Abstract
The diagnosis of Dengue and Chikungunya infections during acute phase is a priority considering emerging pattern and increasing trends of their infections. The present study describes the commercial development and validation of RT-PCR test for the simultaneous detection of of DEN and CHIK viral RNA in a single tube from human plasma samples. Multistep one step RT-PCR assay was developed and validated for detection and discrimination of DEN and CHIK along with exogenous internal control. The test was evaluated for commercial use using 3 different lots to determine analytical sensitivity, specificity, precision and stability. The external clinical evaluation was performed at NABL accredited lab with known positive and negative Chikungunya and Dengue specimens and comparator assay method. The findings showed that the test could identify CHIK and DEN viral nucleic acid in clinical samples within 80 min, without any cross-reactivity. The analytical detection limit of the test was 1.56 copies/µl for both. The clinical sensitivity and specificity was ≥ 98% and provide a high-throughput and screen up to 90 samples in a single run. It is available in a freeze-dried format and can be used in both the manual and automated platforms. This unique combo test, PathoDetect™ “CHIK DEN Multiplex PCR Kit” enables simultaneous, sensitive, specific detection of DENV and CHIKV and serves as “ready to use” platform for commercial use. It would aid the differential diagnosis as early as day 1 of the infection and facilitate screen-and-treat approach.
Keywords: Molecular detection, Multiplex, RT-PCR, CHIKV, DENV
Introduction
Dengue virus (DENV) and Chikungunya virus (CHIKV) are widely distributed in tropical regions of the world, with billions of people living at the risk of infection [1]. Dengue virus (DENV) and Chikungunya virus (CHIKV), the single-stranded positive-sense RNA viruses belonging to the genus Flavivirus and Togavirus, resp., share the same Aedes mosquito vectors [2]. DENV is known to affect the immune system and can progress to dengue hemorrhagic fever and dengue shock syndrome while CHIKV can cause arthralgia, which may persist for long period [3]. It is reported that there has been a shift in the viral genotypes due to mutations in the CHIKV, and has spread to new areas, posing a real threat to temperate areas, such as Europe and the Americas [4]. In the early phase, it can be difficult to distinguish between these two diseases, as neither virus presents any distinctive clinical features, posing a challenge for diagnosis [5].
CHIKV and DENV diagnosis can be approached via several methods such as ELISA, rapid test, virus isolation, antibody screening (IgM, IgG) and conventional RT-PCR. Virus isolation from infected tissue and blood specimens of the infected patients’ need minimum 7 days and BSL-2 laboratory [6]. Diagnosis by immunoglobulin IgM/IgG ELISA assays is reported to have extremely low sensitivity in majority of the CHIKV patients and also has a concern of a cross-reaction with other alphaviruses [7]. Moreover, the patients could have multiple and sequential co-infections with DENV and CHIKV due to the lack of cross-protective neutralizing antibodies against these viruses [8]. Thus, molecular tests are considered to be the most sensitive diagnostics for DENV or CHIKV in the acute settings [9, 10].
Diagnostic testing varies according to the viral type, cost, and time for the detection. Most of the developed tests are not suitable for resource limited settings although DENV or CHIKV are distributed mostly in countries with limited resources [11]. False positive and/or negative results further restrain the implementation of control measures. In addition, information on virus infections is important that may contribute to the surveillance database and assist with molecular epidemiology research, as well as monitoring and aid in control of virus dissemination. A number of molecular tests have been published for the detection of DENV or CHIKV, but the majority of these assays need to be carried out separately [12–15] which adds up to the cost and prolongs turn-around time. Thus, the specific and rapid diagnosis of these viral infection is critical, to halt their progression to atypical complications and facilitate treatment. Early viral detection can provide important information for epidemiological studies of different outbreaks.
The present study describes a single-tube in vitro nucleic acid amplification test, for the simultaneous detection of all genotypes of DENV and CHIKV RNA in human plasma samples. The test was commercialized to aid cost-effective, accessible and rapid testing of DENV or CHIKV.
