14
10.1586/ERM.12.144 205 ISSN 1473-7159 © 2013 Expert Reviews Ltd www.expert-reviews.com Review Malaria diagnostics Malaria is the most devastating parasitic infec- tion in the world, and it causes over 1 mil- lion deaths each year, mainly among children younger than 5 years, while high morbidity also results from the 350–500 million clinical malaria episodes. In particular, Plasmodium falciparum causes high mortality because of vari- ous complications and its increasing resistance to affordable antimalarials [1,2] . Prompt and accu- rate diagnostics are essential for the control and management of malaria in endemic and non- endemic areas [3,4] . In endemic countries, rapid and sensitive diagnostics are needed to identify infected patients and to prevent overprescription of antimalarial drugs on the basis of unreliable clinical diagnoses. In nonendemic countries, the lack of experience among physicians and labora- tory technicians leads to delays and inaccurate diagnoses, which can result in a fatal prognosis for patients [5–7] . The misdiagnosis of malaria may overlook other potentially life-threatening illnesses, and it contributes to the emergence of drug-resistant malaria [8] . It is also increasingly unjustifiable because expensive artemisinin- based combination therapies (ACTs) are rec- ommended as the first-line drug treatment in low-income endemic countries. The high cost of ACTs makes a specific diagnosis of malaria more cost-effective, which necessitates a more accurate diagnostic paradigm. The vast volume of Plasmodium genome infor- mation and revolutionary biotechnological tech- niques are now being applied to the development of new malaria diagnostics [9] . However, there is still a gap between scientific advances and Plasmodium infection diagnostics in the field. Accurate diagnosis is important for the appro- priate treatment of malaria [10] . Existing tools for the diagnosis of malaria include microscopy, molecular tools, parasite antigen/enzyme detec- tion kits (such as rapid diagnostic tests [RDTs]) and fluorochrome methods [3,11–13] . Each of these diagnostic tools has advantages and limitations (TABLE 1) . Light microscopy has relatively high sensitivity and specificity, and it also provides information on the parasite density, stage and species, but this method is labor intensive because it requires well-trained experts, which may result in thera- peutic delays [14–19] . Very long observation peri- ods and considerable expertise are required to make a correct diagnosis by microscopy in cer- tain circumstances, such as low parasitemia and Eun-Taek Han Department of Medical Environmental Biology and Tropical Medicine, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do 200-701, Republic of Korea [email protected] Malaria is one of the most important infectious tropical diseases. Rapid and accurate diagnostics are the basis of therapeutic care for most febrile patients in field clinics and health centers where novel molecular techniques have overcome sensitivity and specificity limitations. Loop- mediated isothermal amplification (LAMP) is a novel molecular method for rapid DNA target amplification with high specificity in isothermal conditions. LAMP provides a simple and rapid diagnostic tool for the early detection and species-specific identification of Plasmodium parasites using the read-out of the LAMP with the naked eye in the field, with no requirement for expensive thermal cyclers. LAMP is readily applicable to clinical diagnosis and active surveillance of malaria parasites in reference laboratories and research centers of endemic countries as a new alternative molecular diagnostic solution. This article summarizes current technical developments, applications, limitations and the future prospects for malaria diagnosis using LAMP in developed countries and endemic areas. KEYWORDS: diagnostics • LAMP • malaria • novel molecular tools • Plasmodium Loop-mediated isothermal amplification test for the molecular diagnosis of malaria Expert Rev. Mol. Diagn. 13(2), 205–218 (2013) For reprint orders, please contact [email protected]

Loop-mediated isothermal amplification test for the molecular diagnosis of malaria

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Page 1: Loop-mediated isothermal amplification test for the molecular diagnosis of malaria

10.1586/ERM.12.144 205ISSN 1473-7159© 2013 Expert Reviews Ltdwww.expert-reviews.com

Review

Malaria diagnosticsMalaria is the most devastating parasitic infec-tion in the world, and it causes over 1 mil-lion deaths each year, mainly among children younger than 5 years, while high morbidity also results from the 350–500 million clinical malaria episodes. In particular, Plasmodium falciparum causes high mortality because of vari-ous complications and its increasing resistance to affordable antimalarials [1,2]. Prompt and accu-rate diagnostics are essential for the control and management of malaria in endemic and non-endemic areas [3,4]. In endemic countries, rapid and sensitive diagnostics are needed to identify infected patients and to prevent overprescription of antimalarial drugs on the basis of unreliable clinical diagnoses. In nonendemic countries, the lack of experience among physicians and labora-tory technicians leads to delays and inaccurate diagnoses, which can result in a fatal prognosis for patients [5–7]. The misdiagnosis of malaria may overlook other potentially life-threatening illnesses, and it contributes to the emergence of drug-resistant malaria [8]. It is also increasingly unjustifiable because expensive artemisinin-based combination therapies (ACTs) are rec-ommended as the first-line drug treatment in

low-income endemic countries. The high cost of ACTs makes a specific diagnosis of malaria more cost-effective, which necessitates a more accurate diagnostic paradigm.

The vast volume of Plasmodium genome infor-mation and revolutionary biotechnological tech-niques are now being applied to the development of new malaria diagnostics [9]. However, there is still a gap between scientific advances and Plasmodium infection diagnostics in the field.

Accurate diagnosis is important for the appro-priate treatment of malaria [10]. Existing tools for the diagnosis of malaria include microscopy, molecular tools, parasite antigen/enzyme detec-tion kits (such as rapid diagnostic tests [RDTs]) and fluorochrome methods [3,11–13]. Each of these diagnostic tools has advantages and limitations (Table 1).

