Prevalence of Plasmodium falciparum Isolates Lacking the Histidine Rich Protein 2 Gene Among Symptomatic Malaria Patients in Kwilu Province, DR. Congo.

Prevalence of Plasmodium falciparum Isolates Lacking the Histidine Rich Protein 2 Gene Among Symptomatic Malaria Patients in Kwilu Province, DR. 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Congo. Yannick Bazitama Munyeku, Alain Abera Musaka, Medard Ernest, Chris Smith, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-79327/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Malaria rapid diagnostic tests have become a primary and critical tool for malaria diagnosis in malaria-endemic countries where Pf HRP2-based RDTs are widely used. However, in the last decade, the accuracy of Pf HRP2-based RDTs has been challenged by the emergence of P. falciparum strains harbouring deletions of the pfhrp2 gene, resulting in false-negative results. In DR. Congo, little is known about the prevalence of the pfhrp2 gene deletion among P. falciparum isolates infecting symptomatic patients, especially in low to moderate transmission areas where pfhrp2 deletion parasites are assumed to emerge and spread. Here we determine the local prevalence and factors associated with pfhrp2 gene deletions among symptomatic malaria patients in the Kwilu province of the Democratic Republic of Congo, a low to moderate malaria transmission area. Methods We used secondary data from a prospective health facility-based cross-sectional study conducted on 684 individuals of all ages, seeking healthcare with symptoms suggestive of malaria from October to December 2018 in 34 randomly selected health facilities. Sociodemographic, malaria prevention and treatment practices, and clinical variables were collected using a pre-tested structured questionnaire. Patients’ medical records were used to collect additional clinical data. Blood was collected for microscopy, Pf HRP2-RDT, and spotted onto Whatman filter paper for downstream genetic analysis. Genomic DNA was extracted and used to perform PCR assays for the detection and confirmation of pf hrp2 gene deletions. Data were entered and analysed using STATA15. Fischer’s exact and the Kruskal-Wallis tests were applied to look for associations between exposures and the pf hrp2 gene deletion with a level of statistical significance set at p < 0.05. Results The overall prevalence of the pf hrp2 gene deletion was 9.2% (95% CI 6.7% – 12.1%). The deletion of the pfhrp2 gene was associated with health zone of origin (p=0.012) and age (p=0.019). Among false-negative Pf HRP2-RDT results, only 9.9% were due to pfhrp2 gene deletion. Conclusion P. falciparum isolates with pfhrp2 gene deletions are relatively common among symptomatic patients in Kwilu province. Further investigations are needed to provide enough evidence for policy change. Meanwhile, the use of RDTs targeting Pf HRP2 and pLDH antigens could limit the spread of deleted isolates. Infectious Diseases Plasmodium falciparum Histidine Rich Protein 2 gene deletion false negative Rapid Diagnostic Test symptomatic patients Figures Figure 1 Figure 2 Background Malaria remains a global health issue despite progress over the last decade. In 2018, there were an estimated 228 million new malaria cases, including 405,000 deaths ( 1 ). Ninety-two percent of malaria cases and 93% of malaria deaths occurred in Africa. Fifteen countries in sub-Saharan Africa and India carried nearly 80% of the global malaria burden of which Nigeria and the Democratic Republic of the Congo accounted for about 35% ( 1 ). The Democratic Republic of Congo (DR. Congo) accounts for 12% of all malaria cases in sub-Saharan Africa ( 1 ). In DR. Congo, malaria is the leading cause of morbidity and mortality, accounting for more than 40% of all outpatient visits, and for 19% of deaths among under five years children ( 2 ). The high burden of malaria in DR. Congo can be explained by the fact that nearly the entire population (97%) lives in high-transmission zones where the most common vector encountered is Anopheles gambiae , and Plasmodium falciparum is the most common species responsible for the majority of severe cases ( 2 , 3 ). An important component of malaria control and elimination is appropriate case management, which is based on early and accurate diagnosis. Accurate diagnosis facilitates appropriate and prompt treatment and minimizes the risk of developing drug resistance. The WHO recommends microscopic examination as the gold standard for malaria diagnosis. However, in rural and semi-urban settings where lack of equipment, reagents, trained and skilled personnel, and electricity can prevent this diagnosis method, the use of rapid diagnostic tests (RDTs) offers an alternative for quick and accurate diagnosis ( 4 , 5 ). RDTs have become a primary and critical tool for malaria diagnosis in the D.R. Congo as well as in Malaria endemic countries. They accounted for nearly 75% of diagnostic testing among suspected cases in Africa in 2017 ( 1 ). Current RDT kits are designed to detect either P. falciparum alone or in combination with other species of human malaria parasites. Three main antigens are detected by malaria RDTs, namely P. falciparum Histidine Rich Protein 2 ( Pf HRP2), parasite lactate dehydrogenase (pLDH), and parasite aldolase (pAldo) ( 6 , 7 ). Pf HRP2, a P. falciparum specific antigen, has the advantage of being highly abundant and heat-stable. Pf HRP2-based RDTs can lead to false-positive results in the case of persistent circulating HRP2 antigen as a result of antimalarial treatment, and false-negative results in individuals whose levels of parasitaemia is under the detection threshold of 200 parasites/µL ( 6 , 7 ). In the last decade, however, some studies have reported false-negative results among individuals infected with P. falciparum parasites presenting a deletion of the P. falciparum histidine-rich protein 2 (pf hrp 2 ) gene. The majority of these studies have also identified co-existing deletions of the P. falciparum histidine-rich protein 3 ( pf hrp 3 ) gene, which produces an antigen that shows some cross-reactivity with HRP2 ( 4 , 5 , 8 – 30 ). While the WHO recommends not to initiate antimalarial treatment without biological evidence, evolutionary selection of P. falciparum isolates with pfhrp2 gene deletions could occur since only positive tested patients will be treated. The non-targeted patients infected by parasites harbouring pfhrp2 gene deletions will facilitate the spread of phrp2 deleted strains, jeopardizing progress towards disease control and elimination in low setting countries. To date, only one study has investigated pf hrp 2 -deleted mutant parasites in DR. Congo, reporting a country-wide prevalence of 6.4% among children under-five years and providing spatial distribution and population genetics of these deletions ( 14 ). However, this nationwide study could not explore clinical differences between pf hrp 2 -deleted and wild type P. falciparum malaria due to limited clinical data and study population (the majority being asymptomatic and under-five), nor was it able to conclude about the relative virulence of pf hrp2-deleted parasites. In order to address the above limitations, we selected Kwilu province which is classified by the DR. Congo National Malaria Control Program (NMCP) as a province at high risk of malaria ( 3 ). Kwilu province is classified in the tropical facies where malaria transmission occurs predominantly during the long rainy season lasting 5 to 8 months, and where the number of infected bites per people per year ranges from 60–400 ( 3 ). Using data from a prospective health facility-based cross-sectional study, we aimed to determine the local prevalence of the pfhrp2 gene deletion among malaria symptomatic patients, and associated clinical, biological, and sociodemographic factors in the Kwilu Province (DR Congo). The aim of this study is to contribute to a better characterization of the prevalence and consequences of pfhrp2 deletions in DR. Congo by providing relevant regional data to improve malaria management and control. Methods Study design and setting This study was aimed at determining the local prevalence and factors associated with pfhrp2 gene deletions. 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 (DR Congo). The Kwilu Province is one of 26 provinces of DR. Congo with an area of 79,906 km 2 . 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 400 km from Kinshasa, the capital of DR. Congo ( 33 ). Bandundu covers an area of 222 km 2 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 26.9 °C ( 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 the Kwilu province, located in the south-west of DR. Congo, at 525 km from Kinshasa and 400 km from Bandundu (Fig. 1). It is the main economic city of the province and a commercial hub that provides access to diamond-rich regions of Kasaï province and Angola. Kikwit covers an area of 92 km 2 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. Ethics, consent, and permissions The study was approved by the Kwilu Province Division of Health, the Kinshasa School of Public Health Ethical Committee, 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. Study population. 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 ( Pf HRP2-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. Sample Size Calculation 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 , 36 ). 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 ( 36 ). 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 . Recruitment method 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 leadinvestigator weighed the number of individuals to recruit per health centre to the average rate of service utilization provided by the National Health Information System. Variables This study used four groups of variables: sociodemographic, malaria prevention, clinical and biological variables. Plasmodium falciparum HRP2 gene deletion ( pfhrp2 ) was the primary outcome variable. Exposure variables were age, sex, health zones, household size, existence of mosquito breeding sites, LLIN ownership, use of LLIN, malaria drug intake, malaria clinical features, parasite density, and microscopy result. Data collection method 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 physician’s or health officer’s clinical examination. Heel or finger-prick blood was collected from each individual. Samples for microscopy were prepared using two drops of blood. Then 50 microliters of blood were applied on Pf HRP2-RDT, and a few drops were spotted onto Whatman filter paper to prepare dried blood spots (DBS). The membranes of spent Pf HRP2-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 4 °C. Malaria RDT screening The CareStart™ Malaria Pf (HRP2) Ag RDT manufactured by Access Bio, Inc., was used for the qualitative detection of malaria histidine-rich protein 2 in the whole blood according to the manufacturer’s instructions (ACCESSBIO, 2018). 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/µl with a false positive rate of 0.9% ( 38 , 39 ) Microscopic diagnosis of malaria 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. Some 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% Giemsa’s 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 8,000 WBC/µL using the following formula: (Number of Parasites counted X 8,000)/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. Extraction of parasite DNA Genomic DNA was extracted from membranes of spent Pf HRP2-RDT cassettes and DBS using the QIAGEN QIAmp®DNA extraction kit according to the manufacturer’s instructions. We also adapted a previously described method to recover DNA from spent RDTs membranes ( 40 ). Detection of P. falciparum infection & pfhrp2 gene deletion 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. This assay was also used to ensure there was sufficient parasite DNA quantity and quality in the samples to discriminate P. falciparum negative samples from samples with pfhrp2 gene deletion, as shown in Fig. 2 . Samples were duplicated and loaded in 96-wells plates along with serially diluted positive controls (1 ng/µl, 0.1 ng/µl, 0.01 ng/µl, 0.001 ng/µl), as well as negative controls containing DNA from blood spots prepared from known malaria negative individuals. We repeated the assay for all discordant duplicates. 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 ( 9 ). We used a lower elongation temperature (68°C) 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 . Table 1 Primer Sequences and PCR conditions for pfhrp2 and pfldh genes amplification Targeted genes Primer sequences (5’ − 3’) Reaction components Cycling conditions LOD (ng/µL) pfhrp2 Exon 1–2, PF3D7_0831800 Outer For: GGTTTCCTTCTCAAAAAATAAAG Rev: TCTACATGTGCTTGAGTTTCG - One Taq 2X Master Mix with standard buffer: 12.5 µL - 10 µM forward primer: 1 µL - 10 µM reverse primer: 1 µL - Nuclease free water: 7.5 µL - DNA template: 3 µL (gDNA or 5X diluted outer PCR product 25 µL reaction volume 95 °C/5 min; 40 cycles of 95 °C/30sec, 55 °C/30sec, 68 °C/30sec 68 °C/5 min 4 °C - ∞ 10 − 5 Inner For: GTATTATCCGCTGCCGTTTTTGCC Rev: CTACACAAGTTATTATTAAATGCGGAA 95 °C/5 min; 40 cycles of 95 °C/30sec, 62 °C/30sec, 68 °C/30sec 68 °C/5 min 4 °C - ∞ pfldh (qPCR) For: ACGATTTGGCTGGAGCAG Rev: GGAACACCTGAATGTTGATG - PowerUp™ SYBR TM Green Master Mix (2X): 12.5 µL - 10 µM forward primer: 0.5 µL - 10 µM reverse primer: 0.5 µL - Nuclease free water: 6.5 µL - DNA template: 2–4 µL 22–24 µL reaction volume 50 °C/2 min; 95 °C/2 min 45 cycles of 95 °C/15sec, 62 °C/1 min, 95 °C/30sec, 60 °C/15sec 10 − 4 LOD: Lower limit of detection, qPCR: quantitative or Real-time PCR PCR product resolution by agarose gel electrophoresis PCR amplicons were separated by electrophoresis on a 2% agarose gel stained with Gel Red® Nucleic Acid Stain 10,000X in water. A total of 12 µL of PCR amplicons (6 µL) and loading dye (6 µL) were loaded onto the gel, which was run for 35 min at 100 V and observed under UV light. A 500 µg/ml Gene Ruler 100 bp DNA Ladder (BioLabs®inc) was loaded onto the same gel to determine the sizes of the resolved fragments. Statistical Analyses Data were entered and analyzed using STATA15. 