Comparative evaluation of two rapid diagnostic test ... - JITMM 2017

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J I T M M Proceedings 6 (2017) 17-25

COMPARATIVE EVALUATION OF TWO RAPID DIAGNOSTIC TEST DEVICES AND REAL-TIME PCR FOR THE DETECTION OF HEPATITIS B SURFACE ANTIGENS IN HUMAN PLASMA: IMPLICATIONS IN BLOOD DONATION SCREENING Kazi Nadim Hasan 1*, Tanzila Wasi1, Nahian Anjum Shejuti1, Aniqa Afzal1, Sohidul Islam1 Department of Biochemistry & Microbiology, School of Health and Life Sciences, North South University, Plot#15, Block B, Bashundhara, Dhaka-1229, Bangladesh

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ABSTRACT

ackground and Aims: Hepatitis B Virus (HBV), a parenterally transmitted infectious virus, is one of the leading causes of hepatitis. Screening for HBV infection is recommended before vaccination and blood transfusion in order to prevent its transmission. Immunochromatography (ICT) based rapid HBsAg kits are popular for the screening of HBV infection as they are faster, cheaper and easier to perform than other methods. This study was aimed to evaluate two rapid HBsAg detection ICT kits for the screening of HBV infection by comparing them with the gold standard real-time polymerase chain reaction (real-time PCR) system that quantifies HBV-DNA.

Materials and Methods: The study comprised 203 samples, of which 103 had detectable and 100 had no detectable HBVDNA by real time PCR. These samples were tested with ICT kits from two companies: OnSite HBsAg Rapid Test (CTK Biotech, Inc., USA) and HBsAg Test Device (EXCEL®, USA). Results: Sensitivity of CTK and EXCEL were found to be 97.1% and 95.1% respectively. Negative predictive value (NPV) of CTK and EXCEL were 97.1% and 95.2% respectively. Both kits showed 100% specificity and 100% positive predictive value (PPV). HBV-DNA status strongly correlated with CTK (r = 0.971, p < 0.001) and with EXCEL (r = 0.952, p < 0.001). Three and five moderate to high HBV load containing samples were undetected by CTK and EXCEL respectively.

Conclusions: The study demonstrates that ICT based Rapid Test Devices might be considered suitable for screening of HBV infection as a point of care test. However, Nucleic Acid Testing for hepatitis B virus (NAT-HBV) should be implemented for donor screening for HBsAg.

Keywords: Hepatitis B virus, Real-time PCR, Rapid Test Devices INTRODUCTION Hepatitis B virus (HBV) infection has been recognized as one of the leading causes of chronic liver diseases including cirrhosis and *Correspondence: Kazi Nadim Hasan PhD Professor Department of Biochemistry & Microbiology School of Health and Life Sciences, North South University, Plot#15, Block B, Bashundhara, Dhaka-1229, Bangladesh Tel: 88-2-01747367550 E-mail: [email protected] Vol. 6 (2017)

hepatocellular carcinoma not only globally but also in Bangladesh (Lee, 1997; Al-Mahtab, 2016). Bangladesh has an intermediate prevalence of hepatitis B with a 4% to 5.4% HBsAg-positive population (Kane, 1994; Rumi et al, 2000; Mahtab et al, 2008). In order to confer high priority for the prevention of HBV transmission the Global Advisory Group to the World Health Organization (WHO) recommended that all countries integrate the hepatitis B vaccine into national immunization programs. In this regard, the hepatitis B vaccine had been introduced into national immunization 17

