Displaying publications 1 - 20 of 33 in total

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  1. Lai MY, Ooi CH, Lau YL
    Malar J, 2021 Mar 25;20(1):166.
    PMID: 33766038 DOI: 10.1186/s12936-021-03707-0
    BACKGROUND: As an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. Under isothermal conditions at 65 °C, the entire procedure takes approximately 30 min to complete. In this study, we establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi.

    METHODS: A total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes.

    RESULTS: LAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2-99.7%) and 100% specific (95% CI 83.2-100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange.

    CONCLUSIONS: These results indicate that SYBR green I LAMP assay is a convenient diagnosis tool for the detection of P. knowlesi in remote settings.

  2. Lai MY, Lau YL
    Am J Trop Med Hyg, 2022 Oct 12;107(4):815-819.
    PMID: 35970289 DOI: 10.4269/ajtmh.22-0136
    We developed a combination of recombinase polymerase and loop-mediated isothermal amplification methods (RAMP) for rapid screening of five human Plasmodium spp. simultaneously. RAMP is a two-stage isothermal amplification method, which consists of a first-stage recombinase polymerase amplification and a second-stage loop-mediated isothermal amplification. Under these two isothermal conditions, five Plasmodium spp. were amplified in less than 40 minutes. We demonstrated RAMP assay with 10-fold better limit of detection than a single (loop-mediated isothermal amplification) LAMP. As compared with microscopy, RAMP assay showed 100% sensitivity (95% CI: 95.65-100.00%) and 100% specificity (95% CI: 69.15-100.00%). The end products were inspected by the color changes of neutral red. Positive reactions were indicated by pink while the negative reactions remained yellow. The combination assay established in this study can be used as a routine diagnostic method for malaria.
  3. Lai MY, Bukhari FDM, Zulkefli NZ, Ismail I, Mustapa NI, Soh TST, et al.
    BMC Infect Dis, 2021 Nov 17;21(1):1162.
    PMID: 34789179 DOI: 10.1186/s12879-021-06876-0
    BACKGROUND: Current assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step.

    METHODS: In the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets.

    RESULTS: Here, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9-96.1%) and 67.4% sensitive (95% CI: 51.5-80.9%) for E gene and RdRp gene, respectively.

    CONCLUSION: Without the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening.

  4. Thong KL, Lai MY, Teh C SJ, Chua KH
    Trop Biomed, 2011 Apr;28(1):21-31.
    PMID: 21602765 MyJurnal
    A PCR-based assay that can simultaneously detect and differentiate five different types of nosocomial bacterial pathogens was developed. Six pairs of selected primers targeting femA (132 bp) and mecA (310 bp) of methicillin-resistant Staphylococcus aureus, gltA (722 bp) of Acinetobacter baumannii, phoA (903 bp) of Escherichia coli, mdh (364 bp) of Klebsiella pneumoniae and oprL (504 bp) of Pseudomonas aeruginosa were used in this study. The conditions were optimized for the multiplex PCR to ensure specific amplification of the selected targets. Sensitivity and specificity tests were also carried out using a blind test approach on 50 bacterial cultures and resulted in 100% for both positive and negative predictive values.
  5. Lai MY, Lau YL
    Parasit Vectors, 2017 Oct 02;10(1):456.
    PMID: 28969712 DOI: 10.1186/s13071-017-2387-y
    BACKGROUND: The identification of receptors or binding partners of Toxoplasma gondii from humans is an essential activity. Many proteins involved in T. gondii invasion have been characterized, and their contribution for parasite entry has been proposed. However, their molecular interactions remain unclear.

    RESULTS: Yeast two-hybrid (Y2H) experiment was used to identify the binding partners of surface antigens of T. gondii by using SAG2 as bait. Colony PCR was performed and positive clones were sent for sequencing to confirm their identity. The yeast plasmids for true positive clones were rescued by transformation into E. coli TOP 10F' cells. The interplay between bait and prey was confirmed by β-galactosidase assay and co-immunoprecipitation experiment. We detected 20 clones interacting with SAG2 based on a series of the selection procedures. Following the autoactivation and toxicity tests, SAG2 was proven to be a suitable candidate as a bait. Thirteen clones were further examined by small scale Y2H experiment. The results indicated that a strong interaction existed between Homo sapiens zinc finger protein and SAG2, which could activate the expressions of the reporter genes in diploid yeast. Co-immunoprecipitation experiment result indicated the binding between this prey and SAG2 protein was significant (Mann-Whitney U-test: Z = -1.964, P = 0.05).

