Displaying publications 61 - 80 of 85 in total

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  1. Evaluation of diagnostic accuracy of loop-mediated isothermal amplification method (LAMP) compared with polymerase chain reaction (PCR) for Leptospira spp. in clinical samples: a systematic review and meta-analysis
    Gunasegar S, Neela VK
    Diagn Microbiol Infect Dis, 2021 Jul;100(3):115369.
    PMID: 33845305 DOI: 10.1016/j.diagmicrobio.2021.115369
    Loop-mediated isothermal amplification (LAMP) test is widely used in molecular diagnostics as a point-of-care technique alternative to traditional PCR especially in resource-limited countries. LAMP has been recently used to diagnose leptospirosis. Therefore, we undertook a systematic review and meta-analysis to compare the accuracy of LAMP with PCR in the diagnosis of leptospirosis. Sixty-one studies were extracted from three international databases and analyzed throughout using the PRISMA guideline. The pooled sensitivity of LAMP and PCR technique was 0.80 (95% CI: 0.58-0.90) and 0.54 (95% CI: 0.35-0.67) respectively indicating that LAMP is more sensitive than PCR. The Q* value of LAMP and PCR-based technique is 274.61 and 397.95, respectively. Among the analyzed studies, significant heterogeneity was observed where I2 is 90.90% for LAMP-based and 86.18% for PCR-based. Our study suggests that LAMP has better diagnostic accuracy than PCR. However, future work should be carried out to reduce heterogeneity as well as to improve and develop effective intervention strategies.
    Matched MeSH terms: Nucleic Acid Amplification Techniques*
  2. Isothermal amplifications - a comprehensive review on current methods
    Glökler J, Lim TS, Ida J, Frohme M
    Crit Rev Biochem Mol Biol, 2021 12;56(6):543-586.
    PMID: 34263688 DOI: 10.1080/10409238.2021.1937927
    The introduction of nucleic acid amplification techniques has revolutionized the field of medical diagnostics in the last decade. The advent of PCR catalyzed the increasing application of DNA, not just for molecular cloning but also for molecular based diagnostics. Since the introduction of PCR, a deeper understanding of molecular mechanisms and enzymes involved in DNA/RNA replication has spurred the development of novel methods devoid of temperature cycling. Isothermal amplification methods have since been introduced utilizing different mechanisms, enzymes, and conditions. The ease with which isothermal amplification methods have allowed nucleic acid amplification to be carried out has had a profound impact on the way molecular diagnostics are being designed after the turn of the millennium. With all the advantages isothermal amplification brings, the issues or complications surrounding each method are heterogeneous making it difficult to identify the best approach for an end-user. This review pays special attention to the various isothermal amplification methods by classifying them based on the mechanistic characteristics which include reaction formats, amplification information, promoter, strand break, and refolding mechanisms. We would also compare the efficiencies and usefulness of each method while highlighting the potential applications and detection methods involved. This review will serve as an overall outlook on the journey and development of isothermal amplification methods as a whole.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  3. Urine as a diagnostic specimen for the detection of Chlamydia trachomatis in Malaysia by ligase chain reaction
    Gaydos CA, Ngeow YF, Lee HH, Canavaggio M, Welsh LE, Johanson J, et al.
    Sex Transm Dis, 1996 9 1;23(5):402-6.
    PMID: 8885072
    BACKGROUND AND OBJECTIVES: Noninvasive urine screening for Chlamydia trachomatis infections offers a valuable public health tool, which could be of vast importance in chlamydial control programs. The authors evaluated a new DNA amplification method, ligase chain reaction (LCR).

    GOALS: The goal was to ascertain whether urine testing could be used as screening method to detect C. trachomatis infections in commercial sex workers, patients at sexually transmitted diseases clinic, and asymptomatic patients in Kuala Lumpur, Malaysia.

    METHODS: First-void urine specimens from 300 men and 300 women were tested by LCR, as well as by a commercially available enzyme immunoassay. The LCR assay amplifies specific sequences within the chlamydial plasmid with ligand-labeled probes, and the resultant amplicons are detected by an automated immunoassay. Specimens with discrepant results were confirmed by another LCR of the specimen that targeted the gene for the major outer membrane protein (OMP1).

