Skip to main content
Download PDF

Exploring association between MBL2 gene polymorphisms and the occurrence of clinical blackwater fever through a case–control study in Congolese children

Malaria Journal volume 19, Article number: 25 (2020) Cite this article

Abstract

Background

Blackwater fever (BWF), one of the most severe and life-threatening forms of falciparum malaria, is characterized by acute massive intravascular haemolysis, often leading to acute renal failure. Thus far, the genetics of the underlying susceptibility to develop BWF is not fully elucidated. Deficiency in the MBL protein, an important component of the innate immune system, has previously been suggested to be a susceptibility factor for the development of severe malaria. This study aimed to evaluate the association between MBL2 gene polymorphisms, known to affect the MBL protein level/activity, and the occurrence of BWF among Congolese children.

Methods

This is a case–control study. Cases were patients with BWF, whereas controls, matched for gender and age, had uncomplicated malaria (UM). Dried blood spot was collected for genotyping.

Results

A total of 129 children were screened, including 43 BWF and 86 UM. The common allele in BWF and UM was A, with a frequency of 76.7 and 61.0%, respectively (OR: 2.67 (0.87–829) and p = 0.079). The frequency of the C allele was 18.6 and 29.1% in BWF and UM groups, respectively, with p = 0.858. Not a single D allele was encountered. Genotype AA was at higher risk for BWF whereas genotypes A0 (AB and AC) were over-represented in UM group (OR: 0.21 (0.06–0.78)) with p = 0.019. Nine haplotypes were observed in this study: 3 high MBL expression haplotypes and 6 low MBL expression haplotype. One new haplotype HYPC was observed in this study. None of these haplotypes was significantly associated with BWF.

Conclusion

This pilot study is a preliminary research on MBL2 gene and infectious diseases in DRC. The study results show a higher risk for BWF in AA. This suggests that future studies on BWF should further investigate the contribution of a strong immune response to the occurrence of BWF.

Background

Mannose-Binding Lectin protein (MBL), encoded by MBL2 gene (Mannose-Binding Lectin soluble 2; OMIM: 154545), is an important component of the innate immune system with 4 main functions, including activation of complement, direct promotion of opsono-phagocytosis, modulation of the inflammatory response, and promotion of apoptosis [1]. There are also other promoter variants that may affect gene expression [2,3,4,5]. The MBL deficiency, also known as ‘dysfunctional MBL’, is one of the most common immune deficiencies in the world [2]. Three non-synonymous single nucleotide substitutions in the exon 1 of MBL2 gene cause dramatic decrease of MBL in heterozygous state or almost complete absence of MBL in homozygous or compound heterozygous state. These include substitutions at codon 52 (CGT =>TGT; p.Arg52Cys, rs5030737), codon 54 (GGC ≥ GAC; p.Gly54Asp, rs1800450) and codon 57 (GCA ≥ GAA, p.Gly57Glu, rs1800451) [6,7,8,9]. Based on the classic MBL2 polymorphisms codification, substitutions at codons 52, 54 and 57 are referred to as D, B and C derived alleles, respectively, whereas the ancestral allele is known as allele A  [10]. Because these three variant alleles cause similar MBL deficiency, the concept of ‘O’ allele is used to describe either of these variants [8].

In addition, 3 substitutions including 2 in the promoter region of MBL2 (-550C/G or rs11003125 and -221G/C or rs7096206) and one in the UTR within the exon 1 (c.4T/C or rs7095891) have been shown to affect the level of MBL protein and influence the outcome of infectious diseases [9, 10]. The derived alleles in the promoter region, the upstream region and the exon 1 have been previously combined into haplotypes [10]. The MBL2 haplotypes HYPA, LYQA, LYPA, and HYQA have been associated with high MBL2 expression. Conversely, haplotypes LXPA, LYPB, LYQC, HYPD, LYPD, HYQC, LXPB, and LYQB showed low MBL2 expression [10]. However, a recent haplotype, termed HYPC, was identified in similar sub-Saharan individuals in a study from Zimbabwe [11].

In the Democratic Republic of Congo (DRC), Plasmodium falciparum is the most severe and lethal species of malaria parasite among children below 5 years of age [12,13,14,15]. The clinical expression of falciparum malaria consists of a wide spectrum, spanning from asymptomatically infected to multiple severe forms depending on multiple factors [16]. Blackwater fever (BWF), one of the life-threatening forms of falciparum malaria, is characterized by acute massive intravascular haemolysis and, usually, acute renal failure which occurs after using quinine in the treatment of malaria [17,18,19,20,21]. Factors such as inadequate malarial immunity, misuse of quinine and G6PD-deficiency have been associated with the occurrence of BWF [22,23,24,25,26]. However, the underlying genetics of the susceptibility to develop BWF is not fully elucidated.

