Study design and setting

We used secondary data from a prospective health facility-based cross-sectional study conducted on individuals of all ages, seeking healthcare from October to December 2018 in 34 randomly selected health facilities of three health zones in the Kwilu Province (D.R. Congo), Fig.1.

The Kwilu Province is one of 26 provinces of D.R. Congo with an area of 79 906 km2. It is divided into five administrative territories: Bagata (including the city of Bandundu), Bulungu (including the city of Kikwit), Gungu, Idiofa, and Masimanimba [31].

The two selected cities (Bandundu and Kikwit) include three of the 24 health zones of the Kwilu Province [31]. They are the two main cities in the province and bear the highest burden of malaria. pfhrp2 gene deletions were previously reported in this region [14, 32].

Bandundu, the capital city of the Kwilu Province, is located 400km from Kinshasa, the capital of D.R. Congo [33]. Bandundu covers an area of 222 km2 with a population estimated at 950 683 as of 2015 [33]. It has a tropical wet and dry climate with two seasons. Heavy rainfalls and constant heat characterize the rainy season while fewer rainfalls are recorded during the dry season. The average annual temperature is 26.9C [33]. Bandundu City has one semi-urban health zone of the same name and 17 health areas, including 11 urban and six rural.

Kikwit is the second-largest city in Kwilu Province, located in the south-west of D.R. Congo, at 525km from Kinshasa and 400km from Bandundu. It is the main economic city of the province and a commercial hub that provides access to diamond-rich regions of Kasa Province and Angola. Kikwit covers an area of 92 km2 with an estimated population of 1 326 068 as of 2016 [34]. The city has a tropical wet and dry climate with a long rainy season from early September through to the end of May and a short dry season from early June to the end of August. Kikwit City has two urban health zones: Kikwit-Nord and Kikwit-Sud.

The study was approved by the Kwilu Province Division of Health (N ADM/707/01/DPS-KLU/CD/JPBK/632/2018), the Kinshasa School of Public Health Ethical Committee (ESP/CE/015/2019) and the School of Tropical Medicine and Global Health Ethical Review Committee.

The study was first explained to all participants, then written and verbal voluntary informed consent was obtained from all study participants including guardian/parents of non-adult participants.

The study population included individuals of all ages seeking health care in health facilities located in the three Health Zones of Bandundu (one) and Kikwit (two) cities. Health facilities included General Reference Hospitals, Reference Health Centres, and Health Centres. The smallest selection units were individuals attending these health facilities with symptoms suggestive of malaria. The study included all individuals seeking care in the selected health facilities with symptoms suggestive of malaria such as fever, headaches, malaise; during the study period for whom a laboratory test (PfHRP2-RDT and/or microscopic examination) was performed. Individuals who failed to meet the inclusion criteria or did not consent to participate in the study were excluded.

The minimum number of subjects required to enrol in this study was calculated based on a previously reported proportion of pfhrp2 gene deletion in the Kwilu Province (3%) and recommendations from WHO for studies on pfhrp2/3 deletion among symptomatic patients [14, 35]. According to the WHO protocol for estimating pfhrp2/3 deletion prevalence, for an expected prevalence of 3.2%, at least 370 individuals with P. falciparum infection are required per sampling domain [35]. In this study, the sampling domain was the Kwilu province, which included 34 health facilities. The study enrolled a total of 684 patients meeting the inclusion criteria of which 491 were positive for P. falciparum using PCR.

The primary study applied a two-stage random sampling to select health centres. At stage one, 27 health centres were randomly selected among the 62 health centres in the targeted areas. For neighbouring health centres, one health centre was randomly selected out of two. In order to increase the chance of catching individuals not respecting the referral system by directly seeking care in high-level health facilities, four reference health centres and three general reference hospitals from the three health zones were included, bringing the total number of selected health facilities to 34 (27 in Kikwit and seven in Bandundu).

At stage two, individuals attending the selected health facilities with symptoms indicative of malaria were recruited. The lead investigator weighed the number of individuals to recruit per health centre to the average rate of service utilization provided by the National Health Information System.

This study used four groups of variables: sociodemographic, malaria prevention, clinical and biological variables. Plasmodium falciparum HRP2 gene deletion (pfhrp2) was the main outcome variable. Explanatory variables were age, sex, health zones, household size, existence of mosquito breeding sites, LLIN (Long Lasting Insecticidal Net) ownership, use of LLIN, malaria drug intake, malaria clinical features, parasite density, and microscopy result.

Potential participants were introduced to the study by a research assistant. After securing consent/assent from the subjects or their guardians, socio-demographic, malaria prevention and treatment practices, and clinical variables were collected using a pre-tested structured questionnaire. Patients medical records were used to collect data from the physicians or health officers clinical examination.

