Strategy to improve malaria surveillance system preventing transfusion-transmitted malaria in blood banks using molecular diagnostic

Malaria is an infectious disease caused by parasites of the Plasmodium genus. In addition to being primarily transmitted by bites of female mosquitoes of Anopheles species, malaria is readily transmitted by blood transfusion through donations collected from asymptomatic or parasitic donors, especially those individuals with low parasite densities [1‐4]. The parasite is released into the bloodstream during its life cycle and will therefore be present in blood donated by infected individuals [5‐7].

The erythrocyte concentrate is the main blood component involved in transfusion-transmitted malaria (TTM), but there are reports of transmission by fresh frozen plasma, cryoprecipitate and platelets concentrate. When preserved in blood units, the parasite remains viable for a long period of time, even after storage. After transfusion, the disease begins in a matter of days or weeks, with severe and often fatal evolution, especially in immunodepressed individuals, young children, pregnant women, and people who have never been exposed to malaria parasites [6, 7].

Haemovigilance is required to identify and prevent the occurrence or recurrence of unwanted transfusion-related transmissions and to increase the safety, efficacy and efficiency of blood transfusions [8]. This vigilance system should include the monitoring of malaria in endemic areas, such as Africa, South America and some areas in Asia, to detect the five species of the Plasmodium genus that infect humans, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi [2, 9].

Malaria transmitted by blood transfusions has been described since the early 1900s. The first report on malaria as a consequence of an artery-to-vein blood transfusion was described in 1910 by Woolsey [5, 10]. However, the first case of TTM following the use of stored blood was described in 1941, and 1756 cases were reported in 49 countries between the years 1950 and 1972 [1]. In the last 40 years, the increased frequency of blood transfusion in medical practice and increase in travelling between countries in which malaria is absent and those where malaria is endemic reveals a clinical and public health problem [4, 8]. Moreover, the urbanization of malaria transmission areas in countries where the disease is endemic has contributed to an increased possibility of accidental transmission of Plasmodium infections through blood transfusion [11]. However, there is a paucity of officially registered data, which depends on appropriate tools for detection of malaria parasites.

An accurate diagnosis of Plasmodium infections is essential for malaria control or eradication [2, 9, 12]. Examination of thick blood smears is performed to detect positivity for Plasmodium, but this test has limited sensitivity to detect asymptomatic infections with low parasitaemia [13]. The lack of precise malaria diagnosis remains an important obstacle that limits the prompt detection and identification of Plasmodium species in the case of individuals with low parasitaemia [14].

In this context, for the first time, an innovative strategy was developed to detect the malaria parasites based on mitochondrial DNA (mtDNA) of malaria parasites, P. falciparum and P. vivax [15]. Efforts have been done to develop tools for disease control, especially to prevent TTM, by reporting the real possibility of transmission and the frequencies of contaminated samples from blood donors who resided in a large urban area within the malaria-endemic region [16]. After development and application of this novel method, other studies have reported the use of mtDNA for malaria diagnosis by nested PCR [17‐21] or loop-mediated isothermal amplification (LAMP) [22, 23], thereby confirming the relevance of this innovative strategy of using mtDNA as a marker for malaria diagnostic tests [15].

Historically, the conventional malaria control has mostly taken aim at P. falciparum, and even its aggressive implementation has few lasting impacts upon endemic P. vivax [9, 12]. In part, it occurs because this resilient species requires multiple fronts of attack, which include the asymptomatic and sub-patent reservoirs [24, 25]. In general, transfusion malaria is not registered by national authorities. Since 2002, the official data reported only 4 cases transmitted by transfusions from the blood banks located in the Brazilian malaria-endemic area [11].

In Brazil, malaria is endemic in the Amazon region, where P. vivax is the main species. In the past two decades, the numbers of malaria cases caused by this parasite have varied between 100,000 and 500,000 per year, representing approximately 85% of all reported cases [26, 27]. Because P. vivax is able to develop latency stages (hypnozoites) in the liver, which leads to relapses, and its life cycle occurs inside the reticulocytes, these factors may contribute to an increase in the number of individuals with low parasite load, which are not usually detected by healthcare facilities [24, 25]. Moreover, in this endemic area, a high prevalence of asymptomatic Plasmodium infection has been reported [28]. This is an aspect that can lead to uncommon ways of transmission, such as TTM [1, 2].

In this study, the performance of molecular approach was improved for detection of malaria parasites via mitochondrial DNA as a careful screening test of blood samples from donors, and compared the more sensitive molecular technique with those used in haemovigilance as the best strategies for detecting P. falciparum, P. vivax and P. malariae. In addition, for the P. vivax parasite, the prevalence of positive samples was determined as units for TTM occurrence upon the transfusion of blood or its components from infected donors living in malaria-endemic urban areas in Brazil.