Light microscopy has relatively high sensitivity and specificity, and it also provides information on the parasite density, stage and species, but this method is labor intensive because it requires well-trained experts, which may result in thera-peutic delays [14–19]. Very long observation peri-ods and considerable expertise are required to make a correct diagnosis by microscopy in cer-tain circumstances, such as low parasitemia and

Eun-Taek HanDepartment of Medical Environmental Biology and Tropical Medicine, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do 200-701, Republic of Korea [email protected]

Malaria is one of the most important infectious tropical diseases. Rapid and accurate diagnostics are the basis of therapeutic care for most febrile patients in field clinics and health centers where novel molecular techniques have overcome sensitivity and specificity limitations. Loop-mediated isothermal amplification (LAMP) is a novel molecular method for rapid DNA target amplification with high specificity in isothermal conditions. LAMP provides a simple and rapid diagnostic tool for the early detection and species-specific identification of Plasmodium parasites using the read-out of the LAMP with the naked eye in the field, with no requirement for expensive thermal cyclers. LAMP is readily applicable to clinical diagnosis and active surveillance of malaria parasites in reference laboratories and research centers of endemic countries as a new alternative molecular diagnostic solution. This article summarizes current technical developments, applications, limitations and the future prospects for malaria diagnosis using LAMP in developed countries and endemic areas.

Keywords: diagnostics • LAMP • malaria • novel molecular tools • Plasmodium

Loop-mediated isothermal amplification test for the molecular diagnosis of malariaExpert Rev. Mol. Diagn. 13(2), 205–218 (2013)

Expert Review of Molecular Diagnostics

© 2013 Expert Reviews Ltd

10.1586/ERM.12.144

1473-7159

1744-8352

Review

For reprint orders, please contact [email protected]

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Table 1. Comparison of Giemsa staining, molecular methods (PCR, loop-mediated isothermal amplification, quantitative nucleic acid sequence-based amplification and mass spectrometry), rapid diagnostic tests and fluorochrome methods (CELL-DYN® full-blood count analysis, microsphere-based assay and fluorescence microscopy) for malaria diagnosis.

Microscopy PCR LAMP QT-NASBA

Nested Real-time

Principle Morphological based Molecular based (DNA) Molecular based (DNA or RNA)

Molecular based (DNA or RNA)

Molecular based (DNA or RNA)

Specificity (species specific)†

Possible Possible Possible Possible Possible

Quantification Gold standard No Yes Yes Yes

Sensitivity‡ 50–100 5 0.7–4 5 0.01–0.1

DNA/RNA purification None Required Required Not absolutely required

Required

Cycle of temperature None Three cycles × 2 Two cycles One cycle One cycle

Temperature for DNA amplification (°C)

None 40–98 40–98 60–65 41

Processing Once Twice Once Once Once

Time required for result (h) 0.5–1 4 2 0.5–1 1

Detection method Eye AGE Eye/graph Eye/graph Eye/graph

Risk of contamination No Possible Low Potentially high§ Possible

Skill level High High Medium to low Medium High

Equipment required Microscopy Machine Machine Minor equipment required

Machine

Cost (US$) Low (0.12–0.40) High (0.35–5) High (0.5) Middle (0.28) High

Mass spectrometry (Hz detection)

Rapid diagnostic tests

Fluorochrome methods

FBC analysis Microsphere assays

Fluorescence microscopy

Principle Molecular-based Immunological-based (pLDH or pHRP2)

Cell-based Antigen-antibody-based (AO/cyscope)

Morphology-based

Specificity (species-specific) No Possible (Pf/non-Pf) No No Possibly high

Quantification Possible Poor No No Possible

Sensitivity‡ 100–1000 100–200 Variable 100 fg/ml of protein 50

DNA purification No No No No No

Cycle of temperature No No No No No

Temperature for DNA amplification

No No No No No

Processing Multiple Single Multiple Multiple Single

Time required for result <1 min/sample 0.3 h <1 min/sample <3 h 5–60 min

Detection method Signal intensity Eye Signal intensity Signal intensity Eye

Risk of contamination Low No Low Low Low

Skill level High Low High High Medium

Equipment required Expensive machine Not required Expensive machine Machine Machine

Cost (US$) High Middle to low (0.6–2.5) Very high Possibly high Middle/low

Future potential Automated, high throughput

First-line screening tool

Automated, high throughput

†Species-specific detection of five human malaria parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi).‡Number of parasites/µl.§High risk of contamination if the product is detected by agarose gel electrophoresis, although it can be minimized by using a closed system.AGE: Agarose gel electrophoresis; AO: Acridine orange; FBC: CELL-DYN® full blood count; Hz: Hemozoine; LAMP: Loop-mediated isothermal amplification; Pf: Plasmodium falciparum; pHRP2: Parasite histidine-rich protein 2; pLDH: Parasite lactate dehydrogenase; QT-NASBA: Quantitative nucleic acid sequence-based amplification; RDT: Rapid diagnostic test.

Han

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mixed infections, after drug treatment and during the chronic phase of infections.

Researchers have developed alternative malaria RDTs on the basis of parasite antigen-captured immunochromatographic tech-nologies to facilitate rapid and accurate malaria diagnosis in areas where laboratory facilities are not available [11,20]. RDTs deliver a similar analytical sensitivity to expert microscopists [21,22], although the sensitivity can vary among products [23], while a species-specific product is commercially differentiate only for P. falciparum or non-P. falciparum.

In an attempt to detect malaria parasites in blood samples, automatic methods have been introduced. Certain fluorescent dyes and antibodies have an affinity or specific interaction for the nucleic acid in the nucleus and proteins of parasites. These techniques have an advantage for automated detection and high-throughput screening in laboratories of hospitals [24–30]; however, are not suitable for field application.

