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Distribution_and_Clinical_Mani.pdf
Distribution and Clinical Manifestations of Cryptosporidium Species and Subtypes in HIV/AIDS Patients in Ethiopia Haileeyesus Adamu1,2, Beyene Petros2, Guoqing Zhang3, Hailu Kassa4, Said Amer1,5, Jianbin Ye1,6,
Yaoyu Feng6*, Lihua Xiao1*
1 Division of Foodborne, Waterborne and Environmental Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 2 Addis Ababa
University, Addis Ababa, Ethiopia, 3 Division of Global HIV/AIDS, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 4 Department of
Public and Allied Health, Bowling Green State University (BGSU), Bowling Green, Ohio, United States of America, 5 Department of Zoology, Faculty of Science, Kafr El
sheikh University, Kafe El Sheikh, Egypt, 6 State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of
Science and Technology, Shanghai, People’s Republic of China
Abstract
Background: Cryptosporidiosis is an important cause for chronic diarrhea and death in HIV/AIDS patients. Among common Cryptosporidium species in humans, C. parvum is responsible for most zoonotic infections in industrialized nations. Nevertheless, the clinical significance of C. parvum and role of zoonotic transmission in cryptosporidiosis epidemiology in developing countries remain unclear.
Methodology/Principal Findings: In this cross-sectional study, 520 HIV/AIDS patients were examined for Cryptosporidium presence in stool samples using genotyping and subtyping techniques. Altogether, 140 (26.9%) patients were positive for Cryptosporidium spp. by PCR-RFLP analysis of the small subunit rRNA gene, belonging to C. parvum (92 patients), C. hominis (25 patients), C. viatorum (10 patients), C. felis (5 patients), C. meleagridis (3 patients), C. canis (2 patients), C. xiaoi (2 patients), and mixture of C. parvum and C. hominis (1 patient). Sequence analyses of the 60 kDa glycoprotein gene revealed a high genetic diversity within the 82 C. parvum and 19 C. hominis specimens subtyped, including C. parvum zoonotic subtype families IIa (71) and IId (5) and anthroponotic subtype families IIc (2), IIb (1), IIe (1) and If-like (2), and C. hominis subtype families Id (13), Ie (5), and Ib (1). Overall, Cryptosporidium infection was associated with the occurrence of diarrhea and vomiting. Diarrhea was attributable mostly to C. parvum subtype family IIa and C. hominis, whereas vomiting was largely attributable to C. hominis and rare Cryptosporidium species. Calf contact was identified as a significant risk factor for infection with Cryptosporidium spp., especially C. parvum subtype family IIa.
Conclusions/Significance: Results of the study indicate that C. parvum is a major cause of cryptosporidiosis in HIV-positive patients and zoonotic transmission is important in cryptosporidiosis epidemiology in Ethiopia. In addition, they confirm that different Cryptosporidium species and subtypes are linked to different clinical manifestations.
Citation: Adamu H, Petros B, Zhang G, Kassa H, Amer S, et al. (2014) Distribution and Clinical Manifestations of Cryptosporidium Species and Subtypes in HIV/AIDS Patients in Ethiopia. PLoS Negl Trop Dis 8(4): e2831. doi:10.1371/journal.pntd.0002831
Editor: Claudia Munoz-Zanzi, University of Minnesota, United States of America
Received September 3, 2013; Accepted March 17, 2014; Published April 17, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This study was supported in part by funds from the Centers for Disease Control and Prevention (CDC), Addis Ababa University, and National Natural Science Foundation of China (No. 31110103901), and a postdoctoral fellowship from the American Society for Microbiology (ASM) and CDC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] (YF); [email protected] (LX)
Introduction
Cryptosporidium is an important protozoan parasite affecting
HIV/AIDS patients, causing diarrhea, wasting syndrome, and
reduced life quality [1]. Since specific therapy or vaccine for the
control of this parasite is not yet available, preventing infections
depends on avoiding exposure to the parasite and maintaining
immune competence. In industrialized nations, access to highly
active antiretroviral therapy (HAART) has significantly reduced
the morbidity and mortality by cryptosporidiosis [2–4]. Nonethe-
less, cryptosporidiosis is still a major threat to AIDS patients who
do not have access to HAART, especially in developing countries
[5–8]. In industrialized nations, transmission of cryptosporidiosis
via contaminated drinking and recreational water and contact
with infected farm animals remains a major public health problem
in both HIV-positive and immunocompetent persons [9–11].
The use of molecular epidemiologic tools has provided new
insights into the diversity of Cryptosporidium species infecting
humans and animals [12]. So far, 26 Cryptosporidium species have
been described [13–17]. Most human cases are caused by C.
hominis and C. parvum [12]. The latter also infects some other
mammals, notably calves and lambs, and is responsible for most
zoonotic infections in humans. Several other Cryptosporidium species
are seen in humans at lower frequency, including C. meleagridis, C.
felis, C. canis, C. ubiquitum, and C. cuniculus [12]. More recently, a
new species, C. viatorum, has been described in 10 travelers
PLOS Neglected Tropical Diseases | www.plosntds.org 1 April 2014 | Volume 8 | Issue 4 | e2831
returning to Great Britain from the Indian subcontinent [17]. This
species appears to be a human-specific pathogen and has since
been found in 2 Swedish travelers to Africa and Latin America
[18]. In children and HIV-positive persons, differences in clinical
manifestations have been observed among different Cryptosporidium
species, especially between C. hominis and C. parvum, with the
former more virulent than the latter [19–21]. In addition,
infections with C. parvum were associated with chronic diarrhea
and vomiting in HIV-positive persons more frequently than was C.
hominis [21].
