REVIEW ARTICLE Year : 2022  |  Volume : 59  |  Issue : 1  |  Page : 29-36

Multiplex loop mediated isothermal amplification (m-LAMP) as a point of care technique for diagnosis of malaria

Supriya Sharma, Jaskirat Singh, Aparajita Sen, Anupkumar R Anvikar
ICMR-National Institute of Malaria Research, New Delhi, India

Date of Submission18-Jul-2021Date of Acceptance13-Oct-2021Date of Web Publication07-Jun-2022

Correspondence Address:
Supriya Sharma
ICMR-National Institute of Malaria Research, Sector-8, Dwarka, New Delhi-110077
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/0972-9062.331409

Diagnosis of malaria is a prominent challenge due to the endemic nature of infection. Malaria poses a great threat to global public health. The disease can be diagnosed by several techniques out of which microscopy is a known gold standard. High sensitivity of molecular techniques is making them more reliable and popular as tools for diagnosis of malaria. However, new methods are required which can fulfill the criteria of being Point of Care Test (POCT) as defined by WHO. Loop-mediated isothermal amplification (LAMP) technique amplifies DNA in an isothermal condition, and surpasses the disadvantages of conventional molecular techniques such as polymerase chain reaction. Multiplex LAMP, a modification of LAMP may emerge as a new POC for malaria diagnosis. This review deals with the use of LAMP and multiplex LAMP in diagnosis of malaria and its prospective use as point of care techniques.

Keywords: Malaria; Diagnosis; Loop mediated isothermal amplification (LAMP); Multiplex; Point of care (POC)

How to cite this article:
Sharma S, Singh J, Sen A, Anvikar AR. Multiplex loop mediated isothermal amplification (m-LAMP) as a point of care technique for diagnosis of malaria. J Vector Borne Dis 2022;59:29-36
How to cite this URL:
Sharma S, Singh J, Sen A, Anvikar AR. Multiplex loop mediated isothermal amplification (m-LAMP) as a point of care technique for diagnosis of malaria. J Vector Borne Dis [serial online] 2022 [cited 2022 Jun 7];59:29-36. Available from: https://www.jvbd.org/text.asp?2022/59/1/29/331409   Introduction 

Various different techniques are used in laboratories for the detection of pathogens, such as detection by serology using enzyme-linked immunosorbent assays (ELISA)[1],[2], morphology using electron microscopy[3],[4],[5], microbial culturing[6], or molecular methods[7]. Even though diagnostic efficacy is subject to numerous variations, in epidemiological disorders timely diagnosis of the pathogen is very important. It requires 24 hours or more for transporting samples from Point of care (POC) to labs and delivering results to the patients[8], and sophisticated infrastructure does not exist in resource limited settings[9],[10]. There is thus an important need of developing effective, sensitive, accurate, and species-specific POC tests. World Health Organization (WHO) has specified criteria for good POC devices, which in short is known as ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, deliverable to end-users)[11],[12]. There are different tests used as POC tests, which include rapid diagnostic strips. These are based on the principle of antigen and antibody interactions, which are often less reliable due to less sensitivity as compared to molecular diagnosis of malaria[13] and less specific in detection of viral serotype in dengue[14] and Zika virus[15]. In view of recent developments, existing field tests are less reliable in many infections including malaria, and techniques with more sensitivity and specificity are required in a more informed manner.

Loop mediated isothermal amplification (LAMP)