Molecular detection methods including nested and real-time PCR, loop-mediated isothermal amplification (LAMP), quantita-tive nucleic acid sequence-based amplification (QT-NASBA) and mass spectrometry were developed and applied for the detection of malaria parasites [13,31–38]. PCR, a typical molecular diagnostic method, has increased the sensitivity and specificity of malaria diagnosis during the identification and differentiation of malaria parasites since the early 1990s [39–41]. PCR can also be useful in a quality-assurance scheme in reference laboratories. Testing sam-ples from routine diagnostic laboratories provides excellent quality controls for comparison with results obtained by conventional microscopy and other diagnostic methods. However, the time lag between sample col-lection, transportation and processing and the notification of diagnostic results to the physician limits the usefulness of PCR in routine clinical practice. High costs and equipment requirements also limit the util-ity of PCR in routine clinical practice and the field clinics where malaria is endemic [42,43]. The many tiresome steps and the requirement for highly skilled techniques, as well as the cost of DNA extraction from blood, thermal cycling and post-PCR analysis, have stimulated scientists to find alternative molecular methods.

An alternative molecular detection method, real-time QT-NASBA, has been developed and applied for species-specific detection of the 18S ribosomal RNA gene (0.1–0.01 parasites per 50 µl of blood samples) within 1 h on isothermal condi-tion at 41°C. Thus, it is simpler and faster than routine PCR condition; however, this method also requires DNA purification for target gene amplification and an expensive real-time analyzer with a commercial-based reaction mixture in field clinics.

LAMP is a new molecular technology that can overcome the disadvantages of PCR. LAMP also eliminates the time-consum-ing and expensive purification of DNA prior to amplification. This technique uses a simple heat treatment of blood samples, and it can amplify target genes without any further requirement for purification [37]. At present, microscopy and RDTs remain the only feasible options for malaria detection in field clinics in many endemic countries.

Microscopic examination as a gold standardAccess to medical care is restricted in many malaria-endemic areas. Medical services exist, but they usually lack laboratory diag-nostic services. Thus, the treatment of malaria is mainly based on clinical diagnosis or self-diagnosis. However, clinical diagnosis is highly inaccurate, even in areas where malaria is a frequent cause of fever, because the signs and symptoms of uncomplicated malaria are nonspecific, and they share common characteristics with other feverish infectious diseases [44,45], while the personal awareness of fever is also unreliable [46,47]. Thus, the routine diag-nostic method for malaria is microscopic examination in most laboratories of endemic and nonendemic areas.

Light microscopy has been considered the ‘gold standard’ for the detection of malaria parasites for approximately 120 years (Figure 1a) [12]. The sensitivity of this method can be excellent, detecting malaria parasite densities as low as five to ten parasites per microliter of blood (~0.0001% parasitemia) [11]. Microscopy can determine the species and the stage of infectious malaria parasites. The parasite density can also be counted and gives

Figure 1. Approaches to the microscopic diagnosis of malaria and four typical malaria diagnostic techniques. LAMP: Loop-mediated isothermal amplification; RDT: Rapid diagnostic test.

‘Goldstandard’malariadiagnosticsin endemiccountries

Fundamentaldiagnostic: 1882

Gold standarddiagnostic: 2012

Giemsa staining PCR RDT LAMP

Loop-mediated isothermal amplification test for the molecular diagnosis of malaria

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additional information on the developmental stage of the para-site to the diagnosis (possible severity if high parasitemia), and serial examinations of blood samples can determine the para-sitological response during chemotherapy. Smear samples pro-vide a permanent record for quality assessments of microscopic diagnosis. Despite these strengths, microscopy has a number of limitations; that is, it is labor intensive and time-consuming, and there can be variations in slide interpretations depending on the level of operator skill, which requires extended training and experience [6,14–19]. Malaria microscopy may be performed infrequently in areas where malaria is not endemic, thus it is so rare that microscopists may lose experience. Although it is very interesting, but certainly not representative of the global situa-tion and not of the situation in nonendemic countries, a single study performed in Canada reported that the diagnosis at pres-entation was missed in 59% of malaria cases and that the main infecting species, Plasmodium vivax, was identified incorrectly in 64% of cases in advanced countries [6]. If the test is ordered, clinicians may decide not to wait for the test results or they may lack confidence in the test results, and they may treat the patient despite negative microscopy [48,49]. Epidemiological surveillance of malaria by microscopy is less suitable for parasite detection in asymptomatic carriers who have the potential for malaria parasite transmission, and it underestimates the number of subjects with concurrent mixed-species infections.

Fluorochrome methodsUsing fluorochrome dye, several technologies were developed for the detection of malaria parasites from field clinical samples; CELL-DYN® full blood count (FBC) analysis [24,25,50], micro-sphere-based assay [51,52] and fluorescence microscopy (Table 1) [26,29]. The malaria pigment (hemozoin)-containing monocytes is rapidly and automatically detected by the FBC analyzer, which distinguishes between white blood cell subsets based on how they scatter multiangle light. Sensitivity, however, is highly variable (48–95%) compared with microscopy and quantification of para-sitemia and speciation were difficult. A major advantage of this technique is that detection of FBC may provide information of possible infection of malaria parasites from unsuspected malaria cases.

Microsphere-based assays have been used for the detection of malaria-specific antibodies from patient samples. The previous study used this sensitivity of multiplex assay to assess the expo-sure to P. falciparum infection in children living in an area of low endemicity and specific IgG antibody response to antigenic candidates were evaluated before and after the period of malaria transmission in endemic countries [52]. This multiplex assay was also a useful technology for serological epidemiology [53]. Several potential advantages are high sensitivity, high-throughput screen-ing, wide range of analysis, potential quantification, use of small sample volume and reproducibility. However, this technology is required to develop and evaluate in each laboratory of the developing countries.

Fluorochrome methods using acridine orange (AO) have usu-ally been used for the detection of malaria parasite nucleic acid

and is used either as a direct-staining technique or combined with a routine method such as a thick blood film [29,54]. The centrifugal quantitative buffy coat combines an AO-coated capillary tube and an internal float to separate layers of white blood cells and platelets using centrifugation [55]. These techniques using AO techniques have been studied under laboratory and field conditions; however, these require specialized equipment to separate the cell layers by centrifugation and/or a good fluorescence microscope. This method is only applicable to well-established laboratories or hos-pitals in endemic and nonendemic areas of developing countries.

