Prevalence of sickle cell disorders and malaria infection in children aged 1-12 years in the Volta Region, Ghana: a community-based study.

BACKGROUND: Alterations in the structure of haemoglobin (Hb) are usually brought about by point mutations affecting one or, in some cases, two codons encoding amino acids of the globin chains. One in three Ghanaians are said to have sickle cell disorders, whereas malaria continues to be one of the leading causes of mortality among children. This study determined the prevalence of sickle cell disorders and malaria infection among children aged 1-12 years in the Volta Region. METHODS: This was a community-based cross-sectional survey that involved 938 children aged 1-12 years selected from three districts, one each from the 3 geographical zones of the Volta Region using a multistage sampling method. Demographic information was collected using a standard questionnaire and anthropometric indices were measured. Isoelectric focusing (IEF) electrophoresis was used to determine the Hb genotypes and sub-microscopic parasites were determined by PCR. RESULTS: The prevalence of sickling screening positive was 16.0% with an overall prevalence of sickle cell disorders being 2.0%. Among the individual genotypes making up the sickle cell disorders, genotype HbSF was the highest (0.9% as compared to 0.2%; HbSS, 0.6%; HbSC and 0.3%; HbSCF). Microscopic Plasmodium falciparum parasitaemia was detected among 5.5% of the children and 14.2% sub-microscopic prevalence by PCR. Children with sickle cell disorders were more likely to have sub-microscopic parasitaemia (AOR = 5.51 95%CI (2.15, 14.10), p < 0.001) as well as anaemia (AOR = 3.03 95% CI (1.04, 8.82), p = 0.042), compared to those with normal genotypes. There was no significant difference observed between sickle cell disorders and growth and development of the children screened. CONCLUSIONS: Sickle cell disorders were significantly associated with sub-microscopic parasitaemia as well as anaemia in this study. Establishment of sickle cell clinics in the district and regional hospitals will help in the management of children with the disorder and also generate a national database on sickle cell disorders. National neonatal screening policies must also be put in place to help in early detection and management of these disorders.

The CYB5R3c .350C>G and G6PD A alleles modify severity of anemia in malaria and sickle cell disease.

Genetic modifiers of anemia in Plasmodium falciparum infection and sickle cell disease (SCD) are not fully known. Both conditions are associated with oxidative stress, hemolysis and anemia. The CYB5R3 gene encodes cytochrome b5 reductase 3, which converts methemoglobin to hemoglobin through oxidation of NADH. CYB5R3c.350C > G encoding CYB5R3T117S , the most frequent recognized African-specific polymorphism, does not have known functional significance, but its high allele frequency (23% in African Americans) suggests a selection advantage. Glucose-6-phosphate dehydrogenase (G6PD) is essential for protection from oxidants; its African-polymorphic X-linked A+ and A- alleles, and other variants with reduced activity, coincide with endemic malaria distribution, suggesting protection from lethal infection. We examined the association of CYB5R3c.350C > G with severe anemia (hemoglobin <5 g/dL) in the context of G6PD A+ and A- status among 165 Zambian children with malaria. CYB5R3c.350C > G offered protection against severe malarial anemia in children without G6PD deficiency (G6PD wild type or A+/A- heterozygotes) (odds ratio 0.29, P = .022) but not in G6PD A+ or A- hemizygotes/homozygotes. We also examined the relationship of CYB5R3c.350C > G with hemoglobin concentration among 267 children and 321 adults and adolescents with SCD in the US and UK and found higher hemoglobin in SCD patients without G6PD deficiency (ß = 0.29, P = .022 children; ß = 0.33, P = .004 adults). Functional studies in SCD erythrocytes revealed mildly lower activity of native CYB5R3T117S compared to wildtype CYB5R3 and higher NADH/NAD+ ratios. In conclusion, CYB5R3c.350C > G appears to ameliorate anemia severity in malaria and SCD patients without G6PD deficiency, possibly accounting for CYB5R3c.350C > G selection and its high prevalence.

Past and current biological factors affecting malaria in the low transmission setting of Botswana: A review.

