Viral infections in homozygous sickle cell children who have received transfusions

INTRODUCTION

Sickle cell disease is the most common genetic disorder in the world.^[1] It affects nearly 300,000 births/year, mainly in sub-Saharan Africa, India and regions with high levels of African immigration. It results from the substitution of glutamic acid by valine at the sixth codon of the B-globin gene, leading to the formation of abnormal haemoglobin S.^[2] This is responsible for red blood cell falciformation and major complications such as vaso-occlusive crises, chronic haemolytic anaemia and increased susceptibility to infections.

Blood transfusions are often essential for the management and/or prevention of these complications, but they expose patients to the risk of viral infections (human immunodeficiency virus [HIV], cytomegalovirus [CMV], hepatitis B and C) due to technical shortcomings in donor screening and in the qualification of phenotyped and leucocyte-depleted blood.^[3] Transfusion–transmissible viral infections (TTVI) pose a significant challenge to transfusion safety, particularly in resource-limited countries.

This risk of infection transmission through transfusion is unavoidable since transfused labile blood products are biological products derived from humans, and the emergence of new pathogens is frequent, thus requiring constant vigilance and individual assessment of transfusion risk.^[4,5] According to the World Health Organization, transfusiontransmissible viral infections (TTVI) are frequent and are responsible for 5.8% of donation rejections.^[6] In China, the overall prevalence of TTVI was 8.96%.^[7]

Significant advances in blood transfusion in developed countries have reduced the residual risk of viral transmission (HIV, hepatitis B virus [HBV], hepatitis C virus [HCV]) to less than one case per million donations. In contrast, sub-Saharan African countries continue to face significant challenges in screening and managing infectious risks. The seroprevalence of these TTVI remains high, ranging from 2% to 16.6%.^[8-10] In India, five children were infected with HIV after a blood transfusion.^[11]

In Congo, a prospective study of patients living with homozygous sickle cell disease showed widespread use of red blood cell concentrates (RBCs) in the management of acute crises, but with limitations in administering non-leucocyte-depleted RBCs due to lack of resources, thereby increasing the risk of alloimmunisation and post-transfusion infections.^[12] Another study on serological monitoring, which included only 19 children with sickle cell disease, noted seroconversion to HIV (1 case), HBV (1 case), C (1 case) and CMV (3 cases) after blood transfusion.^[10] However, very little data specific to the paediatric sickle cell population is available. Therefore, with the aim of contributing to improving the care of children with sickle cell disease, the study sought to determine the seroprevalence of TTVI and identify the associated factors.

MATERIAL AND METHODS

This was a multicentre analytical cross-sectional study conducted from 1 December 2023 to 30 November 2024 in the paediatric departments of the University Hospital Centre and the National Sickle Cell Disease Reference Centre (NSCDRC) in Brazzaville. It involved children aged 1–17 living with major sickle cell syndrome, who were hospitalised or receiving outpatient care, had received RBC transfusions for at least 3 months, and whose parents or legal guardians had consented to participate in the study. These children were divided into two groups based on the results of viral serology testing. Those who tested seropositive were then compared to seronegative children in order to identify factors potentially associated with blood transfusion–transmissible infections (BTTIs).

The choice of study setting was deliberate, given that the paediatric departments of the Brazzaville University Hospital and the National Reference Center for Sickle Cell Disease (CNRDr) are the primary sites for monitoring children with sickle cell disease residing in Brazzaville and the surrounding area. Sampling of patients meeting the inclusion criteria was conducted consecutively and exhaustively on their admission, resulting in a sample size of 187 children with sickle cell disease.