Immunochromatographic RDTsIn most endemic countries, the lack of infrastructure and training has made microscopic diagnosis demanding, which has contrib-uted to a recent increase in the supply of RDTs (Figure 1b) [56,57]. RDTs require no special equipment or stringent storage and trans-portation facilities, so they can be used anywhere by travelers with-out experience or skill. However, RDTs remain at a relatively lower cost than the other way round, and they are restricted mainly to the detection of P. falciparum at lower sensitivities (100–200 parasite/µl) to microscopy. Many currently available RDTs are based on parasite antigens such as HRP-2, lactate dehydrogenase (LDH) and aldolase, which use immunochromatographic tests that have been available for over 10 years [11,12,58]. The majority of commer-cial RDTs detect HRP-2, which is expressed only by P. falciparum parasites, and this test is used for the specific diagnosis of falcipa-rum malaria. A limitation of this test is that HRP-2 can persist in the bloodstream for 7–14 days following chemotherapy in a substantial proportion of individuals, even though these patients no longer have symptoms or parasitemia (as assessed by blood smears) [59]. Therefore, this diagnostic method cannot accurately determine whether a subject has a current or recently treated falci-parum parasite. Another recent concern about this test is that up to 40% of the P. falciparum parasites in parts of South America have an HRP-2 gene deletion, which leads to false-negative results; how-ever, the paper reporting this finding only investigated one country and one area (Peru) with the explicit conclusion that this needs to be investigated in the other parts of South America [60]. Parasite LDH and aldolase are detected in non-HRP-2-based tests, which are commonly used as pan-specific tests that avoid the speciation of P. falciparum and/or nonfalciparum species, including P. vivax [61,62] and Plasmodium knowlesi [63,64]. The LDH test has several advantages as a sensitive measure of the presence of parasites in patient blood samples. However, variable results have been reported in terms of the specificity and sensitivity of different commercially available diagnostic kits [11,12,58]. Recent data show that the use of different anti-LDH antibody combinations facilitates species-specific diagnosis and the detection of four malaria species after the optimization of immunochromatographic LDH tests [65].

Molecular diagnosis of malaria parasitesNucleic acid-based molecular techniques may be alternative meth-ods for malaria diagnosis because they can accurately differenti-ate human-infecting malaria parasite species with an increased detection limit (Figure 1b).

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PCR-based diagnosis was developed recently to identify five human parasites including P. knowlesi parasites, which can be misdiagnosed as P. malariae by microscopy [13,42,66]. However, these advanced PCR-based techniques are beyond the capacity of most malaria-endemic countries because they require labora-tory facilities and training courses, which make these techniques expensive and technically challenging to implement in simple clinical laboratories or field settings (Figure 1b). Highly sensitive diagnostic tools are required to detect asymptomatic patients (with low-level parasitemia) to promote better malaria control with the eventual goal of elimination. Therefore, further efforts are required to develop innovative molecular tools for field tri-als where such tools could complement, or in some situations replace, existing molecular methods for malaria diagnosis, as well as to apply them for operational purposes such as monitoring and evaluation during control and elimination programs.

LAMP: next-generation nucleic acid amplificationHigh-sensitivity & comparable specificity to PCR methodsThe recently developed LAMP method is a relatively simple and field-applicable technique (Figure 2) [67–69]. Parasite DNA is ampli-fied in isothermal conditions using a Bst polymerase with strand displacement properties, which means that sophisticated and expensive thermal cyclers are not required (box 1). Magnesium pyrophosphate, which appears as a precipitate as the reaction progresses, is a byproduct of DNA ampli-fication. The appearance of this precipitate provides a positive indicator of the target DNA amplification reaction. LAMP was shown to amplify DNA with high effi-ciency, amplifying a few copies of DNA to 109 in <1 h [67]. Four LAMP primers (F3 and FIP forward primers; B3c and BIP reverse primers) are used to amplify six specific sites in the target sequence, which makes them highly specific to the target (Figure 3) [67]. The addition of two extra primers (loop primer forward [LPF] and loop primer backward [LPB] primers), known as loop primers, increases the copy number of the amplified product, thereby decreasing the reaction time required (0.5–1 h). Real-time turbidimetry facili-tates the quantification of the template DNA in the reaction, which allows the possibility of analyzing minute quanti-ties of nucleic acids [70]. The addition of manganese ions and a fluorescent metal indicator (calcein) to the reaction allows the modification of fluorescence during the one-step amplification reaction to be visualized within 30–60 min [71]. This method does not require a thermocycler or staff training, so it has the potential to be used as a new molecular diagnostic tool in

developing and developed countries, provided that further modi-fications are made [71]. Moreover, the threat of contamination is reduced greatly because the reaction is carried out in a closed system with no need for agarose gel electrophoresis. The use of gel electrophoresis is optional for detecting the amplification product.

Simple & rapid DNA template preparation from blood samplesOne of the major advantages of LAMP is that amplification is performed with Bst DNA polymerase. Compared with Taq polymerase, which is typically used in PCR, Bst polymerase is more tolerant of inhibitory components present in blood sam-ples. Various sample components (minimal concentrations of hemoglobin, IgG and IgM) can interfere with DNA amplifica-tion during PCR reactions [72,73]. However, LAMP uses Bst poly-merase to amplify the target DNA by strand displacement, so it is less affected by the inhibitory components of clinical samples compared with PCR. Thus, template DNA can be produced by the direct heat treatment of blood samples, with no requirement for time-consuming and expensive DNA extraction using com-mercial kits (Figure 4) [37,38,74,75]. This method is more advanced than other nucleic acid amplification techniques in terms of its simplicity, rapidity, sensitivity and specificity. Therefore, it may facilitate the development of simple diagnostic approaches for

Figure 2. Standard laboratory procedure for loop-mediated isothermal amplification. (A) Blood sample collected from malaria patients; (B) gDNA extracted with a commercial kit; (C) primers designed using the PrimerExplorer program [101]; (D) reaction mixture preparation (gDNA, primers and reagents, including Bst polymerase); (E) incubation of the reaction mixture in isothermal conditions; and (F) detection of the amplified product. gDNA: Genomic DNA.

