Detection of Duffy Blood Group Genotypes and Submicroscopic Plasmodium Infections Using Molecular Diagnostic Assays in Febrile Malaria Patients

Background Malaria remains a severe parasitic disease, posing a significant threat to public health and hindering economic development in sub-Saharan Africa. Ethiopia, a malaria endemic country, is facing a resurgence of the disease with a steadily rising incidence. Conventional diagnostic methods, such asmicroscopy, have become less effective due to low parasite density, particularly among Duffy-negative human populations in Africa. To develop comprehensive control strategies, it is crucial to generate data on the distribution and clinical occurrence of Plasmodium vivax and P. falciparum infections in regions where the disease is prevalent. This study assessed Plasmodium infections and Duffy antigen genotypes in febrile patients in Ethiopia. Methods Three hundred febrile patients visiting four health facilities in Jimma town of southwestern Ethiopia were randomly selected during the malaria transmission season (Apr–Oct). Sociodemographic information was collected, and microscopic examination was performed for all study participants. Plasmodiumspecies and parasitemia as well as the Duffy genotype were assessed by quantitative polymerase chain reaction (qPCR) for all samples. Data were analyzed using Fisher’s exact test and kappa statistics. Results The Plasmodium infection rate by qPCR was 16% (48/300) among febrile patients, of which 19 (39.6%) were P. vivax, 25 (52.1%) were P. falciparum, and 4 (8.3%) were mixed (P. vivax and P. falciparum) infections. Among the 48 qPCR-positive samples, 39 (13%) were negative by microscopy. The results of bivariate logistic regression analysis showed that agriculture-related occupation, relapse and recurrence were significantly associated withPlasmodium infection (P<0.001). Of the 300 febrile patients, 85 (28.3%) were Duffy negative, of whom two had P. vivax, six had P. falciparum, and one had mixed infections.Except for one patient with P. falciparum infection, Plasmodium infections in Duffy-negative individuals were all submicroscopic with low parasitemia. Conclusions The present study revealed a high prevalence of submicroscopic malaria infections. Plasmodium vivax infections in Duffy-negative individuals were not detected due to low parasitemia. Here, we recommend an improved molecular diagnostic tool to detect and characterize plasmodium infections, with the goal of quantifyingP. vivax infection in Duffy-negative individuals.


Background
In malaria-endemic regions where Plasmodium vivax and P. falciparum coexist, P. vivax continues to be the main cause of malaria because existing interventions are primarily focused on P. falciparum.Plasmodium vivax causes severe and fatal outcomes that have reversed the historical notion of benign P. vivax infections 1 .However, despite this burden, P. vivax does not draw as much attention as P. falciparum, particularly in sub-Saharan Africa 2 .
Malaria is a major public health problem in Ethiopia, of which 75% of the landmass of the country is endemic to malaria.The transmission of malaria occurs throughout the year, but it varies based on altitude and seasonality.In Ethiopia, malaria accounts for approximately 60% of all hospital admissions.Many malaria-related illnesses and deaths occur in the most remote, rural areas of the country where there is insu cient health care coverage, poverty, and other socioeconomic issues 3,4,5 .
Ethiopia is one of the few African countries where P. falciparum and P. vivax coexist 6,7 .Plasmodium falciparum accounted for 70% of all malaria cases, and the remaining cases were attributed to P. vivax 8,9 .Efforts to control malaria in Ethiopia have made signi cant progress in recent years.Between 2015 and 2020, malaria cases decreased by approximately 39%, and malariarelated deaths decreased by approximately 34%.While malaria prevalence has shown a slight decline in the past few years, transmission appears to be heterogeneous across the country, with some areas still facing a high malaria burden 10 .
The malaria situation has changed since the COVID-19 pandemic, and such impact varies among nations due to the country's unique epidemiological context, size, and intervention coverage.Based on recent geospatial estimates, the disruption caused by COVID-19 to malaria control in Africa resulted in almost a doubling of malaria mortality in 2020 compared to previous years.Furthermore, this disruption might lead to even more signi cant increases in subsequent years if not properly addressed 11,12 .
Malaria control and elimination strategies heavily depend on timely, accurate diagnosis and effective treatment 13,14 .
Microscopy is a gold standard and common malaria diagnostic test in several African countries because of its high speci city, convenience with a rapid turnaround time, and low cost.However, it has limited sensitivity, especially for low parasitemia infections, which require highly skilled microscopists complemented by molecular assays.An improved point-of-care detection method is critical because transmission caused by low parasitemia infections hinders the progress and goal of malaria elimination 15 .Systematic diagnosis and treatment of individuals with submicroscopic Plasmodium infections as part of the surveillance and intervention strategy would reduce and eliminate the parasite reservoirs that sustain transmission 16,17 .
Another factor that impacts malaria infection is Duffy blood antigens.The Duffy antigen receptor for chemokines (DARC) is encoded by the DARC gene expressed on the surface of red blood cells (RBCs) and plays a role in RBC invasion by the P. vivax parasite 18,19 .Individuals who lack Duffy antigen expression (Duffy-negative individuals) are known to be resistant to P. vivax 20,21 and have a reduced risk of P. vivax infection 22,23 .However, Duffy negativity does not provide complete protection against vivax malaria 24 .Other Plasmodium species, such as P. falciparum, can also infect and cause malaria in Duffy-negative individuals.Additionally, Duffy-negative individuals can be low-parasitemia carriers 25,26 .The parasites remain dormant in the liver and cause relapses later, which can sustain transmission and hinder complete elimination of the disease 27,28,29 .
With the goal of eliminating malaria in Ethiopia and other parts of SSA, there is a pressing need for accurate diagnosis and effective treatment of P. vivax infection with low parasitemia.Understanding the distribution and prevalence of P. vivax can contribute to better implementation of control strategies.This study assessed the performance of molecular assays to detect Plasmodium infection, especially to detect infections with low parasitemia in Duffy-negative individuals, which can hinder progress toward malaria elimination.The ndings can aid policymakers and programs in designing more effective and targeted interventions to combat malaria and work toward its ultimate elimination in the region.