TTM is recognized in many countries where malaria transmission has been eliminated [6]. Although the frequency of this route of transmission is very low in non-endemic malaria areas, it is a substantial problem that involves the risk of receiving a unit of erythrocyte concentrate contaminated with parasites [7, 33]. Transfusion-associated cases of malaria have been reported in many parts of the world, including the USA [1, 3, 34], Canada [35], Spain [36], England [37], and France [38], Therefore, it should be noted that TTM may be implicated in the reintroduction of malaria into areas where it had previously been eradicated.

Conversely, the risk of transmitting malaria by blood transfusion in countries where parasite transmission is endemic can be high, and the healthcare systems clearly need alternative measures to prevented TTM, including tools for detecting the parasite or its components in the blood of potentially infected donors [2, 7, 11].

Although the exact numbers are unknown, the frequency of TTM has been estimated as 1 case per 4 million units donated in non-endemic areas [36, 37]. Moreover, it is worth considering that in the malaria-endemic areas, it is a health problem as reported for Africa [7, 39, 40], Asia [41] and South America, including Brazil, where infected donors were detected in 2 studies that used different molecular approaches to test samples collected in blood banks located in the Amazon region [16, 42]. Besides, TTM is a disease with a high lethality rate: all cases notified in Brazil since 2005 were fatal [43].

Malaria screening is mandatory in endemic regions and the directives of the Brazilian National Health Surveillance Agency (ANVISA) and the Brazilian Ministry of Health require high sensitivity tests. In spite of this, malaria screening in Brazilian blood banks relies on questionnaire data followed by microscopic examination of thick smears, which has several limitations, such as low counts of parasitaemia in asymptomatic patients which may lead to liability to human error [11].

In this study there was an improvement in the methodology developed by Cunha et al., which originally used a strategy based on mtDNA to amplify specific sequences of the Cytochrome C Oxidase genes, such as COXIII and COXI for P. falciparum and P. vivax, respectively. The conventional PCR methodology was changed to a more sensitive and semi-automated technique using one-step qPCR and the same mtDNA sequences. Additionally, mt-qPCR specific to P. malariae was standardized. The conventional PCR identified small amplicons of 273 pb (P. falciparum) and 290 pb (P. vivax) [15] allowing the parasite mtDNA amplification in a shorter time than in the classic nested-PCR based on ribosomal RNA, which has been used for molecular diagnosis of malaria over the past two decades [44].

This new methodology was applied for the detection of 3 Plasmodium species (P. falciparum, P. vivax, and P. malariae) more frequently found as the infecting agents among the 5 species that cause human malaria. The results confirmed that potentially infected volunteers can be among the donors. Although the frequency was low, it represents an even more complicated issue considering that each donation can be distributed over erythrocyte concentrates, platelet concentrates, or serum or plasma/platelets concentrates. Plasmodium falciparum and Plasmodium vivax tests were included, but only samples infected with P. vivax were detected. This can be explained by the fact that this is an area of higher incidence of P. vivax. It is well-characterized as an endemic area where P. vivax causes approximately 85% of malaria episodes registered by the Brazilian Health System [26, 27].

Another contribution was the qPCR designed specifically for detecting mtDNA of P. malariae. This innovative approach, although only preliminarily tested one positive sample, proved to be a suitable screening tool for identification of infected patients. The detection threshold curve varied depending on the infecting species. This suggests that P. malariae, which frequently occur in low parasitaemia, can also be detected by mt-qPCR. Future studies may be conducted to validate this assay in a cross-sectional study. In Brazil, it is necessary because there are certain issues with the transmission of P. malariae that need to be addressed, which include a high prevalence of P. malariae infections in the Brazilian Amazon endemic area [45], as well as a case of TTM after a transfusion of blood donated by an asymptomatic donor infected with this parasite [46]. In both studies, nested-PCR detecting rRNA was used for diagnosis.

The major advantage of using mitochondrial DNA (mtDNA) in malaria diagnosis as originally reported [15] is the largest copy number/cell of the parasite mitochondrial genome, from 20 to 150 copies/parasite [18, 31, 32]. The potentially infectious parasitaemia is largely variable in TTM cases. An estimated value is 10 parasites/unit in 250 mL of erythrocyte concentrate. In this case, a molecular diagnostic method to provide a trustworthy analysis should have a very low parasite detection threshold [33].

