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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*
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*
Assessment of pan-Leishmania detection by recombinase polymerase amplification assay

Louizi C, Khan MAA, Faisal K, Chowdhury R, Ghosh P, Hossain F, et al.

Diagn Microbiol Infect Dis, 2023 Feb;105(2):115862.
PMID: 36493571 DOI: 10.1016/j.diagmicrobio.2022.115862

The spread of vector habitats along with increasing human mobility can introduce atypical Leishmania species and hence can challenge existing diagnostic practices for rapid detection of active infection with species outside the narrow target range. Here we assessed the pan-Leishmania detection ability of isothermal recombinase polymerase amplification (RPA) assays targeting 18S rRNA gene, cathepsin L-like cysteine proteinase B (Cpb) gene, and kinetoplast minicircle DNA (kDNA) regions. While the lowest limit of detection of the 18S rRNA-RPA and Cpb-RPA assays were estimated as 12 and 17 standard DNA molecules, respectively, both assays could amplify genomic DNA of 7 pathogenic Leishmania species. Evaluation of 18S rRNA-RPA and our previously developed kDNA-RPA assays on 70 real-time PCR-positive leishmaniasis samples of varying pathologies resulted in sensitivity rates of 35.71% and 88.57%, respectively, while the combined sensitivity was 98.57%. Combinatorial application of 18S rRNA-RPA and kDNA-RPA assays can be recommended for further diagnostic assessments.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
Cloning of the Aegiceras corniculatum class I chitinase gene (AcCHI I) and the response of AcCHI I mRNA expression to cadmium stress

Wang LY, Wang YS, Cheng H, Zhang JP, Yeok FS

Ecotoxicology, 2015 Oct;24(7-8):1705-13.
PMID: 26044931 DOI: 10.1007/s10646-015-1502-0

Chitinases in terrestrial plants have been reported these are involved in heavy metal tolerance/detoxification. This is the first attempt to reveal chitinase gene (AcCHI I) and its function on metal detoxification in mangroves Aegiceras corniculatum. RT-PCR and RACE techniques were used to clone AcCHI I, while real-time quantitative PCR was employed to assess AcCHI I mRNA expressions in response to Cadmium (Cd). The deduced AcCHI I protein consists of 316 amino acids, including a signal peptide region, a chitin-binding domain (CBD) and a catalytic domain. Protein homology modeling was performed to identify potential features in AcCHI I. The CBD structure of AcCHI I might be critical for metal tolerance/homeostasis of the plant. Clear tissue-specific differences in AcCHI I expression were detected, with higher transcript levels detected in leaves. Results demonstrated that a short duration of Cd exposure (e.g., 3 days) promoted AcCHI I expression in roots. Upregulated expression was also detected in leaves under 10 mg/kg Cd concentration stress. The present study demonstrates that AcCHI I may play an important role in Cd tolerance/homeostasis in the plant. Further studies of the AcCHI I protein, gene overexpression, the promoter and upstream regulation will be necessary for clarifying the functions of AcCHI I.

Matched MeSH terms: Nucleic Acid Amplification Techniques
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
STR data for the AmpFlSTR Profiler loci from the three main ethnic population groups (Malay, Chinese and Indian) in Malaysia

Lim KB, Jeevan NH, Jaya P, Othman MI, Lee YH

Forensic Sci Int, 2001 Jun 01;119(1):109-12.
PMID: 11348801

Allele frequencies for the nine STRs genetic loci included in the AmpFlSTR Profiler kit were obtained from samples of unrelated individuals comprising 139-156 Malays, 149-153 Chinese and 132-135 Indians, residing in Malaysia.

Matched MeSH terms: Nucleic Acid Amplification Techniques/instrumentation; Nucleic Acid Amplification Techniques/methods
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
Fulltext Advances in the Diagnosis of Foot-and-Mouth Disease