Malaria continues to be one of the top infectious agents contributing to morbidity and mortality in sub-Saharan Africa. Annually, Botswana accounts only for a small proportion of cases (<<1%). Despite significantly reduced incidence rate, the country still experiences sporadic outbreaks that hamper the goal of malaria elimination. This review evaluated previous and current biological factors that impact malaria in Botswana, specifically focussing on the vectors, the parasite and the host. This was accomplished via a literature review evaluating these variables in Botswana. Current literature suggests that Anopheles arabiensis is the main malaria vector in the country. Several other potential vectors have been found widely distributed throughout Botswana in high numbers, yet remain largely unstudied with regards to their contribution to the country's malaria burden. We also report the most up to date list of all Anopheles species that have been found in Botswana. Plasmodium falciparum is responsible for the vast majority of symptomatic malaria in the country and some drug resistance markers have been documented for this species. Plasmodium vivax has been reported in asymptomatic subjects, even though a large proportion of the Botswana population appears to be Duffy antigen negative. Very little is known about the true distribution of P. vivax and no point of care testing infrastructure for this species exists in Botswana, making it difficult to tailor treatment to address possible recrudescence or relapse. Due to a genetically diverse population with a substantial Khoisan contribution into the Bantu genetic background, several phenotypes that potentially impact prevalence and severity of malaria exist within the country. These include sickle cell trait, Glucose-6-Phosphate Dehydrogenase deficiency, and Duffy negativity. This review highlights the information that currently exists on malaria in Botswana. It also postulates that a comprehensive understanding of these aforementioned biological factors may help to explain malaria persistence in Botswana.

Robust adaptive immune response against Babesia microti infection marked by low parasitemia in a murine model of sickle cell disease.

The intraerythrocytic parasite Babesia microti is the number 1 cause of transfusion-transmitted infection and can induce serious, often life-threatening complications in immunocompromised individuals including transfusion-dependent patients with sickle cell disease (SCD). Despite the existence of strong long-lasting immunological protection against a second infection in mouse models, little is known about the cell types or the kinetics of protective adaptive immunity mounted following Babesia infection, especially in infection-prone SCD that are thought to have an impaired immune system. Here, we show, using a mouse B microti infection model, that infected wild-type (WT) mice mount a very strong adaptive immune response, characterized by (1) coordinated induction of a robust germinal center (GC) reaction; (2) development of follicular helper T (TFH) cells that comprise ∼30% of splenic CD4+ T cells at peak expansion by 10 days postinfection; and (3) high levels of effector T-cell cytokines, including interleukin 21 and interferon γ, with an increase in the secretion of antigen (Ag)-specific antibodies (Abs). Strikingly, the Townes SCD mouse model had significantly lower levels of parasitemia. Despite a highly disorganized splenic architecture before infection, these mice elicited a surprisingly robust adaptive immune response (including comparable levels of GC B cells, TFH cells, and effector cytokines as control and sickle trait mice), but higher immunoglobulin G responses against 2 Babesia-specific proteins, which may contain potential immunogenic epitopes. Together, these studies establish the robust emergence of adaptive immunity to Babesia even in immunologically compromised SCD mice. Identification of potentially immunogenic epitopes has implications to identify long-term carriers, and aid Ag-specific vaccine development.

Effect of inherited red cell defects on growth of Plasmodium falciparum: An in vitro study.

BACKGROUND & OBJECTIVES: High prevalence of certain polymorphic alleles of erythrocytes in malaria endemic area has been linked to the resistance provided by these alleles against parasitic infestations. Numerous studies undertaken to demonstrate this correlation have generated conflicting results. This study was undertaken to investigate the abilities of various polymorphic erythrocytes to support in vitro growth of Plasmodium falciparum parasites. METHODS: In this study under in vitro condition the ability of P. falciparum parasites to grow was assessed in the erythrocytes obtained from a total of 40 patients with various haemoglobinopathies, such as ß-thalassaemia (ß-Thal), sickle cell anaemia, erythroenzymopathy-like glucose-6-phosphate dehydrogenase deficiency and membranopathy-like hereditary spherocytosis. RESULTS: Significantly reduced in vitro invasion and growth of parasites was seen in the cultures containing abnormal erythrocytes than in control cultures containing normal erythrocytes (P< 0.05). The mean per cent parasitaemia comparison was also carried out among the three polymorphic erythrocyte groups, i.e. ß-Thal, sickle cell anaemia and enzyme-membranopathies. INTERPRETATION & CONCLUSIONS: Erythroenzymopathies and membranopathies were found to provide a more hostile environment for parasites, as the least parasitaemia was observed in these erythrocytes. The present in vitro study showed that P. falciparum did not grow well and did not invade well in erythrocytes obtained from common inherited red cell disorders.

