|Year : 2020 | Volume
| Issue : 2 | Page : 139-145
Evaluation of a NATSpert test developed for low-resource countries for enhancing blood safety
Minal Dakhave Bhosale, Shefali Desai
R and D Department, Mylab Discovery Solutions Pvt. Ltd., Pune, India
|Date of Submission||25-Feb-2020|
|Date of Decision||07-Aug-2020|
|Date of Acceptance||14-Oct-2020|
|Date of Web Publication||13-Nov-2020|
Minal Dakhave Bhosale
R and D Department, Mylab Discovery Solutions Pvt. Ltd., Pune
Source of Support: None, Conflict of Interest: None
Background: NATSpert ID TripleH Detection test is a qualitative multiplex real time PCR test for simultaneous screening of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1/2 in donated blood. Objective of the study was to evaluate NATSpert ID TripleH Detection test, developed indigenously for low resource countries for enhancing blood safety without compromising on blood safety in terms of sensitivity and specificity. Methods: The NATSpert ID TripleH Detection test consists of viral nucleic acid extraction using magnetic beads and multiplex real-time PCR based on hydrolysis probe technology for amplification and detection of the viral target. Quality controls are included for validity of the test. The performance of the test was evaluated for analytical sensitivity and specificity and precision using the World Health Organization international standards. Results: Analytical sensitivity of NATSpert ID TripleH Detection test is 2 IU/ml for HBV, 7 IU/ml for HCV, human immunodeficiency virus-1(HIV-1) M – 17.5 IU/ml, HIV-1O – 18.5 copies/ml, and HIV-2 8.17IU/ml using 1 ml sample input at 95% CI. The NATSpert ID TripleH Detection test detected all three viruses and their genotypes with high repeatability and reproducibility in validation studies. Conclusion: The NATSpert ID TripleH Detection test developed as a cost-effective solution was demonstrated to have the capability to identify either individually or simultaneously the presence of viral targets – HBV, HCV, and HIV in the donated blood. The NATSpert ID Triple H Detection test serves as a highly sensitive, specific, and accurate test for screening of donated blood for transfusion-transmitted viral infections.
Keywords: Blood safety, individual-donation-NAT, real-time polymerase chain reaction
|How to cite this article:|
Bhosale MD, Desai S. Evaluation of a NATSpert test developed for low-resource countries for enhancing blood safety. Glob J Transfus Med 2020;5:139-45
|How to cite this URL:|
Bhosale MD, Desai S. Evaluation of a NATSpert test developed for low-resource countries for enhancing blood safety. Glob J Transfus Med [serial online] 2020 [cited 2021 Jun 15];5:139-45. Available from: https://www.gjtmonline.com/text.asp?2020/5/2/139/300617
| Introduction|| |
Blood and its components are transfused to save innumerable lives, but the quality and safety of the transfused blood and its products is a major public health concern due to transfusion transmitted infections (TTIs). With every unit of blood, there is a 1% chance of transfusion-associated problems, including TTI., Risk of TTI is high in the patients receiving multiple transfusions and undergoing invasive procedures with exposure of circulating system., Safety against three TTI, i.e., human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV), and hepatitis C virus (HCV) is a worldwide concern, in spite of development of highly sensitive and specific serological tests and other surrogate markers because of the time gap between the time of acquiring infection and development of seropositivity (window period [WP]). To address this, various types of Nucleic Acid Test (NAT) were developed in the 1990s. NAT in blood donor screening detects not only WP donors but also those with chronic occult infections which are negative by routine serological screening. It is based on direct amplification and detection of viral nucleic acids, rather than antibody production by the immune system of the infected person. This allows for earlier detection of infection and further decreases the possibility of TTI.
Even in India, with its large population of around 1.23 billion and high prevalence rate of HIV (0.29%), HBV (2%–8%), and HCV (~2%), blood safety is a big challenge. At present, all blood banks follow the National Aids Control Organization guidelines and use the fourth generation, advanced antigen-antibody combined enzyme-linked immunosorbent test (ELISA) to screen blood for HIV, HBV, and HCV. NAT donors' screening is not mandatory in India as of now, and a review published in 2017 stated that NAT screening is carried out in only 2% blood banks and covers only 7% of all collected blood units in India in spite of the fact that the seroprevalence of infections in India is much higher than in the developed world. Major barriers of implementing routine NAT in India are its high cost and lack of technical expertise in most of the blood centers. NATSpert ID TripleH test is our earnest attempt to provide solution to some key issues to facilitate availability of high-quality NAT test at lower cost compared to existing commercially available tests.