Sequence characterization of the 60-kDa glycoprotein (gp60)
gene has been commonly used in subtyping C. hominis and C.
parvum [12]. Differences have been observed in host specificity
among C. parvum subtype families and in virulence among C.
hominis subtype families. Thus, C. parvum subtype family IIa is
commonly found in calves, IId is mostly found in lambs and goat
kids, whereas IIc is mostly found in humans. Within C. hominis,
studies in Peru have shown that subtype family Ib was more
virulent than other subtype families in children, whereas subtype
family Id was more virulent than subtype families Ia and Ie in
HIV-positive patients [20,21].
Cryptosporidiosis is endemic in Ethiopia; occurrence rates of
7.6% to 43.6% were reported in HIV/AIDS patients [22–26].
Some potential risk factors for cryptosporidiosis occurrence
included contamination of drinking water, contact with calves,
living in overcrowded households with many family members, and
poor personal hygiene. Thus far, only one study has genetically
characterized Cryptosporidium spp. from Ethiopia. In the study, 39
of the 41 specimens genotyped had C. parvum, one had C. hominis,
and one had both species. All 12 C. parvum specimens subtyped by
sequence analysis of the gp60 gene belonged to the subtype family
IIa [22].
In the present study, we examined the occurrence of
Cryptosporidium infection in HIV/AIDS patients in Ethiopia and
characterized Cryptosporidium spp. at the species, subtype family,
and subtype levels. We also examined the association between
clinical manifestations and infections with specific Cryptosporidium
species and subtype families. Data generated from the study have
clearly shown a dominance of C. parvum in the study population,
importance of zoonotic transmission in the epidemiology of
cryptosporidiosis in Ethiopia, and differences in clinical manifes-
tations among Cryptosporidium species and subtypes.
Materials and Methods
Ethical statement The research protocol was approved by the Ethical Clearance
Committee of the Addis Ababa University. All study participants
had given written informed consent before enrollment into the
study. When the study participant was a child, written consent was
obtained from his or her parent or guardian. Researchers at the
Centers for Disease Control and Prevention (CDC) had no contact
with patients and no access to personal identifiers. Laboratory
work on the study specimens was covered under CDC IRB
protocol No. 990115: ‘‘Use of residual human specimens for the
determination of frequency of genotypes or sub-types of patho-
genic parasites’’.
Study population and enrollment This study was cross-sectional in nature. It was conducted
between September 2009 and December 2011 in Addis Ababa,
Ethiopia. A total of 520 HIV/AIDS patients were recruited from
in-patients (hospitalized) and outpatients attending the Tikur
Anbessa Hospital, Addis Ababa University, and patients referred
to the study by attending physicians. The criteria for inclusion in
the study were documented HIV infection, the ability to provide
informed consent by the patient or the guardian, and willingness to
provide one stool specimen. A structured questionnaire was used
to collect CD4+ cell counts, demographic data, clinical symptoms, HAART history, antibiotics usage, and animal exposure history.
Each participant was asked to provide a single fresh stool
specimen.
DNA extraction Stool specimens were stored in 2.5% potassium dichromate at
4uC and shipped to the CDC laboratory in Atlanta for screening and molecular characterization of Cryptosporidium spp. by PCR.
After washing the stool specimens twice with distilled water,
genomic DNA was extracted from 0.5 ml of fecal materials using a
FastDNA SPIN Kit for Soil (MP Biomedicals, Solon. OH) and
eluted in 100 ml of reagent-grade water following the manufac- turer-recommended procedures. DNA was stored at 280uC until analyzed by PCR.
Cryptosporidium species detection, genotyping and subtyping
Cryptosporidium oocysts present in the specimens were detected by
nested PCR amplification of an approximate 830 bp fragment of
the small subunit (SSU) rRNA gene as described previously [27].