Description of the test
The present study describes an in vitro nucleic acid amplification test for the detection and discrimination of all genotypes of DENV and CHIKV RNA in human plasma samples. The test uses one step real time reverse transcription polymerase chain reaction (RT-PCR) with hydrolysis probe chemistry that uses the 5´nuclease activity of Taq DNA polymerase and enables the detection of a specific PCR product as it accumulates during PCR cycles. The assay includes validated primers and hydrolysis probe along with the PCR reagents for specific detection of all the genotypes of Chikungunya and dengue in a short time. The test was developed in such as way that it is compatible with manual process and automated platform.
Nucleic acid isolation
Manual method requires liquid handling, nucleic acid extraction, and amplification reagent preparation areas followed by PCR amplification on real time PCR instrument. In manual testing, silica based column was used (Maverick nucleic acid extraction kit, Cat No: EC00001A/B/C) for nucleic acid extraction followed by RT-PCR on QuantStudio 5 machine. Similarly, this diagnostic test was performed with Compact XL automated system with sample to result analysis using sealed prefilled reagent cartridges/plates. Magnetic bead based extraction was carried out (Maverick Nucleic Acid Extraction Kit, EM00001M/L) followed by RT-PCR on Compact Q (Mylab Discovery Solutions, Pvt. Ltd). The PCR formulation was optimized in such a way that it gives similar performance on various available RTPCR platforms. The above systems are compatible with 200 µl plasma or serum sample as a starting material.
The Maverick extraction compositions used in this method are proprietary formulations of Mylab Discovery Solutions. The buffer component contains 850 µl lysis buffer combo (including lysis buffer, isopropanol, proteinase K, and magnetic beads along with IC and carrier RNA), 700 µl wash buffer 1, 900 µl wash buffer two twice, and 90 µl elution buffer. 200 µl K2 EDTA plasma sample was added in lysis buffer combo. General steps include incubation in lysis buffer at 56 °C for 20 min on thermoshaker, followed by washing and elusion. Quantitative RT-PCR is recommended for determination of viral RNA yield.
Internal control
To determine the efficacy of extraction and PCR amplification, internal control was added to the lysis buffer before extraction as a control. A non-competitive and synthetic exogenous IC was chosen to avoid competition with the primer/probe set for nucleic acid detection for CHIK and DEN virus and therefore does not compromise of test sensitivity.
PCR amplification and Detection
All available genomic sequences of CHIK and DEN virus were downloaded from GenBank, and aligned using MEGA-X 10.2.6 (http://www.megasoftware.net/) to find the conserved and variable region. Centre for Disease Control (CDC) and World Health Organization (WHO) guidelines were followed for choosing primers and probes. Based on the alignment, confirmatory genes were selected for primer and probe design. The primer/probe combinations were selected to cover all known genotypes. All sequences were verified for specificity and cross-reactivity “in silico” in a multiplex PCR reaction using Multiple Primer Analyzer (Multiple Primer Analyzer | Thermo Fisher Scientific—IN).
Optimization of the RT-PCR Test
The RT-PCR test was based on hydrolysis probe chemistry for the amplification of conserved regions of CHIK and DEN virus and IC using specific primers/probes. These are usually linked to oligonucleotide probes which bind specifically to the amplified product. The probe for each target gene was tagged with different fluorescent probes such as FAM (Green channel, Ex 493-Em517 nm), VIC/HEX (Yellow Channel, Ex 533-Em559 nm), Cy5 (Orange Channel, Ex 651- Em 670 nm). The FAM, VIC/HEX, Cy5, dyes define DENV, CHIKV targets and Internal control respectively. Monitoring the fluorescence intensities during the PCR run allows the detection and quantitation of the accumulating product in real time and does not require laborious post PCR methods. The PCR buffer was composed of reverse transcriptase and Taq polymerase enzymes that have unique formulation in order to enhance their catalytic activities, minimize use resulting in reduced cost. 8 µl of extracted viral nucleic acid/positive control was added to 17 µl of master mix constituting 6.25 µl of RT and Taq polymerase in PCR buffer components, 3 µl of primer probe mix (upto 10 pmol) for all viral targets along with IC, and 7.75 µl of nuclease-free water.