Study site and sample collection
A total of 300 samples were collected from febrile patients from three health facilities (Jimma Shene Gibe General Hospital, Jimma Higher One Health Center, and Jimma Higher Two Health Center of Jimma town), southwestern Ethiopia.The area is located at Latitude: 7°40'N and Longitude: 36°50'E with an altitude of 1,780 meters; Figure 1).The study was conducted from May to October 2022, and the study design was a cross-sectional study design.Sociodemographic data, including gender, age, occupation, education, and ethnicity, were collected from each study participant.Previous malaria history, antimalarial drug treatment, medical history, and Duffy status were collected for malaria infection risk analysis.For participants aged below 18 years old, the questionnaire was completed by their guardians or parents.A nger-pricked blood sample of approximately 200 µl was collected and preserved on Whatman lter paper 30 .Thin and thick blood smears were prepared for microscopic examination of malaria parasites 31 .A sample was considered malaria-negative if no parasites were detected after examining 200 elds of the thick smear following the standard WHO protocol 32,33 .

Parasite DNA extraction and molecular screening
For each sample, parasite DNA was extracted from a dried blood spot (~50 µl) using the Saponin/Chelex method 34 .Plasmodium species were examined by quantitative PCR of the 18S rDNA gene using species-speci c primers for P. falciparum and P. vivax 35,36 .Ampli cation was performed in a 20 μl reaction mixture containing 2 μl of genomic DNA, 10 μl 2×SYBR Green qPCR Master Mix (Thermo Scienti c), and 0.5 µM primer with an initial denaturation at 95°C for 3 min, followed by 45 cycles at 94°C for 30 sec, 55°C for 30 sec, and 68°C for 1 min with a nal 95°C for 10 sec.This was followed by a melting curve step of temperature ranging from 65°C to 95°C with 0.5°C increments to determine the melting temperature of each ampli ed product.Each assay included positive controls of P. falciparum (MRA-667) and P. vivax (MRA-178) strains, in addition to negative controls including uninfected samples and nuclease-free water.A standard curve was generated from a 10-fold serial dilution of the control plasmids to determine the e ciency of qPCR.Melting curve analyses were performed for each ampli ed sample to con rm speci c ampli cations of the target sequence.Samples yielding a threshold cycle (Ct value) higher than 40 (as indicated in the negative controls) were considered negative for Plasmodium species.Parasite density in a sample was quanti ed with the following equation: GCN sample = 2 E×  , where GCN stands for gene copy number, Ct for the threshold cycle of the sample, and E for ampli cation e ciency 37 . The differences in the log-transformed parasite GCN between the microscopic-positive and microscopic-negative samples were assessed for level of signi cance at p<0.05 by one-tailed t test.