Plasmodium falciparum Merozoite Surface Protein-1 Polymorphisms among Asymptomatic Sickle Cell Anemia Patients in Nigeria.

Asymptomatic malaria (ASM) has been implicated in the development of hemolytic crisis in infected sickle cell anemia (SCA) patients worldwide. This study surveyed steady state SCA Nigerian patients for ASM to investigate the influence of malaria prevention behaviors and age on parasitaemia and multiplicity of infection (MOI). A total of 78 steady SCA patients aged 5 - 27 years on routine care at three health facilities in Lagos were investigated for ASM by light microscopy and PCR with a multiplicity of infection determined by genotyping block 2 of merozoite surface protein 1 (msp1) gene of Plasmodium falciparum (P. falciparum). Use of malaria prevention measures was captured using a semi-structured questionnaire. The prevalence rates of ASM (due to Pf only) by microscopy and PCR were found to be 27.3% and 47.4% respectively (P < 0.05) with a Mean + SEM parasite density of 2238.4 + 464.3 parasites/uL. Five distinct msp1 genotypes [K1 (2), MAD20 (2), RO33 (1)] were detected and significant (P<0.05) disparity in allele frequencies (K1, 91.8%, MAD20, 32.4%; RO33, 18.9%) was found. The overall MOI was 1.43 and 37.8% of infections were polyclonal (P<0.05). ASM was associated with non-use of preventive measures and occurred in 62.1% of SCA patients aged < 10y with lower MOI of 1.3 compared to 38.1% in older patients with a higher MOI of 1.5 (P<0.05). We conclude that PCR improved the diagnosis of ASM among Nigerian SCA patients with infections being of low complexity and associated with non-use of preventive interventions and R033 msp1 allele selection.

A randomized trial of artesunate-amodiaquine versus artemether-lumefantrine in Ghanaian paediatric sickle cell and non-sickle cell disease patients with acute uncomplicated malaria.

BACKGROUND: Sickle cell disease (SCD) is a genetic disorder common in malaria endemic areas. In endemic areas, malaria is a major cause of morbidity and mortality among SCD patients. This suggests the need for prompt initiation of efficacious anti-malarial therapy in SCD patients with acute malaria. However, there is no information to date, on the efficacy or safety of artemisinin combination therapy when used for malaria treatment in SCD patients. METHODS: Children with SCD and acute uncomplicated malaria (n=60) were randomized to treatment with artesunate-amodiaquine (AA), or artemether-lumefantrine (AL). A comparison group of non-SCD children (HbAA genotype; n=59) with uncomplicated malaria were also randomized to treatment with AA or AL. Recruited children were followed up and selected investigations were done on days 1, 2, 3, 7, 14, 28, 35, and 42. Selected clinical and laboratory parameters of the SCD patients were also compared with a group of malaria-negative SCD children (n=82) in steady state. RESULTS: The parasite densities on admission were significantly lower in the SCD group, compared with the non-SCD group (p=0.0006). The parasite reduction ratio (PRR) was lower, clearance was slower (p<0.0001), and time for initial parasitaemia to decline by 50 and 90% were longer for the SCD group. Adequate clinical and parasitological response (ACPR) on day 28 was 98.3% (58/59) in the SCD group and 100% (57/57) in the non-SCD group. Corresponding ACPR rates on day 42 were 96.5% (55/57) in the SCD group and 96.4% (53/55) in the non-SCD group. The fractional changes in haemoglobin, platelets and white blood cell counts between baseline (day 0) and endpoint (day 42) were 16.9, 40.6 and 92.3%, respectively, for the SCD group, and, 12.3, 48.8 and 7.5%, respectively, for the non-SCD group. There were no differences in these indices between AA- and AL-treated subjects. CONCLUSIONS: The parasite clearance of SCD children with uncomplicated malaria was slower compared with non-SCD children. AA and AL showed similar clinical and parasitological effects in the SCD and non-SCD groups. The alterations in WBC and platelet counts may have implications for SCD severity. TRIAL REGISTRATION: Current controlled trials ISRCTN96891086.