Another important point to be considered is the volume of blood needed to obtain the DNA, since the amount of processed specimen determines the limit of detection [33]. Some questions have been raised regarding the volume of blood that should be tested because only a small part of the donor’s total blood volume, which will be transferred to the recipient individual, is included in the molecular analysis. Hence, different concentrations of DNA obtained from P. vivax-infected patients were tested, and the estimated detection threshold was very low (0.000006 parasites/µL) when compared to the detection threshold of the nested PCR (0.004 parasites/µL) [36].

Two research groups discussed this issue and proposed a hypothetical detection threshold, using calculations that offered the hypothetical value as the threshold [33, 36]. Thus, a threshold could be established corresponding to the probability that analysis of a very small volume of the whole blood bag produces a reliable result that may serve as the basis for a conclusion regarding whether the screening could be positive in very low parasitaemia. The qPCR using mtDNA as template for identification of P. vivax proved that it was possible to reach the hypothetical detection threshold to detect parasitaemias as low as 2.2 × 10⁻⁶ parasites/µL, as described by Hänscheid et al. [33] in a commentary on the use of PCR for screening blood donors tested by Benito and Rubio [36]. Our tests showed that mt-qPCR detected P. vivax at 2.4 × 10⁻⁵ parasites/4 µL (6.0 × 10⁻⁶ parasites/µL) density by analysing DNA from a suitable blood volume, thus achieving analytical sensitivity very close to the value estimated almost two decades ago [33], that is, before the routine use of more sensitive molecular techniques such as real-time PCR.

Malaria induced by blood transfusions from asymptomatic carriers is a relevant problem of haemovigilance. A review of the duration of P. falciparum infections has illustrated how these infections have been studied for decades, and based on the data from these studies, it is possible to suggest that for blood transfusion-transmitted malaria, the inoculum should contain approximately 1000 parasites in a blood bag or bottle of approximately 500 mL. Thus, the transmission density approached an average of 2 parasites/mL [5]. A appropriate screening tool for malaria detection in blood units must have a high sensitivity [33]. It must allow the reduction of TTM risk as well as that of falsely deferred blood donors.

Finally, this analysis proved that it was possible to considerably improve the assay to detect the parasite mtDNA, compared to conventional PCR, where the detection threshold was 0.01 parasites/µL [15]. Using mt-qPCR, the achieved threshold was substantially lower, 0.000006 parasites/µL. This confirms the high efficiency of this methodology to detect parasite mtDNA (P. vivax) analysing only 150 µL out of a total of approximately 450 mL of blood.

Prevention of TTM relies entirely on the exclusion of potentially infected donors. However, it can be very difficult since in many cases, the donor screening process is not efficient and fails to obtain accurate information on previous exposure to malaria and identification of the infected donors. It represents a situation that can particularly occur for donors in blood banks of endemic-malaria areas [7, 11, 39].

Analysing the frequency of positive donor detection by molecular diagnostic tools for samples from blood banks from the malaria-endemic area in Brazil, it was identified only two studies up until the present that determined the prevalence of positive samples of Plasmodium species among donors that live in the Amazon region, which varies from 1 to 3%. Both studies included samples collected from donors of the blood banks of the Belém city, and the prevalence observed was similar, 1.34% [16] and 1% [42]. It should also be noted that in these studies with blood bank samples, it was only detected positives for a single species, P. vivax [16], while others reported mixed infection [42]. It can be suggested that mt-qPCR provides a more specific amplification. In a malaria-free area, a non-endemic state located outside the Amazon region, cases of positive samples from blood donors were registered [47]. This motivates an intense discussion that involves technical and epidemiological aspects and exposes how important it is to provide more accurate data, as well as to provide new highly sensitive tools for better explanations of this situation [47‐49].

A suitable screening tool for malaria detection in blood units must have high sensitivity and specificity; it must be fast and reduce the risk of TTM [2, 4]. Considering the limitations of the thick blood smear as a screening method in blood banks, it is suggested that the cost-effectiveness of introducing a molecular diagnostic tool for Plasmodium species in the blood banks of endemic areas or in micro-regions should be evaluated in addition to the possibility of reducing the risks of TTM. Malaria diagnostic research provides a basis for applied advances in malaria surveillance [9, 13‐15]. For this, the laboratorial blood screening can be a critical point in malaria eradication. In the case of TTM, one aspect that must be considered is the low parasitaemia of infected donors.

In conclusion, qPCR is efficient for malaria molecular diagnostics, presenting promising results with good analytic sensitivity. It is a fast and easy molecular methodology to detect mtDNA of the three most frequent malaria parasites, presenting a potential for large-scale use in the prevention of transfusion-transmitted malaria by screening for potential donors as part of malaria haemovigilance in blood banks.