Acute childhood diarrhoea in northern Ghana: epidemiological, clinical and microbiological characteristics.

BACKGROUND: Acute diarrhoea is a major cause of childhood morbidity and mortality in sub-Saharan Africa. Its microbiological causes and clinico-epidemiological aspects were examined during the dry season 2005/6 in Tamale, urban northern Ghana. METHODS: Stool specimens of 243 children with acute diarrhoea and of 124 control children were collected. Patients were clinically examined, and malaria and anaemia were assessed. Rota-, astro-, noro- and adenoviruses were identified by (RT-) PCR assays. Intestinal parasites were diagnosed by microscopy, stool antigen assays and PCR, and bacteria by culturing methods. RESULTS: Watery stools, fever, weakness, and sunken eyes were the most common symptoms in patients (mean age, 10 months). Malaria occurred in 15% and anaemia in 91%; underweight (22%) and wasting (19%) were frequent. Intestinal micro-organisms were isolated from 77% of patients and 53% of controls (P < 0.0001). The most common pathogens in patients were rotavirus (55%), adenovirus (28%) and norovirus (10%); intestinal parasites (5%) and bacteria (5%) were rare. Rotavirus was the only pathogen found significantly more frequently in patients than in controls (odds ratio 7.7; 95%CI, 4.2-14.2), and was associated with young age, fever and watery stools. Patients without an identified cause of diarrhoea more frequently had symptomatic malaria (25%) than those with diagnosed intestinal pathogens (12%, P = 0.02). CONCLUSION: Rotavirus-infection is the predominant cause of acute childhood diarrhoea in urban northern Ghana. The abundance of putative enteropathogens among controls may indicate prolonged excretion or limited pathogenicity. In this population with a high burden of diarrhoeal and other diseases, sanitation, health education, and rotavirus-vaccination can be expected to have substantial impact on childhood morbidity.

Malaria, anemia, and malnutrition in african children--defining intervention priorities.

BACKGROUND: Malaria, anemia, and malnutrition contribute substantially to childhood morbidity in sub-Saharan Africa, but their respective roles and interactions in conferring disease are complex. We aimed to investigate these interactions. METHODS: In 2002, we assessed plasmodial infection, anemia, and nutritional indices in 2 representative surveys comprising >4000 children in northern Ghana. RESULTS: Infection with Plasmodium species was observed in 82% and 75% of children in the rainy and dry season, respectively. The fraction of fever attributable to malaria was 77% in the rainy season and 48% in the dry season and peaked in children of rural residence. Anemia (hemoglobin level, <11 g/dL) was seen in 64% of children and was, in multivariate analysis, associated with young age, season, residence, parasitemia, P. malariae coinfection, and malnutrition (odds ratio [OR], 1.68 [95% confidence interval [CI], 1.38-2.04]). In addition, malnutrition was independently associated with fever (axillary temperature, > or = 37.5 degrees C; OR, 1.59 [95% CI, 1.13-2.23]) and clinical malaria (OR, 1.67 [95% CI, 1.10-2.50]). CONCLUSIONS: Malnutrition is a fundamental factor contributing to malaria-associated morbidity and anemia, even if the latter exhibits multifactorial patterns. Our data demonstrate that malaria-control programs alone may not have the desired impact on childhood morbidity on a large scale without concomitant nutrition programs.

Toll-like receptor (TLR) polymorphisms in African children: Common TLR-4 variants predispose to severe malaria.

Genetic host factors play a substantial role in susceptibility to and severity of malaria, which continues to cause at least one million deaths per year. Recently, members of the toll-like receptor (TLR) family have been shown to be involved in recognition of the etiologic organism Plasmodium falciparum: The glycosylphosphatidylinositol anchor induces signaling in host cells via TLR-2 and -4, whereas hemozoin-induced immune activation involves TLR-9. Binding of microbial ligands to the respective TLRs triggers the release of proinflammatory cytokines via the TLR/IL-1 receptor (TIR) domain and may contribute to the host response in malaria, including cytokine induction and fever. In a case-control study among 870 Ghanaian children, we examined the influence of TLR-2, -4, and -9 polymorphisms in susceptibility to severe malaria. TLR-2 variants common in Caucasians and Asians were completely absent. However, we found a rare previously undescribed mutation (Leu658Pro), which impairs signaling via TLR-2. We failed to detect any polymorphisms within the TLR-9 Toll/IL-1 receptor domain. Two frequent TLR-9 promoter polymorphisms did not show a clear association with malaria severity. In contrast, the TLR-4-Asp299Gly variant occurred at a high rate of 17.6% in healthy controls and was even more frequent in severe malaria patients (24.1%, P < 0.05). Likewise, TLR-4-Thr399Ile was seen in 2.4% of healthy children and in 6.2% of patients (P = 0.02). TLR-4-Asp299Gly and TLR-4-Thr399Ile conferred 1.5- and 2.6-fold increased risks of severe malaria, respectively. These findings suggest TLR4-mediated responses to malaria in vivo and TLR-4 polymorphisms to be associated with disease manifestation.

