https://orcid.org/0000-0003-2159-5667
Figures
Abstract
Malaria elimination efforts in Peru have dramatically reduced the incidence of cases in the Amazon Basin. To achieve the elimination, the detection of asymptomatic and submicroscopic carriers becomes a priority. Therefore, efforts should focus on tests sensitive enough to detect low-density parasitemia, deployable to resource-limited areas and affordable for large screening purposes. In this study, we assessed the performance of the Malachite–Green LAMP (MG-LAMP) using heat-treated DNA extraction (Boil & Spin; B&S MG-LAMP) on 283 whole blood samples collected from 9 different sites in Loreto, Peru and compared its performance to expert and field microscopy. A real-time PCR assay was used to quantify the parasite density. In addition, we explored a modified version of the B&S MG-LAMP for detection of submicroscopic infection in 500 samples and compared the turnaround time and cost of the MG-LAMP with microscopy. Compared to expert microscopy, the genus B&S MG-LAMP had a sensitivity of 99.4% (95%CI: 96.9%– 100%) and specificity of 97.1% (95%CI: 91.9%– 99.4%). The P. vivax specific B&S MG-LAMP had a sensitivity of 99.4% (96.6%– 100%) and specificity of 99.2% (95.5%– 100%) and the P. falciparum assay had a sensitivity of 100% (95%CI: 78.2%– 100%) and specificity of 99.3% (95%CI: 97.3%– 99.8%). The modified genus B&S MG-LAMP assay detected eight submicroscopic malaria cases (1.6%) which the species-specific assays did not identify. The turnaround time of B&S MG-LAMP was faster than expert microscopy with as many as 60 samples being processed per day by field technicians with limited training and utilizing a simple heat-block. The modified B&S MG-LAMP offers a simple and sensitive molecular test of choice for the detection of submicroscopic infections that can be used for mass screening in resources limited facilities in endemic settings nearing elimination and where a deployable test is required.
Citation: Barazorda KA, Salas CJ, Braga G, Ricopa L, Ampuero JS, Siles C, et al. (2021) Validation study of Boil & Spin Malachite Green Loop Mediated Isothermal Amplification (B&S MG-LAMP) versus microscopy for malaria detection in the Peruvian Amazon. PLoS ONE 16(10): e0258722. https://doi.org/10.1371/journal.pone.0258722
Editor: Luzia Helena Carvalho, Instituto Rene Rachou, BRAZIL
Received: May 5, 2021; Accepted: October 4, 2021; Published: October 25, 2021
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by the US DoD Armed Forces Health Surveillance Division (AFHSD), Global Emerging Infections Surveillance (GEIS) Section, PROMIS IDs P0106_18_N6_02 (DKB) and P0143_19_N6_02 (CAJ) from 2018 and 2019. The funders had not role in the study design, data collection and analysis, preparation of the manuscript nor decision to publish. KAB is an employee of Vysnova and this funder provided support in the form of salaries for KAB, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of this author is articulated in the author contributions’ section.
Competing interests: KAB is an employee of Vysnova and reports personal fees outside the submitted work. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Introduction
Malaria is a major problem worldwide with more than 228 million cases and 405,000 deaths in 2018 [1]. Malaria prevention and control efforts have reduced malaria incidence in several countries. However, achieving the elimination goal would require the development of improved diagnostic methods and tools capable of detecting low-density infections that can complement existing interventions [2].
In Peru, it is estimated that at least one third of the population live in malaria endemic areas and are at risk of contracting the disease [3]. Malaria cases are concentrated in the Department of Loreto in the Peruvian Amazon which accounts for 96% of all cases reported in the country during 2017 [4]. Malaria cases are predominantly in rural and riverine communities with very little communication and access to health care.
In this context, the Peruvian Ministry of Health (MoH) developed the plan “Malaria Zero” which is aimed towards malaria elimination in the Amazon region [3,5]. These efforts have dramatically reduced the incidence of cases in the Amazon basin making the detection of asymptomatic and submicroscopic carriers critical in the final stages of elimination as they are known to sustain transmission [6]. Therefore, efforts should focus on tests that are capable of detecting low-density parasitemia, can be performed in resource-limited areas, and are inexpensive enough to screen large populations [2].
Malaria diagnosis in Peru is performed through routine microscopy and rapid diagnostic tests (RDTs) [7]. However, both methods present some challenges to malaria elimination goals because of their inability to detect low parasite density infections. Microscopy requires experienced personnel to provide quality results and is not reliable for detecting low parasite density infections since its limit of detection is about 50 parasites/μL [8–10]. The drawbacks of RDTs are also lower sensitivity with limit of detection of 100–200 parasites/μL [11]. In addition, in Peru there is a relatively high prevalence of parasites that have deleted the P. falciparum-specific genes HRP2 and HRP3 prevalent in up to 41% and 70% of P. falciparum samples, respectively [12] limiting their ability to detect falciparum infections. These limitations make the development of highly sensitive, cost-effective malaria diagnostic tests highly valuable for Peru and similar malaria elimination programs in South America. Early malaria detection will also improve patient care and patient outcomes [1].
