUCTION
HIV-infected individuals in developing
countries such as Malaysia are not only
susceptible to opportunistic infections but
they are also predisposed to a myriad of
enteric pathogens which are endemic in the
tropics. Whilst information on bacterial and
viral infections is more commonly available
to local physicians, unfortunately knowledge
of intestinal parasitic infections (IPIs) among
HIV patients in Malaysia is still very limited.
The lack of knowledge coupled with these
intestinal parasites not being included in
routine diagnostic testing have unknowingly
contributed to these parasites being
perceived as “uncommon”. However, reports
from many regions of the world where HIV/
AIDS is endemic have acknowledged that
intestinal parasitism is widespread among
these populations (Assefa et al., 2009).
Several intestinal parasitic pathogens
which have been reported in HIV patients
include Cryptosporidium parvum, Isospora
belli, microsporidia (Enterocytozoon
bieneusi, Encephalitozoon intestinalis),
Giardia duodenalis, Entamoeba histolytica/
dispar, Cyclospora cayetanensis, Ascaris
lumbricoides, Trichuris trichuria,
hookworms and Strongyloidis stercoralis

Page 2

401
(Ramakrishnan et al., 2007; Gupta et al., 2008;
Farthing et al., 2009). With impaired
immunity especially in patients with low
immune level (CD4 counts < 200 cells/mm3),
infections with intestinal parasites may result
in diarrheal symptoms (Daryani et al., 2009).
Patients with CD4 count of > 180 cells/mm3
usually have self-limiting infections, whereas
most patients with counts < 140 cells/mm3
develop severe and persistent infections
(Current & Garcia, 1991). With the
introduction of HAART which partially
restores the immune function, the incidence
of opportunistic parasite infection such as
cryptosporidiosis has declined (Hung et al.,
2007).
IPIs that are asymptomatic or causes
self-limiting diarrhoea in immunocompetent
individuals can cause profuse diarrhoea in
immunocompromised individuals, generally
accompanied by weight loss, anorexia,
malabsorption and in some cases fever and
abdominal pain (Kurniawan et al., 2009).
Patients with some type of immuno-
compromised condition and those subjected
to immunosuppressive therapy have an
increased probability of acquiring IPIs,
generally with a high degree of morbidity and
mortality in HIV positive individuals
worldwide (Chaisson et al., 1998). The
parasitic infestations are mainly due to
unsanitary conditions (Ramakrishnan et al.,
2007).
Several studies have also investigated
the existence of interaction between HIV
infection and parasitic infections in co-
infected individuals (Assefa et al., 2009). The
most commonly reported include C. parvum,
I. belli, microsporidia, G. duodenalis, E.
histolytica, Cyclospora species (Gupta et al.,
2008) and S. stercoralis. Some of these
parasites may disseminate to other organs
such as the bronchia, bile ducts and liver,
producing symptoms specific to the organs
affected (Botero et al., 2003).
Infection with G. duodenalis affects both
immunocompetent individuals and immuno-
compromised patients, particularly those
with common variable or congenital
hypogammaglobulinaemia and those in
advanced stages of AIDS with prolonged
diarrhoea (Mandell et al., 1995). Parasitic
infections particularly helminths cause
chronic immune activation (Borkow &
Bentwich, 2004) in addition to skewing the
immune response towards T helper-2
immune responses (MacDonald et al., 2002).
In this respect, A. lumbricoides can result
in an often-fatal obstruction and T. trichiura
is associated with chronic dysentery and
rectal prolapse. Though solid evidences are
insufficient, such immune modulation was
shown to increase host susceptibility; thereby,
promoting HIV infection and disease
progression (Brown et al., 2006).
In Malaysia, although IPIs have been
widely reported in immunocompetent
individuals (Li 1990; Chan et al., 1992;
Norhayati et al., 1994; 1997; 1998; DuPont
et al., 1995; Kan, 1998), there is still a lack
of available data among HIV-infected
individuals and whether these parasitic
infections are ubiquitous is not known. In
order to address this, the present study was
undertaken to establish firm data on the
prevalence and pattern of parasitic infections
among these patients. This information will
provide useful insights into the epidemiology
of these infections among HIV-infected
patients in Malaysia, thus facilitating the
understanding of clinical manifestations,
diagnosis and treatment management of
intestinal parasitic diseases.
MATERIALS AND METHODS
Study design and patients selection
The study was conducted from March
2008 to June 2010 on 346 HIV-infected
individuals from three different hospitals in
Malaysia, namely: Hospital Sungai Buloh,
Selangor; University of Malaya Medical
Centre, Kuala Lumpur and Hospital Raja
Perempuan Zainab II, Kelantan. Ethical
clearance and patient’s consent according to
the institutional ethical guidelines (IRB Ref.
No. 655.17, MOH-NMRR ID: # 09-286-3930)
were obtained prior to commencement of
study. Single stool sample from 346 patients
were collected in sterile screw-capped faecal
container with 2.5% potassium dichromate
solution as preservative and stored at 4ºC for
further analysis.