Blood filter paper

gDNA extraction

Primer design by program

Mix with gDNA, primersand reagents (Bst polymerase)

Incubation (62°C, 60 min)

Real-time detection

Amplification

Sampling

DNAextraction

Detection

Reaction mixturepreparation

Loop-mediated isothermal amplification test for the molecular diagnosis of malaria

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many infectious diseases in resource-limited countries, and pos-sible new molecular diagnostic devices. LAMP may be the sim-plest approach for the amplification and subsequent detection of DNA. This simple method requires no specific reagents or a sophisticated instrument manual.

Visual detection of amplified productsThe application of LAMP assays in the field is facilitated by the visual monitoring of amplification based on turbidity or fluo-rescence with the naked eye (Figure 5). A transparent tube con-taining the amplified products allows visualization in the pres-ence of fluorescent intercalating dyes (e.g., ethidium bromide, SYBR® Green I, calcein, cationic polymers, polyethylenimine and hydroxy naphthol blue) with illumination from a UV lamp [71,76,77]. This is another advantage of reading the amplification product without further amplicon processing [78].

In practice, the visual inspection of amplification is based on the observation of precipitates (white color) from byproducts of amplification and a color change following the addition of SYBR Green I to the tube (Figure 5a). Positive amplification is indicated by a change in the white color of tube or the addi-tional dye from orange to green, which can be observed in natural light or UV light with the aid of a handheld UV torch (or a UV transilluminator). The original orange color of the dye remains when there is no amplification. This change in color is permanent, and tubes can be retained for recording purposes. Thus, the presence of fluorescence indicates the pres-ence of amplified target genes, and visual detection can be achieved without opening the tube, thereby avoiding carry over contamination with postamplification products. After incu-bating the reaction mixture, the amplified LAMP products are electrophoresed on agarose gel, followed by staining with ethidium bromide and visualization using a UV transillumina-tor. Agarose gel electrophoresis analysis visualizes the typical electrophoresis pattern of the LAMP-amplified product, which is a ladder pattern rather than a single band because the LAMP method produces various amplified product sizes that consist of repeats of the target sequence connected by loops on the same strand (Figure 5b).

Field application of the LAMP detection method to malaria parasitesReliability & practicality of the LAMP assay in field conditionsThe species-specific LAMP method is relatively simple, but it

can be improved for use in field clinics. The sensitivity and specificity of LAMP for the detection of Plasmodium parasites have been investigated in many studies in endemic and nonendemic countries (Table 2). LAMP targets the parasite small-subunit rRNA (18S rRNA), mitochon-dria (mit), β-tubulin, apical membrane antigen 1 (ama1), Pfs16 and Pfs25 genes to detect Plasmodium parasites [37,38,79–84]. Initially, malaria LAMP assays used primer sets that targeted the 18S rRNA gene, which delivered highly reliable sen-sitivity and specificity in previous studies [37,38,79,84]. 18S rRNA gene-based LAMP detected 50 parasites per microliter of Plasmodium spp., P. falciparum, P. vivax and Plasmodium ovale, and 500 parasites per microliter of P. malariae. Two recent studies [80,82] used the mit gene in a LAMP assay to detect Plasmodium spp., P. falci-parum and P. vivax. A further study [80] compared 18 selected genomic targets, consisting of multiple- and single-copy loci in new malaria LAMP primer sets and found that mit gene-based LAMP detected

Box 1. Characteristics of loop-mediated isothermal amplification.

• Bst polymerase: strand displacement activity

• Isothermal conditions: 60–65°C

• Rapid: 1 h (compared with 3 h for PCR)

• High specificity: six primers (+ two primers)

• High efficiency: 1010 DNA copies h-1

• High sensitivity: ten copies

• Low cost: no special reagents, machines and so on

• One-step amplification from RNA (reverse transcriptase loop-mediated isothermal amplification)

Figure 3. Primer design for loop-mediated isothermal amplification assays, showing the position of the six primers spanning the target gene. Six distinct regions are designated on the target DNA, which are labeled as F3, F2, F1, B1c, B2c and B3 from the 5′ end. The internal primers are referred to as the FIP and the BIP, and both have sense and antisense sequences, which help the formation of a loop. BIP: Backward internal primer; FIP: Forward internal primer; LPB: Loop primer backward; LPF: Loop primer forward.

Target DNA5´3´

5´ 3´

F1cF2cF3c B1 B2 B3

B1c B2c B3cF1F2F3

FIP

F25´3´

F1c

F3 primer

5´F3

3´B2 B1c 5´

5´3´B3

BIP

B3 primer

LPF

LPB

LPF5´ 3´

LPB

3´ 5´

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five parasites per microliter of Plasmodium spp. and P. falciparum parasites within 30–40 min. Different regions of the mit gene were also selected to detect P. vivax, and the results were comparable to those using microscopy and nested PCR [82]. Recently, a P. knowlesi species-specific LAMP detected the β-tubulin gene, and its sensitivity was 100-fold higher than a single PCR assay [80].