The testing was 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 45 cycles of 5 s at 95 °C and 30 s at 60 °C with data collection on.
Methodology
The performance of the assay was determined by evaluating analytical sensitivity, specificity, accuracy, reproducibility and stability [16]. Three batches were prepared for validation and the test was performed in three replicates each in three independent runs, between three different operators and using 3 different lots. The diagnostic evaluation was performed by testing known positive and negative plasma samples.
Analytical sensitivity/Limit of detection
Chikungunya and Dengue in vitro RNA transcript spiked in known negative plasma samples were used for determining analytical sensitivity. A total of 7 concentration levels, with two fold serial dilutions of sample were prepared. LOD studies were performed to determine the lowest detectable concentration of target nucleic acids that can be detected with a positivity rate of ≥ 95%. The LOD of the test was determined by limiting dilution studies of in vitro RNA transcripts of chikungunya and dengue spiked in known negative plasma samples from 1 × 105 copies/µl to 10 copies/µl. A preliminary run was carried out to predetermine LOD. In order to confirm the LOD, serial two fold dilutions of total 7 concentrations of 100 copies/µl to 1.56 copies/µl spanning the predetermined LOD were tested with a total of 20 replicates per concentration in 5 independent runs on QuantStudio 5 (Themo Fischer Scientific) and Compact Q (Mylab Discovery Solutions, Pvt. Ltd). Positive control and no template control were tested in each run for validity of the test. LOD was also verified with clinical samples.
Cross reactivity (analytical specificity)
A total of 22 normal and pathogenic microorganisms were tested using in silico analysis and wet analysis. Chikungunya and Dengue in vitro transcripts of moderate concentration were spiked in negative plasma. Cross reactivity of the PCR test was evaluated by testing the chikungunya and dengue contrived sample with moderately higher concentration of most common pathogens. To estimate the specificity of the test, in silico analysis using the Basic Local Alignment Search Tool (BLAST) managed by the National Center for Biotechnology Information (NCBI) was used to assess the designed primer probe for their inclusivity of all serotypes of chikungunya and Dengue virus (DENV 1–4).
Accuracy
Precision
The known chikungunya and Dengue positive sample with low, moderate and high concentration along with negative sample were used. For precision, three known positive samples for both chikungunya and dengue virus with low (10 copies/µl), moderate (103 copies/µl) and high concentration (105 copies/µl) along with known negative sample were tested.
Trueness
For trueness, 15 known Chikungunya positive samples, 15 known Dengue samples with varying concentration and 30 known negative samples were tested using comparator method. For intra-assay 10 replicates of three known Chikungunya and Dengue positive specimen with low, medium and high concentration along with one negative specimen and positive control were tested in a single run. For inter-assay three known CHIKV and DENV positive specimen with low, medium and high concentration along with one negative specimen and positive control were tested using three different lots and by two different operators. Clinical performance was also verified by enrolling in QCMD Programme (QCMD 2022 Chikungunya Virus EQA Programme—QAV154175_1).
Stability data
The shelf life of the test was determined by real time and accelerated aging technique using three different batches. The real time stability was performed at recommended storage temperature of -20 °C while the accelerated stability was performed at two elevated temperatures at 4 °C and 25 °C for a certain period of time. The prediction of the shelf life, safe storing conditions and quality, was based on application of the rule of Q10 in accordance with the requirements of CLSI EP25-A Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline (2010).
The in-use stability was carried out using on board and open vial (freeze/thaw) stability studies according to the CLSI EP05-A2 guideline on three different batches. For open vial study, the test components were stored at − 20 °C and subjected to 5 freeze thaw cycles (temperature drift of − 20 °C to room temperature) with a duration of 24 h between each cycles. For On board Stability, the final reaction mix prepared after addition of the specimen was stored at room temperature at various time points before loading on to the real time PCR instrument.
