{"paper_id":"dbb64624-c9b9-449a-ba86-141b583dcbba","body_text":"Head-to-head comparison of two loop-mediated isothermal amplification (LAMP) kits for diagnosis of malaria in a non-endemic setting | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Head-to-head comparison of two loop-mediated isothermal amplification (LAMP) kits for diagnosis of malaria in a non-endemic setting Anna-Clara Ivarsson, Elin Fransén, Ioanna Broumou, Anna Färnert, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2984503/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Dec, 2023 Read the published version in Malaria Journal → Version 1 posted 8 You are reading this latest preprint version Abstract Introduction: Light microscopy and rapid diagnostic tests (RDT) have long been the recommended diagnostic methods for malaria. However, in recent years, LAMP techniques have been shown to offer superior diagnostic performance, in particular concerning low-grade parasitaemia, by delivering higher sensitivity and specificity with low laboratory capacity requirements in little more than an hour. In this study we assessed the diagnostic performance of two Loop-mediated Isothermal Amplification (LAMP) kits head-to-head, compared to highly sensitive quantitative real time PCR (qPCR), in a non-endemic setting. Methods: In this retrospective validation study two LAMP kits; Alethia® illumigene Malaria kit and HumaTurb Loopamp™ Malaria Pan Detection (PDT) kit, were evaluated head-to-head for detection of Plasmodium -DNA in 133 biobanked blood samples from suspected malaria cases at the Clinical Microbiology Laboratory of Region Skåne, Sweden to determine their diagnostic performance compared to qPCR. Results: Of the 133 samples tested, qPCR detected Plasmodium DNA in 41 samples (the true positives), and the two LAMP methods detected 41 and 37 of those, respectively. The results from the HumaTurb Loopamp™ Malaria PDT kit were in complete congruence with the qPCR, with a sensitivity of 100% (95% CI 91.40 - 100%) and specificity of 100% (95% CI 96.07 – 100%); and the PPV and NPV were both 100%. The Alethia® illumigene Malaria kit had a sensitivity of 90.24% (95% CI 76.87 – 97.28) and a specificity of 95.65% (95% CI 89.24 – 98.80) as compared to qPCR. The PPV was 90.31% (95% CI 78.06-96.07) and the NPV was 95.62% (95%CI 89.58-98.23) with the malaria positivity rate of 30.8 % in the study material. This performance would give a PPV of 69.75% (95% CI 46.80 – 85.81) and NPV 98.88% (97.20 – 99.56) in a situation where samples tested had a 10% positivity rate, which more accurately resembles the situation in Skåne. Conclusions: This head-to-head comparison showed superior performance of the HumaTurb Loopamp™ Malaria PDT kit compared to the Alethia® illumigene Malaria kit for detection of malaria. Malaria LAMP Loop-mediated isothermal amplification diagnosis. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Management of malaria relies on rapid and accurate diagnosis to secure prompt treatment. The diagnostic methods recommended for malaria diagnosis by the WHO continue to be light microscopy and immunochromatographic rapid diagnostic tests (RDTs)(1). While the sensitivity and specificity of RDTs are about 95% for the most virulent malaria species, P. falciparum , the sensitivity is lower for other Plasmodium species as well as for parasite densities below 200/µL (2). The diagnostic sensitivity and specificity of light microscopy is highly dependent on the skills of the microscopist, something that can be hard to maintain 24/7 in non-endemic settings such as Sweden (3–5). In recent years however, nucleic acid amplification test methods, such as loop mediated isothermal amplification (LAMP), have been tested increasingly in epidemiological studies as well as in clinical settings and have been proposed as a highly sensitive, cost-effective alternative in non-endemic high resource settings (4, 6–10). In LAMP, the DNA-targets are amplified without the temperature cycles of the PCR-technique. Instead, a DNA-polymerase with strand displacement activity makes multiplication of the DNA-target possible at a constant temperature. The sequences targeted by the primers lead to the formation of DNA loops during amplification, which allows a highly sensitive and specific reaction and a shorter time to detection than is usual for PCR. The DNA-amplification reaction forms a white precipitation of magnesium pyrophosphate, detected as turbidity or as fluorescence when exposed to UV light (if dyed with calcein). LAMP methods usually do not quantify the DNA content of the sample, which qPCR methods may through a Cq/CT-value (4, 11). LAMP techniques have been shown to offer superior diagnostic performance to RDT and microscopy, in particular concerning low grade-parasitaemia, and can deliver results with higher sensitivity (97–100%) and specificity (99.2–100%) with low laboratory capacity requirements in a little more than an hour (3–5, 8, 12–19). The high diagnostic performance has been shown to include also non- falciparum species (15, 17, 20). The above-mentioned challenges with current malaria diagnostics and the promising results of LAMP techniques have led us to the question if a LAMP technique could replace RDT and microscopy as a first line, point-of-care test for malaria in southern Sweden. LAMP could then be used to rule out malaria in negative cases and microscopy would only be needed in positive cases to determine Plasmodium species and parasite density. Therefore, this study was designed to assess the diagnostic performance of the internationally available LAMP kits. There are two main LAMP instruments on the international market today: the Alethia® Malaria illumigene (Meridian Bioscience) with reagents in the Malaria or Malaria PLUS kit, and the HumaTurb C + A or Humaloop M (Human Diagnostics Worldwide) with Loopamp™ Malaria Pan Detection kit and separate kits for species identification of P. falciparum or P. vivax (Table 1 ). The aim of this study was to compare these two LAMP instruments and kits by testing the Malaria kit for the Alethia® Malaria illumigene instrument against the Loopamp™ Pan Detection (PDT) kit for the HumaTurb C + A instrument. Hereafter they will be referred to as Alethia® illumigene Malaria kit and HumaTurb Loopamp™ Malaria PDT kit. These two kits have been tested compared to conventional methods in different settings but have to our knowledge not previously been compared head-to-head on the same samples (4, 9). This study was performed as a retrospective laboratory validation study of analytic performance of two LAMP assays on biobanked samples from patients with suspected malaria in Region Skåne. The main research question was which of the two LAMP kits that had the best diagnostic performance compared to highly sensitive qPCR in patients with suspected imported malaria in the non-endemic setting at the Clinical Microbiology Department of Skåne in the South of Sweden. Additionally, we wanted to assess if any of the two LAMP kits could be used as a semiquantitative measure of parasitaemia level. Table 1 – Characteristics of Alethia® illumigene Malaria kit and HumaTurb Loopamp™ Malaria PDT kit. Alethia® illumigene Malaria HumaTurb Loopamp™ Malaria PDT Mean time to result as measured in the study 44 min 60 min Time to result as reported by the manufacturer * < 45 min 50–55 min Mean active time as measured in the study 4 min 10 min Analysis * Qualitative Qualitative + turbidity graph and time to detection Maximum samples per run * 10 16 (up to 94 if expanded with 6 HumaTurb A units) Species identification * Not possible Not possible Validated for species detection P. falciparum, P. vivax, P. ovale, P. malariae. Performance for P. knowlesi established using purified genomic DNA only; whole organism testing has not been performed. P. falciparum, P. vivax, P. ovale, P. malariae. P. knowlesi validated in one study† Read out of the results * Turbidity in Illumipro-10™ incubator or by eye Turbidity in turbidimeter or fluorescence detected by eye Limit of detection* 2.0 p/ µL for P. falciparum and 0.1 p/ µL for P. vivax 1 p/ µL * As described by the manufacturer (11, 24, 26) . † (17) (Table 1 ) Method Study design and study site The performance of two LAMP instruments for detection of Plasmodium -DNA in biobanked blood samples were retrospectively evaluated at the Clinical Microbiology Laboratory of Region Skåne, Sweden to determine their performance compared to highly sensitive qPCR. Performance was defined as sensitivity and specificity compared to qPCR to detect DNA in the first diagnostic blood sample sent to the lab from patients with clinical suspicion of malaria. The Clinical Microbiology Laboratory of Region Skåne is the main laboratory for the Skåne region in the south of Sweden, serving 9 hospitals and covering a population of 1.4 million. The positivity rate of suspected malaria samples at the Clinical Microbiology Laboratory of Region Skåne during the study time was 8–10% yearly (unpublished data from the Laboratory Information System, Region Skåne). The diagnostic method for malaria during the time of the study was RDT (CareStart Malaria HRP 2 /pLDH (Pf/PAN) Combo Test) and light microscopy of thick and thin blood smear films prepared with Giemsa stain according to the current standard at the Clinical Microbiology Laboratory of Region Skåne. The microscopy was performed by an infectious disease specialist at the point of care laboratory, for Plasmodium species and parasitaemia. After diagnosis, whole blood samples (EDTA) are routinely sent to the regional Clinical Microbiology Laboratory without further processing and stored at -80°C in the Region Skåne biobank 136, Klinisk mikrobiologi BD1. Ethics statement The study was approved by the Swedish Ethical Review Authority (Etikprövningsmyndigheten) ID 2020–05249. Study population and sample collection Blood samples sent to the Department of Clinical Microbiology on suspicion of malaria during the period 2018–2020 were selected for the study. These included samples that had been identified by routine light microscopy and/or RDT as malaria positive, and two consecutive negative samples. Exclusion criteria: samples not identified in the bio bank, insufficient volume for testing in the sample and inconclusive information about final malaria diagnosis in the clinical files. Clinical data collection Medical charts from included patients were obtained from Region Skåne medical databases. Epidemiological patient data was retrieved regarding sex, age, country of residence, country of origin, country of exposure, reason for travel, previous malaria episodes, chemoprophylaxis, time of exposure, time from symptom onset, malaria treatment initiated, presence of risk factors and comorbidities and data on parasitaemia and Plasmodium species from microscopy performed by clinicians. LAMP Each of the included frozen whole blood samples obtained from the biobank were tested retrospectively with two LAMP-kits: Alethia® Malaria illumigene Malaria kit (Meridian Bioscience) and HumaTurb C + A Loopamp™ Malaria Pan Detection kit (Human Diagnostics Worldwide). The tests were performed according to the manufacturer’s instructions. The LAMP analysis of Alethia® illumigene Malaria kit is qualitative and is reported as positive, negative, or invalid. HumaTurb Loopamp™ Malaria PDT kit is a qualitative test that also reports time to detection. Neither of the two tested LAMP kits offer species differentiation. The LAMP analyses were performed by research personnel together with staff at the Clinical Microbiology Laboratory of Region Skåne during 2021 and 2022. Results from microscopy were blinded to the research personnel performing the LAMP assays. Invalid results were re-tested once and if the result remained invalid the sample was reported as invalid. Real time PCR A total volume of 50 µL EDTA blood from each sample was used for genomic DNA extraction using the QIAamp DNA blood mini kit (Qiagen) and the protocol ‘DNA purification from Blood or Body fluids (spin protocol)’, according to manufacturer’s instructions. To reach a volume of 200 µL, starting material required for the