High Anti-HCV Seroprevalence and Low Performance of ICT Strip in Diagnosing Hepatitis C Virus Infection in Children in Enugu Metropolis
Maryann C. Ezeilo1, *, Godwill A. Engwa2, *, Romanus I. Iroha3, Damian N. Odimegwu4
1 Department of Microbiology, Godfrey Okoye University, P.M.B 01014, Thinkers Corner, Enugu, Nigeria
2 Department of Chemical Sciences, Godfrey Okoye University, P.M.B 01014, Thinkers Corner, Enugu, Nigeria
3 Department of Microbiology, Ebonyi State University, Abakiliki, Nigeria
4 Department of Pharmaceutical Microbiology and Biotechnology, University of Nigeria Nsukka, Enugu State, Nigeria
The lack of a vaccine for Hepatitis C virus (HCV) places children at a high risk of contracting the infection. It becomes necessary to accurately diagnose this infection for proper treatment as well as identifying potential risk factors for effective management.
This study was conceived to assess the test performance of the commonly used Immunochromatographic test (ICT) strip and identify the associated clinical manifestations and risk factors of HCV in children in Enugu Metropolis.
A cross-sectional study involving randomly selected 270 children below six years of age was conducted in Enugu Nigeria. The subjects were screened for anti-HCV by ICT and Enzyme-Linked Immunosorbent Assay (ELISA) and the demographic, signs and symptoms and risk factors were collected.
A total of 50 out of 270 children were positive for anti-HCV with a seropositivity of 18.5%. ICT strip had a very low sensitivity of 38.00% with an accuracy of 88.52% in detecting anti-HCV. The presence of dark urine was associated (p = 0.01) with HCV infection.
A seroprevalence of 18.5% of Anti-HCV was found in children below six years old in Enugu metropolis and the performance of ICT in diagnosing HCV infection was poor compared to ELISA.
Keywords: Hepatitis C virus, Immunochromatographic test strip, ELISA, seroprevalence, Children, risk factors.
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High Anti-HCV Seroprevalence and Low Performance of ICT Strip in Diagnosing Hepatitis C Virus Infection in Children in Enugu Metropolis
Hepatitis C virus (HCV) is a globally prevalent virus and a leading cause of morbidity and mortality which calls for public health concern [1Cooke GS, Lemoine M, Thursz M, et al. Viral hepatitis and the Global Burden of Disease: a need to regroup. J Viral Hepat 2013; 20(9): 600-1. [http://dx.doi.org/10.1111/jvh.12123] [PMID: 23910643] ]. Recently, the seroprevalence of the disease burden is estimated to be over 185 million infections worldwide as against 170 million infections in early 2000 [2Alter MJ. Prevention of spread of hepatitis C. Hepatology 2002; 36(5)(Suppl. 1): S93-8. [PMID: 12407581] ] with an increase of 2.8% in the last 15 years [3Mohd Hanafiah K, Groeger J, Flaxman AD, Wiersma ST. Global epidemiology of hepatitis C virus infection: New estimates of age-specific antibody to HCV seroprevalence. Hepatology 2013; 57(4): 1333-42. [http://dx.doi.org/10.1002/hep.26141] [PMID: 23172780] ]. Prolong HCV infection can lead to liver cirrhosis, liver failure, hepatocellular cancer, and possibly death [4Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med 2001; 345(1): 41-52. [http://dx.doi.org/10.1056/NEJM200107053450107] [PMID: 11439948] ]. Presently, HCV is the leading cause of death in HIV patients who are on highly active antiretroviral drugs [5Gill J, May M, Lewden C, et al. Causes of death in HIV-1-infected patients treated with antiretroviral therapy, 1996-2006: Collaborative analysis of 13 HIV cohort studies. Clin Infect Dis 2010; 50(10): 1387-96. [http://dx.doi.org/10.1086/652283] [PMID: 20380565] ].