Each child underwent venipuncture, preferably at the elbow, with 4 mL of whole blood collected in tubes containing ethylenediaminetetraacetic acid. The samples were centrifuged at 3,000 rpm for 5 min to obtain plasma. The plasma was then divided into two cryopreservation tubes. The samples were transported to the NSCDRC biomedical analysis laboratory, where they were stored at −20°C for an average of 7 days before serological analysis. HIV testing was performed by combined detection of p24 antigen and anti-HIV1/2 antibodies using Abbott test strips (DETERMINE^™), based on the principle of immunochromatographic migration. The presence of antibodies was confirmed by electrochemiluminescence using the Cobas^® e411 analyser. The principle was based on successive incubations of plasma (in the presence of detergent, biotinylated anti-p24 monoclonal antibodies biotinylated anti-p24 monoclonal antibodies, recombinant HIV-specific antigens/peptides and streptavidin-coated microparticles), followed by aspiration of the reaction mixture into the measuring cell where the microparticles were magnetically captured on the surface of the electrode. Unbound substances were removed with ProCell/ProCell M. Applying voltage to the electrode induced chemiluminescent emission, which was measured by a photomultiplier. Qualitative detection of hepatitis B and C viruses and CMV was performed using immunochromatographic membrane migration tests: AgHBs BIO-Check^™ using anti-AgHBs antibodies immobilised on the test area, anti-HCV BIO-Check^™ antibodies through the pre-attached protein A and the ‘MICROPOINT^™ CMV IgM’ device using purified recombinant CMV antigens as the capture reagent. Interpretation was performed by visual reading: two coloured bands indicated a positive result, the absence of a band in the test zone indicated a negative result, while the band in the control zone validated the test.

For each child, the variables studied concerned sociodemographic characteristics (age, gender, origin, academic performance, level of education, parents’ socioeconomic status); medical history and clinical characteristics (circumstances of disease discovery, haemoglobin profile, transfusion history, number of red blood cell packs transfused), haematological (complete blood count) and virological (anti-HIV1/2 antibodies combined with p24 antigen, anti-HCV antibodies, HBs antigen, anti-CMV immunoglobulin M) characteristics. Polytransfusion was defined as a history of at least three transfusion episodes, regardless of the number of bags received.^[13]

RESULTS

Seroprevalence of viral infections

The overall seroprevalence of TTVI was 8.5% (n = 16). Figure 1 shows the distribution of serological markers for HIV1/2, hepatitis B and C and CMV TTVI according to seropositive sickle cell children and Table 2 shows the distribution of the serological status of sickle cell children who received transfusions by age group. The mean number of transfusion episodes in seropositive transfused sickle cell patients was 2.7 ± 1.6 versus 3.1 ± 2.6 (P = 0.3) and the mean number of RBCs transfused was 2.7 ± 1.7 versus 3.2 ± 2.7 (P = 0.2).

Table 1: Distribution of children with sickle cell disease who received transfusions, by reason for hospitalisation.

Reasons for hospitalisation	n	Percentage
Malaria	123	65,8
Pneumonia	20	10.7
URTI*	13	7
Digestive infection	10	5.4
Bone infection	7	3.7
Bacteraemia	5	2.7
SIRS**	2	1
Adenophlegmon	2	1
Splenectomy	2	1
Splenic sequestration	3	1.6
Total	187	100

*URTI: Upper respiratory tract infection, **SIRS: Systemic inflammatory response syndrome

Table 2: Distribution of children who tested positive by age group.

Age group (years)	n	Children who tested positive	Percentage
(0–5)	48	4	4,3
(5–10)	72	8	11
(10–17)	67	4	6
Total	187	16	8.6

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Figure 1:

Distribution of human immunodeficiency virus (HIV), cytomegalovirus (CMV) and hepatitis B (HBV) and C (HVC) virus. anti-Cytomegolovirus-H immunoglobulin (Anti-CMV) anti-hepatitis B (HBs) antigens

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DISCUSSION

The study conducted amongst 187 children with sickle cell disease who received transfusions in Brazzaville determined the seroprevalence of TTVI. The average age of the children with sickle cell disease who received transfusions in our sample was 8.9 ± 0.6 years; with those aged 5–10 being the most represented, an age group in which complications from the disease are more frequent, as highlighted by other authors.^[14,15] They show academic delay in 2% of cases, unlike Talla in Cameroon, who reports 61% of children with academic delay.^[16] The high proportion of parents with secondary or higher education in this study could positively influence access to and adherence to care, as confirmed by the work of Luboya and Piel on the importance of educational support in the management of patients with sickle cell disease.^[17,18] The main causes of hospitalisation for children with sickle cell disease who have received transfusions remain infections, including malaria, which is widely represented (65.8%), followed by acute lower respiratory infections (10.7%). These results are consistent with the epidemiology of infectious diseases in sub-Saharan Africa, where malaria remains a significant cause of morbidity and mortality in children.^[19-22] Severe anaemia remains the main indication for blood transfusion (98%) in connection with chronic haemolysis, often aggravated by malaria, which is highly endemic in Brazzaville. These results are similar to those reported by McAuley in Kenya and Uyoga, who report a high prevalence of malaria-related haemolytic anaemia in children with sickle cell disease.^[23,24]