Hyposplenism revealed by Plasmodium malariae infection.

BACKGROUND: Hyposplenism, due to splenectomy, inherited red blood cell disorders or acquired conditions such as celiac disease, has an important impact on the severity of malaria, especially in non-immune patients. Conversely, that malaria may reveal functional hyposplenism has not been described previously. METHODS: A 31-year old gardener was diagnosed with an uncomplicated attack of Plasmodium malariae 11 years after leaving the endemic area. In addition to trophozoites and schizonts, thick and thin smears also showed Howell-Jolly bodies, pointing to functional hyposplenism. This was later confirmed by the presence of a calcified spleen in the context of S/ß + sickle-cell syndrome in a patient previously unaware of this condition. CONCLUSION: Malaria may reveal hyposplenism. Although Howell-Jolly bodies are morphologically similar to nuclei of young Plasmodium trophozoite, distinction on smears is based on the absence of cytoplasm and irregular size of Howell-Jolly bodies. In the patient reported here, hyposplenism was revealed by the occurrence of P. malariae infection relatively late in life. Timely diagnosis of hyposplenism resulted in the implementation of appropriate measures to prevent overwhelming infection with capsulated bacteria. This observation highlights the importance of diagnosing hyposplenism in patients with malaria despite the morphological similarities between ring nuclei and Howell-Jolly bodies on thick smears.

The third described case of transfusion-transmitted Babesia duncani.

BACKGROUND: Almost all of the reported US tick-borne and transfusion-associated Babesia cases have been caused by Babesia microti, which is endemic in the Northeast and upper Midwest. We investigated a case caused by B. duncani (formerly, the WA1-type parasite), in a 59-year-old California resident with sickle cell disease (HbSS) whose only risk factor for infection was receipt of red blood cell transfusions. CASE REPORT: The patient's case was diagnosed in September 2008: intraerythrocytic parasites were noted on a blood smear, after a several-month history of increasing transfusion requirements. Molecular and indirect fluorescent antibody (IFA) analyses were negative for B. microti but were positive for B. duncani (IFA titer, 1:1024). The complete 18S ribosomal RNA gene of the parasite was amplified from a blood specimen; the DNA sequence was identical to the sequence for the index WA1 parasite isolated in 1991. The patient's case prompted a transfusion investigation: 34 of 38 pertinent blood donors were evaluated, none of whom tested positive by B. microti IFA. The implicated donor-a 67-year-old California resident-had a B. duncani titer of 1:4096; B. duncani also was isolated by inoculating jirds (Mongolian gerbils) with a blood specimen from March 2009, more than 10 months after his index donation in April 2008. The patient's case was diagnosed more than 4 months after the implicated transfusion in May 2008. CONCLUSIONS: This patient had the third documented transfusion case caused by B. duncani. His case underscores the fact that babesiosis can be caused by agents not detected by molecular or serologic analyses for B. microti.

Heritability of the human infectious reservoir of malaria parasites.

BACKGROUND: Studies on human genetic factors associated with malaria have hitherto concentrated on their role in susceptibility to and protection from disease. In contrast, virtually no attention has been paid to the role of human genetics in eliciting the production of parasite transmission stages, the gametocytes, and thus enhancing the spread of disease. METHODS AND FINDINGS: We analysed four longitudinal family-based cohort studies from Senegal and Thailand followed for 2-8 years and evaluated the relative impact of the human genetic and non-genetic factors on gametocyte production in infections of Plasmodium falciparum or P. vivax. Prevalence and density of gametocyte carriage were evaluated in asymptomatic and symptomatic infections by examination of Giemsa-stained blood smears and/or RT-PCR (for falciparum in one site). A significant human genetic contribution was found to be associated with gametocyte prevalence in asymptomatic P. falciparum infections. By contrast, there was no heritability associated with the production of gametocytes for P. falciparum or P. vivax symptomatic infections. Sickle cell mutation, HbS, was associated with increased gametocyte prevalence but its contribution was small. CONCLUSIONS: The existence of a significant human genetic contribution to gametocyte prevalence in asymptomatic infections suggests that candidate gene and genome wide association approaches may be usefully applied to explore the underlying human genetics. Prospective epidemiological studies will provide an opportunity to generate novel and perhaps more epidemiologically pertinent gametocyte data with which similar analyses can be performed and the role of human genetics in parasite transmission ascertained.

