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# Paper microfluidic implementation of loop mediated isothermal amplification for early diagnosis of hepatitis C virus

## Abstract

The early diagnosis of active hepatitis C virus (HCV) infection remains a significant barrier to the treatment of the disease and to preventing the associated significant morbidity and mortality seen, worldwide. Current testing is delayed due to the high cost, long turnaround times and high expertise needed in centralised diagnostic laboratories. Here we demonstrate a user-friendly, low-cost pan-genotypic assay, based upon reverse transcriptase loop mediated isothermal amplification (RT-LAMP). We developed a prototype device for point-of-care use, comprising a LAMP amplification chamber and lateral flow nucleic acid detection strips, giving a visually-read, user-friendly result in <40 min. The developed assay fulfils the current guidelines recommended by World Health Organisation and is manufactured at minimal cost using simple, portable equipment. Further development of the diagnostic test will facilitate linkage between disease diagnosis and treatment, greatly improving patient care pathways and reducing loss to follow-up, so assisting in the global elimination strategy.

## Introduction

Hepatitis C virus (HCV) is a major cause of liver-related mortality with 71 million people chronically infected globally. Recent advances in direct acting antiviral treatments have improved cure rates to >95%. However, currently an estimated 80% of all infected individuals are unaware of their status due to the asymptomatic nature of infection. Many of these patients will remain undiagnosed until irreversible clinical manifestations, such as liver cirrhosis and hepatocellular carcinoma, develop, contributing to the 400,000 HCV related deaths reported every year1.

The World Health Organisation (WHO) has established a global elimination strategy to reach significant disease reduction targets by 20302,3. The most challenging obstacle in achieving this ambitious goal is the rapid diagnosis of patients and their integration into an appropriate clinical care pathway for treatment3.

Current diagnostic strategies rely on testing for anti-HCV antibodies followed by RNA or core antigen detection4. This two-step process requires a centralised laboratory infrastructure and delays active HCV infection diagnosis by significant periods of time. Successful implementation is also limited by cost, long turnaround times and the high level of expertise required for diagnostic testing. High risk groups, including people-who-inject-drugs (PWID), are often lost to follow-up due to the multiple visits required for HCV diagnosis. Additionally, absent or delayed seroconversion, particularly in immunocompromised patients, may reduce overall rates of diagnosis5.

Most individuals with HCV reside in low and middle-income countries (LMICs) with only limited access to diagnosis1,6. Although recent advances in HCV nucleic acid amplification tests (NAATs) have provided a possible solution for improving the management of HCV diagnosis, the existing diagnostic platforms still pose several limitations, including the high cost and training requirements associated with polymerase chain reaction (PCR) assays, especially when used in decentralised testing7,8. Not only are simpler and cheaper NAATs for point-of-care (POC) testing required in order to improve HCV diagnosis and eliminate the need for follow-up visits3, but importantly, the selection of a universal NAATs’ target is needed. HCV, like many RNA viruses, exhibits high genetic diversity, with eight distinct HCV genotypes and at least 90 different subtypes identified with varied prevalence gobally9,10.

Loop mediated isothermal amplification (LAMP) assays provide high sensitivity and specificity through the use of 4–6 primers, which target 6–8 regions within a sequence of interest11,12. The amplification reaction takes place at a constant temperature between 60–65 °C, offering a cheaper alternative to the traditional PCR assays, with minimal equipment requirements, which has resulted in its use in decentralised (POC) settings to detect the presence of a variety of pathogens13,14,15.

Recently, LAMP assays have also been developed for HCV detection in centralised facilities. Colorimetric outputs have also been developed to enable the detection without bulky optical instrumentation with excellent performance for some genotypes (e.g. Hongjaisee S. et al.16 showed 100% sensitivity with genotype 6). However, these assays showed limited diagnostic performance when used with varied genotypes or low viral loads17,18,19. Their application as POC tests is thus restricted, especially in LMICs20, where a wide variation in the genetic diversity may lead to a lower efficacy of tests.

In this study, we now demonstrate a pan-genotypic HCV LAMP assay, which we validated in a prototype POC diagnostic device as a paper-microfluidic, visually read lateral flow test. The assay was validated as part of a double-blind clinical study of samples from patients with a range of viral loads and genotypes as determined by a highly sensitive gold-standard Abbott RealTime RT-PCR HCV test, within a clinical reference laboratory and our own in-house HCV RT- PCR. We subsequently implemented the LAMP nucleic acid detection strip method into a portable and user-friendly device, with an easy-to-use readout for target detection.