    RESULTS: There were 31 LCR-positive male urine and 37 LCR-positive female urine specimens. The resolved sensitivity and specificity for the LCR of the male urine specimens were 100% and 99.6%, respectively, whereas for female urine specimens, the sensitivity and specificity were 100% and 98.5%, respectively. After resolution of discrepant test results by OMP1 LCR, the prevalence was 10% for men and 11% for women. The urine enzyme immunoassay was not useful in diagnosing C. trachomatis infections in either men or women, as the resolved sensitivities were 10% and 15.2%, respectively. The specificities were 99.6% for men and 98.9% for women.

    CONCLUSIONS: Testing first-void urine specimens by LCR is a highly sensitive and specific method to diagnose C. trachomatis infections in men and women, providing health care workers and public health officials with a new molecular amplification assay that uses noninvasive urine specimens for population-based screening purposes.

    Matched MeSH terms: Nucleic Acid Amplification Techniques*
  4. Fulltext Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample
    Foo PC, Nurul Najian AB, Muhamad NA, Ahamad M, Mohamed M, Yean Yean C, et al.
    BMC Biotechnol, 2020 Jun 22;20(1):34.
    PMID: 32571286 DOI: 10.1186/s12896-020-00629-8
    BACKGROUND: This study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model.

    RESULTS: A set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica. This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites.

    CONCLUSIONS: The analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.

    Matched MeSH terms: Nucleic Acid Amplification Techniques
  5. Development of a thermostabilised triplex LAMP assay with dry-reagent four target lateral flow dipstick for detection of Entamoeba histolytica and non-pathogenic Entamoeba spp
    Foo PC, Chan YY, Mohamed M, Wong WK, Nurul Najian AB, Lim BH
    Anal Chim Acta, 2017 May 08;966:71-80.
    PMID: 28372729 DOI: 10.1016/j.aca.2017.02.019
    This study highlighted the development of a four target nitrocellulose-based nucleic acid lateral flow immunoassay biosensor in a dry-reagent strip format for interpretation of double-labelled double-stranded amplicons from thermostabilised triplex loop-mediated isothermal amplification assay. The DNA biosensor contained two test lines which captured biotin and texas red labelled amplicons; a LAMP internal amplification control line that captured digoxigenin labelled amplicon; and a chromatography control line that validated the functionality of the conjugated gold nanoparticles and membrane. The red lines on detection pad were generated when the gold nanoparticles conjugated antibody bound to the fluorescein labelled amplicons, and the capture agents bound to their specific hapten on the other 5' end of the double-stranded amplicon. The applicability of this DNA biosensor was demonstrated using amoebiasis-causing Entamoeba histolytica simultaneously with the non-pathogenic but morphologically identical Entamoeba dispar and Entamoeba moshkovskii. The biosensor detection limit was 10 E. histolytica trophozoites, and revealed 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. Heat stability test showed that the biosensor was stable for at least 181 days at ambient temperature. This ready-to-use and cold-chain-free biosensor facilitated the post-LAMP analysis based on visualisation of lines on strip instead of observation of amplicon patterns in agarose gel.
    Matched MeSH terms: Nucleic Acid Amplification Techniques*
  6. Fulltext Detection of human papillomavirus in oropharyngeal squamous cell carcinoma
    Fauzi FH, Hamzan NI, Rahman NA, Suraiya S, Mohamad S
    J Zhejiang Univ Sci B, 2021 4 13;21(12):961-976.
    PMID: 33843162 DOI: 10.1631/jzus.B2000161
    Worldwide there has been a significant increase in the incidence of oropharyngeal squamous cell carcinoma (OPSCC) etiologically attributed to oncogenic human papillomavirus (HPV). Reliable and accurate identification and detection tools are important as the incidence of HPV-related cancer is on the rise. Several HPV detection methods for OPSCC have been developed and each has its own advantages and disadvantages in regard to sensitivity, specificity, and technical difficulty. This review summarizes our current knowledge of molecular methods for detecting HPV in OPSCC, including HPV DNA/RNA polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), p16 immunohistochemistry (IHC), and DNA/RNA in situ hybridization (ISH) assays. This summary may facilitate the selection of a suitable method for detecting HPV infection, and therefore may help in the early diagnosis of HPV-related carcinoma to reduce its mortality, incidence, and morbidity.
    Matched MeSH terms: Nucleic Acid Amplification Techniques
  7. An ambient temperature stable and ready-to-use loop-mediated isothermal amplification assay for detection of toxigenic Vibrio cholerae in outbreak settings
    Engku Nur Syafirah EAR, Nurul Najian AB, Foo PC, Mohd Ali MR, Mohamed M, Yean CY
    Acta Trop, 2018 Jun;182:223-231.
    PMID: 29545156 DOI: 10.1016/j.actatropica.2018.03.004
    Cholera, caused by Vibrio cholerae is a foodborne disease that frequently reported in food and water related outbreak. Rapid diagnosis of cholera infection is important to avoid potential spread of disease. Among available diagnostic platforms, loop-mediated isothermal amplification (LAMP) is regarded as a potential diagnostic tool due to its rapidity, high sensitivity and specificity and independent of sophisticated thermalcycler. However, the current LAMP often requires multiple pipetting steps, hence is susceptible to cross contamination. Besides, the strict requirement of cold-chain during transportation and storage make its application in low resource settings to be inconvenient. To overcome these problems, the present study is aimed to develop an ambient-temperature-stable and ready-to-use LAMP assay for the detection of toxigenic Vibrio cholerae in low resource settings. A set of specific LAMP primers were designed and tested against 155 V. cholerae and non-V. cholerae strains. Analytical specifity showed that the developed LAMP assay detected 100% of pathogenic V. cholerae and did not amplified other tested bacterial strains. Upon testing against stool samples spiked with toxigenic V. cholerae outbreak isolates, the LAMP assay detected all of the spiked samples (n = 76/76, 100%), in contrast to the conventional PCR which amplified 77.6% (n = 59/76) of the tested specimens. In term of sensitivity, the LAMP assay was 100-fold more sensitive as compared to the conventional PCR method, with LOD of 10 fg per μL and 10 CFU per mL. Following lyophilisation with addition of lyoprotectants, the dry-reagent LAMP mix has an estimated shelf-life of 90.75 days at room temperature.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  8. The reliability of a rapid molecular detection method in determining the prevalence of rifampicin-resistant Mycobacterium tuberculosis in an urban district health facility in Malaysia
    Ding CH, Ismail Z, Sulong A, Wahab AA, Gan B, Mustakim S, et al.
    Malays J Pathol, 2020 Dec;42(3):401-407.
    PMID: 33361721
    INTRODUCTION: Rifampicin is a key first-line antimycobacterial agent employed for the treatment of pulmonary tuberculosis (PTB). This study sought to obtain prevalence data on rifampicin-resistant Mycobacterium tuberculosis among smear-positive PTB patients in the Klang District of Malaysia.