    CONCLUSIONS: Homo sapiens zinc finger protein was found to interact with SAG2. To improve the understanding of this prey protein's function, advanced investigations need to be carried out.

  6. Lai MY, Abdul Hamid MH, Jelip J, Mudin RN, Lau YL
    Am J Trop Med Hyg, 2024 Feb 27.
    PMID: 38412548 DOI: 10.4269/ajtmh.23-0572
    Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can amplify specific nucleic acids at a constant temperature (63-65°C) within a short period (< 1 hour). In this study, we report the utilization of recombinase-aided LAMP to specifically amplify the 18S sRNA of Plasmodium knowlesi. The method was built on a conventional LAMP assay by inclusion of an extra enzyme, namely recombinase, into the master mixture. With the addition of recombinase into the LAMP assay, the assay speed was executed within a time frame of less than 28 minutes at 65°C. We screened 55 P. knowlesi samples and 47 non-P. knowlesi samples. No cross-reactivity was observed for non-P. knowlesi samples, and the detection limit for recombinase-aided LAMP was one copy for P. knowlesi after LAMP amplification. It has been reported elsewhere that LAMP can be detected through fluorescent readout systems. Although such systems result in considerable limits of detection, the need for sophisticated equipment limits their use. Hence, we used here a colorimetric detection platform for the evaluation of the LAMP assay's performance. This malachite green-based recombinase-aided LAMP assay enabled visualization of results with the naked eye. Negative samples were observed by a change in color from green to colorless, whereas positive samples remained green. Our results demonstrate that the LAMP assay developed here is a convenient, sensitive, and useful diagnostic tool for the rapid detection of knowlesi malaria parasites. This method is suitable for implementation in remote healthcare settings, where centralized laboratory facilities, funds, and clinicians are in short supply.
  7. Lai MY, Ooi CH, Lau YL
    Am J Trop Med Hyg, 2018 03;98(3):700-703.
    PMID: 29260656 DOI: 10.4269/ajtmh.17-0738
    The aim of this study was to develop a recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip method for specific diagnosis of Plasmodium knowlesi. With incubation at 37°C, the 18S rRNA gene of P. knowlesi was successfully amplified within 12 minutes. By adding a specifically designed probe to the reaction solution, the amplified RPA product can be visualized on a LF strip. The RPA assay exhibited high sensitivity with limits of detection down to 10 parasites/μL of P. knowlesi. Nonetheless, it was demonstrated that all P. knowlesi (N = 41) and other Plasmodium sp. (N = 25) were positive while negative samples (N = 8) were negative. Therefore, a combination of RPA and LF strip detection is a highly promising approach with the potential to be suitable for use in resource-limited settings.
  8. Lau YL, Ismail I, Mustapa NI, Lai MY, Tuan Soh TS, Hassan A, et al.
    PeerJ, 2020;8:e9278.
    PMID: 32547882 DOI: 10.7717/peerj.9278
    Background: Highly sensitive real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-CoV-2. However, they are costly. Loop-mediated isothermal amplification (LAMP) assay has emerged as a novel alternative isothermal amplification method for the detection of nucleic acid.

    Methods: A rapid, sensitive and specific real-time reverse transcription LAMP (RT-LAMP) assay was developed for SARS-CoV-2 detection.

    Results: This assay detected one copy/reaction of SARS-CoV-2 RNA in 30 min. Both the clinical sensitivity and specificity of this assay were 100%. The RT-LAMP showed comparable performance with RT-qPCR. Combining simplicity and cost-effectiveness, this assay is therefore recommended for use in resource resource-limited settings.

  9. Lai MY, Ooi CH, Lau YL
    Am J Trop Med Hyg, 2017 Nov;97(5):1597-1599.
    PMID: 28820700 DOI: 10.4269/ajtmh.17-0427
    In this study, we developed a recombinase polymerase amplification (RPA) assay for specific diagnosis of Plasmodium knowlesi. Genomic DNA was extracted from whole blood samples using a commercial kit. With incubation at 37°C, the samples were successfully amplified within 20 minutes. The end product of RPA was further examined by loading onto agarose gel and a specific band was observed with a size of 128 bp. The RPA assay exhibited high sensitivity with limits of detection down to one copy of the plasmid. From the specificity experiments, it was demonstrated that all P. knowlesi samples (N = 45) were positive while other Plasmodium spp. (N = 42) and negative samples (N = 6) were negative. Therefore, the RPA assay is a highly promising approach with the potential to be used in resource-limited settings. This assay can be further optimized for bedside and on field application.
  10. Lai MY, Suppiah J, Thayan R, Ismail I, Mustapa NI, Soh TST, et al.
    Trop Med Health, 2022 Jan 04;50(1):2.
    PMID: 34980275 DOI: 10.1186/s41182-021-00396-y
    BACKGROUND: Current diagnosis of SARS-CoV-2 infection relies on RNA purification prior to amplification. Typical extraction methods limit the processing speed and turnaround time for SARS-CoV-2 diagnostic testing.