NAT is a highly sensitive and advanced technique which has reduced the WP of HBV to 10.34 days, HCV to 1.34 days, and HIV to 2.93 days, but the implementation of NAT in low-resource countries remains a challenge. While the World Health Organization (WHO) recommends diagnostic devices should be affordable, sensitive, and specific, in resource-limited countries, NAT remains highly technically demanding, incurs high costs, and requires dedicated infrastructure facility, equipment, consumables, and technical expertise.
NATSpert TripleH Detection test offers a statistically significant advantage over serology in ability to detect TTI in blood donors. The NAT yield of 1:3829 was in line with other Indian studies.
A sensitive and specific multiplex NATSpert test is described below for the simultaneous detection and discrimination of HBV DNA, HCV RNA, and HIV RNA in individual blood donor samples, blood products, and suspected individuals. The NATSpert test provides viral nucleic acid extraction using magnetic bead technology. The extracted viral nucleic acid is amplified using multiplex real-time polymerase chain reaction (PCR) technology for detection and discrimination of HIV RNA (HIV-1 Groups M, N, and O RNA and HIV-2 RNA), HCV RNA (Genotype 1–6), and HBV DNA (Genotype A–H) using real-time PCR system. The test does not discriminate between HIV-1 Group M, HIV-1 Group O, and HIV-2. The test also incorporates a heterologous Internal Control (IC) to be processed during extraction and real-time PCR for monitoring test performance for each individual sample.
| Materials and Methods|| |
Sensitivity panels for HBV (WHO International Standard 3rd WHO International Standard for HBV for Nucleic Acid Amplification Techniques NIBSC code: 10/264), HCV (WHO International Standard 5th WHO International Standard for HCV NAT NIBSC code: 14/150), HIV-1 (WHO International Standard 3rd HIV-1 International Standard NIBSC code: 10/152), HIV-1 O (Internal calibrators), and HIV-2 (WHO International Standard HIV-2 RNA International Standard NIBSC Code: 08/150) were prepared by diluting standards with certified negative plasma.
Nucleic acid isolation from plasma samples
Nucleic acid isolation procedure was developed using paramagnetic bead technology and was performed on semi-automated extraction machine.
The lysis buffer, wash buffer 1 and 2, and elution buffer used in this method are proprietary formulations of Mylab. They have a unique formulation that increases the catalytic activities of the enzymes and reduces the enzyme unit requirement, resulting in reduced cost.
Buffer component contains 2065 μl lysis buffer combo (including lysis buffer, isopropanol, proteinase K, and magnetic beads along with IC and carrier RNA), 1200 μl wash buffer 1, 1800 μl wash buffer two twice, and 90 μl elution buffer,. 1 ml K2 EDTA plasma sample was added in lysis buffer combo. General steps include incubation of lysis at 56°C for 20 min and washing using wash 1 and 2 buffers, followed by elution at 80°C.
Internal Control (IC) was added to the lysis buffer before extraction as a control to determine the efficacy of extraction and PCR amplification and absence of inhibitory compounds. A noncompetitive and synthetic IC was chosen to avoid competition with the primer/probe set for nucleic acid detection for HIV, HBV, and HCV virus and therefore does not compromise of test sensitivity.
Real-time polymerase chain reaction preparation and amplification/detection
The test was based on hydrolysis probe chemistry for the amplification of conserved regions of HIV, HCV, and HBV and IC using specific primers/probes. The primer/probe combinations were indigenously designed (proprietary formulations, Mylab) to cover all known genotypes and were tested “in silico” as well. The amplified product was detected by the generation of fluorescent signals from target-specific fluorescent probes. Four unique fluorescent dyes were used for HIV, HCV, HBV, and IC target, thus allowing independent identification of all three viruses. All three HIV targets (HIV-M/N, HIV-1O, and HIV-2) were identified using the same dye and were not discriminated from each other. Reverse transcription (RT) and Taq polymerase enzymes in PCR buffer composition have unique formulation to enhance the catalytic activities, thus allowing reduced use of the enzymes resulting in reduced cost.
