Evaluation of the performance of advantage P.f. malaria Card® and advantage malaria Pan + Pf Card®, two rapid diagnostic

Study design and sites

This was a cross-sectional analytical study that took place from December 2019 to February 2020 in Togo. Malaria transmission is stable throughout the country, with two predominant climates: the sub-equatorial with two rainy seasons in the southern part of the country and the tropical with a single rainy season in the northern part. Thus, three sentinel sites for monitoring the effectiveness of ACTs used for the treatment of uncomplicated malaria were used for this evaluation. The Social Medical Center (SMC) of Cacaveli, a public health facility in Lomé, the capital of Togo, was the first site, to which the SMC “UTB Circulaire” was added because of the relatively low patient attendance at the site. The SMC Ahépé, a public health facility in the Maritime region, was the second site, located 66 km from Lomé, to which the hospital “la Providence de Kouvé” was also added. The last site was the Sokodé polyclinic located in the central region approximatively 340 km north of Lomé. The first two sites were sub-equatorial, located respectively in urban and rural areas, and the third site was tropical, located in a semi-urban area.

Study population and sampling

The study population was symptomatic patients suspected of having malaria who were seen in consultation at the different sentinel sites and for whom TTBS was prescribed. Since the sensitivity of RDTs varies according to the parasite density [9, 10] and can reach 100 parasites/μl, which may vary from one product to another [11], blood smear-positive subjects were divided into two groups, a low parasite density group (patients with asexual parasitemia count per microliter between 50 and 1000) and a high parasite density group (those with asexual parasitemia per microliter between 2000 and 10,000). The control group comprised subjects negative for any species of Plasmodium.

Sample size

The sample size calculation methods of Buderer et al. [12] were used. For calculation of sensibility and specificity, we used the formula Np = \(\frac{{{Z}_{a/2}}^{2}\mathrm{se}(1-se)}{{E}^{2}}\) to estimate the number of cases (positives) to include and the formula Nn = \(\frac{{{Z}_{a/2}}^{2}\mathrm{sp}*(1-sp)}{{E}^{2}}\) to estimate the number of controls (negatives). A 90% sensitivity was estimated for low parasitemia and 95% for high parasitemia with a tolerated margin of error (E) of 5% and an accepted risk of error (α) of 5% (Zα/2 at 1.96); the size of positives with low parasitemia for inclusion was 139, and the number of positives with high parasitemia for inclusion was 73. For specificity estimated to 90%, the number of included controls was 139. Therefore, this study should include a total sample size of 351 participants.

Inclusion and non-inclusion criteria

Inclusion criteria were considered by group. Included in the low parasitemia group were patients with asexual parasitemia count per microliter between 50 and 1000; in the high parasitemia group, those with asexual parasitemia per microliter between 2000 and 10000; and in the control group, individuals with negative thick blood smear [13,14,15]. Signed written consent was obtained from each adult patient and the parent/guardian of the children before their enrollment in the study. Any person who did not meet the above criteria and who voluntarily declined to participate in the study was not included in the study population.

Data collection

A structured questionnaire was used to collect information on sociodemographic characteristics, clinical signs presented, history of the disease, and existence of other diseases if applicable.

Laboratory tests

Each patient had a capillary blood sampling for a TTBS. After the microscopy results were known (having parasitemia within a certain range or being malaria negative), a second sample was taken from the included subjects to test the RDTs evaluated for Plasmodium spp. infection, and dried blood spots (DBS) were performed on Wattman type III paper.

Thick and thin blood smear

The thick and thin smears were made on the same slide. Two slides were made. After fixing the thin blood smear with methanol for a few seconds, the first slide was stained by Giemsa 10%, 10 to 15 min, for initial screening (having parasitemia within a certain range or being malaria negative), and the second at 3%, 45 min for detailed examination to obtain definitive results. [16]. After drying, the slides were then read under an immersion microscope with an 100× objective to determine the positivity and identify the plasmodial species and estimate the parasitemia.