Loop-mediated isothermal amplification (LAMP)-based assays were developed to meet the need for a sensitive field-usable assay for numerous diseases including malaria [13–19]. Furthermore, the addition of dyes such as malachite-green facilitates colorimetric readout, thus reducing cost and simplifying the assays.
The Malachite-Green Loop-mediated Isothermal Amplification (MG-LAMP) has shown great performance in field and laboratory-controlled settings [20–22]. We recently demonstrated that this method can be implemented in a reference laboratory in Peru and we documented its usefulness for surveillance studies in areas approaching malaria elimination where submicroscopic infections are challenging to detect by conventional methods like microscopy or RDTs [23].
The optimization of molecular assays for use in resource-limited areas requires simple DNA extraction methods as opposed to conventional DNA extraction kits that are more expensive and require specialized equipment and laboratory space. Methods such as extraction through heat–treated whole blood are a promising alternative method [24,25].
In this study, we assessed the performance and cost of MG-LAMP using heat-treated whole blood (Boil and Spin or B&S) DNA extraction for detection of P. falciparum and P. vivax malaria in field conditions and compared its performance to microscopy as gold standard. A real-time PCR assay (PET-PCR) was used for parasite quantification.
Methods
Study sites and sample collection
The study was carried out in nine study sites in Loreto region which is located in the northwest side of the Peruvian Amazon basin (Fig 1). Samples for this project were obtained from 3 retrospective studies: two passive surveillance studies of febrile disease etiology conducted between 2015 and 2019 in Iquitos, Peru and surroundings communities and from an active surveillance study conducted in 2019 in the Iquitos community of Padre Cocha.
The insert shows the location of the health facilities where samples were collected in Iquitos. Red circles showed the sites for the passive surveillance studies. The community of Padre Cocha (Blue) was a site for the passive and active surveillance study. Map created using open data from OpenStreetMap, under ODbL, under CC BY 2.0 (creativecommons.org/licenses/by/2.0/).
Sample size
The sample size was calculated using the formula for a single proportion assuming a sensitivity of 96% for the MG-LAMP based on previous studies and 95% confidence and 80% power. This resulted in a minimum sample size of 196 samples to be tested.
Passive surveillance studies
A total of 283 whole blood samples were selected from two studies, one conducted in 2015–2019 and another in 2018. The first study was designed for malaria surveillance and 258 samples were used. Inclusion criteria for this study were age greater than 1 year with presence of fever or history of fever during the previous 72 hours and signed informed consent to participate in the study (and assent where appropriate).
The second passive surveillance study was designed for febrile diseases etiology and 25 samples were used. Inclusion criteria included patients 5 years of age or older who presented in outpatient clinics or hospitals with acute, undifferentiated, febrile illness (axillary temperature greater than or equal to 38°C for 7 days duration or less) with specific symptoms [26].
Active surveillance study
Samples from the active surveillance study were collected between July 15th to August 23th 2019 from a cohort study aimed at detecting asymptomatic/submicroscopic malaria infections in the community of Padre Cocha (Fig 1). Inclusion criteria for this study were age greater than or equal to 5 years and resident of Padre Cocha or surroundings areas. Only expert microscopy was available to process all samples from this study.
Ethical considerations
The samples tested in this study were selected from three protocols (NMRCD.2007.0004, NMRCD.2010.0010 and NAMRU6.2018.0008) previously approved by the Institutional Review Board of the U.S Naval Medical Research Unit N°6 (NAMRU-6) in compliance with all applicable federal regulations governing the protection of human subjects. Protocols were also approved by the local Peruvian MoH. Adult participants provided written consent and minors provided written assent and parental/guardian consent.
Training of laboratory staff to perform MG-LAMP in the field site
A standard operating procedure (SOP) was prepared in the reference laboratory at NAMRU-6, Lima and provided to the technicians working in our Iquitos facility before onsite training.
Training on MG-LAMP was provided on-site to two NAMRU-6-Iquitos technicians with basic experience on molecular methods. Before testing the B&S MG-LAMP in the field site, training was conducted in two sessions for two days. The first session was theoretical to address the LAMP chemistry, differences between nested-PCR and advantages of LAMP approaches. The second session was practical, with a first guided experiment by the instructor using positive and negative malaria controls. The second experiment was executed by the technicians under the instructor’s supervision using samples collected during the week.
Sample processing and microscopy
Two levels of microscopy were performed in this study, field and expert microscopy. Two mL of EDTA-whole blood was collected by venipuncture from each participant. Expert microscopy procedure consisted of two thin and thick smears, stained with 10% Giemsa and read by two fully dedicated microscopists in the laboratory at NAMRU-6 Iquitos. A slide was considered negative after reading 200 oil-immersion fields while positive slides were quantified using as reference 6000 WBC/μl of blood. The remaining whole blood was aliquoted in one or two vials and stored at -80°C for further testing. Field microscopy was performed at health-care facilities of each community as per the health facilities’ guidelines. Thick blood smears were prepared, slides were stained for 10 minutes with 10% Giemsa, and 100 fields per slide were read. Results were recorded using the “plus system” following the Peruvian MoH guidelines [7,27].