22 μl of extracted viral nucleic acid/positive control was added to 28 μl of master mix constituting 12.5 μl of RT and Taq polymerase in PCR buffer components, 7.8 μl of primer probe mix for all viral targets along with IC, and 7.7 μl of nuclease-free water. Testing is carried out by multiplex real-time RT-PCR using QuantStudio 5 (Applied Biosystem) real-time PCR machine. The thermal cycling conditions were 50°C, 15 min for reverse transcriptase, and 95°C, 20 s to inactivate the RT enzyme, followed by 50 cycles of 5 sec at 95°C and 30 sec at 60°C.
This complete protocol was also performed with closed fully automated Compact system with a sample to result instrument for NAT testing, using sealed prefilled reagent cartridges.
Performance study on clinical samples
Type of the study
This was a prospective cohort study of blood donors at a Blood Bank in Central India, which is a NABH-accredited standalone blood bank to determine the NAT yield and also determine the sensitivity and specificity of the NATSpert assay.
The ethical clearance for the study was taken from the board of the trust that runs the blood bank, and blood donors were informed, and consent was taken.
All the serologically nonreactive blood donor samples during the study period of June 2017-March 2019 and 77 randomly selected and blinded serologically reactive blood donor samples, totaling to 30,635, were screened on the NATSpert ID TripleH assay.
The study was analyzed using Fisher exact test to find any statistically significant difference in ability to detect TTI using NAT as compared to EIA.
Data analysis and reporting
After completion of the real time PCR run, Ct values were analyzed for positive controls and clinical samples using Quantstudio™ Design and analysis software (V 1.3.1, Thermo Fisher Scientific, Singapore, 2017), built into the Quantstudio™ 5 Real time PCR machine (Thermo Fisher Scientific, Singapore). Results were interpreted as reactive, non- reactive and valid results.
The validation was performed according to the recommended guidelines for validating molecular tests for Infectious Diseases and European guidelines.,, The evaluation of test was done for analytical sensitivity, analytical specificity, precision, and clinical performance.
The analytical sensitivity of the NATSpert ID TripleH Detection test was determined by testing five independent serial dilution series of the 3rd HIV-1 WHO International Standard, WHO International Standard HIV-2 RNA, 5th WHO International Standard for HCV NAT NIBSC, and 3rd WHO International Standard for HBV for Nucleic Acid Amplification Techniques and internal calibrators for HIV-1 O. Dilutions were prepared in HIV/HBV/HCV-negative EDTA plasma and ranged from 107 to 1 IU/ml. Each dilution series was tested in triplicate on different days with 1 ml plasma input volume for extraction. Simultaneous target amplification at low levels of vial loads was assessed using HBV, HCV, and HIV Multiplex 14/198-XXX NIBSC code: 14/198-XXX.
Further, the conversion factor for IU/copies was estimated by comparing the known copies/ml dilution series versus known IU/ml NIBSC standards. Expected versus observed ratio was calculated for the conversion factor.
The probit analysis module of SPSS (Statistics V 19, IBM, US) was used to calculate the limit of detection (LOD). The percent coefficient of variation (% CV) is defined as “(standard deviation/mean) × 100” and used for precision analysis.
The genotype/subtype inclusivity of all relevant genotypes was ensured by testing the following international reference standards: HIV-1 2nd WHO NIBSC Genotype panel # 12/224:041016, 3rd HCV genotype NIBSC WHO Panel (PN 12/172), and 1st HBV WHO NIBSC Genotype panel PE5086/08 V2.
Cross-reactivity for each viral target was determined against blood-borne viruses, bacteria, and fungi.
Accuracy and precision
The accuracy and precision of the NATSpert test were obtained by the analysis of NIBSC standards using two dilutions. The test was performed in replicates in independent runs, between two different operators and using three different lots. The data also were used to determine the intratest and intertest precision. For intratest analysis, 10 replicates of each dilution were tested, while for intertest analysis, three replicates of each dilution in five independent runs on different days were performed. Accuracy was defined as the difference between the measured log concentration and the log nominal concentration.
| Results|| |
The data were generated using semi-automated and automated compact system and showed similar results.