Cryptosporidium species were determined by restriction fragment
length polymorphism (RFLP) analysis of the secondary PCR
products using endonucleases SspI and VspI [27]. PCR products
and restriction fragments were subjected to electrophoresis in
1.5% and 2% agarose gels, respectively, and visualized after
staining with GelRed (Biotium Inc., Hayward, CA). All secondary
PCR products from species other than C. parvum were sequenced
to confirm the identification. Specimens that contained C. parvum
or C. hominis were further subtyped by DNA sequencing of the
nested PCR product of the gp60 gene [28]. Each specimen was
analyzed at least twice by PCR at each locus using 2 ml of the DNA extraction per PCR. As a positive control, C. baileyi DNA
was used in SSU rRNA PCR and C. hominis DNA was used in
Author Summary
The disease burden of Cryptosporidium parvum and role of zoonotic transmission in cryptosporidiosis epidemiology are poorly understood in developing countries. In this study, we examined the distribution and clinical manifes- tations of Cryptosporidium species and subtypes in HIV/ AIDS patients in Addis Ababa, Ethiopia. Using molecular diagnostic tools, we detected Cryptosporidium infection in 26.9% of 520 HIV/AIDS patients studied. We have shown a very high diversity of Cryptosporidium species and subtypes in these patients, but unlike in other developing countries, C. parvum is overwhelmingly the dominant species in the study community, responsible for ,65% Cryptosporidium infections. The common occurrence of C. parvum zoonotic subtype family IIa, combined with calf contact as a significant risk factor, suggest that zoonotic transmission is important in cryptosporidiosis epidemiol- ogy in HIV/AIDS patients in Ethiopia. We have also shown that different Cryptosporidium species and subtypes are linked to different clinical manifestations. Improved hygiene and avoidance of calf contact should be advocat- ed to reduce cryptosporidiosis transmission in HIV/AIDS patients in the study setting.
Cryptosporidiosis in AIDS Patients in Ethiopia
PLOS Neglected Tropical Diseases | www.plosntds.org 2 April 2014 | Volume 8 | Issue 4 | e2831
gp60 PCR. A negative control using DNase-free water was also
included in each PCR run.
Sequence analysis PCR products were sequenced using the forward and reverse
primers of the secondary PCR. An intermediary sequencing
primer gp60-R3 [59-GAG ATA TAT CTT GTT GCG-39] was
also used in the sequencing of gp60 PCR products. DNA
sequencing was done using the ABI BigDye Terminator v. 3.1
Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA)
and an ABI3130 Genetic Analyzer (Applied Biosystems). Sequence
accuracy was confirmed by sequencing of two PCR products from
each positive specimen. Nucleotide sequences obtained were
aligned with reference sequences using the ClustalX 1.81 package
(http://www.clustal.org/) to identify Cryptosporidium species and C.
parvum subtypes. Subtypes of C. parvum and C. hominis were named
based on the established nomenclature system [29]. Unique
sequences generated in this study were deposited in GenBank
under accession numbers AB830575 to AB830590.
Statistical analysis Data from the study were analyzed using the SPSS 20.0 for
Windows software (IBM Corp, Armonk, NY) at three levels of
parasite categorization: presence of Cryptosporidium, species of
Cryptosporidium, and subtype families of C. parvum and C. hominis.
Data from persons infected with low-frequency species were
pooled based on their genetic similarities. Univariate and
multivariate logistic regression modeling were used to analyze
the association between Cryptosporidium infection and clinical
symptoms or risk-factors while adjusting for potential
confounders when the sample size was sufficient. For comparisons
at the species or subtype family level, separate models and different
subsets of the main dataset were run to examine the effect of each
species or subtype family, with Cryptosporidium-negative as the
referent. The Hosmer-Lemeshow test was used to assess the
goodness-of-fit of each multivariate logistic regression model. The
strength of the associations was estimated by odds ratios (OR) and
95% confidence intervals (CI). The association was considered
statistically significant when the P value obtained was smaller
than 0.05.
Results
Study patients and Cryptosporidium occurrence Among the 520 HIV/AIDS patients who participated in this
study, 276 (53.1%) were males and 244 (46.9%) were females. The
median age of the study participants was 41 years (range: 7 months
to 86 years), and the mean CD4+ cell count was 278 cells/mL. Almost one-third (32.9%) of the study patients were bedridden and
hospitalized. Of the 520 stool specimens examined in this study,
140 (26.9%) were positive for Cryptosporidium by the SSU rRNA-
based PCR technique (Table 1). There were no significant
differences in prevalence of Cryptosporidium infection among age
groups and between the male and female gender (Table 1). No
significant association between hospitalization and Cryptosporidium
infection was found; percentages of Cryptosporidium infection in
hospitalized and non-hospitalized patients were 35.7% and 37.4%
(P = 0.83). In addition, no significant difference was observed in
the occurrence of vomiting, diarrhea, age, HAART, antibiotic use,
animal contact and CD4 between inpatients and outpatients.
There was also no association between infections with Cryptospo-
ridium spp. or any specific species and hospitalization rates (data
not shown).
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Cryptosporidiosis in AIDS Patients in Ethiopia
PLOS Neglected Tropical Diseases | www.plosntds.org 3 April 2014 | Volume 8 | Issue 4 | e2831
Cryptosporidium species and C. hominis and C. parvum subtypes
Species determination by RFLP was successful for 128/140
Cryptosporidium-positive specimens (Table 2). Cryptosporidium parvum
(n = 92) and C. hominis (n = 25) were the species most frequently
detected, followed by C. felis (n = 5), C. meleagridis (n = 3), C. canis
(n = 2), and C. parvum and C. hominis co-infection (n = 1). All SSU
rRNA PCR products from non-C. parvum specimens were
sequenced, confirming the RFLP results. In addition, 12
specimens demonstrated a RFLP pattern that was similar to C.
parvum, but with a slightly smaller upper SspI band. DNA
sequences from two of the specimens were identical to a reference
sequence of C. xiaoi (GenBank accession no. JQ413443) and those
from 10 specimens were identical to a reference sequence
(GenBank accession no. HM485434) of C. viatorum, a recently
described species in humans [17].