Two apparently contradictory theories are proposed to explain the involvement of MBL in severe forms of infections such as malaria. On one hand, MBL deficiency is known to be a susceptibility factor for the development of severe infections including malaria [23, 24, 26,27,28,29,30,31,32,33]. On the other hand, MBL deficiency is also thought to be protective against certain complications by preventing excessive activation of the immune response, avoiding thereby deleterious immune-related complications during infections [7, 34, 35]. It has been recently reported that malaria IgG are significantly elevated in BWF [36], which also suggests that unlike other severe forms of malaria, BWF would more likely occur in normal or hyper immune individuals. A straight connection between IgG antibodies and MBL2 alleles have been established in a study on Chlamydia pneumoniae where the mean antibody titre increases with the number of copies of ancestral MBL2 alleles [37]. Although it remains unclear how ancestral MBL2 variants increase antibody titres and whether this matches with known mechanisms of MBL in the immune response, it could be hypothesized that unlike in other severe forms of malaria, people with ancestral MBL2 alleles would be at higher risk to exhibit BWF.

To date, the distribution of MBL2 alleles and their possible association to BWF in the DRC have not been investigated.

Methods

Study aims, design and setting

This study aimed to test the association between MBL2 polymorphisms and Blackwater fever, one of the most severe complications of malaria, and provide the first distribution data for MBL2 haplotypes in Congolese individuals. This is a case–control study conducted over 2 years in 4 medical institutions across Kinshasa, namely University Hospitals of Kinshasa, Kimbanseke Hospital, Bondeko Hospital and General Provincial Hospital of Kinshasa. Sampling methods and case definition are published elsewhere [12]. Altogether, 43 cases and 86 controls were enrolled. Ages for cases and controls ranged from 2 to 15 years.

Clinical evaluation

The medical history was obtained from parents, with particular attention to demographic data, including disease history and medications taken before BWF episode. Clinical data were recorded in a customized pre-tested clinical form. Malaria was confirmed by the presence of parasites on blood thick and film.

Laboratory measurements

Twenty mL of fresh urine were collected from each participant. The presence of haemoglobin in urine was first detected by urinary dip strip (Medi test Combi9, MacheryEur, Paris, France) and then confirmed by spectrometer (Thermo Genesis 10 BIO, New York, USA) using protocol of 3,3′ dimethyl benzidine reagent [38]. The results of urine dip stick were read as either negative (yellow colour) or positive (change in blue colour) 1+, 2+, 3+, which corresponded approximately to haemoglobin concentrations of respectively 0.061 ± 0.0166 mg/L, 0.3986 ± 0.2612 mg/dL and 0.5679 ± 0.27688 mg/L as quantified using the spectrometer.

DNA extraction and MBL2 genotyping

Human MBL2 gene was assessed from genomic DNA. Eight drops of blood were collected on FTA card® WB 120067 (GE Healthcare, Amersham, UK) and stored in a fridge until the transfer to the Institute of Tropical Medicine of Nagasaki University in Japan for DNA testing in the Department of Immunogenetics according to a previously described protocol [39].

DNA samples from 43 cases and 86 controls were examined. The promoter region and exon 1 of MBL2 gene were PCR-amplified and Sanger sequenced. Prior to Sanger sequencing, PCR products were verified by gel electrophoresis to confirm the presence of expected band and exclude unexpected inserts. The PCR mixture contained 17.5 μL of ultra-pure water, 2.5 μL PCR 10 × buffer, 4 μL of dNTPs (2 µmol), 0.4 μL (2 units) of Taq polymerase and 0.8 μL of each primer (2.5 µmol). A disc containing between 5 and 20 ng of DNA was punched from the FTA card and added into the PCR reaction tube. In order to identify technical contaminations, a tube a No DNA template was also included in each run. This consisted of a punch from an unspotted FTA card. After an initial denaturation step of 5 min at 95 °C, 35 amplification cycles were applied including rapid denaturation at 95 °C for 1 min, annealing at 65 °C for 1 min and elongation at 72 °C for 1 min. The reaction ended with a final elongation step at 72 °C for 5 min. PCR product was sequenced by dideoxy termination sequencing using Big-Dye® Terminator version 1.1. Sequencing product was analysed on a 3730 DNA ANALYSER, version 3.0, from HITACHI. Haplotypes were double- and triple-checked using visual inspection of sequencing traces.

Alleles were designated as suggested by Antonarakis et al. [7] for the 3 variants in the Exon 1. The MBL2*B, MBL2*C and other variants alleles were identified as described by Sumiya et al., Lipscombe et al. and Madsen et al. [3,4,5, 9].