specific extraction protocol, 150 µL of PBS 0.01M pH 7.4 was added to 50 µL EDTA blood, according to manufacturer’s recommendation. At the final step, the genomic DNA was incubated for 5 minutes at room temperature prior to elution in 150 µL elution buffer and stored at -20℃. For Plasmodium species detection and identification, a previously published multiplex, probe-based qPCR assay method was used, with slight modifications (21). The method targets the highly conserved Plasmodium 18S rRNA gene and allows the simultaneous detection of four Plasmodium species ( P. falciparum, P. vivax, P. malariae, P. ovale ) using species-specific primers and probes (supplement). Briefly, 5 µL of extracted DNA was used as template in a 25 µL PCR reaction with 1X TaqMan Multiplex master mix (Applied Biosystems), 0.2 µM of primers Fal-F, Viv-F, Mal-F, Ova-F, each, 0,5 µM of primer Plasmo2-R and 0.1 µM of probes Falprobe, Vivprobe, Malaprobe, Ovaprobe, each (all from Thermo Fisher). The cycling conditions were: 95°C for 20 seconds, followed by 45 cycles at 95℃ for 15 seconds and at 60℃ for 1 minute, and the amplification took place in a QuantStudio™ 5 Real-Time PCR system (Applied Biosystems). Each sample was analysed in triplicates. Serial dilutions of P. falciparum , P. vivax , P. malariae and P. ovale samples of known parasitaemia (0.1%, 0.3%, 0.2% and 0.1%, respectively) and non-template negative controls were used in each run. A cycle threshold (CT) value of 40 was used as a cut off to define positive samples. The limit of detection was 0.5-6 parasites. In case of inconsistent qPCR results, i.e., one out of three replicates positive, the qPCR was repeated, and the sample was analysed again in triplicates. CURRENT DIAGNOSTIC METHODS FOR MALARIA IN REGION SKÅNE A blood sample is taken in an EDTA-tube from the suspected patient and is tested with the immunochromatographic rapid diagnostic tests CareStart Malaria HRP2/pLDH (Pf/PAN) Combo Test at the local laboratory at the point of care. The same blood sample is used to prepare, within one hour, two thin- and two thick blood films using Giemsa-stain for light microscopy. The blood smears are immediately prepared with Giemsa-stain in all cases of positive RDT and after agreement with responsible clinician in cases of negative RDT test. The microscopy is performed by an infectious disease specialist at the point of care laboratory, assessing Plasmodium species and parasitaemia. Statistical analysis Sensitivity, specificity, PPV and NPV of LAMP techniques and microscopy plus RDT as compared to qPCR were calculated by the online statistical tool MedCalc (22). Correlations between HumaTurb Loopamp™ Malaria PDT kit time to detection, qPCR CT value and parasitaemia as reported by microscopy was estimated using the Microsoft Excel version 16.66.1 CORREL function to calculate correlation coefficient and Prism to create correlation graphs. Results A total of 51 positive samples from patients diagnosed with malaria by light microscopy and/or RDT between 2018–2020 were identified in the biobank list. To these, 102 consecutive negative samples were selected resulting in a total of 153 samples. Of these, 5 samples could not be located in the biobank leaving a total of 148 samples that were tested with the two LAMP techniques, 47 registered as positive for malaria based on microscopy and/or RDT and 101 as negative. Of these samples, 14 had insufficient amount of material to be analysed with qPCR, resulting in a total of 134 samples analysed by qPCR. The diagnosis of one of these samples was found to be inconclusive with negative results in all diagnostic tests except one of three PCR runs resulting in a high CT value of 40. The final clinical diagnosis of this patient is also unclear in the medical records. Therefore, this sample was excluded, resulting in a total of 133 samples analysed by both qPCR and the two LAMP kits. In total 41 samples were positive by qPCR and 92 were negative. The sample selection flowchart is detailed in Fig. 1 . (Fig. 1 ) Table 2 shows the demographic characteristics of the 47 included positive cases that were tested with the two LAMP kits. The cohort is well balanced by sex and age. Most cases were exposed in Sub Saharan Africa, visiting friends and family, and had not taken malaria chemoprophylaxis. Among the microscopy positive the parasite densities ranged between 0.1–4% and all were positive by RDT. Table 2 – Demographic characteristics of positive cases Characteristics of malaria cases (n = 47) n % Sex Men 27 57% Women 20 43% Age Median age (range) 36 y (6y-75y) Region of infection West Africa 25 53% East Africa 9 19% Central Africa 7 1% Central Asia 3 6% South Asia 3 6% Patient origin from endemic area Yes 35 74% No 12 26% Reason for exposure Visiting friends and family 18 38% Migrant 16 34% Tourist 9 19% Residency in endemic area 4 9% Previous malaria episodes Yes 27 57% No 8 17% No information 12 26% Chemoprophylaxis Yes 5 11% No 39 83% Incomplete prophylaxis 3 6% Parasitaemia % 0.1–0.4 13 28% 0.5–0.9 10 21% 1–1.9 6 13% 2–2.9 2 4% 3–4 4 9% No data 12 26% (Table 2 ) Diagnostic performance Diagnostic performance of Alethia® illumigene Malaria kit and HumaTurb Loopamp™ Malaria PDT kit as compared to RDT + microscopy and the reference method qPCR is presented in Table 3 and Table 4 . The results from the qPCR and HumaTurb Loopamp™ Malaria PDT kit were in complete congruence, resulting in a sensitivity of 100% (95% CI 91.40–100%) and specificity of 100% (95% CI 96.07–100%) for the HumaTurb Loopamp™ Malaria PDT kit as compared to the qPCR. Positive predictive value and negative predictive value were both 100%. When comparing the Alethia® illumigene Malaria kit to qPCR there were 4 false positive and 4 false negative results leading to a sensitivity of 90.24 (95% CI 76.87–97.28) and a specificity of 95.65% (95% CI 89.24–98.80). The positive predictive value was 90.31% (95% CI 78.06–96.07) and the negative predictive value was 95.62% (95% CI 89.58–98.23) in the study material. The samples were collected in such a way that the malaria positivity rate in the study material was 30.8% as opposed to the actual positivity rate in malaria suspected samples in Region Skåne that is around 10%. This difference affects the positive and negative predictive value. With a 10% disease prevalence in tested patients the PPV calculated from our study results would be 69.75% (95% CI 46.80–85.81) and NPV 98.88% (97.20–99.56) for the Alethia® illumigene Malaria kit. The Alethia® illumigene Malaria kit correctly identified 125 of 133 samples as either positive or negative (accuracy 94%) but failed to detect four cases of malaria and misdiagnosed four cases as positive. All four false positive results were negative in qPCR, microscopy and RDT. The clinical diagnosis for the four cases were viral infection or uncertain diagnosis. Out of the four false negative results two were positive in qPCR, microscopy and RDT with a noted parasite density of 0.2%. One of the cases had slightly inconclusive qPCR results with 2 out of 6 replicates (33.3%) positive in two independent qPCR experiments with a CT value of 40 in the first round and 38 in the second, negative microscopy and negative RDT. The clinical presentation of this case was a person with origin from an endemic area that was visiting friends and family and had had symptoms for one day. At presentation the case was diagnosed as non-malaria but three weeks later malaria was diagnosed at another hospital. This case likely had a very low level parasitaemia at the time of testing. The last false negative case was positive in two of three qPCR replicates with a CT value of 40, positive in microscopy and positive in RDT. This case had used oral self-treatment before seeking care and was first deemed as cured but was later reassessed as incompletely cured and put on malaria treatment with oral Atovaquone/proguanil once more which led to clinical improvement. It is likely that this case also had a very low level parasitaemia. In the study samples, the reference method qPCR identified P. falciparum, P. ovale and P. vivax samples along with one mixed infection with P. falciparum and P. ovale. Both LAMP assays correctly identified malaria caused by all species as positive, although one P. vivax sample and three P. falciparum samples were missed by the Alethia® illumigene Malaria kit. The results show that species identification by microscopy was a challenge with only 33 of 41 samples correctly identified by the microscopist as compared to the qPCR (Table 4 ). Most often the misdiagnosis consisted of the false identification of a mixed infection, but one mixed infection with P. falciparum and P. ovale was misdiagnosed in microscopy as only P. falciparum . Two qPCR positive samples were also missed in microscopy and identified as microscopy negative, but in one of the samples the patient had recently gone through successful treatment, and the microscopy is expected to be negative. In four samples out of the 148 analysed with the two LAMP techniques, the first result from the Alethia® illumigene Malaria kit was invalid. Upon retesting, all four samples had valid results. No invalid results were noted with the HumaTurb Loopamp™ Malaria PDT kit. Table 3 - Diagnostic performance of LAMP techniques compared to qPCR. Analysis Sensitivity Specificity % (95% CI) Proportion % (95% CI) Proportion PPV % (in study group) PPV 10% disease prevalence NPV % (in study group) NPV 10% disease prevalence Alethia ® illumigene Malaria 90.24% (76.87 - 97.28) 37/41 95.65% (89.24 - 98.8) 88/92 90.31 % (78.06 - 96.07) 69.75% (46.80 – 85.81) 95.62% (89.58- 98.23) 98.88% (97.20 – 99.56) HumaTurb Loopamp™ Malaria PDT 100% (91.40 - 100) 41/41 100% (96.07 - 100) 92/92 100% 100% 100% 100% Table 4 - Diagnostic performance and species identification of LAMP techniques compared to qPCR, microscopy and RDT. qPCR HumaTurb Loopamp™ Malaria PDT Alethia ® illumigene Malaria Microscopy - correctly identified as pos or neg Microscopy - correct species* RDT -correctly identified as pos or neg Total positive 41 41 37 39† 33 40 P. falciparum 31 31 28 29 25 30 P. ovale 4 4 4 4 4 4 P. vivax 5 5 4 5 4 5 Mix P. falciparum + P. ovale 1 1 1 1 0 1 Negative controls 92 92 88 92 - 91 * For some of the incorrect species’ identification by microscopy, one of the two species reported in the patient file was correct † In one of the samples the patient had recently gone through successful treatment, and the microscopy is expected to be negative (Table 3 ) (Table 4 ) Correlation between HumaTurb Loopamp™ Malaria PDT time to detection and qPCR CT value There was a positive correlation between the HumaTurb Loopamp™ Malaria PDT kit time to detection and the CT value of the qPCR with a correlation coefficient ranging from 0.48–0.75 in the different PCR runs (which were analysed in triplicate for each sample). The correlation coefficient for the mean qPCR CT value was 0.77 (Fig. 2 ). There was a weak negative correlation seen between HumaTurb Loopamp™ Malaria PDT time to detection and level of parasitaemia % as reported in the file at the time of diagnosis (correlation coefficient − 0.14) Fig. 3 , and a moderate negative correlation between mean qPCR CT value and percentage parasitaemia (correlation coefficient − 0.36) (Fig. 4 ). (Fig. 2 ) (Fig. 3 ) (Fig. 4 ) Discussion In this retrospective validation study the performance of two LAMP kits; Alethia® illumigene Malaria kit and HumaTurb Loopamp™ Malaria Pan Detection (PDT) kit, were evaluated head-to-head for detection of Plasmodium DNA in 133 bio banked blood samples from suspected malaria cases at the Clinical Microbiology Laboratory of Region Skåne, Sweden to determine their performance compared to qPCR. The results show that the HumaTurb Loopamp™ Malaria PDT kit had 100% sensitivity and specificity compared to highly sensitive qPCR. The Alethia® illumigene Malaria kit correctly identified 125 of 133 samples as either positive or negative (accuracy 94%) but failed to detect four cases of malaria and misdiagnosed four cases as positive. It is important to note that it is not possible to compare the diagnostic performance of RDT plus