Subtype family data were obtained from 82 (89.1%) of the 92
participants with C. parvum and showed the presence of subtype
families IIa, IIb, IIc, IId, IIe, and a new subtype family genetically
related to If in 71, 1, 2, 5, 1, and 2 persons, respectively. In
contrast, 19 (76%) of the 25 participants with C. hominis were
successfully subtyped and showed the presence of subtype families
Table 2. Distribution of Cryptosporidium species and C. parvum and C. hominis subtypes in HIV/AIDS patients in Ethiopia (n = 140).
Species Subtype family Subtype No. of patients infected
C. parvum 92 (82 subtyped)
IIa 71
IIaA13G2R1 1
IIaA14G2R1 1
IIaA15G2R1 60
IIaA16G2R1 1
IIaA16G3R1 4
IIaA17G2R1 2
IIaA18G2R1 1
IIaA19G1R1 1
IIb
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IIdA24G1 1
IIe
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If-like
If-like 2
C. hominis 25 (19 subtyped)
Id 13
IdA20 10
IdA24 1
IdA26 2
Ib
IbA10G2 1
Ie
IeA11G3T3 5
C. viatorum 10
C. felis 5
C. meleagridis 3
C. canis 2
C. xiaoi 2
C. parvum + C. hominis 1
doi:10.1371/journal.pntd.0002831.t002
Cryptosporidiosis in AIDS Patients in Ethiopia
PLOS Neglected Tropical Diseases | www.plosntds.org 4 April 2014 | Volume 8 | Issue 4 | e2831
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C .
h o
m in
is 2
5 1
8 (7
2 )
1 .5
4 6
(0 .9
9 –
2 .4
2 )
0 .0
5 7
n a
n a
n a
C .
v ia
to ru
m 1
0 7
(7 0
) 1
.2 9
4 (0
.8 2
– 2
.0 4
) 0
.2 6
8 n
a n
a n
a
C .
m e
le a
g ri
d is
/C .
fe li
s/ C
. ca
n is
/C .
xi a
o i
1 2
1 1
(9 1
.7 )
1 .7
8 8
(1 .0
7 –
2 .9
9 )
0 .0
2 7
n a
n a
n a
N o
C ry
p to
sp o
ri d
iu m
3 8
0 1
9 7
(5 1
.8 )
R e
fe re
n t
- -
-
f R
is k
o f
v o
m it
in g
b y
s u
b ty
p e
fa m
il y
* *
C .
p a
rv u
m
II a
7 1
3 8
(5 3
.5 )
1 .0
7 0
(0 .6
4 –
1 .7
8 )
0 .7
9 5
1 .0
6 5
(0 .6
3 –
1 .8
0 )
0 .8
1 6
0 .3
3
II b
/I Ic
/I Id
/I Ie
/I f-
li k
e 1
2 7
(5 8
.3 )
1 .3
0 1
(0 .4
1 –
4 .1
7 )
0 .6
5 8
n a
n a
n a
C .
h o
m in
is
Id 1
3 9
(6 9
.2 )
2 .0
9 0
(0 .6
3 –
6 .9
0 )
0 .2
1 7
n a
n a
n a
Ib /I
e 6
5 (8
3 .3
) 4
.6 4
5 (0
.5 4
– 4
0 .1
3 )
0 .1
2 6
n a
n a
n a
Cryptosporidiosis in AIDS Patients in Ethiopia
PLOS Neglected Tropical Diseases | www.plosntds.org 5 April 2014 | Volume 8 | Issue 4 | e2831
Ib, Id, and Ie in 1, 13, and 5 persons, respectively. Within C.
parvum, 8 subtypes were from the subtype family IIa. This was
followed by subtype families IId with 4, If-like with 2 subtypes, and
IIb, IIc, and IIe with 1 subtype each. Thus, participants in the
study were infected with 14 subtypes of C. parvum. The most
frequently detected subtype was IIaA15G2R1, which was seen in
60 patients. Five subtypes were identified in C. hominis, including
IdA20, IdA26, IdA24, IeA11G3T3, and IbA10G2 in 10, 2, 1, 5,
and 1 patient, respectively (Table 2).
Cryptosporidium species/subtypes and clinical manifestations
Cryptosporidium infection was significantly associated with diar-
rhea in univariate analysis (P,0.001), especially in patients with C.
parvum and C. hominis (P,0.001 and P = 0.012 respectively).
Vomiting was also more often seen in Cryptosporidium-positive than
Cryptosporidium-negative patients (63.6% versus 51.8%; P = 0.018).
Patients with C. hominis or rare species (C. meleagridis/C. felis/C. canis
and C. xiaoi) were more likely to have vomiting compared with
Cryptosporidium-negative patients (P = 0.057 and P = 0.027, respec-
tively; Table 3).