Clinical evaluation
Internal validation was carried out using Quality Control for Molecular Diagnostics (QCMD) samples on 50 Chikungunya positive samples, 20 known Dengue Positive samples and 30 known negative samples. Known Chikungunya (n = 15) and Dengue positive (n = 15) and negative specimens (n = 30) were tested with PathoDetect™ CHIK-DEN Multiplex PCR Kit and abTES™ DEN/CHIKU/ZIKA 3 qPCR I Kit in two independent runs. External evaluation was carried out at NABL accredited laboratory, Metropolis Healthcare Ltd, Mumbai. The external evaluation at ICMR or NABL accredited laboratories is mandatory for the CDSCO approval of new in vitro diagnostic. As Metropolis Healthcare Ltd. lab has NABL scope for chikungunya and dengue testing with sufficient no. of positive and negative samples with certified reports, we have chosen this lab. We have signed a memorandum of understanding (MOU) with this lab for confidentiality.
Results
Sensitivity
Analytical sensitivity/Limit of Detection (LOD) of the test was found to be 1.56 copies/µl at 95% confidence interval for both Chikungunya and Dengue targets (Tables 1, 2). Linearity is shown in Fig. 1.
Table 1.
Linearity for Chikungunya and Dengue targets
| Preliminary Data For Chikungunya and Dengue Targets | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Copies/µl | Log | Chikungunya target | Dengue target | ||||||
| Replicate 1 | Replicate 2 | Replicate 3 | Mean CT |
Replicate 1 | Replicate 2 | Replicate 3 | Mean CT |
||
| 100,000 | 5.0 | 22.888 | 23.253 | 23.328 | 23.16 | 19.368 | 20.423 | 19.119 | 19.64 |
| 10,000 | 4.0 | 26.068 | 26.550 | 26.276 | 26.30 | 23.464 | 23.282 | 23.160 | 23.30 |
| 1000 | 3.0 | 29.592 | 29.869 | 29.912 | 29.79 | 26.695 | 25.840 | 26.386 | 26.31 |
| 100 | 2.0 | 32.651 | 33.165 | 33.245 | 33.02 | 29.406 | 29.027 | 28.924 | 29.12 |
| 10 | 1.0 | 36.728 | 36.386 | 36.873 | 36.66 | 32.668 | 33.053 | 32.483 | 32.73 |
| 1 | 0.0 | 39.034 | 39.630 | 39.083 | 39.25 | 36.396 | 36.855 | 36.250 | 36.50 |
Table 2.
Limit of detection for Chikungunya and Dengue target
| Quantity copies/µl | Chikungunya target | Dengue target | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Mean CT compiled | SD | %CV | Points tested | Hits | Hit rate | Mean CT compiled | SD | %CV | Points tested | Hits | Hit rate | |
| 100 | 32.19 | 0.18 | 0.57 | 20 | 20 | 100 | 29.42 | 0.55 | 1.88 | 20 | 20 | 100 |
| 50 | 33.16 | 0.42 | 1.26 | 20 | 20 | 100 | 30.77 | 1.16 | 3.77 | 20 | 20 | 100 |
| 25 | 34.26 | 0.46 | 1.35 | 20 | 20 | 100 | 31.74 | 0.71 | 2.25 | 20 | 20 | 100 |
| 12.5 | 34.94 | 0.69 | 1.98 | 20 | 20 | 100 | 32.81 | 1.46 | 4.46 | 20 | 20 | 100 |
| 6.25 | 35.75 | 0.92 | 2.57 | 20 | 20 | 100 | 33.55 | 1.24 | 3.70 | 20 | 20 | 100 |
| 3.125 | 36.47 | 0.90 | 2.46 | 20 | 20 | 100 | 34.79 | 1.46 | 4.19 | 20 | 20 | 100 |
| 1.56 | 37.51 | 0.84 | 2.24 | 20 | 19 | 95 | 35.51 | 1.28 | 3.60 | 20 | 19 | 95 |
Fig. 1.