    METHODS: Here, we applied reverse transcription loop-mediated isothermal amplification directly onto human clinical swabs samples to amplify the RNA from SARS-CoV-2 swab samples after processing with chelating resin.

    RESULTS: By testing our method on 64 samples, we managed to develop an RT-LAMP assay with 95.9% sensitivity (95% CI 86 to 99.5%) and 100% specificity (95% CI 78.2-100%).

    CONCLUSION: The entire process including sample processing can be completed in approximately 50 min. This method has promising potential to be applied as a fast, simple and inexpensive diagnostic tool for the detection of SARS-CoV-2.

  11. Amir A, Shahari S, Liew JWK, de Silva JR, Khan MB, Lai MY, et al.
    Acta Trop, 2020 Nov;211:105596.
    PMID: 32589995 DOI: 10.1016/j.actatropica.2020.105596
    Zoonotic cases of Plasmodium knowlesi account for most malaria cases in Malaysia, and humans infected with P. cynomolgi, another parasite of macaques have recently been reported in Sarawak. To date the epidemiology of malaria in its natural Macaca reservoir hosts remains little investigated. In this study we surveyed the prevalence of simian malaria in wild macaques of three states in Peninsular Malaysia, namely Pahang, Perak and Johor using blood samples from 103 wild macaques (collected by the Department of Wildlife and National Parks Peninsular Malaysia) subjected to microscopic examination and nested PCR targeting the Plasmodium small subunit ribosomal RNA gene. As expected, PCR analysis yielded significantly higher prevalence (64/103) as compared to microscopic examination (27/103). No relationship between the age and/or sex of the macaques with the parasitaemia and the Plasmodium species infecting the macaques could be identified. Wild macaques in Pahang had the highest prevalence of Plasmodium parasites (89.7%), followed by those of Perak (69.2%) and Johor (28.9%). Plasmodium inui and P. cynomolgi were the two most prevalent species infecting the macaques from all three states. Half of the macaques (33/64) harboured two or more Plasmodium species. These data provide a baseline survey, which should be extended by further longitudinal investigations that should be associated with studies on the bionomics of the anopheline vectors. This information will allow an accurate evaluation of the risk of zoonotic transmission to humans, and to elaborate effective strategies to control simian malaria.
  12. Lau YL, Lai MY, Fong MY, Jelip J, Mahmud R
    Am J Trop Med Hyg, 2016 Feb;94(2):336-339.
    PMID: 26598573 DOI: 10.4269/ajtmh.15-0569
    The lack of rapid, affordable, and accurate diagnostic tests represents the primary hurdle affecting malaria surveillance in resource- and expertise-limited areas. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cheap diagnostic method. Five species-specific LAMP assays were developed based on 18S rRNA gene. Sensitivity and specificity of LAMP results were calculated as compared with microscopic examination and nested polymerase chain reaction. LAMP reactions were highly sensitive with the detection limit of one copy for Plasmodium vivax, Plasmodium falciparum, and Plasmodium malariae and 10 copies for Plasmodium knowlesi and Plasmodium ovale. LAMP positively detected all human malaria species in all positive samples (N = 134; sensitivity = 100%) within 35 minutes. All negative samples were not amplified by LAMP (N = 67; specificity = 100%). LAMP successfully detected two samples with very low parasitemia. LAMP may offer a rapid, simple, and reliable test for the diagnosis of malaria in areas where malaria is prevalent.
  13. Lai MY, Abdul Hamid M, Jelip J, Mudin RN, Lau YL
    Am J Trop Med Hyg, 2023 Mar 13.
    PMID: 36913921 DOI: 10.4269/ajtmh.22-0657
    This study highlights the development of two lateral flow recombinase polymerase amplification assays for the diagnosis of human malaria. The lateral flow cassettes contained test lines that captured biotin-, 6-carboxyfluorescein, digoxigenin-, cyanine 5-, and dinitrophenyl-labeled amplicons. The overall process can be completed in 30 minutes. Recombinase polymerase amplification coupled with lateral flow had a detection limit of 1 copy/µL for Plasmodium knowlesi, Plasmodium vivax, and Plasmodium falciparum. No cross-reactivity was observed among nonhuman malaria parasites such as Plasmodium coatneyi, Plasmodium cynomolgi, Plasmodium brasilanium, Plasmodium inui, Plasmodium fragile, Toxoplasma gondii, Sarcocystis spp., Brugia spp., and 20 healthy donors. It is rapid, highly sensitive, robust, and easy to use. The result can be read without the need for special equipment and thus has the potential to serve as an effective alternative to polymerase chain reaction methods for the diagnosis of malaria.