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. Fischer’s exact test (for categorical variables) and the Kruskal-Wallis test (for non-normally distributed continuous variables) were applied to look for associations between exposures and the primary outcome ( pf hrp2 gene deletion). We computed the 95% CI for the prevalence of pfhrp2 gene deletion. We considered a p-value less than 0.05 statistically significant. Results Socio-demographic characteristics Of the 684 symptomatic patients who participated in the study; 391 (57.7%) were female, and 287 (42.3%) were male. Kikwit-Nord Health Zone accounted for the majority of patients 362 (52.9%) while Bandundu and Kikwit-Sud Health Zones accounted for 197 (28.8%) and 125 (18.3%) patients, respectively. The median age was nine years, with an interquartile range from 3 to 26 years old. Children under five years old represented 36% of enrolled patients. The median size of household was six, with an interquartile range from five to six. Table 2 summarizes socio-demographic characteristics. Household environment, malaria prevention, and treatment practices The majority (87.7%) of patients/patients’ guardian reported the presence of mosquito breeding sites near their household. Two-thirds of household (64.2%) owned a mosquito bednet, while 49.7% of patients spent the night before the interview under a mosquito bed net. One-third (220) of patients reported prior malaria drug intake. Quinine (13%), Sulfadoxine-Pyrimethamine (6.1%), Artemether-Lumefantrine (4.8%), Artesunate (2.0%), Arteether (1.9%), Artemether (1.8%) were the common drugs taken as illustrated in Table 2 . Table 2 Socio-demographic characteristics, household environment and malaria prevention and treatment practices Characteristic n % Median IQR Sex (n = 678) Female 391 57.7 Male 287 42.3 Health Zone (n = 684) Bandundu 197 28.8 Kikwit-Nord 362 52.9 Kikwit-Sud 125 18.3 Age in years ( n = 680) 9 3–26 5 years 435 64.0 Household Size (n = 684) 6 5–8 Household environment (n = 684) Mosquito Breeding Sites 600 87.7 LLIN ownership 439 64.2 LLIN utilisation 340 49.7 Malaria prevention and treatment practices Prior drug intake (n = 684) 220 32.2 Malaria Drug taken (n = 220) Quinine 89 13.0 Sulfadoxine-Pyrimethamine 42 6.1 Artemether-Lumefantrine 33 4.8 Artesunate 14 2.0 Arteether 13 1.9 Artemether 12 1.8 Arteether-Artemotil 10 1.5 Amodiaquine 4 0.6 Artesunate-Sulfadoxine-Pyrimethamine 2 0.3 Plant extract 1 0.1 For the variable ‘sex’, less than 1% information is missing (six entries). For the variable age, less than 1% information is missing (four entries) Malaria drug taken includes only individuals who reported malaria drug intake at least one week before the survey Clinical and biological features of symptomatic patients At admission, the body temperature of patients ranged from 37 °C to 38 °C (median temperature of 37.5 °C). The common findings of the clinical examination were: A history of fever (81.6%), headaches (41.8%), vomiting (31.4%), rigor (22.8%), fatigue (22.1%) and abdominal pain (20.2%). Among patients with a microscopy positive result, malaria parasite density ranged from 64 parasites /µL to 7200 parasites /µL with a median parasite density of 880 parasites /µL. Table 3 summarizes the distribution of clinical and biological features. Table 3 Clinical and biological features of symptomatic patients Characteristic n % Median IQR Clinical features (n = 684) History of fever 558 81.6 Headache 286 41.8 Vomiting 215 31.4 Rigor 156 22.8 Fatigue 151 22.1 Abdominal pain 138 20.2 Joint Pain 58 8.5 Anorexia 42 6.1 Neckache 34 5.0 Diarrhoea 32 4.7 Pallor 13 1.9 Convulsions 12 1.8 Splenomegaly 5 0.7 Impaired consciousness 3 0.4 Temperature at admission (°C) 37.5 37–38 Biological features ( n = 235) Parasite density (parasites /µL) 880 64–7200 Parasite density accounts for only microscopy positive samples Proportion of pf hrp2 gene deletion in false-negative Pf HRP2-RDT There were 131 false-negative Pf HRP2-RDT results of which only 9.9% could be explained by the pfhrp2 gene deletion, with a 95% CI ranging from 5.4–16.4%. Prevalence of pfhrp2 gene deletion among all symptomatic Pf PCR confirmed cases The overall prevalence of pfhrp2 gene deletion among Pf PCR confirmed symptomatic cases was 9.2%. The highest prevalence was found in Bandundu Health Zone (15.7%), followed by Kikwit-Sud Heath Zone (9.6%) and Kikwit-Nord Health (6.2%). Table 5 shows the distribution of pf hrp2 gene deletion among Pf PCR confirmed cases across Heath Zones. Table 4 Frequency of pfhrp2 gene deletion among all symptomatic Pf PCR confirmed cases Health Zones Subjects P. falciparum infection pf hrp2 gene deletion n Prevalence (95% CI) n Prevalence (95% CI) Overall 684 491 71.8 (68.2–75.1) 45 9.2 (6.7–12.1) Bandundu 197 121 61.4 (54.2–68.2) 19 15.7 (9.7–23.4) Kikwit-Nord 362 276 76.2 (71.5–80.5) 17 6.2 (3.6–9.7) Kikwit-Sud 125 94 75.2 (66.7–82.5) 9 9.6 (4.5–17.4) Socio-demographic, malaria prevention, biological features, and pf hrp2 gene deletion As shown in Table 5 , there was no statistically significant difference in pfhrp2 gene deletion status between males and females (8.5% vs. 9.8%). Conversely, there was a statistically significant difference in pf hrp2 gene deletions status in Bandundu health zone compared to Kikwit-Nord and Kikwit-Sud health zones (p = 0.012). There was a trend towards pfhrp2 gene deletion in older compared to younger patients, but this was not statistically significant (p = 0.079). However, when analyzing age as a continuous variable, the median age among pfhrp2 gene deleted patients was higher than the median age among pfhrp2 gene non-deleted patients (18 years versus 7 years). The Kruskal Wallis test showed strong evidence suggesting that the distributions of age differed by pf hrp2 gene deletion status (p = 0.019). Similarly, there was a trend towards pfhrp2 gene deletion among negative microscopy results, but weak evidence supported this finding (p = 0.079). Clinical features and pfhrp2 gene deletion We found more malaria signs and symptoms among patients infected with parasites not harbouring pfhrp2 gene deletion. However, the difference was not statistically significant (Table 5 ). Table 5 Socio-demographic, malaria prevention, biological features and pf hrp2 gene deletion Characteristic pfhrp2 gene deleted pfhrp2 gene non-deleted p-value n % Median IQR n % Median IQR Sex (n = 487) Female 27 9.8 248 90.2 0.640 Male 18 8.5 194 91.5 Health Zone (n = 491) Bandundu 19 15.7 102 84.3 0.012 Kikwit-Nord 17 6.2 259 93.8 Kikwit-Sud 9 9.6 85 90.4 Age in years (n = 488) 18 4.9–30 7 2.9–19 0.019* 5 years 33 11.1 264 88.9 Household Size (n = 491) 7 6–8 6 5–8 0.388* Mosquito Breeding Sites (n = 491) Absent 8 14.0 49 86.0 0.217 Present 37 8.5 397 91.5 LLIN ownership (n = 491) No 16 9.0 161 91.0 > 0.999 Yes 29 9.2 285 90.8 LLIN utilisation (n = 491) 24 9.5 228 90.5 0.876 No 21 8.8 218 91.2 Yes Prior drug intake (n = 491) No 29 9.0 295 91.0 0.869 Yes 16 9.6 151 90.4 Microscopy (n = 491) Negative 33 11.1 265 88.9 0.079 Positive 12 6.2 181 93.8 Parasite Density (n = 491) 4680 264–14800 1200 112–8080 0,3771* Clinical features (n = 491) Temperature at admission (°C). 37.5 36.4–38.6 37.8 37–38 0.345* History of fever 34 8.4 371 91.6 0.217 Headache 19 9.4 183 90.6 0.875 Vomiting 11 6.6 156 93.4 0.187 Rigor 5 4.4 108 95.6 0.061 Fatigue 9 8.3 99 91.7 0.851 Abdominal pain 5 4.8 100 95.2 0.087 Joint pain 3 7.7 36 92.3 1.000 Anorexia 3 9.4 29 90.6 1.000 Kruskal Wallis test ( * ) has been used to compare distributions for continuous variables (parasite density, age, and household size), Fischer exact test has been used for categorical variables. Discussion We found a prevalence of 9.2% of P. falciparum isolates with a pfhrp2 gene deletion among 684 malaria symptomatic patients in the Kwilu Province (DR Congo). More females participated in this study than males (57% vs. 42.3%). This finding is in keeping with results from the DR. Congo 2013–2014 Demographic and Health Survey(DHS) which reported a sex ratio slightly in favour of females ( 41 ). Surprisingly, children under-five represented 36% of participants. This is in contrast with what might be expected since under-five children are known to be at higher risk of contracting malaria. Despite the burden of malaria in Kwilu province, there is a seasonal trend in transmission, and immunity is acquired later in life, around ten years ( 3 ). Late acquisition of immunity can explain the higher proportion of participants aged more than five years, seeking treatment for symptoms suggestive of malaria and thus enrolled in this study. The median household size of six is similar to findings from the 2013–2014 DHS which reported of median size of 5.7 ( 41 ). There were mosquito breeding sites near households of the majority of participants (87.7%). The survey was conducted during the rainy season, which is characterized by the formation of breeding sites, especially in rural and semi-urban areas with limited public facilities. The use of mosquito bednets was the primary means of bite-prevention. Two-thirds of households (64.2%) owned a bednet, and only 49.7% of participants spent the night before the interview under a mosquito bednet. These results are lower than the previous report from the DHS in Kwilu province. In 2014, 87% of households surveyed in the Kwilu province possessed a bednet, and 69.4% of participants slept under a bednet the night before the interview ( 41 ). Back then, an extensive mosquito bednet distribution campaign was implemented with a higher rate of implementation, especially in Kwilu. However, Mwandagalirwa et al. (2017) recently reported consistent data (72% ownership vs. 45% use) in health zones of Kinshasa province, neighbouring Kwilu province ( 42 ). A low coverage during bednet distribution campaigns can explain the lower proportion of household possessing bed net. Also, bednet usage is known to be higher among under-five children and falls progressively to as low as 34% by the early twenties ( 42 ). Subsequently, the majority of participants (64%) being over five years may account for the majority of participants not using bednets. Among participants who reported prior malaria drug intake, 13% took quinine at least one week before the survey. This finding highlights the poor compliance with the WHO guidelines for the treatment of malaria in the study areas as well as challenges associated with the use of injectable artesunate in areas where quinine is cheaper, easy to use and available. As might be expected, 81.6% of participants reported a history of fever. In many cases, fever is suggestive of malaria but is also reported in several febrile illnesses prevailing in tropical areas. Without a reliable diagnostic tool, a syndromic approach often leads to overtreatment, especially among people living in low to moderate malaria transmission areas ( 43 ). A recent analysis of household survey data from 24 Sub-Saharan Africa countries between 2006 and 2014 showed that 35.7% of all fevers reported by participants were accompanied by malaria infection evidence, but only 10% of these fevers were attributable to malaria ( 44 ). Non-malarial febrile illnesses (NFMI) can coincide with malaria infection, and may lead to over-diagnosis of malaria and underestimation of the burden of associated NFMI ( 44 ). Only 9.9% of false-negative Pf HRP2-RDT results involved parasites with pfhrp2 gene deletions. A similar result (10.6%) has been reported in Nigeria, ( 24 ). Conversely, Wurtz et al . (2013), in Senegal reported a lower proportion (2.4%) while Koita et al . (2012), in Mali and Amoah et al. (2016), in Ghana reported higher proportions, 45% and 23%, respectively ( 4 , 5 , 17 ). This weak proportion shows that pfhrp2 gene deletion is not a major cause of false-negative Pf HRP2-RDT results. Several reasons may explain a false-negative Pf HRP2-RDT result such as poor quality of the test, inappropriate manipulation and interpretation, low parasite density, excess of circulating parasite antigens creating a prozone-like effect, and genetic polymorphisms in the target antigen ( 6 , 23 , 45 , 46 ). Even though the proportion of Pf HRP2-RDT false-negative results due to pfhrp2 gene deletion has surpassed the 5% threshold set by the WHO, requiring a subnational change in malaria RDTs, the required number of P. falciparum isolates ( 37 ) to include per health facility in the sampling domain was not reached. Therefore, further investigations are needed to provide enough evidence for policy change. The overall prevalence of P. falciparum isolates with pfhrp2 gene deletion was 9.2%. This prevalence is higher than the previously reported national prevalence of 6.4% and the local prevalence of 3% ( 14 ). The present study exclusively enrolled subjects with symptoms suggestive of malaria and thus more likely to be infected while the previous one included more asymptomatic subjects. Secondly, Kwilu province is located in a low to moderate transmission area where immunity is acquired later in life. This condition of reduced host immunity is favourable to infection by parasites harbouring pfhrp2 gene deletion, which can survive and spread ( 18 ). Thirdly, the prevalence of parasites harbouring pfhrp2 gene deletion has been shown to be higher in low to moderate transmission area in the beginning of