Diarrhea was significantly associated with infections with C.
parvum subtype family IIa compared with Cryptosporidium-negative
patients (78.9%; P = 0.003). However, there were no significant
differences in diarrhea occurrence between patients infected with
C. hominis subtype families and Cryptosporidium-negative patients.
Patients infected with different C. parvum and C. hominis subtype
families also did not show significant differences in the occurrence
of vomiting compared to Cryptosporidium-negative patients (P.0.05;
Table 3).
Multivariable modeling was attempted for diarrhea and
vomiting occurrence in order to adjust for age, gender, type of
patients and clinical parameters (HAART and CD4+); however, it did not reveal major differences compared with the crude OR
(Table 3).
Cryptosporidium species/subtypes and risk factors After controlling for other potential risk factors, history of any
contact with animals was associated with overall Cryptosporidium
infections (OR = 1.6; P = 0.04), and with C. parvum (OR = 2.5;
P = 0.002) and its subtype family IIa (OR = 2.1; P = 0.02) in
particular. This association was mostly due to contact with calves
(OR = 1.6 and P = 0.02 for overall Cryptosporidium infection;
OR = 1.8 and P = 0.01 for C. parvum; and OR = 2.0 and P = 0.01
for C. parvum subtype family IIa; Table 4). However, there was no
significant association between Cryptosporidium infection and age,
gender, HAART history, CD4+ cell counts, antibiotics use, or type of patients (P = 0.56, 0.13, 0.59, 0.90, 0.84, and 0.83, respectively).
No significant association was found between Cryptosporidium
infection and CD4+ cell counts or HAART history at the species and subtype levels (Tables S1 and S2).
Discussion
The present findings showed that (1) Ethiopian HIV/AIDS
patients were infected with a diverse population of Cryptosporidium
species, including the unusual species C. viatorum and C. xiaoi; (2) C.
parvum was the most frequently detected species; and (3)
Cryptosporidium species or subtype families were associated with
different clinical manifestations. The dominance of C. parvum in
this study is in agreement with the previous observation in a small
study in Ethiopia [22]. It is, however, in sharp contrast with studies
of human cryptosporidiosis in other developing countries where C.
hominis dominates [12,30,31]. In Europe, the two species are
T a
b le
3 .
C o
n t.
P a
ra m
e te
r T
o ta
l p
a ti
e n
ts P
a ti
e n
ts w
it h
s y
m p
to m
(% )
C ru
d e
A d
ju s
te d
# G
o o
d n
e s
s o
f fi
t# #
O R
(9 5
% C
I) *
P -v
a lu
e O
R (9
5 %
C I)
P -v
a lu
e P
-v a
lu e
N o
C ry
p to
sp o
ri d
iu m
3 8
0 1
9 7
(5 1
.8 )
R e
fe re
n t
- -
-
# M
u lt
iv a
ri a
b le
lo g
is ti
c re
g re
ss io
n s
w e
re p
e rf
o rm
e d
to a
d ju
st th
e p
o te
n ti
a l
c o
n fo
u n
d e
rs in
c lu
d in
g a
g e
, g
e n
d e
r, H
A A
R T
, C
D 4
, a
n d
ty p
e o
f p
a ti
e n
ts w
h e
n sa
m p
le si
ze w
a s
su ff
ic ie
n t.
n a
: sa
m p
le si
ze w
a s
to o
sm a
ll fo
r m
u lt
iv a
ri a
te lo
g is
ti c
re g
re ss
io n
a n
a ly
si s.
# #
H o
sm e
r a
n d
L e
m e
sh o
w te
st w
a s
a p
p li
e d
to te
st th
e g
o o
d n
e ss
o f
fi t
o f
m u
lt iv
a ri
a te
lo g
is ti
c s
re g
re ss
io n
m o
d e
ls .
*9 5
% C
I: 9
5 %
c o
n fi
d e
n c
e in
te rv
a ls
. **
F o
r e
a c
h C
ry p
to sp
o ri
d iu
m sp
e c
ie s
o r
su b
ty p
e fa
m il
y ,
p a
ti e
n ts
w it
h th
e sp
e c
ie s
o r
su b
ty p
e fa
m il
y w
e re
ta k
e n
a s
‘‘p o
si ti
v e
’’, p
a ti
e n
ts w
h o
w e
re n
o t
in fe
c te
d a
t a
ll w
e re
ta k
e n
a s
‘‘n e
g a
ti v
e ’’
(r e
fe re
n t)
, w
h il
e p
a ti
e n
ts in
fe c
te d
w it
h o
th e
r sp
e c
ie s
o r
su b
ty p
e fa
m il
ie s
w e
re n
o t
in c
lu d
e d
in th
is sp
e c
if ic
m o
d e
l. d
o i:1
0 .1
3 7
1 /j
o u
rn a
l.p n
td .0
0 0
2 8
3 1
.t 0
0 3
Cryptosporidiosis in AIDS Patients in Ethiopia
PLOS Neglected Tropical Diseases | www.plosntds.org 6 April 2014 | Volume 8 | Issue 4 | e2831
T a
b le
4 .