DARC genotyping
An approximately 500-bp fragment of the human DARC gene that encompasses the SNP position rs2814778 (-67T>C) located in the promoter region was ampli ed following established protocols 38 .Ampli cations were conducted in a mixture containing 2 μl of genomic DNA, 7 μl of Taqman Fast Advanced Master Mix (Thermo Fisher), 0.54 µM of each forward and reverse primer and 0.54 µM of each Probe C-FAM and T-HEX.The reactions were performed with an initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 30 sec, 58°C for 30 sec, and 65°C for 40 sec, with a nal 2-min extension at 65°C.An allelic discrimination plot was used to distinguish Duffy genotypes.DARC genotypes were con rmed by PCR and Sanger sequencing for all Duffy-negative and a subset of Duffy-positive samples (Figure 2).

Data analyses
All data were analyzed using SPSS software package version 21.0.Descriptive statistics were used to summarize the sociodemographic characteristics of the study participants.Bivariate logistic regression was used to determine the association of malaria infection with independent variables.The odds ratio and the corresponding 95% CI were calculated to determine the strength of the association.A p value < 0.05 was considered statistically signi cant during the analysis.

Demographic and socioeconomic factors
Of the 300 study participants, 126 (42.0%) were males and 174 (58.0%) were females (Table 1).The median age of the participants in the study was 24 years (range = 1-85 years).Approximately 29% (n = 86) of the participants were engaged in agriculture and open eld work (farmers, construction workers, guardsmen, soldiers, and gardeners), and the remaining (n =214, 71.3%) were o ce clerks, teachers, shopkeepers, full-time students, preschool children, and housekeepers (Table 1).The results of bivariate logistic regression analysis showed that agriculture-related occupation, relapse and recurrence were signi cantly associated with Plasmodium infection (P<0.001).Individuals with a history of malaria infection who failed to comply with the complete antimalaria drug prescription protocol were found to be at a higher risk of Plasmodium infections.Conversely, those who diligently adhered to the complete course of anti-malaria drugs prescribed were found to be at a lower risk of Plasmodium infection (OR: 0.3; CI: 0.1-0.72;P=0.009; Table 1).Individuals who had agriculture-related occupations were 27-fold at higher risk of malaria infection than individuals who were engaged in other occupations (OR: 27.36; CI: 9.3-80.8;P<0.001;

Plasmodium detection in Duffy-negative patients by microscopy and qPCR
The frequencies of Duffy-negative and Duffy-positive genotypes among all participants were 85 (28.3%) and 215 (71.6%), respectively.These ndings underscore the importance of the Duffy blood group in regard to malaria susceptibility and other health outcomes.Speci cally, 41 males and 44 females were found to be Duffy negative, while 85 males and 130 females were Duffy positive, indicating a higher proportion of females in this group.These results suggest that further research on the relationship between the Duffy blood group and health outcomes is needed to better understand the implications of these ndings.Both P. vivax and P. falciparum infections were observed in Duffy-negative patients (Table 2).This study revealed that Duffy-positive individuals were at higher risk of having P. vivax than Duffy-negative individuals (OD: 7.65 [1.37-42.7]P=0.020 and OD: 6.18 [1.1-34.7]P=0.038, respectively) (Table 2).However, no such association was found for P. falciparum or mixed (P.vivax and P. falciparum) infections among the different Duffy genotypes.Prevalence of submicroscopic infections A higher number of malaria-positive samples were detected by qPCR (n= 48; 16.0%) than by microscopy (n=29; 9.6%; Table 3).Among qPCR-positive samples, 19 (39.58%) had P. vivax, 25 (52.08%) had P. falciparum, and 4 (8.33%) had mixed infections (P.falciparum and P. vivax.Interestingly, only 4 out of 19 P. vivax samples and 3 out of 25 P. falciparum samples were identi ed by microscopy from qPCR-positive samples.No mixed infections were identi ed (Table 3).Moreover, P. falciparum and P. vivax infections were con rmed by qPCR (n=39, 13%) to be negative by microscopy and were submicroscopic Plasmodium infections.

