Establishment and application of a novel isothermal amplification assay for rapid detection of chloroquine resistance (K76T) in Plasmodium falciparum

Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloroquine resistance transporter (Pfcrt) gene. The point mutation at codon 76 (K76T), which has been observed in more than 91% of P. falciparum isolates in India, is the major determinant of CQ resistance. To overcome the limitations and challenges of traditional methods, in this investigation we developed an easy to use loop mediated isothermal amplification (LAMP) protocol for rapid detection of the K76T mutation associated with CQ resistance in P. falciparum with naked eye visualization. In- house designed primers were synthesized and optimized to specifically distinguish the CQ resistant mutants of P. falciparum. The LAMP reaction was optimal at 61 °C for 60 min and calcein dye was added prior to amplification to enable visual detection. We demonstrate the detection limit of <2 ng/μl respectively, supporting the high sensitivity of this calcein based LAMP method. To the best of our knowledge this is the first report on the establishment of an easy, reliable and cost effective LAMP assay for rapid and specific detection of highly CQ resistance in P. falciparum malaria.

(FIP, BIP) primers, identifying six distinct regions on the target DNA 8 . In case of blood parasite detection, LAMP has been found superior to the PCR based techniques, especially due to its stability against inhibitory substance present in blood 12 , easy single step amplification reaction, visual detection and high amplification efficiency 13 .
In recent studies, LAMP assay has also been successfully applied for molecular detection and diagnostics of bacterial 14,15 , viral 16,17 fungal [18][19][20] and parasitic diseases 21,22 in both animals and plants. Several studies investigate loop mediated isothermal amplification assay (LAMP) for the detection of malaria parasite in humans [23][24][25][26][27][28][29] . However the application of LAMP in the field of anti-malarial drug resistance detection has not been tested so far. In fact early detection of anti-malarial drug resistance in an endemic population is one of the key requirements to plan any pilot surveillance project for control/treatment of malaria, but currently available molecular tools face challenges and limitations of rapid detection in the field setup, requiring optimal laboratory setup with trained man power and cost effectiveness. In this investigation we optimized and tested the ability of LAMP strategy as a novel, reliable and economic tool for rapid CQ resistance detection in field settings.
We developed a field based LAMP method for the detection of CQ resistance in P. falciparum caused by K76T point mutation of the Pfcrt gene as a molecular marker. Our data provide strong evidence that this new LAMP method could be used for early warning of CQ resistant P. falciparum in field conditions. We believe that pre-establishment of a novel isothermal amplification molecular assay for K76T detection could also be useful to develop similar technique valuable for the other important molecular markers like K13-propeller region (especially mutation at codon C580Y, R539T, and Y439H) for large-scale surveillance of artemisinin resistance in P. falciparum 30,31 .

Results
Optimization and specificity of in-house designed LAMP Primers. LAMP primers were designed for Pfcrt gene encoding K76T point mutation to detect CQ resistance, five primers set (Supplementary Table 1) was designed by using the software Primer explorer V4 program. For the accuracy and specificity of LAMP method the optimization of LAMP primers is essential task. To increase the specificity of LAMP primers for CQ resistance detection, one mismatch was inserted to the forward inner primer (FIP) enabling specific amplification at the target position so the primers would specifically amplify the target position K76T (Fig. 1A,B). Five sets of primers were synthesized and tested for appropriate set of LAMP primers to differentiate the K76T mutant and wild type genomic DNA of P. falciparum. Out of five sets only one primer set (FIPM4) qualified for accurate detection of the K76T mutants associated with CQ resistance (Fig. 2). Calcein dye induced color change to bright yellow (positive reaction) in the LAMP in K76T mutant was easy to visual discrimination, when compared to light orange color (negative reaction) in the wild type ( Fig. 2A). In post visual discrimination we further reconfirmed the data by gel electrophoresis showing, ladder like pattern in mutant strain (positive control) but missing in wild type (negative control) on 3% agarose gel (Fig. 2B). These results indicated that our in-house designed primer set could be used to distinguish the CQ resistant K76T mutant of P. falciparum.