## Results

### Optimisation of HCV LAMP primers

Previously published LAMP primers were selected based on evidence of a low limit of detection from HCV RNA (50 IU/mL)17. In this study, the use of an additional accelerating primer (AP, Table 1) ensured improved sensitivity and specificity across several HCV genotypes. We analysed an alignment file of over 200 sequences of major HCV genotypes and subtypes21. The majority of the observed mismatches were in the middle of the primers (Fig. S1). We only noted one mismatch within the last base pair of the 3′ end of the backward loop primer (BLP), within genotype 3 sequences. Focussing on genotype 3 and in order to further improve the previously published assay, we removed the cytosine mismatch at the 3′ end of BLP. Additionally, a cytosine was added at the beginning of the primer, in order to conserve the primer melting temperature (Fig. S1 and Table 1). The removal of the cytosine mismatch and the subsequent use of the new primer improved the time to positivity by 21% in genotype 3 (Fig. S2). As a confirmation, the new BLP was also compared to the original BLP performance on genotype 1 targets, which, did not result in significant differences in the assay performance as there were no mismatches within this genotype.

### Clinical sensitivity and specificity

Using optimised primers, we conducted a double-blind study of LAMP on RNA and cDNA from samples with a range of viral loads and from different HCV genotypes, including a recently identified genotype 7a (Table 2)22.

The performance of HCV LAMP using RNA samples (RT-LAMP) directly and HCV LAMP using cDNA as template were compared with a highly sensitive in-house HCV RT- PCR by genotype and viral load (Tables 2 and 3). The in-house RT-PCR assay has been characterised previously as a quantitative assay (qRT-PCR23).

HCV RT-LAMP and HCV LAMP assays detected 96 and 97 HCV samples out of 100 respectively, comparable with the RT-PCR assay which detected 96/100 samples. The false negatives correlated well between the three assays (in two cases false negatives were due to low viral loads, ≤3.95 log10 IU/mL). RT-LAMP failed to detect a single genotype 4 sample with high viral load (5.64 log10 IU/mL). RT-LAMP and LAMP assays both exhibited high specificity, correctly identifying 91 and 90 control samples out of 100 respectively, although our in-house RT-PCR assay had a higher specificity with no false positives.

### Time to detection

In order to evaluate the impact of genotype and viral load on the performance of both HCV RT-LAMP and LAMP assay, we defined samples as positive when the fluorescence signal was at least ten standard deviations above the mean baseline fluorescence of the positive control. The time to positive was then determined as described in Fig. S3. The majority of positive samples were detected within 30 min (Fig. 1a, b). For RT-LAMP, genotype 3 and 4 detection took longer than genotype 1 and 2. Similarly, for cDNA, genotype 3 positivity occurred later than all other genotypes. There was an inverse relationship between time to detection and viral load and significant differences occurred between the 1.70–3.95 log10 IU/mL group, 4.40–4.85 log10 IU/mL group and the 6.05–6.97 log10 IU/mL group for both RNA and cDNA (Fig. 1c, d).

Since most samples were detected within the first 30 min, we assessed if this would be a suitable detection time period. Receiver operating characteristic (ROC) curves for both LAMP and the RT-PCR were used to determine the efficacy by which the assays would distinguish between HCV-positive and control samples at different time points or cycles (Fig. 2, Fig. S4 and Tables S12). Both RT-LAMP and LAMP had areas under the curve of 0.97 (Fig. 2A, B), showing close statistical similarity to the RT-PCR assay (Fig. S4 and Table S1). The cut-off of <29 min for RT-LAMP was equivalent to 95.0% sensitivity (95% CI: 88.7–98.4%) and 94.0% specificity (95% CI: 87.4%–97.8%) (Fig. 2C and Table S2). LAMP had the same sensitivity at <29.75 min, but the specificity was slightly lower—92.0% (95% CI: 84.8–96.5%) (Fig. 2D and Table S3). To achieve the specificity of 98%, recommended by the WHO, (98.0%, 95% CI of 93.0–99.8%), the cut-off value would be set to <24.75 min for RT-LAMP and <26.75 min for LAMP, resulting in a sensitivity of 90.0% (95% CI of 82.4–95.1%) and 92.0% (95% CI of 84.8–96.5%) for RT-LAMP and LAMP, respectively (Fig. 2C, D, Tables S13)24.