Loop mediated isothermal amplification, more commonly known as LAMP, is a novel technique for gene amplification, which mitigates the limitations of molecular techniques like Polymerase Chain Reaction (PCR). This technique was first developed and reported by Notomi et al in 2000. It uses four primers which are target specific and recognize six distinct sites. It couples amplification with detection and provides various read out methods like observation with naked eyes or fluorescence in reaction chamber[16] or by gel electrophoresis. LAMP is a rapid and specific nucleic acid amplification technique[17],[18]. It is more robust than PCR. Robustness of this technique has been proven by various workers when compared to other pre-existing molecular techniques. Techniques like Self-Sustained Sequence Replication (3SR), Nucleic Acid Sequence-Based Amplification (NASBA), Standard Displacement Amplification (SDA), Rolling Circle Amplification (RCA) and most prominently Polymerase Chain Reaction (PCR), are among the pre-existing molecular techniques. Reagents of LAMP can be dried and stored at 56°C for 30 days[19]. This ability makes it a good technique to be used as a POC device in an environment with limited resources[20]. Different researchers used LAMP with other technologies according to their need, for example Microfluidic LAMP chip[21], a one-step Real Time LAMP which is 100 times more sensitive than RT PCR[21]. A microfluidic capillary is used for performing LAMP, real time fluorogenic on chip LAMP, direct LAMP technique without genomic extraction[22], Multiplex LAMP detection[23].

Sample preparation for LAMP

Sample preparation in a molecular technique is a very crucial and lengthy step, and is usually performed manually. Yield and quality of nucleic acid depend on this step. There are different methods used for isolation of DNA and an ideal method should be rapid, high throughput, reliable and have good quality product isolation. LAMP is a technique which can be performed in unprocessed or poorly processed samples, no DNA extraction is required[24],[25]. Bst DNA polymerase which is used in LAMP is resistant to presence of anticoagulants, hemin, N-acetylcystein, NaCl and other PCR inhibiting substances unlike Taq polymerase. Therefore, it can be performed without the isolation of DNA, which is primarily helpful in healthcare center where a rapid diagnostic tool is required[26].

Designing of LAMP primers

Designing of primers is a very crucial step in LAMP method as primers should be highly specific and sensitive. Six primers (2 outer, 2 inner and 2 loop primers) are needed for LAMP, which recognizes 8 distinct sites [Figure 1]. Software used for designing primers are LAMP Designer software version 1.10 (PREMIER Biosoft)[27] and Primer Explore[28]. Factors like base composition, GC content and secondary structure formation should be taken under consideration.

Figure 1: LAMP primers. FIP and BIP (Inner primers) are made up of F2 (B2) and F1c (B1c). F3 and B3 are designed for outer regions. Loop primers are designed between F1c (B1c) and F2c (B2c)

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Post amplification detection

Post amplification the outcome of LAMP reactions is visualized by several ways. LAMP reaction generates high amounts of white magnesium pyrophosphate precipitate due to which it can be observed either directly by naked eye or by adding SYBR green to the product[29][Figure 2]A. Detection and quantification of very less quantity of nucleic acid can be done by Real Time turbidimetry or by visually observing a colored precipitate of poly-ethylenimine (PEI) and amplicon complex[29]. Other colorimetric methods include addition of Hydroxy Napthol Blue (HNB) indicator which does not affect the amplification reaction, and can be performed in a microtitre plate. A microtiter plate absorbance reader can be used for simple quantification[30]. Adding manganese and calcein, a fluorescent metal indicator before amplification, results in highly sensitive visual detection[18]. Product of LAMP reaction can also be visualized with the help of Gel electrophoresis technique which is a traditional technique for visualization of PCR products [Figure 2]B.