Malaria in patients with sickle cell anemia: burden, risk factors, and outcome at the outpatient clinic and during hospitalization.

Approximately 280,000 children are born with sickle cell anemia (SCA) in Africa annually, yet few survive beyond childhood. Falciparum malaria is considered a significant cause of this mortality. We conducted a 5-year prospective surveillance study for malaria parasitemia, clinical malaria, and severe malarial anemia (SMA) in Dar-es-Salaam, Tanzania, between 2004 and 2009. We recorded 10,491 visits to the outpatient clinic among 1808 patients with SCA and 773 visits among 679 patients without SCA. Similarly, we recorded 691 hospital admissions among 497 patients with SCA and 2017 in patients without SCA. Overall, the prevalence of parasitemia was lower in patients with SCA than in patients without SCA both at clinic (0.7% vs 1.6%; OR, 0.53; 95% CI, 0.32-0.86; P = .008) and during hospitalization (3.0% vs 5.6%; OR, 0.46; 95% CI, 0.25-0.94; P = .01). Furthermore, patients with SCA had higher rates of malaria during hospitalization than at clinic, the ORs being 4.29 (95% CI, 2.63-7.01; P < .001) for parasitemia, 17.66 (95% CI, 5.92-52.71; P < .001) for clinical malaria, and 21.11 (95% CI, 8.46-52.67; P < .001) for SMA. Although malaria was rare among patients with SCA, parasitemia during hospitalization was associated with both severe anemia and death. Effective treatment for malaria during severe illness episodes and further studies to determine the role chemoprophylaxis are required.

Comparison of Plasmodium falciparum growth in sickle cells in low oxygen environment and candle-jar.

The first successful in vitro cultivation of Plasmodium falciparum in sickle cells in a gas mixture containing 3% oxygen, 4% carbon dioxide and 93% nitrogen has been reported recently, contradicting earlier claims that the parasite does not multiply continuously in sickle cell trait (HbAS) and sickle cell anemia (HbSS) erythrocytes at low oxygen tension. The present study extends that report by growing three P. falciparum strains in erythrocytes from four different sickle cell trait and four sickle cell anemia donors. Because P. falciparum is known to grow normally in sickle cells when incubated in a candle-jar estimated to contain 15-18% oxygen, we have also compared the growth at 3% oxygen with that in a candle-jar. For convenience, we also refer to the 3% oxygen and the candle-jar as low and high oxygen environment, respectively. The three P. falciparum strains were first grown continuously in low oxygen environment for at least 1 month in erythrocytes from one HbAS carrier. These stock cultures were then used to infect erythrocytes from additional three HbAS carriers and four HbSS patients. Results of the experiments showed that parasite growth and hemozoin production in HbAS erythrocytes in low oxygen environment were comparable to those obtained in the candle-jar. There was growth retardation in HbSS erythrocytes in low oxygen environment, but some of the parasites survived and eventually produced high parasitemia levels. Continuous cultivation of different P. falciparum strains in HbAS erythrocytes is necessary for investigation of possible molecular differences between malaria parasites in sickle cells and those in HbAA erythrocytes.

Malaria parasite density and splenic status by ultrasonography in stable sickle-cell anaemia (HbSS) children.

BACKGROUND: To determine the relationship, if any, between malaria parasitaemia, parasite density and presence/size of the spleen, using abdominal ultrasonography in stable sickle cell anaemia subjects aged 6 to 15 years. METHODS: A prospective study of one hundred consecutive sickle cell anaemia (SCA) and 100 matched healthy HbAA controls aged 6 to 15 years was undertaken. The presence of malaria parasite, and parasite density were determined using thick blood film. Splenic status was determined by using abdominal ultrasound. None of the children was symptomatic for malaria. RESULTS: The prevalence of autosplenectomy and splenomegaly were 20% and 27% respectively in SCA subjects compared to 0% and 4% respectively in HbAA controls. Thirty percent and 34% of the SCA and controls respectively had malaria parasitaemia. In SCA subjects, the parasite density ranged from 33 to 4000 per microl with a mean of 1071.10 +/- 895.5 per microl. In HbAA controls, the parasite density ranged from 180 to 5150 per microl with a mean of 1759 +/- 1382.87 per microl. The difference in parasite density between SCA subjects and HbAA was significant p<0.05. The parasite densities were relatively higher among SCA with splenomegaly and normal spleen sizes compared to SCA subjects with autosplenectomy. CONCLUSIONS: The prevalence of malaria parasitaemia is lower in healthy SCA subjects than in HBAA controls. Sickle cell anaemia subjects have lower malaria parasite density. Autosplenectomy may be a positive adaptation in SCA subjects with effective innate immunity to malaria.