Linearity for Chikungunya and Dengue targets
Cross-reactivity
The result of the in silico and wet analysis suggested that the test was specific for the detection of all the serotypes of Chikungunya and Dengue virus with no expected cross reactivity to the human genome, other pathogenic viruses or human micro flora. A potential cross-reactivity was tested using the control group listed in Table 3.
Table 3.
Testing the specificity of the Test with potentially cross-reactive pathogens
| Control group | Laboratory testing | In silico analysis |
|---|---|---|
| done | ||
| Human immunodeficiency virus 1 (HIV-1) | ✓ | ✓ |
| Human immunodeficiency virus 2 (HIV-2) | ✓ | ✓ |
| Hepatitis C virus (HCV) | ✓ | ✓ |
| Hepatitis B virus (HBV) | ✓ | ✓ |
| Cytomegalovirus (CMV) | ✓ | ✓ |
| Epstein-Barr virus (EBV) | ✓ | ✓ |
| Hepatitis A virus (HAV) | ✓ | ✓ |
| Hepatitis D virus (HDV) | ✓ | ✓ |
| Hepatitis E virus (HEV) | ✓ | ✓ |
| Malaria | ✓ | ✓ |
| Herpes simplex virus 1 (HSV-1) | ✓ | ✓ |
| Herpes simplex virus 2 (HSV-2) | ✓ | ✓ |
| Human T-lymphotropic virus I (HTLV-I) | ✓ | ✓ |
| Human T-lymphotropic virus II (HTLV-II) | ✓ | ✓ |
| Parvovirus B19 | ✓ | ✓ |
| Mycobacterium tuberculosis | ✓ | ✓ |
| Staphylococcus aureus | ✓ | ✓ |
| Staphylococcus epidermidis | ✓ | ✓ |
| Stenotrophomonas maltophilia | ✓ | ✓ |
| Escherichia coli | ✓ | ✓ |
| Enterobacter faecalis | ✓ | ✓ |
| Klebsiella oxytoca | ✓ | ✓ |
Accuracy
The test was found to be comparable. All the known positive samples were found to be positive while all the known negative samples were found to be negative indicating the trueness of the kit.
Precision data
The SD and percentage coefficient of variations (% CV) was calculated to assess the precision of the replicates as well as the assay performance. Day to day, lot to lot and operator to operator reproducibility was studied.
Results showed % CV and SD within permissible limits of < 5% for Chikungunya and Dengue targets (Tables 4, 5).
Table 4.
Precision data
| Precision combined data for Chikungunya | ||||||
|---|---|---|---|---|---|---|
| Concentration (Copies/ul) | Standard variation | % Coefficient Variation | ||||
| Log 5 (105 Copies/ul) |
Log 3 (103 Copies/ul) |
Log 1 (10 Copies/ul) |
Log 5 (105 Copies/ul) |
Log 3 (103 Copies/ul) |
Log 1 (10 Copies/ul) |
|
| Intra assay | 0.24 | 0.22 | 1.33 | 1.03 | 0.74 | 3.66 |
| Inter Assay | 0.23 | 0.22 | 0.42 | 1.01 | 0.76 | 1.16 |
| Inter Operator | 0.18 | 0.13 | 0.03 | 0.79 | 0.43 | 0.09 |
Table 5.