HCV is transmitted through infected blood or blood-derived body fluids. Thus, the likely primary means of HCV transmission especially in developing countries include blood transfusion from unscreened donors, exposure of blood through the use of contaminated or inadequately sterilized medical instruments and needles for surgical and dental procedures, performing activities that cause skin breaks such as tattooing, ear and body piercing, the use of unsterilized objects for rituals and traditional medicine such as circumcision, scarification, face marks, acupuncture etc [2Alter MJ. Prevention of spread of hepatitis C. Hepatology 2002; 36(5)(Suppl. 1): S93-8. [PMID: 12407581] , 6 Report of a WHO Consultation Organization in Collaboration with the Viral Hepatitis Prevention Board, Antwerp, Belgium. 1999. Global surveillance and control of hepatitis C. J Viral Hepatitis 6(1): 35-47.].
HCV is highly genetically diverse with 7 main genotypes, of which each genotype has about 67 sub-types [7Simmonds P, Alberti A, Alter HJ, et al. A proposed system for the nomenclature of hepatitis C viral genotypes. Hepatology 1994; 19(5): 1321-4. [http://dx.doi.org/10.1002/hep.1840190538] [PMID: 8175159] , 8Smith DB, Bukh J, Kuiken C, et al. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: Updated criteria and genotype assignment web resource. Hepatology 2014; 59(1): 318-27. [http://dx.doi.org/10.1002/hep.26744] [PMID: 24115039] ]. With this high genetic diversity, the development of a vaccine as well as some pan-genotypic drugs has been a major challenge [9Timm J, Roggendorf M. Sequence diversity of hepatitis C virus: Implications for immune control and therapy. World J Gastroenterol 2007; 13(36): 4808-17. [http://dx.doi.org/10.3748/wjg.v13.i36.4808] [PMID: 17828811] ]. As such, there is no existing vaccine for HCV, although clinical trials for potential vaccines are ongoing [10Swadling L, Klenerman P, Barnes E. Ever closer to a prophylactic vaccine for HCV. Expert Opin Biol Ther 2013; 13(8): 1109-24. [http://dx.doi.org/10.1517/14712598.2013.791277] [PMID: 23651228] ]. The lack of a vaccine places children at a high risk of contracting HCV since at this stage in life, their immune system is not fully developed. More so, children are at high risk of prolong HCV infection to develop to chronic liver disease and other liver complications [11Liang TJ, Hepatitis B. Hepatitis B: the virus and disease. Hepatology 2009; 49(5)(Suppl.): S13-21. [http://dx.doi.org/10.1002/hep.22881] [PMID: 19399811] ]. With this vulnerability of children to HCV infection and no available vaccine, effective management of the disease will depend on routine screening of infected individuals for proper treatment. Also, identifying the possible means of transmission of the virus will help prevent contraction of the infection.
The most commonly used screening tool for HCV infection in Nigeria today is the Immunochromatographic test (ICT) strips because it is very rapid with results obtained in less than 10 minutes, relatively cheap and affordable, and doesn't require expertise in performing the test. Though developed by different diagnostic and pharmaceutical companies across the world, the mode of action of these ICTs is similar and is based on the common principle of antibody present in plasma or serum to migrate upward by capillary action and react with recombinant antigen present on the chromatographic membrane of the strip thereby generating a colour line in the test region. Usually, the manufacturers claim that these test strips have relatively high sensitivity, specificity and accuracy but querying performances have been reported [12Comănescu C, Aramă V, Grancea C, et al. The performance of a rapid test for anti-hcv screening in oral fluids. Roum Arch Microbiol Immunol 2015; 74(1-2): 40-5. [PMID: 26727853] ] as the very low level of the anti-HCV antibodies may not be detected by the strips. More so, in case of poor diagnosis, a presumptive diagnosis can aid clinicians to screen individuals for vital signs of clinical manifestations. Thus, it is necessary to routinely assess the performance of the commonly used ICT test for HCV screening and also, identify the associated signs and symptoms as well as potential risk factors for effective management of the disease.
2. MATERIALS AND METHODS
2.1. Study Area
This study was a hospital based cross-sectional study carried out at Enugu State University Teaching Hospital (ESUTH) and Favor Child Pediatrics Hospital in Enugu. Enugu is the capital of Enugu State located in South Eastern Nigeria with latitude and longitude coordinates of 6.459964 and 7.548949.