Short communication: high prevalence of the cytochrome P450 2C8*2 mutation in Northern Ghana.

Recently, Ghana has changed the first-line treatment of uncomplicated malaria from chloroquine to amodiaquine (AQ) plus artesunate. AQ may cause adverse events such as agranulocytosis and hepatoxicity. The pro-drug AQ is transformed by cytochrome P450 CYP2C8 to the active metabolite N-desethylaminodiaquine. Several polymorphic variants of CYP2C8 are known, some with reduced activity. In 200 randomly selected children from Northern Ghana, we determined the allele frequencies of the CYP2C8 variants CYP2C8*1 (wild type), CYP2C8*2, CYP2C8*3, and CYP2C8*4. We did not detect CYP2C8*3 and CYP2C8*4, but CYP2C8*2 showed an allele frequency of 0.1675. AQ metabolism in patients with CYP2C8*2 may be impaired, and with an increase of AQ based treatment the risk of severe adverse events may mount.

Plasmodium falciparum dhfr but not dhps mutations associated with sulphadoxine-pyrimethamine treatment failure and gametocyte carriage in northern Ghana.

Both use of sulphadoxine-pyrimethamine (SP) and SP-resistance of Plasmodium falciparum are increasing in sub-Saharan Africa. Mutations in the P. falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes can predict treatment failure of SP, however, the degree of this relationship varies regionally. In northern Ghana, pre-treatment dhfr/dhps genotypes were examined in 126 children and associations with PCR-corrected SP treatment outcome and gametocyte carriage were analysed. SP treatment failure within 4 weeks of follow-up occurred in 28%. Among all pre-treatment isolates, the dhfr triple mutation (Ile-51 + Arg-59 + Asn-108) was detected in 47%. Compared with dhfr wildtype parasites, the presence of the dhfr triple mutation increased the risk of treatment failure tenfold. Likewise, parasite clearance was delayed in the presence of dhfr variants. Dhfr mutants and dhps Gly-437 were selected in treatment failure isolates. Gametocytaemia 1 week following treatment was strongly associated with dhfr mutations. Remarkably, this was also true for the prevalence of gametocytes at recruitment. Dhps alleles did neither influence treatment outcome nor gametocyte carriage. In northern Ghana, the prevalence of the dhfr triple mutation can be used as a tool to screen for and to monitor SP resistance. The lack of association between dhps alleles and SP treatment outcome suggests a minor role of these molecular markers in this region at present.

Concurrence of Plasmodium falciparum dhfr and crt mutations in northern Ghana.

BACKGROUND: Both chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) are failing drugs in much of sub-Saharan Africa. Previous findings suggest an association between resistance to CQ and to SP in vivo, in vitro, and on the molecular level. METHODS: In 126 Ghanaian children with uncomplicated malaria, associations between mutations conferring resistance in the Plasmodium falciparum dihydrofolate reductase (dhfr; SP) and chloroquine resistance transporter (crt; CQ) genes, concentrations of residual antimalarial drugs, and gametocyte carriage were examined. RESULTS: Mutant dhfr alleles and the CQ-resistance allele crt T76 were strongly associated with each other. Isolates exhibiting the dhfr triple mutation seven times more likely also contained crt T76 parasites as compared to isolates without the dhfr triple variant (P = 0.0001). Moreover, both, isolates with the dhfr triple mutation (adjusted OR, 3.2 (95%CI, 1.0-10.4)) and with crt T76 (adjusted OR, 14.5 (1.4-150.8)) were associated with an increased likelihood of pre-treatment gametocytaemia. However, crt T76 did not correlate with gametocytaemia following SP treatment and no selection of crt T76 in SP treatment failure isolates was observed. CONCLUSION: These results confirm an association between CQ and SP resistance markers in isolates from northern Ghana. This could indicate accelerated development of resistance to SP if CQ resistance is already present, or vice versa. Considering the enhanced transmission potential as reflected by the increased proportion of isolates containing gametocytes when resistant parasites are present, co-resistance can be expected to spread in this area. However, the underlying mechanism leading to this constellation remains obscure.

