Immunological testing for malaria and blood donor deferral: the experience of the Ca’ Granda Polyclinic Hospital in Milan

Abstract

Background.

Current European regulations require a deferral period of 6 months or 3 years, depending on the risk of exposure, for prospective blood donors at risk of malaria. This period may be reduced to 4 months if an immunological or molecular genomic test is negative at each donation, but Italian regulations have not adopted this provision. As cases of transfusion-transmitted malaria have been recorded in medical literature in blood donors deferred for 3 years and not tested, the Immunohaematology and Transfusion Centre of the Ca’ Grande Polyclinic Hospital in Milan decided to introduce immunological testing for all donors at risk of malaria.

Materials and methods.

Four hundred and twelve blood donors at risk of malaria, who had lived in a malarial area during the first 5 years of life or for more than 6 consecutive months, were tested for malarial antibodies using an enzyme immunoassay kit. The kit (Malaria EIA, Newmarket, UK) uses four recombinant antigens specific for P. falciparum and P. vivax and with cross-reactivity for P. ovale and P. malariae. The kit detects total immunoglobulin antibodies against P. falciparum and P. vivax and shows 80% cross-reactivity with P. ovale and 67% with P. malariae. Antibody-positive samples were further checked by an immunochromatographic test for P. falciparum, P. vivax, P. ovale and P. malariae antigens and by haemoscopy (thin film and thick smear).

Results.

Italian citizens accounted for 16.8% (69/412) of the whole group of donors examined. We found that 8.7% of the donors who were classified as being at risk of malaria were positive for total immunoglobulin antibodies. Only one Italian citizen resulted positive for the test. The positive candidates were deferred from blood donation. None of the antibody-positive donors was confirmed positive by the immunochromatographic test and by haemoscopy.

Conclusion.

The introduction of a malarial screening test in the assessment of blood donor eligibility may increase the safety of blood donations, but could further reduce blood availability. If immunological testing were to be accepted nationally as a valid method of assessing the risk of malaria, more than 90% of the donors who are currently deferred for 3 years could be accepted 4 months after their last visit to an endemic area, thus increasing the availability of blood

Introduction

Malaria infection is caused by a blood protozoa of the genus Plasmodium. Usually malaria is transmitted by the bite of an infected Anopheles mosquito, but cases of transfusion-transmitted malaria (TTM) have been recorded since 1911¹. TTM can be acquired through blood components such as red cell concentrates, platelets², leucocytes³, fresh-frozen plasma⁴ and frozen red blood cells⁵. TTM is very rare in countries in which malaria is not endemic, such as Italy, where the incidence of TTM ranges from 0 to 2 cases per million blood donations⁶. Nevertheless, this disease has an impact on the resources of Italian Blood Banks because over the last few years more and more prospective donors have been coming from malaria endemic areas either as tourists, immigrants or workers. Malaria is currently re-emerging in many areas where it had been eradicated in the past and, consequently, the number of subjects coming from malaria areas has risen. In Italy, the deferral criteria for blood donors at risk of malaria are defined by the Decree of the Ministry of Health dated March 3^rd, 2005, published in the Gazzetta Ufficiale N.85, April 13^th, 2005⁷. Whole blood donation is deferred for 3 years in potential donors who lived the first 5 years of their life in malaria endemic areas, and for 6 months in potential donors returning from an endemic area and those who have been infected previously. Italian laws comply with current European regulations⁸, which require a deferral period of 6 months or 3 years depending on the risk of exposure for prospective blood donors at risk. This period may, however, be reduced to 4 months if an immunological or molecular genomic test is negative at each donation. This provision has not been adopted in Italy. Given that cases of TTM have been recorded in blood donors who were deferred for 3 years but not tested⁹, the Immunohaematology and Transfusion Centre of Milan decided to introduce immunological testing for donors at risk of malaria.

Materials and methods

Materials

From February 1^st, 2007 to June 30^th, 2010, we evaluated 412 blood donors at risk of malaria, because of having lived in a malarial area during the first 5 years of life or for more than 6 consecutive months, for malarial antibodies using an enzyme immunoassay (EIA) kit. Blood from each donor enrolled was collected by venipuncture into a 3 mL serum tube with no additives. For each donor identified as being positive by the EIA, some whole blood drops were collected by pricking the finger of the donor, and examined by optical microscopy. Another 3 mL of whole blood were collected by venipuncture into a test-tube containing EDTA for a rapid diagnostic test of malaria. Italian citizens accounted for 16.8% (69/412) of the whole group of donors examined.

Methods

The malaria antibody EIA (Newmarket, UK; Bio-Rad Italia) is based on binding of anti-Plasmodium antibodies present in a serum sample to antigens immobilised on a solid phase. The antigens are four recombinant types specific for P. falciparum with cross-reactivity for P. ovale and P. malariae (one MSP1 and two MSP2) and one specific antigen for P. vivax (MSP1). The test detects total immunoglobulin (Ig) antibodies against P. falciparum and P. vivax and shows 80% cross-reactivity with P. ovale and 67% with P. malariae. The malaria antibody EIA was performed as recommended by the manufacturer.