The LAMP method has been devel-oped to detect f ive human malaria parasites of Plasmodium spp.; that is, P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi (Table 2). The P. falciparum LAMP (PfLAMP) method [37] was established first, which was fol-lowed by the development of Plasmodium spp. and four species-specif ic LAMP assays with species-specif ic primer sets for use with clinical samples [38]. The Plasmodium genus-specif ic LAMP had 98.5–100% sensitivity and 94.3–100% specif icity compared with microscopic examinations in two previous reports [80]. The sensitivity (five parasites/µl) of mit gene-based LAMP assays was higher than an 18S rRNA gene-based LAMP assay (50 parasites/µl) for the detection of Plasmodium spp. and P. falciparum parasites. PfLAMP has also been com-pared with three diagnostic tools; that is, microscopy, RDT (PfHRP-2) and PCR [37,80,85–88].

PfLAMP was established to analyze heat-treated patient blood samples (boil-ing for 5 min) in field malaria research [37]. Previous PfLAMP studies have reported high sensitivity (92.0–100%) and specific-ity (91.7–100%) [37,86–88], with the excep-tion of one report (73.1–77.6% sensitivity and 89.6–100% specificity) [85]. The sensi-tivity and specificity of PfLAMP is similar to PCR-based methods when compared with microscopic examination as the ref-erence method, and similar to the results obtained using template DNA extracted with commercial QIAmp® Blood Mini kits (Qiagen, Hilden, Germany), with heat treatment and/or Chelex®-100 (Bio-Rad, CA, USA). Reverse transcriptase (RT)-LAMP was developed for the clini-cal detection of P. falciparum gametocytes using nested RT-PCR as the gold standard [81]. Pfs16 and Pfs25 gene-based RT-LAMP

Figure 4. Field-based clinical procedure for loop-mediated isothermal amplification. (A) Malaria patient blood sample mixed with distilled water; (B) heating at 95°C for 5 min in boiling water; (C) centrifugation (Model Micro 12™, Hanil Science Industrial, Ganeung, Korea, 110 v) at 2000 rpm for 5 min; (D) after centrifugation, the supernatant is mixed with the commercially available reaction mixture (Loopamp® DNA Amplification kit, Cat. # LMP206 (US$5.31/test, Eiken Co, Ltd., Tokyo, Japan) or ready-made reaction mixture prepared with individual reagents (US$0.28/test) [37,72] and kept at -20°C; (E) incubation in a water bath (110 v) for 1 h at about 62 ± 2°C in range; and (F) read-out of product using the naked eye or a Loopamp real-time turbidimeter (LA500, Eiken Chemical Co., Tokyo, Japan). gDNA: Genomic DNA.

Heating(95°C, 5 min)

Centrifuge

Reactionmixture

+ primers,gDNA

Visibleread-out

Water bath

Incubation

62°C, 1 h

Figure 5. Visual readout of loop-mediated isothermal amplification. (A) After incubation in a closed system, the byproducts of LAMP (magnesium pyrophosphate) or a fluorescent metal indicator (calcein) are monitored using the naked eye, a turbidimeter, a UV transilluminator or a black light with/without fluorescent dye. (B) Agarose gel electrophoresis analysis visualizes the typical electrophoresis pattern of the LAMP-amplified product, which is a ladder pattern rather than a single band because the LAMP method produces various sizes of the amplified product that consist of repeats of the target sequence connected by a loop on the same strand. LAMP: Loop-mediated isothermal amplification; M: Size marker.

Positive Negative Fluorescentdye

Agarose gel analysis

Naked eyeor

turbidometer

UVTransilluminator

-

+

+

+ UV light

Productstructure

Detection methodsProduct

M Positive

Detection methodsProduct

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Table 2. Performance assessments of loop-mediated isothermal amplification in the diagnosis of Plasmodium species in previous studies.

Species Samples (n)

Sensitivity (%)

Specificity (%)

Reference test

DNA extraction method

Detection method

Country Notes Ref.

Plasmodium spp.

68 98.5 94.3 Microscopy Heat-Chelex Turbidimeter Thailand Results comparable to nested PCR

[38]

Plasmodium spp.

31 100 100 Microscopy Heat Turbidimeter Various WHO international standard samples

[80]

Plasmodium falciparum

102 95.0 99.0 Nested PCR Heat Turbidimeter Thailand Results comparable to nested PCR

[37]

P. falciparum 16 100100

10094.3

Nested PCRMicroscopy

Heat-Chelex Turbidimeter Thailand Results comparable to nested PCR

[38]

P. falciparum 115 76.176.179.177.673.1

89.683.358.3100100

Nested PCRNested PCRNested PCRRDT (HRP-2)Microscopy

Qiagen Mini prepHeatHeat

GelGelNaked eye

Thailand Heat treatment and gel or naked eye detection

[85]

P. falciparum 137 98.5 Microscopy Organic solvent Turbidimeter Thailand, Zimbabwe

Comparison heat treatment from FTA® card filter paper samples

[86]

P. falciparum 49 98.092.0

10093.0

Nested PCRMicroscopy

Qiagen Mini prep Turbidimeter Thailand [87]

P. falciparum 94 98.996.7

10091.7

Nested PCRMicroscopy

Qiagen Mini prep RealAmp detector

Various RealAmp detecting the amplified product via fluorescence

[88]

P. falciparum 3015

100100

98.1100

Nested RT-PCRNested RT-PCR

TRIzol® Turbidimeter Thailand RT-LAMPResults comparable to nested RT-PCR

[81]

P. falciparum 19 94.7 100 Microscopy Heat Naked eye/turbidimeter

Thailand [75]

P. falciparum 30 93.3 100 Microscopy Heat Turbidimeter Various Results comparable to nested PCR

[80]

Plasmodium vivax

38 97.489.4

10094.3

Nested PCRMicroscopy

Heat-Chelex Turbidimeter Thailand Results comparable to nested PCR

[38]

P. vivax 23 95.7 88.9 Nested PCR Qiagen Mini prep Turbidimeter Thailand Only 65% sensitivity and 98% specificity compared with microscopy

[87]

P. vivax 117 98.393.3

100100

MicroscopyMicroscopy

Qiagen Mini prepHeat

Naked eyeNaked eye

Korea Results comparable to nested PCR

[79]

LAMP: Loop-mediated isothermal amplification; RDT: Rapid diagnostic test; RT-LAMP: Reverse-transcriptase loop-mediated isothermal amplification.