microscopy versus the LAMP assays with this study design, since positive samples in this study were included on the base of current laboratory methods for the malaria diagnosis in Skåne (based on RDT and microscopy). In our data, there seems to be a correlation between qPCR CT value, which previously has been shown to correlate with level of parasitaemia (23), and time to detection with the HumaTurb Loopamp™ Malaria PDT kit but our sample size is too small to draw any significant conclusions. This is an area where further research could be beneficial, as levels of parasitaemia may be difficult to measure for inexperienced microscopists. This might explanain for the weak correlation between reported level of parasitaemia and qPCR CT-values or time to detection with the HumaTurb Loopamp™ Malaria PDT kit. A study comparing time to detection in the HumaTurb Loopamp™ Malaria PDT kit with level of parasitaemia by microscopy performed by an accredited laboratory with experienced staff would be of interest in order to evaluate the possible use of time to detection as an indirect measure of parasitaemia. The laboratory analyses were performed by trained biomedical scientists as well as non-laboratory trained hospital staff that had gone through a short training and it is possible that the non-BMA staff made handling errors during the testing which contributed to contamination. However, since these tests are planned to be run at the point of care it is important that non expert BMA can run the tests with maintained accuracy and quality. As the LAMP technique in the non-endemic setting often will be used as a first diagnostic test to rule out malaria, the 4 false negative results by the Alethia® illumigene Malaria kit are problematic. As described above, two of these samples probably had a very low parasite density, which could have been below the limit of detection for the Alethia® illumigene Malaria kit. This is in line with previous studies that have found that the limit of detection for Alethia® illumigene Malaria kit lies at PCR CT values of 37–40 (13). In two of the samples, however, the parasite density was 0.2% (CT values 25 and 26) and no obvious reason for failure to detect Plasmodium DNA can be found. It is possible that the freezing of the samples for 2–4 years has damaged the parasite DNA, and thus leads to failure to detect the DNA, but this would most likely have been seen in the HumaTurb Loopamp™ Malaria PDT kit as well. Other possible reasons for false negative results could be the presence of inhibitors or the handling of the sample during the LAMP process. Strengths and limitations A strength of this study is the comparison of two LAMP methods with both highly sensitive qPCR as well as “field microscopy” at the point of care. The results show that species identification by microscopy was a challenge with only 33 of 41 samples being correctly identified by the microscopist. Most often the misdiagnosis consisted of the false identification of a mixed infection but one mixed infection with P. falciparum and P. ovale was misdiagnosed in microscopy as only P. falciparum . The clinical reality in Sweden and many other non-endemic countries is that the microscopy is done at the point of care by the infectious disease doctor on call that might perform malaria microscopy very rarely and might have had the most recent training in the technique a very long time ago. This makes both sensitivity, species determination, and assessment of parasitaemia a challenge, and may have affected a possible correlation between actual level of parasitaemia and time to detection by the HumaTurb Loopamp™ Malaria PDT kit. The problems associated with maintaining diagnostic skills adequately high for doctors on call during nighttime and weekends is one of the main reasons more sensitive methods are needed. This study is performed on samples that were frozen for 2–4 years which does not accurately represent the test material that will be used in practice which might lead to unforeseen differences in diagnostic performance. However, according to the manufacturers, both kits should be able to handle frozen samples (11, 24). The malaria positivity rate in this material is 31% which is very much higher than it will be in practical use at the labs in most non-endemic countries (3, 14, 19, 25). This affects the PPV and NPV. The lower positivity rate in the real-life setting increases the NPV as compared to what is shown in this study material (Table 3 ), which means that the clinical use of the Alethia® illumigene Malaria kit might be underestimated in our material. The selection of samples by positive results in RDT and microscopy also means that there are very few low parasite density infections in the studied group, which might lead to an overestimation of the diagnostic accuracy of both LAMP instruments in our study. However, one case that presented with fever since only one day that was negative in RDT and microscopy and later found to be malaria positive, was diagnosed as positive by HumaTurb Loopamp™ Malaria PDT kit and qPCR, indicating the superior diagnostic sensitivity of these molecular diagnostic tools compared to RDT and microscopy recommended by the WHO (10). The cost-effectiveness has not been assessed in this study and is highly dependent on local factors, why it ideally should be done in each setting. Added value of this study This study is the first study, to our knowledge, to compare the performance of the two leading LAMP instruments for diagnosis of malaria on the same material. And the results show a superior performance of the HumaTurb Loopamp™ Malaria PDT kit. Conclusion In this head-to-head retrospective validation study of the diagnostic performance of two LAMP kits compared to qPCR, the HumaTurb Loopamp™ Malaria PDT kit was shown to have a better diagnostic performance than the Alethia® illumigene Malaria kit. The high diagnostic sensitivity and specificity of HumaTurb Loopamp™ Malaria PDT kit with a NPV of 100% in this study, along with ease of use and time to result around one hour makes this analysis suitable as a first line point of care diagnostic test for malaria in non-endemic high resource settings globally. However, the inability of these methods to perform full species differentiation and grade of parasitaemia mean that they cannot replace microscopy in clinical diagnosis as this data is crucial for accurate treatment. Declarations Availability of data and materials The datasets supporting the conclusions of this article are included within the article and its additional files. Competing interests The authors have no financial or non-financial competing interests. Funding The LAMP instruments and reagent kits needed for the evaluation were provided by both manufacturers without cost during the time of laboratory analysis. Authors' contributions Study idea and study design by S.K.S. and AC.I. Laboratory LAMP analyses were performed by E.F. and AC.I. qPCR analyses were performed by I.B and A.F. Data acquisition, analysis and interpretation of data was done by AC.I., with support from all authors. 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LAMP kit for diagnosis of non-falciparum malaria in Plasmodium ovale infected patients. Malaria Journal. 2017;16(1). Charpentier E, Benichou E, Pagès A, Chauvin P, Fillaux J, Valentin A, et al. Performance evaluation of different strategies based on microscopy techniques, rapid diagnostic test and molecular loop-mediated isothermal amplification assay for the diagnosis of imported malaria. Clinical Microbiology and Infection. 2020;26(1):115-21. Piera KA, Aziz A, William T, Bell D, González IJ, Barber BE, et al. Detection of Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax using loop-mediated isothermal amplification (LAMP) in a co-endemic area in Malaysia. Malaria Journal. 2017;16(1). De Koninck A-S, Cnops L, Hofmans M, Jacobs J, Van den Bossche D, Philippé J. Diagnostic performance of the loop-mediated isothermal amplification (LAMP) based illumigene® malaria assay in a non-endemic region. Malaria Journal. 2017;16(1):418. Marti H, Stalder C, González IJ. Diagnostic accuracy of a LAMP kit for diagnosis of imported malaria in Switzerland. Travel Medicine and Infectious Disease. 2015;13(2):167-71. Nolasco O, Infante B, Contreras-Mancilla J, Incardona S, Ding XC, Gamboa D, et al. Diagnosis of Plasmodium vivax by Loop-Mediated Isothermal Amplification in Febrile Patient Samples from Loreto, Perú. The American Journal of Tropical Medicine and Hygiene. 2020;103(4):1549-52. Shokoples SE, Ndao M, Kowalewska-Grochowska K, Yanow SK. Multiplexed Real-Time PCR Assay for Discrimination of <i>Plasmodium</i> Species with Improved Sensitivity for Mixed Infections. Journal of Clinical Microbiology. 2009;47(4):975-80. MedCalc Software. Free statistical calculators. Diagnostic test evaluation calculator: MedCalc Software Ltd; 2022 [cited 2022 October 23]. Available from: https://www.medcalc.org/calc/diagnostic_test.php. Ballard E, Wang CYT, Hien TT, Tong NT, Marquart L, Pava Z, et al. A validation study of microscopy versus quantitative PCR for measuring Plasmodium falciparum parasitemia. Tropical Medicine and Health. 2019;47(1). Meridian Bioscience. Alethia Malaria and Malaria PLUS DNA Amplification Assays, User Information. Cincinnati, Ohio, USA: Meridian Bioscience; 2020. Burdino E, Calleri G, Ghisetti V. Added value of loop-mediated isothermal amplification technology (LAMP) in real life for the diagnosis of malaria in travellers. Journal of Travel Medicine. 2019;26(7):taz052. Human Diagnostics Worldwide. Frequently Asked Questions. Malaria-LAMP Wiesbaden, Germany: Human Diagnostics Worldwide; 2019 [Available from: https://www.human.de/fileadmin/content/02_Products/04_Molecular_DX/Documents/FAQ_Malaria-LAMP_print_and_view_rev.004.pdf. Additional Declarations No competing interests reported. Supplementary Files SupplementsHeadtoheadcomparisonoftwoloopmediatedisothermalamplificationLAMPkitsfordiagnosisofmalariainanonendemicsetting.docx Cite Share Download PDF Status: Published Journal Publication published 13 Dec, 2023 Read the published version in Malaria Journal → Version 1 posted Editorial decision: Major revision 19 Sep, 2023 Reviews received at journal 07 Aug, 2023 Reviewers agreed at journal 17 Jul, 2023 Reviewers agreed at journal 31 May, 2023 Reviewers invited by journal 31 May, 2023 Editor assigned by journal 27 May, 2023 Submission checks completed at journal 27 May, 2023 First submitted to journal 26 May, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-2984503\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":204347629,\"identity\":\"e2d25a2e-4e66-49ba-88bf-4b3d10596f2e\",\"order_by\":0,\"name\":\"Anna-Clara 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3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":34699,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCorrelation between HumaTurb Loopamp™ Malaria PDT time to detection and parasitaemia.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2984503/v1/666246474641409603a83f11.jpg\"},{\"id\":37674376,\"identity\":\"ad897268-3a65-4e69-8cea-745edf0458b5\",\"added_by\":\"auto\",\"created_at\":\"2023-05-30 15:30:18\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":41619,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCorrelation between mean qPCR CT value and 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15:30:29\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":19183,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementsHeadtoheadcomparisonoftwoloopmediatedisothermalamplificationLAMPkitsfordiagnosisofmalariainanonendemicsetting.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2984503/v1/a6c8ace86c8886dcaecef03a.