key: cord-0760365-h5ipyj2p
authors: Promlek, Thanyarat; Thanunchai, Maytawan; Phumisantiphong, Uraporn; Hansirisathit, Tonsan; Phuttanu, Chayanit; Dongphooyao, Sunisa; Thongsopa, Wipawee; Nuchnoi, Pornlada
title: Performance of colorimetric RT-LAMP as a diagnostic tool for SARS-CoV-2 infection during the fourth wave of COVID-19 in Thailand
date: 2021-12-25
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.12.351
sha: 43d01d1be511a91c56deb40b0b394b2855e4b879
doc_id: 760365
cord_uid: h5ipyj2p

Background: Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, poses an ongoing global threat, particularly in low-immunization coverage regions. Thus, rapid, accurate, and easy-to-perform diagnostic methods are in urgent demand to halt the spread of the virus. Objective: We aimed to validate the clinical performance of the FastProof™ 30 min-TTR SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP) method using leftover ribonucleic acid (RNA) samples extracted from 315 nasopharyngeal swabs. The sensitivity and specificity of RT-LAMP were determined in comparison with RT-PCR. Result: Out of 315 nasopharyngeal swabs, viral RNA was detected in 154 (48.9%) samples by RT-PCR assay. Compared with RT-PCR, overall sensitivity and specificity of RT-LAMP were 81.82% (95% CI: 74.81–87.57) and 100% (95% CI: 97.73–100), respectively. A 100% positivity rate was achieved in samples with cycle threshold (Ct) <31 for RT-PCR targeting the ORF1ab gene. However, samples with Ct >31 accounted for false-negative results by RT-LAMP in 28 samples. Conclusion: RT-LAMP reliably detected viral RNA with high sensitivity and specificity and has potential application for mass screening of patients with acute COVID-19 infection, when viral load is high.

Coronavirus disease 2019 is an acute infection of the respiratory tract caused by a recently emerged coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was first detected in Wuhan, China, in late 2019, and continues to cause major public health concerns. The virus spread worldwide and has been declared a pandemic, as there were more than 194 million confirmed cases and over 4 million deaths in 222 countries as of July 2021 (World Health Organization., n.d.) . Currently, there are different platforms of COVID-19 vaccines available, which have been distributed globally. Yet, the number of new confirmed cases continues to rise in many countries, especially in regions with low-vaccination rates.

In Thailand, the number of daily positive COVID-19 cases and deaths remain high due to insufficient vaccine coverage and the impact of SARS-CoV-2 variants. At the time of manuscript preparation, the fourth COVID-19 wave driven by the delta variant is surging. This variant is highly contagious and rapidly transmitted among vaccinated and unvaccinated people. However, accurate and reliable diagnostic tests for SARS-COV-2 infection and nonpharmaceutical measures could decelerate the spread of SARS-CoV-2 by allowing faster identification and separation of infected individuals from uninfected individuals. Reverse transcriptase-polymerase chain reaction (RT-PCR), a gold-standard laboratory test, has been used routinely for diagnosis since the beginning of the outbreak due to its high sensitivity and specificity (Corman et al., 2020) . However, the method is time-consuming and requires costly equipment and trained personnel, which is not applicable for mass testing during the fourth-wave COVID-19 crisis (Feng et al., 2020; Shen et al., 2020) .

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid, reliable alternative molecular test. This method amplifies specific genes at a constant temperature of 60-65°C for a duration of 30 min. RT-LAMP reactions can be detected via various readouts, including gel electrophoresis, turbidity, fluorescence, and visual color changes (Amaral et al., 2021; Ganguli et al., 2020; Inaba et al., 2020; Nawattanapaiboon et al., 2021; Silva et al., 2021) . Therefore, RT-LAMP could overcome the drawbacks of RT-PCR in terms of fast turnaround time and affordable reagents and instruments, which would enable large-scale rapid testing (Jiang et al., 2020) . Colorimetric RT-LAMP has been established for naked eye observation. This approach has been well validated in real-life clinical settings and was demonstrated to achieve 95%-100% specificity and 90%-100% sensitivity, based on the cycle threshold (Ct) values representing viral ribonucleic acid (RNA) levels (Kitagawa et al., 2020; Nawattanapaiboon et al., 2021) . Decreased sensitivity was frequently found in samples with low viral RNA copies (Silva et al., 2021; Subsoontorn et al., 2020) . Colorimetric FastProof™ RT-LAMP was developed as an alternative rapid, reliable, and cost-effective diagnostic test for COVID-19, which provides support for mass testing and active case detection in regions with low resources. In this study, we therefore validated the performance of the colorimetric RT-LAMP method compared to the gold-standard RT-PCR method using the same 315 COVID-19suspected clinical samples.