the rainy season, which is the case for the present study ( 47 ). A similar prevalence has been reported in Eritrea (9.7%) and Kenya (9%) ( 13 , 48 ). However, lower prevalence has been reported in Mali (2%), Senegal (2.4%), Mozambique (1,4%) and higher prevalence in Nigeria (17%), Ghana (36%) and Zambia (37.5%) ( 4 , 15 – 17 , 24 , 25 ). The difference in study design and methodology used for deletion confirmation (nested PCR vs. qPCR of a single-copy gene) could explain the discrepancy. Publications using nested PCR for confirmation of deletion tend to overestimate the prevalence while amplification of a single-copy gene by real-time PCR is recommended for appropriate deletion call ( 49 , 50 ). The first protocol concerning phrp2/3 deletions was released in 2014 ( 51 ). Subsequently, the WHO released a second protocol in 2018 for estimating phrp2/3 deletions among symptomatic patients ( 36 ). Finally, Parr et al . (2018) revised the existing protocols and released a streamlined protocol taking into account challenges faced by previous authors ( 50 ). There was a statistically significant difference in pfhrp2 gene deletion prevalence in Bandundu health zone compared to Kikwit-Nord and Kikwit-Sud health zones (p = 0.012). Variations in pfhrp2 gene deletion status within regions and countries have been previously reported and depend on several factors including level of transmission and magnitude of Pf HRP2-RDT use ( 10 , 12 , 20 , 52 ). Further analysis of population genetics may clarify this finding. There was a trend towards pfhrp2 gene deletion in older compared to younger (under-five) patients, but this was not statistically significant (p = 0.079). However, when analyzing age as a continuous variable, the Kruskal Wallis test showed strong evidence suggesting that the distributions of age differed by pf hrp2 gene deletion status (p = 0.019). This may suggest that the risk of being infected with parasites harboring pfhrp2 deletion increases with age while traditionally under-five years old are at high risk of contracting malaria compared to the older group. Similarly, there was a trend towards pfhrp2 gene deletion among negative microscopy results, but weak evidence supported this finding (p = 0.079). Microscopic examination is based on morphological aspects of the parasite and might not be influenced by genetic traits such as gene deletions. Even considering that Pf HRP2 is known to be involved in the formation of hemozoin, this is not the only morphological feature allowing parasite detection by microscopic examination. We found more malaria signs and symptoms among patients infected with parasites not harbouring pf hrp2 gene deletions. However, the difference was not statistically significant. This study could only identify 45 P. falciparum isolates with pfhrp2/3 gene deletions, and a larger sample size may be required to detect differences and provide evidence of association. This study is the first to provide the local prevalence of P. falciparum isolates with pfhrp2/3 gene deletion among symptomatic patients in this region. The availability of clinical, biological, and sociodemographic data allowed exploration of differences between infection by pfhrp2 -deleted and wild-type P. falciparum parasites. However, the limited sample size preculded identification of predictors of pfhrp2 gene deletion and did not allow us to make solid conclusions regarding differences in pathology between pfhrp2 deleted and wild-type parasites The selection of study sites based on known burden of malaria and sociodemographic characteristics may have introduced a selection bias making the sample not representative of the whole province within which the level of transmission varies. A health facility-based design is undoubtedly the best choice to recruit symptomatic patients, but the low service utilization rate and insufficient public subsidies allocated to malaria management may have prevented some subjects with the characteristic of interest to attend health facilities and thus to be enrolled in the study. The DR. Congo National Malaria Control Program has adopted a five year (2016–2020) strategic plan with goals including the diagnosis of at least 80% of fever cases and the treatment of all positive diagnosed cases with ACT. Increased service utilization, the significant use of Pf HRP2 based RDTs as primary diagnostic tools and co-infections with non-malaria febrile illnesses could lead to the initiation of a selective treatment favoring pfhrp2 deleted parasites. There is a need, therefore, to establish a surveillance system for pfhrp2 deleted mutants as a part of malaria control programs. Such a surveillance system should be strengthened with reliable diagnostic tools such as molecular point of care testing to ensure efficient and evidence-based allocation of resources to disease control programs. In routine practice, clinicians should investigate other febrile illnesses despite a positive RDT result to minimize failure in disease management. Conclusion We found a local prevalence of 9.2% of P. falciparum isolates with a pfhrp2 gene deletion among symptomatic patients. These isolates explained only 9.9% of Pf HRP2-RDT false-negative results, suggesting that factors other than pfhrp2 gene deletion are of significant importance in the false-positivity rates of Pf HRP2-based RDTs. Even though the proportion of false-negative Pf HRP2-RDT results due to the pfhrp2 deletion has surpassed the 5% threshold set by the WHO for a subnational change in malaria RDTs, further regional investigations with appropriate sampling are needed to provide enough evidence for policy change. Meanwhile, the use of RDTs targeting Pf HRP2 and pLDH antigens could limit the spread of deleted isolates. List of Abbreviations °C Degree centigrade ACT Artemisinin-based Combination Therapy Ag Antigen CAID Cellules d’Analyses des Indicateurs de Developpement/Unit for development indicators analysis D.R. Congo Democratic Republic of Congo DBS Dried Blood Spot DNA Deoxyribonucleic Acid ELISA Enzyme-linked Immunosorbent Assay g/dL gram per deciliter HIV/AIDS Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome IQR Inter Quartile Range kDa kiloDalton LLIN Long Lasting Insecticidal Net LOD Limit of Detection mmHg millimeter mercury mmol/L Millimole per liter ng nanogram NMFI Non-Malaria Febrile Illness pAldo Parasite Aldolase PCR Polymerase Chain Reaction Pf HRP1 Plasmodium falciparum Histidine Rich Protein 1 pfhrp2 Plasmodium falciparum Histidine Rich Protein 2 gene Pf HRP2 Plasmodium falciparum Histidine Rich Protein 2 pf HRP2-RDT Plasmodium falciparum Histidine Rich Protein 2 based Rapid Diagnostic Test Pf HRP3 Plasmodium falciparum Histidine Rich Protein 3 pfldh Plasmodium falciparum lactate dehydrogenase gene PfPCR Plasmodium falciparum Polymerase Chain Reaction pLDH Parasite Lactate Dehydrogenase PNLP Programme National de Lutte contre le Paludisme / National Malaria Control Programme QBC Quantitative Buffy Coat qPCR quantitative Polymerase Chain Reaction RBC Red Blood Cell RDT Rapid Diagnostic Test SP Sulfadoxine-Pyrimethamine USAID United States Agency for International Development WBC White Blood Cell WHO World Health Organization µL microliter Declarations Ethical approval and consent to participate 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. Written and verbal voluntary informed consent was obtained from all study participants including guardian/parents of non-adult participants. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during this study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the Nagasaki University School of Tropical Medicine and Global Health and the Malaria Unit of the Nagasaki Institute of Tropical Medicine. R.C. is supported by Japanese Society for the Promotion of Science (JSPS), Japan Grant-in-Aid for Scientific Research Nos. 16K21233 and 19K07526 Author’s contributions YBM, AAM and PMM conceived and designed the study; YBM, AAM & ME performed experiments and data analysis; YBM, RLC and CS interpreted the results and wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank all patients and are grateful to the Division Provinciale de Santé du Kwilu (DPS-Kwilu) for assistance with samples collection. The authors are also grateful to Carole Ihomi Nkoy, Joel Imponge, and Agathe Bikupe Nkoy for their valuable contribution in data entry and field logistics. References World Health Organization. World malaria Report 2019. 2019. 238 p. USAID P. 2019 | U.S. President’s Malaria Initiative, 13th Annual Report to Congress. 2019;(May). PNLP, KSPH, Swiss KSPH, INRB, INFORM (2014). An epidemiological profile of malaria in the Democratic Republic of Congo. A report prepared for the Federal Ministry of Health, Democratic Republic of Congo, the Roll Back Malaria Partnership and the Department for International Development, UK. 2014;(September). Amoah LE, Abankwa J, Oppong A. Plasmodium falciparum histidine rich protein-2 diversity and the implications for PfHRP 2: Based malaria rapid diagnostic tests in Ghana. 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Implications of parasites lacking plasmodium falciparum histidine-rich protein 2 on Malaria morbidity and control when rapid diagnostic tests are used for diagnosis. J Infect Dis. 2017;215(7):1156–66. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-79327","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":2641372,"identity":"7ccdf2d5-5afc-4ff3-b926-7d54f3f9f035","order_by":0,"name":"Yannick Bazitama Munyeku","email":"","orcid":"","institution":"Ministère de la Santé, Direction des Laboratoires de Santé","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yannick","middleName":"Bazitama","lastName":"Munyeku","suffix":""},{"id":2641373,"identity":"f8fd9ed7-8957-493d-8b48-7384fbbd7a06","order_by":1,"name":"Alain Abera Musaka","email":"","orcid":"","institution":"Division Provinciale de la Santé du Kwilu","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alain","middleName":"Abera","lastName":"Musaka","suffix":""},{"id":2641374,"identity":"c7f163c4-22f4-4b43-8fd8-4c40b5b50811","order_by":2,"name":"Medard Ernest","email":"","orcid":"","institution":"National Institute of Allergy and Infectious Diseases Laboratory of Malaria and Vector Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Medard","middleName":"","lastName":"Ernest","suffix":""},{"id":2641375,"identity":"34ce872a-febe-44b8-81a4-2a8ec4d5cdc1","order_by":3,"name":"Chris Smith","email":"","orcid":"","institution":"Nagasaki University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chris","middleName":"","lastName":"Smith","suffix":""},{"id":2641376,"identity":"a5c6d2cc-83ef-4bb0-9a9a-ce91c13f156e","order_by":4,"name":"Paul Mankadi Mansiangi","email":"","orcid":"","institution":"Institut Supérieur des Techniques Médicales (ISTM)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Paul","middleName":"Mankadi","lastName":"Mansiangi","suffix":""},{"id":2641377,"identity":"736abb51-1ffd-4a5d-b9d6-1c4ba9b4d6d8","order_by":5,"name":"Richard Culleton","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8808-5771","institution":"Ehime University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Culleton","suffix":""}],"badges":[],"createdAt":"2020-09-17 10:20:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-79327/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-79327/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":2580174,"identity":"0357debc-bf3a-4804-9942-25d2b6b3ae04","added_by":"auto","created_at":"2020-09-24 15:59:13","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":805526,"visible":true,"origin":"","legend":"Map of the study sites \nNote: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.","description":"","filename":"MunyekuFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-79327/v1/MunyekuFigure1.jpg"},{"id":2580175,"identity":"d16855ca-31d5-4b81-91f8-3e94e2a2687f","added_by":"auto","created_at":"2020-09-24 15:59:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":711590,"visible":true,"origin":"","legend":"Pfhrp2 gene deletion testing pipeline","description":"","filename":"MunyekuFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-79327/v1/MunyekuFigure2.png"},{"id":15669604,"identity":"c35d4136-f0dc-4242-8875-6370c066b7cd","added_by":"auto","created_at":"2021-11-18 13:54:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":939116,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-79327/v1/3e62bfb9-cd46-4a86-8eef-4a1cfb5a2df6.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePrevalence of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e Isolates Lacking the Histidine Rich Protein 2 Gene Among Symptomatic Malaria Patients in Kwilu Province, DR. Congo.\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eMalaria remains a global health issue despite progress over the last decade. In 2018, there were an estimated 228\u0026nbsp;million new malaria cases, including 405,000 deaths (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Ninety-two percent of malaria cases and 93% of malaria deaths occurred in Africa. Fifteen countries in sub-Saharan Africa and India carried nearly 80% of the global malaria burden of which Nigeria and the Democratic Republic of the Congo accounted for about 35% (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Democratic Republic of Congo (DR. Congo) accounts for 12% of all malaria cases in sub-Saharan Africa (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In DR. Congo, malaria is the leading cause of morbidity and mortality, accounting for more than 40% of all outpatient visits, and for 19% of deaths among under five years children (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The high burden of malaria in DR. Congo can be explained by the fact that nearly the entire population (97%) lives in high-transmission zones where the most common vector encountered is \u003cem\u003eAnopheles gambiae\u003c/em\u003e, and \u003cem\u003ePlasmodium falciparum\u003c/em\u003e is the most common species responsible for the majority of severe cases (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn important component of malaria control and elimination is appropriate case management, which is based on early and accurate diagnosis. Accurate diagnosis facilitates appropriate and prompt treatment and minimizes the risk of developing drug resistance.