Malaria diagnosis

World Health Organization (WHO) labels malaria as a life-threatening disease and reported 219 million cases of malaria in 87 countries in 2017. Malaria is a curable disease if treated immediately and accurately. Thus, it requires sensitive and specific methods of diagnosis[32]. There are several methods of diagnosis of malaria. Among them, microscopy is the conventional method. It is done by staining red blood corpuscles in thick and thin smears which allows observation of the morphology of parasites present in blood[33]. But few drawbacks of microcopy are that it requires skilled microscopist and is time consuming, and therefore results in delayed treatment[34]. Immunochromatographic dipstick tests, also known as rapid diagnostic tests (RDTs) give rapid results and do not require microscopic examination[13]. Lactose Dehydrogenase (LDH) and Histidine Rich Protein-2 (HRP-2) are two most common proteins used to detect malaria via RDT. Rapid Diagnostic Tests however are less sensitive and less specific[35]. Nowadays, ultrasensitive RDTs as being used in African countries which has a higher sensitivity but a lower specificity[36]. The presence of a faint band in Pan RDTs and the absence of HRPII gene in few parasites leading to false negative results in some RDTS are major challenges for RDT use[37]. Still, they have been widely used as diagnostic tools. Molecular diagnosis is more relevant when it comes to malaria infection. Techniques based on PCR are more sensitive and specific and are developed to differentiate between different species of Plasmodium[38],[39]. It can detect less than 5 parasites/μl[40]. However, a problem with PCR is that it is not rapid[35], requires high-cost equipment which cannot be provided in clinics of field areas where the disease is endemic[40], and often fails to detect below 20 parasites/μl[41] reported less sensitivity of nested PCR. However, some efforts are made to use PCR in field settings by adding some modifications like PCR-LDR (PCR-Ligase detection reaction assay)[42], nested PCR-HRM (nested PCR high resolution melting analysis)[43], and PCR-enzyme linked ELISA[44]. [Table 1] represent comprehensive view of comparison of commonly used method for disease detection with multiplex LAMP.

Table 1: Comparison of commonly used techniques in malaria diagnosis with multiplex LAMP

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Malaria and LAMP

WHO has recommended a detection limit of 2 parasites/μl[50], studies show this detection limit[26],[51],[52]. Several studies investigated LAMP technique and its aspects on diagnosis of all human Plasmodium infections[52]. Poon et al. (2006) reported 1/10th cost of LAMP assay when compared with conventional PCR while diagnosing Plasmodium falciparum. Various reports state more than 95% sensitivity and 100% specificity of LAMP assay in diagnosis of Plasmodium species[32],[53],[54],[55],[56],[57],[58] in their review compared the merits and demerits of LAMP with those of RDT’s for malaria diagnosis, and reported that LAMP has more advantages over currently used RDT’s. LAMP has potential to be used as a novel, rapid diagnostic assay for diagnosis of malaria[32]. A high throughput LAMP has been developed and combined with photo-spectrometry for objective confirmation of visually identified HNB color change as an indicative of presence of Plasmodium[10]. For diagnosis of malaria, LAMP has been evaluated in field settings and 98.3% sensitivity and 100% specificity were reported[56],[59]. R reported meta-analysis of seven studies using LAMP against reference microscopy and reported sensitivity of 98% and specificity of 97%. When PCR was used as a reference method, sensitivity was 96% and specificity was 91%. Oriero et al. (2015) in their review predicted LAMP as the best molecular technique in near future, except that it is expensive than microscopy and RDT at present[60].

Other isothermal amplification methods

Recently, various methods have been developed for diagnosis of pathogens and can be used for diagnosis of malaria. Nucleic acid sequence-based amplification (NASBA) introduced by Kievits et al. (1991)[61], targets RNA by using a cocktail containing the enzymes T7 polymerase, reverse transcriptase and Rnase H, and results in more than 8 fold amplification. Thermophilic helicasedependent amplification (tHDA) is another isothermal technique which was first introduced in 2004. It amplifies DNA at 65°C and has very high sensitivity and specificity[62]. Other potential methods which can be used are single primer isothermal amplification[27], transcription-mediated amplification (TMA)[63], isothermal and chimeric primer-initiated amplification of nucleic acids[64], recombinase polymerase amplification (RPA)[21],[64], exponential amplification reaction, strand displacement methods such as strand displacement amplification (SDA)[65], self-sustained sequence replication reaction[66], cross-priming amplification[67], rolling circle amplification[19], and smart amplification (SmartAmp)[68],[69]. A precise optimization is required for using these techniques in diagnosis as POC.