### Analytical sensitivity and end-point detection

Analytical sensitivity of HCV LAMP was assessed using serial dilutions of plasmids containing HCV sub-genomic replicon over 40 min by three different detection methods; nucleic acid detection strips, gel electrophoresis and changes in fluorescence over time. The concentration of HCV ranged between 2.6 log10 copies/reaction and 4.1 log10 copies/reaction. Figure 3 shows the mechanism of strip detection (Fig. 3a), the assembly of the lateral flow device (Fig. 3b), and the results from the analytical sensitivity experiments (Fig. 3c). The lower limit of detection was the same for each method, below 2.6 log10 copies/reaction (broadly equivalent to a detection threshold of 398 copies/reaction, which is lower than the recommended ≤3000 IU/mL limit of detection by WHO)2.

The nucleic acid detection strips used in this study offered an easy-to-interpret pregnancy test-like result with two bands for a positive reaction and one (control) band for negative. The gel electrophoresis revealed a characteristic smear with ladder-like banding pattern for all samples except the negative control. The size of the initial bands (274 bp) correlate with the region between F3/B3 representing the initial stem loop formation of the reaction. As the reaction proceeded, larger constructs were created, with random termination, generating a other bands with a smear-like pattern11,12,17. The results correlated with the nucleic acid detection strips. There was an inverse relationship between viral load and time to positivity and all positive reactions were statistically different from the negative control (p < 0.0001). The double-blind study revealed that optimal sensitivity and specificity occur at <30 min with a lower limit of detection of 417 IU/mL.

Nucleic acid detection strips are easy to visualise and interpret and are known to correlate well with other detection methods and were thus used as the basis for the development of a low-cost, microfluidic HCV test, which could be used either in bedside and/or POC settings. An enclosed lateral flow device containing a LAMP reaction chamber, valves and detection strips was manufactured in-house using methods adapted from previous studies (shown in Fig. 3b and Fig. S5)15. The LAMP chamber was incubated in a heat block for 30 min at 65 °C by inserting the device upright (thus ensuring that the lateral flow strips were held away from high temperatures, Fig. S6). At the end of the reaction, the amplicons, generated within the LAMP chamber were eluted onto the lateral flow strips by pressing a blister pack/finger pump containing running buffer. The amplicons, labelled with both FITC and biotin ligand binding sites, contacted the end of the lateral flow devices and then were carried along the paper strips by capillarity, where they interacted with conjugation pads. This prototype HCV test device offers a cheap and user-friendly detection method together with the high sensitivity and specificity of the LAMP reaction.

To characterise the performance of the lateral flow detection devices, we tested a further 40 patient samples (20 HCV-positive and 20 healthy controls) on this platform and compared the results to our in-house qPCR assay (see ‘Methods’ for details). Results show no false positives (20/20 negatives detected correctly, Supplementary Table S4). Three clinically positive samples did not provide any Ct value on qPCR, indicative of either or both of low viral loads or degradation of the RNA from the additional freeze-thaw cycles and longer storage. 14/17 positive samples were also identified correctly by the LAMP on lateral flow devices. Two of the false negative samples had Ct values above 30 for our in house qPCR, which is beyond our threshold for detection, indicating low viral loads. Consequently, only one sample (Ct 29) was negative for the lateral flow device, whilst being positive for qPCR, showing excellent agreement, in line with the results obtained for fluorescence read-outs and demonstrating the potential for this low-cost and user-friendly method.

## Discussion

In this study, we aimed to develop a cheap, sensitive and specific bedside test for HCV. Using optimised primers, we validated its potential as a future POC/bedside test in a double-blind study of clinical samples with varied viral loads and comprising all major genotypes, including a recently identified genotype 7 sample22. Both RT-LAMP and LAMP performed well in this study, exceeding the standards set out by WHO for POC tests24. The samples were diagnosed by comparison with the gold standard Abbott RealTime HCV PCR assay within a validated clinical reference laboratory and further compared with an in-house RT-PCR assay. A small number of samples that were not detected by RT-PCR were also missed by the LAMP assay, possibly due to low viral load.