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Head-to-head comparison of two loop-mediated isothermal amplification (LAMP) kits for diagnosis of malaria in a non-endemic setting\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eManagement of malaria relies on rapid and accurate diagnosis to secure prompt treatment. The diagnostic methods recommended for malaria diagnosis by the WHO continue to be light microscopy and immunochromatographic rapid diagnostic tests (RDTs)(1). While the sensitivity and specificity of RDTs are about 95% for the most virulent malaria species, \\u003cem\\u003eP. falciparum\\u003c/em\\u003e, the sensitivity is lower for other \\u003cem\\u003ePlasmodium\\u003c/em\\u003e species as well as for parasite densities below 200/µL (2). The diagnostic sensitivity and specificity of light microscopy is highly dependent on the skills of the microscopist, something that can be hard to maintain 24/7 in non-endemic settings such as Sweden (3–5). In recent years however, nucleic acid amplification test methods, such as loop mediated isothermal amplification (LAMP), have been tested increasingly in epidemiological studies as well as in clinical settings and have been proposed as a highly sensitive, cost-effective alternative in non-endemic high resource settings (4, 6–10). In LAMP, the DNA-targets are amplified without the temperature cycles of the PCR-technique. Instead, a DNA-polymerase with strand displacement activity makes multiplication of the DNA-target possible at a constant temperature. The sequences targeted by the primers lead to the formation of DNA loops during amplification, which allows a highly sensitive and specific reaction and a shorter time to detection than is usual for PCR. The DNA-amplification reaction forms a white precipitation of magnesium pyrophosphate, detected as turbidity or as fluorescence when exposed to UV light (if dyed with calcein). LAMP methods usually do not quantify the DNA content of the sample, which qPCR methods may through a Cq/CT-value (4, 11).\\u003c/p\\u003e\\n\\u003cp\\u003eLAMP techniques have been shown to offer superior diagnostic performance to RDT and microscopy, in particular concerning low grade-parasitaemia, and can deliver results with higher sensitivity (97–100%) and specificity (99.2–100%) with low laboratory capacity requirements in a little more than an hour (3–5, 8, 12–19). The high diagnostic performance has been shown to include also non-\\u003cem\\u003efalciparum\\u003c/em\\u003e species (15, 17, 20). The above-mentioned challenges with current malaria diagnostics and the promising results of LAMP techniques have led us to the question if a LAMP technique could replace RDT and microscopy as a first line, point-of-care test for malaria in southern Sweden. LAMP could then be used to rule out malaria in negative cases and microscopy would only be needed in positive cases to determine \\u003cem\\u003ePlasmodium\\u003c/em\\u003e species and parasite density. Therefore, this study was designed to assess the diagnostic performance of the internationally available LAMP kits. There are two main LAMP instruments on the international market today: the Alethia® Malaria illumigene (Meridian Bioscience) with reagents in the Malaria or Malaria PLUS kit, and the HumaTurb C + A or Humaloop M (Human Diagnostics Worldwide) with Loopamp™ Malaria Pan Detection kit and separate kits for species identification of \\u003cem\\u003eP. falciparum\\u003c/em\\u003e or \\u003cem\\u003eP. vivax\\u003c/em\\u003e (Table \\u003cspan refid=\\\"Tab1\\\"\\u003e1\\u003c/span\\u003e). The aim of this study was to compare these two LAMP instruments and kits by testing the Malaria kit for the Alethia® Malaria illumigene instrument against the Loopamp™ Pan Detection (PDT) kit for the HumaTurb C + A instrument. Hereafter they will be referred to as Alethia® illumigene Malaria kit and HumaTurb Loopamp™ Malaria PDT kit. These two kits have been tested compared to conventional methods in different settings but have to our knowledge not previously been compared head-to-head on the same samples (4, 9).\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was performed as a retrospective laboratory validation study of analytic performance of two LAMP assays on biobanked samples from patients with suspected malaria in Region Skåne. The main research question was which of the two LAMP kits that had the best diagnostic performance compared to highly sensitive qPCR in patients with suspected imported malaria in the non-endemic setting at the Clinical Microbiology Department of Skåne in the South of Sweden. Additionally, we wanted to assess if any of the two LAMP kits could be used as a semiquantitative measure of parasitaemia level.\\u003c/p\\u003e\\n\\u003cdiv\\u003e\\n \\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv\\u003eTable 1\\u003c/div\\u003e\\n \\u003cdiv\\u003e\\n \\u003cp\\u003e– Characteristics of Alethia® illumigene Malaria kit and HumaTurb Loopamp™ Malaria PDT kit.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003eAlethia® illumigene Malaria\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003eHumaTurb Loopamp™ Malaria PDT\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eMean time to result as measured in the study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003e44 min\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003e60 min\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eTime to result as reported by the manufacturer *\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt; 45 min\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003e50–55 min\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eMean active time as measured in the study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003e4 min\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003e10 min\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eAnalysis *\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003eQualitative\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003eQualitative + turbidity graph and time to detection\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eMaximum samples per run *\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003e16 (up to 94 if expanded with 6 HumaTurb A units)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eSpecies identification *\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003eNot possible\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003eNot possible\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eValidated for species detection\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eP. falciparum, P. vivax, P. ovale, P. malariae.\\u003c/em\\u003e Performance for \\u003cem\\u003eP. knowlesi\\u003c/em\\u003e established using purified genomic DNA only; whole organism testing has not been performed.\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eP. falciparum, P. vivax, P. ovale, P. malariae. \\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eP. knowlesi validated in one study†\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eRead out of the results *\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003eTurbidity in Illumipro-10™ incubator or by eye\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003eTurbidity in turbidimeter or fluorescence detected by eye\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\n \\u003cp\\u003eLimit of detection*\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\n \\u003cp\\u003e2.0 p/ µL for P. falciparum and 0.1 p/ µL for P. vivax\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\n \\u003cp\\u003e1 p/ µL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003ctfoot\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"3\\\"\\u003e\\u003cem\\u003e* As described by the manufacturer (11, 24, 26) .\\u003c/em\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"3\\\"\\u003e\\u003cem\\u003e† (17)\\u003c/em\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tfoot\\u003e\\n \\u003c/table\\u003e\\n\\u003c/div\\u003e\\n\\u003cp\\u003e(Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\"\\u003e1\\u003c/span\\u003e)\\u003c/p\\u003e\"},{\"header\":\"Method\",\"content\":\"\\u003cp\\u003eStudy design and study site\\u003c/p\\u003e\\u003cp\\u003eThe performance of two LAMP instruments for detection of \\u003cem\\u003ePlasmodium\\u003c/em\\u003e-DNA in biobanked blood samples were retrospectively evaluated at the Clinical Microbiology Laboratory of Region Skåne, Sweden to determine their performance compared to highly sensitive qPCR. Performance was defined as sensitivity and specificity compared to qPCR to detect DNA in the first diagnostic blood sample sent to the lab from patients with clinical suspicion of malaria.\\u003c/p\\u003e\\u003cp\\u003eThe Clinical Microbiology Laboratory of Region Skåne is the main laboratory for the Skåne region in the south of Sweden, serving 9 hospitals and covering a population of 1.4\\u0026nbsp;million. The positivity rate of suspected malaria samples at the Clinical Microbiology Laboratory of Region Skåne during the study time was 8–10% yearly (unpublished data from the Laboratory Information System, Region Skåne). The diagnostic method for malaria during the time of the study was RDT (CareStart Malaria HRP\\u003csub\\u003e2\\u003c/sub\\u003e/pLDH (Pf/PAN) Combo Test) and light microscopy of thick and thin blood smear films prepared with Giemsa stain according to the current standard at the Clinical Microbiology Laboratory of Region Skåne. The microscopy was performed by an infectious disease specialist at the point of care laboratory, for \\u003cem\\u003ePlasmodium\\u003c/em\\u003e species and parasitaemia. After diagnosis, whole blood samples (EDTA) are routinely sent to the regional Clinical Microbiology Laboratory without further processing and stored at -80°C in the Region Skåne biobank 136, Klinisk mikrobiologi BD1.\\u003c/p\\u003e\\u003cp\\u003eEthics statement\\u003c/p\\u003e\\u003cp\\u003e The study was approved by the Swedish Ethical Review Authority (Etikprövningsmyndigheten) ID 2020–05249.\\u003c/p\\u003e\\u003cp\\u003eStudy population and sample collection\\u003c/p\\u003e\\u003cp\\u003eBlood samples sent to the Department of Clinical Microbiology on suspicion of malaria during the period 2018–2020 were selected for the study. These included samples that had been identified by routine light microscopy and/or RDT as malaria positive, and two consecutive negative samples.\\u003c/p\\u003e\\u003cp\\u003eExclusion criteria: samples not identified in the bio bank, insufficient volume for testing in the sample and inconclusive information about final malaria diagnosis in the clinical files.\\u003c/p\\u003e\\u003cp\\u003eClinical data collection\\u003c/p\\u003e\\u003cp\\u003eMedical charts from included patients were obtained from Region Skåne medical databases. Epidemiological patient data was retrieved regarding sex, age, country of residence, country of origin, country of exposure, reason for travel, previous malaria episodes, chemoprophylaxis, time of exposure, time from symptom onset, malaria treatment initiated, presence of risk factors and comorbidities and data on parasitaemia and \\u003cem\\u003ePlasmodium\\u003c/em\\u003e species from microscopy performed by clinicians.\\u003c/p\\u003e\\u003ch2\\u003eLAMP\\u003c/h2\\u003e\\u003cp\\u003eEach of the included frozen whole blood samples obtained from the biobank were tested retrospectively with two LAMP-kits: Alethia® Malaria illumigene Malaria kit (Meridian Bioscience) and HumaTurb C + A Loopamp™ Malaria Pan Detection kit (Human Diagnostics Worldwide). The tests were performed according to the manufacturer’s instructions. The LAMP analysis of Alethia® illumigene Malaria kit is qualitative and is reported as positive, negative, or invalid. HumaTurb Loopamp™ Malaria PDT kit is a qualitative test that also reports time to detection. Neither of the two tested LAMP kits offer species differentiation. The LAMP analyses were performed by research personnel together with staff at the Clinical Microbiology Laboratory of Region Skåne during 2021 and 2022. Results from microscopy were blinded to the research personnel performing the LAMP assays. Invalid results were re-tested once and if the result remained invalid the sample was reported as invalid.