Nasopharyngeal swabs were obtained from 315 suspected COVID-19 patients and preoperative patients at Vajira Hospital, Navamindradhiraj University, Bangkok, Thailand in July 2021. Briefly, nasopharyngeal swabs collected from acute respiratory infection clinic (ARI) were subjected to RNA extraction and followed by RT-PCR in the laboratory. After completing the routine operation, all RT-PCR positive RNA samples regardless of Ct values and five percent of RT-PCR negative RNA samples were taken daily for RT-LAMP testing. The study protocol was approved by the Institutional Review Board of the Faculty of Medicine Vajira Hospital, Navamindradhiraj University (Study code 126/64 E); COA: 131/2564.

The specimens were collected in 2 mL viral transport media (VTM) (Dewei Medical Equipment Co., Ltd., China) and were immediately transported at a temperature of 2°C-8°C to the Biomolecular Laboratory at Vajira Hospital.

RNA was extracted from 200 µl of VTM samples using the Zybio Nucleic Acid Extraction Kit (magnetic bead method) (Zybio Inc., China) according to the manufacturer's instructions. From each sample, 50 µl of viral RNA was eluted and used as a template for detecting SARS-CoV-2 infection by RT-PCR and RT-LAMP. China) was used for amplification. The amplification reaction consisted of one cycle at 50°C for 30 min and one cycle at 95°C for 1 min for reverse transcription and reverse transcription denaturation, respectively. Next, 45 cycles were repeated at 95°C for 15 seconds and 60°C for 31 seconds with DNA polymerase. The result was analyzed using ABI 7500 software, in which a Ct value less than 40 for both target genes was defined as a positive result.

RT-LAMP detection of the N genes of SARS-CoV-2 and human RNAse P gene as an internal control was performed using the FastProof™ 30 min-TTR SARS-CoV-2 RT-LAMP Kit (Zenostic Co., Ltd., Thailand) according to the manufacturer's instructions. The total volume was 20 µl, which consisted of 10 µl Color LAMP-TTR, 5 µl primer mix-TTP, and 5 µl extracted RNA sample. The reaction was incubated at 65°C for 30 minutes, in a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A positive result was determined by observing the colors of the reaction changes from pink to yellow. Positive and negative controls were employed in all assays.

Descriptive data was presented in the following formats: percent, median, range, mean, and standard deviation (SD). The sensitivity and specificity of RT-LAMP in detecting SARS-CoV-2 were analyzed using GraphPad Prism 6.0 (GraphPad Prism Software Inc., San Diego, CA, USA) and MedCalc's diagnostic test evaluation, an online statistical calculator.

Nasopharyngeal swabs were collected from a total of 315 COVID-19 suspected cases and preoperative patients in July 2021 at Vajira Hospital, a tertiary hospital in Bangkok, Thailand.

Patient characteristics were retrospectively reviewed from medical records, as shown in Table 1 .

The median age of the study population was 40 (range: <1-92) years; 186 (59%) were females and 129 (41%) were males. Out of 315 subjects, 196 presented illnesses, 110 had no symptoms, and 9 were underreported. Common symptoms included fever, cough, sore throat, runny nose, muscle aches, headache, loss of taste or smell, diarrhea, difficulty breathing, and pneumonia, which appeared 0-30 days after exposure or direct contact with COVID-19 confirmed cases. The (Table 1 ). This result indicated that the ORF1ab gene was less sensitive than the N gene for detecting SARS-CoV-2. Among 154 RT-PCR positive cases, 124 (80.5%) cases were symptomatic, and 30 (19.5%) cases were asymptomatic (Table S1 ).

To validate the performance of the colorimetric RT-LAMP method, all 315 leftover RNA samples were assessed by RT-LAMP, of which 126 and 189 samples were found to be positive and negative, respectively. Compared to RT-PCR, the overall sensitivity and specificity of RT-LAMP were 81.82% (95% CI: 74.81-87.57) and 100% (95% CI: 97.73-100), respectively ( . 1) .