The overall seroprevalence of BTTIs is 8.5%; the agents detected are CMV (2.1%), HIV (2.1%) and HCV (3.7%). No cases of HBs antigen positivity were found.

This high overall seroprevalence is similar to that reported in African studies of children living with sickle cell disease, ranging from 5% to 12%.^[25-28] It remains higher than the rates observed in developed countries, where transfusion safety measures have reduced this risk to <1%.^[29,30] It is higher in children aged 5–10 years (11%), an age group in which complications of the disease are more frequent and blood transfusions are increasingly repeated, as reported by Fiawoo in Togo.^[14]

The HCV infection rate found in 3.7% of children in this series is comparable to certain data from Cameroon (3.9%)^[9] and Burkina Faso (2.8%),^[3] but lower than that reported in Nigeria: 15.8%.^[31] This rate is significantly higher than the prevalence rates reported in Western countries (<0.1%), as shown in Pirenne’s study.^[32] The lack of systematic screening for this infection, the high frequency of intra-family blood donations in low-resource countries and the non-use of leucocyte-depleted blood products could explain these high prevalences. This is common amongst children with sickle cell disease who receive transfusions in Africa, as highlighted by Kissou et al.^[3] It is therefore necessary to strengthen virological screening of donations, ideally using molecular biology, which is still not widely available in countries with limited resources.^[33]

With regard to HIV, the seroprevalence of 2.1% observed is lower than that reported by other authors (7.9%; 11.5%),^[31,34] but remains a cause for concern as it reflects the persistence of a transfusion risk despite efforts to make blood safe in Congo. Human immunodeficiency virus (HIV) screening using combined tests antigens (Ag)/antibodies (Ab) improves detection but does not eliminate the risk associated with the ‘serological window’. This result is similar to those reported by Kitenge in the Democratic Republic of Congo, who found a prevalence of 2%.^[35] He thus highlights the residual risk of virus transmission during transfusion qualification practices. Systematic screening of donors is still only partial. This risk of post-transfusion infections is currently minimal in Europe thanks to the strengthening of transfusion safety procedures.^[30,36]

CMV infection accounts for 2.1% of cases in our series. This result is consistent with data in the literature highlighting the significant role of CMV in morbidity amongst transfused patients in Africa, due to the high prevalence of the virus in the general population and the frequent lack of systematic screening.^[37,38] Diop and Pirenne had already pointed out in 2021 that this virus is underestimated in transfusion screening. The use of irradiated blood products, as in industrialised countries, could reduce the risk of CMV transmission in immunocompromised patients, including those with sickle cell disease.^[14,39,40]

Unlike certain African studies^[41-43] in which HBV is often more prevalent than HCV, no positive cases were noted. The existence of a screening programme for this infection, but above all the introduction and widespread use of the hepatitis B vaccine in the Expanded Program on Immunization in Congo, could explain these results.^[40] In Europe, vaccination campaigns and strict screening have almost completely eradicated post-transfusion cases of HBV.^[33,37]

Positive children with TTVI more frequently presented with mucosal pallor and physical asthenia, associated with lower haemoglobin levels (P <0.05). This confirms that TTVI aggravates the clinical fragility of already vulnerable children with sickle cell disease.^[46]

CONCLUSION

The overall prevalence of viral infections amongst transfused sickle cell patients is high in Brazzaville, reflecting the vulnerability of this population group. The main infections found are HCV, HIV and CMV. These infections occur more frequently in children with sickle cell disease who have an altered clinical condition and low haemoglobin levels, requiring increased vigilance in transfusion screening. The high prevalence of viral infections amongst children with sickle cell disease who receive transfusions requires that transfusion safety activities be strengthened. A further haemovigilance study is necessary.