Page 3

402
Stool analysis
Stool samples were subjected to macro-
scopic examination. Stool consistency was
graded by these categories; formed, soft and
watery. These samples were concentrated
by formal-ether sedimentation technique
and the detection of intestinal parasites was
examined by wet saline mount and iodine
preparation for the presence of ova, larvae
and (oo)cysts under 100x and 400x
microscopy observation. Modified Ziehl-
Neelsen acid fast stain was used for the
staining of Cryptosporidium and other
coccidian parasites and examined under
400x. Cryptosporidium oocysts appeared as
bright rose-pink sphere (5 ± 1µm) on a pale
green background.
Data collection
A standardized form was used to collect
information, which includes: (1) socio-
demographic characteristics (gender, age
and race), (2) clinical and immunological
information and (3) treatment regime
(antiretroviral drugs). Diarrhoea was defined
as three or more watery or loose stools in 24-
hour period. Other types of clinical data such
as CD4 count and type of opportunistic
infections were obtained from the medical
record with patient’s consent and permission
from health authorities.
Statistical analysis
Data entry and analysis was performed using
SPSS Version 13 software (Statistical Package
for the Social Sciences) programme for
Windows (SPSS, Chicago, IL, USA).
Descriptive statistics were mainly used to
describe the characteristics of the population
under study such as age and gender, including
the prevalence of intestinal parasitic
infections. Statistical significance of
differences in proportions was evaluated by
Pearson’s Chi-square test and student t-test.
A significant level of a p-value < 0.05 was
used for all tests. Univariate analysis was
used to identify the potential risk factors
between each variable.
RESULTS
Descriptive analysis
Faecal samples were collected from a total
of 346 HIV patients, with 262 samples
obtained from Hospital Sungai Buloh
(national HIV referral centre for infectious
diseases), 63 samples from Hospital Raja
Perempuan Zainab II and 21 samples from
University Malaya Medical Centre (UMMC).
There were 26 (7.5%) children with age
ranging from 1 to 13 years with a mean age of
3 years, and 320 (92.5) adults with age
ranging from 29 to 42 years with a mean age
of 36 years. With regards to gender, there
were 302 (87.3%) males and 44 (12.7%)
females. There were 185 (53.5%) Malay, 77
(22.3%) Chinese, 42 (12.1%) Indians, 39
(11.3%) foreigners (i.e., Myanmarese) and 2
(0.6%) indigenous East Malaysian. Based on
stool macroscopic examination, there were
30 (8.7%) diarrheic stools (symptomatic)
whilst 316 (91.3%) stools were formed or soft
(asymptomatic).
Since some patients did not give consent
for their clinical records to be accessed,
clinical information such as mode of HIV
transmission, CD4 count, highly active
antiretroviral therapy (HAART) and the
presence of opportunistic infections were
only obtained from 253 patients. Of these 253
patients, 118 (46.6%) were IVDU (intravenous
drug users), 39 (15.4%) were heterosexuals,
11 (4.3%) were homosexuals and there were
82 (32.4%) patients who chose not to disclose
their mode of transmission. There were 189
(74.7% of 253) patients having CD4 counts
< 200 cells/mm3, 49 (19.4%) having CD4
counts > 200 cells/mm3 whilst 15 (0.6%)
patients did not have any record on CD4
count. At the time of sample collection, there
were 119 (47% of 253) patients who were on
HAART (highly active anti-retroviral
therapy) and 133 (52.6%) who were not on
HAART due to reasons such as side effects,
non compliance and late presentation.

Page 4

403
Prevalence of intestinal parasitic
infections (IPIs) among HIV patients
Microscopic examination of IPIs revealed an
overall prevalence of 37.9% (131) among 346
HIV patients. There were 65 patients (18.8%
of 346) who harboured protozoan parasites
and 26 (7.5%) harbouring helminthes whilst
40 (11.6%) patients had both protozoa and
helminthes. Protozoan parasites detected
included E. histolytica/dispar in 58 patients
(16.8% of 346), Cryptosporidium spp. in
43 (12.4%), I. belli in 35 (10.1%), Cyclospora
spp. in 17 (4.9%) and G. duodenalis in 11
(3.2%). Helminthes observed included A.
lumbricoides in 48 patients (13.9%), T.
trichiura in 22 (6.4%) and hookworm in 2
(0.6%) (Table 1). Parasitic infections in
HIV patients were observed to be mostly
mixed infections (66 of 346, 19%) while 64
(18.5%) were single infection. Among the
mixed infection cases, combination of E.
histolytica/dispar and A. lumbricoides is the
most common while for single infection,
Cryptosporidium infection was found to
be the most prevalent. Most infected cases
(118 of 131, 90.1%) were asymptomatic with
reference to the consistency of the stool.
Based on individual analysis of parasitic
infection, there is no significant association
between diarrhoea and IPIs. Student t- test
analysis showed that there were significant
risk of parasitic infection in asymptomatic
(non-diarrheic) patients (p=0.000).
With regards to IPIs according to age,
those aged 13 years and above (118 of 131;
90.1%) had higher prevalence rate compared
to those 12 years and below (13, 9.9%) (Table
2). A majority of those 12 years and below
had single infections which include protozoan
and helminth infections (Table 2). There was
only 1 case of mixed infection with both
protozoan and helminth in the 12 years and
below age group. In contrast, for those aged
13 years and above, 58 (49.1% of 118) had
single infection with protozoa, followed by
mixed infection of both protozoa and
helminthes (39, 33%) while single helminth
infection was the lowest (21, 17.8%). Results
showed that those above 13 years were
harbouring greater diversity of parasites
compared to those of 12 years and below
(Table 2). Parasites which were found in
those above 13 years and not in those of
below 12 years include Cyclospora species,
G. duodenalis and hookworms.
Based on gender, there were 114 (87% of
131) male and 17 (13%) female positive with
IPIs (Table 2). In male patients, protozoan
infections 57 (50% of 114) were more
common, followed by mixed infection of both
protozoa and helminthes (37, 32.4%) and
lastly helminth infections (20, 17.5%) (Table
2). Similarly for female HIV-infected patients,
protozoan infections 8 (47% of 17) were also
more prevalent, followed by single helminth
infection (6, 35.3%) and mixed infection of
both protozoa and STH was the lowest
(3, 17.6%). Male were infected with more
types of parasites whereas female had E.
histolytica/dispar, Cryptosporidium spp.,
I. belli, A. lumbricoides, and T. trichiura
infections. Similar with the findings based
on age, parasites which were found in male
but not in female were Cyclospora spp., G.
duodenalis and hookworms.
IPIs were also evaluated based on the
different ethnic groups in Malaysia (Table 2).
Those with IPIs consisted of 72 Malay (55%
of 131), 31 Chinese (23.6%), 16 foreigners
(i.e., Myanmarese) (12.2%) and 12 Indian
(9.2%) (Table 2). For both Malay and Chinese,
it was observed that protozoan infections
Table 1. Prevalence of intestinal parasitic
infections (IPIs) according to species among HIV
patients (n=346)
Type of speciesNo.%
Protozoa
E. histolytica/dispar
Cryptosporidium spp.
I. belli
Cyclospora spp.
G. duodenalis
58
43
35
17
11
16.8
12.4
10.1
4.9
3.2
Helminthes
A. lumbricoides
T. trichiura
Hookworms
48
22
13.9
6.4
0.62
Protozoa
Helminthes
Protozoa + Helminthes
65
26
40
18.8
7.5
11.6
Total
131 37.9