\\u003c/p\\u003e\\u003cp\\u003eReal time PCR\\u003c/p\\u003e\\u003cp\\u003eA total volume of 50 µL EDTA blood from each sample was used for genomic DNA extraction using the QIAamp DNA blood mini kit (Qiagen) and the protocol ‘DNA purification from Blood or Body fluids (spin protocol)’, according to manufacturer’s instructions. To reach a volume of 200 µL, starting material required for the specific extraction protocol, 150 µL of PBS 0.01M pH 7.4 was added to 50 µL EDTA blood, according to manufacturer’s recommendation. At the final step, the genomic DNA was incubated for 5 minutes at room temperature prior to elution in 150 µL elution buffer and stored at -20℃.\\u003c/p\\u003e\\u003cp\\u003eFor \\u003cem\\u003ePlasmodium\\u003c/em\\u003e species detection and identification, a previously published multiplex, probe-based qPCR assay method was used, with slight modifications (21). The method targets the highly conserved \\u003cem\\u003ePlasmodium\\u003c/em\\u003e 18S rRNA gene and allows the simultaneous detection of four \\u003cem\\u003ePlasmodium\\u003c/em\\u003e species (\\u003cem\\u003eP. falciparum, P. vivax, P. malariae, P. ovale\\u003c/em\\u003e) using species-specific primers and probes (supplement). Briefly, 5 µL of extracted DNA was used as template in a 25 µL PCR reaction with 1X TaqMan Multiplex master mix (Applied Biosystems), 0.2 µM of primers Fal-F, Viv-F, Mal-F, Ova-F, each, 0,5 µM of primer Plasmo2-R and 0.1 µM of probes Falprobe, Vivprobe, Malaprobe, Ovaprobe, each (all from Thermo Fisher). The cycling conditions were: 95°C for 20 seconds, followed by 45 cycles at 95℃ for 15 seconds and at 60℃ for 1 minute, and the amplification took place in a QuantStudio™ 5 Real-Time PCR system (Applied Biosystems). Each sample was analysed in triplicates. Serial dilutions of \\u003cem\\u003eP. falciparum\\u003c/em\\u003e, \\u003cem\\u003eP. vivax\\u003c/em\\u003e, \\u003cem\\u003eP. malariae\\u003c/em\\u003e and \\u003cem\\u003eP. ovale\\u003c/em\\u003e samples of known parasitaemia (0.1%, 0.3%, 0.2% and 0.1%, respectively) and non-template negative controls were used in each run. A cycle threshold (CT) value of 40 was used as a cut off to define positive samples. The limit of detection was 0.5-6 parasites. In case of inconsistent qPCR results, i.e., one out of three replicates positive, the qPCR was repeated, and the sample was analysed again in triplicates.\\u003c/p\\u003e\\u003ch2\\u003eCURRENT DIAGNOSTIC METHODS FOR MALARIA IN REGION SKÅNE\\u003c/h2\\u003e\\u003cp\\u003eA blood sample is taken in an EDTA-tube from the suspected patient and is tested with the immunochromatographic rapid diagnostic tests CareStart Malaria HRP2/pLDH (Pf/PAN) Combo Test at the local laboratory at the point of care. The same blood sample is used to prepare, within one hour, two thin- and two thick blood films using Giemsa-stain for light microscopy. The blood smears are immediately prepared with Giemsa-stain in all cases of positive RDT and after agreement with responsible clinician in cases of negative RDT test. The microscopy is performed by an infectious disease specialist at the point of care laboratory, assessing \\u003cem\\u003ePlasmodium\\u003c/em\\u003e species and parasitaemia.\\u003c/p\\u003e\\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e\\u003cp\\u003eSensitivity, specificity, PPV and NPV of LAMP techniques and microscopy plus RDT as compared to qPCR were calculated by the online statistical tool MedCalc (22). Correlations between HumaTurb Loopamp™ Malaria PDT kit time to detection, qPCR CT value and parasitaemia as reported by microscopy was estimated using the Microsoft Excel version 16.66.1 CORREL function to calculate correlation coefficient and Prism to create correlation graphs.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003eA total of 51 positive samples from patients diagnosed with malaria by light microscopy and/or RDT between 2018\\u0026ndash;2020 were identified in the biobank list. To these, 102 consecutive negative samples were selected resulting in a total of 153 samples. Of these, 5 samples could not be located in the biobank leaving a total of 148 samples that were tested with the two LAMP techniques, 47 registered as positive for malaria based on microscopy and/or RDT and 101 as negative. Of these samples, 14 had insufficient amount of material to be analysed with qPCR, resulting in a total of 134 samples analysed by qPCR. The diagnosis of one of these samples was found to be inconclusive with negative results in all diagnostic tests except one of three PCR runs resulting in a high CT value of 40. The final clinical diagnosis of this patient is also unclear in the medical records. Therefore, this sample was excluded, resulting in a total of 133 samples analysed by both qPCR and the two LAMP kits. In total 41 samples were positive by qPCR and 92 were negative. The sample selection flowchart is detailed in Fig. \\u003cspan\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e\\n\\u003cdiv id=\\\"Sec7\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e(Fig.\\u0026nbsp;\\u003c/strong\\u003e\\u003cspan\\u003e\\u003cstrong\\u003e1\\u003c/strong\\u003e\\u003c/span\\u003e\\u003cstrong\\u003e)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eTable \\u003cspan\\u003e2\\u003c/span\\u003e shows the demographic characteristics of the 47 included positive cases that were tested with the two LAMP kits. The cohort is well balanced by sex and age. Most cases were exposed in Sub Saharan Africa, visiting friends and family, and had not taken malaria chemoprophylaxis. Among the microscopy positive the parasite densities ranged between 0.1\\u0026ndash;4% and all were positive by RDT.\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n \\u003ctable id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv\\u003eTable 2\\u003c/div\\u003e\\n \\u003cdiv\\u003e\\n \\u003cp\\u003e\\u0026ndash; Demographic characteristics of positive cases\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eCharacteristics of malaria cases (n\\u0026thinsp;=\\u0026thinsp;47)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003en\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eSex\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eMen\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e57%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eWomen\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e43%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAge\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eMedian age (range)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e36 y (6y-75y)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eRegion of infection\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eWest Africa\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e25\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e53%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eEast Africa\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e19%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eCentral Africa\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e1%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eCentral Asia\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e6%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eSouth Asia\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e6%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePatient origin from endemic area\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eYes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e74%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eNo\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e26%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eReason for exposure\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eVisiting friends and family\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e18\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e38%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eMigrant\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e16\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e34%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eTourist\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e19%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eResidency in endemic area\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e9%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePrevious malaria episodes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eYes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e57%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eNo\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e17%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eNo information\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e26%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eChemoprophylaxis\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eYes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e11%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eNo\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e39\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e83%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eIncomplete prophylaxis\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e6%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eParasitaemia %\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.1\\u0026ndash;0.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e13\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e28%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.5\\u0026ndash;0.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e21%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1\\u0026ndash;1.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e13%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2\\u0026ndash;2.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e4%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3\\u0026ndash;4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e9%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eNo data\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e26%\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec8\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e(Table\\u0026nbsp;\\u003c/strong\\u003e\\u003cspan\\u003e\\u003cstrong\\u003e2\\u003c/strong\\u003e\\u003c/span\\u003e\\u003cstrong\\u003e)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eDiagnostic performance\\u003c/p\\u003e\\n \\u003cp\\u003eDiagnostic performance of Alethia\\u0026reg; illumigene Malaria kit and HumaTurb Loopamp\\u0026trade; Malaria PDT kit as compared to RDT\\u0026thinsp;+\\u0026thinsp;microscopy and the reference method qPCR is presented in Table\\u0026nbsp;\\u003cspan\\u003e3\\u003c/span\\u003e and Table\\u0026nbsp;\\u003cspan\\u003e4\\u003c/span\\u003e.\\u003c/p\\u003e\\n \\u003cp\\u003eThe results from the qPCR and HumaTurb Loopamp\\u0026trade; Malaria PDT kit were in complete congruence, resulting in a sensitivity of 100% (95% CI 91.40\\u0026ndash;100%) and specificity of 100% (95% CI 96.07\\u0026ndash;100%) for the HumaTurb Loopamp\\u0026trade; Malaria PDT kit as compared to the qPCR. Positive predictive value and negative predictive value were both 100%. When comparing the Alethia\\u0026reg; illumigene Malaria kit to qPCR there were 4 false positive and 4 false negative results leading to a sensitivity of 90.24 (95% CI 76.87\\u0026ndash;97.28) and a specificity of 95.65% (95% CI 89.24\\u0026ndash;98.80). The positive predictive value was 90.31% (95% CI 78.06\\u0026ndash;96.07) and the negative predictive value was 95.62% (95% CI 89.58\\u0026ndash;98.23) in the study material. The samples were collected in such a way that the malaria positivity rate in the study material was 30.8% as opposed to the actual positivity rate in malaria suspected samples in Region Sk\\u0026aring;ne that is around 10%. This difference affects the positive and negative predictive value. With a 10% disease prevalence in tested patients the PPV calculated from our study results would be 69.75% (95% CI 46.80\\u0026ndash;85.81) and NPV 98.88% (97.20\\u0026ndash;99.56) for the Alethia\\u0026reg; illumigene Malaria kit.