Data management and analysis

Alleles and genotypes frequencies were obtained by direct scoring of electropherogram. Data were recorded using the software Epi Info 7. All analyses were carried out using SPSS 18.0. All records were crosschecked with the original data sheets before the analysis. A non-conditional model was used. This was a binary logistic regression including covariates, anti-malaria drugs, MBL2 gene polymorphism, G6PD and parasitaemia. Multivariate logistic regression analysis was used to evaluate associations between MBL2 haplotypes/genotypes/alleles and the BWF. Odds ratio and confidence intervals were calculated. All tests were two-sided, and the level of significance was set at p < 0.05.

Results

A total of 129 Congolese children were investigated, including 43 cases and 86 controls. Sixty-eight were girls (52.7%) and 61 boys (47.3%). The mean age was 8.75 ± 3.73 years for all the study population, 8.62 ± 3.84 years and 8.55 ± 3.77 years, respectively, for cases and controls (uncomplicated malaria, UM), only 8 cases (18.6%) were below 5 years, which is the most vulnerable period for severe malaria, versus 20 patients (23.26%) in the control group. The majority of BWF cases (38 cases) occurred during the rainy season (88.4%) and 5 (11.6%) occurred during the dry season. Low parasitaemia was associated to BWF OR: 3.31 (1.41–7.79) with p = 0.005 (Table 1).

Table 1 Socio-demographic features of patients in the study population
Full size table

Using a non-conditional model, a binary logistic regression, including covariant, anti-malaria drugs, MBL2 gene polymorphism, G6PD and parasitaemia, it was observed that MBL2*AB or AC is protective factor in the development of BWF. OR: 0.09 (0.01–0.63), with p = 0.015. The association with quinine intake and low parasitaemia, observed in this study (Table 2), was already published [12].

Table 2 Determinant factors of Blackwater fever occurrence
Full size table

The association between alleles and genotypes, and each of the 2 clinical groups was also assessed. The A allele was the most common in BWF group as well as in the UM group with allele frequency of 76.7 and 61.0%, respectively, and the difference was not statistically significant, OR: 2.67 (0.87–8.29 and p = 0.079 (Table 3). Conversely, the C allele frequency was 0.186 and 0.291 in BWF and UM groups, respectively, and the difference was not statistically significant (p = 0.853). Not a single D allele was encountered in the present study population (Table 3). Regarding the genotypes; the proportion of homozygote’s AA was higher in the BWF group (72.0%) compared to the UM (50.0%). Conversely, the 00 genotype was proportionately more frequent in the UM (27.9%) than in BWF (18.6%) (Table 3). A0 genotype is significantly over-represented in UM population compared to BWF patients, OR: 0.21 (0.06–0.78) with p = 0.019 (Table 3).

Table 3 Alleles and genotypes Frequencies for the 3 polymorphisms in the Exon 1
Full size table

Nine haplotypes were encountered in this study cohort, including 3 high MBL expression haplotypes and 6 low MBL expression haplotypes (Table 4). The high expression MBL2*LYQA haplotype was the most prevalent haplotype in BWF as well as in UM, with 46.3 and 39.5%, respectively. Low MBL expression haplotypes were; MBL2*HYPB; MBL2*HYPC; MBL2*LYQC (Y16578); MBL2*LYPC, MBL2*LYPB (Y16579); MBL2*LXPA and were not significant. Only MBL2*LYQA haplotype was consistently over-represented in UM group, but not significantly (Table 4). None of the groups deviated from the Hardy–Weinberg expectations [40] as showed in Table 3.

Table 4 MBL2 haplotypes (promoter region and exon1) and risk assessment
Full size table

Discussion

The present study investigated whether some alleles, genotypes or haplotypes were significantly over-represented or under-represented in patients with BWF compared to those with UM. A cohort of 129 patients was recruited from 4 hospitals across Kinshasa. Only a few of them were within higher risk group to develop severe malaria, meaning below 5 years of age, as described in many studies. However, the majority of recruited patients was at risk for BWF as this form of malaria is mostly observed in older children and adults [12, 13, 17, 21, 40,41,42,43,44,45].

Allele frequency

MBL2*A allele was the most common allele within the 2 groups compared to each of the derived alleles individually. However, when considered together, null alleles (allele 0) were more frequent among patients with UM compared to those with BWF, with allele frequencies of 0.39 and 0.233, respectively. 0 includes B, C and D alleles (Table 3). MBL2*C was the most frequent in both groups. Bellamy et al. [46] reported also a higher frequency of the MBL2*C in in the population of The Gambia. Compared to the other null alleles, the MBL2*C has been demonstrated to be extremely common in sub-Saharan Africans with a population frequency of 0.30, whereas the MBL2*B was predominant in Europeans, in Asians and in indigenous people of South America with population frequencies of 0.13, 0.20 and 0.50, respectively [3, 5, 46]. None of the alleles observed in the study population presented a significant preferential distribution between the 2 groups.