The cut-off value was calculated by the average optical density (OD) value of the three negative controls plus 0.100. All results are reported as an odds ratio of sample/cut-off. A reaction was considered negative when this ratio had a value of 1 or less, and positive reaction if the ratio was 1 or more. This sample to cut-off ratio is proportional to the amount of bound antibody¹⁰. The test does not distinguish between IgG and IgM, or between antibodies such as P. falciparum, P. vivax. P. ovale and P. malariae. The assay was carried out manually. Positive donors were investigated for Plasmodia stages in the blood stream using haemoscopy (thin film and thick smear stained with Giemsa)¹¹ and a rapid diagnostic test for evidence of malaria antigens¹² (Figures 1 and 2). Four thin films and four thick smears were prepared for each positive donor. Each stained preparation was observed for at least 20 minutes by an expert microscopist using an optical microscope at a 1,000x magnification¹³. Positive donors were also assayed for malaria antigens in whole blood using the Core Malaria Pan/Pv/Pf^© rapid diagnostic test (FGM, Italy). Monoclonal antibodies for detecting specific antigens for malaria Plasmodia (HRP 2 for P. falciparum, P. vivax-specific pLDH and pan-specific pLDH for all human malarial Plasmodia) are immobilised on the membrane in the test region. The device was used, the tests were performed and the results evaluated following the manufacturer’s recommendations.

Figure 1.

A rapid diagnostic test for malaria showing a positive result for P. falciparum. In addition to the control band, pink/purple bands appear at the Pf and Pan regions, respectively. The Pv region, specific for P. vivax antigen (P. vivax pLDH) is negative. The whole blood sample is dispensed in well A (5 μL). In well B (out of the shot) at the bottom of the device, four drops of the clearing buffer with conjugate antibody were dispensed. Core Malaria Pan/Pv/Pf^® RDT (FGM - Italia). Photograph by Romualdo Grande and Vito Carta.

Figure 2.

Rapid diagnostic test positive, from left, for P. vivax, P. ovale/P. malariae, and P. falciparum (the last two). Pan malaria antigen can be visible for P. vivax and P. falciparum (the second from right). Core Malaria Pan/Pv/Pf^® RDT (FGM - Italia). Photograph by Romualdo Grande and Vito Carta.

Results

Of the 412 donors considered at risk of malaria (Table I), 36 (8.7%) proved positive for total Ig antibodies. The EIA was positive for only one Italian citizen (1.45% of the Italian donors examined). None of these positive EIA results was confirmed by the immunochromatographic test and haemoscopy for malaria antigens and Plasmodia blood stages. The candidates who were positive in the EIA were definitively deferred from making further blood donations (Figure 3). Among the positive group, 17 candidate donors (42.2%) had a high sample/cut-off ratio of greater than 5 (Table II). The threshold value was chosen arbitrarily for this study.

Table I.

Percentages of donors positive for malarial antibodies according to the enzyme immunoassay.

Total donors evaluated in this study	412 (100%)
N. of positive cases	36 (8.7%)
N. of negative cases	376 (91.3%)

Figure 3.

Algorithm used for deferring donors at risk of malaria. Donors' sera are tested using an EIA incorporating three antigens specific for P. falciparum and one specific for P. vivax. The sensitivity and specificity of the EIA were reported by Kitchen et al.¹⁰. Blood products entering the therapeutic circuit tested negative or were not repeat reactive in the EIA and/or haemoscopy and/or the rapid diagnostic test for antigenaemia. In all other cases, the products were discarded and the blood donor deferred to his physician for information, investigations and/or counselling.

Table II.

Percentage of positive donors with a high (>5) S/CO ratio.

Total number of positive cases	36 (100%)
Donors with S/CO ratio >5	17 (42.2%)
Donors with S/CO ratio =1 to =5	19 (57.8%)

Total positives=number of donors positive by the malaria antibody EIA. S/CO ratio=odds ratio OD sample(S)/cut-off (CO)+0.100. Samples with a S/CO=1 were considered positive; a S/CO ratio >5 was considered high.

Discussion

The introduction of a malarial screening test to assess the eligibility of potential blood donors may increase the safety of blood donation, but may also reduce the availability of blood. In this study we used the same EIA as that adopted by the National Blood Service in England¹⁴. This test, distributed and imported into Italy by Bio-Rad Italia, was developed by Newmarket Laboratories in conjunction with the Parasitology Reference Laboratory, Hospital for Tropical Diseases, London, and the National Transfusion Microbiology Reference Laboratory, National Blood Service, England, and is based on an EIA sandwich format incorporating four recombinant antigens for P. falciparum and P. vivax. The performance of the assay was found to be superior to that of an immunofluorescence antibody test¹⁰.