The LOD of the NATSpert ID TripleH detection test for each viral target was evaluated using sensitivity panels described in Materials and Methods. The final LOD was determined by probit analysis module of SPSS. Overall, the LOD (with 95% CI) of the NATSpert test was 2 IU/ml for HBV, 7 IU/ml for HCV, 17.5 IU/ml for HIV-1M, 18.5 copies/ml for HIV-1O, and 8.17 IU/mL for HIV-2 [Table 1]. Conversion factors calculated for HIV-1, HCV, and HBV are 1.57, 1.13 and 4.53 copies/IU, respectively [Figure 1], [Figure 2], [Figure 3].
Test performance for simultaneous target detection
The ability of NATSpert test to simultaneously detect HBV DNA, HCV, and HIV-1 RNA in the same sample was evaluated using the NIBSC multiplex standard. Studies have attributed the nominal unitage is of <50 IU/ml HBV, <50 IU/ml HCV, and <200 IU/ml HIV (package insert). The NIBSC standard was run in triplicate in three different runs and specific amplification for each virus was observed. The results are summarized in [Table 2].
Genotype/subtype inclusivity of all relevant genotypes was tested using international reference standards with 100% detection rate. Known HBV clinical samples were tested to verify the inclusivity of HBV Genotype H and pre-core mutant [Table 3], [Table 4], [Table 5].
Accuracy and precision
Coefficient of Variation (CV) was determined for intratest and intertest variation with < 10%.
The results are summarized in [Table 6].
Performance study on clinical samples
Our study showed that 85 blood donor samples of 30,635 units were reactive on NAT screening, whereas only 77 of these were reactive on serological screening. Significantly, there were eight reactive results on NAT screening that were nonreactive on serologic testing (NAT yield of 1: 3829), of which four were HCV reactive and two each were HBV and HIV reactive [Table 8].
|Table 8: Total number of seroreactive and individual donor multiplex nucleic acid amplification test reactive samples|
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On statistical analysis using Fisher's exact test, the NATSpert ID TripleH assay was found to offer a statistically significant advantage over EIA in ability to detect TTI in blood donors (P < 0.05, Fisher's exact test) [Table 9].
| Discussion|| |
Although advanced testing techniques are now becoming available and are being adopted at many centers, the risk of contracting transfusion-transmitted infections after transfusion of blood or components still persists.
Reports from developed countries have shown the limited value of NAT blood screening in improving blood safety. The Scottish BTS reported a NAT yield rate for HIV and HCV of 1 per 1.9 and 0.77 million donations. Reports on NAT yield of screening 3.6 million blood donations from continental Europe for HBV, HCV, and HIV-1 were 1 per 0.6 million donations for HBV, HCV and 1 per 1.9 million for HIV-1, This is primarily due to the low prevalence of HIV-1, HBV, and HCV in these countries. In contrast to this, the prevalence of these viral infections in resource-limited countries is generally high. Most of the reports of NAT screening in these countries showed NAT yield as high as 1:60 blood donation for HBV and 1/3100 blood donations for HCV. Also of importance in the consideration of NAT blood screening in resource-limited areas is to assessing infectivity for HCV and HBV., India is in the intermediate zone of HBV endemicity, with HBsAg prevalence among the general population ranging from 2% to 8%.,
In India and around the world, fully automated multiplex nucleic acid amplification technology (NAT) enabling ID or minipool of 6 (MP6) screening for HIV, HBV, and HCV is provided by two major commercial players (Grifols and Roche). Grifols offers three triplex NAT assays, ProcleixUltrio, ProcleixUltrio Plus, and ProcleixUltrio Elite and Roche offers Cobas TaqScreen MPX and Cobas TaqScreen MPX v2.0.
Griffols assays are based on transcription-mediated amplification (TMA), whereas Roche assays are based on PCR/RT-PCR technology.,,,,, These assays involve three main steps: (a) specimen preparation, (b) amplification, and (c) detection.
Before the commercialization of NAT testing kits used for blood screening, it is very important to determine whether the manufactured kits meet the prescribed safety and effectiveness standards. Analytical sensitivity, specificity, precision, reproducibility, and repeatability are the most important parameters of these standards.
The analytical sensitivity determines the ability of a test method to differentiate between two very close concentration of any analyte and the LOD. The 95% LOD of Ultrio Elite Assay, Cobas MPX V 2.0 assay and NATSpert ID test are mentioned in [Table 7]. Taken together, it can be said that all three tests have comparable 95% LOD.