2.2. Study Population
The study population consisted of children with minor illnesses and fever who attended the hospitals. Subjects who met the criteria for inclusion which includes children less than 71 months old (less than six years) and whose parents and guardians provided a written informed consent were randomly selected and recruited for the study. Within the period of February to August 2017, a total of 270 children were recruited based on the calculated sample size using the formula: N = Z2pq/d2, where z = Normal standard deviation at 1.96 which corresponds to 95% confidence interval; p = Prevalence of Hepatitis C virus infection; q = 1-p; and d = degree of accuracy/ precision expected = 0.05.
2.3. Data Collection
After the recruitment, a structured interviewer-administered questionnaire on the predisposing factors to HCV infection was used to obtain participants data. The data collected include demographic, socioeconomic, background knowledge about HCV, its screening, associated signs and symptoms as well as risk factors and preventive measures.
2.4. Sample Collection and Serological Screening for Anti-HCV
After the participants were interviewed, three millilitres of blood was collected through venous puncture and transferred into sterile plain bottles. The blood was then centrifuged at 1500 rpm for 5 minutes to obtain the serum. The serum was aliquot and transported to the National Arbovirus and Vectors Research Center (NAVRC) Enugu for Enzyme-Linked Immunosorbent Assay (ELISA) analysis and later to the Microbiology laboratory of Godfrey Okoye University Enugu for ICT assay. Anti-HCV antibody was screened using anti-HCV ELISA test kits by Fortress Diagnostics Limited, United Kingdom and HCV one step Hepatitis C test ICT strip by Biogate Laboratories Ltd, Canada according to the manufacturer’s protocol.
2.5. Data Analysis
The seropositivity (%) of anti-HCV was calculated as (presence of anti-HCV / total population) x100. The specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of ICT test for HCV infection was calculated from true positive (TP), true negative (TN), false negative (FN) and false positive (FP). Sensitivity was calculated as TP/(TP + FN) x100, specificity as TN/(TN + FP) x100, NPV as TN/(TN + FN) x100, PPV as TP/(TP + FP) x100 and accuracy as ((TP+ TN)/sample size) x100.
2.6. Statistical Analysis
Data was analyzed using Statistical Package for Social Science (SPSS) version 16. Results were expressed in frequencies and proportion, and presented in tables. Pearson chi-square (χ2) test was used to determine the association between ICT and ELISA tests in diagnosis HCV infections. Also, χ2 was used to determine the association of various parameters including socio-demographic characteristics as well as signs and symptoms with HCV infections. The odd ratio (OR) was generated using a binomial logistic regression model to determine the independent contribution of risk factors to HCV infection. All statistical analysis employed a two-sided test and 95% confidence interval (CI) for p-value determination. A p-value ≤ 0.05 was considered statistically significant.
Among the 270 subjects, 50 were tested positive for anti-HCV by ELISA giving a seropositivity of 18.5%. The ELISA assay was more effective and reliable than the ICT strips in diagnosing anti-HCV as it detected 50 positive subjects as against 19 for ICT strips. As such ICT test had a low potential in detecting anti-HCV positive subjects with a sensitivity of 38.00% (19/50), but was very effective in screening subjects without HCV as all anti-HCV negative subjects were confirmed negative by ELISA, resulting to a specificity of 100%. More so, the PPV, NPV and accuracy of ICT test were 100%, 87.65% and 88.52%, respectively. The performances of the two tests are summarized in Table 1. As shown in Table 2, the seroprevalence of anti-HCV was insignificantly greater (χ2: 0.014; p = 0.514) in females (11.1%) than males (7.4%), and thus was not associated with gender. Also, though the prevalence of HCV infection was greatest (5.6%) in the age group of 48-59 months (4 years+), HCV was not associated (χ2: 2.099; p = 0.914) with age group. The presence of HCV infection in children was not associated with the patients/guardians level of education (χ2: 5. 246; p = 0.073) and marital status (χ2: 2.234; 0.525). Evaluating clinical signs and symptoms of subjects showed dark urine to associate significantly (p< 0.05) with HCV infection while no subject with jaundice was HCV positive. Moreover, fever, joint pain, itchy skin, loss of appetite, fatigue and abdominal pains did not show any significant association (p> 0.05) with the infection (Table 3). Potential risk factors of the parents or guardians of patients such as the presence of tattoo mark, alcohol intake, sharing of items, dental procedure and blood transfusion were observed to be associated significantly (p<0.05) with HCV infection with OR of 6.000, 1.898, 3.195, 2.184 and 2.984, respectively. Among these factors, the presence of a tattoo mark showed the greatest risk.