Page 5

404
Table 2. Types of intestinal parasites detected in HIV patients according to age, gender and ethnic group (n = 131)
Age group (Years)
< 12
(n = 13)
Gender
Male
(n = 114)
Ethnic Groups
Chinese
(n = 31)
Parasites > 13
(n = 118)
Female
(n = 17)
Malay
(n = 72)
Indian
(n = 12)
Foreigners
(n = 16)
Protozoa
Entamoeba histolytica/
dispar
Cryptosporidium spp.
Isospora belli
Cyclospora spp.
Giardia duodenalis
2 (15.3) 56 (47.5)54 (47.4) 4 (23.5)30 (41.7) 15 (48.4)7 (58.3)6 (37.5)
4 (30.8)
1 (7.7)
0 (0)
0 (0)
39 (33.0)
34 (28.8)
17 (14.4)
11 (9.3)
39 (34.2)
30 (26.3)
17 (14.9)
11 (9.6)
4 (23.5)
5 (29.4)
0
0
24 (33.3)
13 (18.0)
9 (12.5)
6 (8.3)
10 (32.2)
13 (41.9)
3 (9.6)
3 (9.6)
4 (33.3)
4 (33.3)
3 (25.0)
0
5 (31.2)
5 (31.2)
2 (12.5)
2 (12.5)
Helminthes
Ascaris lumbricoides
Trichuris trichiuria
Hookworm
4 (30.8)
2 (15.3)
0
44 (37.2)
20 (17.0)
2 (1.7)
43 (37.7)
17 (14.9)
2 (1.7)
5 (29.4)
5 (29.4)
0
24 (33.3)
10 (13.8)
1 (1.4)
10 (32.2)
7 (22.5)
1 (3.2)
3 (25.0)
2 (16.6)
0
11 (68.7)
3 (18.7)
0
Protozoa
Helminthes
Protozoa + Helminthes
7 (53.8)
5 (38.5)
1 (7.6)
58 (49.1)
21 (17.8)
39 (33.0)
57 (50.0)
20 (17.5)
37 (33.0)
8 (47.0)
6 (35.3)
3 (17.6)
38 (52.7)
16 (22.2)
18 (25.0)
17 (55.0)
5 (16.1)
9 (29.0)
7 (58.3)
0
5 (41.6)
3 (18.8)
5 (31.2)
8 (50.0)
were more common (38 of 72, 52.7% and 17
of 31, 55% respectively), followed by mixed
infection of both protozoa and helminthes
(18 of 72, 25% and 9 of 31, 29% respectively)
and lastly helminth infections (16 of 72, 22.2%
and 5 of 31, 16.1% respectively) (Table 2).
Interestingly, among Indians, there were only
protozoan infection (7 of 12, 58.3%) and
mixed infections (5 of 12, 41.6%). There was
no single helminth infection among Indians.
As for foreigners, mixed infection of both
protozoa and helminthes were the highest
(8 of 16, 50%), followed by helminth infection
(5 of 16, 31.2%) and protozoan infection (3 of
16, 18.8%). Further evaluation of types of
parasites observed that Malay and Chinese
had greater diversity of parasites harboured.
It was interesting to note that Indian only had
E. histolytica/dispar, Cryptosporidium spp.,
I. belli and A. lumbricoides infections. As
for foreigners, all types of parasites found in
Malay and Chinese were also found in them
except for hookworms (Table 2).
As mentioned above, information such as
mode of HIV transmission, CD4 count, highly
active antiretroviral therapy (HAART) and
the presence of opportunistic infections can
only be based on 253 patients. Of these, 98
were infected with IPIs. For the mode of HIV
transmission, 48 of 98 (48.9%) were IVDU, 19
(19.4%) were heterosexual and 5 (5.1%) were
homosexual. There were 26 patients who
were not sure how they acquired the HIV
infection (termed as “unknown”) (Table 3).
Results showed that IVDU, heterosexual and
“unknown” harboured all types of parasites.
Cryptosporidium spp., G. duodenalis and
hookworm parasites were not found among
homosexual. With regards to CD4 count, out
of a total 238 patients, 84 had CD4 counts
< 200 cells/mm3 and 14 had CD4 counts >
200 cells/mm3. It was noted that patients
with CD4 counts < 200 cells/mm3 had a
significantly higher risk of parasitic
infections (Table 4). Patients with CD4 counts
< 200 cells/mm3 had a significantly high risk
of acquiring parasitic infections. The CD4
count was in the range of 2-525 cells/mm3
with the mean value 92.3% cells/mm3. Out of
253 patients, 238 had CD4 count information
whilst 15 patients did not have any record on
CD4 count. In addition to 98 IPIs positive
patients, 85.7%, (84) of patients with IPIs were
found to have CD4 counts < 200 cells/mm3
and only 14.3%, (14) of the patients have CD4
counts > 200 cells/mm3 at the time of
diagnosis (p<0.05) (Table 4). Based on CD4
classification, it was observed that the
number of patients infected with IPIs ranged
from 49% (48 of 98) in the category of CD4
counts of < 50 cells/mm3 group, 18.3% (18
of 98) and 17.3% (17 of 98) in CD4 counts of
51-100 cells/mm3 and 101-200 cells/mm3
respectively, whilst 11.2% (11 of 98) in CD4

