No evidence of false-negative Plasmodium falciparum rapid diagnostic results in Monrovia, Liberia

The study was conducted at the Outpatients Department, Emergency and Antenatal Consultation of the not-for-profit Saint Joseph’s Catholic Hospital (SJCH) in Congo Town neighbourhood, Monrovia. The SJCH provides general services to the population in Monrovia. Although the SJCH applies a cost recovery system for the general public, the institution has a charity arm to subsidize healthcare-related costs for the most deprived.

In the time-period 21 March to 21 July 2019, all patients who presented at the facilities with fever (temperature ≥ 37.5 °C) or history of fever during the preceding week, as well as pregnant women attending ANC for the first time during their pregnancy (irrespective of their fever status), were eligible for inclusion in the study. No individuals meeting inclusion criteria were excluded based on their race, social or economic status, religion, ethnic affiliation, nationality, political affiliation, or sexual orientation.

Eligible patients were invited to participate and informed of the study objectives and specimen collection procedures. After providing written informed consent, they were queried on basic socio-demographic and malaria prevention-related data. Their forehead temperature was measured with an infrared thermometer. For the participants attending their first ANC, the gestational age was assessed by date of last menstrual period and by measurement of fundal height. Data were manually captured by the recruiting research team using individual standardized paper-based case report forms.

Participants were finger pricked for malaria testing. Five μl of blood were used to perform malaria testing using Paracheck Pf® (Orchid Biomedical Systems, Goa, India), a PfHRP2-based malaria RDT. Another 5 μl of blood was used to prepare a thick blood film for malaria parasite microscopy examination. If present, malaria parasites were detected and semi-quantified through the microscopic examination of Field’s stained thick blood film. A total of 9 technicians were in charge of the microscopic diagnosis. The diagnosis was made by a single technician without double confirmation. In case of any doubt, the expert opinion of the laboratory manager was used for a final decision. A 50-μl blood drop was spotted on Whatman 903 filter papers, dried for 24 h and stored in plastic bags with silica gel at -20ºC. One of the prepared filter papers was shipped to the Barcelona Institute of Global Health (Barcelona, Spain) for molecular detection of P. falciparum. All the samples with microscopy-, and RDT-discrepant results, plus a 25% random selection of the rest of samples, were selected for molecular assessment. DNA was extracted from filter papers following the Chelex method [21] and used for quantitative real-time PCR targeting P. falciparum 18S ribosomal RNA (rRNA) gene [21, 22]. In brief, purified DNA templates were amplified in an ABI PRISM 7500 Real-Time System (Applied Biosystems) following a previously described method [13]. Briefly, a 20-μl PCR mixture was performed using 5 μl of template, 10 μl of 2 × TaqMan® Universal PCR Master Mix (Applied Biosystems), a 300-nM concentration of each primers specific for 18S rRNA gene of P. falciparum, and a 150-nM concentration of probe labelled with 6-carboxy-fluorescein (FAM) as a reporter and 6-carboxytetramethylrhodamine (TAMRA) as a quencher. Amplification and detection were performed under the following conditions: 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The results were automatically analysed by the ABI Prism SDS2.0.6 software. Each specimen was run in duplicate. Parasitaemia was calculated by extrapolation against a standard curve of 5 serially diluted points prepared with known numbers of 3D7 ring-infected erythrocytes [23]. Samples without amplification (no Ct detected) were considered negative. A negative control (uninfected erythrocytes) and a blank control (no template) were run in all reactions.

Samples positive by microscopy and negative by the 18S P. falciparum qPCR were analysed by qPCR for Plasmodium spp using one set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium [24]. Amplification and detection of the amplified product were performed on an ABI Prism 7500 (Applied Biosystems). A negative control (uninfected erythrocytes) and a blank control (no template) as well as 4 positive controls (one for P. falciparum, P. vivax, P. ovale, and P. malariae) were included. The sample was considered positive by identifying the threshold cycle number (Ct) at which normalized reporter dye emission raised above background noise. If the fluorescent signal did not increase within 40 cycles (Ct = 40), the sample was considered negative.

Samples that were positive by 18S rRNA qPCR but negative by PfHRP2-RDT were subjected to multiplex qPCR assay for detection of pfhrp2 and pfhrp3 following previously described method [25] with minor modifications. In brief, an ABI 7500 real-time machine was used, with the following QSY probes (Applied Biosystems): Pfhrp2_probe 6FAM 5′-ATGCAAAAGGACTTAATTTAAATAAGAGATT-3′; Pfhrp3_probe VIC 5′-ACAATTCCCATACTTTACATCATGCA-3′; Pfldh_probe ABY 5′-GTAATAGTAACAGCTGGATTTACCAAGGCCCCA-3′; HumanTuBB_P JUN 5′-TTAACGTGCAGAACAAGAACAGCAGCT-3′.