Table 1 Comparison between ICT and ELISA techniques in diagnosing HCV.
Table 2 Association of anti-HCV seroprevalence with some study characteristic of participants.
Table 3 Association of anti-HCV seroprevalence with signs and symptoms of participants.
Table 4 Binomial logistic regression analyses for potential risk factors of HCV infection based on ELISA assay.
HCV infection is a life-threatening viral infection and a leading cause of liver disease, thus, a major public health problem in the world [13Lee MH, Yang HI, Yuan Y, L’Italien G, Chen CJ. Epidemiology and natural history of hepatitis C virus infection. World J Gastroenterol 2014; 20(28): 9270-80. [PMID: 25071320] , 142017. WHO. WHO. Fact sheet. Hepatitis B & C, 2017. Accessed online April]. With no existing vaccine against HCV [15Swadling L, Klenerman P, Barnes E. Ever closer to a prophylactic vaccine for HCV. Expert Opin Biol Ther 2013; 13(8): 1109-24. [http://dx.doi.org/10.1517/14712598.2013.791277] [PMID: 23651228] ], young children remain the most vulnerable infectious group of the society. As such, effective diagnosis especially in young children is important for prompt treatment. ICT strips are commonly used for routine screening of HCV infected subject, thus, it was necessary to assess their performance. In this study, ICT strip showed a poor performance characterised by a low sensitivity of 38.0% in detecting HCV when compared to ELISA assay as the standard. Though this strip had a high specificity (100%) and accuracy (84. 52%), the low sensitivity suggests that some infected subjects may not be diagnosed by the strip. The fact that ICT strips are routinely used for screening HCV infection poses a major challenge as false negative subjects may harbour the infection for a long time which may develop to chronic liver disease or hepatocellular carcinoma [16Shepard CW, Finelli L, Alter MJ. Global epidemiology of hepatitis C virus infection. Lancet Infect Dis 2005; 5(9): 558-67. [http://dx.doi.org/10.1016/S1473-3099(05)70216-4] [PMID: 16122679] ]. ELISA has been considered very reliable in diagnosing HCV infection but its routine use may not be feasible due to its high cost and longer time of analysis compared to ICT strips. Thus, periodical assessment of the performance of ICT strips by health practitioners is necessary to identify the most reliable ICT strip for routine use. More so, ELISA should be used as a confirmatory test when indiscriminate result of ICT strips is found.
Based on ELISA assay, anti-HCV was found to have a seropositivity of 18.5% and was higher compared to other previous studies conducted in children in Nigeria [17Waje T, Dadah JA, Muhammad Y, Orukotan A, Ladan Z. Prevalence of Hepatitis C Infections Among the Outpatient Population of Selected Hospitals Within Kaduna City, Nigeria. Pub Hlth Int 2016; 1(1): 33-44.-19Prevalence of Hepatitis C virus among patients attending state specialist hospital Maiduguri, Nigeria. J Appl Sci Res 2014; 1(4): 274-8.]. This seroprevalence was far higher than the reported rate of 2.1%-2.8% in Sub-Saharan Africa and the highest rate of 2.8% in West African sub-region [20Layden JE, Phillips R, Opare-Sem O, et al. Hepatitis C in sub-saharan Africa: urgent need for attention. Open Forum Infect Dis 2014; 1(2): ofu065. [http://dx.doi.org/10.1093/ofid/ofu065] [PMID: 25734135] ]. Also, this prevalence is high compared to other findings across Africa such as 4.3% in Northern Ethiopia [21Ataklti HA, Tseheye AD, Rashmi B, Konjit G, Muthpandian S, Araya GW. Seroprevalence and associated Risk Factors for Hepatitis C Virus Infection among Voluntary Couseling Testing and anti Retroviral Treatment Clinic Attendants in Adwa Hospital, Northern Ethiopia. BMC Res Notes 2014; 9: 121.] and 1.8% in Ghana [22Lokpo SY, Osei-Yeboah J, Norgbe GK, et al. 2017. A 5 Year Retrospective Study at the Ho Municipal Hospital, Ghana. Hepatitis Res Treatment ; 2017: Article ID 6174743, 7 pages] but comparable to 16.0% in Rwanda [23Esperence U, Fabien N, John K, Naomi M. 2016. Prevalence of Hepatitis C Virus Infection and its Risk Factors among Patients Attending Rwanda Military Hospital, Rwanda ; Hindam BioMed Res Intern 5841272 ]. The high seroprevalence of this infection in children suggest that the transmission rate has increased and thus calls for public health concern. More so, the low seroprevalence of anti-HCV was observed in children below 23 months old while high seroprevalence was found in children between 48-71 months of age but there was no significant relationship between age and seroprevalence of anti-HCV (χ2 = 2.185, p > 0.05). This suggest that older children may be at a higher risk of contracting the infection probably because they are more active than the younger ones so they can play, move around and thus gets exposed to the infection easily.