High levels of circulating cardiac proteins indicate cardiac impairment in African children with severe Plasmodium falciparum malaria.

A role of the heart in the pathophysiology of severe Plasmodium falciparum malaria has recently been suggested. The objective of the present study was to substantiate this finding in a large group of African children and to correlate results with metabolic conditions in these children. Furthermore, the impact of a potential cardiac impairment on outcome in severe cases was assessed. Results may have important implications on the currently ongoing debate on fluid management in severe malaria patients. Plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), heart-type fatty acid-binding protein (H-FABP), myoglobin and creatine kinase muscle-brain (CK-MB) were compared in 400 African children with severe and mild falciparum malaria. Plasma levels of these markers were correlated with lactate and glucose blood levels, indicators for hypovolemia, and with clinical outcome. Children suffering from severe malaria and children who died (n = 22) exhibited high to very high levels of cardiac markers, respectively. Cardiac factors themselves were not predictive of fatal outcome, while, in multivariate analysis, lactic acidosis was the most important biochemical predictor of death in the severe malaria group. Lactic acidosis and hypoglycemia, however, result in cardiac impairment as defined by elevated levels of circulating cardiac proteins. Our results point to hypovolemia as a major underlying cause of lactic acidosis and hypoglycemia in African children with severe falciparum malaria. These deleterious metabolic conditions contribute to myocardial affection which was evident but not predictive per se of fatal outcome.

Limited influence of haptoglobin genotypes on severe malaria in Ghanaian children.

Haptoglobin (Hp) polymorphisms in sub-Saharan Africa have been associated with an increased risk of severe malaria. However, available data are inconclusive. We examined the role of Hp polymorphisms in susceptibility to Plasmodium falciparum infection and to severe malaria in northern Ghana. Three groups each of 290 age and sex-matched children with severe malaria, children with asymptomatic P. falciparum infection and aparasitaemic healthy controls were studied. Hp typing was based on PCR. In all children, Hp1-1, Hp2-1, and Hp2-2 occurred in 32.4%, 54.1%, and 13.5%, respectively. The prevalence of the Hp genotypes did not differ significantly between groups. However, Hp2 alleles were least common in healthy children (0.379), more frequent in parasitaemic controls (0.402), and most common in severe malaria patients (0.434; = 3.7; P = 0.06). In matched pair analysis, no Hp genotype increased the risk of severe malaria. However, using Hp1-1 as a reference, children with Hp2-2 exhibited a slightly increased risk of severe malaria (odds ratio, 1.6; P = 0.04). These results indicate that Hp polymorhisms may have a rather limited influence on the development of severe malaria.

Age-dependent effect of plasma nitric oxide on parasite density in Ghanaian children with severe malaria.

Nitric oxide (NO) has toxic properties against Plasmodium falciparum. While high blood levels have been associated with protection against severe malarial disease, they may also contribute to the pathophysiology of cerebral malaria and severe anaemia. Promoter variants in the inducible nitric oxide synthase (iNOS) gene have been shown to influence NO concentrations and disease manifestation. However, findings are conflicting. We examined associations of plasma NO metabolites (NOx) with symptoms of severe malaria, particularly malarial anaemia and cerebral malaria, and with iNOS promoter variants. In 210 Ghanaian children with severe malaria, we measured plasma nitrite, nitrate, and S-nitrosothiol, and genotyped the iNOS promoter variants -954G-->C, -1173C-->T, and the -2.5 kb (CCTTT)(n) microsatellite. NOx levels decreased with age. In young children (<24 months), high NOx was associated with reduced parasite density. This was not seen in patients of 24-48 months of age and reversed in older children. Subgroup analysis revealed that in children with severe anaemia but without cerebral involvement (prostration, impaired consciousness, convulsions), high NOx levels correlated with low parasitaemia (P = 0.02). In these children, elevated NOx levels were also associated with the iNOS-954C-->T/(CCTTT)(8) haplotype (P = 0.03). No association between NOx or iNOS genotypes and cerebral malaria was observed. Our findings suggest that in young children with severe malaria NOx reduces parasitaemia. This effect wanes at higher ages and may reflect a predominance of unspecific immune responses to infection in early childhood. This finding may have importance for the understanding of associations between iNOS variants and severe malaria in regions of differing disease manifestation.