\u003c/p\u003e \u003cp\u003eThe WHO recommends microscopic examination as the gold standard for malaria diagnosis. However, in rural and semi-urban settings where lack of equipment, reagents, trained and skilled personnel, and electricity can prevent this diagnosis method, the use of rapid diagnostic tests (RDTs) offers an alternative for quick and accurate diagnosis (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRDTs have become a primary and critical tool for malaria diagnosis in the D.R. Congo as well as in Malaria endemic countries. They accounted for nearly 75% of diagnostic testing among suspected cases in Africa in 2017 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Current RDT kits are designed to detect either \u003cem\u003eP. falciparum\u003c/em\u003e alone or in combination with other species of human malaria parasites. Three main antigens are detected by malaria RDTs, namely \u003cem\u003eP. falciparum\u003c/em\u003e Histidine Rich Protein 2 (\u003cem\u003ePf\u003c/em\u003eHRP2), parasite lactate dehydrogenase (pLDH), and parasite aldolase (pAldo) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePf\u003c/em\u003eHRP2, a \u003cem\u003eP. falciparum\u003c/em\u003e specific antigen, has the advantage of being highly abundant and heat-stable. \u003cem\u003ePf\u003c/em\u003eHRP2-based RDTs can lead to false-positive results in the case of persistent circulating HRP2 antigen as a result of antimalarial treatment, and false-negative results in individuals whose levels of parasitaemia is under the detection threshold of 200 parasites/\u0026micro;L (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the last decade, however, some studies have reported false-negative results among individuals infected with \u003cem\u003eP. falciparum\u003c/em\u003e parasites presenting a deletion of the \u003cem\u003eP. falciparum\u003c/em\u003e histidine-rich protein 2 \u003cem\u003e(pf\u003c/em\u003ehrp\u003cem\u003e2\u003c/em\u003e) gene. The majority of these studies have also identified co-existing deletions of the \u003cem\u003eP. falciparum\u003c/em\u003e histidine-rich protein 3 (\u003cem\u003epf\u003c/em\u003ehrp\u003cem\u003e3\u003c/em\u003e) gene, which produces an antigen that shows some cross-reactivity with HRP2 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile the WHO recommends not to initiate antimalarial treatment without biological evidence, evolutionary selection of \u003cem\u003eP. falciparum\u003c/em\u003e isolates with \u003cem\u003epfhrp2\u003c/em\u003e gene deletions could occur since only positive tested patients will be treated. The non-targeted patients infected by parasites harbouring \u003cem\u003epfhrp2\u003c/em\u003e gene deletions will facilitate the spread of \u003cem\u003ephrp2\u003c/em\u003e deleted strains, jeopardizing progress towards disease control and elimination in low setting countries.\u003c/p\u003e \u003cp\u003eTo date, only one study has investigated \u003cem\u003epf\u003c/em\u003ehrp\u003cem\u003e2\u003c/em\u003e-deleted mutant parasites in DR. Congo, reporting a country-wide prevalence of 6.4% among children under-five years and providing spatial distribution and population genetics of these deletions (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). However, this nationwide study could not explore clinical differences between \u003cem\u003epf\u003c/em\u003ehrp\u003cem\u003e2\u003c/em\u003e-deleted and wild type \u003cem\u003eP. falciparum\u003c/em\u003e malaria due to limited clinical data and study population (the majority being asymptomatic and under-five), nor was it able to conclude about the relative virulence of \u003cem\u003epf\u003c/em\u003ehrp2-deleted parasites.\u003c/p\u003e \u003cp\u003eIn order to address the above limitations, we selected Kwilu province which is classified by the DR. Congo National Malaria Control Program (NMCP) as a province at high risk of malaria (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Kwilu province is classified in the tropical facies where malaria transmission occurs predominantly during the long rainy season lasting 5 to 8 months, and where the number of infected bites per people per year ranges from 60\u0026ndash;400 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Using data from a prospective health facility-based cross-sectional study, we aimed to determine the local prevalence of the \u003cem\u003epfhrp2\u003c/em\u003e gene deletion among malaria symptomatic patients, and associated clinical, biological, and sociodemographic factors in the Kwilu Province (DR Congo).\u003c/p\u003e \u003cp\u003eThe aim of this study is to contribute to a better characterization of the prevalence and consequences of \u003cem\u003epfhrp2\u003c/em\u003e deletions in DR. Congo by providing relevant regional data to improve malaria management and control.\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eStudy design and setting\u003c/h2\u003e\n\u003cp\u003eThis study was aimed at determining the local prevalence and factors associated with \u003cem\u003epfhrp2\u003c/em\u003e gene deletions. 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 (DR Congo).\u003c/p\u003e\n\u003cp\u003eThe Kwilu Province is one of 26 provinces of DR. Congo with an area of 79,906\u0026nbsp;km\u003csup\u003e2\u003c/sup\u003e. It is divided into five administrative territories: Bagata (including the city of \u003cstrong\u003eBandundu\u003c/strong\u003e), Bulungu (including the city of \u003cstrong\u003eKikwit\u003c/strong\u003e), Gungu, Idiofa, and Masimanimba (\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe two selected cities (Bandundu and Kikwit) include three of the 24 health zones of the Kwilu Province (\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e). They are the two main cities in the province and bear the highest burden of malaria. \u003cem\u003epfhrp2\u003c/em\u003e gene deletions were previously reported in this region (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eBandundu, the capital city of the Kwilu province, is located 400\u0026nbsp;km from Kinshasa, the capital of DR. Congo (\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e). Bandundu covers an area of 222\u0026nbsp;km\u003csup\u003e2\u003c/sup\u003e with a population estimated at 950,683 as of 2015 (\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e). 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 26.9\u0026nbsp;\u0026deg;C (\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e). Bandundu city has one semi-urban health zone of the same name and 17 health areas, including 11 urban and six rural.\u003c/p\u003e\n\u003cp\u003eKikwit is the second-largest city in the Kwilu province, located in the south-west of DR. Congo, at 525\u0026nbsp;km from Kinshasa and 400\u0026nbsp;km from Bandundu (Fig.\u0026nbsp;1). It is the main economic city of the province and a commercial hub that provides access to diamond-rich regions of Kasa\u0026iuml; province and Angola. Kikwit covers an area of 92\u0026nbsp;km\u003csup\u003e2\u003c/sup\u003e with an estimated population of 1,326,068 as of 2016 (\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e). 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.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eEthics, consent, and permissions\u003c/h2\u003e\n\u003cp\u003eThe study was approved by the Kwilu Province Division of Health, the Kinshasa School of Public Health Ethical Committee, and the School of Tropical Medicine and Global Health Ethical Review Committee.\u003c/p\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003ch2\u003eStudy population.\u003c/h2\u003e\n\u003cp\u003eThe 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 (\u003cem\u003ePf\u003c/em\u003eHRP2-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.\u003c/p\u003e\n\u003ch2\u003eSample Size Calculation\u003c/h2\u003e\n\u003cp\u003eThe minimum number of subjects required to enrol in this study was calculated based on a previously reported proportion of \u003cem\u003epfhrp2\u003c/em\u003e gene deletion in the Kwilu province (3%) and recommendations from WHO for studies on \u003cem\u003epfhrp2/3\u003c/em\u003e deletion among symptomatic patients (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e). According to the WHO protocol for estimating \u003cem\u003epfhrp2/3\u003c/em\u003e deletion prevalence, for an expected prevalence of 3.2%, at least 370 individuals with \u003cem\u003eP. falciparum\u003c/em\u003e infection are required per sampling domain (\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e). 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 \u003cem\u003eP. falciparum\u003c/em\u003e.\u003c/p\u003e\n\u003ch2\u003eRecruitment method\u003c/h2\u003e\n\u003cp\u003eThe 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).\u003c/p\u003e\n\u003cp\u003eAt stage two, individuals attending the selected health facilities with symptoms indicative of malaria were recruited. The leadinvestigator weighed the number of individuals to recruit per health centre to the average rate of service utilization provided by the National Health Information System.\u003c/p\u003e\n\u003ch2\u003eVariables\u003c/h2\u003e\n\u003cp\u003eThis study used four groups of variables: sociodemographic, malaria prevention, clinical and biological variables. \u003cem\u003ePlasmodium falciparum\u003c/em\u003e HRP2 gene deletion (\u003cem\u003epfhrp2\u003c/em\u003e) was the primary outcome variable. Exposure variables were age, sex, health zones, household size, existence of mosquito breeding sites, LLIN ownership, use of LLIN, malaria drug intake, malaria clinical features, parasite density, and microscopy result.\u003c/p\u003e\n\u003ch2\u003eData collection method\u003c/h2\u003e\n\u003cp\u003ePotential 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\u0026rsquo; medical records were used to collect data from the physician\u0026rsquo;s or health officer\u0026rsquo;s clinical examination.\u003c/p\u003e\n\u003cp\u003eHeel or finger-prick blood was collected from each individual. Samples for microscopy were prepared using two drops of blood. Then 50 microliters of blood were applied on \u003cem\u003ePf\u003c/em\u003eHRP2-RDT, and a few drops were spotted onto Whatman filter paper to prepare dried blood spots (DBS).\u0026nbsp;The membranes of spent \u003cem\u003ePf\u003c/em\u003eHRP2-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 4\u0026nbsp;\u0026deg;C.\u003c/p\u003e\n\u003ch2\u003eMalaria RDT screening\u003c/h2\u003e\n\u003cp\u003eThe CareStart\u0026trade; Malaria \u003cem\u003ePf\u003c/em\u003e (HRP2) Ag RDT manufactured by Access Bio, Inc., was used for the qualitative detection of malaria histidine-rich protein 2 in the whole blood according to the manufacturer\u0026rsquo;s instructions (ACCESSBIO, 2018).\u003c/p\u003e\n\u003cp\u003eThe test membrane strip is pre-coated with a \u003cem\u003eP. falciparum\u003c/em\u003e HRP2 specific monoclonal antibody as a single line across the test strip. The reported panel detection score is 91.0% at 200 parasites/\u0026micro;l with a false positive rate of 0.9% (\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e)\u003c/p\u003e\n\u003ch2\u003eMicroscopic diagnosis of malaria\u003c/h2\u003e\n\u003cp\u003eA 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. Some slides went through another quality control in the vector control laboratory of the Kinshasa School of Public Health.\u003c/p\u003e\n\u003cp\u003eThick 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% Giemsa\u0026rsquo;s 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 8,000 WBC/\u0026micro;L using the following formula: (Number of Parasites counted X 8,000)/Number of counted WBC.\u003c/p\u003e\n\u003cp\u003eParasite 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.\u003c/p\u003e\n\u003ch2\u003eExtraction of parasite DNA\u003c/h2\u003e\n\u003cp\u003eGenomic DNA was extracted from membranes of spent \u003cem\u003ePf\u003c/em\u003eHRP2-RDT cassettes and DBS using the QIAGEN QIAmp\u0026reg;DNA extraction kit according to the manufacturer\u0026rsquo;s instructions. We also adapted a previously described method to recover DNA from spent RDTs membranes (\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e\n\u003ch2\u003eDetection of \u003cem\u003eP. falciparum\u003c/em\u003e infection \u0026amp;\u003cem\u003e pfhrp2\u003c/em\u003e gene deletion\u003c/h2\u003e\n\u003cp\u003eTo confirm \u003cem\u003eP. falciparum\u003c/em\u003e infection, we designed specific primers targeting a 226 base pair region of the \u003cem\u003eP. falciparum\u003c/em\u003e lactate dehydrogenase (\u003cem\u003epfldh\u003c/em\u003e) gene and performed a real-time PCR assay. This assay was also used to ensure there was sufficient parasite DNA quantity and quality in the samples to discriminate \u003cem\u003eP. falciparum\u003c/em\u003e negative samples from samples with \u003cem\u003epfhrp2\u003c/em\u003e gene deletion, as shown in \u003cstrong\u003eFig.\u0026nbsp;2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eSamples were duplicated and loaded in 96-wells plates along with serially diluted positive controls (1\u0026nbsp;ng/\u0026micro;l, 0.1\u0026nbsp;ng/\u0026micro;l, 0.01\u0026nbsp;ng/\u0026micro;l, 0.001\u0026nbsp;ng/\u0026micro;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.