Wong CL, Yong CY, Ong HK, Ho KL, Tan WS

Front Vet Sci, 2020;7:477.
PMID: 32974392 DOI: 10.3389/fvets.2020.00477

Foot-and-mouth disease (FMD) is a devastating livestock disease caused by foot-and-mouth disease virus (FMDV). Outbreaks of this disease in a country always result in conspicuous economic losses to livestock industry and subsequently lead to serious socioeconomic damages due to the immediate imposition of trade embargo. Rapid and accurate diagnoses are imperative to control this infectious virus. In the current review, enzyme-linked immunosorbent assay (ELISA)-based methods used in FMD diagnosis are extensively reviewed, particularly the sandwich, liquid-phase blocking, and solid-phase competition ELISA. The differentiation of infected animals from vaccinated animals using ELISA-based methods is also highlighted, in which the role of 3ABC polyprotein as a marker is reviewed intensively. Recently, more studies are focusing on the molecular diagnostic methods, which detect the viral nucleic acids based on reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated isothermal amplification (RT-LAMP). These methods are generally more sensitive because of their ability to amplify a minute amount of the viral nucleic acids. In this digital era, the RT-PCR and RT-LAMP are progressing toward the mobile versions, aiming for on-site FMDV diagnosis. Apart from RT-PCR and RT-LAMP, another diagnostic assay specifically designed for on-site diagnosis is the lateral flow immunochromatographic test strips. These test strips have some distinct advantages over other diagnostic methods, whereby the assay often does not require the aid of an external device, which greatly lowers the cost per test. In addition, the on-site diagnostic test can be easily performed by untrained personnel including farmers, and the results can be obtained in a few minutes. Lastly, the use of FMDV diagnostic assays for progressive control of the disease is also discussed critically.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext Real-time PCR evaluation of seven DNA extraction methods for the purpose of GMO analysis

Jasbeer, K., Son, R., Mohamad Ghazali, F., Cheah, Y.K.

International Food Research Journal, 2009;16(3):329-341.
MyJurnal

Successful DNA amplification is vital for the detection of specific DNA targets in feeds, and this in return depends on the ability of DNA extraction methods to produce good quality DNA. In this study, seven methods were compared for DNA extraction from feeds using quantitative polymerase chain reaction (PCR) of single copy maize (Zea mays) endogenous hmg (high mobility group) gene. Relative levels of hmg were used to evaluate the DNA quality. Spectrophotometer determination of DNA was also carried out to assess DNA yield and DNA purity, while electrophoretic analysis of genomic DNA extracts was carried out to investigate DNA integrity. The findings illustrate that the DNA extraction methods have a significant effect on DNA quality. Statistically, the Epicentre method extracted the highest DNA yield while the Wizard method had the lowest DNA yield with high DNA purity and integrity. However, the Wizard method recovered the most amplifiable DNA per reaction, indicating that template quality and integrity had greater influence over hmg amplification than DNA yield.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext Colorimetric detection of SARS-CoV-2 by uracil-DNA glycosylase (UDG) reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Lai MY, Bukhari FDM, Zulkefli NZ, Ismail I, Mustapa NI, Soh TST, et al.

Int J Infect Dis, 2022 Jul;120:132-134.
PMID: 35472524 DOI: 10.1016/j.ijid.2022.04.036

OBJECTIVES: Preventing reverse transcription loop-mediated isothermal amplification (RT-LAMP) carryover contamination could be solved by adding deoxyuridine triphosphate (dUTP) and uracil-DNA glycosylase (UDG) into the reaction master mix.
METHODS: RNA was extracted from nasopharyngeal swab samples by a simple RNA extraction method.
RESULTS: Testing of 77 samples demonstrated 91.2% sensitivity (95% confidence interval [CI]: 78-98.2%) and 100% specificity (95% confidence interval: 92-100%) using UDG RT-LAMP.
CONCLUSION: This colorimetric UDG RT-LAMP is a simple-to-use, fast, and easy-to-interpret method, which could serve as an alternative for diagnosis of SARS-CoV-2 infection, especially in remote hospitals and laboratories with under-equipped medical facilities.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
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
Dual-Functional Tetrahedron Multivalent Aptamer Assisted Amplification-Free CRISPR/Cas12a Assay for Sensitive Detection of Salmonella

Qiao Z, Xue L, Sun M, Ma N, Shi H, Yang W, et al.

J Agric Food Chem, 2024 Jan 10;72(1):857-864.
PMID: 38134022 DOI: 10.1021/acs.jafc.3c07582

Salmonellosis continues to impose a significant economic burden globally. Rapid and sensitive detection of Salmonella is crucial to preventing the outbreaks of foodborne illnesses, yet it remains a formidable challenge. Herein, a dual-functional tetrahedron multivalent aptamer assisted amplification-free CRISPR/Cas12a assay was developed for Salmonella detection. In the system, the aptamer was programmatically assembled on the tetrahedral DNA nanostructure to fabricate a multivalent aptamer (TDN-multiApt), which displayed a 3.5-fold enhanced avidity over the monovalent aptamer and possessed four CRISPR/Cas12a targeting fragments to amplify signal. Therefore, TDN-multiApt could directly activate Cas12a to achieve the second signal amplification without any nucleic acid amplification. By virtue of the synergism of high avidity and cascaded signal amplifications, the proposed method allowed the ultrasensitive detection of Salmonella as low as 7 cfu mL-1. Meanwhile, this novel platform also exhibited excellent specificity against target bacteria and performed well in the detection of various samples, indicating its potential application in real samples.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

