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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 39  |  Issue : 4  |  Page : 217-221

Detection of hepatitis C virus antibody in the saliva of children and adolescents with b-thalassemia


1 Department of Pediatrics, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Date of Submission04-Dec-2014
Date of Acceptance06-Dec-2014
Date of Web Publication25-Mar-2015

Correspondence Address:
Jonair H Labib
Department of Pediatrics, Thalassemia Center, Ain Shams University, Cairo 11566
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-1067.153962

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  Abstract 

Introduction Children with b-thalassemia (BTM) are at risk of acquiring hepatitis C virus (HCV) infections because of their need for multiple blood transfusions. The aim of this study was to evaluate the new rapid antibody test (OraQuick) for detection of HCV antibody in the saliva of BTM major and intermedia patients with known and unknown anti-HCV antibody status.
Patients and methods The present study included 150 children and adolescents suffering from BTM enrolled from the Hematology and Oncology Clinic of the Children's Hospital of Ain Shams University. A full medical history, clinical examination, salivary swab detection of anti-HCV in saliva, blood sampling for complete blood count, and liver enzyme assessment were performed. Anti-HCV testing by enzyme-linked immunosorbent assay and HCV RNA testing by PCR were carried out in those studied patients who had not undergone such tests earlier.
Results There was significant correlation between HCV status and the total number of blood transfusions. A significant fall in the incidence of HCV infection after 2004 was proven. The sensitivity and specificity of both, the OraQuick test and the enzyme-linked immunosorbent assay test, were the same (97.8 and 100%, respectively).
Conclusion The OraQuick HCV Rapid Antibody Test using saliva proved to have a high sensitivity and specificity. Therefore, it could be considered a noninvasive, easy, rapid, and reliable test for screening of patients suspected to have HCV infection.

Keywords: Anti-hepatitis C virus in saliva; b-thalassemia; hepatitis C


How to cite this article:
El-Sayed MH, Labib JH, Abdel-Hamid DH, Helaly HR. Detection of hepatitis C virus antibody in the saliva of children and adolescents with b-thalassemia. Egypt J Haematol 2014;39:217-21

How to cite this URL:
El-Sayed MH, Labib JH, Abdel-Hamid DH, Helaly HR. Detection of hepatitis C virus antibody in the saliva of children and adolescents with b-thalassemia. Egypt J Haematol [serial online] 2014 [cited 2019 Nov 13];39:217-21. Available from: http://www.ehj.eg.net/text.asp?2014/39/4/217/153962


  Introduction Top


b-Thalassemia (BTM) patients are at high risk for acquiring hepatitis C virus (HCV) infection because of the frequent transfusions they receive [1],[2]. Their longstanding liver iron overload, if superimposed with HCV infection, makes them susceptible to liver cirrhosis and/or hepatocellular carcinoma [3]. Therefore, early diagnosis of HCV infection among them is significantly important. The OraQuick HCV Rapid Antibody Test was approved by the US Food and Drug Administration to be used with blood in 2010. The test can also detect anti-HCV antibodies in salivary fluid [4]. Our study aimed to evaluate its sensitivity and specificity in the saliva of BTM patients with known and unknown anti-HCV status.


  Patients and methods Top


Patients

The present study was a cross-sectional study that included 150 children and adolescents suffering from BTM major and intermedia. Patients were recruited from the Pediatric Hematology and Oncology Clinic of Ain Shams University (Cairo, Egypt). Eligibility criteria included diagnosis of BTM major or intermedia, age 1-19 years, resident of Cairo, and undergoing regular follow-up at the clinic. Patients with both known and unknown HCV RNA status or anti-HCV status were eligible to participate in the study. The exclusion criteria were end-stage organ failure, hemodynamic compromise, and major psychological disturbances. The study complied with the World Medical Association Declaration of Helsinki of 1975, as revised in 2000, regarding ethical conduct of research involving human participants. The Pediatric Department Board Committee approved the study protocol in September 2011. Patients were enrolled during the period from October 2011 to December 2012. Eligible patients and/or their guardians were asked for their consent, which was obtained after being fully informed about the study's aims and procedures. The study recruiter interviewed the mothers or guardians of eligible children below the age of 10 years, and the patients themselves beyond that age. The recorded patient data included age, sex, diagnosis of the original disease, symptoms related to hepatitis illness, including jaundice, pallor, easy fatigability, edema and hematemesis, blood and blood product transfusion frequency, age at onset of transfusion, total number of transfusions, complications of transfusion, surgical interventions, and HCV status if known to the patients and/or their guardians. The study team carried out baseline clinical examination of the enrolled patients for signs of liver disease, such as jaundice, hepatomegaly, splenomegaly and ascites. In addition, salivary swabs, blood samples for complete blood count, liver enzyme assessment, anti-HCV tests, and HCV RNA tests were carried out. The enzyme-linked immunosorbent assay (ELISA) and PCR tests were conducted for patients who had not undergone them before the study (n = 106). PCR testing for HCV RNA was used as the reference test for the newly developed OraQuick test.