    MATERIALS AND METHODS: A total of 103 patients from the Chest Clinic of Hospital Tengku Ampuan Rahimah with sputum smears positive for acid-fast bacilli were included in this cross-sectional study. All sputa were tested using Xpert MTB/RIF to confirm the presence of M. tuberculosis complex and detect rifampicin resistance. Sputa were also sent to a respiratory medicine institute for mycobacterial culture. Positive cultures were then submitted to a reference laboratory, where isolates identified as M. tuberculosis complex underwent drug susceptibility testing (DST).

    RESULTS: A total of 58 (56.3%) patients were newly diagnosed and 45 (43.7%) patients were previously treated. Xpert MTB/RIF was able to detect rifampicin resistance with a sensitivity and specificity of 87.5% and 98.9%, respectively. Assuming that a single resistant result from Xpert MTB/RIF or any DST method was sufficient to denote resistance, a total of 8/103 patients had rifampicinresistant M. tuberculosis. All eight patients were previously treated for PTB (p<0.05). The overall prevalence of rifampicin resistance among smear-positive PTB patients was 7.8%, although it was 17.8% among the previously treated ones.

    CONCLUSION: The local prevalence of rifampicin-resistant M. tuberculosis was particularly high among previously treated patients. Xpert MTB/RIF can be employed in urban district health facilities not only to diagnose PTB in smear-positive patients, but also to detect rifampicin resistance with good sensitivity and specificity.