Wong YP, Othman S, Lau YL, Radu S, Chee HY

J Appl Microbiol, 2018 Mar;124(3):626-643.
PMID: 29165905 DOI: 10.1111/jam.13647

Loop-mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro-organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro-organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real-time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro-organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Specific, sensitive, and rapid diagnosis of active toxoplasmosis by a loop-mediated isothermal amplification method using blood samples from patients

Lau YL, Meganathan P, Sonaimuthu P, Thiruvengadam G, Nissapatorn V, Chen Y

J Clin Microbiol, 2010 Oct;48(10):3698-702.
PMID: 20660217 DOI: 10.1128/JCM.00462-10

Loop-mediated isothermal amplification (LAMP), a rapid nucleic acid amplification method, was developed for the clinical diagnosis of toxoplasmosis. Three LAMP assays based on the SAG1, SAG2, and B1 genes of Toxoplasma gondii were developed. The sensitivities and specificities of the LAMP assays were evaluated by comparison with the results of conventional nested PCR. The LAMP assays were highly sensitive and had a detection limit of 0.1 tachyzoite, and no cross-reactivity with the DNA of other parasites was observed. Blood was collected from 105 individuals to test the LAMP assays: 40 patients with active toxoplasmosis, 40 negative controls, and 25 patients with other parasitic infections. The SAG2-based LAMP (SAG2-LAMP) had a greater sensitivity (87.5%) than the SAG1-LAMP (80%), B1-LAMP (80%), and nested PCR (62.5%). All the LAMP assays and nested PCR were 100% specific. This is the first report of a study which applied the LAMP method to diagnose toxoplasmosis from human blood samples. Due to its simplicity, sensitivity, and specificity, LAMP is suggested as an appropriate method for routine diagnosis of active toxoplasmosis in humans.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext A generic assay for whole-genome amplification and deep sequencing of enterovirus A71

Tan le V, Tuyen NT, Thanh TT, Ngan TT, Van HM, Sabanathan S, et al.

J Virol Methods, 2015 Apr;215-216:30-6.
PMID: 25704598 DOI: 10.1016/j.jviromet.2015.02.011

Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Thanarajoo SS, Kong LL, Kadir J, Lau WH, Vadamalai G

J Virol Methods, 2014 Jun;202:19-23.
PMID: 24631346 DOI: 10.1016/j.jviromet.2014.02.024

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Specific, sensitive and rapid detection of human plasmodium knowlesi infection by loop-mediated isothermal amplification (LAMP) in blood samples

Lau YL, Fong MY, Mahmud R, Chang PY, Palaeya V, Cheong FW, et al.

Malar J, 2011;10:197.
PMID: 21774805 DOI: 10.1186/1475-2875-10-197

The emergence of Plasmodium knowlesi in humans, which is in many cases misdiagnosed by microscopy as Plasmodium malariae due to the morphological similarity has contributed to the needs of detection and differentiation of malaria parasites. At present, nested PCR targeted on Plasmodium ssrRNA genes has been described as the most sensitive and specific method for Plasmodium detection. However, this method is costly and requires trained personnel for its implementation. Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method was developed for the clinical detection of P. knowlesi. The sensitivity and specificity of LAMP was evaluated in comparison to the results obtained via microscopic examination and nested PCR.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
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*
Fulltext Loop-mediated isothermal amplification (LAMP) test for diagnosis of uncomplicated malaria in endemic areas: a meta-analysis of diagnostic test accuracy