Page 6

405
Table 3. IPIs in HIV patients according to mode of transmission (n=98)
IVDU
n = 48
Heterosexual
n = 19
Homosexual
n = 5
Unknown
n = 26
Protozoa
E. histolytica/dispar
Cryptosporidium spp.
I. belli
Cyclospora spp.
G. duodenalis
24 (50.0)
20 (41.6)
12 (25.0)
09 (18.7)
07 (14.5)
10 (52.6)
07 (36.8)
05 (26.3)
02 (10.5)
01 (5.2)
2 (40.0)
–
3 (60.0)
1 (20.0)
–
12 (46.1)
06 (23.0)
10 (38.5)
02 (7.6)
02 (7.6)
Helminthes
A. lumbricoides
T. trichiura
Hookworm
18 (37.5)
06 (12.5)
01 (50.0)
07 (36.8)
03 (15.7)
01 (50.0)
3 (60.0)
1 (20.0)
–
08 (30.7)
06 (23.0)
–
Table 4. IPIs in HIV patients in relation to CD 4 cell counts (n=98)
No. with parasites (%)
Parasite
N
0-50
cells/mm3
51-100
cells/mm3
101-200
cells/mm3
201-400
cells/mm3
> 400
cells/mm3
infected
Protozoa
E. histolytica/dispar
Cryptosporidium spp.
I. belli
Cyclospora spp.
G. duodenalis
49
33
31
14
10
21 (42.9)
18 (54.5)
14 (45.2)
07 (50.0)
06 (60.0)
11 (22.4)
03 (9.1)
04 (12.9)
03 (21.4)
01 (10.0)
9 (18.4)
6 (18.2)
6 (19.4)
2 (14.3)
1 (10.0)
7 (14.3)
4 (12.1)
7 (22.6)
2 (14.3)
2 (20.0)
1 (2.0)
2 (6.1)
0
0
0
Helminthes
A. lumbricoides
T. trichiura
Hookworm
37
16
02
20 (54.1)
06 (37.5)
00
08 (21.6)
03 (18.8)
01 (50.0)
5 (13.5)
5 (31.3)
1 (50.0)
2 (5.4)
2 (12.5)
0
2 (5.4)
0
0
* CD4 count < 200 cells/mm3 84 (85.7%); p=0.044, OR (95% CI) = 2.0 (1.01–3.96)
counts of 201-400 cells/mm3 and only 3.0%
(3 of 98) in CD4 > 400 cells/mm3. It was
observed that E. histolytica/dispar, A.
lumbricoides, Cryptospordium spp. and
Isospora spp. were more common as compare
to other parasites. The maximum parasitic
isolation was in the group of patients who had
CD counts below 200 cells/mm3 (Table 4).
Interestingly, 63 (64.3% of 98) patients
who had IPIs also had concurrent
opportunistic infections (OIs) with the most
common being Mycobacterium tuberculosis
infection (37, 58.7% of 63), cerebral
toxoplasmosis (11, 17.5%), candidiasis (9,
14.3%) and disseminated or extrapulmonary
M. tuberculosis infection (8, 12.7%).
DISCUSSION
In Malaysia, IPIs have been widely reported
in immunocompetent individuals (Norhayati
et al., 1994, 1997, 1998; Kan et al., 1998; Chan
et al., 1992; Li, 1990; DuPont et al., 1995),
however there is still a lack of available data
among HIV-infected individuals as only
information on Cryptosporidium infection is
currently available among Malaysian HIV
individuals (Kamel et al., 1994; Lim et al.,
2005; Zaidah et al., 2008). In the present study,
the overall prevalence of IPIs among HIV-
infected patients were found to be
considerably higher (37.9% of 346 patients)
compared to studies carried out among

