Hindawi Publishing Corporation
Journal of Tropical Medicine
Volume 2011, Article ID 598341, 6 pages
RiskFactors and Seroprevalenceof Hepatitis Camong
Patients Hospitalized at Mulago Hospital, Uganda
J. I.O’Reilly,1P. Ocama,2C.K. Opio,2A.Alfred,2E.Paintsil,1
1Yale University School of Medicine, New Haven, CT 06510, USA
2Makerere University Medical School, Kampala, Uganda
Correspondence should be addressed to J. I. O’Reilly, firstname.lastname@example.org
Received 5 April 2011; Accepted 21 June 2011
Academic Editor: Thomas R. Unnasch
Copyright © 2011 J. I. O’Reilly et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The emergence of hepatitis C virus (HCV) and its associated sequelae in Africa is a cause for significant concern. Human
immunodeficiency virus (HIV) positive patients are at an increased risk of contracting HCV infection due to similar risk factors
and modes of transmission. We investigated the seroprevalence of hepatitis C in hospitalized HIV-positive and HIV-negative
patients in Mulago Hospital, an academic hospital in Uganda. Blood samples were first tested for HCV antibodies, and positive
tests were confirmed with HCV RNA PCR. We enrolled five hundred patients, half HIV-positive and half HIV negative. Overall,
13/500 patients (2.6%) tested positive for HCV antibodies. There was no difference in HCV antibody detection among HIV-
positive and HIV-negative patients. Out of all risk factors examined, only an age greater than 50 years was associated with HCV
infection. Traditional risk factors for concurrent HIV and HCV transmission, such as intravenous drug use, were exceedingly rare
in Uganda. Only 3 of 13 patients with detectable HCV antibodies were confirmed by HCV RNA detection. This result concurs
with recent studies noting poor performance of HCV antibody testing when using African sera. These tests should be validated in
the local population before implementation.
Hepatitis C virus (HCV) infection is a significant cause of
morbidity and mortality worldwide; however, the prevalence
and burden of HCV in sub-Saharan Africa have received
little attention. World Health Organization data gathered
from more than 130 countries estimated that more than 170
to a worldwide prevalence of 3% . The prevalence of HCV
infection in the general population in sub-Saharan Africa is
estimated at 3% to 5.3% . Despite this high prevalence,
active surveillance for HCV is rarely performed. This is
due partly to resource constraints, unreliability of available
serological tests, the high cost of nucleic acid testing (PCR)
and confirmatory testing, and unaffordable cost of treatment
Studies of HCV prevalence in Uganda have yielded
inconsistent results . The earliest study showed a 2.5%
prevalence by antibody testing. Two studies of HCV in HIV-
infected patients gave an HCV prevalence of 0.6% and 2.9%
[6, 7]. However, none of these studies confirmed antibody
positivity by testing for HCV RNA or a recombinant
reported an initial HCV antibody prevalence of 4.0%, but
only 0.6% after RIBA confirmation . Another study
among individuals with sickle cell disease found a 4%
antibody prevalence in children and 8% in their mothers .
However, only 11% and 8% of the seropositive children and
mothers, respectively, had confirmation by RT-PCR. There
is also a significant difference in the sensitivities of available
serological tests . For example, in one study of prenatal
screening of Ugandan women, there was 16.6% antibody
prevalence identified during the initial screening . Upon
retesting with a different antibody test, less than half of these
were positive. Most recently a study of hospitalized patients
from Kampala showed discordant results between hepatitis
C rapid assays, standard EIA testing, and the confirmatory
HCV RNA testing . Anti-HCV antibody testing was
2 Journal of Tropical Medicine
positive in 13% of patients by one or both of these initial
tests. However, only 29% of these samples had detectable
HCV RNA on confirmatory testing.
In the developed world, hepatitis C is strongly associated
with human immunodeficiency virus due to shared risk
factors and modes of transmission [3, 12]. Much like
HIV, HCV is transmitted through blood-to-blood contact.