Discussion
The Ethiopian government aims to eliminate malaria by 2030.However, submicroscopic infections, relapses of P. vivax, and residual transmission present tremendous challenges to this goal.Submicroscopic infections contribute to malaria transmission.The prevalence of submicroscopic malaria infections detected in this study was higher than that previously reported in low-endemic areas such as Malo (9.7%) and Bonga (10%) of southwest Ethiopia 39 but lower compared to regions in southwestern Saudi Arabia, Senegal, and Uganda, where submicroscopic malaria infections were more than 23% 40,41,42 .
Infected individuals with submicroscopic parasitemia have the potential to serve as infection reservoirs in the community and other malaria-free areas as these infections are not detected and treated with antimalarial drugs 39 .
Failure to detect submicroscopic infections, especially in the case of P. vivax in Duffy-negative individuals, poses a signi cant challenge to malaria control and elimination efforts.These infections can remain unnoticed and untreated, leading to persistent and ongoing transmission in the affected communities.Duffy-negative individuals can serve as parasite reservoirs for new infections to emerge and circulate in Duffy-positive individuals 43 .Our ndings showed that 28% of the febrile patients were Duffy negative.This proportion is slightly higher than an earlier report of ~20% homozygous Duffy-negative patients in Harar and Jimma 44 but was much lower than the proportion of Duffy-negative patients in West and Central Africa (>97%) 45,46 .Surveillance that relies on detecting symptomatic cases and carrying out routine blood smears or RDTs might underestimate the true prevalence of P. vivax in Duffy-negative individuals.Duffy-negative individuals can be infected with P.
vivax, as well as other malaria parasite species such as P. falciparum as mono-or mixed-infections 47 .Outside Africa, in the Brazilian Amazon, aP.In the vivax endemic area, a small percentage of Duffy-negative individuals have been infected with P.
vivax 48 , suggesting that alternative invasion pathways may exist for this parasite.Mixed infections involving P. vivax and P. falciparum, as well as other mixed infections of malaria parasite species, were detected in both Duffy-positive and Duffynegative individuals 49 .Given that P. vivax infections in Duffy-negative individuals have low parasite densities, a sensitive and molecular diagnostic assay such as qPCR is needed to accurately detect these infections and compare P. vivax epidemiology in malaria endemic countries.
It is not surprising that individuals who had a previous history of malaria infection with not taking all anti-malaria drugs in the previous prescriptions had a signi cantly higher risk for malaria infection than those who took all anti-malaria drugs in the previous prescriptions.This study provides compelling evidence that the complete course of antimalaria drugs prescribed should be diligently followed to reduce the risk of malaria infection.The risk of relapse or reinfection could be affected by the complex immune responses developed after an initial infection.When a person is infected with malaria, the immune system produces speci c antibodies and immune cells that target the parasites.While such immune responses recognize and eliminate parasite strains that cause the initial infection 50 , some strains may evade the immune system and remain dormant in the liver 51 .These parasites can persist in the body at low levels, leading to a chronic low-grade infection.In individuals who have had a previous malaria infection, the presence of residual parasites can lead to a faster and more robust immune response upon reinfection.This phenomenon is known as a "premunition" response 52 .The high levels of antibodies and immune cells generated in response to the rst infection can sometimes contribute to an exaggerated immune response, leading to increased in ammation and tissue damage in subsequent infection.The duration of dormancy varies depending on the parasite strain, but it is generally thought to be several months 53 .Therefore, individuals who have experienced a recent malaria infection, particularly within the previous 4-6 months, are more likely due to dormant parasites in the liver.This increases their susceptibility to reinfection compared to individuals who have never been infected or have a more distant history of infection.It is important to note that the risk of malaria infection can also depend on the complex interactions between the immune system and the characteristics of malaria parasites, including its ability to persist at low levels, evade immune detection, speci c strain of the parasites, the intensity of malaria transmission in a given area, the use of preventive measures such as bed nets or insecticide spraying, and the individual's overall health and immune status.These factors can further in uence the risk of malaria reinfection in individuals with a previous infection history.

Conclusion
Submicroscopic malaria infections contribute to persistent transmission of the disease.The present study revealed that submicroscopic infection in Duffy-negative individuals was high.Microscopy failed to detect a signi cant number of Plasmodium infections in Duffy-negative individuals.However, molecular diagnostic assays such as qPCR could detect more submicroscopic infections in Duffy-negative individuals than microscopy.Our ndings support the notion that P. vivax can infect Duffy-negative individuals.Therefore, improved diagnostic methods are critical to detect submicroscopic Plasmodium infections in Duffy-negative individuals.This could reduce reservoir infections, which enhances malaria elimination efforts.
Abbreviations COR Crude odds ratio GCN

Figure 3
Figure 3 presents the distribution of the Duffy genotype in relation to parasite density.Overall, a signi cant difference was observed in the parasite density among the Duffy phenotypes (p < 0.05).Notably, individuals with the Duffy-positive phenotype (C/T) exhibited signi cantly higher P. falciparum parasite density than individuals with Duffy-negative phenotypes (CC, T/T) (p < 0.003).

Figure 1 Map
Figure 1

Table 1 .
). Demographic characteristics and Risk factors associated with clinical malaria among malaria suspected individuals in

Table 2 .
The distributions of malaria cases by Duffy genotype using qPCR among malaria-suspected individuals in Ethiopia.

Table 3 .
Prevalence of submicroscopic and missed infections among malaria-suspected individuals in Ethiopia.