Repeatability data
| Concentration (copies/ul) | Log 5 (105 Copies/ul) | Log 3 (103 Copies/ul) | Log 1 (10 Copies/ul) | |||
|---|---|---|---|---|---|---|
| Mean of runs | Chikungunya | Dengue | Chikungunya | Dengue | Chikungunya | Dengue |
| Inter-assay Data Run 1 | ||||||
| Replicate 1 | 23.44 | 22.03 | 29.54 | 28.61 | 34.43 | 34.11 |
| Replicate 2 | 22.99 | 22.27 | 29.29 | 28.71 | 36.38 | 36.30 |
| Replicarte 3 | 23.07 | 21.77 | 29.54 | 27.34 | 37.85 | 36.09 |
| Mean | 23.17 | 22.02 | 29.46 | 28.22 | 36.22 | 35.50 |
| SD | 0.20 | 0.20 | 0.12 | 0.63 | 1.40 | 0.99 |
| % CV | 0.84 | 0.92 | 0.40 | 2.22 | 3.86 | 2.78 |
| Inter-assay Data Run 2 | ||||||
| Replicate 1 | 23.72 | 22.32 | 29.80 | 28.89 | 34.71 | 34.43 |
| Replicate 2 | 23.24 | 22.56 | 29.53 | 29.01 | 36.77 | 36.59 |
| Replicarte 3 | 23.34 | 22.09 | 29.81 | 27.75 | 37.19 | 36.39 |
| Mean | 23.43 | 22.32 | 29.71 | 28.55 | 36.22 | 35.80 |
| SD | 0.20 | 0.19 | 0.13 | 0.57 | 1.08 | 0.97 |
| % CV | 0.87 | 0.86 | 0.44 | 1.99 | 2.99 | 2.72 |
| Inter-assay Run 3 | ||||||
| Replicate 1 | 23.12 | 21.67 | 29.25 | 28.27 | 34.12 | 33.76 |
| Replicate 2 | 22.71 | 21.93 | 29.02 | 28.36 | 35.96 | 35.97 |
| Replicarte 3 | 22.76 | 21.41 | 29.22 | 26.75 | 35.93 | 35.73 |
| Mean | 22.86 | 21.67 | 29.16 | 27.79 | 35.34 | 35.15 |
| SD | 0.18 | 0.21 | 0.10 | 0.74 | 0.86 | 0.99 |
| % CV | 0.79 | 0.99 | 0.36 | 2.66 | 2.43 | 2.82 |
Reproducibility
Day to day, lot to lot and operator to operator reproducibility was studied (Table 6).
Table 6.
Reproducibility data
| Concentration (copies/ul) | Log 5 (105 Copies/ul) | Log 3 (103 Copies/ul) | Log 1 (10 Copies/ul) | |||
|---|---|---|---|---|---|---|
| Mean of runs | Chikungunya | Dengue | Chikungunya | Dengue | Chikungunya | Dengue |
| Inter lot data | ||||||
| Lot 1 | 23.17 | 22.02 | 29.46 | 28.22 | 36.22 | 35.50 |
| Lot 2 | 23.43 | 22.32 | 29.71 | 28.55 | 36.22 | 35.80 |
| Lot 3 | 22.86 | 21.67 | 29.16 | 27.79 | 35.34 | 35.15 |
| Mean | 23.15 | 22.01 | 29.44 | 28.19 | 35.93 | 35.49 |
| SD | 0.23 | 0.27 | 0.22 | 0.31 | 0.42 | 0.27 |
| % CV | 1.01 | 1.21 | 0.76 | 1.10 | 1.16 | 0.75 |
| Inter operator data | ||||||
| Operator 1 | 23.53 | 21.67 | 29.71 | 27.79 | 36.16 | 35.15 |
| Operator 2 | 23.17 | 22.32 | 29.46 | 28.55 | 36.22 | 35.80 |
| Mean | 23.35 | 22.00 | 29.58 | 28.17 | 36.19 | 35.48 |
| SD | 0.18 | 0.33 | 0.13 | 0.38 | 0.03 | 0.32 |
| % CV | 0.79 | 1.49 | 0.43 | 1.35 | 0.09 | 0.91 |
Results showed % CV and SD within permissible limits of < 5% for Chikungunya and Dengue targets.
Interfering substances
The susceptibility of the assay to interference by elevated levels of endogenous substances was evaluated. Chikungunya and dengue negative samples and samples containing 1000 copies/µl of Chikungunya and dengue virus RNA were tested. No interference in the performance of the assay was observed in the presence of the haemoglobin (500 mg/dl), lipids (800 mg/dl) and bilirubin (< 15 mg/dl) for all positive and negative samples tested. Heparin (≥ 10 IU/ml) was found to affect the PCR.
Stability data
Accelerated and real-time stability studies and stress tests were performed in order to determine shelf life, storage and transport conditions. The shelf-life of the test was found to be 12 months.