It has been hypothesized that *B, *C and *D alleles are positively selected in order to reduce susceptibility or mortality due to certain infectious diseases [5, 24, 34]. This study did not identify the MBL2*D allele within the 2 groups. This allele has been detected with frequencies up to 0.05 in the northeast of Africa, in Europe and India [3, 10]. Hence, the absence of the MBL2*D may simply indicate a low admixture with European and Indian in the Congolese population examined in this study [47,48,49].

MBL2 genotypes and BWF

MBL might protect against severe disease forms but not against BWF

Multiple genetic epidemiological studies reported that the presence MBL2 derived alleles and genotypes are associated with an increased risk to infections [4, 28, 29, 32, 50] and might be considered as a prognostic marker in various infectious conditions [29, 32, 51, 52]. Functional studies showed that heterozygotes for a MBL2 variant produces low concentration of MBL protein and this may hamper the phagocytosis of bacteria or parasites, thereby allowing the replication of the pathogen [24, 28, 48, 53, 54]. Based on this group of studies, one would expect individuals with ancestral MBL2 AA alleles to be protected against BWF, a severe phenotype. Unlike in other severe forms of malaria, such association was not observed in this study.

Homozygotes for ancestral MBL2 alleles are at higher risk for BWF

The other wildly supported theory is that low levels of functional MBL may decrease excessive activation of the immune response and enhance survival in some patients [5, 34, 35]. Therefore, low levels of functional MBL protects against severe complications triggered by the host immune response. In this study individuals with AA genotype had higher risk for BWF as compared to A0 genotype, which is consistent with the second theory. This observation and the previously reported elevated levels of malaria IgG in BWF suggests that BWF might be caused mainly by excessive activation of the immune response. The current results do not formally exclude the role of MBL deficiency in the occurrence of BWF since 00 individuals presented with intermediate risk for BWF as compared to A0.

Haplotypes

The present study revealed 9 haplotypes, including 3 high MBL expression haplotypes (Table 4) and 6 low expression haplotypes. The LYQA haplotype was the most prevalent haplotype both in BWF and UM group with, respectively, 45.5 and 39.5%, followed by MBL2⃰ LYQC in UM population with 23.3%. In Gabon, Boldt et al. defined 14 new haplotypes and reported that MBL2*LYQC, MBL2*LYQA and MBL2*LYPA were the most prevalent haplotypes in the children population [55]. A new haplotype HYPC only described in Zimbabwe individuals was observed in 2 patients: one BWF and one UM. A study in India reported that the MBL2*LYPA haplotypes confers protection, whereas MBL2*LXPA increases the malaria risk. These findings in Indian populations demonstrate that MBL2 functional variants are strongly associated with malaria and infection severity [10]. However, no significant association was find between BWF and a particular haplotype.

Parasitaemia and BWF

Lower parasitaemia was observed in BWF patients. Considering that quinine intake offers a strong clearance of parasite, low parasitaemia observed in BWF may be secondary to the quinine intake that triggers BWF occurrence. In that prospect, the time between quinine intake and the occurrence of BWF may influence parasitaemia. However, this timing remains unclear since reported time-lapses range from 12 h to multiple days after treatment [56,57,58]. Another reason for low parasitaemia in BWF could be the activity of the immune system in AA individuals. The active immune response would provide a good clearance of parasite and accessorily cause BWF. Further studies may be needed to investigate this hypothesis.

Limitations of the study

The major limitation of this study is the small sample size. Although BWF is rare in the study setting, the small sample size may have influenced the statistical calculations. Another limitation was that the investigation of G6PD polymorphisms, and the complement activation and MBL protein were not measured. In addition, no data exist in the community about the frequency of MBL2 polymorphism in the country. Despite these limitations, these data provide insights into the relationship between MBL protein level/activity and BWF, and could form a basis for further studies in a large Congolese population.

Conclusion

This pilot study is a preliminary research on MBL2 gene and infectious diseases in DRC. The result shows a higher risk for BWF in AA. This suggests that future studies on BWF should further investigate the contribution of a strong immune response to the occurrence of BWF.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

DRC:

Democratic Republic of Congo

UM:

uncomplicated malaria

BWF:

blackwater fever

MBL:

Mannose Binding Lectin protein

MBL2 :

Mannose Binding Lectin gene

References

  1. Naito H, Ikeda A, Hasegawa K, Oka S, Uemura K, Kawasaki N, et al. Characterization of human serum mannan-binding protein promoter. J Biochem. 1999;126:1004–12.