J I T M M PROCEEDINGS

programs in Bangladesh (Bangladesh MDG Progress Report, 2009). Bangladesh introduced the hepatitis B vaccine into the routine childhood vaccination schedule. (Health and Science Bulletin, icddr,b 2013). Screening of HBV infection by detecting hepatitis B virus surface antigen (HBsAg) in every pregnant woman has been practiced in Bangladesh to prevent perinantal transmission. In addition, in order to prevent horizontal transmission and transmission by donated blood, screening for HBsAg before vaccination and blood transfusion had also been practiced for many years in Bangladesh. About 350 million of more than 2 billion HBV infected individuals worldwide suffer from chronic infection, which is defined by having the presence of hepatitis B surface antigen (HBsAg) in the serum for more than six months (WHO, 2002; Zukerman, 1996). Chronic infection can eventually lead to cirrhosis, hepatic decompensation or hepatocellular carcinoma (Fattovich G et al 2008; Yang et al, 2011). As chronically infected individuals may remain undetected for many years, it increases the possibility of spreading disease transmission. HBV infection is spreading rapidly in developing countries due to lack of health education, illiteracy, poverty and a lack of awareness of the need for HBV vaccination (Khan et al, 2011). Therefore, it is important for apparently healthy individuals to undergo screening for HBV before blood donation and vaccination, ensuring prompt treatment and avoidance of transmission. Early and accurate detection of HBV infection using sensitive and specific methods allow investigators to evaluate the status of HBV infection and develop strategies to prevent transmission. Different methods are available for the differential diagnosis of HBV infection including Enzyme Immuno Assay (EIA), Realtime polymerase chain reaction (Real-time PCR), PCR and Immunochromatography (ICT) based Rapid Test Devices. Real-time PCR and PCR are the most sensitive and reliable method of choice to evaluate the status of HBV infection. EIA is considered the most sensitive and widely used 18

method for the screening of hepatitis B surface antigen (HBsAg) in serum (Yue-Ping et al, 2015; Ghosh et al, 2015). Both PCR and EIA methods are expensive and are used in well-equipped major tertiary care hospitals, reference centers or central blood banks. ICT based Rapid Test Devices have been developed for the qualitative detection of HBsAg in human serum with expected sensitivity and specificity similar to EIA as a gold standard. These tests are intended for field survey diagnosis, emergencies, home testing as well as blood screening before transfusion. Rapid HBV test devices have become very popular and are used widely as they are less expensive and do not need highly skilled manpower or well-equipped infrastructure. In Bangladesh, they are found useful in resource limited settings and as point of care tests, making them widely popular for the screening of HBV infection. In addition, they are found to be especially convenient for testing blood donors during blood donation drives or blood transfusions, which do not allow time for time consuming tests (Hayder et al, 2012; Chameera et al, 2013; Mutocheluh et al, 2014; Mbanya, 2013; Chevaliez et al, 2008). The risk of transfusion-transmitted HBV infection was dramatically reduced after the development of ICT based HBsAg tests to screen blood donations in the late 1960s and 1970s (Schreiber et al, 1996). Bangladesh has also observed dramatic reductions in the prevalence of HBV infection due to implementation of screening of HBsAg using Rapid Test Devices as point of care tests and on blood donations. However, it has been found by many studies that HBV transmission by blood can occur from asymptomatic donors who have not yet developed HBsAg (i.e., WP) or from chronic cases where serological markers are not detected while having HBV-DNA (occult hepatitis B) ( Allain, 2004; Allain, 2004; Brojer et al, 2006; Candotti et al, 2008; Chevrier et al, 2007; Yoshikawa et al, 2007). Therefore, HBsAg screening by Rapid Test Devices might impose an obstacle in gaining complete success in preventing HBV transmission by blood donations. Comparison of the sensitivity and specificity of Vol. 6 (2017)