The open vial study data indicated that the test is stable up-to 4 freeze thaw cycle.
On board stability data indicated that the reaction mix is stable up-to 45 min after addition of the template till loading on the instrument.
Clinical data
Results of both the recommended comparator study (NABL data) and clinical independent evaluation indicate the test has 100% sensitivity and specificity (Table 7). The clinical performance was found to be satisfactory with > 98% score match.
Table 7.
Clinical validation data
| Specimen type n = |
Number of samples | PathoDetect™ CHIK-DEN Multiplex PCR Kit | abTES™DEN/CHIKU/ZIKA 3 qPCR I Kit | |||
|---|---|---|---|---|---|---|
| Reactive | Non-reactive | Reactive | Non-reactive | Reactive | Non-reactive | |
| Chikungunya Plasma Sample | 15 | 15 | 15 | 15 | 15 | 15 |
| Dengue Plasma Sample | 15 | 15 | 15 | 15 | 15 | 15 |
| Total | 30 | 30 | 30 | 30 | 30 | 30 |
| %CI | 100% | 100% | 100% | 100% | ||
Comparison of nucleic acid extraction process between manual and automated methods
We have verified the performance of Nucleic acid Extraction process using Manual method and Automated CompactXL platform. The performance characteristics such as sensitivity, specificity and turnaround time were comparable between the two methods. However, nucleic acid extraction using Automated Compact XL platform has several benefits including pipette and expert-free handling, high throughput analysis, low risk of contamination with ease of operations.
Discussion
The present study describes the development and validation of the Multiplex real time PCR test for the direct detection of all genotypes of DENV and CHIKV RNA in human plasma or serum samples. This test was designed for commercial use with quality objectives. The test presents several novel characteristics such as: a. highly sensitive and specific detection of CHIKV and DENV in a single tube, b. inclusion of positive and negative controls for quality check, c. inclusion of internal control gene to assess the performance of both nucleic acid extraction and real time PCR, d. cost effective and rapid test, e. fast results within 80 min after PCR start.
CHIKV and DENV have similar epidemiology, transmission cycles and clinical symptoms [5], thus the tests that screen for these viruses separately are preferrable at the initial patient presentation. With the advents in the field of molecular diagnostics over the past decade, more reliable assays have been developed using two-step RT-PCR, nested RT-PCR, quantitative real-time RT-PCR (qRT-PCR), and nucleic acid sequence-based amplification (NASBA) for the identification of viral pathogens and have gradually become the preferred standard methods over virus isolation and sero-confirmation.
This study describes a qualitative in vitro diagnostic test for the direct detection of all genotypes of DENV and CHIKV RNA in human plasma or serum samples. It is a single-tube, single-step test based on RT-PCR for amplification of specific regions of the target genome using hydrolysis probe chemistry. In real-time PCR, the amplified product is detected via fluorescent dyes. These are usually linked to oligonucleotide probes which bind specifically to the amplified product. Monitoring the fluorescence intensities during the PCR run allows the detection and quantitation of the accumulating product in real time and does not require laborious post PCR methods. The PCR mix contains all the components including HotStart RT, Fast Taq polymerase, primers and hydrolysis probes in a single PCR reaction tube.
Several tests have been developed for the detection of DENV and CHIKV for Lab development testing of DENV and CHIKV, either separately or simultaneously in a single tube [14, 15, 17–21]. All these RT-PCRs appear to perform well, as reported but some of these assays have either used duplex approach, a three- or four-step cycling method, which was found to be associated with increased turnaround time. SYBR green-based method were shown to produce non-specific reaction products such as primer-dimers and nonspecific amplification of other unrelated gene products increasing background and false positives [22]. Some of these assays need additional post-amplification processes such as gel electrophoresis, visualization which adds to the time and cost. Further, complete validation along with stability testing is crucial for its commercial utility. The protocol has been developed so as to have robust sensitive and stable product for commercial utility.