Rapid diagnostic tests

Two types of Advantage brand RDTs were evaluated: Advantage P.f. Malaria Card^® (IR016025), which is specific for Plasmodium falciparum, and Advantage Malaria Pan + Pf Card^® (IR231025), which can detect P. falciparum, P. malariae, P. vivax, and P. ovale (J. Mitra & Co. Pvt. Ltd.). Both RDTs are based on the immunochromatographic technique: Advantage P.f. Malaria Card®, using a monoclonal anti-Pf HRP2 antibody, detects HRP2 antigen, specific for P. falciparum, and Advantage Malaria Pan + Pf Card^®, in addition to the P. falciparum-specific anti-Pf HRP2 monoclonal antibody, detects pLDH (plasmodium lactate dehydrogenase) antigen, which is common to all plasmodial species, using a Plasmodium-specific anti-Pan pLDH monoclonal antibody. Both RDTs were performed simultaneously in the laboratory by study staff for each enrolled subject according to the manufacturer’s instructions as well as the interpretation of the results. Four µicroliters of fresh blood from finger prick using the inverted up (by touching the base of the inverted cup into the blood drop) was immediately placed in the sample well, and then three drops of the assay buffer were added to the buffer well. The results were read at 20 min.

Real-time PCR assay

Plasmodium DNA was extracted by the heating method described by Miura et al. [17]; the Qiagen^® kit was used according to the manufacturer’s instructions to validate the heating extraction method. Real-time PCR was performed using primers, probes, and reaction conditions described by Shokoples et al. [18] and Divis et al. [19] with the following modification; the fluorophores for the P. falciparum probes were replaced with Cy5-BHQ-1 [20]. Two separate reactions were performed: (i) a screening reaction for the presence of Plasmodium species with Plasmodium genus-conserved primer pair (Plasmo1 and Plasmo2) and Plasprobe to detect a conserved region of the Plasmodium 18S ssu rRNA gene of all five human malaria parasites [21]; (ii) a monoplex PCR for the detection of P. falciparum using species-specific forward primer paired with Plasmo2 and species-specific probes [18]. Briefly, the screening and monoplex assays were performed with a final volume of 25 μl containing 5 μl template DNA, 12.5 μl QuantiFast Multiplex PCR master mix (Qiagen, Germany), and 7.5 μl pooled primer and probe mix. All assays were performed under standard conditions (1 cycle of 95°C for 5 min; 45 repeated cycles of 95°C for 30 s and 60°C for 30 s) with the CFX96 Real-time PCR machine (Bio-Rad, USA).

Quality control

Duplicated reading was done by two experienced microscopists for each TTBS [22]. If the coefficient of variation in parasite density estimate was > 5%, a third reading was performed by another independent microscopist [16]. If there was a difference between the study sites’ parasitemia and results found by the quality control, parasitemia of the quality control was considered. The estimated parasitemia was used to constitute the three groups.

RDT results are considered valid and interpreted only in the presence of the control line at the end of the test, following the manufacturer’s instructions.

A cutoff of 40 cycles was used to define PCR-positive samples. The test panel included several controls: (i) negative sample extraction as a negative control, (ii) β2-macroglobulin (β2M) target amplification (cycle threshold < 40) as a positive extraction control for the sample, and (iii) a positive reference control to detect any variation between runs and non-template control for each of the master mixes [20, 21].

Training of site team members and supervisors was conducted to standardize work methods and ensure the smooth running of the activity, especially for filling out questionnaires and conducting RDTs, TTBS, and filter paper sampling.

Endpoints

Thick/thin blood smear and PCR were considered the reference methods in this evaluation to which both RDTs were compared. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) are the estimated performance indicators of these two RDTs. Sensitivity and PPV were calculated for low and high parasitemia.

Data management and analysis

Data were recorded on register forms, entered in a Microsoft Excel database (Microsoft Corp, Redmond, Washington, USA), and analyzed using EpiInfoTM version 3.5.1 software. Sensitivity, specificity, and positive and negative predictive values of RDTs were determined using microscopy (or PCR), using 2 × 2 contingency tables. Exact 95% confidence intervals (95% CI) were calculated for each measure listed above.

Ethical considerations

The study protocol obtained ethical clearance from the Bioethics Committee for Health Research (CBRS) of Togo (no. 046/2019/CBRS of November 21, 2019) before its implementation. In addition, signed consent was obtained from adults and children’s parents/guardians. Any patient detected positive by at least one of the methods was referred to the clinicians at the sites for free management with an antimalarial drug available through the National Malaria Control Program.