DNA extraction methods
Two DNA extraction methods were performed for all the samples tested, i) boil and spin method (B&S) was used only for the MG-LAMP and ii) Qiagen™ kit was used for PET-PCR and nested-PCR. The B&S DNA extraction method was performed as previously described [24,25,28] with a minor modification in the elution step to concentrate the obtained DNA. Briefly, an aliquot of 60 μL of whole blood was transferred into a 1.5 mL tube containing 60 μL of extraction buffer (400 mM NaCl, 40 mM Tris pH 6.5, 0.4% SDS). The mixture was incubated for 5 min using a heat-block set at 95°C followed by centrifugation for 3 min at 10,000g. Finally, we prepared a 1:4 dilution consisting of 30 μL of clear supernatant mixed with 90 μL of ultra-pure water. Extracted DNA was immediately used or stored at -20°C for no longer than a week. DNeasy Blood & Tissue kit (Qiagen)™ was used for DNA extraction for PET-PCR method. All samples were processed at NAMRU-6 Lima following the manufacturer’s instruction using 200 μL of EDTA-whole blood. DNA was eluted in 70μL of elution buffer and used immediately for the amplification reactions; the remaining volume was stored at -20°C.
Malachite green LAMP assay (MG-LAMP)
For the passive surveillance studies MG-LAMP was performed using the B&S DNA (B&S MG-LAMP). The primers used are listed in S1 Table. All samples were first tested with the genus-specific MG-LAMP assay. The genus positive samples were further tested by the species-specific assays for both P. falciparum and P. vivax. Each assay was carried out using individually capped tube strips to avoid cross-contamination among samples. The final volume of each reaction was 20 μL, which contained 2X in-house LAMP buffer (40 mM Tris-HCL pH 8.8, 20 mM KCL, 16 mM MgSO4, 20 mM (NH4)SO4, 0.2% Tween-20, 1.5M Betaine, 2 mM of DNTP’s each, 0.004% Malachite Green dye (MG), 320U/mL of Bst DNA polymerase (New England Biolabs, Ipswich, MA) and 5 μL of B&S DNA template, as previously described [20].
Samples collected from the active surveillance study were processed using a modified version of the original MG-LAMP master mix in a final volume of 25 μL containing 2X in-house LAMP buffer, 0.0048% Malachite Green dye (MG), 320U/mL of Bst DNA polymerase and 10 μL of B&S DNA template (1.6x of target DNA in comparison with the MG-LAMP used on the passive surveillance component).
To maintain the Quality Control (QC) of the in-house buffer, a set of positive controls were tested every time a new LAMP buffer batch was prepared before testing field samples. All amplification reactions were done for 1 hour at 63°C using a mini heat block (Gene Mate, Bio Express, Utah, US). PCR reaction tube strips were removed and kept at room temperature for 15 minutes before being scored for amplification by two independent readers. A positive result was defined by the presence of a light blue-green color, while the negative samples were colorless. If the color obtained after amplification was grey or uncertain, a second reaction was performed.
Photo-induced electron transfer-PCR (PET-PCR)
To quantify parasite density and assess the limit of detection of the B&S MG-LAMP, all samples were tested in duplicates using a Plasmodium genus PET-PCR as previously described with a slight modification on the cut-off value [29]. All genus positive samples were further tested by singleplex reactions to detect P. falciparum and P. vivax as previously described [21,29]. Samples were considered positive if they had a threshold cycle (Ct) of 41 or below and negative if they had no Ct value or a Ct value above 41. This cut-off was determined using clinical samples.
Nested-PCR
The 18srRNA-nested PCR was chosen for species identification of specimens collected in the active surveillance study because of its high sensitivity and specificity [30].The first reaction amplified the 18srRNA subunit ribosomal (ssrRNA) gene and the second reaction amplified specific regions for each plasmodium species (P. falciparum and P. vivax). Both PCR reactions were performed in 50 μl of final volume. For the first reaction 5 μl of DNA template was used, while 5 μl of the amplified product were used in the second reaction. Results were visualized in a 2% agarose gel stained with GelRed®
Analytical sensitivity of B&S MG-LAMP
To assess the limit of detection (LoD) of the B&S MG-LAMP we used serial dilutions Plasmodium control strains. For P. falciparum, we performed continuous in vitro culture of the 3D7 reference strain following the same procedure previously described [23]. For P. vivax we used a clinical specimen with 121,500 parasites/μL. For both controls, we performed tenfold dilutions starting from 10,000 parasites/μL to 1 parasite/μL with two-fold dilutions from 10 parasites/μL to 1.25 parasites/μL. These controls were also used for training laboratory staff in Iquitos.
Turn-around time and cost analysis
We used 8 samples to estimate the required processing time, turn-around time (TAT) and costs of expert microscopy and B&S MG-LAMP for the specific identification of Plasmodium genus and species. Cost-analysis included the costs required for sample preparation (preparation of slides and staining for microscopy) and DNA extraction and amplification supplies (for B&S MG-LAMP). Salaries were not included in the calculations.
Quality assessment
To evaluate the limitations and accuracy of our study, we followed the guidelines of the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) which evaluate four areas of bias (patient selection, index test, gold standard method, and flow/timing) [31]. Concerns regarding applicability were also evaluated in three areas: patient selection, index test, and reference standards and where also evaluated to have low, unclear or high risk of bias.