  14. Lai MY, Abdul-Majid N, Lau YL
    Acta Parasitol, 2019 Sep;64(3):575-581.
    PMID: 31165984 DOI: 10.2478/s11686-019-00066-4
    Toxoplasma gondii is one of the most successful human pathogens. To eliminate the infection, identification of receptors or binding partners from humans is indeed urgent. T. gondii surface antigen is the ultimate component involved during the attachment of parasite into host cell. However, mechanism of invasion between SAG and host-cell membrane remains unclear. Yeast two-hybrid experiment was used to identify the binding partners from cDNA human library by using T. gondii SAG1 as bait. Mated yeast cells were plated on DDO/X plates to confirm only prey plasmid that expressing interacting protein was selected. We detected 39 clones interacted with SAG1 based on a series of the selection procedures. After colony PCR, only 29 clones were positive and subsequently sent for sequencing. The yeast plasmids for true positive clones were rescued by transformation into E. coli TOP 10F' cells. Twenty-two clones were further examined by small-scale Y2H experiment. The results indicated that a strong interaction existed between Homo sapiens lysine-rich coil-coiled and SAG1 protein, which could activate the expressions of the reporter genes in diploid yeast. Co-immunoprecipitation experiment result indicated the binding between this prey and SAG1 protein was significant (Mann-Whitney U test, Z = - 1.964, P = 0.05). H. sapiens lysine-rich coil-coiled protein was found to be interacted with SAG1. This prey protein may serve as the potential drug target in vaccination study.
  15. Lai MY, Rafieqin N, Lee PYL, Amir Rawa MS, Dzul S, Yahaya N, et al.
    Trop Biomed, 2021 Sep 01;38(3):248-253.
    PMID: 34362867 DOI: 10.47665/tb.38.3.065
    Through the regional control programme, Malaysia has been successfully reducing the incidence of Plasmodium falciparum and Plasmodium vivax infections. However, the incidence of zoonotic malaria Plasmodium knowlesi infection is increasing and now has been the major cause of malaria in Malaysia especially Malaysian Borneo. The emergence of knowlesi infection has threatened the malaria elimination programme which the government aims to reduce the overall malaria infections by 2020. Unlike other benign human Plasmodium spp., P. knowlesi can cause fatal infections. The aim of this study was to determine the incidence and distribution of five human malaria parasites including P. knowlesi in Peninsular Malaysia and Malaysian Borneo. A total of 112 blood samples were collected from seven states and district hospitals in Peninsular Malaysia and Malaysian Borneo from year 2015 to 2016. The samples were examined by microscopy and further confirmed by nested PCR assay targeting 18S rRNA gene of Plasmodium spp. Following the nested PCR assays, a total of 54 (48.2%) samples were positive for P. knowlesi infections, 12 (10.7%) cases were positive for P. vivax infections, followed by 7 (6.3%) cases of P. falciparum and 4 (3.5%) cases of P. malariae. There were 3 cases (2.7%) of mixed infections (P. knowlesi/P. vivax). However, no cases were identified as P. ovale. A total of 32 (28.6%) cases were found as negative infections. LoopMediated Isothermal Amplification Assay (LAMP) was performed to confirm inconclusive results produced by microscopy and nested PCR. P. knowlesi showed the highest prevalence in Sarawak (n= 30), Sabah (n=13), Pulau Pinang (n=5) and Pahang (n=6). PCR and LAMP was not able to detect a large number of microscopy positive samples due to DNA degradation during storage and shipping. Among all the states involved in this study, the highest prevalence of P. knowlesi infection was found in Sabah and Sarawak.
  16. Wong KC, Lai MY, De Silva JR, Cheong FW, Fong MY, Lau YL
    Trop Biomed, 2021 Jun 01;38(2):143-148.
    PMID: 34172703 DOI: 10.47665/tb.38.2.051
