Prevalence of Asymptomatic Malaria Parasitemia among Blood Donors in Cape Coast, Ghana: A Cross-Sectional Study

Background Malaria is an important transfusion-associated infection in many parts of the world, particularly in sub-Saharan Africa, where it is endemic. We studied the prevalence of malaria parasites among blood donors in the Cape Coast Metropolitan Area. Methods A malaria parasite examination was added to the blood donor screening protocol for 240 voluntary and replacement blood donors (224 males and 16 females) between December 2020 and July 2021. Results Overall, 2.5% (6/240) had Plasmodium falciparum trophozoites detected in their blood sample. The remaining had no parasites detected. Four of the 148 who passed the blood donor screening tests were infected. The remaining two with malaria parasites failed one screening test. These included one donor with “hepatitis B + P. falciparum” and another with “syphilis + P. falciparum” parasite coinfection. All blood donors who had malaria parasites detected in their blood were males. Most donors, 45.8% (110/240), were in the 26–35 age group, with the highest prevalence of 1.3% (3/240). Blood group O was predominant (75.0%, 180/240), followed by B (12.9%, 31/240), A (11.3%, 27/240), and AB (0.8%, 2/240). All malaria parasites detected were among individuals with blood group O. Moreover, 96.3% (231/240) were rhesus-positive and had the highest prevalence of 2.1% (5/240). Conclusions Screening of blood donors in Ghana does not include malaria, although there is the potential for transmission through blood products. Malaria transmission via blood transfusion remains an issue of public health concern, as indicated in the results of this current study. We recommend studies on malaria prevention, pretransfusion and posttransfusion, and pathogen reduction technology.


Introduction
Te World Malaria Report estimates 241 million malaria cases in 2020 and 227 million in 2019 [1]. Malaria is known to cause 1.5-2.7 million deaths worldwide, particularly in Africa [2]. It is estimated that over 95% of malaria cases and 96% of malaria deaths are concentrated in Africa [3]. It is a disease with very high morbidity and mortality rates in Africa [4]. Malaria claimed the lives of an estimated 445,000 people worldwide, with Africa accounting for roughly 91% of the total [3]. It is one of the vector-borne diseases that can be transmitted through blood transfusion, aside from chagas, toxoplasmosis, leishmaniasis, babesiosis, and microflariasis [5].
Te causative organism for malaria is the Plasmodium species, which are P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. All four human malaria parasites (P. falciparum, P. vivax, P. ovale, and P. malariae) could be transmitted through blood transfusion [6]. Plasmodium falciparum is responsible for the severest form of malaria and is the most prevalent in sub-Saharan Africa [4]. Some Plasmodium species can survive in stored blood for seven to 40 days, depending on the species.
Blood donors are not routinely screened for malaria parasites before donation, even though the World Health Organization recommends that all donor blood be tested for malaria in most malaria-endemic countries in sub-Saharan Africa [7]. Tus, there is an increased potential for malaria parasite transmission to blood recipients and clinical diseases [8].
Blood transfusions can be lifesaving for individuals who have lost large volumes of blood. A blood transfusion may be required in serious accidents, gynaecological haemorrhages, surgery, stem cell transplants, symptomatic anaemia, and cancer [9]. Although blood is used to save lives, it could do more harm than good if not screened thoroughly to prevent the transmission of diseases [10]. Te most afected groups include children under fve years, pregnant women, victims of serious blood loss in road trafc accidents, and immunosuppressive patients [7]. Blood and its products are essential in emergencies for every healthcare system. However, using blood products may be complicated by the risks of adverse immunological reactions and transmission of other blood-borne pathogens [11].
Since blood donors may have malaria parasites, there could be a risk of transmission to vulnerable blood recipients. Te intraerythrocytic stage of the parasite can be transmitted by transfusion of any blood component containing infected cells. An initial report on malaria resulting from a blood transfusion was published in 1911 [12]. Some clinical manifestations of malaria include headache, generalized body pain, especially in the back and limbs, anorexia, nausea, chills, and fever [11].
Te National Blood Transfusion Service of Ghana stated in 2010 that the country requires 250,000 pints of blood annually. While there are some studies regarding malaria parasitemia in donor blood around the middle belt of Ghana [12][13][14], it is uncertain how many transfused blood units contain malaria parasites in the southern part of Ghana. In sub-Saharan Africa, malaria parasitemia in blood donors ranges from 0.6 to 50% [4]. We conducted this preliminary study to add to existing data and determine the occurrence of malaria parasitemia among blood donors in Cape Coast, the capital of the Central Region of Ghana.