Statistical methods
Sensitivity and specificity with 95% confidence intervals (95% CI) were calculated for field microscopy and B&S MG-LAMP using expert microscopy results as the gold standard test. Statistical differences between expert microscopy and B&S MG-LAMP were determined using the non-parametric McNemar test. The Cohen’s Kappa coefficient [32] was used to estimate agreement between diagnostic tests and the area under the receiver operating characteristic (ROC) curve was used to compare the performance between tests. Analysis of PET-PCR quantification was performed on log-transformed data to compare with microscopy quantification. Data were analyzed using Stata, version, 13 (StataCorp LP, College station, TX).
Results
Analytical limit of detection of B&S MG-LAMP
The analytical LoD for the genus B&S MG-LAMP was one parasite/μL while that of the P. falciparum and P. vivax was 2.5 parasites/μL for both assays with 100% concordance between two independent readers (Fig 2). Regarding the modified B&S MG-LAMP used in the active surveillance component, the LoD for the genus and species-specific assays was 0.5 parasites/μL.
The analytical limit of detection of genus (A) and P. vivax (B) B&S MG-LAMP was determined using 10-fold (104−1.0 parasite/μL) and 2-fold (5–1.25 parasite/μL) serial dilution of well-quantified P. falciparum (genus) and P. vivax samples, as indicated on the tubes. Colorimetric B&S MG-LAMP results can be visualized with the naked-eye, blue/green indicate positive whereas colorless, negative. NC = no-template control. The LoD of the genus-specific B&S MG-LAMP assay was 1 parasite/μL and that of P. vivax was 2.5 parasite/μL.
Malaria species detection in the passive surveillance study
A total of 283 samples were used in this study (S2 Table): All the 283 samples were tested with the Plasmodium genus- specific MG-LAMP using DNA obtained by the B&S extraction method. A total of 67 samples were tested at NAMRU-6 laboratory in Lima, while the remaining 216 were tested at field laboratory in Iquitos. A flow chart of all laboratory tests performed is shown in S1 Fig).
As summarized in Table 1, field microscopy identified 109 negatives and 174 positives (18 P. falciparum and 156 P. vivax). Expert microscopy identified 105 negatives and 178 positives (15 P. falciparum, 162 P. vivax and 1 mixed P. falciparum/P. vivax) with a mean asexual parasitemia of 796 parasites/μL (range: 12–69,235 parasites/μL) and mean gametocytemia of 72.46 gametocytes/μL (range: 12–1,235 gametocytes/μL).
The genus B&S MG-LAMP identified 180 Plasmodium spp and 103 negatives whereas the species-specific B&S MG-LAMP identified 179 positives (17 P. falciparum and 162 P. vivax).
Diagnostic performance of microscopy and B&S MG-LAMP
Using expert microscopy as gold standard, field microscopy had a sensitivity for detecting either Plasmodium spp. of 97.2% (95%CI: 93.6–99.1%) and specificity of 99% (95%CI: 94.8% - 100%). Regarding the species-specific detection, field microscopy had a sensitivity for P. vivax of 96.3% (95%CI: 92.1%– 98.6%) and specificity of 100% (95%CI: 97%– 100%). For P. falciparum, field microscopy had a sensitivity of 93.3% (95%CI: 68.1%– 99.8%) and specificity of 98.5% (95%CI: 96.2%– 99.6%) (Fig 3, S3 Table). The genus B&S MG-LAMP had a sensitivity of 99.4% (95%CI: 96.9%– 100%) and specificity of 97.1% (95%CI: 91.9%– 99.4%). The P. vivax specific B&S MG-LAMP had a sensitivity of 99.4% (96.6%– 100%) and specificity of 99.2% (95.5%– 100%) and the P. falciparum assay had a sensitivity of 100% (95%CI: 78.2%– 100%) and specificity of 99.3% (95%CI: 97.3%– 99.8%) (Fig 3, S3 Table).
Agreement of B&S MG-LAMP and field microscopy compared with expert microscopy
The McNemar test did not show statistical differences between the results of the B&S MG-LAMP and expert microscopy for the detection of Plasmodium spp, P. vivax and P. falciparum (p> 0.05). However, statistical differences were found between field microscopy and B&S MG-LAMP for the detection of P. vivax (p<0.05), but there were no differences for the detection of genus and P. falciparum. Cohen’s kappa showed that B&S MG-LAMP presented a kappa of 0.97 (95%CI: 0.94–0.99) and field microscopy of 0.93 (95%CI: 0.89–0.98). The area under the ROC curve for the B&S MG-LAMP was 0.99 while for field microscopy was 0.97 (S2 Fig).