Selvarajah D, Naing C, Htet NH, Mak JW

Malar J, 2020 Jun 19;19(1):211.
PMID: 32560728 DOI: 10.1186/s12936-020-03283-9

BACKGROUND: The global malaria decline has stalled and only a few countries are pushing towards pre-elimination. The aim of the malaria elimination phase is interruption of local transmission of a specified malaria parasite in a defined geographical area. New and improved screening tools and strategies are required for detection and management of very low-density parasitaemia in the field. The objective of this study was to synthesize evidence on the diagnostic accuracy of loop-mediated isothermal amplification (LAMP) test for the detection of malaria parasites among people living in endemic areas.
METHODS: This study adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis for Diagnostic Test Accuracy (PRISMA-DTA) guideline. Relevant studies in the health-related electronic databases were searched. According to the criteria set for this study, eligible studies were identified. The quality of included studies was evaluated with the use of a quality assessment checklist. A summary performance estimates such as pooled sensitivity and specificity were stratified by type of LAMP. Bivariate model for data analyses was applied. Summary receiver operating characteristics plots were created to display the results of individual studies in a receiver operating characteristics space. Meta-regression analysis was performed to investigate the sources of heterogeneity among individual studies.
RESULTS: Twenty-seven studies across 17 endemic countries were identified. The vast majority of studies were with unclear risk of bias in the selection of index test. Overall, the pooled test performances were high for Pan LAMP (sensitivity: 0.95, 95% CI 0.91 to 0.97; specificity: 0.98, 95% CI 0.95 to 0.99), Plasmodium falciparum (Pf) LAMP (sensitivity: 0.96, 95% CI 0.94 to 0.98; specificity: 0.99, 95% CI 0.96 to 1.00) or for Plasmodium vivax (Pv) LAMP from 6 studies (sensitivity: 0.98, 95% CI 0.92 to 0.99; specificity: 0.99, 95% CI 0.72 to 1.00). The area under the curve for Pan LAMP (0.99, 95% CI 0.98-1.00), Pf LAMP (0.99, 95% CI 0.97-0.99) and Pv LAMP was (1.00, 95% CI 0.98-1.00) indicated that the diagnostic performance of these tests were within the excellent accuracy range. Meta-regression analysis showed that sample size had the greatest impact on test performance, among other factors.
CONCLUSIONS: The current findings suggest that LAMP-based assays are appropriate for detecting low-level malaria parasite infections in the field and would become valuable tools for malaria control and elimination programmes. Future well-designed larger sample studies on LAMP assessment in passive and active malaria surveillances that use PCR as the reference standard and provide sufficient data to construct 2 × 2 diagnostic table are needed.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Detection of Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax using loop-mediated isothermal amplification (LAMP) in a co-endemic area in Malaysia

Piera KA, Aziz A, William T, Bell D, González IJ, Barber BE, et al.

Malar J, 2017 01 13;16(1):29.
PMID: 28086789 DOI: 10.1186/s12936-016-1676-9

BACKGROUND: Plasmodium knowlesi is the most common cause of malaria in Malaysia. However, microscopic diagnosis is inaccurate and rapid diagnostic tests (RDTs) are insufficiently sensitive. PCR is sensitive and specific but not feasible at a district level. Loop-mediated isothermal amplification (LAMP) shows potential with only basic requirements. A commercially available LAMP assay, the Eiken Loopamp™ MALARIA Pan Detection kit, is sensitive for Plasmodium falciparum and Plasmodium vivax, but has not previously been evaluated for P. knowlesi. This study aims to determine the sensitivity of this LAMP assay for detecting P. knowlesi infection.
METHODS: Study participants included 73 uncomplicated malaria patients with PCR species confirmation: 50 P. knowlesi, 20 P. falciparum and 3 P. vivax. Nineteen malaria-negative, non-endemic area controls were also included. The sensitivity of the Eiken Loopamp™ MALARIA Pan Detection kit (Pan LAMP) for detecting each Plasmodium species was evaluated. Sensitivity and specificity of the Eiken Loopamp™ MALARIA Pf Detection kit (Pf LAMP) for P. falciparum were also determined. The limit of detection for each LAMP assay was evaluated, with results compared to PCR. All P. knowlesi patients were also tested by CareStart™ (Pf/VOM) and OptiMAL-IT™ (Pan/Pf) RDTs.
RESULTS: The sensitivity of the Pan LAMP assay was 100% for P. knowlesi (95% CI 92.9-100), P. falciparum (95% CI 83.2-100), and P. vivax (95% CI 29.2-100). The Pf LAMP was 100% sensitive and specific for P. falciparum detection, with all P. knowlesi samples having a negative reaction. LAMP sensitivity was superior to both RDTs, with only 10 and 28% of P. knowlesi samples testing positive to CareStart™ and OptiMAL-IT™, respectively. Limit of detection using the Pan LAMP for both P. knowlesi and P. vivax was 2 parasites/μL, comparable to PCR. For P. falciparum both the Pan LAMP and Pf LAMP demonstrated a limit of detection of 20 parasites/μL.
CONCLUSIONS: The Eiken Loopamp™ MALARIA Pan Detection kit is sensitive for detection of P. knowlesi in low parasitaemia clinical infections, as well as P. falciparum and P. vivax. However, a P. knowlesi-specific field assay in a simpler format would assist correct species identification and initiation of optimal treatment for all malaria patients.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*

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