\u003c/p\u003e\n\u003cp\u003eFor detection of the \u003cem\u003epfhrp2\u003c/em\u003e 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 \u003cem\u003epfhrp2\u003c/em\u003e as previously described (\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e). We used a lower elongation temperature (68\u0026deg;C) to improve PCR sensitivity, \u003cem\u003epfhrp2\u003c/em\u003e being AT-rich, and increased the number of cycles to 40. We used genomic DNA from Dd2 (\u003cem\u003epfhrp2\u003c/em\u003e negative) and 3D7 (\u003cem\u003epfhrp2\u003c/em\u003e positive) as controls for all assays.\u003c/p\u003e\n\u003cp\u003eWe 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.\u003c/p\u003e\n\u003cp\u003eReaction components for both real-time and nested PCR are summarized in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003ePrimer Sequences and PCR conditions for \u003cem\u003epfhrp2 and pfldh\u003c/em\u003e genes amplification\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTargeted genes\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePrimer sequences (5\u0026rsquo; \u0026minus;\u0026thinsp;3\u0026rsquo;)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eReaction components\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCycling conditions\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLOD\u003c/p\u003e\n\u003cp\u003e(ng/\u0026micro;L)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003epfhrp2\u003c/em\u003e Exon 1\u0026ndash;2, PF3D7_0831800\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOuter\u003c/p\u003e\n\u003cp\u003eFor: GGTTTCCTTCTCAAAAAATAAAG\u003c/p\u003e\n\u003cp\u003eRev: TCTACATGTGCTTGAGTTTCG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e- One \u003cem\u003eTaq\u003c/em\u003e 2X Master Mix with standard buffer: 12.5\u0026nbsp;\u0026micro;L\u003c/p\u003e\n\u003cp\u003e- 10\u0026nbsp;\u0026micro;M forward primer: 1\u0026nbsp;\u0026micro;L\u003c/p\u003e\n\u003cp\u003e- 10\u0026nbsp;\u0026micro;M reverse primer: 1\u0026nbsp;\u0026micro;L\u003c/p\u003e\n\u003cp\u003e- Nuclease free water: 7.5\u0026nbsp;\u0026micro;L\u003c/p\u003e\n\u003cp\u003e- DNA template: 3\u0026nbsp;\u0026micro;L (gDNA or 5X diluted outer PCR product\u003c/p\u003e\n\u003cp\u003e25\u0026nbsp;\u0026micro;L reaction volume\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95\u0026nbsp;\u0026deg;C/5\u0026nbsp;min;\u003c/p\u003e\n\u003cp\u003e40 cycles of 95\u0026nbsp;\u0026deg;C/30sec, 55\u0026nbsp;\u0026deg;C/30sec, 68\u0026nbsp;\u0026deg;C/30sec\u003c/p\u003e\n\u003cp\u003e68\u0026nbsp;\u0026deg;C/5\u0026nbsp;min\u003c/p\u003e\n\u003cp\u003e4\u0026nbsp;\u0026deg;C - \u0026infin;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eInner\u003c/p\u003e\n\u003cp\u003eFor: GTATTATCCGCTGCCGTTTTTGCC\u003c/p\u003e\n\u003cp\u003eRev: CTACACAAGTTATTATTAAATGCGGAA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95\u0026nbsp;\u0026deg;C/5\u0026nbsp;min;\u003c/p\u003e\n\u003cp\u003e40 cycles of 95\u0026nbsp;\u0026deg;C/30sec, 62\u0026nbsp;\u0026deg;C/30sec, 68\u0026nbsp;\u0026deg;C/30sec\u003c/p\u003e\n\u003cp\u003e68\u0026nbsp;\u0026deg;C/5\u0026nbsp;min\u003c/p\u003e\n\u003cp\u003e4\u0026nbsp;\u0026deg;C - \u0026infin;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003epfldh\u003c/em\u003e (qPCR)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFor: ACGATTTGGCTGGAGCAG\u003c/p\u003e\n\u003cp\u003eRev: GGAACACCTGAATGTTGATG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e- PowerUp\u0026trade; SYBR \u003csup\u003eTM\u003c/sup\u003e Green Master Mix (2X): 12.5\u0026nbsp;\u0026micro;L\u003c/p\u003e\n\u003cp\u003e- 10\u0026nbsp;\u0026micro;M forward primer: 0.5\u0026nbsp;\u0026micro;L\u003c/p\u003e\n\u003cp\u003e- 10\u0026nbsp;\u0026micro;M reverse primer: 0.5\u0026nbsp;\u0026micro;L\u003c/p\u003e\n\u003cp\u003e- Nuclease free water: 6.5\u0026nbsp;\u0026micro;L\u003c/p\u003e\n\u003cp\u003e- DNA template: 2\u0026ndash;4\u0026nbsp;\u0026micro;L\u003c/p\u003e\n\u003cp\u003e22\u0026ndash;24\u0026nbsp;\u0026micro;L reaction volume\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e50\u0026nbsp;\u0026deg;C/2\u0026nbsp;min;\u003c/p\u003e\n\u003cp\u003e95\u0026nbsp;\u0026deg;C/2\u0026nbsp;min\u003c/p\u003e\n\u003cp\u003e45 cycles of 95\u0026nbsp;\u0026deg;C/15sec, 62\u0026nbsp;\u0026deg;C/1\u0026nbsp;min, 95\u0026nbsp;\u0026deg;C/30sec, 60\u0026nbsp;\u0026deg;C/15sec\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eLOD: Lower limit of detection, qPCR: quantitative or Real-time PCR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003ePCR product resolution by agarose gel electrophoresis\u003c/h2\u003e\n\u003cp\u003ePCR amplicons were separated by electrophoresis on a 2% agarose gel stained with Gel Red\u0026reg; Nucleic Acid Stain 10,000X in water. A total of 12\u0026nbsp;\u0026micro;L of PCR amplicons (6\u0026nbsp;\u0026micro;L) and loading dye (6\u0026nbsp;\u0026micro;L) were loaded onto the gel, which was run for 35\u0026nbsp;min at 100\u0026nbsp;V and observed under UV light. A 500\u0026nbsp;\u0026micro;g/ml Gene Ruler 100\u0026nbsp;bp DNA Ladder (BioLabs\u0026reg;inc) was loaded onto the same gel to determine the sizes of the resolved fragments.\u003c/p\u003e\n\u003ch2\u003eStatistical Analyses\u003c/h2\u003e\n\u003cp\u003eData were entered and analyzed using STATA15. 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.\u003c/p\u003e\n\u003cp\u003eFischer\u0026rsquo;s exact test (for categorical variables) and the Kruskal-Wallis test (for non-normally distributed continuous variables) were applied to look for associations between exposures and the primary outcome (\u003cem\u003epf\u003c/em\u003ehrp2 gene deletion). We computed the 95% CI for the prevalence of \u003cem\u003epfhrp2\u003c/em\u003e gene deletion. We considered a p-value less than 0.05 statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eSocio-demographic characteristics\u003c/h2\u003e\n\u003cp\u003eOf the 684 symptomatic patients who participated in the study; 391 (57.7%) were female, and 287 (42.3%) were male. Kikwit-Nord Health Zone accounted for the majority of patients 362 (52.9%) while Bandundu and Kikwit-Sud Health Zones accounted for 197 (28.8%) and 125 (18.3%) patients, respectively.\u003c/p\u003e\n\u003cp\u003eThe median age was nine years, with an interquartile range from 3 to 26\u0026nbsp;years old. Children under five years old represented 36% of enrolled patients. The median size of household was six, with an interquartile range from five to six. Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes socio-demographic characteristics.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eHousehold environment, malaria prevention, and treatment practices\u003c/h2\u003e\n\u003cp\u003eThe majority (87.7%) of patients/patients\u0026rsquo; guardian reported the presence of mosquito breeding sites near their household. Two-thirds of household (64.2%) owned a mosquito bednet, while 49.7% of patients spent the night before the interview under a mosquito bed net. One-third (220) of patients reported prior malaria drug intake. Quinine (13%), Sulfadoxine-Pyrimethamine (6.1%), Artemether-Lumefantrine (4.8%), Artesunate (2.0%), Arteether (1.9%), Artemether (1.8%) were the common drugs taken as illustrated in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eSocio-demographic characteristics, household environment and malaria prevention and treatment practices\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCharacteristic\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003en\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e%\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMedian\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIQR\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;678)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFemale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e391\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e57.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e287\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e42.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHealth Zone\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;684)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBandundu\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e197\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e28.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKikwit-Nord\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e362\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e52.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKikwit-Sud\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAge in years (\u003c/strong\u003en\u0026thinsp;=\u0026thinsp;680)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u0026ndash;26\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt; 5\u0026nbsp;years\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e245\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e36.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt; 5\u0026nbsp;years\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e435\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHousehold Size\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;684)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u0026ndash;8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHousehold environment\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;684)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMosquito Breeding Sites\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e600\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e87.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLLIN ownership\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e439\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLLIN utilisation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e340\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e49.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMalaria prevention and treatment practices\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePrior drug intake (n\u0026thinsp;=\u0026thinsp;684)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e220\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e32.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMalaria Drug taken (n\u0026thinsp;=\u0026thinsp;220)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQuinine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e89\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSulfadoxine-Pyrimethamine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eArtemether-Lumefantrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eArtesunate\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eArteether\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eArtemether\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eArteether-Artemotil\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAmodiaquine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eArtesunate-Sulfadoxine-Pyrimethamine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePlant extract\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eFor the variable \u0026lsquo;sex\u0026rsquo;, less than 1% information is missing (six entries). For the variable age, less than 1% information is missing (four entries)\u003c/p\u003e\n\u003cp\u003eMalaria drug taken includes only individuals who reported malaria drug intake at least one week before the survey\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eClinical and biological features of symptomatic patients\u003c/h2\u003e\n\u003cp\u003eAt admission, the body temperature of patients ranged from 37\u0026nbsp;\u0026deg;C to 38\u0026nbsp;\u0026deg;C (median temperature of 37.5\u0026nbsp;\u0026deg;C). The common findings of the clinical examination were: A history of fever (81.6%), headaches (41.8%), vomiting (31.4%), rigor (22.8%), fatigue (22.1%) and abdominal pain (20.2%). Among patients with a microscopy positive result, malaria parasite density ranged from 64 parasites /\u0026micro;L to 7200 parasites /\u0026micro;L with a median parasite density of 880 parasites /\u0026micro;L. Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e summarizes the distribution of clinical and biological features.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eClinical and biological features of symptomatic patients\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCharacteristic\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003en\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e%\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMedian\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIQR\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eClinical features\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;684)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHistory of fever\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e558\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHeadache\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e286\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e41.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eVomiting\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e215\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e31.