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had a higher sensitivity but similar specifi city when compared with nested RT-PCR. Given the low correlation between LAMP and PCR, RDT and microscopy in a previous study [85], further optimization is required for falciparum malaria parasite detection in field clinics.

A P. vivax LAMP assay was also developed successfully, which targeted the 18S rRNA and mit genes to detect parasites in blood filter paper samples using a heat-extraction method [38,79,82,84]. The limit of detection for P. vivax was 30–50 parasites per micro-liter [38,79], with 89.4–98.3% sensitivity and 88.9–100% specific-ity compared with microscopy and PCR using template DNA from heat-treated samples. The LAMP results using a simple heating method for P. vivax parasite detection were comparable to DNA extraction with commercial kits.

P. malariae and P. ovale LAMP delivered 75.0–100% sensitivity and 100% specificity with heat-Chelex-treated DNA samples, and the results were comparable with microscopy and PCR [38]. Two previous studies of P. knowlesi LAMP detected the β-tubulin and

ama1 genes [83,89] with high sensitivity in <30 min using samples with parasite densities of five parasites per microliter. When cou-pled with a heat-treatment method, the total reaction time may be technically sufficient to perform both the examinations and evaluations within 1 h.

The simplicity of template DNA preparation was studied using an FTA® Card test (DNA filter paper, WhatmanTM, GE Healthcare, PA, USA) and melting curve analysis [86]. The LAMP DNA template preparation method can reduce the risk of con-tamination compared with agarose gel electrophoresis, which was not recommended after adapting the melting curve analysis as a simple read-out.

In practice, a portable handheld detector device is applicable for field clinics in endemic areas; however, it requires high cost. Lucchi et al. developed a portable ‘RealAmp detector’, which combined an isothermal amplification platform (heating block) and a fluorescent detector for end point use (to acquire real-time data) in a single compact device for LAMP assays [88].

Table 2. Performance assessments of loop-mediated isothermal amplification in the diagnosis of Plasmodium species in previous studies (cont.).

Species Samples (n)

Sensitivity (%)

Specificity (%)

Reference test

DNA extraction method

Detection method

Country Notes Ref.

P. vivax 89 98.3 100 Microscopy Heat Naked eye China Developed color of LAMP using SYBR® Green I; results comparable to nested PCR

[82]

P. vivax 39 97.4 100 Microscopy Heat Naked eye/turbidimeter

Thailand Comparison with heat treatment of heparinized capillary tube samples

[75]

P. vivax 164 97.6 100 Microscopy Heat-Chelex Naked eye China Results comparable to nested PCR

[84]

Plasmodium malariae

10 100 100 Nested PCR Heat-Chelex Turbidimeter Thailand Results comparable to nested PCR

[43]

75.0 100 Microscopy

Plasmodium ovale

7 100 100 Nested PCR Heat-Chelex Turbidimeter Thailand Results comparable to nested PCR

[43]

100 100 Microscopy

Plasmodium knowlesi

Microscopy Heat Turbidimeter Experimental infection of a monkey

[89]

P. knowlesi 74 100 100 Nested PCR Qiagen Naked eye Malaysia LAMP combined with DNA filter paper and melting curve analysis

[83]

LAMP: Loop-mediated isothermal amplification; RDT: Rapid diagnostic test; RT-LAMP: Reverse-transcriptase loop-mediated isothermal amplification.

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To improve the detection sensitivity of the amplified product, a LAMP assay using an intercalating dye, SYBR Green I [85,88], or a fluorescent metal indicator, calcein [71], can be used to measure the end reaction using a UV light or a real-time fluorescence reader. To prevent contamination during the addition of the SYBR Green dye after amplification, Tao et al. opted for the pre-addition of a microcrystalline wax-dye capsule containing the SYBR Green I in the LAMP reaction tube before the initiation of the reac-tion [82]. In a field test of the fluorescent LAMP assay, the sensi-tivity was lower than normal LAMP with heat-treated samples in field clinics in Thailand [Jetsumon S, MVRC, Mahidol University,

Thailand; Unpublished Data]. This may require confirmation with a large number of samples in field clinics. These results support the development of rapid and objective analysis for the diagnosis of clinical malaria samples in the field as an RDT in the near future.

Parasite detection from mosquitoesLAMP could be used for the detection and differentiation of Plasmodium species in mosquitoes and applied to vector-control activities, which is a promising field that requires development and evaluation. A rapid test for intramosquito stages may be another advantage of LAMP. Previous results suggest that LAMP may be applicable to mosquito studies using dipstick tests for the detection of sporozoites in anopheline mosquitoes [90–93]. Recently, LAMP was also applied to the detection of Plasmodium berghei oocysts and sporozoites in Anopheles stephensi mosquitoes with high sensitivity [94]. LAMP detected the parasite in template DNA samples, which were extracted from whole mosquito bodies [94]. More recently, a multiplex fluorescence-based LAMP assay was developed for the simultaneous sensitive and specific detection of multiple parasites in mosquitoes [95]. Both of these studies reported highly promising findings, which demonstrated the possible future integration of LAMP to provide a simple and cost-effective molecular approach for use in parasite-infected mosquito control programs.