Rapid Test Devices and EIA for detecting HBsAg have been carried out by many researchers (Lau et al, 2003; Whang and Um, 2005; Sato et al, 1996), however, no comparative study was carried out using quantitative PCR method as a gold standard in Bangladesh. This study aimed to evaluate two widely used HBsAg rapid test devices by comparing them with Real-time PCR measured HBV-DNA as the gold standard. In this way, the study intended to see whether HBsAg Rapid Test Devices could provide an effective method in the screening of blood donors who might have HBV – DNA. MATERIALS AND METHODS The study comprised 203 samples, of which 103 were chronic HBV infected and 100 were young healthy individuals. The study was conducted maintaining the ethical guidelines of the 1975 Declaration of Helsinki. Informed consent was taken from each of the individuals from whom samples were obtained. About 5 ml of blood was drawn from individuals and collected in a tube containing EDTA. Plasma were separated after 15 minutes at room temperature by bench centrifuge and stored at -400°C until further analyses. Real-time PCR: Quantification of HBVDNA was performed in all chronic HBV positive individuals (n = 103) using Real-time PCR kits (RoboGene® HBV DNA Quantification Kit, CE – IVD, Analytic Jena AG, Jena, Germany) on a Smart Cycler II platform (Cepheid™ SmartCycler™ Real-Time Thermal Cycler, USA) according to the manufacturers’ instructions. HBsAg EIA: All healthy individuals were tested for HBsAg using fourth generation ELISA kit (Murex HBsAg, 4th generation, Murex Biotech Limited, UK). HBsAg ICT: All studied samples were tested for the detection of HBsAg using two ICT based Rapid Test Devices: OnSite HBsAg Rapid Test (CTK Biotech, Inc., USA) and HBsAg Test Device (EXCEL®, USA). A few drops of plasma were used for this test. The presence of HBsAg was Vol. 6 (2017)

determined by two red colored lines appearing on the antibody pre-coated region in the kit: the test region “T” and the control region “C”. The negative results were determined by the appearance of red line only the “C” region. Each test took about 10-15 minutes to perform. Statistical analysis: Accuracy, sensitivity, specificity, positive and negative predictive value (PPV, NPV), and Pearson’s correlation (r) value were calculated by comparison with Real-time PCR results as the gold standard (Irwig et al, 2002). RESULTS Results obtained from Rapid Test Devices were compared with the results from Real-time PCR and EIA. It was found that among 103 Real-time PCR reactive individuals, 3 were found non-reactive by the CTK device and 5 by the EXCEL device. 100 Real-time PCR/EIA non-reactive individuals were also all found non-reactive by both devices (Table 1). True positive and true negative values were evaluated for detecting HBsAg in 203 individuals using CTK and EXCEL rapid devices by comparing with real-time PCR as gold standard with sensitivity and specificity of 100%. It was found that CTK had higher true positives than EXCEL. True negative results were same in both devices (Table 2). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Pearson’s Correlation (r) values of two devices used in the study were calculated with their 95% confidence intervals (CI) in comparison to the Real-time PCR method as gold standard. CTK HBsAg rapid device was found to have higher sensitivity and NPV than EXCEL. Both the HBsAg rapid devices have the same specificity and PPV. Gold standard Real-time PCR results correlated significantly with both CTK and EXCEL, however, a stronger correlation was found with CTK ( HBV-DNA vs CTK-ICT: r = 0.97, p < 0.001; HBV-DNA vs EXCEL-ICT: r = 0.95, p < 0.001) ( Table 3). 19


Table 1 Comparison of Rapid HBsAg kit with Real-time PCR Real-time PCR (Gold Standard)/EIA Rapid HBsAg Devices

Reactive

Non-Reactive

(n = 103)

(n = 100)

Total

CTK Reactive Non-Reactive

100 03

0 100

100 103

EXCEL Reactive Non-Reactive

98 05

0 100

98 105

Table 2 True Positive and True Negative values for CTK and EXCEL compared to Real-time PCR Parameters

True Positive

True Negative

Total No. of samples

CTK

100 (97.1%)

100 (100%)

203

EXCEL

98 (95.1%)

100 (100%)

203

PCR

103 (100%)

100 (100%)

203

Table 3 Evaluation of CTK and EXCEL results compared to real-time PCR Parameter Sensitivity (%)