    CAS  PubMed  Google Scholar 

  2. Giubergia V, Salim M, Fraga J, Castiglioni N, Sen L, Castanos C, et al. Post-infectious bronchiolitis obliterans and mannose-binding lectin insufficiency in Argentinean children. Respirology. 2015;20:982–6.

    PubMed  Google Scholar 

  3. Madsen HO, Garred P, Kurtzhals JA, Lamm LU, Ryder LP, Thiel S, et al. A new frequent allele is the missing link in the structural polymorphism of the human mannan-binding protein. Immunogenetics. 1994;40:37–44.

    CAS  PubMed  Google Scholar 

  4. Sumiya M, Super M, Tabona P, Levinsky RJ, Arai T, Turner MW, et al. Molecular basis of opsonic defect in immunodeficient children. Lancet. 1991;337:1569–70.

    CAS  PubMed  Google Scholar 

  5. Lipscombe RJ, Sumiya M, Hill AV, Lau YL, Levinsky RJ, Summerfield JA, et al. High frequencies in African and non-African populations of independent mutations in the mannose binding protein gene. Hum Mol Genet. 1992;1:709–15.

    CAS  PubMed  Google Scholar 

  6. Garred P, Larsen F, Seyfarth J, Fujita R, Madsen HO. Mannose-binding lectin and its genetic variants. Genes Immun. 2006;7:85.

    CAS  PubMed  Google Scholar 

  7. Antonarakis SE. Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat. 1998;11:1–3.

    CAS  Google Scholar 

  8. Eisen DP, Minchinton RM. Impact of mannose-binding lectin on susceptibility to infectious diseases. Clin Infect Dis. 2003;37:1496–505.

    CAS  PubMed  Google Scholar 

  9. Madsen HO, Garred P, Thiel S, Kurtzhals JA, Lamm LU, Ryder LP, et al. Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein. J Immunol. 1995;155:3013–20.

    CAS  PubMed  Google Scholar 

  10. Jha AN, Sundaravadivel P, Singh VK, Pati SS, Patra PK, Kremsner PG, et al. MBL2 variations and malaria susceptibility in Indian populations. Infect Immun. 2014;82:52–61.

    PubMed  PubMed Central  Google Scholar 

  11. Mhandire K, Pharo G, Kandawasvika GQ, Duri K, Swart M, Stray-Pedersen B, et al. How does mother-to-child transmission of HIV differ among African populations? Lessons from MBL2 genetic variation in Zimbabweans. OMICS. 2014;18:454–60.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Bodi JM, Nsibu CN, Longenge RL, Aloni MN, Akilimali PZ, Tshibassu PM, et al. Blackwater fever in Congolese children: a report of clinical, laboratory features and risk factors. Malar J. 2013;12:205.

    PubMed  PubMed Central  Google Scholar 

  13. Delacollette C, Taelman H, Wery M. An etiologic study of hemoglobinuria and blackwater fever in the Kivu Mountains, Zaire. Ann Soc Belg Med Trop. 1995;75:51–63.

    CAS  PubMed  Google Scholar 

  14. Ministère du Plan et Suivi de la Mise en Oeuvre de la Révolution et de la Modernité Modernité, Ministère de la Santé Publique. Enquête Démographique et de la Santé. République Démocratique du Congo, 2013.

  15. Mokoli MV, Nseka MN, Lepira FB, Sumaili EK, Bukabau J. Profil clinico-bio-morphologique et évolutif de l’insuffisance rénale aiguë aux Cliniques Universitaires de Kinshasa. Annales Africaines de Médecine. 2007;1:11–9.

    Google Scholar 

  16. Nebie I, Diarra A, Ouedraogo A, Soulama I, Bougouma EC, Tiono AB, et al. Humoral responses to Plasmodium falciparum blood-stage antigens and association with incidence of clinical malaria in children living in an area of seasonal malaria transmission in Burkina Faso, West Africa. Infect Immun. 2008;76:759–66.

    CAS  PubMed  Google Scholar 

  17. Aloni MN, Nsibu CN, Meeko-Mimaniye M, Ekulu PM, Bodi JM. Acute renal failure in Congolese children: a tertiary institution experience. Acta Paediatr. 2012;101:e514–8.

    PubMed  Google Scholar 

  18. Assounga AG, Assambo-Kieli C, Mafoua A, Moyen G, Nzingoula S. Etiology and outcome of acute renal failure in children in congo-brazzaville. Saudi J Kidney Dis Transpl. 2000;11:40–3.

    CAS  PubMed  Google Scholar 

  19. Bruneel F, Gachot B, Wolff M, Bedos JP, Regnier B, Danis M, et al. Blackwater fever. Presse Med. 2002;31:1329–34 (in French).