Page 7

406
immunocompetent patients (6.9% to 19.2%)
(Nor Aza et al., 2003; Jamaiah & Rohela,
2005).
Globally, information on IPIs in HIV
patients are largely concentrated in Asia,
Africa and some parts of Middle Europe
(Wiwanitkit et al., 2001; Boral et al., 2004;
Adesiji et al., 2007; Assefa et al., 2009;
Kurniawan et al., 2009; Kulkarni et al., 2009;
Berenji et al., 2010). Prevalence rates of
IPIs among HIV individuals similar to the
present study were also observed in India
(35% of 137 patients) (Kulkarni et al., 2009),
Turkey (47% of 38 patients) (Boral et al.,
2004) and Thailand (50% of 60 patients)
(Wiwanitkit et al., 2001). However, studies
reported among HIV-infected individuals in
Ethiopia (59.8% of 214) (Assefa et al., 2009),
Iran (67.7% of 51) (Berenji et al., 2010),
Nigeria (79.3% of 150) (Adesiji et al., 2007)
and Indonesia (84.3% of 318 patients) have
recorded much higher prevalence of IPIs
(Kurniawan et al., 2009).
Intestinal parasites noted in the present
HIV-infected patients were protozoa such as
E. histolytica/dispar (16.8%), Cryptos-
poridium spp. (12.4%), I. belli (10.1%),
Cyclospora spp. (4.9%) and G. duodenalis
(3.2%), and helminthes which include A.
lumbricoides (13.9%), T. trichiura (6.4%)
and hookworms (0.6%). These percentages
were in accordance with data from Ethiopia
(Assefa et al., 2009), whereby the most
frequently detected parasites were
Cryptosporidium spp. (11.4%), I. belli
(12.2%), E. histolytica/dispar (26.5%), A.
lumbricoides (12.4%), T. trichiura (5.6%).
Studies from Brazil (Silva et al., 2007),
Colombia (Botero et al., 2003), Cuba
(Escobedo and Núñez 1999), India
(Ramakrishnan et al., 2007; Gupta et al.,
2008), Italy (Peruzzi et al., 2006), Iran
(Daryani et al., 2009) and Venezuela (Certad
et al., 2005) have also noted protozoan
infections to be more frequent than helminth
infections among HIV patients.
Current study showed that the most
common combination of multiple infections
is of E. histolytica/dispar and A.
lumbricoides whilst for single infection,
Cryptosporidium infection was found to be
the most prevalent. In Italy, Peruzzi et al.
(2006) also reported more mixed infections
comprising of infections with Blastocystis
hominis, E. histolytica and G. duodenalis
especially in foreigners as compared to
Italian patients. Similarly, in Ethiopia (Assefa
et al., 2009), there were higher rate (27.2%)
of mixed infection occurring among HIV
positive patients compared to HIV negative
individuals (OR=2.0; 95% CI 1.2 to 3.6).
Berenji et al. (2010) also found that in Iran,
there were 12.9% of multiple parasitic
infections in HIV infected patients.
The high prevalence of Cryptospori-
dium species noted in this study (12.4%) was
in agreement with previous study carried out
among HIV positive IVDU drug users (Kamel
et al., 1994) and in hospitalized patients in
Kota Bharu (Zaidah et al., 2008). However,
another Malaysian study reported a lower
prevalence of cryptosporidiosis among HIV
patients (i.e., 3%) (Lim et al., 2005). Globally
the prevalence rate of cryptosporidiosis is
from 3-50% (Pedersen et al., 1996; Florez et
al., 2003). The prevalence of cryptospori-
diosis in HIV patients also varies among
studies, depending on where the study was
conducted, the age of population studied,
the stage of disease and the laboratory
methods used (Chhin et al., 2006). A number
of studies indicated that the prevalence of
cryptospori-diosis in HIV-infected patients
was in the range of 15% or below (Hunter &
Nichols, 2002). However, our results are in
accordance with the study carried out in
other parts of the world such as Iran (Daryani
et al., 2009;), Brazil (Botero et al., 2003),
India (Ramakrishnan et al., 2007), Thailand
(Nuchjangreed et al., 2008; Srisuphanunt
et al., 2008), Cambodia (Chhin et al., 2006),
Ethiopia (Assefa et al., 2009) and Nigeria
(Okodua et al., 2003).
The overall prevalence of IPIs in the
present study was higher in patients above
13 years of age as compared to those 12 years
and below. The majority (118 patients) of
patients in our study was aged 29-42 years,
however; age distribution revealed that
patients older than 35 years had increase risk
of infection. Such age related increase in IPIs
prevalence has been previously reported in
adults as well (Certad et al., 2005; Assefa
et al., 2009). As there were very limited