In our experience at the Immunohaematology and Transfusion Centre of Milan, the introduction of a malarial screening test of potential donors with a specific risk of malaria increased the number of permanent deferrals of blood donors. In our study, 36 donors resulted positive for malaria serology and were, consequently, excluded from all donation programmes permanently. In the UK and France donors negative for malaria by immunological tests would have been allowed to donate blood, because any donor can be readmitted 4 months after being in an endemic area if such tests are negative. This practice complies with European guidelines^14,15. In Italy the situation is different because national guidelines do not recognise any diagnostic method for reducing the deferral time following the last visit to an endemic area. In accordance with Italian guidelines, the 36 donors who tested positive in the screening test and had lived in endemic areas for the first 5 years of their lives would have been admitted to the donor programme as they were considered to be in good health at the end of their deferral time. Our decision was, however, to defer all them permanently. The Italian authorities require only that certain clinical criteria are fulfilled in order to re-admit a patient to the donor programme. Further laboratory investigations, such as specific serology for malaria are not mandatory. Serological assays and/or polymerase chain reaction-based techniques are used by some centres⁹: to date, only one centre in Northern Italy has used the same immunoassay as us. Clinical criteria alone are not sufficient to ensure completely safe donations.

At present, the main risk of introducing malaria parasites into the blood supply in countries where malaria is non-endemic comes from semi-immune individuals. Semi-immunity develops in those individuals who generally live in areas with high entomological inoculation rates and are thus multiply exposed to malaria¹⁶ on a regular basis. A dynamic balance develops in the individual, who is generally well and in good health despite the simultaneous presence of both a high titre of antibodes and very low level of parasitaemia. Very low level parasitaemia can remain undetected by optical microscopy; the expected sensitivity of detection of parasitaemia on a thick blood smear by an experienced microscopist is approximately about 50 parasites/μL, which is equivalent to 0.001% infected red blood cells¹⁷. Milne et al., however, found that the sensitivity of detection in most routine diagnostic laboratories is about 10-fold lower (average 0.01% infected red blood cells, 500 parasites/μL)¹⁸. It is important to note that expatriates who have been resident in malarial areas for long periods of time can also become semi-immune. All donors who resulted positive in our study were individuals who had lived in areas of endemic malaria during infancy and/or for at least 5 continuous years. Many of them (42.2%, Table II) had a high antibody titre. The positive donors were negative for haemoscopy (thick smear and thin film) and a rapid diagnostic test. The study raises two very important questions: (i) are high-titre antibody-positive donors (Table II) likely to transmit malaria and (ii) are they semi-immune individuals?

We cannot answer these questions for our potential donors because additional molecular assays such as nucleic acid testing were not performed. We highlight that microscopic techniques and the rapid diagnostic test lack the required sensitivity to detect a very low parasitaemia such as 0.00004 parasites/μL. Although the sensitivity of polymerase chain reaction tests has been reported to be as low as 0.004 parasites/μL, this is 100-less than the required sensitivity¹⁹. To determine whether an individual has a very low level of parasitaemia, for example 1 parasite/mL of whole blood (below the reported sensitivity of current polymerase chain reaction tests), a unit of blood would need to contain at least 200 parasites. However, 10 parasites are enough to transmit malaria through a mosquito-infected bite^20,21. Cases of TTM with negative haemoscopy and positive high antibody titre have been recorded in the literature²².

Serological tests on new donors at risk of malaria could contribute to safer blood donations. This could be useful to reduce the wastage of blood products due to travel-based restrictions. For example, Chiodini et al.²³ estimated that approximately 40,000 units of whole blood were discarded annually as a result of the UK National Blood Service Strategy before the introduction of a new donor deferral strategy based on serological markers plus travel-restricted criteria¹⁶. In the USA, which has a markedly lower rate of imported malaria than the UK, the wastage has been estimated to be 50,000 blood donations annually²⁴. In Italy and in USA, the results of EIA or other indirect diagnostic tests are not recognised for readmission to blood donation programmes. In Italy the percentage of blood donors deferred each year is not known on a national basis. However, given differences in travelling habits of blood donors, figures may vary considerably according to the location of blood centres in the Italian territory, being highest in metropolitan areas (5% in Milan), intermediate in smaller urban centres (0–5%) and lowest in rural areas (0–1%)⁶. The current European Regulations⁸ require a deferral period of 6 months or 3 years depending on the risk of exposure of prospective blood donors. This period may be reduced to 4 months if an immunological or molecular genomic test is negative at each donation.

Since France and the UK have adopted this reduction in compliance with the European Regulations, it could been worthwhile re-evaluating Italy’s current policy of donor deferral for malaria. Lengthy deferral increases the risk of donors not returning. If the Italian policy were to be adapted in compliance with European Regulations, a high percentage of the 412 donors screened for antimalarial antibodies (91.3%) could be readmitted to the donor programme sooner than the current deferral time, reducing the risk of donors abandoning the programme.