    CAS  PubMed  Google Scholar 

  20. Das BS. Renal failure in malaria. J Vector Borne Dis. 2008;45:83–97.

    CAS  PubMed  Google Scholar 

  21. Kunuanunua TS, Nsibu CN, Gini-Ehungu JL, Bodi JM, Ekulu PM, Situakibanza H, et al. Acute renal failure and severe malaria in Congolese children living in Kinshasa, Democratic Republic of Congo. Nephrol Ther. 2013;9:160–5 (in French).

    PubMed  Google Scholar 

  22. Daubrey-Potey T, Die-Kacou H, Kamagate M, Vamy M, Balayssac E, Yavo JC. Blackwater fever during antimalarial treatment in Abidjan (West Africa): report of 41 cases. Bull Soc Pathol Exot. 2004;97:325–8 (in French).

    PubMed  Google Scholar 

  23. Garred P, Nielsen MA, Kurtzhals JA, Malhotra R, Madsen HO, Goka BQ, et al. Mannose-binding lectin is a disease modifier in clinical malaria and may function as opsonin for Plasmodium falciparum-infected erythrocytes. Infect Immun. 2003;71:5245–53.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Madsen HO, Satz ML, Hogh B, Svejgaard A, Garred P. Different molecular events result in low protein levels of mannan-binding lectin in populations from southeast Africa and South America. J Immunol. 1998;161:3169–75.

    CAS  PubMed  Google Scholar 

  25. WHO Group. Guidelines for the Treatment of Malaria. 3rd ed. Geneva: World Health Organization; 2015.

    Google Scholar 

  26. Steffensen R, Thiel S, Varming K, Jersild C, Jensenius JC. Detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (MBL) gene by polymerase chain reaction with sequence-specific primers. J Immunol Methods. 2000;241:33–42.

    CAS  PubMed  Google Scholar 

  27. Casanova JL, Abel L. Human Mannose-binding Lectin in Immunity: friend, Foe, or Both? J Exp Med. 2004;199:1295–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Garred P, Madsen HO, Balslev U, Hofmann B, Pedersen C, Gerstoft J, et al. Susceptibility to HIV infection and progression of AIDS in relation to variant alleles of mannose-binding lectin. Lancet. 1997;349:236–40.

    CAS  PubMed  Google Scholar 

  29. Garred P, Madsen HO, Hofmann B, Svejgaard A. Increased frequency of homozygosity of abnormal mannan-binding-protein alleles in patients with suspected immunodeficiency. Lancet. 1995;346:941–3.

    CAS  PubMed  Google Scholar 

  30. Gordon AC, Waheed U, Hansen TK, Hitman GA, Garrard CS, Turner MW, et al. Mannose-binding lectin polymorphisms in severe sepsis: relationship to levels, incidence, and outcome. Shock. 2006;25:88–93.

    CAS  PubMed  Google Scholar 

  31. Sullivan KE, Wooten C, Goldman D, Petri M. Mannose-binding protein genetic polymorphisms in black patients with systemic lupus erythematosus. Arthritis Rheum. 1996;39:2046–51.

    CAS  PubMed  Google Scholar 

  32. Summerfield JA, Ryder S, Sumiya M, Thursz M, Gorchein A, Monteil MA, et al. Mannose binding protein gene mutations associated with unusual and severe infections in adults. Lancet. 1995;345:886–9.

    CAS  PubMed  Google Scholar 

  33. Summerfield JA, Sumiya M, Levin M, Turner MW. Association of mutations in mannose binding protein gene with childhood infection in consecutive hospital series. BMJ. 1997;314:1229–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Garred P, Thiel S, Madsen HO, Ryder LP, Jensenius JC, Svejgaard A. Gene frequency and partial protein characterization of an allelic variant of mannan binding protein associated with low serum concentrations. Clin Exp Immunol. 1992;90:517–21.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Takahashi K, Gordon J, Liu H, Sastry KN, Epstein JE, Motwani M, et al. Lack of mannose-binding lectin-A enhances survival in a mouse model of acute septic peritonitis. Microbes Infect. 2002;4:773–84.

    CAS  PubMed  Google Scholar 

  36. Bodi J, Nsibu C, Longenge R, Aloni M, Akilimali P, Tshibassu P, et al. High IgG1 malaria antibodies level in children is a possible risk factor of blackwater fever: a case-control study. Pediatr Health Res. 2018;3:9.

    Google Scholar 

  37. Monsey L, Best LG, Zhu J, DeCroo S, Anderson MZ. The association of mannose binding lectin genotype and immune response to Chlamydia pneumoniae: the Strong Heart Study. PLoS ONE. 2019;14:e0210640.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Assoumanou MG, Akpona SA. Dosage de l’hémoglobine urinaire par un réactif 3,3′ diméthylbenzidine: mise au point technique. Int J Biol Chem Sci. 2011;5:11.