Intravenous drug use and the sharing of virus-contaminated
needles constitute the primary risk factor for cotransmission
in the developed world. In some communities of IV drug
approaches 90% . Other risk factors include accidental
needle stick injuries, transfusion of contaminated blood and
blood products, as well as nonsterile tattooing practices
. In addition, vertical transmission from mother-to-
child occurs in both hepatitis C and HIV . Sexual
contact is another important mode of transmission for HIV
in both the developing and the industrialized world .
Unlike HIV, sexually transmission of HCV has received
mixed reviews in the literature . However, it is plausible
that certain sexual practices that may involve exposure
to blood, such as unprotected anal sex, may increase the
risk for HCV transmission . Recent epidemiological
studies have revealed that certain medical interventions,
such as nonsterile needle injections of antiparasitic drugs
in Africa during the colonial era (1920s–1950s), might
have contributed to high prevalence of chronic blood-borne
pathogens such as HCV [19, 20].
There is also evidence that concurrent HCV infection
makes it more difficult for HIV-infected patients to tolerate
highly active antiretroviral therapy (HAART), leading to
worse outcomes [21–23]. HIV has been shown to accelerate
the progression of HCV-related sequelae such as cirrhosis
and hepatocellular carcinoma [24–26]. Given the recent
successful implementation of highly active antiretroviral
therapy in Uganda, patients with HIV can live significantly
longer and have less chance of developing AIDS-related
illnesses [27, 28]. In the absence of these typical oppor-
tunistic infections, chronic comorbid illnesses will become
a greater cause for concern. Currently, HCV is generally not
monetary considerations. We therefore sought to investigate
used universally in Uganda. A positive correlation between
HIV and HCV would suggest that HIV-infected patients be
treated with a higher degree of suspicion for HCV infection.
Our study compared cohorts of HIV-infected and HIV-non-
infected patients for hepatitis C in an inpatient setting at an
academic hospital in Uganda and to describe any risk factors
associated with HCV infection in this cohort.
2.1. Study Participants. The study was conducted in Mulago
admits over 12,000 patients a year, nearly half of whom are
HIV positive. Patients 18 years old and above with a known
invited to participate in the study. HIV testing is normally
performed on all willing patients of unknown serostatus in
an HIV voluntary counseling and testing (VCT) program
provided on all the wards of Mulago Hospital. Five hundred
in this study. One half of the patients enrolled were HIV-
seropositive, and the other half were HIV-seronegative.
Translators were utilized for any patient unable to speak
After obtaining written informed consent, patients had
approximately 10mL of blood drawn for HCV testing. We
also administered a questionnaire to all patients in order to
define demographic, epidemiologic, and clinical variables.
The study participants were not given any monetary
incentives and those found to be HCV infected were not
offered treatment as there are no national guidelines for
HCV treatment. Patients testing positive for HCV infection,
either by EIA or by PCR, were informed of their status
and its ramifications by an experienced gastroenterologist
and were invited to attend the gastroenterology clinic.
They were also informed that the final tests to confirm
infection would be delivered to them when the result became
This study was approved by the Makerere University
Faculty of Medicine Research and Ethics committee Review
Board, the Uganda Council for Science and Technology, and
the Yale University School of Medicine Human Investigation
2.2. Laboratory Analysis. Blood samples collected from study
participants initially were tested for hepatitis C antibody
using a Bioline rapid test kit (Bioline Medical Systems, Pasig
City, Philippines). This rapid HCV antibody test was the
preferred method of HCV detection in Mulago Hospital.
The remaining blood was centrifuged within 4 hours of
to 5 volumes of RNAlater solution in accordance with
Ambion protocol (Austin, Texas, USA). RNAlater was used
to preserve viral RNA during transport. The solution was
stored at −20◦C before transport to Yale University School of
Medicine for further analysis. Per RNAlater protocol samples
can be thawed during transit and refrozen without affecting
the amount or integrity of recoverable RNA.
RNA was extracted from the previously described sam-
ples using a Qiagen QIAamp Viral Kit (Valencia, California,
USA). Extracted RNA was stored at −20◦C until RT-PCR
could be performed. Primers from the core region of the
HCV genome were used to amplify the extracted RNA using
a Qiagen one step RT-PCR kit as previously described .