A ss
o c
ia ti
o n
b e
tw e
e n
a n
im a
l c
o n
ta c
t a
n d
in fe
c ti
o n
w it
h C
ry p
to sp
o ri
d iu
m sp
e c
ie s
o r
C .
p a
rv u
m a
n d
C .
h o
m in
is su
b ty
p e
s in
H IV
/A ID
S p
a ti
e n
ts in
E th
io p
ia *.
P a
ra m
e te
r T
o ta
l P
a ti
e n
ts
P a
ti e
n ts
w it
h a
n im
a l
c o
n ta
c t
n (%
) C
ru d
e A
d ju
s te
d #
G o
o d
n e
s s
o f
fi t#
#
O R
(9 5
% C
I) *
* P
-v a
lu e
O R
(9 5
% C
I) P
-v a
lu e
P -v
a lu
e
A n
im a
l c
o n
ta c
t h
is to
ry b
y C
ry p
to sp
o ri
d iu
m
C ry
p to
sp o
ri d
iu m
1 4
0 1
0 8
(7 7
.1 )
1 .5
5 6
(1 .0
1 –
2 .4
0 )
0 .0
4 6
1 .5
7 7
(1 .0
1 –
2 .4
6 )
0 .0
4 4
0 .8
1
N o
C ry
p to
sp o
ri d
iu m
3 8
0 2
4 7
(6 5
) R
e fe
re n
t -
- -
A n
im a
l c
o n
ta c
t h
is to
ry b
y C
ry p
to sp
o ri
d iu
m s
p e
c ie
s #
# #
C .
p a
rv u
m 9
2 7
5 (8
1 .5
) 2
.3 7
6 (1
.3 5
– 4
.1 9
) 0
.0 0
3 2
.4 9
5 (1
.3 9
– 4
.4 8
) 0
.0 0
2 0
.4 9
C .
h o
m in
is 2
5 2
1 (8
4 )
1 .6
8 1
(0 .9
8 –
2 .9
0 )
0 .0
6 2
n a
n a
n a
C .
v ia
to ru
m 1
0 6
(6 0
) 0
.9 3
1 (0
.6 1
– 1
.4 3
) 0
.7 4
4 n
a n
a n
a
C .
m e
le a
g ri
d is
/C .
fe li
s/ C
. ca
n is
/C .
xi a
o i
1 2
5 (4
1 .7
) 0
.7 8
8 (0
.5 9
– 1
.0 5
5 )
0 .1
0 8
n a
n a
n a
N o
C ry
p to
sp o
ri d
iu m
3 8
0 2
4 7
(6 5
) R
e fe
re n
t -
- -
A n
im a
l c
o n
ta c
t h
is to
ry b
y s
u b
ty p
e fa
m il
y #
# #
C .
p a
rv u
m
II a
7 1
5 7
(8 0
.3 )
2 .1
9 2
(1 .1
8 –
4 .0
8 )
0 .0
1 2
2 .1
2 4
(1 .1
3 –
3 .9
8 )
0 .0
1 9
0 .5
5
II b
/I Ic
/I Id
/I Ie
/I f-
li k
e 1
2 9
(7 5
) 1
.6 1
5 (0
.4 3
– 6
.0 7
) 0
.4 7
4 n
a n
a n
a
C .
h o
m in
is
Id 1
3 1
2 (9
2 .3
) 6
.4 6
2 (0
.8 3
– 5
0 .2
4 )
0 .0
8 1
n a
n a
n a
Ib /I
e 6
4 (6
6 .7
) 1
.0 7
7 (0
.1 9
– 5
.9 6
) 0
.7 2
9 n
a n
a n
a
N o
C ry
p to
sp o
ri d
iu m
3 8
0 2
4 7
(6 5
) R
e fe
re n
t -
- -
C a
lf c
o n
ta c
t h
is to
ry b
y C
ry p
to sp
o ri
d iu
m
C ry
p to
sp o
ri d
iu m
1 4
0 5
6 (4
0 )
1 .6
7 9
(1 .1
2 –
2 .5
2 )
0 .0
1 2
1 .6
2 8
(1 .0
8 –
2 .4
6 )
0 .0
2 1
0 .5
5
N o
C ry
p to
sp o
ri d
iu m
3 8
0 1
0 8
(2 8
.4 )
R e
fe re
n t
- -
-
C a
lf c
o n
ta c
t h
is to
ry b
y C
ry p
to sp
o ri
d iu
m s
p e
c ie
s #
# #
C .
p a
rv u
m 9
2 4
0 (4
3 .5
) 1
.9 3
7 (1
.2 2
– 3
.1 0
) 0
.0 0
6 1
.8 3
9 (1
.1 4
– 2
.9 7
) 0
.0 1
3 0
.6 9
C .
h o
m in
is 2
5 1
5 (6
0 )
1 .9
4 4
(1 .2
9 –
2 .9
5 )
0 .0
0 2
n a
n a
n a
C .
v ia
to ru
m 1
0 1
(1 0
) 0
.6 5
4 (0
.3 3
– 1
.3 1
) 0
.2 3
0 n
a n
a n
a
C .
