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

Malaria rapid diagnostic tests have become a primary and critical tool for malaria diagnosis in malaria-endemic countries where PfHRP2-based RDTs are widely used. However, in the last decade, the accuracy of PfHRP2-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. secondary data from prospective in Genomic extracted and used pfhrp2 Data Kruskal-Wallis

PfHRP2, a P. falciparum speci c antigen, has the advantage of being highly abundant and heat-stable. PfHRP2-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 (pfhrp2) gene. The majority of these studies have also identi ed co-existing deletions of the P. falciparum histidine-rich protein 3 (pfhrp3) gene, which produces an antigen that shows some cross-reactivity with HRP2 (4,5,.
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 pfhrp2-deleted mutant parasites in DR. Congo, reporting a country-wide prevalence of 6.4% among children under-ve years and providing spatial distribution and population genetics of these deletions (14). However, this nationwide study could not explore clinical differences between pfhrp2-deleted and wild type P. falciparum malaria due to limited clinical data and study population (the majority being asymptomatic and under-ve), nor was it able to conclude about the relative virulence of pfhrp2-deleted parasites.
In order to address the above limitations, we selected Kwilu province which is classi ed by the DR. Congo National Malaria Control Program (NMCP) as a province at high risk of malaria (3). Kwilu province is classi ed 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.

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 ve 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 rst 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 (PfHRP2-RDT and/or microscopic examination) was performed. Individuals who failed to meet the inclusion criteria or did not consent to participate in the study were excluded.

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

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 o cer's clinical examination.
Heel or nger-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 PfHRP2-RDT, and a few drops were spotted onto Whatman lter paper to prepare dried blood spots (DBS). The membranes of spent PfHRP2-RDT cassettes and the DBSs were individually stored in plastic bags, sealed with a desiccant at room temperature before being shipped to the Institute of Tropical Medicine in Nagasaki (NEKKEN) where they were refrigerated at 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 speci c 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 xed with methanol and dried. Then the whole slide was stained with 10% Giemsa's solution for ten minutes and nally washed off with distilled water and air-dried. Stained smears were examined under a microscope for malaria parasite identi cation. 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 PfHRP2-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 con rm P. falciparum infection, we designed speci c primers targeting a 226 base pair region of the P. falciparum lactate dehydrogenase (p dh) gene and performed a real-time PCR assay. This assay was also used to ensure there was su cient 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 ampli cation a third time and counted two consistent results as the nal result.
Reaction components for both real-time and nested PCR are summarized in Table 1. 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.  Prevalence of pfhrp2 gene deletion among all symptomatic PfPCR con rmed cases The overall prevalence of pfhrp2 gene deletion among PfPCR con rmed 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 pfhrp2 gene deletion among PfPCR con rmed cases across Heath Zones. Socio-demographic, malaria prevention, biological features, and pfhrp2 gene deletion As shown in Table 5, there was no statistically signi cant difference in pfhrp2 gene deletion status between males and females (8.5% vs. 9.8%). Conversely, there was a statistically signi cant difference in pfhrp2 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 signi cant (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 pfhrp2 gene deletion status (p = 0.019).
Similarly, there was a trend towards pfhrp2 gene deletion among negative microscopy results, but weak evidence supported this nding (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 signi cant (Table 5).

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 nding 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-ve represented 36% of participants. This is in contrast with what might be expected since under-ve 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 ve years, seeking treatment for symptoms suggestive of malaria and thus enrolled in this study. The median household size of six is similar to ndings 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-ve children and falls progressively to as low as 34% by the early twenties (42). Subsequently, the majority of participants (64%) being over ve 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 nding 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). This weak proportion shows that pfhrp2 gene deletion is not a major cause of false-negative PfHRP2-RDT results. Several reasons may explain a false-negative PfHRP2-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 PfHRP2-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).
The rst 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 signi cant 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 PfHRP2-RDT use (10,12,20,52). Further analysis of population genetics may clarify this nding.
There was a trend towards pfhrp2 gene deletion in older compared to younger (under-ve) patients, but this was not statistically signi cant (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 pfhrp2 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-ve 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 nding (p = 0.079). Microscopic examination is based on morphological aspects of the parasite and might not be in uenced by genetic traits such as gene deletions. Even considering that PfHRP2 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 pfhrp2 gene deletions. However, the difference was not statistically signi cant. 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 rst 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 identi cation 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 insu cient 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 ve 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 signi cant use of PfHRP2 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 e cient 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 PfHRP2-RDT false-negative results, suggesting that factors other than pfhrp2 gene deletion are of signi cant importance in the falsepositivity rates of PfHRP2-based RDTs. Even though the proportion of false-negative PfHRP2-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 PfHRP2 and pLDH antigens could limit the spread of deleted isolates.  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.  Map of the study sites Note: 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.