In the present study, the enzyme combinations (Reverse transcriptase, Taq polymerase Enzyme), primer selection, primer probe mixture [Primer probe specific for Chikungunya (VIC), Dengue (FAM) and internal control target (Cy5)], enzyme concentration, and thermal profile designed for the multiplex PCR increased the specificity and sensitivity of the test for the simultaneous detection of the DENV and CHIKV from blood sample. Primers and probes were designed so that they could be used under similar amplification conditions but provide immediate viral identity response. Further, automated platform eliminates the need of pipette and technical experts as well as requirement of pipette and liquid handling.
The linear dynamic range in the assay covers seven log10 copies of nucleic acid template, where the lower limit was set at the LOD CT of 37.51 for CHIKV and 35.51 for DENV. In order to control for tube-to-tube variation in the assay, the acceptance criterion for the standard curve slope was set within -3.0 to -3.7 with the correlation coefficient greater than 0.98. The assay includes an exogenous internal control to identify possible PCR inhibition and to confirm the integrity of the reagents and nucleic acid. For precision study, three known positive samples for both CHIKV and DENV with low (10 copies/µl), moderate × (103 copies/µl) and high concentration (105 copies/µl) along with known negative sample were tested. The assay was highly reproducible for detecting virus at concentrations ranging from 105 to 1 gene copy numbers and LOD was found to be 1.56 copies/µl. In-silico study was performed to check the homology of primer/probes with all sequences published in NCBI database. The tested pathogens were not reactive, and no cross-reactivity observed with mixed infection. The assay was found to have high analytical specificity, and no false-positive results were seen from testing of relatively high concentrations of related flavivirus and alphavirus RNAs. Furthermore, low intra-assay and inter-assay variabilities were seen in CT values over a wide range of concentrations, with two different operators and three lots on different days. The intra and inter assays presented optimal coefficients of variation < 5% for Dengue and Chikungunya. We have evaluated the diagnostic application of the multiplex RT-PCR assay using 30 plasma samples representing all four serotypes of DENV and CHIKV. A total of 60 samples (15 Chikungunya positive, 15 Dengue positive and 30 negative specimens) were tested with both PathoDetect™ CHIK-DEN Multiplex PCR Kit and abTESTM DEN/CHIKU/ZIKA 3 qPCR I Kit in two independent runs. Proficiency testing was carried out using QCMD Panel. Three independent runs were carried out in triplicates on QuantStudio5 and Compact Q machines. For the detection of DENV and CHIKV, the multiplex RT-PCR showed 100% sensitivity and specificity.
This diagnostic test can be performed with Compact XL automated system. The Compact XL is a sample-to-PCR ready system, enabled with automated barcode reading, auto-feed protocol and self sanitization, eliminating the huge infrastructure and capital cost of the molecular lab thus aids in increasing the efficiency of molecular diagnostic for commercialization. The cost difference between Mylab manufactured assay and currently used commercial assays in India is approximately INR. 850.
Conclusion
The test named “PathoDetect™ CHIK-DEN Multiplex PCR Kit” offers a one-step multiplex real time RT PCR for simultaneous detection and differentiation of dengue and chikungunya virus from clinical samples. The developed assay is highly sensitive and specific for the detection of two clinically indistinguishable diseases, within 80 min including nucleic acid extraction. Early-stage diagnosis of viral nucleic acid would greatly help in disease management. The assay would be useful for differential diagnosis of CHIKV and DENV in a situation where a number of other clinically indistinguishable infectious diseases like malaria, rickettsia and leptospira might occur. It has implications for molecular epidemiological surveillance of Chikungunya or Dengue infections in epidemic and endemic regions.
Acknowledgements
We thank the R&D team for their contribution in executing the projects till completion. We also thank Dr. Amrita Khaire for critically evaluating the manuscript drafts.
Author contributions
MD, contributed in the designing the product and manuscript writing. GM and HS executed experiments and analysed the data. All the authors agreed to the publication of the final manuscript.
Funding
This work was funded by Mylab Discovery Solutions Pvt. Ltd, Pune, India.
Declarations
Conflict of interest
The authors declare that there are no conflicts of interest.
Footnotes
Publisher's Note
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References
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