Multiplex LAMP

Multi target diagnosis like multiplex LAMP amplifies several target sequences using multiple primers in a single reaction. To standardize the components and remove primer dimer, designing of primers should be done carefully. It offers rapid detection and lesser steps when compared with (quantitative LAMP (qLAMP) and other molecular techniques like PCR and RT-PCR. Various researchers reported diagnosis in 20 minutes or less using Multiplex LAMP. Yang et al. (2018) reported detection of Acinetobacter baumannii in less than 21 min without any pre-requisite time for DNA purification[4]. Fowler et al. (2014) reported detection and discrimination of foot and mouth disease (FMD) from other vesicular diseases within 10 minutes using multiplex LAMP. Wang et al. (2015) developed multiplex LAMP technique with detection time of 12 minutes or less[71]. Multiplex LAMP has high detection sensitivity and has been reported by various researchers as shown in [Table 2]. This detection limit is much higher when compared with other robust molecular techniques like PCR, RT-PCR, qPCR[5],[50],[52],[71].

Table 2: Comparison of sensitivity and specificity of multiplex LAMP (m-LAMP).

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Application of m-LAMP

Multiplex LAMP has a huge variety of applications due to its advantages. Major application of multiplex LAMP is in diagnosis of bacterial[4],[52], parasitological[51],[72],[73] and viral[50],[74],[75] diseases at early stages, which further leads to better treatment of the infection. In veterinary sciences, FMD is a fatal viral disease which can be rapidly diagnosed by Multiplex LAMP[70]. Gong et al. (2012) had developed Multiplex LAMP for identifying Sulfonamide resistance genes in clinical enterobacteriaceae isolates which had been isolated from animals in poultry farms[76]. Multiplex LAMP is also used for detection of pathogenic yeast contamination in dairy products. The method should be sensitive and specific as fermented dairy products normally contain yeast cells[77].

Shortcoming of m-LAMP

Despite having many advantages, m-LAMP comes with the similar loopholes as of LAMP. Complex primer design is one of the demerits of LAMP making it the most crucial step of the assay[26]. Due to presence of 4–6 primers, self-priming is a major drawback of LAMP which eventually results in false positive results. This can be overcome by optimizing assay conditions such as concentration of primers, dNTP’s, polymerase and cutoff time[78]. There are reports which have suggested different corrective actions to overcome the shortfall of LAMP reactions.

Advances of multiplex LAMP

Mao et al. (2018) coupled multiplex LAMP with microfluidic systems and developed a multiplex microfluidic chip with visual detection for identification of malaria related species[72]. They showed that it is a highly productive method with sufficient level of sensitivity and specificity. Multiplex LAMP when combined with paper origami-based microfluidic techniques resulted in a highly precise method, which can be used in underdeveloped areas for the diagnosis of malaria. Cost of multiplex assay is less than USD 2 and can be reduced at industrial scale[73]. Real time multiplex LAMP has been developed with the addition of endonuclease enzyme, a flourophore and a quencher and shown to produce a faster result than real time PCR[52],[71]. Multiplex Reverse Transcription LAMP is coupled with cascade invasive reaction and nanoparticle hybridization, and results in fast results by visual detection, making subtyping of Influenza virus easy[50],[51]. Combined multiplex LAMP with lateral flow biosensors using principle of immunoassay resulted in rapid detection and easy interpretation of results[51].

  Conclusion 

Malaria is a life-threatening illness with no existing in use vaccines, and thus correct diagnosis has a crucial role in prophylaxis and treatment of the disease. At present, lateral flow diagnostics are the most used detection methods. However, they have their own pros and cons. Multiplex LAMP is a modification of LAMP that can be used as a POC for malaria diagnosis. It is a molecular-based technique, which is quantitative, species-specific, requires moderate skill, time saving and is economical. Although, it is a well-established diagnostic method, but it can be improved in terms of reducing errors that arise from sample contamination. It is also recommended to incorporate it to test devices that are portable as well as disposable, to be easily used in field conditions. Multiple pathogens detection in a single test, such as one multiplex method to detect the markers of multiple diseases such as dengue and chikungunya, along with malaria may enhance its utility in future.

Conflict of interest: None

  Acknowledgements 

This manuscript is approved by Publication Committee of ICMR-NIMR with approval no - RIC-28/2021. Supriya Sharma is thankful to Indian Council of Medical Research for RA fellowship and all authors are grateful to ICMR-NIMR for overall support.

 

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