Study Site. Te Cape Coast Metropolitan Hospital
(CCMH) is a 98-bed government hospital located at 24 Beulah Road (5.1021°N, 1.2597°W), Cape Coast, Central Region. Te CCMH is equipped with a district-level medical laboratory, obstetrics and gynaecology, paediatrics, male/female wards, antenatal and postnatal care, public health, nutrition, herbal medicine units, a theatre, and a mortuary. Te recent COVID-19 pandemic has led to the addition of a new COVID-19 ward, a COVID-19 PCR testing centre, and an oxygen gas plant. Te hospital's OPD attendance is about 100-150 patients during the day's peak hours (8 : 00 am-2 : 00 pm). Between July and August, when it typically rains, is when malaria cases are at their highest in Cape Coast.
Cape Coast is the regional capital of the Central Region and shares borders with the Gulf of Guinea to the south, Komenda-Edina-Eguafo-Abbrem (KEEA) Municipal to the west, Abura-Asebu-Kwamankese (AAK) District to the east, and Twifo-Hemang Lower Denkyira District to the north ( Figure 1). Facts from the 2021 population and housing census show that the Metropolis has a population of 189,925 people, with 92,790 men and 97,135 women.

Inclusion Criteria.
To be included, participants (volunteers, predeposit, and replacement donors) had blood pressure measurements between 60-90 and 90-140 mmHg (diastolic/systolic) and body weight greater than or equal to 50 kg. Adequate haemoglobin concentration level (>12.5 g/ dl for females and >13.5 g/dl for males) was also considered an important inclusion criterion. Te participants were visibly healthy asymptomatic individuals who showed no signs of malaria, such as fever in the cold or sweating, headache, and clinical signs of anaemia, joint pain, generalized weakness, and vomiting. Both frst-time and repeat donors were included. However, repeat donors should not have donated blood in less than the past three months.

Exclusion Criteria.
All blood donors who did not satisfy the abovementioned descriptions and had tattoos and other visible skin rashes or bruises and responded to having drunk alcohol in the past 12-24 hours or engaged in direct smoking of any form were automatically excluded. Also excluded from the study were those donors who took any antimalarial drugs within the last two weeks before the blood test. Participants who were taking drugs for high blood pressure were excluded. Tough systolic/diastolic measurements may be normal, the bleeding process may trigger a hypertension crisis. Females who were menstruating or breastfeeding were not included. Tose who did not consent at any stage of the screening process were also excluded.

Sampling Method.
Tis study design was cross-sectional. Te participant selection was passive and purposeful, and samples were collected daily from December 2020 through July 2021, until the sample size was achieved. Tese blood donors comprised 224 males and 16 females between the ages of 16 and 45.

Sample Size Estimation.
Our sample size was based on a similar study and population setting conducted by Ofosu et al. at the Asamankese Government Hospital, Eastern Region, Ghana, where a sample size of 240 was estimated [4]. Sample size, n � N/[1 + N(e 2 )], where N is the approximate number of donors screened within the year and e is the margin of error (5%). In our laboratory, an average of 580 blood donors is screened annually. Terefore, n � 580/[1 + 580(0.05 2 )] � 236.73 -240 blood donors.