\\u003c/p\\u003e\\n \\u003cp\\u003eThe Alethia\\u0026reg; illumigene Malaria kit correctly identified 125 of 133 samples as either positive or negative (accuracy 94%) but failed to detect four cases of malaria and misdiagnosed four cases as positive. All four false positive results were negative in qPCR, microscopy and RDT. The clinical diagnosis for the four cases were viral infection or uncertain diagnosis. Out of the four false negative results two were positive in qPCR, microscopy and RDT with a noted parasite density of 0.2%. One of the cases had slightly inconclusive qPCR results with 2 out of 6 replicates (33.3%) positive in two independent qPCR experiments with a CT value of 40 in the first round and 38 in the second, negative microscopy and negative RDT. The clinical presentation of this case was a person with origin from an endemic area that was visiting friends and family and had had symptoms for one day. At presentation the case was diagnosed as non-malaria but three weeks later malaria was diagnosed at another hospital. This case likely had a very low level parasitaemia at the time of testing. The last false negative case was positive in two of three qPCR replicates with a CT value of 40, positive in microscopy and positive in RDT. This case had used oral self-treatment before seeking care and was first deemed as cured but was later reassessed as incompletely cured and put on malaria treatment with oral Atovaquone/proguanil once more which led to clinical improvement. It is likely that this case also had a very low level parasitaemia.\\u003c/p\\u003e\\n \\u003cp\\u003eIn the study samples, the reference method qPCR identified \\u003cem\\u003eP. falciparum, P. ovale\\u003c/em\\u003e and \\u003cem\\u003eP. vivax\\u003c/em\\u003e samples along with one mixed infection with \\u003cem\\u003eP. falciparum\\u003c/em\\u003e and \\u003cem\\u003eP. ovale.\\u003c/em\\u003e Both LAMP assays correctly identified malaria caused by all species as positive, although one \\u003cem\\u003eP. vivax\\u003c/em\\u003e sample and three \\u003cem\\u003eP. falciparum\\u003c/em\\u003e samples were missed by the Alethia\\u0026reg; illumigene Malaria kit. The results show that species identification by microscopy was a challenge with only 33 of 41 samples correctly identified by the microscopist as compared to the qPCR (Table\\u0026nbsp;\\u003cspan\\u003e4\\u003c/span\\u003e). Most often the misdiagnosis consisted of the false identification of a mixed infection, but one mixed infection with \\u003cem\\u003eP. falciparum\\u003c/em\\u003e and \\u003cem\\u003eP. ovale\\u003c/em\\u003e was misdiagnosed in microscopy as only \\u003cem\\u003eP. falciparum\\u003c/em\\u003e. Two qPCR positive samples were also missed in microscopy and identified as microscopy negative, but in one of the samples the patient had recently gone through successful treatment, and the microscopy is expected to be negative.\\u003c/p\\u003e\\n \\u003cp\\u003eIn four samples out of the 148 analysed with the two LAMP techniques, the first result from the Alethia\\u0026reg; illumigene Malaria kit was invalid. Upon retesting, all four samples had valid results. No invalid results were noted with the HumaTurb Loopamp\\u0026trade; Malaria PDT kit.\\u003c/p\\u003e\\n \\u003cp\\u003eTable 3 - Diagnostic performance of LAMP techniques compared to qPCR.\\u003c/p\\u003e\\n \\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"680\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"13.80323054331865%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAnalysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"22.173274596182086%\\\" colspan=\\\"2\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eSensitivity\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"20.851688693098385%\\\" colspan=\\\"2\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eSpecificity\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.160058737151248%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.160058737151248%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"9.691629955947137%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.160058737151248%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"13.782991202346041%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e% (95% CI)\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eProportion\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"9.67741935483871%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e% (95% CI)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eProportion\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePPV % (in study group)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePPV 10% disease prevalence\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"9.67741935483871%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNPV % (in study group)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNPV 10% disease prevalence\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"13.782991202346041%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAlethia\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026reg;\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;illumigene Malaria\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e90.24%\\u0026nbsp;\\u003cbr\\u003e\\u003cem\\u003e(76.87 - 97.28)\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e37/41\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"9.67741935483871%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e95.65%\\u0026nbsp;\\u003cbr\\u003e\\u003cem\\u003e(89.24 - 98.8)\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e88/92\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e90.31 %\\u003cbr\\u003e\\u003cem\\u003e(78.06 - 96.07)\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e69.75% (46.80 \\u0026ndash; 85.81)\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"9.67741935483871%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e95.62%\\u003cbr\\u003e\\u003cem\\u003e(89.58- 98.23)\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e98.88% \\u003cem\\u003e(97.20 \\u0026ndash; 99.56)\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"13.782991202346041%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eHumaTurb Loopamp\\u0026trade; Malaria PDT\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e100%\\u003cbr\\u003e\\u003cem\\u003e(91.40 - 100)\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e41/41\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"9.67741935483871%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e100%\\u003cbr\\u003e\\u003cem\\u003e(96.07 - 100)\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e92/92\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e100%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e100%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"9.67741935483871%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e100%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"11.143695014662757%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e100%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n\\u003c/div\\u003e\\n\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTable 4 - Diagnostic performance and species identification of LAMP techniques compared to qPCR, microscopy and RDT.\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"16.363636363636363%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.380165289256198%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eqPCR\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.87603305785124%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eHumaTurb Loopamp\\u0026trade; Malaria PDT\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.537190082644628%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAlethia\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026reg;\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;illumigene Malaria\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"13.71900826446281%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eMicroscopy - correctly identified as pos or neg\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.231404958677686%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eMicroscopy - correct species*\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.892561983471074%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eRDT -correctly identified as pos or neg\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"16.363636363636363%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTotal positive\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.380165289256198%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e41\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.87603305785124%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e41\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.537190082644628%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e37\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"13.71900826446281%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e39\\u0026dagger;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.231404958677686%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e33\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.892561983471074%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e40\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"16.363636363636363%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eP. falciparum\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.380165289256198%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e31\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.87603305785124%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e31\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.537190082644628%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e28\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"13.71900826446281%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e29\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.231404958677686%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e25\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.892561983471074%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e30\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"16.363636363636363%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eP. ovale\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.380165289256198%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.87603305785124%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.537190082644628%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e4\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"13.71900826446281%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.231404958677686%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.892561983471074%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"16.363636363636363%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eP. vivax\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.380165289256198%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.87603305785124%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.537190082644628%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"13.71900826446281%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.231404958677686%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.892561983471074%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"16.363636363636363%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eMix P. falciparum + P. ovale\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.380165289256198%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.87603305785124%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.537190082644628%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"13.71900826446281%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.231404958677686%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.892561983471074%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"16.363636363636363%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eNegative