CTK

95% CI

EXCEL

95% CI

97.1

(93.8 – 100)

95.1

(91.0 – 99.3)

Specificity (%)

100

(100 – 100)

100

(100 – 100)

PPV (%)

100

(100 – 100)

100

(100 – 100)

NPV (%)

97.1

(93.8 – 100)

95.2

(91.2 – 99.3)

Pearson correlation (r) (%)

97.1

(96.2 – 97.8)

95.2

(93.7 – 96.3)

Although a significant correlation was observed between rapid test devices used in the study with the Real-time PCR results, five moderate to high HBV-DNA containing samples were found undetected by EXCEL and three by CTK HBsAg device. Moderate to high HBV-DNA containing sample results by real-time PCR that were found negative by EXCEL and CTK are shown in Figure 20

1a. Samples with curves above the FAM threshold cycle (Ct) signified positive results and those below signified negative results. Five samples with identification numbers (ID) 2, 48, 61, 86 and 91were found to have HBV-DNA levels of 26336 IU/ml, 119713 IU/ml, 89880 IU/ml, 3287 IU/ml and 2630852 IU/ml respectively. Quantification of positive samples was obtained by comparing Vol. 6 (2017)


them with quantitation standards (QS) with known concentrations used during Real-time PCR run. Four QS were run during each batch of sample quantification in the Real-time PCR. HBV-DNA concentrations for QS1 were 50000000 IU/ml, QS2 500000 IU/ml, QS3 12500 IU/ml and QS4

500 IU/ml. Among five samples, sample IDs 2, 48, 61, 86 and 91 could not be detected by EXCEL and 2, 86, 91 could not be detected by CTK. Positive results for the HBsAg ICT kits were achieved with two colored lines and negative results were achieved with one colored line (Figure 1b). 1a

1b ID

FAM Ct

HBV-DNA IU/ml

EXCEL HBsAg

CTK HBsAg

240 2

30.89

26336

48

28.55

119713

61

29.0

89880

86

34.0

3287

91

23.8

2630852

QS 1

19.19

50000000

QS 2

26.23

500000

QS3

32.4

12500

QS4

36.81

500

TC

CT

Fig. 1- 1a: Quantitative Real-time PCR results of five samples with Quantitation Standards (QS). 1b: HBV-DNA concentrations of five samples. Negative HBsAg results (one red line) of Rapid Test Device experiments.

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DISCUSSION Rapid and accurate detection is essential for preventing the transmission and treatment decision of HBV infection. Therefore it is necessary to evaluate the assays used to detect HBV infection before vaccination and blood transfusion. Several methods of HBV detection exist for the diagnosis of acute or chronic hepatitis B and the assessment of prognosis. Such methods may use immunological, molecular and histological assays including ELISA, real-time PCR and liver biopsy respectively (Chevaliez and Pawlotsky, 2008). Rapid detection ICT kits can be ideal alternatives for diagnosis. However, a major concern in using these kits is their variable degrees of sensitivity and specificity. Therefore it is important to evaluate these kits against highly accurate testing methods such as real-time PCR and ELISA before choosing them for routine use. Immunochromatography based rapid assays used for HBsAg detection may not have the same accuracy indices in every region due to the differences in the prevalence of HBV infection in a given population. The prevalent subtype of HBV infecting a population can be different. Some ICT kits may not detect certain subtypes of HBV giving a false negative result (Chameera et al, 2013; Scheiblauer et al, 2010). Rapid ICT kits giving more false positive results are better for diagnosis than those that give more false negative results (Ansari et al, 2007). A positive result is usually followed by more accurate testing methods like real-time PCR to confirm the presence of infection. A negative test result is seldom retested, considering the costs of retesting in resource limiting settings. Hence, choosing a test with high sensitivity and NPV is more important than choosing a test with high specificity and PPV for routine use. ICT results should be interpreted carefully, because some positive samples containing a low reactivity by quantitative real-time PCR can produce weak color in the ICT kit, which may be misinterpreted as a negative result (Ansari et al, 2007). In some cases, even higher viral loads of 22