    Google Scholar 

  39. Dean FB, Nelson JR, Giesler TL, Lasken RS. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 2001;11:1095–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Gobbi F, Audagnotto S, Trentini L, Nkurunziza I, Corachan M, Di Perri G. Blackwater fever in children, Burundi. Emerg Infect Dis. 2005;11:1118–20.

    PubMed  PubMed Central  Google Scholar 

  41. Bouldouyre MA, Dia D, Carmoi T, Fall KB, Chevalier B, Debonne JM. A mild blackwater fever. Med Mal Infect. 2006;36:343–5 (in French).

    PubMed  Google Scholar 

  42. Khandelwal V, Udawat H, Kumhar MR, Goyal RK. Blackwater fever treated with artemether. J Assoc Physicians India. 2001;49:1191–2.

    CAS  PubMed  Google Scholar 

  43. Rogier C, Imbert P, Tall A, Sokhna C, Spiegel A, Trape JF. Epidemiological and clinical aspects of blackwater fever among African children suffering frequent malaria attacks. Trans R Soc Trop Med Hyg. 2003;97:193–7.

    PubMed  Google Scholar 

  44. Tran TH, Day NP, Ly VC, Nguyen TH, Pham PL, Nguyen HP, et al. Blackwater fever in southern Vietnam: a prospective descriptive study of 50 cases. Clin Infect Dis. 1996;23:1274–81.

    CAS  PubMed  Google Scholar 

  45. WHO. Malaria Policy Advisory Committee to the WHO: conclusions and recommendations of September 2012 meeting. Malar J. 2012;2012(11):424.

    Google Scholar 

  46. Bellamy R, Ruwende C, McAdam KP, Thursz M, Sumiya M, Summerfield J, et al. Mannose binding protein deficiency is not associated with malaria, hepatitis B carriage nor tuberculosis in Africans. QJM. 1998;91:13–8.

    CAS  PubMed  Google Scholar 

  47. Bernig T, Taylor JG, Foster CB, Staats B, Yeager M, Chanock SJ. Sequence analysis of the mannose-binding lectin (MBL2) gene reveals a high degree of heterozygosity with evidence of selection. Genes Immun. 2004;5:461–76.

    CAS  PubMed  Google Scholar 

  48. Boldt AB, Messias-Reason IJ, Meyer D, Schrago CG, Lang F, Lell B, et al. Phylogenetic nomenclature and evolution of mannose-binding lectin (MBL2) haplotypes. BMC Genet. 2010;11:38.

    PubMed  PubMed Central  Google Scholar 

  49. Verdu P, Barreiro LB, Patin E, Gessain A, Cassar O, Kidd JR, et al. Evolutionary insights into the high worldwide prevalence of MBL2 deficiency alleles. Hum Mol Genet. 2006;15:2650–8.

    CAS  PubMed  Google Scholar 

  50. Guo SW, Thompson EA. Performing the exact test of Hardy–Weinberg proportion for multiple alleles. Biometrics. 1992;48:361–72.

    CAS  PubMed  Google Scholar 

  51. Garred P, Harboe M, Oettinger T, Koch C, Svejgaard A. Dual role of mannan-binding protein in infections: another case of heterosis? Eur J Immunogenet. 1994;21:125–31.

    CAS  PubMed  Google Scholar 

  52. Garred P, Madsen HO, Halberg P, Petersen J, Kronborg G, Svejgaard A, et al. Mannose-binding lectin polymorphisms and susceptibility to infection in systemic lupus erythematosus. Arthritis Rheum. 1999;42:2145–52.

    CAS  PubMed  Google Scholar 

  53. Hoal-Van Helden EG, Epstein J, Victor TC, Hon D, Lewis LA, Beyers N, et al. Mannose-binding protein B allele confers protection against tuberculous meningitis. Pediatr Res. 1999;45(4 Pt 1):459–64.

    CAS  PubMed  Google Scholar 

  54. Santos IK, Costa CH, Krieger H, Feitosa MF, Zurakowski D, Fardin B, et al. Mannan-binding lectin enhances susceptibility to visceral leishmaniasis. Infect Immun. 2001;69:5212–5.

    CAS  PubMed  Google Scholar 

  55. Boldt AB, Petzl-Erler ML. A new strategy for mannose-binding lectin gene haplotyping. Hum Mutat. 2002;19:296–306.

    PubMed  Google Scholar 

  56. Sher A, Ed. Hemoglobinuria (Black Water Fever) in severe falciparum malaria—a case report. In: 17th International Congress on Infectious Diseases; 2016.