Blood sampling was performed under complete aseptic conditions in a specially prepared room in the clinic. Samples were collected and transferred in appropriate containers to the Ain Shams University Central Laboratory, where they were properly analyzed. Testing for serum HCV antibody was carried out by ELISA (third generation ELISA) using a Murex Anti-HCV Kit (ABBOTT, Innogenetics, Murex, Monolisa version 4.0; Australia) [5]. The test detected antibodies directed against NS3, NS4, NS5, and core regions of the HCV genome using synthetic peptides. Result analysis was carried out according to the manufacturer's instructions, and was available after 1 week.

Detection of serum HCV RNA was performed by real-time reverse transcriptase PCR (RT-PCR) using Stratagene Mx3000p Real Time Thermocycler (Corbett Research, Sydney, Australia). Synthetic positive-strand and negative-strand HCV RNAs were transcribed in vitro and purified extensively to remove DNA. RNA was diluted in normal cell RNA such that all samples contained 1 mg of cell RNA. RT-PCR was performed for both negative-strand and positive-strand RNA [6]. PCR test results were declared after 2 weeks. Appropriate healthcare referrals were provided for those who were newly diagnosed HCV-infected patients.

Salivary swab collection

The OraQuick HCV Rapid Antibody Test swab was manufactured by OraSure Technologies Inc. (Bethlehem, Pennsylvania, USA). Specific precautions were followed to ensure accuracy of the results. The study team made sure that the tested patient did not eat, drink, or chew gum for at least 15 min before testing. Patients were asked to wait for at least 30 min before testing if they had used any oral care products. The OraQuick test swab was removed from the pouch without touching the Flat Pad. The lower and upper outer gums were swabbed completely all around (only one time) without swabbing the roof of the mouth, cheeks, or tongue. The test device was inserted into the developer solution vial and left for 20-40 min, after which test results were available and disclosed to the patients in the same setting. A nonreactive test result meant that HCV antibodies were not detected in the specimen. A reactive test result meant that HCV antibodies had been detected in the specimen [7].

Statistical analysis

All data were entered and analyzed using statistical package for the social sciences (SPSS, version 11; SPSS Inc., Chicago, Illinois, USA) using double data entry to ensure quality control. Quantitative variables were described as mean, SD, and range. Qualitative variables were represented by number and percentage. Thec2 -test was applied to compare qualitative variables in different groups. The unpaired t-test was used to compare quantitative variables in parametric data. The Mann-Whitney test was used instead of the unpaired t-test for nonparametric data. Spearman's correlation test was used to rank positive and negative variables versus each other. The P level of significance was considered at less than 0.05.


  Results Top


The study included 150 index cases; 45 of them had HCV infection (30% of studied cases). More than half of thalassemia patients were female, as shown in [Table 1].
Table 1: Demographic and clinical data of the studied patients

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The majority of patients with thalassemia underwent their first blood transfusion before 2005. Nearly one-quarter of patients suffered from various complications of blood transfusion, such as fever, skin rash, and loin pain. Patients who suffered from these complications had higher incidence of HCV infection. Nearly half of the patients with transfusion complications had HCV infection (40%), whereas approximately one-fourth of the rest had HCV infection. The decreased incidence of HCV infection with absence of transfusion complications was statistically significant [Table 2].
Table 2: The relation of hepatitis C virus infection to different risk factors

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There was a significant correlation between HCV-positive status and the total number of blood transfusions. The earliest date of the first blood transfusion in our patient cohort was in the year 1995. Among patients who received their first blood transfusion before 2004, more than one-third (38.1%) had HCV infection, whereas among patients receiving blood transfusion after 2004 less than one-sixth (15.1%) had HCV infection. Thus, there was a significant fall in the incidence of HCV infection in the latter group of patients.

Previous surgical interventions seemed to have a significant association with HCV infection. Nearly half (44.8%) of the patients who had undergone surgical interventions in the past suffered from HCV infection, compared with less than one-tenth (9.5%) of patients who did not undergo any surgical intervention.

A comparison of the results of OraQuick, ELISA, and PCR [Table 3] showed one false-negative result of HCV infection on OraQuick test and one false-negative result on ELISA in two different patients. Both tests had 97.8% sensitivity, 100% specificity, 100% positive predictive value, and 99.1% negative predictive value.
Table 3: The relation of OraQuick and enzyme-linked immunosorbent assay results to the polymerase chain reaction results

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  Discussion Top


Viral hepatitis is arguably the most significant public health problem facing Egypt today. Infection with HCV occurs after exposure to infected blood or other body fluids containing blood [8]. Children with BTM are at risk of acquiring several viral infections by virtue of their need for multiple blood transfusions. Of these viral infections, HCV infection is particularly important [9].