The performance of RT-LAMP in detecting SARS-CoV-2 in symptomatic patients versus asymptomatic patients was further evaluated. Among the 126 true positive results, RT-LAMP was able to detect SARS-CoV-2 in symptomatic patients with 87.10% sensitivity, while the sensitivity was slightly decreased in asymptomatic patients (60%). For 28 false negative results, RT-LAMP could not detect SARS-CoV-2 in 16 and 12 symptomatic and asymptomatic patients, respectively (Table 3 ). These results suggested that RT-LAMP assay exhibited high accuracy for determining SARS-CoV-2 infection, especially in symptomatic patients.

The correlation between the positivity rate by RT-LAMP and four Ct ranges was explored ( Table 2 ). All samples with Ct values <31 in RT-PCR had a 100% positivity rate by RT-LAMP. A decrease in the positivity rate of Ct values≥31 which declined to 53.85%, was obvious, whereas for samples with Ct values ≥36, the positivity rate decreased to 15.79%. The 28 samples that were discordant with the RT-PCR results were false negatives due to Ct values >31 (range: 31.52-39.96). Our finding demonstrated that RT-LAMP could effectively detect the SARS-CoV-2 RNA in samples with low to moderate Ct values.

Rapid identification of infection is the key to control the spread of COVID-19. Thus, fast, simple, and reliable approaches are needed to speed up disease screening and surveillance in large-scale testing. RT-LAMP is a promising method for achieving rapid, accurate, and easy-to-perform diagnostic methods. However, the quality of RT-LAMP depends on the quality of reagents, primer design, and target genes of the SARS-CoV-2 that are used for detection. Importantly, the RT-LAMP test kit for the SARS-CoV-2 diagnosis should be authorized by US Launching this test kit in the in vitro diagnostics market could eliminate concerns about delayed international reagent shipping and the costs of expensive RT-PCR tests, which supports the feasibility of using colorimetric RT-LAMP in low-to-middle income countries, where immunization coverage may be poor.

In this study, we validated the performance of this colorimetric RT-LAMP kit compared to RT-PCR to assure that it could be used as an alternative diagnostic test and/or screening test for COVID-19 detection in the real-world clinical setting. The overall sensitivity and specificity of RT-LAMP were 81.82% and 100%, respectively. The greatest sensitivity of 100% was shown at Ct values lower than 31, which dramatically decreased to 15.79% at Ct values higher than 36, indicating that for the low viral load, RT-LAMP was much less sensitive. The correlation between the Ct values of patients and the disease severity revealed that a Ct values greater than 30 was associated with low mortality and Ct value over 33 may not potentially transmit diseases (Magleby et al., 2021; La Scola et al., 2020) . On the other hand, low viral loads probably appeared in the preincubation period and later increased within a few days of testing negative.

In our study, the RT-LAMP assay was three times faster and cheaper than the gold standard RT-PCR, which helped to minimize time and cost in diagnosis. However, this kit requires staff without vision problems and laboratory equipment for the RNA extraction step and preparation of the reaction mixture; thus, it may not be accessible for remote health clinics. The SARS-CoV-2 Rapid Antigen Test (RAT) kit, which is based on immunochromatography, becomes popular in many countries, including Thailand. The RAT kit is a fast and simple test, which is more accessible for patients than the RT-LAMP. Currently, there are large numbers of SARS-CoV-2 rapid antigen test kits with varying quality available in the market. Even the kits produced by well-known manufacturers had lower efficacy for samples with Ct values of RT-PCR above 24-28 (Platten et al., 2021) . The performance of Standard™ Q COVID-19 Ag kit was evaluated in a Thai healthcare facility showing 98.33% sensitivity and 98.73% specificity.

This high sensitivity could be due to adding the extra step of reducing viscosity in samples.

Additionally, two-thirds of the tested samples had Ct values<28 (Chaimayo et al., 2020) . Thus, the RAT has both advantages and drawbacks: it is easy to perform and affordable at-home test kits are preferable, but the drawbacks are the high rate of false negative results arising from low viral loads and the lack of proper specimen processing. Still, RT-LAMP has advantages over RAT in terms of sensitivity and specificity. The RT-LAMP kit's sensitivity and specificity in our setting are better than RAT because the overall sensitivity and specificity of RAT in previous reports were 68.4% and 99.4%, respectively (Khandker et al., 2021) .

