Study design
A cross-sectional multi-site study was conducted on individuals clinically suspected of having P. falciparum malaria seeking medical care at health facilities. The study was designed considering WHO guidelines for surveillance of hrp2/3 deletions published in 2018 [9]. Screening for P. falciparum infection was conducted using routine HRP2-based RDT in accordance with National Malaria Control Programme malaria diagnosis policy, together with light microscopy (LM) as a confirmatory test.
Study site
The study was conducted in four malaria endemic districts located along the border of Gia Lai (Krong Pa and Ia Pa districts) and Dak Lak (Ea Kar and Krong Nang districts) provinces, Central Highlands, Vietnam (Fig. 1). A total of 33 community health centres (CHC) were included: 21 in Gia Lai and 12 in Dak Lak. The area is characterized by a tropical climate and hilly geography with partially forested areas. The main occupation is agriculture in forest fields (maize, manioc, rice), which are often located far from villages of residence and require overnight stays in the forest. Malaria transmission mainly occurs in forested areas and is perennial with a peak between September and December and the lowest incidence from February to April. Both provinces have the highest burden of malaria in the country. In years 2018, 2019 and 2020, there were 946, 1804 and 530 malaria cases in Gia Lai and 733, 638 and 124 malaria cases in Dak Lak, respectively (National Institute of Malariology, Parasitology and Entomology, NIMPE). Plasmodium falciparum represented 70% of reported cases.
Sample size
Due to the absence of conclusive preliminary evidence on false-negative RDT prevalence or pfhrp2 deletions in the country, an initial sample size of 370 symptomatic P. falciparum patients per sampling domain was targeted as recommended in WHO guidelines [9]. This sample size would be adequate to demonstrate that a pfhrp2/3 deletion prevalence is below the 5% threshold for an expected population prevalence of 3.2% with 95% confidence. The four districts across the two provinces were considered as a single sampling domain.
Patients and enrolment procedures
Between June 2019 and June 2021 participants were recruited from individuals self-presenting at CHC with symptoms requiring a test for malaria, based on the criteria of the medical staff. Routine testing was conducted using SD Bioline Malaria Pf/Pv Antigen (Abbott) and LM, as further detailed below. Patients with confirmed P. falciparum diagnosis by LM and/or their parents/guardians were informed about the study objectives and invited to participate. Individuals with P. vivax malaria or other non-falciparum species by LM, including mixed infections, and those presenting signs or symptoms of severe malaria were excluded [12]. A clear explanation was provided in Vietnamese language and signed informed consent (or parental/guardian permission for those under 18 years of age and ≥ 12 months old and assent for those between 10 and 17 years of age) was obtained. For all consenting individuals, three dried blood spots (DBSs) containing 50 μl of blood each were obtained from a second finger prick, air-dried for 24 h and stored in silica gel containing bags, before being transferred to NIMPE (Hanoi, Vietnam) for molecular analysis. After sample collection, patients were treated for malaria as per Vietnam’s Ministry of Health (MoH) national guidelines, and asked to complete the short questionnaire on malaria symptoms and treatment, prevention habits and risk behaviour.
Light microscopy
All microscopy evaluations at point-of-care were conducted by expert microscopists (WHO Level 1 or equivalent). Blood slides were stained with Giemsa and examined at a magnification of 1000×. Parasite density was determined by counting the number of asexual parasites per 200 white blood cells (WBCs) with a hand tally counter. If more than 500 parasites were counted before reaching 200 WBCs, the count was stopped after completion of the field. Density was expressed as the number of asexual parasites per µl of blood, calculated by dividing the number of asexual parasites by the number of WBCs and corrected by the estimated WBCs density (typically 8000 per µl). A blood slide was considered negative when examination of 1000 WBCs revealed no asexual parasites. All blood films were stored in boxes and shipped to NIMPE (Hanoi, Vietnam) for double reading by WHO Level 1 microscopists to confirm results from the field and adjust the final diagnosis, if necessary. A third reading was conducted only in case of discrepancy (i.e., disagreement in species identification, in positive vs negative, or in parasite density estimation > 25%).
Quantitative PCR
DNA was extracted at NIMPE from 5 mm punches of DBS using QIAamp DNA 96 Blood kit (Qiagen), following manufacturer’s instructions and a final elution in 200 μl of water. A duplex quantitative real-time polymerase chain reaction (qPCR) targeting 18S ribosomal gene of P. falciparum and P. vivax was conducted to confirm species identified by LM [13]. Briefly, a 7.5 µl mix was prepared using primers and probes from QuantiNova Probe RT-PCR Kit (Qiagen) in a 7500 Real-Time PCR System (Applied Biosystems). Ct value > 40 was considered negative. Samples with no amplification in the Pf–Pv specific qPCR were checked for presence of other Plasmodium species using the generic QMAL qPCR assay [14].