To optimize the final reaction, pfhrp2 primers and probe were increased to 900 nM and 250 nM, respectively. TaqMan® Multiplex Master Mix was used. The thermocycling conditions were 20 s at 95 °C, followed by 45 cycles of 3 s at 95 °C and 33 s at 60 °C. Samples with HumTuBB positive and pfldh positive but negative for pfhrp2 or pfhrp3 were considered as pfhrp2-deleted and pfhrp3-deleted, respectively. Samples with HumTuBB positive but pfldh negative were defined as having a parasitaemia too low to be detected by this method. Finally, samples with a HumTuBB negative result were considered to be invalid and indicated the need to repeat the DNA extraction and/or PCR experiment.

The study participants were assigned with a sequential unique identification number (UIN) that linked the signed consent forms to the case report forms. The case report forms did not include personal identifiers and were used to collect socio-demographic and malaria care-related data. The laboratory technologists were oriented to document all laboratory test results and report both the blood film and RDT results in standard reporting forms. All information contained in the case report forms was double-captured (by a trained laboratory technician and by a member of the research team) into a research database built in Microsoft Excel. The point-of-access to the Excel spreadsheets were designed to protect the confidentiality and integrity of the data and included authorization, authentication, auditing, and availability features to safeguard the access and usage of the data. The Excel spreadsheets were in a password-protected computer sited at the SJCH laboratory.

Data entered into Excel was converted to STATA (version 8.0, STATA Corporation, College Station, TX, USA) for further analyses. The sensitivity, specificity, false negative, and false positive values of Paracheck Pf® were calculated using microscopy as the gold standard. Briefly, sensitivity was calculated as the proportion of positive test results against true positives; specificity was calculated as a proportion of negative test results against true negatives. Negative RDT results were considered false-negatives if microscopy result was positive. Positive RDTs were considered false-positives if microscopy was negative. True false-negatives and false-positives were considered if the qPCR was positive and negative, respectively. Proportions were compared using Chi2 test and differences with a probability of less than 0.05 (P < 0.05) were accepted as significant.

One-thousand and forty participants meeting inclusion criteria were invited to participate between 21 March and 21 July, 2019, all of whom consented. Of the 1040 enrolled participants, two discontinued their participation before blood specimen collection. The analysis presented is based on data from 1038 participants (951 febrile individuals and 87 pregnant women) with peripheral blood samples available and analysed for P. falciparum infection. From the 951 febrile participants (Table 1), 459 were males (48%), 330 (35%) under 5 years of age, 387 (41%) above 20 years, and 541 (57%) reported primary education or below. Use of insecticide-treated nets and household indoor residual spraying was reported by 441 (46%) and 532 (56%) of the study participants, respectively. Three-hundred and sixty-five (38%) presented with fever and 725 (76%) reported fever during the preceding week. Malaria signs and symptoms ranged from 13% in the case of diarrhoea (n = 119) to 44% in the case of headache (n = 420). Seven-hundred and six of the study participants (74%) reported history of a previous malaria episode.

Out of the 87 women at first ANC visit, 4 (5%) and 7 (8%) had fever or reported any sign/symptom of malaria at their first ANC visit, respectively (Table 2).

One-hundred and eighty-six (19.6%) and 200 (21.0%) of the 951 febrile participants had a P. falciparum-positive result by RDT and microscopy, respectively (Table 1). Positivity rate increased with age and with the reporting of joint pain, chills and shivers, headache, vomiting and weakness. In contrast, it was lower in patients with cough and nausea compared to those with other signs of malaria. The positivity rate among pregnant women at first ANC visit was 5.7% (5 out of 87 women) and 8.0% (7 out of 87) by RDT and microscopy, respectively (Table 2). None of the clinical variables tested were associated with positivity by RDT, microscopy or qPCR.

Compared to microscopy (Table 3), RDT sensitivity was 89% (185/207), with a false negativity rate of 11% (22/207). RDT specificity was 99% (825/831) and the false positivity rate was < 1% (6/831). Among the 28 samples with discordant RDT and microscopy results, 21 (5 RDT-positive but microscopy-negative, and 16 RDT-negative but microscopy-positive) were available for molecular analysis to screen for P. falciparum DNA using qPCR. Three (60%) of the 5 samples found to be positive by RDT but negative by microscopy were negative by qPCR, while 2 (40%) of them were positive by qPCR. The 16 (100%) samples, which were positive by microscopy but negative by RDT, were also negative by qPCR. Among the 22 samples positive by microscopy and negative by the 18S P. falciparum qPCR, only one was positive for Plasmodium spp. (Ct = 39.8) [24]. Among the randomly selected 224 samples that were negative by RDT and microscopy, 14 (6.2%) were confirmed positive by qPCR. Plasmodium falciparum densities (as quantified by qPCR) were higher among RDT-positive infections (n = 72, geometric mean: 767.9 parasites/μL; SD: 2,938.2) than RDT-negative infections (n = 14, 2.1, SD: 2.9; p < 0.001). The 14 samples were tested for the presence of hrp2/3 deletions using a multiplex qPCR assay that targets pfhrp2, pfhrp3, pfldh_ HumanTuBB_ [25]. The 14 samples were positive for the human gene (HumTuBB), but only 3 amplified pfldh. Among these 3 samples with enough parasitaemias for the detection of single copy parasite genes (pfpldh), all were positive for hrp2 and hrp3.