m e
le a
g ri
d is
/C .
fe li
s/ C
. ca
n is
/C .
xi a
o i
1 2
0 n
a n
a n
a n
a n
a
N o
C ry
p to
sp o
ri d
iu m
3 8
0 1
0 8
(2 8
.4 )
R e
fe re
n t
- -
-
C a
lf c
o n
ta c
t h
is to
ry b
y s
u b
ty p
e fa
m il
y #
# #
C .
p a
rv u
m
II a
7 1
3 3
(4 6
.5 )
2 .1
8 7
(1 .3
0 –
3 .6
7 )
0 .0
0 3
2 .0
0 1
(1 .1
8 –
3 .4
0 )
0 .0
1 0
0 .9
8
II b
/I Ic
/I Id
/I Ie
/I f-
li k
e 1
2 3
(2 5
) 0
.8 3
9 (0
.2 2
– 3
.1 6
) 0
.7 9
6 n
a n
a n
a
C .
h o
m in
is
Id 1
3 9
(6 9
.2 )
5 .6
6 7
(1 .7
1 –
1 8
.7 9
) 0
.0 0
2 n
a n
a n
a
Ib /I
e 6
1 (1
6 .7
) 0
.5 0
4 (0
.0 6
– 4
.3 6
) 0
.5 2
6 n
a n
a n
a
Cryptosporidiosis in AIDS Patients in Ethiopia
PLOS Neglected Tropical Diseases | www.plosntds.org 7 April 2014 | Volume 8 | Issue 4 | e2831
almost evenly distributed, with C. parvum being more prevalent in
some reports [32] and C. hominis in others [33]. Data from this
study also indicate that the recently established species C. viatorum
is more widely distributed than believed, and humans can be
infected occasionally with the sheep and goat parasite C. xiaoi. As
expected, the prevalence of cryptosporidiosis in HIV+ patients (26.9%) in this study is substantially higher than in the largely
healthy persons (8.7%) in a previous study conducted in the same
area [22].
Differences in geographical distribution of C. parvum and C.
hominis are generally considered a reflection of differences in
infection sources and transmission routes [12]. C. hominis is
transmitted almost exclusively among humans, whereas C. parvum,
especially its IIa subtype family, is more likely transmitted
zoonotically. In industrialized nations, C. parvum infections have
often been linked to contact with farm animals, and C. hominis
infections to contact with children with diarrhea [34]. Both species
have been associated with drinking-water outbreaks [33]. The
predominance of C. hominis in most developing countries suggests
that anthroponotic transmission is more important than zoonotic
transmission in cryptosporidiosis epidemiology in developing
countries in general [12]. In contrast, the dominance of C. parvum
IIa subtype family in Ethiopia in HIV/AIDS patients suggests that
unlike in other developing countries, Cryptosporidium infection in
Ethiopia is mostly transmitted zoonotically. Previously, it was
shown that that small numbers of C. parvum infections seen in
humans in developing countries were mostly caused by the
anthroponotic subtype family IIc [12].
Results of the risk factor analysis support the role of zoonotic
transmission in cryptosporidiosis epidemiology in HIV/AIDS
patients in Ethiopia. Even though the present study was performed
in an urban area, more than 50% of patients had contact with
animals, as households in the study area usually have farm animals
living inside the residence. Thus, in this study, animal contact,
especially with calves, was a significant risk factor for Cryptosporid-
ium, especially C. parvum and its IIa subtype family. The
distribution of C. parvum subtypes in this study reinforces the likely
occurrence of zoonotic transmission, as the majority of C. parvum
infections were caused by IIa subtypes (71/82 specimens
subtyped), especially its IIaA15G2R1 subtype (60/82 specimens
subtyped), which is a dominant C. parvum subtype in calves around
the world [12]. In contrast, the anthroponotic C. parvum subtype
family IIc was seen in only 2/82 of C. parvum infections.
In addition to IIa, C. parvum subtype family IId was also
identified in five Ethiopian HIV/AIDS patients. Subtype family
IId of C. parvum is generally considered a sheep and goat parasite
[35], but has been found at high frequency in calves in China,
Egypt, and Sweden [36–38]. In this study, only one patient
infected with IId had contact with sheep in this study. Genotyping
and subtyping studies of domestic animals from the study area and
additional case-control-studies are needed to support the conclu-
sion on the importance of zoonotic transmission in cryptosporid-
iosis epidemiology in Ethiopia.
In agreement with previous observations elsewhere [19–21],
data from the study suggest that different Cryptosporidium species
and subtypes are linked to different manifestations of cryptospo-
ridiosis. As expected, cryptosporidiosis in our study was signifi-
cantly associated with the occurrence of diarrhea. This association,
however, was largely attributable to C. parvum and C. hominis; other
species, including the newly described C. viatorum, were less
pathogenic than these two species. Likewise, the significant
association between Cryptosporidium infection and the occurrence
of vomiting was also largely attributable to C. hominis and some less
frequent species (C. meleagridis, C. canis, C. felis and C. xiaoi); C.