    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  9. Long Fragment Polymerase Chain Reaction
    Chua EW, Maggo S, Kennedy MA
    Methods Mol Biol, 2017;1620:65-74.
    PMID: 28540699 DOI: 10.1007/978-1-4939-7060-5_3
    Polymerase chain reaction (PCR) is an oft-used preparatory technique in amplifying specific DNA regions for downstream analysis. The size of an amplicon was initially limited by errors in nucleotide polymerization and template deterioration during thermal cycling. A variant of PCR, designated long-range PCR, was devised to counter these drawbacks and enable the amplification of large fragments exceeding a few kb. In this chapter we describe a protocol for long-range PCR, which we have adopted to obtain products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  10. Polydimethylsiloxane-Paper Hybrid Lateral Flow Assay for Highly Sensitive Point-of-Care Nucleic Acid Testing
    Choi JR, Liu Z, Hu J, Tang R, Gong Y, Feng S, et al.
    Anal Chem, 2016 06 21;88(12):6254-64.
    PMID: 27012657 DOI: 10.1021/acs.analchem.6b00195
    In nucleic acid testing (NAT), gold nanoparticle (AuNP)-based lateral flow assays (LFAs) have received significant attention due to their cost-effectiveness, rapidity, and the ability to produce a simple colorimetric readout. However, the poor sensitivity of AuNP-based LFAs limits its widespread applications. Even though various efforts have been made to improve the assay sensitivity, most methods are inappropriate for integration into LFA for sample-to-answer NAT at the point-of-care (POC), usually due to the complicated fabrication processes or incompatible chemicals used. To address this, we propose a novel strategy of integrating a simple fluidic control strategy into LFA. The strategy involves incorporating a piece of paper-based shunt and a polydimethylsiloxane (PDMS) barrier to the strip to achieve optimum fluidic delays for LFA signal enhancement, resulting in 10-fold signal enhancement over unmodified LFA. The phenomena of fluidic delay were also evaluated by mathematical simulation, through which we found the movement of fluid throughout the shunt and the tortuosity effects in the presence of PDMS barrier, which significantly affect the detection sensitivity. To demonstrate the potential of integrating this strategy into a LFA with sample-in-answer-out capability, we further applied this strategy into our prototype sample-to-answer LFA to sensitively detect the Hepatitis B virus (HBV) in clinical blood samples. The proposed strategy offers great potential for highly sensitive detection of various targets for wide application in the near future.
    Matched MeSH terms: Nucleic Acid Amplification Techniques
  11. An integrated lateral flow assay for effective DNA amplification and detection at the point of care
    Choi JR, Hu J, Gong Y, Feng S, Wan Abas WA, Pingguan-Murphy B, et al.
    Analyst, 2016 05 10;141(10):2930-9.
    PMID: 27010033 DOI: 10.1039/c5an02532j
    Lateral flow assays (LFAs) have been extensively explored in nucleic acid testing (NAT) for medical diagnostics, food safety analysis and environmental monitoring. However, the amount of target nucleic acid in a raw sample is usually too low to be directly detected by LFAs, necessitating the process of amplification. Even though cost-effective paper-based amplification techniques have been introduced, they have always been separately performed from LFAs, hence increasing the risk of reagent loss and cross-contaminations. To date, integrating paper-based nucleic acid amplification into colorimetric LFA in a simple, portable and cost-effective manner has not been introduced. Herein, we developed an integrated LFA with the aid of a specially designed handheld battery-powered system for effective amplification and detection of targets in resource-poor settings. Interestingly, using the integrated paper-based loop-mediated isothermal amplification (LAMP)-LFA, we successfully performed highly sensitive and specific target detection, achieving a detection limit of as low as 3 × 10(3) copies of target DNA, which is comparable to the conventional tube-based LAMP-LFA in an unintegrated format. The device may serve in conjunction with a simple paper-based sample preparation to create a fully integrated paper-based sample-to-answer diagnostic device for point-of-care testing (POCT) in the near future.
    Matched MeSH terms: Nucleic Acid Amplification Techniques*
  12. Fulltext Identification of Mycobacterium tuberculosis-specific DNA marker using MAMA-PCR
    Chin Kai Ling, Jaeyres Jani, Zainal Arifin Mustapha
    Malaysian Journal of Medicine and Health Sciences, 2019;15(106):36-36.
    MyJurnal
    Introduction: Tuberculosis (TB), commonly caused by Mycobacterium tuberculosis (Mtb), is one of the ten leading causes of death worldwide. The gold standard, microbiological culture for detection and differentiation of mycobac-teria are time-consuming and laborious. The use of fast, easy and sensitive nucleic acid amplification tests (NAATs) for diagnosis of TB remains challenging because there is a high degree of homology within Mtb complex (MTBC) members and absence of target genes in the genome of some strains. This study aimed to identify new candidate genetic marker and to design specific primers to detect Mtb using in silico methods. Methods: Using Basic Local Alignment Search Tool (BLAST) program, Mtb H37Rv chromosome reference genome sequence was mapped with other MTBC members and a single nucleotide polymorphism (SNP) at Rv1970 was found to be specific only for Mtb strains. Mismatch amplification mutation assay (MAMA) combine with polymerase chain reaction (PCR) was used as an alternative method to detect the point mutation. MAMA primers targeting the SNP were designed using Primer-BLAST and the PCR assay was optimized via Taguchi method. Results: The assay amplified a 112 bp gene fragment and was able to detect all Mtb strains, but not the other MTBC members and non-tuberculous Mycobacte-ria. The detection limit of the assay was 60 pg/μl. Conclusion: Bioinformatics has provided predictive identification of many new target markers. The designed primers were found to be highly specific at single-gene target resolution for detection of Mtb.