The analytical specificity determines the ability of the test to detect all the genetic variants and inability to detect closely related or any other nonrelated analyte. The results of study aimed at determining the ability to detect HIV 1, HIV 2, HCV, and HBV genetic variants showed that NATSpert ID test detected all major genotypes of HIV, HBV, and HCV in genotype coverage, for HIV-1, genotype M, N, and O, for HBV, genotype A–H, and for HCV, genotype 1–6 were detected. Furthermore, it had no cross-reactivity with any of the nonreactive viral and nonviral agents tested. These results were similar to Grifols and Roche tests., NATSpert ID showed acceptable repeatability and reproducibility for the results when tested for different lots, days, and operators.
In the first multicentric study, a total of 12,224 samples along with their serological results were obtained from eight blood banks in India and were tested individually manually by Procleix Ultrio test for HIV 1, HCV, and HBV. The study observed eight NAT yield cases. According to a study from the western part of India, combined NAT yield (NAT reactive/seronegative) for HIV, HCV, and HBV was 0.034% (1 in 2972 donations) which is high when compared to studies from developed countries. In another study conducted in North India, 18,354 donors were tested by both ID-NAT and fourth-generation ELISA, 7 were found to be NAT-positive but ELISA-negative (NAT yield) for HBV and HCV. The prevalence of NAT yield cases among routine donors was 1 in 2622 donations tested (0.038%) In our own study, during the study period of 2017–2019 at external evaluation site, all 70 seropositive specimens detected positive, while of 30,558 prescreened seronegative specimens, the NAT yield found was 2 each for HBV and HIV and 4 of HCV.
Commercially available NAT tests have their own limitations. TMA-based test involves two step of detection and does not discriminate between HIV, HCV, and HBV. It requires a supplementary discriminatory viral test for the detection of HIV RNA, HCV RNA, and HBV DNA. Thus, the turnaround time is longer. In case of MP-NAT test, although it is based on multi dye RT PCR technique, the sensitivity is compromised due to pooled samples and requires the second test to confirm positivity by ID testing, thus increasing the reporting time.
In contrast, NATSpert ID testing utilizes an aliquot of each sample tested individually, with no dilution due to addition of noninfectious sample. It provides faster results than MP-NAT since it does not require balanced sample pooling and resolution of positive samples. Some recent data from Southeast Asia highlight this advantage. With occult HBV infection frequency of 1:40,000, ID-NAT detected HBV-DNA in blood samples at levels of <100 IU/ml. If the same samples were tested in mini pools of 6 with an analytical sensitivity of 5 IU/ml, 70.7% of samples would have been not detected. Recent reports also show that in case of HBV, apart from initial WP, a second WP exists at 75–85 days post infection, when HBsAg is no longer detectable and anti-HBsAg not yet detectable, but HBV NAT is positive during this period. The ID-NAT test is important in patients who receive multiple blood transfusions for diseases such as thalassaemia and hemophilia. Such patients require repeated lifelong blood transfusion and are at higher risk of being infected with serious TTIs.
Taken together, we present a NATSpert ID testing solution that gives highly sensitive and specific real-time PCR technology for testing of individual donors, blood products, or suspected individuals where multiple analytes can be detected and identified in a single test, thus improving workflow turnaround time and sensitivity compared to the two currently available commercial kits. Furthermore, Mylab's proprietary buffer formulations offer a significant cost advantage.
| Conclusion|| |
The NATSpert ID TripleH test demonstrated excellent sensitivity and specificity in viral target detection and discrimination. It is sensitive, specific, user-friendly, rapid, robust, and affordable. The test is suitable to screen specimen in individual ID format and furthermore, pliable for automation. The performance of NATSpert ID TripleH is expected to be of great significance to low-resource countries, as it resolves most of the hurdles currently faced by them in screening blood donors for HIV, HBV, and HCV, given the prevalence of HIV, HBV, and HCV in these countries.
The authors would like to thank Ms. Aditi Kavimandan and Reshma Naiknaware for technical assistance and Mr. Ranjit Desai and Ms. Guari Metkar for the execution of experiments.
Financial support and sponsorship
Conflicts of interest
The authors of this study are full time employees of Mylabs Discovery Solutions Pvt Ltd.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]