Optimization of standard conditions and validation of LAMP reaction.
For the optimization of LAMP reaction the initial experiments was performed by using genomic DNA of the mutant control (RKL9), wild type control strain (3D7, NF54, MRC-2) and fifty well characterized samples of P. falciparum. To optimize the LAMP assay, a color change in the reaction was monitored in response to different temperature range from 57-65 °C with pre-defined time interval of 40-120 minutes. We observed a highest intensity of color change at 61 °C ( Fig. 3A) with an optimal time of 60 min (Fig. 4A) which was successfully corroborated by gel Standardization of LAMP reaction and sensitivity detection. Following microscopic verification next we evaluated the detection limit of LAMP using serially diluted samples. Our results shows LAMP could detect a limit of 10 −7 fold dilution, accounting 7 parasites/μ l supporting the high sensitivity of LAMP technique. The parasite samples chosen for these dilutions contained initial densities ranging between 0.02 and 1 parasitized cells per 100 red blood cells (referred to as 0.02% and 1% parasitemia, respectively), as determined by microscopic examination. DNA concentration was quantified by using spectrophotometer (Nanodrop Technologies), where a tenfold dilutions were used to perform the reaction. The lowest concentration (< 2 ng/μ l) of standard DNA detection was successfully observed in 10 −7 fold dilution (Fig. 5). This confirmation of LAMP sensitivity was monitored by calcein induced color change visualization at different dilutions ( Fig. 6A) followed by reconfirmation with 3% gel electrophoresis ( LAMP specificity and accuracy. The above demonstration of the successful LAMP assay optimization clearly differentiated the mutant (RKL9) and wild type (3D7, NF54, MRC2) control strains. To better evaluate the efficiency and specificity we further assessed the calcein dye fluorescence in the presence of UV light ( Fig. 7A and B). Our results showed dark black coloration appearance in the positive LAMP amplification DNA samples having K76T mutant, reconfirmed by ladder like pattern on gel electrophoresis, but no amplification in the negative reaction (Fig. 7C). A comparative assessment of each LAMP experiments with the inclusion of negative, positive   (Pre-validated strains) control samples not only showed high specificity of our test, but also confirmed the 100% accuracy, as validated by sequencing analysis (given below).

LAMP validation, stability and repeatability test.
Finally, to validate the result of positive LAMP reaction, the final amplification products including mutant control strain RKL9 were subjected to gold standard method of DNA sequencing. Our results showed 100% identity to DNA sequences when compared to the standard mutant and wild type strains (Fig. 8), that optimized LAMP to specifically amplify the Pfcrt gene region having K76T mutation. To test the stability, robustness and repeatability of the LAMP technique, 36 known K76T mutants and 14 known wild type samples (with the wild type and mutant controls) from Tripura region (Table S2 and Fig. 8) were tested three times independently by LAMP. All the mutant strains of P. falciparum were found positive including the mutant control RKL9 and wild type strains were negative including the wild type control. Analysis was based on calcein-visualization or gel electrophoresis (Figs 2 and 7). Sensitivity, specificity and accuracy of LAMP were compared and statistically analysed with PCR-RFLP and gold standard sequencing results ( Table 1). The highest agreement with sequencing gold standard was achieved with the LAMP visual interpretation and LAMP-gel (100%, kappa (κ ) = 1.0), showed a similar level of agreement with sequencing ( Table 1). The visual interpretation by LAMP seemed to be high with kappa value 1.0 respectively. The κ statistics (range 1, to − 1) measures the observed percentage of agreement between tests against what might be expected by chance, ranging from < 0 = poor agreement to 1 = perfect agreement. On the basis of results it suggested that establishment  Application of LAMP for monitoring chloroquine resistant P. falciparum as a field based method. The aim of this study was to demonstrate the application of LAMP for rapid monitoring of chloroquine resistant P. falciparum in the field conditions. Our technique is based on simple one step amplification procedure, where; results could be analyzed by naked eye within 60 min without using thermal cycler and other standard lab practices. To further verify the application of LAMP, we analyzed a total of 50 well characterized P. falciparum samples including 36 mutants (K76T) and 14 wild type strains. In each experiment we included, the   (Table S2). The results detected by LAMP were in full agreement to the result of sequencing gold standard method (Table S2 and Fig. 8); verifying that current optimized LAMP strategy is feasible to detect the CQ resistant K76T mutants with limited amount of DNA samples and can be used for rapid monitoring of CQ resistant population of P. falciparum in field.