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRigor\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e156\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFatigue\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e151\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAbdominal pain\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e138\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eJoint Pain\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e58\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAnorexia\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNeckache\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDiarrhoea\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePallor\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eConvulsions\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSplenomegaly\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpaired consciousness\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTemperature at admission (\u0026deg;C)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e37.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e37\u0026ndash;38\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eBiological features (\u003c/strong\u003en\u0026thinsp;=\u0026thinsp;235)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eParasite density (parasites /\u0026micro;L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e880\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64\u0026ndash;7200\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eParasite density accounts for only microscopy positive samples\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eProportion of \u003cem\u003epf\u003c/em\u003ehrp2 gene deletion in false-negative \u003cem\u003ePf\u003c/em\u003eHRP2-RDT\u003c/h2\u003e\n\u003cp\u003eThere were 131 false-negative \u003cem\u003ePf\u003c/em\u003eHRP2-RDT results of which only 9.9% could be explained by the \u003cem\u003epfhrp2\u003c/em\u003e gene deletion, with a 95% CI ranging from 5.4\u0026ndash;16.4%.\u003c/p\u003e\n\u003ch2\u003ePrevalence of \u003cem\u003epfhrp2\u003c/em\u003e gene deletion among all symptomatic\u003cem\u003e Pf\u003c/em\u003ePCR confirmed cases\u003c/h2\u003e\n\u003cp\u003eThe overall prevalence of \u003cem\u003epfhrp2\u003c/em\u003e gene deletion among \u003cem\u003ePf\u003c/em\u003ePCR confirmed symptomatic cases was 9.2%. The highest prevalence was found in Bandundu Health Zone (15.7%), followed by Kikwit-Sud Heath Zone (9.6%) and Kikwit-Nord Health (6.2%). Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e shows the distribution of \u003cem\u003epf\u003c/em\u003ehrp2 gene deletion among \u003cem\u003ePf\u003c/em\u003ePCR confirmed cases across Heath Zones.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eFrequency of \u003cem\u003epfhrp2\u003c/em\u003e gene deletion among all symptomatic \u003cem\u003ePf\u003c/em\u003ePCR confirmed cases\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eHealth Zones\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSubjects\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eP. falciparum\u003c/em\u003e infection\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003epf\u003c/em\u003ehrp2 gene deletion\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003en\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePrevalence (95% CI)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003en\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePrevalence (95% CI)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOverall\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e684\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e491\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e71.8 (68.2\u0026ndash;75.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.2 (6.7\u0026ndash;12.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBandundu\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e197\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e121\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e61.4 (54.2\u0026ndash;68.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15.7 (9.7\u0026ndash;23.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKikwit-Nord\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e362\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e276\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.2 (71.5\u0026ndash;80.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.2 (3.6\u0026ndash;9.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKikwit-Sud\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75.2 (66.7\u0026ndash;82.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.6 (4.5\u0026ndash;17.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eSocio-demographic, malaria prevention, biological features, and \u003cem\u003epf\u003c/em\u003ehrp2 gene deletion\u003c/h2\u003e\n\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, there was no statistically significant difference in \u003cem\u003epfhrp2\u003c/em\u003e gene deletion status between males and females (8.5% vs. 9.8%). Conversely, there was a statistically significant difference in \u003cem\u003epf\u003c/em\u003ehrp2 gene deletions status in Bandundu health zone compared to Kikwit-Nord and Kikwit-Sud health zones (p\u0026thinsp;=\u0026thinsp;0.012). There was a trend towards \u003cem\u003epfhrp2\u003c/em\u003e gene deletion in older compared to younger patients, but this was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.079). However, when analyzing age as a continuous variable, the median age among \u003cem\u003epfhrp2\u003c/em\u003e gene deleted patients was higher than the median age among \u003cem\u003epfhrp2\u003c/em\u003e gene non-deleted patients (18\u0026nbsp;years versus 7\u0026nbsp;years). The Kruskal Wallis test showed strong evidence suggesting that the distributions of age differed by \u003cem\u003epf\u003c/em\u003ehrp2 gene deletion status (p\u0026thinsp;=\u0026thinsp;0.019). Similarly, there was a trend towards \u003cem\u003epfhrp2\u003c/em\u003e gene deletion among negative microscopy results, but weak evidence supported this finding (p\u0026thinsp;=\u0026thinsp;0.079).\u003c/p\u003e\n\u003ch2\u003eClinical features and \u003cem\u003epfhrp2\u003c/em\u003e gene deletion\u003c/h2\u003e\n\u003cp\u003eWe found more malaria signs and symptoms among patients infected with parasites not harbouring \u003cem\u003epfhrp2\u003c/em\u003e gene deletion. However, the difference was not statistically significant (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab5\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eSocio-demographic, malaria prevention, biological features and \u003cem\u003epf\u003c/em\u003ehrp2 gene deletion\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCharacteristic\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003epfhrp2\u003c/em\u003e gene deleted\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003epfhrp2\u003c/em\u003e gene non-deleted\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ep-value\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003en\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e%\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMedian\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIQR\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003en\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e%\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMedian\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIQR\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;487)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFemale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e248\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e90.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.640\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e194\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHealth Zone\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;491)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBandundu\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e102\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e84.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.012\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKikwit-Nord\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e259\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKikwit-Sud\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e85\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e90.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAge\u003c/strong\u003e in years (n\u0026thinsp;=\u0026thinsp;488)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.9\u0026ndash;30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.9\u0026ndash;19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.019*\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt; 5\u0026nbsp;years\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e179\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.079\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt; 5\u0026nbsp;years\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e264\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e88.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHousehold Size\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;491)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u0026ndash;8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u0026ndash;8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.388*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMosquito Breeding Sites\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;491)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e86.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.217\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePresent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e397\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eLLIN ownership\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;491)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e161\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;0.999\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e285\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e90.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eLLIN utilisation\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;491)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e228\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e90.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.876\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e218\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ePrior drug intake\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;491)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e295\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.869\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e151\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e90.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopy\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;491)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e265\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e88.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.079\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e181\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eParasite Density\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;491)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4680\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e264\u0026ndash;14800\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1200\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e112\u0026ndash;8080\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0,3771*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eClinical features\u003c/strong\u003e (n\u0026thinsp;=\u0026thinsp;491)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTemperature at admission (\u0026deg;C).\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e37.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e36.4\u0026ndash;38.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e37.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e37\u0026ndash;38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.345*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHistory of fever\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e371\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.217\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHeadache\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e183\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e90.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.875\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eVomiting\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e156\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.187\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRigor\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e108\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.061\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFatigue\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e99\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.851\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAbdominal pain\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.087\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eJoint pain\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAnorexia\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e90.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"10\" align=\"left\"\u003e\n\u003cp\u003eKruskal Wallis test (\u003cstrong\u003e*\u003c/strong\u003e) has been used to compare distributions for continuous variables (parasite density, age, and household size), Fischer exact test has been used for categorical variables.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe found a prevalence of 9.2% of \u003cem\u003eP. falciparum\u003c/em\u003e isolates with a \u003cem\u003epfhrp2\u003c/em\u003e gene deletion among 684 malaria symptomatic patients in the Kwilu Province (DR Congo).