Discordant results
Of the 178 positive samples, nine were shown to be mixed infections by PET-PCR. Expert microscopy detected one mixed infection which was shown to be P. falciparum infection by the other methods. The B&S MG-LAMP and field microscopy detected eight and seven, respectively, of the nine mixed infections as single infections, detecting only the predominant species (S4 Table). The median species parasitemia in the mixed infections as determined by PET-PCR was 60.3 (0.2–72 parasites/uL) for P. falciparum and 0.7 (0.3–10,830 parasites/ uL) for P. vivax. Eight out of the 9 missed mixed infections had parasite densities below the limits of detection of the B&S MG-LAMP and microscopy (S4 Table). In addition, eight samples diagnosed as P. vivax by the PET-PCR were found to be negative by the B&S MG-LAMP and field microscopy while expert microscopy detected only one of these (S4 Table). The mean parasitemia for these samples was 0.6 parasites/uL.
B&S MG-LAMP performance in the active surveillance study
A total of 500 participants were enrolled on the active surveillance study with a mean age of 33 ± 20 years old and 57% were females. Malaria-like symptoms in the previous three days before enrollment were reported by 21% of participants. These symptoms included headache (85%), malaise (39%), fever (29%) and chills (28%). Expert microscopy detected one P. vivax case (0.2%) while the genus B&S MG-LAMP assay detected eight positive cases (1.6%) (Table 2). The range of parasites/mL of the 8 positive samples was 1.3–45.0 parasites/μL as determined by genus PET-PCR. Nested-PCR identified these as 7 P. vivax and 1 P. falciparum (Table 2). However, the species-specific B&S MG-LAMP did not identify any positive samples. One out of eight positive subjects reported fever during the previous 3 days before enrollment (12.5%).
The remaining 493 samples were negative.
Turnaround time (TAT) and Cost Analysis
For ease of estimation, the TAT and cost analysis was estimated using 8 samples because the strip of PCR tubes used in the MG-LAMP has 8 samples (Table 3). The processing of a total of 8 samples was about $2 cheaper by expert microscopy compared to B&S MG-LAMP and the overall TAT was shorter by B&S MG-LAMP compared to expert microscopy (Table 3). The total number of samples the technicians were able to process per day was evaluated during the processing of samples from the active surveillance study: the technicians increased the number of samples processed daily by B&S MG-LAMP from 12 samples to 60 samples per day. All 500 samples were processed in 12 working days by two lab technicians.
Discussion
Malaria transmission in several countries in the Americas has been gradually declining due to renewed efforts towards malaria elimination [1]. Under this context, there is a great need for highly sensitive tools to detect asymptomatic or low parasite density carriers that are frequently missed by current RDTs and microscopy [12,33–35]. Furthermore, these new tools need to be tailored to the epidemiology and conditions of endemic settings in each country so that they can better complement and inform targeting of malaria interventions [2].
In this study, we assessed the performance of a simplified MG-LAMP using heat-treated whole blood versus field and expert microscopy. Our results from the passive surveillance studies showed that overall, the B&S MG-LAMP has a similar performance compared to both levels of microscopy. This can be explained by the fact that the mean asexual parasitemia in the passive surveillance study was 796 parasites/μL which is within the detection limits of both microscopy and MG-LAMP. In addition, the malaria control program in Peru has invested heavily in quality microscopy even in health centers/diagnostic post greatly improving the quality of microscopy even in the field settings [3]. This suggests that both levels of microscopy are good options for case management in Peru. In contrast, the modified B&S MG-LAMP used in the active surveillance study was superior to field and expert microscopy in the detection of asymptomatic low-density infections.
The use of PET-PCR for parasite quantification allowed us to estimate the parasitemia on the positive genus samples missed by the species B&S MG-LAMP on the passive surveillance component. Our results showed that in all these cases, parasite densities were below the analytical LoD of the B&S MG-LAMP. Furthermore, in the case of missed mixed infections, the B&S MG-LAMP detected the parasite with a higher parasite density and missed the other species as they occurred at extremely low-parasite densities, below the detection limit of B&S MG-LAMP. However, it is also possible that this failure was caused by a limitation of the primer target used in the MG-LAMP or the method of signal detection as previously observed [19,21,23].
These are important issues to address since misdiagnosis in mixed infections can lead to inappropriate treatment management in addition to increased relapse risk for P. vivax infections as previously shown [36]. Our results showed that false negative samples by B&S MG-LAMP were classified as submicroscopic infections by PET-PCR. This scenario highlights the need for continuing developing methods for highly sensitive detection which are key in areas undergoing elimination such as Peru. In these regions, accurate detection of submicroscopic cases prevents ongoing transmission by tackling hotspots of submicroscopic and asymptomatic reservoirs that are responsible of continuing transmission [3,37]. Nonetheless, the modified B&S MG-LAMP assay tested on the active surveillance study has the potential for detection of submicroscopic and asymptomatic infections. However, more thorough testing of its performance is needed.
Heat-treatment has been utilized in previous studies as an alternative to the more expensive DNA extraction kits for LAMP [38–40]. The LoD of the genus and species-specific B&S MG-LAMP assays observed in this study was similar to that observed using the QIAGEN DNA extraction kit in previous studies [13,23]. This shows that the use of the B&S unpurified DNA extraction does not impact the performance of the MG-LAMP reaction dye [41]. In addition, unlike Taq polymerase, Bst polymerase used in LAMP is not affected by inhibitors of conventional PCR such as haemoglobin [42,43], making LAMP easily adaptable to easier DNA extraction methods such as B&S. However, the B&S DNA template should be utilized soon after the extraction in order to avoid enzyme degradation [38]. Our B&S DNA extraction protocol included a final DNA concentration step during the elution step which improved the performance of the MG-LAMP compared to the original protocol [21,44].