PCR products were separated by agarose gel electrophoresis
positive for HCV RNA by RT-PCR were confirmed by
HCV sequence analysis. PCR products were purified using a
Qiagen QIAquick PCR purification kit, and sequenced at the
Keck Biotechnology Resource Laboratory at Yale University.
version 3.0 ELISA test kit (Ortho-Clinical Diagnostics, New
Journal of Tropical Medicine3
Table 1: Demographic Characteristics of the Study Population.
(n = 250)
(n = 250)
1.6% (4)4.0% (10)0.177
2.3. Statistics. Data were analyzed using statistical analysis
system (SAS) version 9.1 (SAS Institute Inc., Cary, North
Carolina, USA). Demographic and epidemiologic character-
istics of the study sample were described using descriptive
statistics. For comparisons between HCV and HIV infection,
as well as other variables, a two-sided Z-test to compare
proportions was used at an α of 0.05.
From January to February 2008, we enrolled 500 patients
who attended the Mulago Hospital inpatient service, of
whom 235/500 (47%) were male. The median age of the
patients was 35 years. One half (250/50) of the patients
were HIV positive. In the subgroup analysis shown in
Table 1, patients aged 31–50 were significantly more likely
to have HIV, and those over 50 years of age significantly
less likely. The patients who identified themselves as farmers
or unemployed were significantly more likely to have HIV
less likely to be HIV positive.
Overall 16 of the 500 (3.2%) patients tested positive for
HCV antibodies using the Bioline rapid assay. Subsequently,
all seropositive samples were retested with a confirmatory
Ortho HCV 3.0 immunoassay. 13 of the 16 positive HCV
antibody samples also tested positive by this immunoassay
(2.6%). HIV status was not associated with HCV antibody
detection in patients. There was no difference in HCV
antibody detection among HIV negative (7 cases) and
HIV positive (6 cases). Confirmation of HCV viremia was
performed by the detection of HCV RNA using RT-PCR
on all 13 HCV-positive antibody samples. HCV RNA was
detected in only 3 of the 13 (23%) samples. Of these, 2
were detected in HIV-positive patients, and 1 was detected
in HIV-negative patients. In addition, a cohort who tested
negative for the presence of HCV antibodies were tested for
the presence of HCV RNA. Interestingly, 5 of 89 samples
randomly selected from the HCV antibody-negative HIV-
positive patients tested positive for HCV RNA. All HCV
antibody-negative HIV-negative patients tested negative for
As indicated in Table 2, quantitative analysis demon-
strated that participants with ages greater than 50 were
significantly associated with HCV antibody detection. Blood
donors, patients receiving transfusions, patients with previ-
ous surgeries, and patients with tattoos or ritual scars were
also more likely to have HCV. However, these other risk
factors were not sufficiently powered to achieve statistical
Samples that had tested positive for HCV RNA were sent
for RNA sequencing. These samples were matched against
known databases for HCV. All amplifiable sequences were
matched to genotype 1B.
Our results indicate much variability between the screen-
ing and confirmatory testing for hepatitis C in Uganda.
Screening for hepatitis C is performed by testing for
antibodies in patients using commercially available assays.
The confirmatory testing is PCR to detect HCV RNA
viremia. Acute hepatitis C infection is generally thought to
progress to chronic hepatitis C 75% of the time . In
this case, we would expect only 75% of antibody testing
to be confirmed by PCR, since a quarter of patients will
of 13 HCV-antibody positive samples, or 23%, subsequently
tested positive for active HCV infection. As previous studies
testing for hepatitis C infection in Uganda have shown,
confirmatory testing. Seremba et al.  were only able to
confirm 29% of positive samples with RNA testing, and even
showed marked discrepancies between different antibody
tests. In our study, we initially tested for HCV using the
Bioline rapid assay used in Mulago Hospital. Subsequently,
13 of 16 samples also tested positive with the Ortho third
generation anti-HCV ELISA, which was chosen because it
is a widely used immunoassay. Thus the Bioline rapid assay
appears to be adequate in this population compared to
other available immunoassays. However, antibody testing in
Uganda may be a poor indicator of active HCV infection .