controls\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.380165289256198%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e92\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"14.87603305785124%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e92\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"15.537190082644628%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e88\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"13.71900826446281%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e92\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.231404958677686%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e-\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"12.892561983471074%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e91\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e* For some of the incorrect species\\u0026rsquo; identification by microscopy, one of the two species reported in the patient file was correct\\u003cbr\\u003e\\u0026nbsp;\\u0026dagger; In one of the samples the patient had recently gone through successful treatment, and the microscopy is expected to be negative\\u003c/p\\u003e\\n\\u003cdiv id=\\\"Sec9\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e(Table\\u0026nbsp;\\u003c/strong\\u003e\\u003cspan\\u003e\\u003cstrong\\u003e3\\u003c/strong\\u003e\\u003c/span\\u003e\\u003cstrong\\u003e)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cdiv id=\\\"Sec10\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e(Table\\u0026nbsp;\\u003c/strong\\u003e\\u003cspan\\u003e\\u003cstrong\\u003e4\\u003c/strong\\u003e\\u003c/span\\u003e\\u003cstrong\\u003e)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eCorrelation between HumaTurb Loopamp\\u0026trade; Malaria PDT time to detection and qPCR CT value\\u003c/p\\u003e\\n \\u003cp\\u003eThere was a positive correlation between the HumaTurb Loopamp\\u0026trade; Malaria PDT kit time to detection and the CT value of the qPCR with a correlation coefficient ranging from 0.48\\u0026ndash;0.75 in the different PCR runs (which were analysed in triplicate for each sample). The correlation coefficient for the mean qPCR CT value was 0.77 (Fig.\\u0026nbsp;\\u003cspan\\u003e2\\u003c/span\\u003e). There was a weak negative correlation seen between HumaTurb Loopamp\\u0026trade; Malaria PDT time to detection and level of parasitaemia % as reported in the file at the time of diagnosis (correlation coefficient \\u0026minus;\\u0026thinsp;0.14) Fig.\\u0026nbsp;\\u003cspan\\u003e3\\u003c/span\\u003e, and a moderate negative correlation between mean qPCR CT value and percentage parasitaemia (correlation coefficient \\u0026minus;\\u0026thinsp;0.36) (Fig.\\u0026nbsp;\\u003cspan\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec11\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e(Fig.\\u0026nbsp;\\u003c/strong\\u003e\\u003cspan\\u003e\\u003cstrong\\u003e2\\u003c/strong\\u003e\\u003c/span\\u003e\\u003cstrong\\u003e)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cdiv id=\\\"Sec12\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e(Fig.\\u0026nbsp;\\u003c/strong\\u003e\\u003cspan\\u003e\\u003cstrong\\u003e3\\u003c/strong\\u003e\\u003c/span\\u003e\\u003cstrong\\u003e)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cdiv id=\\\"Sec13\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e(Fig.\\u0026nbsp;\\u003c/strong\\u003e\\u003cspan\\u003e\\u003cstrong\\u003e4\\u003c/strong\\u003e\\u003c/span\\u003e\\u003cstrong\\u003e)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIn this retrospective validation study the performance of two LAMP kits; Alethia\\u0026reg; illumigene Malaria kit and HumaTurb Loopamp\\u0026trade; Malaria Pan Detection (PDT) kit, were evaluated head-to-head for detection of \\u003cem\\u003ePlasmodium\\u003c/em\\u003e DNA in 133 bio banked blood samples from suspected malaria cases at the Clinical Microbiology Laboratory of Region Sk\\u0026aring;ne, Sweden to determine their performance compared to qPCR. The results show that the HumaTurb Loopamp\\u0026trade; Malaria PDT kit had 100% sensitivity and specificity compared to highly sensitive qPCR. The Alethia\\u0026reg; illumigene Malaria kit correctly identified 125 of 133 samples as either positive or negative (accuracy 94%) but failed to detect four cases of malaria and misdiagnosed four cases as positive.\\u003c/p\\u003e \\u003cp\\u003eIt is important to note that it is not possible to compare the diagnostic performance of RDT plus microscopy versus the LAMP assays with this study design, since positive samples in this study were included on the base of current laboratory methods for the malaria diagnosis in Sk\\u0026aring;ne (based on RDT and microscopy).\\u003c/p\\u003e \\u003cp\\u003eIn our data, there seems to be a correlation between qPCR CT value, which previously has been shown to correlate with level of parasitaemia (23), and time to detection with the HumaTurb Loopamp\\u0026trade; Malaria PDT kit but our sample size is too small to draw any significant conclusions. This is an area where further research could be beneficial, as levels of parasitaemia may be difficult to measure for inexperienced microscopists. This might explanain for the weak correlation between reported level of parasitaemia and qPCR CT-values or time to detection with the HumaTurb Loopamp\\u0026trade; Malaria PDT kit. A study comparing time to detection in the HumaTurb Loopamp\\u0026trade; Malaria PDT kit with level of parasitaemia by microscopy performed by an accredited laboratory with experienced staff would be of interest in order to evaluate the possible use of time to detection as an indirect measure of parasitaemia.\\u003c/p\\u003e \\u003cp\\u003eThe laboratory analyses were performed by trained biomedical scientists as well as non-laboratory trained hospital staff that had gone through a short training and it is possible that the non-BMA staff made handling errors during the testing which contributed to contamination. However, since these tests are planned to be run at the point of care it is important that non expert BMA can run the tests with maintained accuracy and quality.\\u003c/p\\u003e \\u003cp\\u003eAs the LAMP technique in the non-endemic setting often will be used as a first diagnostic test to rule out malaria, the 4 false negative results by the Alethia\\u0026reg; illumigene Malaria kit are problematic. As described above, two of these samples probably had a very low parasite density, which could have been below the limit of detection for the Alethia\\u0026reg; illumigene Malaria kit. This is in line with previous studies that have found that the limit of detection for Alethia\\u0026reg; illumigene Malaria kit lies at PCR CT values of 37\\u0026ndash;40 (13). In two of the samples, however, the parasite density was 0.2% (CT values 25 and 26) and no obvious reason for failure to detect \\u003cem\\u003ePlasmodium\\u003c/em\\u003e DNA can be found. It is possible that the freezing of the samples for 2\\u0026ndash;4 years has damaged the parasite DNA, and thus leads to failure to detect the DNA, but this would most likely have been seen in the HumaTurb Loopamp\\u0026trade; Malaria PDT kit as well. Other possible reasons for false negative results could be the presence of inhibitors or the handling of the sample during the LAMP process.\\u003c/p\\u003e \\u003cp\\u003eStrengths and limitations\\u003c/p\\u003e \\u003cp\\u003eA strength of this study is the comparison of two LAMP methods with both highly sensitive qPCR as well as \\u0026ldquo;field microscopy\\u0026rdquo; at the point of care. The results show that species identification by microscopy was a challenge with only 33 of 41 samples being correctly identified by the microscopist. Most often the misdiagnosis consisted of the false identification of a mixed infection but one mixed infection with \\u003cem\\u003eP. falciparum\\u003c/em\\u003e and \\u003cem\\u003eP. ovale\\u003c/em\\u003e was misdiagnosed in microscopy as only \\u003cem\\u003eP. falciparum\\u003c/em\\u003e. The clinical reality in Sweden and many other non-endemic countries is that the microscopy is done at the point of care by the infectious disease doctor on call that might perform malaria microscopy very rarely and might have had the most recent training in the technique a very long time ago. This makes both sensitivity, species determination, and assessment of parasitaemia a challenge, and may have affected a possible correlation between actual level of parasitaemia and time to detection by the HumaTurb Loopamp\\u0026trade; Malaria PDT kit. The problems associated with maintaining diagnostic skills adequately high for doctors on call during nighttime and weekends is one of the main reasons more sensitive methods are needed.\\u003c/p\\u003e \\u003cp\\u003eThis study is performed on samples that were frozen for 2\\u0026ndash;4 years which does not accurately represent the test material that will be used in practice which might lead to unforeseen differences in diagnostic performance. However, according to the manufacturers, both kits should be able to handle frozen samples (11, 24). The malaria positivity rate in this material is 31% which is very much higher than it will be in practical use at the labs in most non-endemic countries (3, 14, 19, 25). This affects the PPV and NPV. The lower positivity rate in the real-life setting increases the NPV as compared to what is shown in this study material (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e), which means that the clinical use of the Alethia\\u0026reg; illumigene Malaria kit might be underestimated in our material. The selection of samples by positive results in RDT and microscopy also means that there are very few low parasite density infections in the studied group, which might lead to an overestimation of the diagnostic accuracy of both LAMP instruments in our study. However, one case that presented with fever since only one day that was negative in RDT and microscopy and later found to be malaria positive, was diagnosed as positive by HumaTurb Loopamp\\u0026trade; Malaria PDT kit and qPCR, indicating the superior diagnostic sensitivity of these molecular diagnostic tools compared to RDT and microscopy recommended by the WHO (10).\\u003c/p\\u003e \\u003cp\\u003eThe cost-effectiveness has not been assessed in this study and is highly dependent on local factors, why it ideally should be done in each setting.\\u003c/p\\u003e \\u003cp\\u003eAdded value of this study\\u003c/p\\u003e \\u003cp\\u003eThis study is the first study, to our knowledge, to compare the performance of the two leading LAMP instruments for diagnosis of malaria on the same material. And the results show a superior performance of the HumaTurb Loopamp\\u0026trade; Malaria PDT kit.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eIn this head-to-head retrospective validation study of the diagnostic performance of two LAMP kits compared to qPCR, the HumaTurb Loopamp\\u0026trade; Malaria PDT kit was shown to have a better diagnostic performance than the Alethia\\u0026reg; illumigene Malaria kit. The high diagnostic sensitivity and specificity of HumaTurb Loopamp\\u0026trade; Malaria PDT kit with a NPV of 100% in this study, along with ease of use and time to result around one hour makes this analysis suitable as a first line point of care diagnostic test for malaria in non-endemic high resource settings globally. However, the inability of these methods to perform full species differentiation and grade of parasitaemia mean that they cannot replace microscopy in clinical diagnosis as this data is crucial for accurate treatment.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eAvailability of data and materials\\u003c/h2\\u003e\\n\\u003cp\\u003eThe datasets supporting the conclusions of this article are included within the article and its additional files.\\u003c/p\\u003e\\n\\u003ch2\\u003eCompeting interests\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors have no financial or non-financial competing interests.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003ch2\\u003eFunding\\u003c/h2\\u003e\\n\\u003cp\\u003eThe LAMP instruments and reagent kits needed for the evaluation were provided by both manufacturers without cost during the time of laboratory analysis.\\u003c/p\\u003e\\n\\u003ch2\\u003eAuthors\\u0026apos; contributions\\u003c/h2\\u003e\\n\\u003cp\\u003eStudy idea and study design by S.K.S. and AC.I. Laboratory LAMP analyses were performed by E.F. and AC.I. qPCR analyses were performed by I.B and A.F.