    Matched MeSH terms: Nucleic Acid Amplification Techniques
  13. Haplotype diversity of 17 Y-chromosomal STRs in three native Sarawak populations (Iban, Bidayuh and Melanau) in East Malaysia
    Chang YM, Swaran Y, Phoon YK, Sothirasan K, Sim HT, Lim KB, et al.
    Forensic Sci Int Genet, 2009 Jun;3(3):e77-80.
    PMID: 19414156 DOI: 10.1016/j.fsigen.2008.07.007
    17 Y-STRs (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635 or Y-GATA C4, DYS392, Y-GATA H4, DYS437, DYS438 and DYS448) have been analyzed in 320 male individuals from Sarawak, an eastern state of Malaysia on the Borneo island using the AmpFlSTR Y-filer (Applied Biosystems, Foster City, CA). These individuals were from three indigenous ethnic groups in Sarawak comprising of 103 Ibans, 113 Bidayuhs and 104 Melanaus. The observed 17-loci haplotypes and the individual allele frequencies for each locus were estimated, whilst the locus diversity, haplotype diversity and discrimination capacity were calculated in the three groups. Analysis of molecular variance (AMOVA) indicated that 87.6% of the haplotypic variation was found within population and 12.4% between populations (fixation index F(ST)=0.124, p=0.000). This study has revealed that the indigenous populations in Sarawak are distinctly different to each other, and to the three major ethnic groups in Malaysia (Malays, Chinese and Indians), with the Melanaus having a strikingly high degree of shared haplotypes within. There are rare unusual variants and microvariants that were not present in Malaysian Malay, Chinese or Indian groups. In addition, occurrences of DYS385 duplications which were only noticeably present in Chinese group previously was also observed in the Iban group whilst null alleles were detected at several Y-loci (namely DYS19, DYS392, DYS389II and DYS448) in the Iban and Melanau groups.
    Matched MeSH terms: Nucleic Acid Amplification Techniques
  14. DNA Switch: Toehold-Mediated DNA Isothermal Amplification for Dengue Serotyping
    Chan SK, Kuzuya A, Choong YS, Lim TS
    SLAS Discov, 2019 01;24(1):68-76.
    PMID: 30063871 DOI: 10.1177/2472555218791743
    The inherent ability of nucleic acids to recognize a complementary pair has gained wide popularity in DNA sensor applications. DNA molecules can be produced in bulk and easily incorporated with various nanomaterials for sensing applications. More complex designs and sophisticated DNA sensors have been reported over the years to allow DNA detection in a faster, cheaper, and more convenient manner. Here, we report a DNA sensor designed to function like a switch to turn "on" silver nanocluster (AgNC) generation in the presence of a specific DNA target. By defining the probe region sequence, we are able to tune the color of the AgNC generated in direct relation to the different targets. As a proof of concept, we used dengue RNA-dependent RNA polymerase conserved sequences from all four serotypes as targets. This method was able to distinguish each dengue serotype by generating the serotype-respective AgNCs. The DNA switch was also able to identify and amplify the correct target in a mixture of targets with good specificity. This strategy has a detection limit of between 1.5 and 2.0 µM depending on the sequence of AgNC. The DNA switch approach provides an attractive alternative for single-target or multiplex DNA detection.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  15. Fulltext Rapid electrochemical detection of coronavirus SARS-CoV-2
    Chaibun T, Puenpa J, Ngamdee T, Boonapatcharoen N, Athamanolap P, O'Mullane AP, et al.
    Nat Commun, 2021 02 05;12(1):802.
    PMID: 33547323 DOI: 10.1038/s41467-021-21121-7
    Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
  16. Fulltext A simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (HtLAMP) assay for malaria elimination
    Britton S, Cheng Q, Sutherland CJ, McCarthy JS
    Malar J, 2015;14:335.
    PMID: 26315027 DOI: 10.1186/s12936-015-0848-3
    To detect all malaria infections in elimination settings sensitive, high throughput and field deployable diagnostic tools are required. Loop-mediated isothermal amplification (LAMP) represents a possible field-applicable molecular diagnostic tool. However, current LAMP platforms are limited by their capacity for high throughput.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  17. Fulltext Sensitive Detection of Plasmodium vivax Using a High-Throughput, Colourimetric Loop Mediated Isothermal Amplification (HtLAMP) Platform: A Potential Novel Tool for Malaria Elimination
    Britton S, Cheng Q, Grigg MJ, Poole CB, Pasay C, William T, et al.
    PLoS Negl Trop Dis, 2016 Feb;10(2):e0004443.
    PMID: 26870958 DOI: 10.1371/journal.pntd.0004443
    INTRODUCTION: Plasmodium vivax malaria has a wide geographic distribution and poses challenges to malaria elimination that are likely to be greater than those of P. falciparum. Diagnostic tools for P. vivax infection in non-reference laboratory settings are limited to microscopy and rapid diagnostic tests but these are unreliable at low parasitemia. The development and validation of a high-throughput and sensitive assay for P. vivax is a priority.