The need for a rapid, reliable, and noninvasive test for screening of HCV infection in those patients is of supreme importance. Hence, the aim of this study was to evaluate the new rapid antibody test (OraQuick) for detection of HCV antibody in the saliva of BTM patients with known and unknown anti-HCV antibody status.

Among the studied 150 BTM cases, nearly one-third were infected with HCV (n = 45, 30%). Mollah et al. [10] conducted a study to assess the prevalence of transfusion-transmitted infections among 152 thalassemic children in Bangladesh. The percentage of HCV-infected patients was lower than ours (12.5%). This can be attributed to the higher prevalence of HCV infection in the Egyptian population [10].

Omar et al. [11] documented that the prevalence of HCV infection in 174 Egyptian BTM patients followed up in the Cairo University Pediatric Hematology Department was higher than that at our center (51.7%). Meanwhile, their study, unlike ours, revealed a nonsignificant correlation between the frequency of transfusion and HCV infection [11]. We found that HCV-positive thalassemia patients had a significantly higher number of blood transfusions compared with those with an HCV-negative status.

Studies in Indian multitransfused thalassemic patients reported that prevalence of anti-HCV antibodies varied from 19 to 68% [12],[13]. An Iranian study conducted by Ghaderi and Agahi [14] found the prevalence of HCV infection in BTM major patients to be 68.5%. Similar studies in different parts of Iran have indicated a high rate of HCV infection in thalassemia patients, from 19.3 to 55.9% [14]. The main reason for this wide range of HCV infection rates could be attributed to the type of blood screening modality, the sensitivity of the tests performed, or the generation of laboratory techniques used for detection of HCV infection [15].

Our study showed that there was a significant decline in the percentage of HCV-infected patients who started to receive blood transfusion after 2004 in relation to those who started before that year (from over half of patients to less than one-sixth contracted HCV infection after 2004). This may reflect the improved accuracy of blood-donor screening programs and the use of more sensitive generations of ELISA and recombinant immunoblot assay in blood banks. Another Egyptian study found a significant reduction in the prevalence of HCV in Egyptian thalassemic patients followed up in Cairo University from 1995 until 2010. The percentage of HCV-infected patients reduced from 71% in 1995 to 51.7% in 2010. The decline in incidence was attributed to the strict application of blood-donor screening protocols for viral antibodies [11].

The presence of transfusion complications was not related to the HCV status of the patients, but surgical interventions had a significant association with positive HCV status. The percentage of HCV-infected patients who underwent previous surgical interventions was significantly higher than those who did not. This reflected the considerable risk of transmission of HCV particles during various surgical interventions, which is in accordance with the observation of other authors [16],[17],[18].

A comparison of the results of OraQuick, ELISA, and PCR tests for the detection of HCV infection revealed the same specificity, sensitivity, and positive and negative predictive values for both the OraQuick and ELISA tests. Drobnik et al. [19], who conducted a study among 503 high-risk adult patients in New York City (USA), stated that the results of OraQuick matched PCR results in six of the seven instances in which the rapid test and the ELISA were discordant [19]. The study by Lee et al. [7], which included 2206 adult patients at risk for HCV or with signs and/or symptoms of hepatitis, found similar results to ours. In that study, the OraQuick test sensitivity of oral fluid was 98.1% and the positive and negative predictive values were 99.3 and of 99.0%, respectively [7].

Other tests to detect HCV antibodies in saliva have been introduced and evaluated. Yaari et al. [20] reported in 2006 that saliva testing for anti-HCV antibodies in hemodialysis patients using the 'ImmunoComb II kit' yielded a sensitivity of 100% and a specificity that was 'similar or better' when compared with serum testing. However, in the ImmunoComb II test the concentration of saliva specimens had to be increased and the incubation period had to be prolonged to overnight, at low temperature (4°C) [21],[22]. Thus, OraQuick remained more rapid and easier to perform, which makes OraQuick an ideal screening test. Rapid testing offers the chance for people who may not otherwise be tested to learn their HCV status without being exposed to the inconveniences associated with phlebotomy and retiming to the test site to receive results. In addition, at-risk and infected individuals can be aware of their HCV status, an issue that may greatly affect their lives [19],[23],[24]. This in turn may reduce the future morbidity and the associated healthcare burden caused by progression of liver disease in individuals with unidentified HCV infection [19]. The use of rapid HCV tests in centers with limited financial resources will allow for improvements in practice. Testing oral fluid rather than blood also eliminates the risk of staff exposure to blood-borne pathogens. However the OraQuick test will not change the need for anti-HCV-positive individuals to receive PCR testing and medical care [19],[25],[26].

In conclusion, the OraQuick rapid antibody test for detection of salivary HCV antibodies is shown to be an easy-to-use, high-sensitivity, point-of-care test that may increase testing opportunities and enable identification of more asymptomatic HCV-infected patients who could benefit from early therapeutic intervention.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
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