\\u003c/p\\u003e\\n\\u003cp\\u003eData acquisition, analysis and interpretation of data was done by AC.I., with support from all authors. The manuscript was primarily written by AC.I. and S.K.S., but all authors were involved in drafting the manuscript and revising it critically for important intellectual content.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eWHO. WHO Guidelines for Malaria. Geneva: World Health Organisation; 2023.\\u003c/li\\u003e\\n\\u003cli\\u003eWHO. Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 8 (2016\\u0026ndash;2018). Geneva: World Health Organisation; 2018.\\u003c/li\\u003e\\n\\u003cli\\u003eCheaveau J, Nguyen H, Chow B, Marasinghe D, Mohon AN, Yuan H, et al. Clinical Validation of a Commercial LAMP Test for Ruling out Malaria in Returning Travelers: A Prospective Diagnostic Trial. Open Forum Infectious Diseases. 2018;5(11).\\u003c/li\\u003e\\n\\u003cli\\u003eMorris U, Aydin-Schmidt B. Performance and Application of Commercially Available Loop-Mediated Isothermal Amplification (LAMP) Kits in Malaria Endemic and Non-Endemic Settings. Diagnostics. 2021;11(2):336.\\u003c/li\\u003e\\n\\u003cli\\u003eFrickmann H, Hinz R, Rojak S, Bonow I, Ruben S, Wegner C, et al. Evaluation of automated loop-mediated amplification (LAMP) for routine malaria detection in blood samples of German travelers \\u0026ndash; A cross-sectional study. Travel Medicine and Infectious Disease. 2018;24:25-30.\\u003c/li\\u003e\\n\\u003cli\\u003eWHO. WHO Evidence Review Group on Malaria Diagnosis in Low Transmission Settings Meeting report. Geneva: WHO; 2013.\\u003c/li\\u003e\\n\\u003cli\\u003eOyegoke OO, Maharaj L, Akoniyon OP, Kwoji I, Roux AT, Adewumi TS, et al. Malaria diagnostic methods with the elimination goal in view. Parasitology Research. 2022.\\u003c/li\\u003e\\n\\u003cli\\u003eLjolje D, Abdallah R, Lucchi NW. Detection of malaria parasites in samples from returning US travelers using the Alethia\\u0026reg; Malaria Plus LAMP assay. BMC Research Notes. 2021;14(1).\\u003c/li\\u003e\\n\\u003cli\\u003eSelvarajah D, Naing C, Htet NH, Mak JW. Loop-mediated isothermal amplification (LAMP) test for diagnosis of uncomplicated malaria in endemic areas: a meta-analysis of diagnostic test accuracy. Malaria Journal. 2020;19(1).\\u003c/li\\u003e\\n\\u003cli\\u003eWHO. WHO Guidelines for Malaria. Geneva: World Health Organisation; 2022.\\u003c/li\\u003e\\n\\u003cli\\u003eHuman Diagnostics Worldwide. Products. Malaria-LAMP Germany: Human Diagnostics Worldwide,; 2022 [Available from: https://www.human.de/products/molecular-dx/malaria-lamp/.\\u003c/li\\u003e\\n\\u003cli\\u003eRypien C, Chow B, Chan WW, Church DL, Pillai DR. Detection of Plasmodium Infection by the illumigene Malaria Assay Compared to Reference Microscopy and Real-Time PCR. Journal of Clinical Microbiology. 2017;55(10):3037-45.\\u003c/li\\u003e\\n\\u003cli\\u003eHartmeyer GN, Hoegh SV, Skov MN, Kemp M. Use of Loop-Mediated Isothermal Amplification in a Resource-Saving Strategy for Primary Malaria Screening in a Non-Endemic Setting. The American Journal of Tropical Medicine and Hygiene. 2019;100(3):566-71.\\u003c/li\\u003e\\n\\u003cli\\u003ePolley SD, Gonz\\u0026aacute;lez IJ, Mohamed D, Daly R, Bowers K, Watson J, et al. Clinical Evaluation of a Loop-Mediated Amplification Kit for Diagnosis of Imported Malaria. The Journal of Infectious Diseases. 2013;208(4):637-44.\\u003c/li\\u003e\\n\\u003cli\\u003eCuadros J, Martin Ram\\u0026iacute;rez A, Gonz\\u0026aacute;lez IJ, Ding XC, Perez Tanoira R, Rojo-Marcos G, et al. LAMP kit for diagnosis of non-falciparum malaria in Plasmodium ovale infected patients. Malaria Journal. 2017;16(1).\\u003c/li\\u003e\\n\\u003cli\\u003eCharpentier E, Benichou E, Pag\\u0026egrave;s A, Chauvin P, Fillaux J, Valentin A, et al. Performance evaluation of different strategies based on microscopy techniques, rapid diagnostic test and molecular loop-mediated isothermal amplification assay for the diagnosis of imported malaria. Clinical Microbiology and Infection. 2020;26(1):115-21.\\u003c/li\\u003e\\n\\u003cli\\u003ePiera KA, Aziz A, William T, Bell D, Gonz\\u0026aacute;lez IJ, Barber BE, et al. Detection of Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax using loop-mediated isothermal amplification (LAMP) in a co-endemic area in Malaysia. Malaria Journal. 2017;16(1).\\u003c/li\\u003e\\n\\u003cli\\u003eDe Koninck A-S, Cnops L, Hofmans M, Jacobs J, Van den Bossche D, Philipp\\u0026eacute; J. Diagnostic performance of the loop-mediated isothermal amplification (LAMP) based illumigene\\u0026reg; malaria assay in a non-endemic region. Malaria Journal. 2017;16(1):418.\\u003c/li\\u003e\\n\\u003cli\\u003eMarti H, Stalder C, Gonz\\u0026aacute;lez IJ. Diagnostic accuracy of a LAMP kit for diagnosis of imported malaria in Switzerland. Travel Medicine and Infectious Disease. 2015;13(2):167-71.\\u003c/li\\u003e\\n\\u003cli\\u003eNolasco O, Infante B, Contreras-Mancilla J, Incardona S, Ding XC, Gamboa D, et al. Diagnosis of Plasmodium vivax by Loop-Mediated Isothermal Amplification in Febrile Patient Samples from Loreto, Per\\u0026uacute;. The American Journal of Tropical Medicine and Hygiene. 2020;103(4):1549-52.\\u003c/li\\u003e\\n\\u003cli\\u003eShokoples SE, Ndao M, Kowalewska-Grochowska K, Yanow SK. Multiplexed Real-Time PCR Assay for Discrimination of \\u0026lt;i\\u0026gt;Plasmodium\\u0026lt;/i\\u0026gt; Species with Improved Sensitivity for Mixed Infections. Journal of Clinical Microbiology. 2009;47(4):975-80.\\u003c/li\\u003e\\n\\u003cli\\u003eMedCalc Software. Free statistical calculators. Diagnostic test evaluation calculator: MedCalc Software Ltd; 2022 [cited 2022 October 23]. Available from: https://www.medcalc.org/calc/diagnostic_test.php.\\u003c/li\\u003e\\n\\u003cli\\u003eBallard E, Wang CYT, Hien TT, Tong NT, Marquart L, Pava Z, et al. A validation study of microscopy versus quantitative PCR for measuring Plasmodium falciparum parasitemia. Tropical Medicine and Health. 2019;47(1).\\u003c/li\\u003e\\n\\u003cli\\u003eMeridian Bioscience. Alethia Malaria and Malaria PLUS DNA Amplification Assays, User Information. Cincinnati, Ohio, USA: Meridian Bioscience; 2020.\\u003c/li\\u003e\\n\\u003cli\\u003eBurdino E, Calleri G, Ghisetti V. Added value of loop-mediated isothermal amplification technology (LAMP) in real life for the diagnosis of malaria in travellers. Journal of Travel Medicine. 2019;26(7):taz052.\\u003c/li\\u003e\\n\\u003cli\\u003eHuman Diagnostics Worldwide. Frequently Asked Questions. Malaria-LAMP Wiesbaden, Germany: Human Diagnostics Worldwide; 2019 [Available from: https://www.human.de/fileadmin/content/02_Products/04_Molecular_DX/Documents/FAQ_Malaria-LAMP_print_and_view_rev.004.pdf.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"malaria-journal\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"malj\",\"sideBox\":\"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)\",\"snPcode\":\"12936\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12936/3\",\"title\":\"Malaria Journal\",\"twitterHandle\":\"@malariajournal\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Malaria, LAMP, Loop-mediated isothermal amplification, diagnosis.\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-2984503/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-2984503/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eIntroduction:\\u003c/strong\\u003e Light microscopy and rapid diagnostic tests (RDT) have long been the recommended diagnostic methods for malaria. However, in recent years, LAMP techniques have been shown to offer superior diagnostic performance, in particular concerning low-grade parasitaemia, by delivering higher sensitivity and specificity with low laboratory capacity requirements in little more than an hour. In this study we assessed the diagnostic performance of two Loop-mediated Isothermal Amplification (LAMP) kits head-to-head, compared to highly sensitive quantitative real time PCR (qPCR), in a non-endemic setting.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMethods:\\u003c/strong\\u003e In this retrospective validation study two LAMP kits; Alethia® illumigene Malaria kit and HumaTurb Loopamp™ Malaria Pan Detection (PDT) kit, were evaluated head-to-head for detection of \\u003cem\\u003ePlasmodium\\u003c/em\\u003e-DNA in 133 biobanked blood samples from suspected malaria cases at the Clinical Microbiology Laboratory of Region Skåne, Sweden to determine their diagnostic performance compared to qPCR.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults:\\u003c/strong\\u003e Of the 133 samples tested, qPCR detected \\u003cem\\u003ePlasmodium\\u003c/em\\u003e DNA in 41 samples (the true positives), and the two LAMP methods detected 41 and 37 of those, respectively. The results from the HumaTurb Loopamp™ Malaria PDT kit were in complete congruence with the qPCR, with a sensitivity of 100% (95% CI 91.40 - 100%) and specificity of 100% (95% CI 96.07 – 100%); and the PPV and NPV were both 100%. The Alethia® illumigene Malaria kit\\u003cem\\u003e \\u003c/em\\u003ehad a sensitivity of 90.24% (95% CI 76.87 – 97.28) and a specificity of 95.65% (95% CI 89.24 – 98.80) as compared to qPCR. The PPV was 90.31% (95% CI 78.06-96.07) and the NPV was 95.62% (95%CI 89.58-98.23) with the malaria positivity rate of 30.8 % in the study material. This performance would give a PPV of 69.75% (95% CI 46.80 – 85.81) and NPV 98.88% (97.20 – 99.56) in a situation where samples tested had a 10% positivity rate, which more accurately resembles the situation in Skåne.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusions:\\u003c/strong\\u003e This head-to-head comparison showed superior performance of the HumaTurb Loopamp™ Malaria PDT kit compared to the Alethia® illumigene Malaria kit for detection of malaria.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Head-to-head comparison of two loop-mediated isothermal amplification (LAMP) kits for diagnosis of malaria in a non-endemic setting\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2023-05-30 15:29:45\",\"doi\":\"10.21203/rs.3.rs-2984503/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Major revision\",\"date\":\"2023-09-19T11:16:46+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2023-08-08T01:43:35+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"cde3c3b5-92cd-4eae-b9fa-c408656bc58b\",\"date\":\"2023-07-17T13:55:42+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"0a5adea8-816c-44c8-a2f6-7d4ef7cbca0a\",\"date\":\"2023-05-31T14:13:36+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2023-05-31T14:08:31+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2023-05-27T05:19:43+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2023-05-27T05:19:43+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Malaria Journal\",\"date\":\"2023-05-26T08:06:39+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"malaria-journal\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"malj\",\"sideBox\":\"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)\",\"snPcode\":\"12936\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12936/3\",\"title\":\"Malaria Journal\",\"twitterHandle\":\"@malariajournal\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"59eb3607-d1f8-4c45-bfdd-429b9126bc73\",\"owner\":[],\"postedDate\":\"May 30th, 2023\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2023-12-18T15:08:32+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-2984503\",\"link\":\"https://doi.org/10.1186/s12936-023-04809-7\",\"journal\":{\"identity\":\"malaria-journal\",\"isVorOnly\":false,\"title\":\"Malaria Journal\"},\"publishedOn\":\"2023-12-13 15:02:18\",\"publishedOnDateReadable\":\"December 13th, 2023\"},\"versionCreatedAt\":\"2023-05-30 15:29:45\",\"video\":\"\",\"vorDoi\":\"10.1186/s12936-023-04809-7\",\"vorDoiUrl\":\"https://doi.org/10.1186/s12936-023-04809-7\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-2984503\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-2984503\",\"identity\":\"rs-2984503\",\"version\":[\"v1\"]},\"buildId\":\"7rjqhiLT3MXkJMwkYKINL\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}