    METHODS: A high-throughput LAMP assay targeting a P. vivax mitochondrial gene and deploying colorimetric detection in a 96-well plate format was developed and evaluated in the laboratory. Diagnostic accuracy was compared against microscopy, antigen detection tests and PCR and validated in samples from malaria patients and community controls in a district hospital setting in Sabah, Malaysia.

    RESULTS: The high throughput LAMP-P. vivax assay (HtLAMP-Pv) performed with an estimated limit of detection of 1.4 parasites/ μL. Assay primers demonstrated cross-reactivity with P. knowlesi but not with other Plasmodium spp. Field testing of HtLAMP-Pv was conducted using 149 samples from symptomatic malaria patients (64 P. vivax, 17 P. falciparum, 56 P. knowlesi, 7 P. malariae, 1 mixed P. knowlesi/P. vivax, with 4 excluded). When compared against multiplex PCR, HtLAMP-Pv demonstrated a sensitivity for P. vivax of 95% (95% CI 87-99%); 61/64), and specificity of 100% (95% CI 86-100%); 25/25) when P. knowlesi samples were excluded. HtLAMP-Pv testing of 112 samples from asymptomatic community controls, 7 of which had submicroscopic P. vivax infections by PCR, showed a sensitivity of 71% (95% CI 29-96%; 5/7) and specificity of 93% (95% CI87-97%; 98/105).

    CONCLUSION: This novel HtLAMP-P. vivax assay has the potential to be a useful field applicable molecular diagnostic test for P. vivax infection in elimination settings.