The TAT of B&S MG-LAMP was faster than expert microscopy with as many as 60 samples being processed per day by field technicians with limited training and utilizing a simple heat-block. With additional heat-blocks, this number of samples can be increased to allow the screening of many more samples. The cost per sample of the B&S MG-LAMP was calculated to be about $1.2 compared to $0.94 for expert microscopy. This cost is much lower compared to other molecular methods such as PCR-based assays [45]. Therefore, the B&S MG-LAMP offers a test of choice for mass screening and detection of submicroscopic infections due to i) minimal expertise required, ii) reduced cost iii) potential for increased throughput with portable heat-blocks for large scale screening which is a limitation for microscopic diagnosis and iv) increased sensitivity over field and expert microscopy as shown in our active surveillance study.
Using the QUADAS-2 tool, we evaluated that there was a low bias risk for sample identification by the operators as they did not have access to individual sample identification nor study file details. For the reference B&S MG-LAMP test, samples were tested in batches of 30 specimens at the end of the week for the passive surveillance studies or 1 month after enrolment of the active surveillance.
In summary, our study shows that the modified B&S MG-LAMP is a simpler molecular test that can be implemented in resources limited facilities in endemic settings nearing for elimination and where a deployable test is required [19] such as points of entry and in support of active surveillance studies. The increasing availability of such tests in the field will help in the detection of parasite reservoirs that can be targeted with interventions to help interrupt transmission. Our study also highlights the need of maintaining good malaria microscopy skills that can deliver quality results. We validated a methodology that aimed to be compliant with the WHO [46], to solve the urgent need for new tools to support the eradication efforts.
Supporting information
S1 Fig. Flow chart of all methods performed in the study.
https://doi.org/10.1371/journal.pone.0258722.s001
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S2 Fig. ROC curve comparison with expert microscopy.
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S1 Table. List of primers used for the B&S MG-LAMP.
https://doi.org/10.1371/journal.pone.0258722.s003
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S2 Table. Individual test results for passive surveillance component.
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S3 Table. Comparison of the B&S MG-LAMP and field microscopy with expert microscopy.
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S4 Table. Mixed infections results.
Comparison of results of mixed infections (Pf/Pv) performed by each methodology.
https://doi.org/10.1371/journal.pone.0258722.s006
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Acknowledgments
We would like to thank Dr. Venkatachalam Udhayakumar for his support in reviewing the article and express our gratefulness to Naomi W. Lucchi for her support in conceptualization, writing and reviewing the draft and added valuable suggestions to obtain this final version. She and Dragan Ljolje also worked in improving the current methodology. Finally, we thank the laboratory team in Iquitos for their continuous hard work in collecting samples and to the anonymous volunteers who kindly contributed to this outcome.
Ethical approval
The study protocols were approved by the Naval Medical Research Center Unit 6 Institutional Review Board in compliance with all applicable Federal regulations governing the protection of human subjects.
Disclaimer: The views expressed in this article reflect the results of research conducted by the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the United States Government.
References
- 1.
WHO. World malaria report World Health Organization. 2019: Available from: https://www.who.int/publications/i/item/9789241565721.
- 2. mal ERARCPoTfME. malERA: An updated research agenda for diagnostics, drugs, vaccines, and vector control in malaria elimination and eradication. PLoS medicine. 2017;14(11):e1002455. pmid:29190291; PubMed Central PMCID: PMC5708606.
- 3.
Ministerio de Salud del Perú DGdIEeSP. Plan Malaria cero—Periodo 2017–2021. Lima 2017: Available from: https://www.gob.pe/institucion/minsa/normas-legales/189846-244-2017-minsa.
- 4.
Centro Nacional de Epidemiologia PyCdE-M. Lima- 2019: Available from: http://www.dge.gob.pe/portal/docs/vigilancia/sala/2019/SE02/malaria.pdf.
- 5. Ordoñez. Situacion Epidemiologica de la Malaria en el Peru—SE 42. Boletin Epidemiologico del Peru.2019. p. 1069–71.
- 6. mal ERARCPoCtR, Measuring T. malERA: An updated research agenda for characterising the reservoir and measuring transmission in malaria elimination and eradication. PLoS medicine. 2017;14(11):e1002452. pmid:29190279; PubMed Central PMCID: PMC5708619.
- 7.
(INS) INdS. Norma Técnica de Salud para el control de calidad del diagnóstico microscópico de Malaria. Lima—Perú2010. p. 41.
- 8. Eugenia Lo GZ, Winny Oo, Yaw Afrane, Andrew Githeko, Guiyun Yan Low Parasitemia in Submicroscopic Infections Significantly Impacts Malaria Diagnostic Sensitivity in the Highlands of Western Kenya. PLoS ONE. 2015;10(3). pmid:25816298
- 9. Ohrt C, Purnomo , Sutamihardja MA, Tang D, Kain KC. Impact of microscopy error on estimates of protective efficacy in malaria-prevention trials. J Infect Dis. 2002;186(4):540–6. Epub 2002/08/27. pmid:12195382.