A randomized, placebo-controlled, double-blind trial on sulfadoxine-pyrimethamine alone or combined with artesunate or amodiaquine in uncomplicated malaria.

The therapeutic efficacy of sulfadoxine-pyrimethamine (SP) alone, SP plus amodiaquine (AQ), and SP plus artesunate (AS) was assessed in a randomized, placebo-controlled, and double-blind trial among 438 children with uncomplicated Plasmodium falciparum malaria in northern Ghana. Clinical and parasitological responses were monitored for 28 days following treatment; 86%, 98% and 97% of SP-, SP + AQ-, and SP + AS-treated patients achieved adequate clinical and parasitological response (ACPR) within 2 weeks, respectively. Parasite clearance was better with SP + AS than with SP or SP + AQ treatment but re-infections were more common. Polymerase chain reaction (PCR)-corrected rates of ACPR at day 28 were 72.2% for SP, 94.1% for SP + AQ (P < 0.0001), and 94.5% for SP + AS (P < 0.0001). Gametocyte prevalence and density 1 week after treatment were highest in children treated with SP, and lowest in patients receiving SP + AS. No severe adverse events attributable to study medication were observed. In northern Ghana, more than one of four children suffered SP treatment failure within 4 weeks. Both SP + AQ and SP + AS are efficacious alternative therapeutic options in this region. Although SP + AS and SP + AQ treatments have virtually identical cure rates, rapid parasite clearance and pronounced gametocidal effects are the advantages of the former, whereas cost and a lower rate of late re-infections are those of the latter.

iNOS promoter variants and severe malaria in Ghanaian children.

Nitric oxide is an important mediator in the host defence against Plasmodium falciparum malaria. It has antiparasitic effects in vitro. However, its role in clinical disease remains controversial. Polymorphisms in the inducible nitric oxide synthase promoter (iNOS; -954G-->C, -1173C-->T, -2.6 kb CCTTT(n) microsatellite) may influence susceptibility to and severity of malaria. We tested this hypothesis in a case-control study among Ghanaian children with severe malaria (SM) and asymptomatic parasitaemia, respectively, and in healthy controls. In this study, the respective frequencies of iNOS-954G-->C and -1173C-->T did not differ between groups but > or =13 microsatellite copies were associated with SM. -954G-->C and -1173C-->T were in linkage disequilibrium with CCTTT(8) and CCTTT(13), respectively. -954G-->C/CCTTT(8) protected against hyperparasitaemia whereas -1173C-->T/CCTTT(13) increased fatality. These findings suggest that iNOS promoter haplotypes rather than single nucleotide polymorphisms are associated with malaria in Ghanaian children.

Manifestation and outcome of severe malaria in children in northern Ghana.

The symptoms of severe malaria and their contribution to mortality were assessed in 290 children in northern Ghana. Common symptoms were severe anemia (55%), prostration (33%), respiratory distress (23%), convulsions (20%), and impaired consciousness (19%). Age influenced this pattern. The fatality rate was 11.2%. In multivariate analysis, circulatory collapse, impaired consciousness, hypoglycemia, and malnutrition independently predicted death. Children with severe malaria by the current World Health Organization (WHO) classification, but not by the previous one (1990), showed relatively mild clinical manifestations and a low case fatality rate (3.2%). In hospitalized children with severe malaria in northern Ghana, severe anemia is the leading manifestation, but itself does not contribute to mortality. In this region, malnutrition and circulatory collapse were important predictors of fatal malaria. The current WHO criteria serve well in identifying life-threatening disease, but also include rather mild cases that may complicate the allocation of immediate care in settings with limited resources.

Hemoglobin C and resistance to severe malaria in Ghanaian children.