Discussion
In recent years LAMP strategy has been recognized as a unique and valuable molecular tool enabling fast, simple, accurate and cost effective diagnosis and detection of various bacterial, viral, fungal and parasitic diseases 11,[14][15][16][17][18][19][20][21][22][23][24]26,32,33 . However the application of LAMP strategy to detect anti-malarial drug resistance in P. falciparum remains un-reported. To test and validate its suitability of malaria resistance evaluation, we developed a field based simple, easy to use and rapid LAMP assay, enabling highly chloroquine resistant P. falciparum detection. For a successful LAMP method, it is always challenging to design and identify highly specific primers, quantifying various conditions and possibilities of one step target DNA amplification. Initially, to identify K76T mutation in Pfcrt gene responsible for CQ resistance in P. falciparum, we designed and tested a set of five primers; while only one primer set (FIPM4) out of five was found to be most appropriate for the detection of K76T mutation in CQ resistant P. falciparum (Table S1 and Fig. 2). Selected loop primers not only accelerated the LAMP reaction by shortening the reaction time in comparison to the original method 34 ; but also distinguished the mutant from the wild types, simply by one mismatch addition to the forward inner primer (FIP).
On the basis of selected most appropriate LAMP primer set, the reaction conditions and the concentration of reagents were optimized for successful LAMP assay establishment. In fact, the principle of LAMP results visualization is mainly based on the production of pyrophosphate ions, which binds to metal indicator and form a white precipitate. Therefore the naked eye visualization of LAMP results is possible by adding metal ion as indicator or fluorescent dye i.e. calcein, hydroxynaphthol blue (HNB), SYBR green, calcium chloride, magnesium pyrophosphate and CuSO 4 etc 35 . Though most of fluorescent intercalating dyes (SYBR green, Picogreen, EvaGreen etc.) can be added after the completion of LAMP reaction, but re-opening of reaction tubes and adding dye may have greater chance of contamination and false results. To avoid the chances of contamination, in this study we added calcein dye in the reaction mix prior to incubation, therefore the reaction was completed in a closed system and the color change detection system doesn't require any costly equipment. For a successful LAMP reaction simple naked eye visualization of color change from light orange to bright yellow for positive samples, was further confirmed through gel electrophoresis.
As compared to traditional PCR based methods, the developed LAMP is not only easy to use, faster, without requiring any costly instruments and sophisticated lab settings, but the results are also easier to assess with naked eye. Furthermore, in our protocol we observed that a successful LAMP reaction needing an optimal temperature 61 °C incubation, and could be finished within 60 min, for the detection of K76T mutation in P. falciparum. Because LAMP is conducted isothermally, requiring heat block of constant temperature and thus it should be useful even for those laboratory conditions unfamiliar with PCR based methods or other molecular analysis. Our independent validation by DNA sequencing of each LAMP experimental data provide strong evidence of robustness, repeatability, specificity of our in-house optimized LAMP protocol with high accuracy and sensitivity, requiring very limited amount i.e. < 2 ng/μ l of genomic DNA.
Abdul Ghani 36 suggested the application and possibilities of LAMP as POCTs for the diagnosis of malaria where; several studies followed to evaluate the LAMP assay in the field of malaria diagnosis [23][24][25][26]33 . However evaluation of antimalarial drug resistance using LAMP remains un-tested, though it has been well tested to detect resistance in bacteria [37][38][39] . Towards this goal, our newly developed LAMP was found highly sensitive, specific and efficient as compare to other PCR based molecular methods for rapid and easy detection of CQ resistance in P. falciparum.
In conclusion a field based LAMP assay with calcein visualization was established and demonstrated to be simple, easy to use, more sensitive, specific, and found to be very feasible to detect the K76Y mutation in CQ resistant samples of P. falciparum than previously used PCR based methods. Therefore the application of this method can be potentially useful for monitoring and early warning of CQ resistance in P. falciparum malaria, it could be an effective approach to facilitate anti-malarial resistance detection and tracking in resource-limited countries afflicted with the disease, so that correct and timely treatment can be given to reduce morbidity and mortality associated with the disease. There is need to field test the assay on a larger sample size to assess suitability in field conditions. Moreover we also believe that current optimized technique could be very helpful for rapid Genomic DNA extraction and Quantification. Genomic DNA was extracted from P. falciparum samples and control strains by using the QIAamp DNA Blood Mini/Maxi Kit (Qiagen) as per manufacturer's protocol.
In order to quantify the minimum DNA concentration (ng/μ l), optical density (OD) of extracted DNA samples were checked by using spectrophotometer (Nanodrop Technologies). A tenfold serial dilutions (10 −1 to 10 −7 ) were used to determine the minimum detected DNA concentration. The LAMP sensitivity was determined up to 10 −7 fold dilution (< 2 ng/μ l DNA).
In-house design LAMP primers. Five sets of primers for LAMP assay were designed by using Primer explorer V4 software (http://primerexplorer.jp/e/). The sequence of P. falciparum (GenBank accession number M19173.1) were used to designed the target, we have designed the parameters for most widely reported mutation (K76T) in Pfcrt gene associated with molecular detection of CQ resistance. LAMP primers sets and their sequence alignment used in this study are given in Table S1 and Fig. 1.