Levels of malaria specific immunoglobulin G in Nigerian sickle cell disease patients with and without splenomegaly.

Fifty patients with sickle cell disease(SCD) reporting for routine clinical evaluation and fifty normal individuals at the University College Hospital (UCH) community were recruited into this study. They were matched for age and sex. Patients were aged 15 to 54 years. Forty-four (88.0%) of the patients were haemoglobin S while 6 (12.0%) were haemoglobin S + C. Blood samples were collected and examined for malaria parasite. Malaria specific Immunoglobulin G (pf-IgG) absorbance reading was estimated by Enzyme linked immunosorbent assay in all the patients. Of the forty-four patients with sickle cell anaemia, twenty-two had palpable spleen while the remainder (twenty-two) did not have palpable spleen, and four of the six with Haemoglobulin S + C had palpable enlarged spleens. Spleens were graded using the Hacketts grading system. Five (10%) had grade I splenic size, seventeen (34.0%) had splenic size grade II, one (2%) had grade III, two (4%) had grade IV splenic size and one (2%) had grade V splenic size. Malarial specific. Immunoglobulin G (pf-IgG) was estimated by the Enzyme linked immunosorbent assay. Thick blood films stained with Giemsa were examined microscopically for malaria parasite. The result revealed the mean plasmodium falciparum specific pf-Ig G absorbance reading in patients was 1.081 +/- 0.66 while that of the control was 1.037 +/- 0.38. The difference was not statistically significant (p > 0.05). The high mean pf-IgG indicated intensive malarial exposure and higher impact of malaria infection in patients with sickle cell disease in this environment.

Sickle hemoglobin instability: a mechanism for malarial protection.

Heterozygosity for the mutant sickle hemoglobin confers protection from severe Plasmodium falciparum malaria. It is here proposed that this protection derives from the instability of sickle hemoglobin, which clusters red cell membrane protein band 3 and triggers accelerated removal by phagocytic cells. This explanation requires that sickle trait cells manifest greater hemoglobin instability than normal red cells, something that could derive from their content of sickle hemoglobin. The mechanism also implicates splenic function as a determinant of the protective effect.

The Haldane malaria hypothesis: facts, artifacts, and a prophecy.

Heterozygous thalassemia and sickle cell disease produce mild hematological symptoms but provide protection against malaria mortality and severe malaria symptoms. Two explanations for resistance are considered in the literature - impaired growth of the parasite or enhanced removal by the host immune cells. A critical overview of studies that connect malaria resistance with impaired intra-erythrocytic growth is presented. All studies are fraught with two kinds of bias. The first one resides in the impossibility of reproducing the in vivo situation in the simplified model in vitro. The second stems from the generalized use of RPMI 1640 culture medium. RPMI 1640 has critically low levels of several amino acids; is devoid of hypoxanthine (essential for parasite growth) and adenine; and is low in reduced glutathione. Analysis of representative studies indicates that impaired parasite growth in heterozygous red blood cells (RBCs) may derive from nutrient limitations and, therefore, possibly be of artefactual origin. This conclusion seems plausible because studies were performed with RPMI 1640 medium at relatively high hematocrit and for prolonged periods of time. Mutations considered are particularly sensitive to nutrient deprivation because they have higher metabolic demands due to permanent oxidant stress related to unpaired globin chains, sickle hemoglobin and high levels of membrane-free iron. In addition, non-parasitized AS- and thalassemic-RBCs are dehydrated and microcytic. Thus, the number of metabolically active elements per unit of blood volume is remarkably larger in mutant RBCs compared to normocytes. The latter point may represent a confirmation of Haldane's prophetic statement: 'The corpuscles of the anaemic heterozygotes are smaller than normal, and more resistant to hypotonic solutions. It is at least conceivable that they are also more resistant to attacks by the sporozoa which cause malaria.'