\u003c/p\u003e\n\u003cp\u003eMore females participated in this study than males (57% vs. 42.3%). This finding is in keeping with results from the DR. Congo 2013\u0026ndash;2014 Demographic and Health Survey(DHS) which reported a sex ratio slightly in favour of females (\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e). Surprisingly, children under-five represented 36% of participants. This is in contrast with what might be expected since under-five children are known to be at higher risk of contracting malaria. Despite the burden of malaria in Kwilu province, there is a seasonal trend in transmission, and immunity is acquired later in life, around ten years (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e). Late acquisition of immunity can explain the higher proportion of participants aged more than five years, seeking treatment for symptoms suggestive of malaria and thus enrolled in this study. The median household size of six is similar to findings from the 2013\u0026ndash;2014 DHS which reported of median size of 5.7 (\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThere were mosquito breeding sites near households of the majority of participants (87.7%). The survey was conducted during the rainy season, which is characterized by the formation of breeding sites, especially in rural and semi-urban areas with limited public facilities.\u003c/p\u003e\n\u003cp\u003eThe use of mosquito bednets was the primary means of bite-prevention. Two-thirds of households (64.2%) owned a bednet, and only 49.7% of participants spent the night before the interview under a mosquito bednet. These results are lower than the previous report from the DHS in Kwilu province. In 2014, 87% of households surveyed in the Kwilu province possessed a bednet, and 69.4% of participants slept under a bednet the night before the interview (\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e). Back then, an extensive mosquito bednet distribution campaign was implemented with a higher rate of implementation, especially in Kwilu. However, Mwandagalirwa \u003cem\u003eet al.\u003c/em\u003e (2017) recently reported consistent data (72% ownership vs. 45% use) in health zones of Kinshasa province, neighbouring Kwilu province (\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e). A low coverage during bednet distribution campaigns can explain the lower proportion of household possessing bed net. Also, bednet usage is known to be higher among under-five children and falls progressively to as low as 34% by the early twenties (\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e). Subsequently, the majority of participants (64%) being over five years may account for the majority of participants not using bednets.\u003c/p\u003e\n\u003cp\u003eAmong participants who reported prior malaria drug intake, 13% took quinine at least one week before the survey. This finding highlights the poor compliance with the WHO guidelines for the treatment of malaria in the study areas as well as challenges associated with the use of injectable artesunate in areas where quinine is cheaper, easy to use and available.\u003c/p\u003e\n\u003cp\u003eAs might be expected, 81.6% of participants reported a history of fever. In many cases, fever is suggestive of malaria but is also reported in several febrile illnesses prevailing in tropical areas. Without a reliable diagnostic tool, a syndromic approach often leads to overtreatment, especially among people living in low to moderate malaria transmission areas (\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e). A recent analysis of household survey data from 24 Sub-Saharan Africa countries between 2006 and 2014 showed that 35.7% of all fevers reported by participants were accompanied by malaria infection evidence, but only 10% of these fevers were attributable to malaria (\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e). Non-malarial febrile illnesses (NFMI) can coincide with malaria infection, and may lead to over-diagnosis of malaria and underestimation of the burden of associated NFMI (\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eOnly 9.9% of false-negative \u003cem\u003ePf\u003c/em\u003eHRP2-RDT results involved parasites with \u003cem\u003epfhrp2\u003c/em\u003e gene deletions. A similar result (10.6%) has been reported in Nigeria, (\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e). Conversely, Wurtz \u003cem\u003eet al\u003c/em\u003e. (2013), in Senegal reported a lower proportion (2.4%) while Koita \u003cem\u003eet al\u003c/em\u003e. (2012), in Mali and Amoah \u003cem\u003eet\u003c/em\u003e al. (2016), in Ghana reported higher proportions, 45% and 23%, respectively (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThis weak proportion shows that \u003cem\u003epfhrp2\u003c/em\u003e gene deletion is not a major cause of false-negative \u003cem\u003ePf\u003c/em\u003eHRP2-RDT results. Several reasons may explain a false-negative \u003cem\u003ePf\u003c/em\u003eHRP2-RDT result such as poor quality of the test, inappropriate manipulation and interpretation, low parasite density, excess of circulating parasite antigens creating a prozone-like effect, and genetic polymorphisms in the target antigen (\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eEven though the proportion of \u003cem\u003ePf\u003c/em\u003eHRP2-RDT false-negative results due to \u003cem\u003epfhrp2\u003c/em\u003e gene deletion has surpassed the 5% threshold set by the WHO, requiring a subnational change in malaria RDTs, the required number of \u003cem\u003eP. falciparum\u003c/em\u003e isolates (\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e) to include per health facility in the sampling domain was not reached. Therefore, further investigations are needed to provide enough evidence for policy change.\u003c/p\u003e\n\u003cp\u003eThe overall prevalence of \u003cem\u003eP. falciparum\u003c/em\u003e isolates with \u003cem\u003epfhrp2\u003c/em\u003e gene deletion was 9.2%.\u003c/p\u003e\n\u003cp\u003eThis prevalence is higher than the previously reported national prevalence of 6.4% and the local prevalence of 3% (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e). The present study exclusively enrolled subjects with symptoms suggestive of malaria and thus more likely to be infected while the previous one included more asymptomatic subjects. Secondly, Kwilu province is located in a low to moderate transmission area where immunity is acquired later in life. This condition of reduced host immunity is favourable to infection by parasites harbouring \u003cem\u003epfhrp2\u003c/em\u003e gene deletion, which can survive and spread (\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e). Thirdly, the prevalence of parasites harbouring \u003cem\u003epfhrp2\u003c/em\u003e gene deletion has been shown to be higher in low to moderate transmission area in the beginning of the rainy season, which is the case for the present study (\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eA similar prevalence has been reported in Eritrea (9.7%) and Kenya (9%) (\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e). However, lower prevalence has been reported in Mali (2%), Senegal (2.4%), Mozambique (1,4%) and higher prevalence in Nigeria (17%), Ghana (36%) and Zambia (37.5%) (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e). The difference in study design and methodology used for deletion confirmation (nested PCR vs. qPCR of a single-copy gene) could explain the discrepancy. Publications using nested PCR for confirmation of deletion tend to overestimate the prevalence while amplification of a single-copy gene by real-time PCR is recommended for appropriate deletion call (\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe first protocol concerning \u003cem\u003ephrp2/3\u003c/em\u003e deletions was released in 2014 (\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e). Subsequently, the WHO released a second protocol in 2018 for estimating \u003cem\u003ephrp2/3\u003c/em\u003e deletions among symptomatic patients (\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e). Finally, Parr \u003cem\u003eet al\u003c/em\u003e. (2018) revised the existing protocols and released a streamlined protocol taking into account challenges faced by previous authors (\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThere was a statistically significant difference in \u003cem\u003epfhrp2\u003c/em\u003e gene deletion prevalence in Bandundu health zone compared to Kikwit-Nord and Kikwit-Sud health zones (p\u0026thinsp;=\u0026thinsp;0.012). Variations in \u003cem\u003epfhrp2\u003c/em\u003e gene deletion status within regions and countries have been previously reported and depend on several factors including level of transmission and magnitude of \u003cem\u003ePf\u003c/em\u003eHRP2-RDT use (\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e). Further analysis of population genetics may clarify this finding.\u003c/p\u003e\n\u003cp\u003eThere was a trend towards \u003cem\u003epfhrp2\u003c/em\u003e gene deletion in older compared to younger (under-five) patients, but this was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.079). However, when analyzing age as a continuous variable, the Kruskal Wallis test showed strong evidence suggesting that the distributions of age differed by \u003cem\u003epf\u003c/em\u003ehrp2 gene deletion status (p\u0026thinsp;=\u0026thinsp;0.019). This may suggest that the risk of being infected with parasites harboring \u003cem\u003epfhrp2\u003c/em\u003e deletion increases with age while traditionally under-five years old are at high risk of contracting malaria compared to the older group.\u003c/p\u003e\n\u003cp\u003eSimilarly, there was a trend towards \u003cem\u003epfhrp2\u003c/em\u003e gene deletion among negative microscopy results, but weak evidence supported this finding (p\u0026thinsp;=\u0026thinsp;0.079). Microscopic examination is based on morphological aspects of the parasite and might not be influenced by genetic traits such as gene deletions. Even considering that \u003cem\u003ePf\u003c/em\u003eHRP2 is known to be involved in the formation of hemozoin, this is not the only morphological feature allowing parasite detection by microscopic examination.\u003c/p\u003e\n\u003cp\u003eWe found more malaria signs and symptoms among patients infected with parasites not harbouring \u003cem\u003epf\u003c/em\u003ehrp2 gene deletions. However, the difference was not statistically significant. This study could only identify 45 \u003cem\u003eP. falciparum\u003c/em\u003e isolates with \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletions, and a larger sample size may be required to detect differences and provide evidence of association.\u003c/p\u003e\n\u003cp\u003eThis study is the first to provide the local prevalence of \u003cem\u003eP. falciparum\u003c/em\u003e isolates with \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletion among symptomatic patients in this region. The availability of clinical, biological, and sociodemographic data allowed exploration of differences between infection by \u003cem\u003epfhrp2\u003c/em\u003e-deleted and wild-type \u003cem\u003eP. falciparum\u003c/em\u003e parasites. However, the limited sample size preculded identification of predictors of \u003cem\u003epfhrp2\u003c/em\u003e gene deletion and did not allow us to make solid conclusions regarding differences in pathology between \u003cem\u003epfhrp2\u003c/em\u003e deleted and wild-type parasites\u003c/p\u003e\n\u003cp\u003eThe selection of study sites based on known burden of malaria and sociodemographic characteristics may have introduced a selection bias making the sample not representative of the whole province within which the level of transmission varies. A health facility-based design is undoubtedly the best choice to recruit symptomatic patients, but the low service utilization rate and insufficient public subsidies allocated to malaria management may have prevented some subjects with the characteristic of interest to attend health facilities and thus to be enrolled in the study.\u003c/p\u003e\n\u003cp\u003eThe DR. Congo National Malaria Control Program has adopted a five year (2016\u0026ndash;2020) strategic plan with goals including the diagnosis of at least 80% of fever cases and the treatment of all positive diagnosed cases with ACT. Increased service utilization, the significant use of \u003cem\u003ePf\u003c/em\u003eHRP2 based RDTs as primary diagnostic tools and co-infections with non-malaria febrile illnesses could lead to the initiation of a selective treatment favoring \u003cem\u003epfhrp2\u003c/em\u003e deleted parasites.\u003c/p\u003e\n\u003cp\u003eThere is a need, therefore, to establish a surveillance system for \u003cem\u003epfhrp2\u003c/em\u003e deleted mutants as a part of malaria control programs. Such a surveillance system should be strengthened with reliable diagnostic tools such as molecular point of care testing to ensure efficient and evidence-based allocation of resources to disease control programs.\u003c/p\u003e\n\u003cp\u003eIn routine practice, clinicians should investigate other febrile illnesses despite a positive RDT result to minimize failure in disease management.