- 10. Kiggundu M, Nsobya SL, Kamya MR, Filler S, Nasr S, Dorsey G, et al. Evaluation of a comprehensive refresher training program in malaria microscopy covering four districts of Uganda. Am J Trop Med Hyg. 2011;84(5):820–4. pmid:21540396; PubMed Central PMCID: PMC3083754.
- 11. Mwesigwa J, Slater H, Bradley J, Saidy B, Ceesay F, Whittaker C, et al. Field performance of the malaria highly sensitive rapid diagnostic test in a setting of varying malaria transmission. Malaria journal. 2019;18(1):288. Epub 2019/08/29. pmid:31455349; PubMed Central PMCID: PMC6712604.
- 12. Gamboa D, Ho MF, Bendezu J, Torres K, Chiodini PL, Barnwell JW, et al. A large proportion of P. falciparum isolates in the Amazon region of Peru lack pfhrp2 and pfhrp3: implications for malaria rapid diagnostic tests. PloS one. 2010;5(1):e8091. pmid:20111602; PubMed Central PMCID: PMC2810332.
- 13. Krampa FD, Aniweh Y, Awandare GA, Kanyong P. Recent Progress in the Development of Diagnostic Tests for Malaria. Diagnostics. 2017;7(3). pmid:28925968; PubMed Central PMCID: PMC5617953.
- 14. Serra-Casas E, Guetens P, Chiheb D, Gamboa D, Rosanas-Urgell A. A pilot evaluation of alternative procedures to simplify LAMP-based malaria diagnosis in field conditions. Acta Tropica. 2019;200:105125. pmid:31394079
- 15. Lucchi NW, Ndiaye D, Britton S, Udhayakumar V. Expanding the malaria molecular diagnostic options: opportunities and challenges for loop-mediated isothermal amplification tests for malaria control and elimination. Expert Rev Mol Diagn. 2018;18(2):195–203. Epub 2018/01/23. pmid:29353522.
- 16. Cunningham JA, Thomson RM, Murphy SC, de la Paz Ade M, Ding XC, Incardona S, et al. WHO malaria nucleic acid amplification test external quality assessment scheme: results of distribution programmes one to three. Malaria journal. 2020;19(1):129. pmid:32228615
- 17. Cheaveau J, Mogollon DC, Mohon MAN, Golassa L, Yewhalaw D, Pillai DR. Asymptomatic malaria in the clinical and public health context. Expert Review of Anti-infective Therapy. 2019;17(12):997–1010. pmid:31718324
- 18. Selvarajah 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):211. pmid:32560728
- 19. Picot S, Cucherat M, Bienvenu AL. Systematic review and meta-analysis of diagnostic accuracy of Loop-mediated isothermal amplification (LAMP) methods compared to microscopy, PCR, and rapid diagnostic tests, for malaria diagnosis. International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases. 2020. Epub 2020/07/14. pmid:32659450.
- 20. Lucchi NW, Ljolje D, Silva-Flannery L, Udhayakumar V. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites. PloS one. 2016;11(3):e0151437. pmid:26967908; PubMed Central PMCID: PMC4788150.
- 21. Kudyba HM, Louzada J, Ljolje D, Kudyba KA, Muralidharan V, Oliveira-Ferreira J, et al. Field evaluation of malaria malachite green loop-mediated isothermal amplification in health posts in Roraima state, Brazil. Malaria journal. 2019;18(1):98. pmid:30909912; PubMed Central PMCID: PMC6434790.
- 22. Mohon AN, Getie S, Jahan N, Alam MS, Pillai DR. Ultrasensitive loop mediated isothermal amplification (US-LAMP) to detect malaria for elimination. Malaria journal. 2019;18(1):350–. pmid:31619258.
- 23. Barazorda KA, Salas CJ, Bishop DK, Lucchi N, Valdivia HO. Comparison of real time and malachite-green based loop-mediated isothermal amplification assays for the detection of Plasmodium vivax and P. falciparum. PLoS One. 2020;15(6):e0234263. pmid:32525900
- 24.
FIND E, HTD. Manual of Standard Operating Procedures for malaria LAMP. 2012.
- 25. Aninagyei E, Smith-Graham S, Boye A, Egyir-Yawson A, Acheampong DO. Evaluating 18s-rRNA LAMP and selective whole genome amplification (sWGA) assay in detecting asymptomatic Plasmodium falciparum infections in blood donors. Malaria journal. 2019;18(1):214. pmid:31234871; PubMed Central PMCID: PMC6591871.
- 26. Forshey BM, Guevara C, Laguna-Torres VA, Cespedes M, Vargas J, Gianella A, et al. Arboviral etiologies of acute febrile illnesses in Western South America, 2000–2007. PLoS neglected tropical diseases. 2010;4(8):e787–e. pmid:20706628.
- 27.
OMS. Bases del diagnostico microscópico del paludismo 1991;(2).
- 28. Cuadros J, Martin Ramirez A, Gonzalez 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):20. pmid:28061871; PubMed Central PMCID: PMC5219760.