HBV-DNA positive samples remain undetected, as seen in this study. Several studies done in various countries previously have shown different ICT kits to have high sensitivity and specificity. In a study reported in India, 3 different HBsAg ICT kits: Hepacard, Crystal and SD BIOLINE, were evaluated, all of them showing 100% sensitivity and 100% specificity (Maity et al, 2012). In another study in Iran, 6 different ICT kits: Acon, Atlas, Intec, Blue Cross, DIMA and Cortex, showed sensitivity between 97.5-99.2% and specificity between 97.599.2% compared to quantitative real-time PCR as gold standard testing method (Ansari et al, 2007). A study in Pakistan used 3 different ICT kits: Acon, Intec and Determine, showed sensitivity of 95-98% and specificity of 100% compared to ELISA as gold standard (Hayder et al, 2012). On the other hand, some evaluation studies have noted ICT kits as having high specificity but low sensitivity. One in Sri Lanka reported the evaluation of two ICT kits: OnSite and Cortex, using ELISA as gold standard, with sensitivity of 80% and 60% respectively. The specificity was 100% for both (Chameera et al, 2013). In Pakistan, two ICT kits: One Check and Accurate, using ELISA as gold standard, and found sensitivity to be 53% and 50% respectively. The specificity was 100% and 95% respectively (Khan et al, 2011). In Ghana, evaluation of 3 ICT kits: Abon Kit, Accutell, Wondfo, showed sensitivity of 50-59.1% and specificity of 99.2-99.3% compared to ELISA as gold standard (Mutocheluh et al, 2014). This study was performed using real-time PCR HBV-DNA as the gold standard instead of ELISA. This is because ELISA only represents exposure to HBV by detecting HBsAg, whereas real-time PCR represents infection status by detecting HBVDNA, making real-time PCR a more accurate and superior method of HBV detection. For a better evaluation of the two ICT kits, a higher sample size could be used in the study. ICT for HBsAg detection must be validated by real-time PCR before being used in resource limited settings. It is also recommended to validate them by evaluating their effectiveness in the Vol. 6 (2017)


detection of different genotypes and subtypes of HBV that are known to have infected a given population before initiating routine use (Chameera et al, 2013). Bangladesh has been trying to increase awareness of hepatitis B and implement vaccination among the general population. To prevent transmission of HBV infection further, the sensitivity of any tests performed to detect HBV infection before vaccination and blood transfusion must be 100%. In this study 3 positive samples were undetected by CTK and 5 positive samples remained undetected by EXCEL. Therefore, it is important to use nucleic acid based rapid diagnostic kits to detect HBV infection with 100% sensitivity and specificity. Implementation of NAT-HBV is especially useful to detect HBV infection during the seronegative window period of early acute phase and the occult HBV infection of chronic stage, as serological tests cannot detect HBsAg during these stages (Hans and Marwaha, 2014; Seo et al, 2015). With increased detection of HBV infection by NAT-HBV before blood donation, the transmission risk of HBV can be completely reduced. In conclusion, the study demonstrates that ICT based Raid Test Devices are suitable to be used for the screening of Hepatitis B infection as a point of care test. However, in order to prevent HBV transmission from “occult” HBV infection in some donors, rapid Nucleic Acid Testing for hepatitis B virus (NAT-HBV) should be implemented for blood screening. ACKNOWLEDGEMENTS We gratefully acknowledge the assistance and support from Dr. Sujauddin Ahmed and other staff of North South University Medical Center during collection of blood samples from participants. We are grateful to students of the University who gave their consent to participate in this study. We are greatly indebted to the Conference Travel and Research Grant Committee (CTRG), North South University for providing funds (Faculty Research Grant 2013 – 2014) to carry out this study. Vol. 6 (2017)

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