Hemoglobin (Hb) C has been reported to protect against severe malaria. It is unclear whether relative resistance affects infection, disease, or both. Its extent may vary between regions and with disease pattern. We conducted a case-control study of children with severe malaria, asymptomatic parasitemic children, and healthy children in Ghana. HbAC did not prevent infection but reduced the odds of developing severe malaria and severe anemia. Protection was stronger with HbAS. The frequencies of HbCC and HbSC decreased, from healthy children to asymptomatic parasitemic children to children with severe malaria. These data support the notion that natural selection of HbC occurs because of the relative resistance it confers against severe malaria but argue against the notion that HbC offers resistance to infection.

Alpha(+)-thalassemia protects African children from severe malaria.

The high frequency of alpha(+)-thalassemia in malaria-endemic regions may reflect natural selection due to protection from potentially fatal severe malaria. In Africa, bearing 90% of global malaria morbidity and mortality, this has not yet been observed. We tested this hypothesis in an unmatched case-control study among 301 Ghanaian children with severe malaria and 2107 controls (62% parasitemic). In control children, alpha(+)-thalassemia affected neither prevalence nor density of Plasmodium falciparum. However, heterozygous alpha(+)-thalassemia was observed in 32.6% of controls but in only 26.2% of cases (odds ratio [OR], 0.74; 95% confidence interval [CI], 0.56-0.98). Protection against severe malaria was found to be pronounced comparing severe malaria patients with parasitemic controls (adjusted OR in children < 5 years of age, 0.52; 95% CI, 0.34-0.78) and to wane with age. No protective effect was discernible for homozygous children. Our findings provide evidence for natural selection of alpha(+)-thalassemia in Africa due to protection from severe malaria.

Allelic dimorphism of the erythrocyte binding antigen-175 (eba-175) gene of Plasmodium falciparum and severe malaria: Significant association of the C-segment with fatal outcome in Ghanaian children.

BACKGROUND: The erythrocyte binding antigen-175 (EBA-175) on Plasmodium falciparum merozoites mediates sialic acid dependent binding to glycophorin A on host erythrocytes and, therefore, plays a crucial role in cell invasion. Dimorphic allele segments have been found in its encoding gene with a 342 bp segment present in FCR-3 strains (F-segment) and a 423 bp segment in CAMP strains (C-segment). Possible associations of the dimorphism with severe malaria have been analysed in a case-control study in northern Ghana. METHODS: Blood samples of 289 children with severe malaria and 289 matched parasitaemic but asymptomatic controls were screened for eba-175 F- and C-segments by nested polymerase chain reaction. RESULTS: In children with severe malaria, prevalences of F-, C- and mixed F-/C-segments were 70%, 19%, and 11%, respectively. The C-segment was found more frequently in severe malaria cases whereas mixed infections were more common in controls. Infection with strains harbouring the C-segment significantly increased the risk of fatal outcome. CONCLUSION: The results show that the C-segment is associated with fatal outcome in children with severe malaria in northern Ghana, suggesting that it may contribute to the virulence of the parasite.

Chloroquine blood concentrations and molecular markers of chloroquine-resistant Plasmodium falciparum in febrile children in northern Ghana.

Plasmodium falciparum malaria is a predominant reason for health care utilization among children in sub-Saharan Africa. Despite the spread of resistance, chloroquine (CQ) is the most commonly used antimalarial. Little is known about the pattern of CQ use and resistance to the drug prior to attendance at a health care facility, and its impact on clinical presentation in children attending health care facilities in endemic regions. In a cross-sectional study among 840 febrile children presenting at a primary health care facility in northern Ghana from September to December 2000, CQ blood levels were measured by enzyme-linked immunosorbent assay and parasite isolates were genotyped for the CQ resistance markers pfcrt T76 and pfmdr1 Y86. Plasmodium falciparum was present in 95% by polymerase chain reaction and CQ was detected in 64% of the children. Concentrations of CQ in blood ranged from 31 to 3897 nmol/L (median 198 nmol/L). The pfcrt T76 and pfmdr1 Y86 resistance markers were detected in 84% and 57% of the isolates, respectively, and were selected by CQ. A significant trend for higher frequencies of the resistance markers with increasing CQ concentrations was observed. In this typical primary health care setting in sub-Saharan Africa, CQ use prior to attendance at a health care facility and CQ-resistant P. falciparum are frequent. As CQ selects resistant P. falciparum genotypes, CQ should be omitted as a first-line drug even in primary health care facilities when self-treatment with CQ is common.