Page 8

407
information of IPIs in HIV patients those of 12
years and below whether within Malaysia or
globally, it was interesting to note from this
study that infected patients 12 years and
below, generally have single infection
(protozoa or helminth infection). Co-infection
with both protozoa and helminth was only
observed in one patient. This study showed
that in patients above 13 years were
harbouring greater diversity of parasites
compared to those 12 years and below.
Parasites found in those 12 years and
below included E. histolytica/dispar,
Cryptosporidium spp., I. belli, A.
lumbricoides, and T. Trichiura whereas;
Cyclospora spp., G. duodenalis and
hookworm which were found in patients
above 13 years were not seen in those 12
years and below. The prevalence of IPIs in
HIV patients also varies among studies,
depending on the location of study, the age of
population studied, the stage of disease and
the laboratory methods used (Chhin et al.,
2006).
The present results showed that CD4
count could be used as a potential predicting
indicator of protozoa and helminthes
infections as those with of CD4 count < 200
cells/mm3 were significantly at higher risk
of acquiring parasitic infection (p < 0.05).
Further observation based on CD4
classification highlighted that a majority of
IPIs were found in HIV-infected individuals
having CD4 counts of < 50 cells/mm3.
Although IPIs occurred in all CD4 groups, low
number of parasitic infections were seen in
those with CD4 counts > 400 cells/mm3.
These findings were consistent with other
studies that showed parasitic infection
usually occurs in patients with CD4 counts
of < 100cells/mm3 (Pedersen et al., 1996; Zali
et al., 2004; Chhin et al., 2006; Assefa et al.,
2009; Kurniawan et al., 2009). A study in
Ethiopia mentioned six-fold higher infection
rate of IPIs at CD4 counts of < 200 cells/mm3
compared with individuals having counts of
> 500 cells/mm3 (OR = 6.3; 95% CI 2.6 to 15.1)
(Assefa et al., 2009). In India, researchers
determined the pattern of IPIs in different
categories of CD4 count on the basis of
diarrhoea status (i.e., acute and chronic) and
it was reported that IPIs were significantly
associated with chronic diarrhoea among
those with CD4 count of < 50 cells/mm3
(Daryani et al., 2009). Another study which
looked at the types of parasites based on
CD4 count in HIV patients with diarrhoea
noted that Cryptosporidium spp. had the
highest prevalence (56.5%, p=0.037) and E.
histolytica had 28.9% infection (p=0.007)
among those in CD4 counts < 200 cells/mm3
group; both were significant compared with
the other CD4 groups (CD4 counts 200–500
cells/mm3 and CD4 < 500 cells/mm3)
(Sadraei et al., 2005). Similarly, HIV
individuals in Indonesia with CD4 cell counts
of < 50/mm3 were observed to harbor more
types of intestinal parasites such as
Blastocystis, Cyclospora, Cryptosporidium,
Giardia, Entamoeba and Ascaris compared
to those with CD4 count 51-100 cells/mm3
and 101-200 cells/mm3 group (Kurniawan et
al., 2009).
Unfortunately, individual statistical
analysis on the association of opportunistic
infection with each intestinal parasite was
not significant. Analysis of association
between IPIs and OIs were observed,
interestingly, 63 (64.3% of 98) patients who
had IPIs also had concurrent OIs with the most
common being M. tuberculosis, cerebral
toxoplasmosis, candidiasis and disseminated
or extrapulmonary M. tuberculosis infection.
It is striking that in almost every study from
developing regions, M. tuberculosis infection
is the most frequently identified OIs in HIV
patients (Swaminathan & Narendran, 2008;
Cain et al., 2009), but none of these studies
reported any association of IPIs and OIs in
HIV patients.
In conclusion, the high prevalence of IPIs
(37.9%) in the present study highlighted the
ubiquitousness of these infections among HIV-
infected individuals particularly in those with
lower CD4 T-cell counts, with or without
diarrhoea. The high rates of IPIs, along with
profound immunosuppression, may increase
risk of developing symptomatic disease such
as OIs with mixed infections. With these
indications of high prevalence of IPIs in
asymptomatic HIV patients, routine
screening is utterly crucial and should be
instituted in most laboratories at the primary
care level, for early treatment and better

Page 9

408
management of gastrointestinal illnesses
among HIV individuals.
Acknowledgments. We would like to thank
Dr. Mohammed AK Mahdy and Mr. Romano
Ngui for their technical assistant and kind
support. This study was funded by the
research grant from University of Malaya-
50603 Kuala Lumpur, Malaysia (Research
grant No. PS007/2008B, University of
Malaya).
REFERENCES
Adesiji, Y.O., Lawal, R.O., Taiwo, S.S.,
Fayemiwo, S.F. & Adeyeba, O.A. (2007).
Cryptosporidiosis in HIV infected patients
with diarrhea in Osun state SouthWestern
Nigeria. European Journal of General
Medicine 4: 119-122.
Assefa, S., Erko, B., Medhin, G., Assefa, Z. &
Shimelis, T. (2009). Intestinal parasitic
infections in relation to HIV/AIDS status,
diarrhea and CD4 T-cell count. BMC
Infectious Disease 9: 155 (doi: 10.1186/
1471-2334-9-155).
Berenji, F., Sarvghad, M.R., Fata, A.M.,
Hosseininejad,
Ganjbakhsh, M. & Izadi, J.R. (2010). A
study of the prevalence of intestinal
parasitic infection in HIV positive
individuals in Mashhad, Northeast Iran.
Jundishapur Journal of Microbiology 3:
61-65.
Boral, O.B., Uysal, H., Alan, S. & Nazlican, O.
(2004). Investigation of intestinal
parasites in AIDS patients. Mikrobiyoloji
Bulteni 38: 121-128.
Borkow, G. & Bentwich, Z. (2004). Chronic
immune activation associated with
chronic helminthic and human immuno-
deficiency virus infections: Role of hypo-
responsiveness and energy. Clinical
Microbiology Review 17: 1012-1030.
Botero, H.J., Castanol, A., Montoya, M.N.,
Ocampo, N.E., Hurtado, M.I. & Lopera,
M.M. (2003). A preliminary study of the
prevalence of intestinal parasites in
immunocompromised patients with and
without gastrointestinal manifestations.
Z., Saremi, E.,
Revista do Instituto de Medicina
Tropical de São Paulo 45: 197-200.
Brown, M., Mawa, P.A., Kaleebu, P. & Elliott,
A.M. (2006). Helminths and HIV infection:
epidemiological observations on
immunological hypotheses. Parasite
Immunology 28: 613-623.
Cain, K.P., Anekthananon, T., Burapat, C.,
Akksilp, S., Mankhatitham, W., Srinak, C.,
Nateniyom, S., Sattayawuthipong, W.,
Tasaneeyapan, T. & Varma, J.K. (2009).
Causes of Death in HIV-infected Persons
Who Have Tuberculosis, Thailand.
Emerging Infectious Disease 15: 258-
264.
Certad, G., Arenas-Pinto, A., Pocaterra, L.,
Ferrara, G., Castro, J., Bello, A. & Nunez,
L. (2005). Cryptosporiodiosis in HIV
infected Venezuelan adults is strongly
associated with acute or chronic
diarrhea. American Journal of Tropical
Medicine and Hygiene 73: 54-57.
Chaisson, R.E., Gallant, J.E., Keruly, J.C.
& Moore, R.D. (1998). Impact of
opportunistic disease on survival in
patients with HIV infection. AIDS 12: 29-
33.
Chan, L., Kan, S.P. & Bundy, D.A.P. (1992). The
effect of reported chemotherapy on the
prevalence and intensity infection of
Ascaris lumbricoides and Trichuris
trichiura. Southeast Asian Journal of
Tropical Medicine and Public Health 23:
228-234.
Chhin, S., Harwell, J.I., Bell, J.D., Rozycki, G.,
Ellman, T., Barnett, J.M., Ward, H.,
Reinert, S.E. & Puhatch, D. (2006).
Etiology of chronic diarrhea in
antiretroviral-naive patients with HIV
infection admitted to Norodom Sihanuk
hospital. Phnom Penh, Cambodia.
Clinical Infectious Diseases 43: 925-932.
Current, W.L. & Garcia, L.S. (1991).
Cyptosporidiosis. Clinical Microbiology
Review 4: 325-358.
Daryani, A.M., Sharif, M., Meigouni, F.,
Mahmoudi, B., Rafiei, A., Gholami, S.,
Khalilian, A., Gohardehi, S. & Mirabi, A.M.
(2009). Prevalence of intestinal parasites
and profile of CD4+ counts in HIV+/AIDS
people in North of Iran, 2007-2008.