  57. Lon C, Spring M, Sok S, Chann S, Bun R, Ittiverakul M, et al. Blackwater fever in an uncomplicated Plasmodium falciparum patient treated with dihydroartemisinin–piperaquine. Malar J. 2014;13:96.

    PubMed  PubMed Central  Google Scholar 

  58. Rodriguez-Valero N, Castro P, Martinez G, Marco Hernandez J, Fernandez S, Gascon J, et al. Blackwater fever in a non-immune patient with Plasmodium falciparum malaria after intravenous artesunate. J Travel Med. 2018;25:e101093.

    Google Scholar 

Download references

Acknowledgements

The authors are thankful to all children and parents who participated to this study, and to Nasir Nshuaib for the quantification of malaria IgG1 antibodies (Department of Immunogenetics, Nagasaki University, Japan). The authors thank all colleagues, nurses and lab technicians involved in sample and data collection. The authors are grateful to Prof Fons Verdonck of the KU Leuven Alumni for his support.

Data distribution

Anonymized genomic data can be obtained upon request to the corresponding author.

Funding

This research was supported by the University of Nagasaki through the Grant-in-Aid for Scientific Research (B) 17H04072 (2017–2021) of KAKENHI; and the Katholieke Universiteit Leuven (Belgium) through the scholarship program for young Congolese researchers (Bourses chaires scientifiques pour jeunes Congolais).

Author information

Authors and Affiliations

  1. Department of Pediatrics, Emergency and Intensive Care Unit, University Hospital of Kinshasa, Faculty of Medicine, University of Kinshasa, Kinshasa, Democratic Republic of Congo

    Joseph M. Bodi, Célestin N. Nsibu & Roland L. Longenge

  2. Department of Pediatrics, Haemato-oncology and Nephrology Unit, University Hospital of Kinshasa, Faculty of Medicine, University of Kinshasa, Kinshasa, Democratic Republic of Congo

    Michel N. Aloni

  3. Division of Biostatistics and Epidemiology, School of Public Health, University of Kinshasa, Kinshasa, Democratic Republic of Congo

    Pierre Z. Akilimali & Patrick K. Kayembe

  4. Division of Malaria Control (DOMC), Ministry of Health, Nairobi, Kenya

    Ahmeddin H. Omar

  5. Department of Microbiology, Katholieke Universiteit de Leuven, Brussels, Belgium

    Jan Verhaegen

  6. Department of Pediatrics, Gastroenterology and Neurology Unit, University Hospital of Kinshasa, Faculty of Medicine, University of Kinshasa, Kinshasa, Democratic Republic of Congo

    Pierre M. Tshibassu

  7. Département des Sciences Biomédicales et Précliniques, GIGA-R, Laboratoire de Génétique Humaine, University of Liège, Liège, Belgium

    Aimé Lumaka

  8. Center for Human Genetics, Department of Pediatrics, Faculty of Medicine, University of Kinshasa, Kinshasa, Democratic Republic of Congo

    Prosper T. Lukusa & Aimé Lumaka

  9. Department of Immunogenetics, Institute of Tropical Medicine (Nekken), University of Nagasaki, Nagasaki, Japan

    Kenji Hirayama

Authors
  1. Joseph M. Bodi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Célestin N. Nsibu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Roland L. Longenge
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michel N. Aloni
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pierre Z. Akilimali
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Patrick K. Kayembe
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ahmeddin H. Omar
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jan Verhaegen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Pierre M. Tshibassu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Prosper T. Lukusa
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Aimé Lumaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Kenji Hirayama
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

CNN, PMT, MNA, PLT, JV and JMB, conceived, designed, deployed and directed the case–control study at the Department of Pediatrics at Kinshasa university hospital and wrote the manuscript. RLL carried out patient recruitment and follow-up, sample collection, storage and transport. JMB and MNA wrote the first draft of the manuscript. KH, JV, AZL and PTL brought very precious corrections. PKK and PPA analysed data. AO edited the English corrections. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Joseph M. Bodi.

Ethics declarations

Ethics approval and consent to participate

All information about this study was provided to parents in local languages. Written informed consent from parents for each patient in this study has been obtained. The Ethics Committee of Public Health School of University of Kinshasa approved the protocol under the number ESP/CE/027B/2011.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bodi, J.M., Nsibu, C.N., Longenge, R.L. et al. Exploring association between MBL2 gene polymorphisms and the occurrence of clinical blackwater fever through a case–control study in Congolese children. Malar J 19, 25 (2020). https://0-doi-org.brum.beds.ac.uk/10.1186/s12936-020-3100-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://0-doi-org.brum.beds.ac.uk/10.1186/s12936-020-3100-8

Keywords

Malaria Journal

ISSN: 1475-2875

Contact us