Current limitations & the need for further developmentThe validation of the robustness, performance and utility of the LAMP assay has a long way to go in field trials as a point-of-care (POC) test. Microscopic examination could identify the presence of parasites at an average of 50–100 parasite per microliter with morphological findings [13]; however, LAMP does not differenti-ate among the various development stages within infected eryth-rocyte. High sensitivity of LAMP is similar to that of the PCR method. LAMP is monitored density of parasites, likely real-time PCR, among samples by real-time monitoring system. In practical sample collection methods, blood filter paper (Whatman FTA Card [GE Healthcare] or filter paper [Schleicher and Schuell no. 903]) is widely used; however, template DNA preparation from those of samples is lengthy compared with direct boiling of whole-blood samples in a heparinized (capillary) tube with distilled water at a 1:1 (v/v) ratio. Therefore, it requires at least 1 h for fast amplification from template DNA preparation to visual inspection of results and the cost of time included template preparation (transfer of sample and centrifugation), reaction mixture preparation and incubation in water bath-optimized temperature for at least 30–40 min. Without

standard DNA extraction by commercial kits, the sensitivity and specificity of the reaction needs to be validated from variable reaction conditions. It may be limited by the difficulty of visualization of pre-cipitates, especially at lower target DNA concentrations. Therefore, attempts have been made to use the intercalating dye SYBR Green I to measure the end reaction using a UV light [82,84,85,87] or conven-tional real-time PCR fluorescence readers [70,88]. However, it would be necessary to overcome false-positives from a previous study [85] with modifications of the LAMP method [86].

In addition, the use of sophisticated equipment for diagnostic applications is not feasible in many field settings due to the lack of appropriate infrastructure. Therefore, there is a need for a simple field-usable method that can afford a quicker and objective read-out for the diagnosis of malaria using the LAMP method. The util-ity of a simple portable device (tube scanner or turbidi meter) was designed from both the amplification platform (heating block) and fluorescent detection unit (hand-held UV lamp) for end point use (with the ability to acquire real-time data); however, it may need to improve cost-effective device as portable one being used in field clinics. The reagents, including reaction mixture was required for shipment, storage and transportation at cold chain for conduction of LAMP assays in remote malaria clinics, where primary diagnosis is conducted by microscopy. The total cost of the LAMP reaction including primer design and reagents is US$0.28 for an individual reaction test [37]. Commercial reaction mixtures (US$5.31/test, Eiken Co, Ltd., Tokyo, Japan) are available and, alternatively, a reaction mixture may be prepared from individual reagents by the researcher in each laboratory with similar sensitivity and at a lower cost. Another current drawback of the technology compared with real-time PCR is the inability to carry out multiplex LAMP. Therefore, to diagnose malaria and differentiate between the species, one needs to carry out several LAMP reactions, making the technol-ogy more complicated and more expensive. However, the wider field application of LAMP for malaria diagnosis requires that several con-siderations are addressed: the variable condition of template DNA after the heat treatment of blood samples and the reduction of the sample preparation time; the high risk of irreversible contamination if users lack an adequate understanding of the LAMP mechanism, especially from agarose gels after electrophoresis in the laboratory; and the technical-specific training for performance of tests such as molecular-based tests to pipette the very small quantities of all the different component primers, buffers, template DNA and so on.

Expert commentarySimple, rapid, specific, sensitive and low-cost diagnostic methods will have a significant impact in reducing the incidence, morbid-ity and mortality of malaria parasites in endemic countries. Three diagnostic tools – that is, microscopy, PCR and RDTs – are used widely, and PCR has the highest sensitivity and specificity. In addi-tion, new molecular tools are required with RDT functions to facilitate rapid visual detection. Novel LAMP-based molecular technologies have been successfully applied to the detection of malaria parasites with high sensitivity, simplicity, rapidity and high efficiency in endemic and nonendemic countries. As a pos-sible future POC test, or something similar, LAMP simplifies

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the molecular diagnosis of malaria and provides the following advantages: high sensitivity and specificity, which are similar to PCR; the detection and differentiation of Plasmodium species; the development of rapid on-site molecular diagnostic tests; simple read-out results and visual detection with the naked eye or using fluorescent dyes; and cost–effectiveness for the early diagnosis, prompt treatment and post-treatment monitoring of malaria. In addition, the best possible sensitivity may be more relevant in nonendemic countries than in semi-immune adults in endemic countries. Other aspects in endemic countries include: diagnosis in acutely ill nonsevere malaria cases before ACT treatment in remote areas, diagnosis of malaria in pregnant women, future detection of asymptomatic parasitemia because of eradication, diagnosis in a child with cerebral malaria in a hospital setting and so on.

Five-year viewFollowing the development of high-sensitivity and -specificity nested PCR techniques, new isothermal amplification techniques have been introduced that enhance the performance of existing techniques. However, there is a need to develop more accurate

and sensitive methods for the diagnosis of malaria. LAMP-based technologies provide simplicity, rapidity and high specificity for the detection of malaria parasites above certain detection limits in patient samples and mixed malaria infections. Malaria LAMP can detect Plasmodium spp., including five human malaria parasites. The detection of drug-resistant malaria parasites is another reason for the development of rapid and cost-effective molecular tech-niques; however, this requires further study. As a POC test for the molecular diagnosis of malaria, a small single-use device has been developed that combines a real-time reader, a sensitive naked-eye signal detector, agarose gel electrophoresis and an incubator for the isothermal amplification of target genes.

Financial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• Advances in molecular techniques over the past 20 years have led to new diagnostic techniques for malaria parasites.

• The loop-mediated isothermal amplification (LAMP) assay is as sensitive and specific as nested PCR, and both the methods are more efficient for parasite detection and identification than microscopy.

• The LAMP reaction delivers results within 30–40 min, and positive assays are easy to identify based on a visual examination of the reaction tube.

• A simple and low-cost LAMP protocol might provide an alternative to microscopy for the routine screening of malaria parasites.

• In many studies, malaria LAMP tools have proven useful for molecular diagnostics, and they might be applicable in field clinics in the future.

ReferencesPapers of special note have been highlighted as:• of interest•• of considerable interest

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