The target product profile for diagnosis of HCV as recommended by WHO requires a minimum diagnostic specificity of >98% and sensitivity of >90–95% which was fulfilled by RT-LAMP at a cut-off of <25 min22. However, the optimal analytical sensitivity of RT-LAMP assay, exceeding the WHO requirements, was reached at a cut-off of <30 min. The small number of false positive samples detected by LAMP versus RT-PCR could represent a slightly increased risk of cross-contamination or the formation of primer-dimer structures detected by LAMP. Overall, false positives did not occur commonly in this study, as in other HCV LAMP studies17,18,19,25.

The analytical sensitivity of the assay at 40 min was ~2.6 log10 copies/reaction. In the original study from which we adapted our primers, the authors report a limit of detection of 50 IU/mL17. Both studies show that the assay falls well within the WHO’s criteria for a point-of-care test (3000 IU/mL or below)2.

### HCV RT-PCR assay

An in-house RT-PCR with primer and probe sequences based on the 5′UTR (JFH1- primer 16; 5′-TCTGCGGAACCGGTGAGTAC-3′, JFH1-primer 17; 5′-GCACTCGCAAGCACCCTAT-3′, FAM probe; 6-FAM-AAAGGCCTTGTGGTACTG-MGB) was used for comparison, whilst the Abbott real-time RT-PCR assay was used as a gold-standard HCV detection reference assay. Each master mix consisted of 2× TaqMan Fast Universal Mix (Thermo Fisher Scientific), 18 µM forward and reverse primers, 5 µM probe and 1 µL cDNA template in a final volume of 10 µL. The run consisted of 20 s hold at 95 °C followed by 40 cycles of denaturation for 20 s at 95 °C and amplification for 30 s at 60 °C.

### Statistical analysis

Normal distribution of data was determined in GraphPad Prism version 7 using D’Agostino & Person normality test. A parametric one-way ANOVA was used for normally distributed data and a non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons test was used for other data types. ROC curves were plotted on the same software. Data were considered significant if p value was less than or equal to 0.05.

### Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

## Data availability

The raw data, sequence alignment, gel blots and images that support the findings of this study are also available in University of Glasgow’s Enlighten: Research Data with the identifier: https://doi.org/10.5525/gla.researchdata.1127Source data are provided with this paper.

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## Acknowledgements

The authors would like to Dr. Shantimoy Kar and Dr. Xiaoxiang Yan from the James Watt School of Engineering at the University of Glasgow for their inputs on the design of the lateral flow device. This study was funded in part by Engineering and Physical Sciences Research Council, Grant Number: EP/M508056/1, Medical Research Council Grant Numbers: G0801566, G0901213–92157, MC_PC_16045, MC_UU_12014/1, MR/K013491/1 and the Wellcome Trust, Grant Number: 102789/Z/13/A. We also acknowledge the support of NHS Research Scotland (NRS) Greater Glasgow and Clyde Biorepository.

## Author information

Authors

### Contributions

E.C.T., J.M.C. and J.R. supervised the project. A.B.S. determined the viral loads and genotypes of the HCV samples by the Abbott RealTime PCR. A.B.S. and R.G. provided and anonymised the samples for the double-blind studies. C.D. and S.R.S. extracted RNA and conducted cDNA synthesis of all samples for the double-blind study. W.M.W. performed the LAMP and in-house RT-PCR assays for the double-blind studies. W.W.M., A.G., G.X. and Z.Y. designed the lateral flow devices. W.W.M. and A.G. manufactured and assembled the lateral flow devices. W.W.M., P.J., J.M.C. and J.R. analysed the data. W.W.M. wrote the original manuscript. E.C.T., J.M.C. and J.R. provided revisions and all authors reviewed and edited the manuscript.

### Corresponding authors

Correspondence to Emma C. Thomson or Jonathan M. Cooper.

## Ethics declarations

### Competing interests

The authors declare no competing interests.

Peer review information Nature Communications thanks Maria-Nefeli Tsaloglou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Witkowska McConnell, W., Davis, C., Sabir, S.R. et al. Paper microfluidic implementation of loop mediated isothermal amplification for early diagnosis of hepatitis C virus. Nat Commun 12, 6994 (2021). https://doi.org/10.1038/s41467-021-27076-z

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• DOI: https://doi.org/10.1038/s41467-021-27076-z