T a
b le
4 .
C o
n t.
P a
ra m
e te
r T
o ta
l P
a ti
e n
ts
P a
ti e
n ts
w it
h a
n im
a l
c o
n ta
c t
n (%
) C
ru d
e A
d ju
s te
d #
G o
o d
n e
s s
o f
fi t#
#
O R
(9 5
% C
I) *
* P
-v a
lu e
O R
(9 5
% C
I) P
-v a
lu e
P -v
a lu
e
N o
C ry
p to
sp o
ri d
iu m
3 8
0 1
0 8
(2 8
.4 )
R e
fe re
n t
- -
-
*C o
n ta
c ts
w it
h sp
e c
if ic
a n
im a
l sp
e c
ie s
o th
e r
th a
n c
a tt
le w
e re
n o
t si
g n
if ic
a n
t ri
sk fa
c to
rs in
th is
st u
d y
. **
9 5
% C
I: 9
5 %
c o
n fi
d e
n c
e in
te rv
a ls
. #
M u
lt iv
a ri
a b
le lo
g is
ti c
re g
re ss
io n
s w
e re
p e
rf o
rm e
d to
a d
ju st
th e
p o
te n
ti a
l c
o n
fo u
n d
e rs
in c
lu d
in g
a g
e ,
g e
n d
e r,
H A
A R
T ,
C D
4 ,
a n
d ty
p e
o f
p a
ti e
n ts
w h
e n
sa m
p le
si ze
w a
s su
ff ic
ie n
t. n
a :
sa m
p le
si ze
w a
s to
o sm
a ll
fo r
m u
lt iv
a ri
a te
lo g
is ti
c re
g re
ss io
n a
n a
ly si
s. #
# H
o sm
e r
a n
d L
e m
e sh
o w
te st
w a
s a
p p
li e
d to
te st
th e
g o
o d
n e
ss o
f fi
t o
f m
u lt
iv a
ri a
te lo
g is
ti c
s re
g re
ss io
n m
o d
e ls
. #
# #
F o
r e
a c
h C
ry p
to sp
o ri
d iu
m sp
e c
ie s
o r
su b
ty p
e fa
m il
y ,
p a
ti e
n ts
w it
h th
e sp
e c
ie s
o r
su b
ty p
e fa
m il
y w
e re
ta k
e n
a s
‘‘p o
si ti
v e
’’, p
a ti
e n
ts w
h o
w e
re n
o t
in fe
c te
d a
t a
ll w
e re
ta k
e n
a s
‘‘n e
g a
ti v
e ’’
(r e
fe re
n t)
, w
h il
e p
a ti
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Cryptosporidiosis in AIDS Patients in Ethiopia
PLOS Neglected Tropical Diseases | www.plosntds.org 8 April 2014 | Volume 8 | Issue 4 | e2831
parvum and C. viatorum were largely not associated with vomiting.
Within C. parvum, the IIa subtype family also appeared to be more
associated with the occurrence of diarrhea than other C. parvum
subtype families. We did not observe any effect of CD4+ cell counts and HAART on the occurrence of cryptosporidiosis in
HIV/AIDS patients in this study. This was probably largely the
result of severe overall immunodeficiency in the study population,
as reflected by the low mean CD4+ cell counts (278 cells/mL) and very high hospitalization rate (32.9%) and occurrence of
cryptosporidiosis (26.9%).
In conclusion, Ethiopian HIV/AIDS patients with low CD4+ cell counts had an extremely high occurrence of Cryptosporidium
infection, even when they were on HAART. Although the majority
of cryptosporidiosis cases were caused by C. parvum, there was a high
diversity of Cryptosporidium species, with a significant number of cases
caused by the newly recognized C. viatorum. These Cryptosporidium
spp. and C. parvum subtypes were linked to different clinical
manifestations. Therefore, improved hygiene and avoidance of calf
contact among this population should be advocated to reduce the
occurrence of Cryptosporidium infections, especially those caused by C.
parvum IIa subtypes of calves.
Supporting Information
Checklist S1 STROBE checklist.
(PDF)
Table S1 Association between CD4+ cell count and infection with Cryptosporidium species or C. parvum and C. hominis subtypes in
HIV/AIDS patients in Ethiopia*.
(DOCX)
Table S2 Association between HAART history and infection
with Cryptosporidium species or C. parvum and C. hominis subtypes in
HIV/AIDS patients in Ethiopia*.
(DOCX)
Acknowledgments
We thank the Tikur Anbessa Hospital for supporting the sample
collection and CD4 cell count, and Na Li, Hongwei Zhang, Theresa
Dearen, and Dawn Roellig for technical assistance. We also thank
the anonymous reviewer for suggestions on statistical analyses of
the data.
The findings and conclusions in this report are those of the authors and
do not necessarily represent the views of the Centers for Disease Control
and Prevention.
Author Contributions
Conceived and designed the experiments: HA LX BP HK. Performed the
experiments: HA SA JY. Analyzed the data: HA GZ LX. Contributed
reagents/materials/analysis tools: LX YF JY. Wrote the paper: HA LX
YF.
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