Performance of the procedure for ultra-rapid extraction and loop-mediated isothermal amplification (PURE-LAMP) method to detect malaria in Haiti

Study sites

Based on data from the national malaria control program, three departments (main administrative divisions in Haiti) with the highest positivity rates in 2016 and early 2017 were selected as study sites. They were Nippes, Sud, and Grand’Anse in the southwestern part of the country (Fig. 1), with respective annual parasite indexes of 2.5, 4.4 and 22.3 in 2016 and 2.9, 7.6, and 18.4 in 2017 per 1000 population. Sud and Grand’Anse shared 63.6% of the malaria cases registered in 2016 in the country. Although a descending or at least stable incidence trend has been registered in the other 7 departments since 2015, the three chosen departments have shown an increasing or stable trend. Recruitment was carried out in small coastal cities with less than 50,000 inhabitants and which were located remotely from the capital cities of each department. At the start of the study, the sequels of hurricane Matthew that had hit 10 months earlier (October 2016) could still be spotted in a recovering environment.

Fig. 1figure 1

Location of the study area. Haiti is located in the Americas. The three departments (administrative divisions) of the study are highlighted in grey, and the others are shown in white. The number of participants by department, year and nested PCR diagnosis, is shown based on recruitment setting, hospital or community

Light microscopy is dictated as the gold standard for malaria diagnosis by the Haitian Ministry of Health; however, after allowing the alternative use of rapid diagnostic tests (RDT) in recent years, they are more widely used [4, 25, 26]. Besides the availability of these tests and malaria treatment free of charge for patients, vector control activities had been carried out under the national malaria control program.

Recruitment

People aged 1 year and over were recruited following two approaches: (i) hospital-based recruitment targeting participants with fever on the day of their hospital visit or anytime up to 2 weeks earlier, and (ii) community-based recruitment targeting the general population during social gatherings. There was no criterion for fever. Most social group participants were afebrile, but some people recruited there reported a history of fever.

Recruitment was carried out simultaneously at the same three health facilities, Centre de santé de Baradères (Nippes), Hôpital de Port-à-Piment (Sud) and Hôpital de la communauté de Dame-Marie (Grand’Anse), during the summers of 2017 (early August to early September) and 2018 (late July to late August). Community-based recruitment was carried out in Port-à-Piment, Port-Salut (Sud) and Petite Rivière de Dame-Marie. At the point of recruitment, participants had to submit a consent form, answers to a short questionnaire about demography, and their fever history and preventive practices, and venous blood samples were collected by a qualified staff member either in ethylenediaminetetraacetic acid (EDTA)- or heparin-containing blood tubes. After blood collection, the participants were tested immediately with an SD Bioline Malaria Ag Pf/Pan RDT (Standard Diagnostics, Inc., Suwon, the Republic of Korea) in accordance with the manufacturer’s guidelines, and blood smears were prepared and 100 μl of blood was seeded on filter paper (Whatman™ FTA™ classic cards; GE Healthcare, Tokyo, Japan) and air dried. After drying, each card was stored in a plastic bag containing a small desiccant bag. The participants were treated immediately if the RDT result was positive. Blood smears were fixed, stained and transported to the same laboratory along with the filter papers. The other tests (PURE-LAMP, PCR, microscopy) were conducted afterwards.

This study used venous blood to avoid double invasion of the participants as the majority seeking care were also predicted to require other blood tests. A small volume (100 μl) was used to prepare dried blood spots to approximate capillary blood sampling.

Microscopy

Two microscopists based at a Haitian reference laboratory visualized thick and thin Giemsa-stained smears using a × 1000 light microscope for each participant. Three hundred microscopic fields of the thick smear were visualized before declaring a negative sample. Parasite density was determined as percentage of parasitized red blood cells after observing about 5000 of them using the thin smear. Any positive was seen by a third microscopist who also assessed 10% of the negatives. The microscopists were blinded to the other test results of the participants.

PURE-LAMP

DNA was extracted with the Loopamp™ PURE DNA Extraction Kit from three dried blood spots with a diameter of 3 mm following the manufacturer’s guidelines and as reported elsewhere [16]. Reactions were carried in two tubes for all samples with the Loopamp™ MALARIA Pan/Pf Detection Kit, a Pan (targeting Plasmodium spp.) and a Pf (P. falciparum-specific) tube for each. Negative and positive controls (provided in the kit) were also included in each run.

The presence of amplified DNA inside the reaction tubes may be recognized in two speedy ways: (i) by fluorescence under UV excitation due to calcein unquenched during reactions, and (ii) by turbidity due to the precipitation of magnesium pyrophosphate [27]. In this study, only end-point evaluation by fluorescence under UV excitation was conducted for samples collected in heparin tubes giving a dichotomous positive or negative result; while real-time turbidity was measured with a Loopamp EXIA real-time turbidimeter (Eiken Chemical, Tokyo, Japan) for hospital samples collected in EDTA blood tubes, giving a turbidity threshold time (tt) for amplified samples. This was done to exclude any possibility of false positives due to EDTA chelating properties.

DNA extraction for PCR

DNA samples were extracted from three dried blood spots with a diameter of 3 mm using a Maxwell® RSC DNA FFPE Kit (Promega, Madison, WI, USA) and the automated Maxwell® RSC Instrument (Promega, Madison, WI, USA). Minor modifications to the manufacturer’s guidelines can be found in a previous report [28]. DNA was eluted in 50 μl of elution buffer and stored at 4 °C.

Nested PCR

The reference test in this study was a nested PCR system targeting the Plasmodium 18S ribosomal RNA gene [29]. This method generally implies a secondary step with species-specific primer sets for all human Plasmodium. However, secondary PCRs in this study were conducted only with a primer set specific to P. falciparum, which is the species expected in Haiti. For discrepant samples between PURE-LAMP ( +) and nested PCR (−), both tests were repeated, and the number of secondary PCR cycles was increased from 20 to 30. The nested PCR diagnosis was updated if a previously negative sample became positive at this step.

Statistical analysis

Assuming 5% positivity rate among febrile participants, PURE-LAMP sensitivity of 97%, specificity of 99% [16], at least 895 participants samples had to be analyzed to estimate the performance of PURE-LAMP with a marginal error of 5% and confidence level of 95%.

Data analysis was performed with Stata (StataCorp, TX, USA). Sensitivity, specificity, positive predictive value and negative predictive value with their respective 95% confidence intervals (CIs) and kappa statistics were estimated with nested PCR as the reference.

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