Page 10

409
Pakistan Journal of Biological Sciences
12: 1277-1281.
DuPont, H.L., Chapell, C.L., Sterling, C.R.,
Okhuysen, P.C., Rose, J.B. & Jakubowski,
W. (1995). The infectivity of Cryptospori-
dium parvum in healthy volunteers. New
England Journal of Medicine 332: 855-
859.
Escobedo, A.A. & Núñez, F.A. (1999).
Prevalence of intestinal parasites in
Cuban acquired immunodeficiency
syndrome (AIDS) patients. Acta Tropica
15: 125-130.
Farthing, M.G.J, Cevallos, A.M. & Kelly, P.
(2009). Intestinal protozoa. In: Manson’s
Tropical Disease Ed. Gordon C. Cook &
Alimuddin I. Zumla. 22nd Edition.
Saunders Elsevier.
Florez, A.C., Gracia, D.A., Moncada, L. &
Beltran, M. (2003). Prevalence of
Microsporidia and other intestinal
parasites with HIV infection. Biomedica
23: 274-282.
Gupta, S., Narang, S., Nunavath, V. & Singh, S.
(2008). Chronic diarrhea in HIV patients:
Prevalence of coccidian parasites.
Indian Journal of Medial Microbiology
26: 172-175.
Hung, C.C., Tsaihong, J.C., Lee, Y.T., Deng, H.Y.,
Hsiao, W.H., Chang, S.Y., Chang, S.C. &
Su, K.E. (2007). Prevalence of intestinal
infection due to Cryptospordium species
among Taiwanese patients with human
immunodeficiency virus infection.
Journal of Formosan Medical Associa-
tion 106: 31-35.
Hunter, P.R. & Nichols, G. (2002). Epidemio-
logy and clinical features Cryptospori-
dium infection in immunocompromised
patients. Clinical Microbiology Review
15: 145-154.
Jamaiah, I. & Rohela, M. (2005). Prevalence
of intestinal parasites among members
of the public in Kuala Lumpur, Malaysia.
Southeast Asian Journal of Tropical
Medicine and Public Health 36: 68-71.
Kamel, A.G.M., Maning, N., Arulmainathan,
S., Murad, S., Nasuruddin, A., Lai, K.P.
(1994). Cryptosporidiosis among HIV
positive intravenous drug users in
Malaysia. Southeast Asian Journal of
Tropical Medicine and Public Health 25:
650-653.
Kan, S.P. (1998). Soil-transmitted helmin-
thiases among inhabitants of an oil palm
plantation in West Malaysia. Journal of
Tropical Medicine and Hygiene 92: 263-
269.
Kulkarni, S.V., Kairon, R., Sane, S.S.,
Padmawar, P.S., Kale, V.A., Thakar, M.R.,
Mehendale, S.M. & Risbud, A.R. (2009).
Opportunistic parasitic infections in HIV/
AIDS patients presenting with diarrhea
by the level of immunesuppression.
Indian Journal of Medical Research
130: 63-66.
Kurniawan, A., Karyadi, T., Dwintasari, S.W.,
Sari, I.P., Yunihastuti, E., Djauzi, S. &
Smith, H.V. (2009). Intestinal parasitic
infections in HIV/AIDS patients
presenting with diarrhea in Jakarta,
Indonesia. Transactions of the Royal
Society of Tropical Medicine and
Hygiene 103: 892-898.
Li, C.F. (1990). Hookworm infection and
protein energy malnutrition: Tansverse
evidence from two Malaysian ecological
groups. Tropical and Geographical
Medicine 42: 8-12.
Lim, Y.A.L., Rohela, M., Sim, B.L.H., Jamaiah,
I. & Nurbayah, M. (2005). Prevalence of
Cryptosporidiosis in HIV-infected
patients in Kajang Hospital, Selangor.
Southeast Asian Journal of Tropical
Medicine and Public Health 36: 30-32.
MacDonald, A.S., Araujo, M.I. & Pearce, E.J.
(2002). Immunology of parasite helminth
infections. Infection and Immunity 70:
427-433.
Mandell, G., Bennet, J. & Dolin, R. (1995).
Giardia lamblia. In: principles and
practices of infectious diseases, 4th ed.
New York, Churchill Livingstone, pp.
2487-2493.
Nor Aza, Ashley, S. & Albert, J. (2003).
Parasitic infections in human com-
munities living on the fringes of the
crocker range park sabah, Malaysia.
ASEAN Review of Biodiversity and
Environmenatal Conservation http://
www.arbec.com.my/pdf/art11janmar
03.pdf