    Normocyte binding protein Xa (NBPXa) has been implied to play a significant role in parasite invasion of human erythrocytes. Previous phylogenetic studies have reported the existence of three types of NBPXa for Plasmodium knowlesi (PkNBPXa). PkNBPXa region II (PkNBPXaII) of type 1, type 2 and type 3 were expressed on mammalian cell surface and interacted with human and macaque (Macaca fascicularis) erythrocytes. The binding activities of PkNBPXaII towards human and macaque erythrocytes were evaluated using erythrocyte-binding assay (EBA). Three parameters were evaluated to achieve the optimal protein expression of PkNBPXaII and erythrocyte binding activity in EBA: types of mammalian cells, post transfection time and erythrocyte incubation time. COS-7, HEK-293, and CHO-K1 cells showed successful expression of PkNBPXaII, despite the protein expression is weak compared to the positive control. COS-7 was used in EBA. All three types of PkNBPXaII showed rosette formation with macaque erythrocytes but not with human erythrocytes. Future studies to enhance the PkNBPXaII expression on surface of mammalian cells is indeed needed in order to elucidate the specific role of PkNBPXaII in erythrocytes invasion.
  17. Lai MY, Ooi CH, Jaimin JJ, Lau YL
    Am J Trop Med Hyg, 2020 06;102(6):1370-1372.
    PMID: 32228783 DOI: 10.4269/ajtmh.20-0001
    The incidence of zoonotic malaria, Plasmodium knowlesi, infection is increasing and now is the major cause of malaria in Malaysia. Here, we describe a WarmStart colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Plasmodium spp. The detection limit for this assay was 10 copies/µL for P knowlesi and Plasmodium ovale and 1 copy/µL for Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae. To test clinical sensitivity and specificity, 100 microscopy-positive and 20 malaria-negative samples were used. The WarmStart colorimetric LAMP was 98% sensitive and 100% specific. Amplification products were visible for direct observation, thereby eliminating the need for post-amplification processing steps. Therefore, WarmStart colorimetric LAMP is suitable for use in resource-limited settings.
  18. Zen LPY, Lai MY, Lau YL
    Trop Biomed, 2020 Dec 01;37(4):1124-1128.
    PMID: 33612764 DOI: 10.47665/tb.37.4.1124
    The LAMP assay, amplifies the target DNA rapidly, with 10-fold greater sensitivity than conventional PCR. The greater sensitivity also comes with greater risks of contamination. To overcome this issue, the current project includes either uracil DNA glycosylase (UDG) or a mineral oil overlay in the LAMP assay. Our results indicated that UDG or a mineral oil overlay can effectively prevent carryover contamination in the LAMP assay for the detection of human malaria. By incorporating these preventative methods, contamination can be eliminated and LAMP can potentially be used in the field; and point of care diagnosis for human malaria.
  19. Lau YL, Ismail IB, Mustapa NIB, Lai MY, Tuan Soh TS, Haji Hassan A, et al.
    PLoS One, 2021;16(1):e0245164.
    PMID: 33406112 DOI: 10.1371/journal.pone.0245164
    Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.
  20. Mallepaddi PC, Lai MY, Podha S, Ooi CH, Liew JW, Polavarapu R, et al.
    Am J Trop Med Hyg, 2018 09;99(3):704-708.
    PMID: 29943720 DOI: 10.4269/ajtmh.18-0177
    The present study aims to develop a method for rapid diagnosis of malaria using loop-mediated isothermal amplification (LAMP) combined with a lateral flow device (LFD). By adding the biotin-labeled and fluorescein amidite-labeled loop primers to the LAMP reaction solution, the end product can be visualized on a LFD. The entire procedure takes approximately 42 minutes to complete, LAMP assay exhibited high sensitivity, as the detection limit was 0.01 pg/μL for all five Plasmodium species. It was demonstrated that all Plasmodium knowlesi (N = 90) and Plasmodium vivax (N = 56) were positively amplified by LAMP-LFD assay, whereas healthy donor samples (N = 8) were negative. However, not all mixed infections were positive, and other infected nonmalaria samples were negative. Loop-mediated isothermal amplification-LFD represents a robust approach with potential suitability for use in resource-constrained laboratories. We believe that LAMP-LFD has a potential to be developed as point-of-care diagnostic tool in future.
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