\u003c/p\u003e"},{"header":"Conclusion","content":" \u003cp\u003eWe found a local prevalence of 9.2% of \u003cem\u003eP. falciparum\u003c/em\u003e isolates with a \u003cem\u003epfhrp2\u003c/em\u003e gene deletion among symptomatic patients. These isolates explained only 9.9% of \u003cem\u003ePf\u003c/em\u003eHRP2-RDT false-negative results, suggesting that factors other than \u003cem\u003epfhrp2\u003c/em\u003e gene deletion are of significant importance in the false-positivity rates of \u003cem\u003ePf\u003c/em\u003eHRP2-based RDTs. Even though the proportion of false-negative \u003cem\u003ePf\u003c/em\u003eHRP2-RDT results due to the \u003cem\u003epfhrp2\u003c/em\u003e deletion has surpassed the 5% threshold set by the WHO for a subnational change in malaria RDTs, further regional investigations with appropriate sampling are needed to provide enough evidence for policy change. Meanwhile, the use of RDTs targeting \u003cem\u003ePf\u003c/em\u003eHRP2 and pLDH antigens could limit the spread of deleted isolates.\u003c/p\u003e "},{"header":"List of Abbreviations","content":"\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u0026deg;C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eDegree centigrade\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eACT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eArtemisinin-based Combination Therapy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eAg\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eAntigen\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eCAID\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eCellules d\u0026rsquo;Analyses des Indicateurs de Developpement/Unit for development indicators analysis\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eD.R. Congo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eDemocratic Republic of Congo\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eDBS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eDried Blood Spot\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eDNA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eDeoxyribonucleic Acid\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eELISA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eEnzyme-linked Immunosorbent Assay\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eg/dL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003egram per deciliter\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eHIV/AIDS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eHuman Immunodeficiency Virus/Acquired Immunodeficiency Syndrome\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eIQR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eInter Quartile Range\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003ekDa\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003ekiloDalton\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eLLIN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eLong Lasting Insecticidal Net\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eLOD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eLimit of Detection\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003emmHg\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003emillimeter mercury\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003emmol/L\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eMillimole per liter\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eng\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003enanogram\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eNMFI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eNon-Malaria Febrile Illness\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003epAldo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eParasite Aldolase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003ePCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003ePolymerase Chain Reaction\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cem\u003ePf\u003c/em\u003eHRP1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003e\u003cem\u003ePlasmodium falciparum\u003c/em\u003e Histidine Rich Protein 1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cem\u003epfhrp2\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003e\u003cem\u003ePlasmodium falciparum \u003c/em\u003eHistidine Rich Protein 2 gene\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cem\u003ePf\u003c/em\u003eHRP2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003e\u003cem\u003ePlasmodium falciparum\u003c/em\u003e Histidine Rich Protein 2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cem\u003epf\u003c/em\u003eHRP2-RDT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003e\u003cem\u003ePlasmodium falciparum\u003c/em\u003e Histidine Rich Protein 2 based Rapid Diagnostic Test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cem\u003ePf\u003c/em\u003eHRP3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003e\u003cem\u003ePlasmodium falciparum\u003c/em\u003e Histidine Rich Protein 3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cem\u003epfldh\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003e\u003cem\u003ePlasmodium falciparum lactate dehydrogenase gene\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cem\u003ePfPCR\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003e\u003cem\u003ePlasmodium falciparum \u003c/em\u003ePolymerase Chain Reaction\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003epLDH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eParasite Lactate Dehydrogenase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003ePNLP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eProgramme National de Lutte contre le Paludisme / National Malaria Control Programme\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eQBC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eQuantitative Buffy Coat\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eqPCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003equantitative Polymerase Chain Reaction\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eRBC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eRed Blood Cell\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eRDT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eRapid Diagnostic Test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eSP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eSulfadoxine-Pyrimethamine\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eUSAID\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eUnited States Agency for International Development\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eWBC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eWhite Blood Cell\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eWHO\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003eWorld Health Organization\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u0026micro;L\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"454\"\u003e\n\u003cp\u003emicroliter\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Kwilu Province Division of Health (N\u0026deg; 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.\u003c/p\u003e\n\u003cp\u003eWritten and verbal voluntary informed consent was obtained from all study participants including guardian/parents of non-adult participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Nagasaki University School of Tropical Medicine and Global Health and the Malaria Unit of the Nagasaki Institute of Tropical Medicine. R.C. is supported by Japanese Society for the Promotion of Science (JSPS), Japan Grant-in-Aid for Scientific Research Nos. 16K21233 and 19K07526\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYBM, AAM and PMM conceived and designed the study; YBM, AAM \u0026amp; ME performed experiments and data analysis; YBM, RLC and CS interpreted the results and wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank all patients and are grateful to the Division Provinciale de Sant\u0026eacute; du Kwilu (DPS-Kwilu) for assistance with samples collection. The authors are also grateful to Carole Ihomi Nkoy, Joel Imponge, and Agathe Bikupe Nkoy for their valuable contribution in data entry and field logistics.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Health Organization. World malaria Report 2019. 2019. 238 p.\u003c/li\u003e\n\u003cli\u003eUSAID P. 2019 | U.S. President\u0026rsquo;s Malaria Initiative, 13th Annual Report to Congress. 2019;(May).\u003c/li\u003e\n\u003cli\u003ePNLP, KSPH, Swiss KSPH, INRB, INFORM (2014). An epidemiological profile of malaria in the Democratic Republic of Congo. A report prepared for the Federal Ministry of Health, Democratic Republic of Congo, the Roll Back Malaria Partnership and the Department for International Development, UK. 2014;(September).\u003c/li\u003e\n\u003cli\u003eAmoah LE, Abankwa J, Oppong A. Plasmodium falciparum histidine rich protein-2 diversity and the implications for PfHRP 2: Based malaria rapid diagnostic tests in Ghana. 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Acta Trop. 2015;152.\u003c/li\u003e\n\u003cli\u003eAbdallah JF, Okoth SA, Fontecha GA, Mejia Torres RE, Banegas EI, Matute ML, et al. Prevalence of pfhrp2 and pfhrp3 gene deletions in Puerto Lempira, Honduras. Malar J. 2015;14(1):1\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eViana GMR, Okoth SA, Silva-Flannery L, Barbosa DRL, De Oliveira AM, Goldman IF, et al. Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia. PLoS One. 2017;12(3):1\u0026ndash;13.\u003c/li\u003e\n\u003cli\u003eOkoth SA, Abdallah JF, Ceron N, Adhin MR, Chandrabose J, Krishnalall K, et al. Variation in Plasmodium falciparum histidine-rich protein 2 (Pfhrp2) and Plasmodium falciparum histidine-rich protein 3 (Pfhrp3) gene deletions in Guyana and Suriname. PLoS One. 2015;10(5):1\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eDorado EJ, Okoth SA, Montenegro LM, Diaz G, Barnwell JW, Udhayakumar V, et al. 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J Infect Dis. 2017;215(7):1156\u0026ndash;66.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Plasmodium falciparum Histidine Rich Protein 2, gene deletion, false negative Rapid Diagnostic Test, symptomatic patients","lastPublishedDoi":"10.21203/rs.3.rs-79327/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-79327/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eMalaria rapid diagnostic tests have become a primary and critical tool for malaria diagnosis in malaria-endemic countries where \u003cem\u003ePf\u003c/em\u003eHRP2-based RDTs are widely used. However, in the last decade, the accuracy of \u003cem\u003ePf\u003c/em\u003eHRP2-based RDTs has been challenged by the emergence of P. falciparum strains harbouring deletions of the\u003cem\u003e pfhrp2\u003c/em\u003e gene, resulting in false-negative results. In DR. Congo, little is known about the prevalence of the \u003cem\u003epfhrp2 \u003c/em\u003egene deletion among P. falciparum isolates infecting symptomatic patients, especially in low to moderate transmission areas where \u003cem\u003epfhrp2\u003c/em\u003e deletion parasites are assumed to emerge and spread. Here we determine the local prevalence and factors associated with \u003cem\u003epfhrp2 \u003c/em\u003egene deletions among symptomatic malaria patients in the Kwilu province of the Democratic Republic of Congo, a low to moderate malaria transmission area.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eWe used secondary data from a prospective health facility-based cross-sectional study conducted on 684 individuals of all ages, seeking healthcare with symptoms suggestive of malaria from October to December 2018 in 34 randomly selected health facilities. Sociodemographic, malaria prevention and treatment practices, and clinical variables were collected using a pre-tested structured questionnaire. Patients’ medical records were used to collect additional clinical data. Blood was collected for microscopy, \u003cem\u003ePf\u003c/em\u003eHRP2-RDT, and spotted onto Whatman filter paper for downstream genetic analysis. Genomic DNA was extracted and used to perform PCR assays for the detection and confirmation of \u003cem\u003epf\u003c/em\u003ehrp2 gene deletions. Data were entered and analysed using STATA15. Fischer’s exact and the Kruskal-Wallis tests were applied to look for associations between exposures and the \u003cem\u003epf\u003c/em\u003ehrp2 gene deletion with a level of statistical significance set at p \u0026lt; 0.05.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe overall prevalence of the \u003cem\u003epf\u003c/em\u003ehrp2 gene deletion was 9.2% (95% CI 6.7% – 12.1%). The deletion\u003cem\u003e \u003c/em\u003eof the \u003cem\u003epfhrp2\u003c/em\u003e gene was associated with health zone of origin (p=0.012) and age (p=0.019). Among false-negative \u003cem\u003ePf\u003c/em\u003eHRP2-RDT results, only 9.9% were due to \u003cem\u003epfhrp2\u003c/em\u003e gene deletion.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eP. falciparum isolates \u003c/em\u003ewith \u003cem\u003epfhrp2\u003c/em\u003e gene deletions are relatively common among symptomatic patients in Kwilu province. Further investigations are needed to provide enough evidence for policy change. Meanwhile, the use of RDTs targeting \u003cem\u003ePf\u003c/em\u003eHRP2 and pLDH antigens could limit the spread of deleted isolates.\u003c/p\u003e","manuscriptTitle":"Prevalence of Plasmodium falciparum Isolates Lacking the Histidine Rich Protein 2 Gene Among Symptomatic Malaria Patients in Kwilu Province, DR. Congo.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-09-24 15:59:12","doi":"10.21203/rs.3.rs-79327/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c8eb7baa-8d63-4f36-8da4-728b25b20da6","owner":[],"postedDate":"September 24th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":601775,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"2020-12-10T23:24:48+00:00","versionOfRecord":[],"versionCreatedAt":"2020-09-24 15:59:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-79327","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-79327","identity":"rs-79327","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}