Our findings are concordant with several colorimetric RT-LAMP studies that reported sensitivity of 70%-90% and specificity of 99%-100% (Nawattanapaiboon et al., 2021; Silva et al., 2021; Subsoontorn et al., 2020) . (Silva et al., 2021) ; the unskilled nasopharyngeal swab collections resulted in viral loads that were too low despite the samples being collected during the early phase of infection (Mallett et al., 2020; Zou et al., 2020) ; and the pH > 8 of the elution buffer in the RNA purification step interfered with the color changes in the RT-LAMP reactions. These issues could contribute to misinterpretation Nawattanapaiboon et al., 2021) . A lack of actual viral loads in RNA copies per microliter unit is a limitation of this study. Direct swab-to-RT-LAMP without an RNA purification step as well as testing with other respiratory specimens such as saliva or sputum should be performed in further studies. Considered together, colorimetric RT-LAMP exhibited satisfactory performance. The assay has good sensitivity and specificity and is able to detect high-to-moderate viral RNA levels, which frequently appear at the early phase of symptom onset.

This study demonstrated that the FastProof™ 30 min-TTR SARS-CoV-2 RT-LAMP Kit provides rapid and simple molecular testing that has high specificity and sensitivity. It has the potential to be used as an alternative screening tool to help speed up SARS-CoV-2 diagnosis.

However, because the sensitivity of RT-LAMP at low viral loads is not comparable to that of RT-qPCR, negative results could not rule out SARS-CoV-2 infection; hence, negative results in patients with COVID-19 symptoms should be confirmed by RT-qPCR. Tables   Table 1 

Samplesno. 315 

A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

Rapid SARS-CoV-2 antigen detection assay in comparison with realtime RT-PCR assay for laboratory diagnosis of COVID-19 in Thailand

Detection of 2019 -nCoV by RT-PCR

A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples

Molecular diagnosis of COVID-19: challenges and research needs

Low clinical performance of the Isopollo COVID-19 detection kit (M Monitor, South Korea) for RT-LAMP SARS-CoV-2 diagnosis: A call for action against low quality products for developing countries

Rapid isothermal amplification and portable detection system for SARS-CoV-2

RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2

Diagnostic accuracy of LAMP versus PCR over the course of SARS-CoV-2 infection

Development and Validation of a Rapid, Single-Step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) System Potentially to Be Used for Reliable and High-Throughput Screening of COVID-19

Diagnostic accuracy of rapid antigen test kits for detecting SARS-CoV-2: A systematic review and meta-analysis of 17,171 suspected COVID-19 patients

Evaluation of rapid diagnosis of novel coronavirus disease (COVID-19) using loop-mediated isothermal amplification

Impact of Severe Acute Respiratory Syndrome Coronavirus 2 Viral Load on Risk of Intubation and Mortality Among Hospitalized Patients With Coronavirus Disease

At what times during infection is SARS-CoV-2 detectable and no longer detectable using RT-PCR based tests?: A systematic review of individual participant data

Colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a visual diagnostic platform for the detection of the emerging coronavirus SARS-CoV-2

Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards

Recent advances and perspectives of nucleic acid detection for coronavirus

Can a field molecular diagnosis be accurate? A performance evaluation of colorimetric RT-LAMP for the detection of SARS-CoV-2 in a hospital setting

The diagnostic accuracy of isothermal nucleic acid point-of-care tests for human coronaviruses: A systematic review and meta-analysis

Viral Load in upper respiratory specimens of infected patients

median (range) 40 yr (<1-92 yr)

Mean ± SD of Ct value of ORF1ab gene (min, max)

Mean ± SD of Ct value of N gene (min, max)

Results of RT-LAMP (FastProof™ 30 min-TTR SARS-CoV

We would like to thank Faculty of Medicine Vajira Hospital for providing samples and patient information. 

The authors declare no conflicts of interest.

The study protocol was approved by the Institutional Review Board of the Faculty of Medicine Vajira Hospital, Navamindradhiraj University (Study code 126/64 E); COA: 131/2564.