    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  18. Fulltext A Sensitive, Colorimetric, High-Throughput Loop-Mediated Isothermal Amplification Assay for the Detection of Plasmodium knowlesi
    Britton S, Cheng Q, Grigg MJ, William T, Anstey NM, McCarthy JS
    Am J Trop Med Hyg, 2016 07 06;95(1):120-2.
    PMID: 27162264 DOI: 10.4269/ajtmh.15-0670
    The simian parasite Plasmodium knowlesi is now the commonest cause of malaria in Malaysia and can rapidly cause severe and fatal malaria. However, microscopic misdiagnosis of Plasmodium species is common, rapid antigen detection tests remain insufficiently sensitive and confirmation of P. knowlesi requires polymerase chain reaction (PCR). Thus available point-of-care diagnostic tests are inadequate. This study reports the development of a simple, sensitive, colorimetric, high-throughput loop-mediated isothermal amplification assay (HtLAMP) diagnostic test using novel primers for the detection of P. knowlesi. This assay is able to detect 0.2 parasites/μL, and compared with PCR has a sensitivity of 96% for the detection of P. knowlesi, making it a potentially field-applicable point-of-care diagnostic tool.
    Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
  19. Streptococcus gallolyticus infection: A neglected marker for colorectal cancer?
    Bin Ismail CMKH, Bin Mohammad Aidid E, Binti Hamzah HA, Bin Shalihin MSE, Bin Md Nor A
    Arab J Gastroenterol, 2023 Aug;24(3):163-167.
    PMID: 37156704 DOI: 10.1016/j.ajg.2023.02.002
    BACKGROUND AND STUDY AIMS: Colorectal cancer (CRC) is the second most common cancer in Malaysia and mostly detected at advanced stages due to lack of awareness of CRC symptoms and signs. CRC pathogenesis is multifactorial, and there is ambiguous evidence on association of Streptococcus gallolyticus infection with CRC that needs further attention. Thus, a case-control study was conducted to determine whether S. gallolyticus infection is a predictor for CRC occurrence among patients attending Sultan Ahmad Shah Medical Centre@IIUM (SASMEC@IIUM).

    PATIENTS AND METHODS: A total of 33 stool samples from patients diagnosed with CRC and 80 from patients without CRC attending surgical clinic of SASMEC@IIUM were collected and analyzed with iFOBT test and PCR assay to detect S. gallolyticus.

    RESULTS: In this study, the proportion of S. gallolyticus infection was higher among patients with CRC (48.5%) compared with the control group (20%). Univariate analysis shows that occult blood in stool, S. gallolyticus infection and family history were significantly associated with the development of CRC (P 

    Matched MeSH terms: Nucleic Acid Amplification Techniques
  20. Direct detection of Strongyloides infection via molecular and antigen detection methods
    Balachandra D, Ahmad H, Arifin N, Noordin R
    Eur J Clin Microbiol Infect Dis, 2021 Jan;40(1):27-37.
    PMID: 32729057 DOI: 10.1007/s10096-020-03949-x
    Laboratory diagnosis of Strongyloides infections can be grouped into direct and indirect detection methods, and a combination of the two methods is often needed to reach an accurate and timely diagnosis. This review focuses on non-conventional direct detection via molecular and antigen detection assays. Conventional PCR is the most commonly used molecular diagnostic for Strongyloides. Real-time PCR is accurate and highly sensitive for quantitative and qualitative analysis. Meanwhile, PCR-RFLP can efficiently distinguish human and dog isolates of S. stercoralis, S. fuelleborni (from monkey), and S. ratti (from rodent). Loop-mediated isothermal amplification (LAMP) amplifies DNA isothermally with high specificity, efficiency, and rapidity, and has potential for point-of-care (POC) translation. As for antigen detection assay, coproantigen detection ELISAs for strongyloidiasis traditionally relied on raising rabbit polyclonal antibodies against the parasite antigens for use as capture or detection reagents. Subsequently, hybridoma technology using animals has enabled the discovery of monoclonal antibodies specific to Strongyloides antigens and was utilised to develop antigen detection assays. In recent times, phage display technology has facilitated the discovery of scFv antibody against Strongyloides protein that can accelerate the development of such assays. Improvements in both direct detection methods are being made. Strongyloides molecular diagnostics is moving from the detection of a single infection to the simultaneous detection of soil-transmitted helminths. Meanwhile, antigen detection assays can also be multiplexed and aptamers can be used as antigen binders. In the near future, these two direct detection methods may be more widely used as diagnostic tools for strongyloidiasis.
    Matched MeSH terms: Nucleic Acid Amplification Techniques
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