Though more than half of individuals with early HCV infection are usually asymptomatic, chronic infection for over 30 days may tend to manifest symptoms [24Jaffray CE, Flint LM. Blood-borne viral diseases and the surgeon. Curr Probl Surg 2003; 40(4): 195-251. [http://dx.doi.org/10.1067/msg.2003.4] [PMID: 12624594] ]. Dark urine (p = 0.01) was found to be associated with HCV while fever, jaundice, itchy skin, joint pain, depression or weight loss, loss of appetite, fatigue and abdominal pain were not associated with the infection. Dark urine is a clinical manifestation commonly observed in individuals with chronic liver disease, thus it may be likely that some of the HCV infected individuals may develop liver disease.
With high seroprevalence of anti-HCV, is it believed that the rate of transmission may have increased thus it was necessary to identify the possible associated risk factors for transmission. Several risk factors such as multiple sex partners, practice of homosexuality, too much alcohol intake, intravenous drug use, presence of tattoo or tribal marks, sharing of personal items such as toothbrush, body piercing, history of any liver disease, blood transfusion, dental procedure and history of surgery etc. have been documented from previous studies to be associated with HCV infection [25Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. MMWR Recomm Rep 1998; 47(RR-19): 1-39. [PMID: 9790221] -27Hyams KC, Riddle J, Rubertone M, et al. Prevalence and incidence of hepatitis C virus infection in the US military: a seroepidemiologic survey of 21,000 troops. Am J Epidemiol 2001; 153(8): 764-70. [http://dx.doi.org/10.1093/aje/153.8.764] [PMID: 11296148] ] and thus could be a potential source of transmission from parents/guardians to children. Thus, our assessment of such risk factors in this study showed the presence of tattoo mark, shared items, alcohol intake, dental procedure and blood transfusion as potential risk factors of HCV infection. This finding concords with previous studies which have equally shown these factors as potential means of transmission of HCV infection [21Ataklti HA, Tseheye AD, Rashmi B, Konjit G, Muthpandian S, Araya GW. Seroprevalence and associated Risk Factors for Hepatitis C Virus Infection among Voluntary Couseling Testing and anti Retroviral Treatment Clinic Attendants in Adwa Hospital, Northern Ethiopia. BMC Res Notes 2014; 9: 121., 23Esperence U, Fabien N, John K, Naomi M. 2016. Prevalence of Hepatitis C Virus Infection and its Risk Factors among Patients Attending Rwanda Military Hospital, Rwanda ; Hindam BioMed Res Intern 5841272 , 28Chlabicz S, Grzeszczuk A, Prokopowicz D. Medical procedures and the risk of iatrogenic hepatitis C infection: case-controlled study in north-eastern Poland. J Hosp Infect 2004; 58(3): 204-9. [http://dx.doi.org/10.1016/j.jhin.2004.06.014] [PMID: 15501335] ].