Genotyping of pfhrp2 and pfhrp3
The regions covering exon 2 of both pfhrp2 and pfhrp3 genes were assessed by nested polymerase chain reaction (PCR) amplification using primers from Baker et al. [15] and HotStarTaq Plus Master Mix Kit (Qiagen), under the optimized cycling conditions described in Parr et al. [16]. DNA obtained from DBS of laboratory strains 3D7 (no deletions), Dd2 (pfhrp2-deletion; pfhrp3-wild type), HB3 (pfhrp2-wild type; pfhrp3-deletion) were used as positive controls (kindly provided by Pau Cisteró, ISGlobal, Barcelona). Nested PCR products were visualized in a 2% agarose gel and samples were considered positive if a band was observed in the 600-1000 bp range (in 3D7, expected products are 790 bp for pfhrp2 and 698 bp for pfhrp3). DNA samples testing negative for pfhrp2 and/or pfhrp3 were further analysed by nested PCR of P. falciparum glutamine-rich protein (pfglurp) [17]. Negative result in pfglurp confirmatory PCR was interpreted as low quality/insufficient DNA and invalidated pfhrp2 or pfhrp3 negative result as evidence of deletion. A random selection of ≈ 10% of the samples (n = 40) was shipped for external quality assessment of pfhrp2 and pfglurp nested PCRs at IMPE-Quy Nhon laboratory (Quy Nhon, Binh Dinh Province, Vietnam); results agreement was 100% (40/40) for both assays.
pfhrp2 sequencing
pfhrp2-exon2 PCR products were shipped to Genewiz (Suzhou, China) for DNA purification and bidirectional Sanger sequencing. Analysis was conducted in BioEdit 7.0.5. Quality of chromatograms of both forward and reverse sequences was inspected by eye, forward sequence and reverse complement of reverse sequence were aligned, and a consensus sequence was generated at overlapping regions. Nucleotide sequences were translated to amino acids and a multiple sequence alignment was performed using Clustal Omega in EMBL-EBI server (https://www.ebi.ac.uk/Tools/msa/clustalo/). pfhrp2sequence of the 3D7 strain (PF3D7_0831800) was included as reference.
The number and type of amino acid sequence repeats were identified based on the classification developed by Baker et al. [15]. The number of type 2 and type 7 repeats at each sequence was used to calculate type 2× type 7 score and classify sequences for their predicted sensitivity in RDT detection as type A (score > 100, “very sensitive”), type B (score 50–99; “sensitive”) or type C (< 50; “low or non-sensitive” group) [3, 15].
HRP2 levels by ELISA
The levels of HRP2 protein were measured in all RDT negative/LM positive samples and a random selection of RDT positive samples. Protein elution was conducted from 2 × 5 mm punch of DBS (equivalent to 11.2 μl of blood or 5.6 μl of plasma, [18]), with a final elution in 1120 μl of buffer (1/200) [19]. Quantitative ELISA-based detection was conducted using Quantimal™ CELISA Ultra-sensitive PfHRP2 Malaria (Cellabs, Australia) according to manufacturer’s instructions [20]. Samples were tested in duplicate. Optical density (OD) values were read in a spectrophotometer at 450 nm/620 nm, and cut-off was set at negative control OD + 0.1. HRP2 concentration in plasma was estimated as previously described [21]. HRP2 concentration was calculated by interpolating OD in the standard curve of recombinant HRP2 provided in the kit (0.01–10 ng/ml). Samples with positive OD but below the limit of quantification of final standards were given a value of 0.01 ng/ml. Results were adjusted for specimen pre-dilution and expressed as estimated concentration in plasma.
Definitions and statistical analysis
Prevalence of suspected false-negative HRP2-RDT results among symptomatic patients with P. falciparum malaria was determined from patient case report form data by dividing the number of patients testing RDT-negative and LM-positive by the total number of symptomatic P. falciparum cases. The prevalence of false-negative HRP2-RDT caused by hrp2/3 deletions was determined as the number of pfhrp2 or pfhrp3 deletions divided by the total number of symptomatic P. falciparum cases. Differences between point estimates across sociodemographic and parasitological variables were tested using chi-square or Fisher’s exact test. Mann–Whitney/Kruskal Wallis test was used to compare parasite densities between population groups.