Sample Collection and
Testing. Te venepuncture technique was used. Venous blood was collected using 5 ml disposable syringes ftted with needles. Te blood was dispensed into 5 ml EDTA tubes and mixed gently.
Laboratory analysis determined the donors' blood group and serological tests (hepatitis B, hepatitis C, syphilis, and HIV). Tick and thin flms were prepared with 6 µl and 2 µl of blood on grease-free labelled slides using a smooth-edged slide spreader. Te thin flm side of each slide was fxed in absolute methanol and air-dried. All smears were then stained with 10% Giemsa for 10 minutes. Te slides were washed with bufered water and air-dried on drying racks. Te sexual and asexual stages of the Plasmodium parasites were identifed using 100× light microscope magnifcation. Te tile method was used to determine a blood donor's ABO group. Tree drops each of anti-sera A (anti-A), anti-B, and anti-D were applied to a clean and dry white ceramic tile using a Pasteur pipette. Equal amounts of thoroughly mixed donor blood were mixed with the antisera over a 1.5 cm diameter area on the tile. After 2 minutes of incubation at room temperature, the mixtures on the tile were examined for agglutination. Data on the age and sex of each donor were documented during blood collection.
2.6. Statistical Analysis. Data entry and analysis were carried out using Statistical Package for the Social Sciences (IBM ® SPSS, Version 25.0, https://www.ibm.com), software for Windows. Te data extracted were analysed and presented using descriptive statistics according to age, sex, blood group, and malaria parasitemia outcome. Binary logistic regression analysis on the malaria microscopy test outcome (dependent variable) and the independent variables listed above did not yield signifcant associations (p > 0.05). Te numbers in the subgroups were not enough to test for associations. Terefore, the table for the regression analysis was not included in the results.

Ethical Consideration.
Blood donors whose data were used in this article voluntarily agreed to have their anonymized information recorded through informed consent. Te hospital granted permission for this study, provided that the data were only used for research and learning purposes, would help improve the management of patients in the future, would cause no harm under the Declaration of Helsinki (1964), and was part of the requirements for allied health professionals working in the hospital's laboratory to renew licenses. Participants screened out of the blood donation process were provided adequate support through the hospital's counselling unit and treated.
Six of the 240 individuals examined were infected, indicating an overall prevalence of 2.5%. Tese six (2.5%) individuals were infected with Plasmodium falciparum. Of  (Table 2). Most disqualifed individuals were reactive for syphilis (22.1%) (53/240), followed by hepatitis B (13.3%) (32/240). One blood donor (0.4%) tested positive for HIV. One of the positive donors for hepatitis B was also coinfected with malarial parasites. One blood donor who was reactive to syphilis had malaria parasites (Table 3).