Heel or finger-prick blood was collected from each individual. Samples for microscopy were prepared using two drops of blood. Then 50l of blood were applied on PfHRP2-RDT, and a few drops were spotted onto Whatman filter paper to prepare dried blood spots (DBS).

The membranes of spent PfHRP2-RDT cassettes and the DBSs were individually stored in plastic bags, sealed with a desiccant at room temperature before being shipped to the Institute of Tropical Medicine in Nagasaki (NEKKEN) where they were refrigerated at 4C.

The CareStart Malaria Pf (HRP2) Ag RDT (Access Bio, Inc., Somerset, New Jersey, USA) was used for the qualitative detection of malaria histidine-rich protein 2 in the whole blood according to the manufacturers instructions [ACCESSBIO, 2018, Somerset, New Jersey, USA]).

The test membrane strip is pre-coated with a P. falciparum HRP2 specific monoclonal antibody as a single line across the test strip. The reported panel detection score is 91.0% at 200 parasites/l with a false positive rate of 0.9% [36, 37]

A team of four medical technologists read the slides in the laboratories of health facilities where samples were collected. When a health facility did not have the necessary equipment to perform the examination, slides were read at the nearest laboratory possessing adequate equipment. For quality assurance, one expert microscopist randomly selected positive and negative slides to cross-check results. In the case results were not concordant, another reading was performed. About five percent of slides went through another quality control in the vector control laboratory of the Kinshasa School of Public Health.

Thick and thin smears were made on the same slide. The part of the slide containing the thin smear was fixed with methanol and dried. Then the whole slide was stained with 10% Giemsas solution for ten minutes and finally washed off with distilled water and air-dried. Stained smears were examined under a microscope for malaria parasite identification. For positive slides, malaria parasites were counted against 200 white blood cells (WBC), and parasite density was calculated based on a total of 8000 WBC/l using the following formula: (Number of Parasites counted8000)/Number of counted WBC.

Parasite density calculation was immediately performed when 100 parasites were counted against 200 WBC. However, in the case that fewer than 100 parasites were counted against 200 WBC, the count continued until 500 WBC.

Genomic DNA was extracted from membranes of spent PfHRP2-RDT cassettes and DBS using the QIAGEN QIAmpDNA extraction kit (company, city, country) according to the manufacturers instructions. We also adapted a previously described method to recover DNA from 197 spent RDTs membranes from Bandundu Health Zone [38].

To confirm P. falciparum infection, we designed specific primers targeting a 226 base pair region of the P. falciparum lactate dehydrogenase (pfldh) gene and performed a real-time PCR assay (Table 1). This assay was also used to ensure there was sufficient parasite DNA quantity in the samples to discriminate P. falciparum negative samples from samples with pfhrp2 gene deletion, as shown in Fig.2.

pfhrp2 gene deletion testing pipeline

Samples were duplicated and loaded in 96-wells plates along with serially diluted positive controls (gDNA from in vitro cultured P. falciparum strain 3D7) (1ng/l, 0.1ng/l, 0.01ng/l, 0.001ng/l), as well as negative controls containing DNA from blood spots prepared from known malaria negative individuals. We repeated the assay for all discordant duplicates and counted three consistent results as the final result.

For detection of the pfhrp2 gene, we performed a nested PCR assay using primers targeting a 228 base pair fragment spanning exon 1, the intron, and a portion of exon 2 of pfhrp2 as previously described (Table 1) [9]. We used a lower elongation temperature (68C) to improve PCR sensitivity, pfhrp2 being AT-rich, and increased the number of cycles to 40. We used genomic DNA from Dd2 (pfhrp2 negative) and 3D7 (pfhrp2 positive) as controls for all assays.

We repeated the nested PCR for all negative results. In the case of discordant results, we performed the amplification a third time and counted two consistent results as the final result.

Reaction components for both real-time and nested PCR are summarized in Table 1.

PCR amplicons were separated by electrophoresis on a 2% agarose gel stained with Gel Red Nucleic Acid Stain 10 000in water. A total of 12l of PCR amplicons (6l) and loading dye (6l) were loaded onto the gel, which was run for 35min at 100V and observed under UV light. A 500g/ml Gene Ruler 100bp DNA Ladder (BioLabsinc, Ipswich, USA) was loaded onto the same gel to determine the sizes of the resolved fragments.

Data were entered and analyzed using STATA15 (StataCorp LLC, Lakeway, Texas, USA). Tables have been used to describe categorical variables. Continuous variables have been summarized using median and interquartile ranges. Proportions have been used to summarize categorical variables.

Fischers exact test (for categorical variables) and the KruskalWallis test (for non-normally distributed continuous variables) were applied to look for associations between potential explanatory variables and the main outcome (pfhrp2 gene deletion). We computed the 95% Confidence Interval (95% CI) for the prevalence of pfhrp2 gene deletion. We considered a P-value less than 0.05 statistically significant.

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