- 29. Lucchi NW, Narayanan J, Karell MA, Xayavong M, Kariuki S, DaSilva AJ, et al. Molecular diagnosis of malaria by photo-induced electron transfer fluorogenic primers: PET-PCR. PloS one. 2013;8(2):e56677. pmid:23437209; PubMed Central PMCID: PMC3577666.
- 30. Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown KN. Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Molecular and biochemical parasitology. 1993;58(2):283–92. pmid:8479452.
- 31. Whiting P, Rutjes AW, Reitsma JB, Bossuyt PM, Kleijnen J. The development of QUADAS: a tool for the quality assessment of studies of diagnostic accuracy included in systematic reviews. BMC Med Res Methodol. 2003;3:25. Epub 2003/11/11. pmid:14606960; PubMed Central PMCID: PMC305345.
- 32. Cohen J. Weighted kappa: nominal scale agreement with provision for scaled disagreement or partial credit. Psychol Bull. 1968;70(4):213–20. Epub 1968/10/01. pmid:19673146.
- 33. Kobayashi T, Gamboa D, Ndiaye D, Cui L, Sutton PL, Vinetz JM. Malaria Diagnosis Across the International Centers of Excellence for Malaria Research: Platforms, Performance, and Standardization. Am J Trop Med Hyg. 2015;93(3 Suppl):99–109. pmid:26259937; PubMed Central PMCID: PMC4574279.
- 34. Koita OA, Doumbo OK, Ouattara A, Tall LK, Konare A, Diakite M, et al. False-negative rapid diagnostic tests for malaria and deletion of the histidine-rich repeat region of the hrp2 gene. Am J Trop Med Hyg. 2012;86(2):194–8. pmid:22302847; PubMed Central PMCID: PMC3269266.
- 35. Mendoza NM, Cucunuba ZM, Aponte S, Gonzalez NE, Bernal SD. [Field evaluation for diagnostic accuracy of the rapid test SD Bioline Malaria Antigen Pf/Pv(R) in Colombia]. Biomedica. 2013;33(4):587–97. pmid:24652214.
- 36. Douglas NM, Nosten F, Ashley EA, Phaiphun L, van Vugt M, Singhasivanon P, et al. Plasmodium vivax recurrence following falciparum and mixed species malaria: risk factors and effect of antimalarial kinetics. Clin Infect Dis. 2011;52(5):612–20. pmid:21292666; PubMed Central PMCID: PMC3060895.
- 37. Rovira-Vallbona E, Contreras-Mancilla JJ, Ramirez R, Guzman-Guzman M, Carrasco-Escobar G, Llanos-Cuentas A, et al. Predominance of asymptomatic and sub-microscopic infections characterizes the Plasmodium gametocyte reservoir in the Peruvian Amazon. PLoS Negl Trop Dis. 2017;11(7):e0005674. Epub 2017/07/04. pmid:28671944; PubMed Central PMCID: PMC5510906.
- 38. Sattabongkot J, Tsuboi T, Han ET, Bantuchai S, Buates S. Loop-mediated isothermal amplification assay for rapid diagnosis of malaria infections in an area of endemicity in Thailand. Journal of clinical microbiology. 2014;52(5):1471–7. pmid:24574279; PubMed Central PMCID: PMC3993686.
- 39. Tao ZY, Zhou HY, Xia H, Xu S, Zhu HW, Culleton RL, et al. Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection. Parasites & vectors. 2011;4:115. pmid:21693031; PubMed Central PMCID: PMC3127850.
- 40. Viana GMR, Silva-Flannery L, Lima Barbosa DR, Lucchi N, do Valle SCN, Farias S, et al. Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil. PloS one. 2018;13(7):e0200492. Epub 2018/07/12. pmid:29995953; PubMed Central PMCID: PMC6040774.
- 41. Sekikawa TAKAHIRO, KAWASAKI Y., KATAYAMA Y., & IWAHORI K. Suppression of Bst DNA polymerase inhibition by nonionic surfactants and its application for Cryptosporidium parvum DNA detection. Jpn. J. Water Treat. Japanese Journal of Water Treatment Biology 2008;44:203–8.
- 42. Zhu Q, Gao Y, Yu B, Ren H, Qiu L, Han S, et al. Self-priming compartmentalization digital LAMP for point-of-care. Lab on a chip. 2012;12(22):4755–63. pmid:22986619.
- 43. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors—occurrence, properties and removal. Journal of applied microbiology. 2012;113(5):1014–26. pmid:22747964.
- 44. Zhang Y, Yao Y, Du W, Wu K, Xu W, Lin M, et al. Development of loop-mediated isothermal amplification with Plasmodium falciparum unique genes for molecular diagnosis of human malaria. Pathog Glob Health. 2017;111(5):247–55. pmid:28683669; PubMed Central PMCID: PMC5560202.
- 45. Shehata N, Kohli M, Detsky A. The cost-effectiveness of screening blood donors for malaria by PCR. Transfusion. 2004;44(2):217–28. pmid:14962313.
- 46.
WHO. Strategic Advisory Group on Malaria Eradication (SAGme) 2020. Available from: https://www.who.int/news-room/detail/20-04-2020-who-advisory-body-releases-malaria-eradication-report.