Discussion
Malaria, in general, is not only a serious threat because posttransfusion malaria can exacerbate recipients' already poor health, but it can also be fatal. Terefore, the need for efective donor selection guidelines cannot be overstated [15]. Tis study sought to determine the prevalence of malaria infection among blood donors in Cape Coast. Blood donors were individuals who had visited the laboratory to either donate blood for a relative, replace blood for a relative, or in walk randomly to donate blood voluntarily. Te overall prevalence of malaria parasitemia among blood donors was 2.5%, while that among those who passed the screening test and donated was 1.7%.
All malaria-positive blood donors in this study were infected with Plasmodium falciparum, which is not a surprising or solitary fnding. Tis fnding is congruent with other studies where P. falciparum was either the predominant or the only Plasmodium species detected [11]. Plasmodium falciparum is the most endemic malaria species in Ghana and sub-Saharan Africa, and it is recognized as the severest form of malaria and is responsible for most malaria deaths [12,16,17].
Te prevalence in this study is comparable to another conducted among blood donors at the Asamankese Government Hospital in Ghana, which was 2.7% [4]. As well, our prevalence is lower when compared to similar studies such as 4.1% in Maiduguri, Nigeria [18], 6.8% in Zaria, Nigeria [19], 7.5% in Kaduna, Nigeria [11], and 13.0% in Kumasi, Ghana [12]. Tis study's prevalence was lower when compared to some other studies, such as 28.0% in Jos [20] and Lagos, Nigeria [21], 30.2% in Nnewi, Nigeria [22], 51.5% in Abakaliki, Nigeria [2], and 67.5% in Port Harcourt, Nigeria [16]. Our fndings are also in contrast to what has been reported in some countries, such as 0.76% in Dhaka City, Bangladesh [23], 0.58% in Peshawar, Pakistan [24], and 0.00% on microscopic examination in India [10]. Te different geographical locations and levels of endemicity could account for the variations in the proportions.
Typically, more males than females volunteer to donate blood. Most females do not meet the haemoglobin concentration requirement for donating blood. Tis phenomenon, therefore, accounts for the large diference between male and female participants in this study. Te high male participation in blood donation could account for the higher prevalence of malaria in males (2.5%) than in females (0.0%). Te higher prevalence rate observed for the males in this study is in harmony with Muntaka and Opoku-Okrah [12] and Mbanugo and Emenalo [25]. Still, it difers from Otajevwo's [26] fndings and Vlassof and Bonilla's [27], where females had a higher Plasmodium parasite infection rate than males. Te highest prevalence was among those 26-35 years old (1.3%, 3/ 240), as observed by Okocha et al. [22].
Although the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) methods for all these diseases are available commercially, they are only available in tertiary care health institutions in Ghana. As laboratories in Ghana continue to participate in the SLMTA (Strengthening Laboratory Management Trough Accreditation) programme [28], introduced in 2009, challenges with infrastructure and upgrading medical laboratories with modern equipment to facilitate the use of more sensitive testing will be available in resource-limited settings as well.    Journal of Tropical Medicine Te most common blood group was "O," and the least common was "AB." Our study is consistent with most reports showing the dominance of blood group O [20,29] among blood donors. All positive malaria cases were among blood group O individuals. To a large extent, this outcome is biased because blood group O is the most sought-after; therefore, we perceive that individuals with blood group O mostly volunteer to support their friends and relatives. Tis reason could account for the observation in this study. Our opinion difers from that of Abah and Joe-Clif [7]. Tey hypothesized that blood group O might protect against severe malaria and even prevent people with blood type O from dying. Tey concluded from their study that this reasoning could illustrate why blood group O is the predominant group infected with malaria parasites. Rhesus-positive donors accounted for 96.3% of the total participants and constituted 2.1% (5/240) of the total prevalence. Te rhesus-negative participants had a prevalence of 0.4% (1/240).
In 2005, Kitchen et al. published a review on Plasmodium species in 100 transfusion-transmitted malaria (TTM) case reports with varying occurrences of the following: P. falciparum (45%), P. malariae (30%), P. vivax (16.0%), P. ovale (4%), P. knowlesi (2%), and a mixed infection comprising P. falciparum and P. malariae (1%) [30]. Natural infection through the bite of an infected female Anopheles mosquito and TTM vary markedly, with the former going through the liver stage, which stimulates host defense cells against malaria parasites and gives the naive host time to produce a much more directed protective immunity. Te risk of complications rises when infected blood transfusions directly release malaria parasites into the recipient's bloodstream, inhibiting the activation of innate immunity [31]. Tus, malaria presents a serious complication in blood transfusions despite being rare. Te presence of asymptomatic blood donors who are primarily "partially immune" and have very low parasite loads is the main issue with TTM. Due to this, donor screening using thick and thin blood smears, the gold standard for diagnosing malaria, might not be the most suitable [32].
Although not sufciently supported and implemented, exciting novel technologies are on the horizon in western nations. Te most prevalent and virulent form of malaria in sub-Saharan Africa, P. falciparum, is highly sensitive to inactivation by photochemical treatment with amotosalen and long-wavelength ultraviolet light [33,34]. Tese pathogen reduction measures have the potential to lower TTM.

Strength of the Study
(1) Tests were performed immediately with the screening sample and not from stored blood to ensure that the parasite and red blood cell biological features did not deteriorate.

Limitations of the Study
(1) Although three WHO-trained malaria microscopists thoroughly examined blood flms in our laboratory, much more sensitive methods are required for comparison. Tese methods will enable us to screen blood that the "gold standard" might miss. (2) Sampling was purposeful; hence, we perceive that most blood donors knew their blood group and might have had experience with blood donation. We envisage a diferent outcome in a nonhealth facility or mobile session blood donation exercise. (3) To realize our turnaround time for the blood donation, we could not administer questionnaires to investigate other relevant information. We relied on routine hospital records for information.

Conclusion
A 2.5% malaria parasite prevalence among asymptomatic blood donors suggests a risk to blood recipients, particularly those susceptible to malaria. Tis outcome suggests the possibility of transfusion-related malaria. In the regular assessment of potential blood donors, we propose that all blood earmarked for transfusion be screened for malaria parasites and labelled negative or positive. Malaria-infected blood may be useful to patients whose circumstances permit malaria prevention therapy. Te reason is that malaria is curable, and rejecting low-intensity malaria-infected blood might exacerbate the country's present donor blood shortage. Tere is, however, an urgent need, in the short term, to determine prophylactic and treatment protocols for posttransfusion malaria in endemic regions, including Ghana.

Data Availability
All the data in this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.