Studies comparing different antibody tests have noted poor
performance when using sera from Africans using the Ortho
anti-HCV 2nd and 3rd generation EIA test kits [3, 9]; these
tests are normally very sensitive . In this case, antibody
tests developed based on sera from developed countries may
not sufficiently capture the seroepidemiology of the disease.
The hepatitis C epidemic is maturing in a subregion with a
wide genetic diversity, so poor detection of this virus using
narrow-spectrum tests may be expected. There remains the
possibility that a larger percentage of Ugandan patients clear
4 Journal of Tropical Medicine
Table 2: Risk factor analysis of study population.
Percentage P value
IV drug use
Sex for money
the disease; however, this seems unlikely to account for all of
the noted discrepancy.
Analysis of the demographics of HIV infection between
our two cohorts reveals interesting findings. The HIV
prevalence between the younger subgroups in each cohort
was similar, but the middle-aged subgroup was significantly
more likely to have contracted HIV. This correlates with
an increased opportunity to acquire infection as one ages.
The older subgroup was significantly less likely to have HIV,
which may be due to decreased sexual risk factors, but is
more likely due to the mortality associated with chronic HIV
infection. Decreased prevalence of HIV infection was seen in
students, a younger, more educated subgroup. In contrast,
more HIV infection was seen in those less educated: farmers
and the unemployed.
There are several limitations to our study and its results.
us a more accurate determination of antibody status. Since
all samples must be transported overseas for PCR analysis,
RNA degradation was possible despite precautions taken,
Although the custom primer used during PCR had been
validated internally at Yale, to our knowledge it has never
been used on African sera. Finally, HCV RNA PCR is a
technically demanding process, which may cause both false
positives and false negatives to occur.
Given these constraints, it is informative to evaluate the
results of antibody testing. The prevalence of hepatitis C
infection detected by antibody testing was between 2.6%
and 3.2% in this study, depending on the assay used. The
lack of clear association between HCV and HIV infection
is likely due to the fact that some risk factors commonly
seen in the West, namely, injection drug use, are not
common practice in most African countries. HIV currently
is primarily transmitted by sexual contact in Uganda; the
likelihood of HCV transmission though sexual contact is
very low. The one risk factor shown in this study for
HCV infection, an age over 50, is unlikely to be associated
with HIV because elderly patients who contracted this HIV
decades ago would have died by now since HAART was not
an association between blood borne infections and the use
of contaminated medical instruments and unsterile practices
in the past [19, 20]. These risks are primarily confined to
older generations before safe practices were instituted. The
data also suggest that risk factors for blood transmission,
such as tattooing, transfusions, and surgery increase risk for
HCV transmission. However, the study was not sufficiently
powered to show statistical significance for these variables.
It is notable that 5 of 84 HIV-positive patients who
initially tested negative for HCV antibodies were found
to have HCV viremia. This phenomenon seems limited
to HIV-positive patients, as none of the 25 HCV/HIV-
negative patients tested had detectable HCV RNA. HIV-
positive patients in Uganda are usually identified very late
in the course of disease progression, with low CD4 counts
and symptomatic AIDS-related illnesses. There is evidence
to suggest that in patients with HIV-associated immune
In summary, the seroprevalence noted in this study
appears to greatly overestimate the prevalence of HCV
viremia in this cohort of Ugandan hospital patients. This
discrepancy may be due to disease clearance or false positives
in antibody testing, possibly related to chronic stimulation
of the immune system from a variety of tropical infectious
diseases. There remains an urgent need for these tests to
be validated in each population prior to clinical utilization.
An analysis determining if African sera tested for HCV
Journal of Tropical Medicine5
antibodies can be accurately and cost-effectively confirmed
by RNA PCR testing is also needed. Finally, it is currently
unknown how the progression of HIV in Ugandan patients
affects the detection of HCV antibodies in these patients.
These issues would benefit greatly from a further study.
The authors report no conflict of interests. This study was
funded through the Wilbur G. Downs Fellowship at Yale
University. The authors would like to thank the patients and
and the administrators of Makerere University. The authors
would also like to thank Amisha Patel for her assistance in
the Yale virology lab.
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