Specificity of LAMP primers.
To improve the specificity of the in-house designed LAMP primers, one base-pair mismatche was introduced within three nucleotides of the 3' end of the forward inner primer (FIP) (Fig. 1). Thus five sets of LAMP primers were synthesized and tested to distinguish the Pfcrt K76T mutants from the wild type. The genomic DNA of mutant control strain RKL9 and wild type control strain 3D7, NF-54, MRC2 were tested to validate the accuracy of LAMP primers. The LAMP was performed at the given standard optimum conditions; each LAMP product was analyzed by gel electrophoresis on 3.0% agarose gel. In addition, the LAMP amplification product visualized by naked eye with the calcein color change reaction from light orange to bright yellow if the required product was present in contrast it remain light orange in the absence of product.
Optimization of standard LAMP reaction and conditions. The standard LAMP reaction was opti- °C to determine the optimal reaction temperature and the LAMP was also performed at different time intervals of 40, 60, 80, 100 and 120 min to determine the optimal reaction time. The standard LAMP assay conditions were optimized at 61 °C for 60 min and then at 90 °C for 7 min to terminate the reaction. The LAMP assay was based on the assessment of calcein color change reaction and pattern of band in gel electrophoresis. To confirm the stability each experiment was repeated thrice having three independent replicates.

Confirmation of the LAMP results by Sequencing.
For the validation and accuracy of LAMP results, CQ resistant samples having Pfcrt K76T mutation and all the wild types were confirmed through commercial available sequence service.
Statistical analysis. All statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software version 10. Sensitivity, specificity and accuracy of LAMP and PCR-RFLP were comparable with results of sequencing gold standard reference test. In this study we evaluated the LAMP assay using two different methods (sequencing and PCR-RFLP) and each experiment was repeated thrice, leading to three practical modalities for assessing a positive test result.