Page 11

410
Norhayati, M., Noor Hayati, M.I., Oothhuman,
P., Azizi, O., Fatima, M.S., Ismail, G. &
Muzain, M.Y. (1994). Enterobius
vermicularis infection among children
aged 1-8 years in a rural area in Malaysia.
Southeast Asian Journal of Tropical
Medicine and Public Health 25: 494-497.
Norhayati, M., Zainudin, B., Mohammod, C.G.,
Oothuman, P., Azizi, O. & Fatmah, M.S.
(1997). The prevalence of Trichuris,
Ascaris and hookworm infections in
Orang Asli children. Southeast Asian
Journal of Tropical Medicine and
Public Health 9: 272-276.
Norhayati, M., Pengabean, M., Oothhuman, P.
& Fatima, M.S. (1998). Prevalence and
some risk factors of Giardia duodenalis
infection in rural communities in
Malaysia. Southeast Asian Journal of
Tropical Medicine and Public Health 29:
735-738.
Nuchjangreed, C., Boonrod, K., Ongerth, J. &
Karanis, P. (2008). Prevalence and
molecular characterization of human
and bovine Cryptosporidium isolates in
Thailand. Parasitology Research 103:
1347-1353.
Okodua, M., Adeyeba, O.A., Tatfeng, Y.M. &
Okpala, H.O. (2003). Age and sex
distribution of intestinal parasitic
infection among HIV infected subjects in
Abeokuta, Nigeria. Online Journal of
Health and Allied Sciences 4: 3.
Pedersen, C., Danner, S., Lazzarin, A., Glauser,
M.P., Weber, R., Katlama, C., Barton, S.E.
& Lundgren, J.D. (1996). Epidemiology
of cryptospordiosis among European
AIDS patients. Genitourinary Medicine
72: 128-131.
Peruzzi, S., Gorrini, C., Piccolo, G., Calderaro,
A., Dettori, G. & Chezzi, C. (2006).
Prevalence of intestinal parasites in the
area of Parma during the year 2005. Acta
Biomedica 77: 147-151.
Ramakrishnan, K., Shenbagarathai, R.,
Uma, A., Kavitha, K., Rajendran, R. &
Thirumalaikolundusubramanian, P.
(2007). Prevalence of Intestinal parasitic
infestation in HIV/AIDS patients with
diarrhea in Madurai city, South India.
Japanese Journal of Infectious Diseases
60: 209-210.
Sadraei, J., Rizvi, M.A. & Baveja, U.K. (2005).
Diarrhea, CD4+ cell counts and
opportunistic protozoa in Indian HIV-
infected patients. Parasitology Research
97: 270-273.
Silva, O.M.B., Oliveira, L.R., Presende, J.C.,
Peghini, B.C., Ramirez, L.E., Silva, E.L.
& Correia, D. (2007). Seasonal profile
and level of CD4 lymphocytes in the
occurrence of cryptosporidiosis and
cystoisosporidiosis in HIV/AIDS patients
in the Triângulo Mineiro region, Brazil.
Revista da Sociedade Brasileira de
Medicina Tropical 40: 512-515.
Srisuphanunt, M., Suvedyathavorn, V.,
Suputtamongkol, Y., Arnantapunpong, S.,
Wiwanitkit, V., Satitvipawee, P. &
Tansuoasawadikul, S. (2008). Potential
risk factors for Cryptosporidium
infection among HIV/AIDS patients in
central areas of Thailand. Journal of
Public Health 16: 173-182.
Swaminathan, S. & Narendran, G. (2008). HIV
and tuberculosis in India. Journal of
Biological Sciences 33: 527-537.
Wiwanitkit, V. (2001). Intestinal parasitic
infections in Thai HIV-infected patients
with different immunity status. BMC
Gastroenterology 1: 3.
Zaidah, A.R., Chan, Y.Y., Asma, S.H., Abdullah,
S., Nurhaslindawati, A.R., Salleh, M.,
Zeehaida, M., Lalitha, P., Mustafa, M. &
Ravichandran, M. (2008). Detection of
Cryptosporidium parvum in HIV-
infected patients in Malaysia using
molecular approach. Southeast Asian
Journal of Tropical Medicine and
Public Health 39: 511-516.
Zali, M.R., Jafari-Mehr, A., Rezaian, M.,
Meamar, A.R., Vaziri, S. & Mohraz, M.
(2004). Prevalence of intestinal parasitic
pathogenes among HIV-positive in-
dividuals in Iran. Japanese Journal of
Infectious Diseases 57: 268-270.