This study showed a high anti-HCV seroprevalence of 18.5% in children below 6 years of age and this infection was associated with dark urine. Also, the presence of tattoo mark, sharing of items, alcohol intake, dental procedure and blood transfusion were found to be potential risk factors of transmission from parents/ guardians to children. ICT strip showed a poor performance in diagnosing HCV infection compared to ELISA assay with a sensitivity of 38%. The poor performance of ICT strip calls for general public health concern as false negative subjects are at risk of liver disease and eventually death due to prolong unidentified infection. Thus, a more effective diagnosis such as ELISA should be done for confirmation of inconclusive diagnosis by ICT strips especially when a subject is manifesting clinical signs and symptoms. Effective control measures should be undertaken by the society, health practitioners, stakeholder, public health advocates, etc. for the prevention of the risk factors and better management of HCV infection.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
The study design was reviewed and approved by the ethical committee of ESUTH Enugu, Nigeria with approval no: ESUTHP/C-MAC/RA/034/165.
HUMAN AND ANIMAL RIGHTS
No Animals were used in this research. All human research procedures followed were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2013.
CONSENT FOR PUBLICATION
Written informed consent was obtained from all parents or guardians willing to include their children in the study.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.
The authors are thankful to the medical students of ESUTH Enugu for their assistance in data collection and blood collection. Also, the authors are grateful to Mr. Emmanuel Ogudu of the National Arbovirus and Vectors Research Center (NARVC) Enugu for his assistance in conducting ELISA assay.
Alter MJ. Prevention of spread of hepatitis C. Hepatology 2002; 36(5)(Suppl. 1): S93-8. [PMID: 12407581]
Mohd Hanafiah K, Groeger J, Flaxman AD, Wiersma ST. Global epidemiology of hepatitis C virus infection: New estimates of age-specific antibody to HCV seroprevalence. Hepatology 2013; 57(4): 1333-42. [http://dx.doi.org/10.1002/hep.26141] [PMID: 23172780]
Gill J, May M, Lewden C, et al. Causes of death in HIV-1-infected patients treated with antiretroviral therapy, 1996-2006: Collaborative analysis of 13 HIV cohort studies. Clin Infect Dis 2010; 50(10): 1387-96. [http://dx.doi.org/10.1086/652283] [PMID: 20380565]
Report of a WHO Consultation Organization in Collaboration with the Viral Hepatitis Prevention Board, Antwerp, Belgium. 1999. Global surveillance and control of hepatitis C. J Viral Hepatitis 6(1): 35-47.
Smith DB, Bukh J, Kuiken C, et al. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: Updated criteria and genotype assignment web resource. Hepatology 2014; 59(1): 318-27. [http://dx.doi.org/10.1002/hep.26744] [PMID: 24115039]
Waje T, Dadah JA, Muhammad Y, Orukotan A, Ladan Z. Prevalence of Hepatitis C Infections Among the Outpatient Population of Selected Hospitals Within Kaduna City, Nigeria. Pub Hlth Int 2016; 1(1): 33-44.
Isa MA, Kolo BS, Ibrahim A, Bulakarima A, Dawud H. Prevalence of Hepatitis C Virus among Children Attending University of Maiduguri Teaching Hospital, Nigeria. Int J Sci Res Methodol Human 2015; 2(2): 14-21.
Prevalence of Hepatitis C virus among patients attending state specialist hospital Maiduguri, Nigeria. J Appl Sci Res 2014; 1(4): 274-8.
Ataklti HA, Tseheye AD, Rashmi B, Konjit G, Muthpandian S, Araya GW. Seroprevalence and associated Risk Factors for Hepatitis C Virus Infection among Voluntary Couseling Testing and anti Retroviral Treatment Clinic Attendants in Adwa Hospital, Northern Ethiopia. BMC Res Notes 2014; 9: 121.
Lokpo SY, Osei-Yeboah J, Norgbe GK, et al. 2017. A 5 Year Retrospective Study at the Ho Municipal Hospital, Ghana. Hepatitis Res Treatment ; 2017: Article ID 6174743, 7 pages
Esperence U, Fabien N, John K, Naomi M. 2016. Prevalence of Hepatitis C Virus Infection and its Risk Factors among Patients Attending Rwanda Military Hospital, Rwanda ; Hindam BioMed Res Intern 5841272
Hyams KC, Riddle J, Rubertone M, et al. Prevalence and incidence of hepatitis C virus infection in the US military: a seroepidemiologic survey of 21,000 troops. Am J Epidemiol 2001; 153(8): 764-70. [http://dx.doi.org/10.1093/aje/153.8.764] [PMID: 11296148]