Antibodies to the Plasmodium falciparum rhoptry protein RAP-2/RSP-2 in relation to anaemia in Cameroonian children.

Previous studies have implicated reactive antibodies to the low molecular weight rhoptry-associated proteins (RAP-1, RAP-2/RSP-2 and RAP-3) in erythroid cell destruction during Plasmodium falciparum infection. In this pilot study, the frequency, specificity and functional capacity of naturally acquired anti-RAP-2/RSP-2 antibodies were investigated in the sera of anaemic and nonanaemic malaria-infected Cameroonian children. All sera recognized RAP-2/RSP-2 by FACS, irrespective of the clinical status of the subjects. However, the anaemic children showed higher levels of IgG antibodies than the nonanaemic group, while both groups showed similar levels of IgM antibodies. Only few individuals had detectable levels of RAP-2/RSP-2-specific IgG1 and IgG3 subclass antibodies, while no IgG2 and IgG4 subclass antibodies were detected in these subjects. By ELISA, the anaemic group tended to show higher levels of antibodies to RAP-2/RSP-2 regarding all antibody classes tested, except for IgG4 and IgE. Unexpectedly, sera from the nonanaemic group activated complement to a greater extent than those from the anaemic group. These results need to be confirmed in extended studies but indicate that the effector functions of the RAP-2/RSP-2-reactive antibodies may be more important than their amounts. Such antibodies could play a role in both immunity and pathogenesis during P. falciparum infection.

Febrile status, malarial parasitaemia and gastro-intestinal helminthiases in schoolchildren resident at different altitudes, in south-western Cameroon.

In the many areas where human malaria and helminthiases are co-endemic, schoolchildren often harbour the heaviest infections and suffer much of the associated morbidity, especially when co-infected. In one such area, the Buea district, in south-western Cameroon, two cross-sectional surveys, together covering 263 apparently healthy schoolchildren aged 4-12 years, were recently conducted. The prevalences of fever, malarial parasitaemia and intestinal helminth infections, the seroprevalences of anti-Plasmodium falciparum IgG and IgE and anti-glycosylphosphatidylinositol (anti-GPI) IgG, plasma concentrations of total IgE, and the incidence of anaemia were all investigated. The mean (S.D.) age of the study children was 7.56 (1.82) years. Overall, 156 (59.3%) of the children were found parasitaemic, with a geometric mean parasitaemia of 565 parasites/microl. Parasitaemia and fever were significantly associated (P=0.042). The children who lived at low altitude, attending schools that lay 400-650 m above sea level, had significantly higher parasitaemias than their high-altitude counterparts (P<0.01). At low altitude, the children attending government schools had significantly higher parasitaemias than their mission-school counterparts (P=0.010). Of the 31 children (11.9%) found anaemic, 22 (70.4%) had mild anaemia and none had severe anaemia. A significant negative correlation (r=-0.224; P=0.005) was observed between haemoglobin concentration and level of parasitaemia. Infection with Plasmodium appeared to reduce erythrocyte counts (P=0.045), a condition that was exacerbated by co-infection with helminths (P=0.035). Plasma concentrations of total IgE were higher in the children found to be excreting helminth eggs than in those who appeared helminth-free, while levels of anti-P. falciparum IgE were higher in the children with low-grade parasitaemias than in those with more intense parasitaemias. Levels of anti-GPI IgG increased with age and were relatively high in the children who lived at low altitude and in those who were aparasitaemic. The survey results confirm that asymptomatic malarial parasitaemia frequently co-exists with helminth infections in schoolchildren and indicate links with fever, altitude and school type. Immunoglobulin E may play a role in immune protection against helminthiasis whereas anti-GPI antibodies may be important in the development of antimalarial immunity in such children. In Cameroon, as in other areas with endemic malaria, control programmes to reduce the prevalences of infections with intestinal helminths and malarial parasites in schoolchildren, which may effectively reduce the incidence of anaemia, are clearly needed.

Malaria parasitemia and systemic cytokine bias in pregnancy.

OBJECTIVES: To investigate the effects of age, gravidity, and gestational age on peripheral malaria parasitemia and functional T helper (Th) cell heterogeneity in pregnant women. METHODS: Maternal age, gravidity, and gestational age were recorded and peripheral venous blood collected from 175 women attending antenatal clinics in south western Cameroon between March and September 2002. The blood was checked for malaria parasitemia by light microscopy and plasma levels of interleukin (IL)-4 and human interferon (IFN)-gamma were measured by indirect enzyme-linked immunosorbent assay. RESULTS: Malaria parasites were detected in 45 (25.4%) of 174 women, with rates similar for different age groups, trimesters of pregnancy, and gravidity. The geometric mean parasite density was 565, and parasite density was significantly higher in younger than in older women. For all groups combined, the mean IL-4 level was significantly higher than the mean IFN-gamma (P=0.0004), irrespective of the presence and density of malaria parasites, gravidity except for women in their first trimester of pregnancy and grandmultiparas, who had similar levels of IFN-gamma and IL-4. In general, the cytokine profile was biased toward Th2-type of responses in 112 (84.3%) of 132 women. CONCLUSIONS: In this study the ability to control malaria parasitemia during pregnancy was found to be predominantly age dependent, suggesting naturally acquired immunity. Furthermore, the systemic cytokine profile was found to be biased towards Th2 responses, a prerequisite for a successful pregnancy. This pattern was unaffected by maternal age, gestational age, gravidity, or parasitemia.

The effect of maternal, umbilical cord and placental malaria parasitaemia on the birthweight of newborns from South-western Cameroon.

AIM: The impact of maternal, umbilical cord and placental malaria parasitaemia on the incidence of low birthweight was investigated in pregnant women reporting for delivery at the Mutengene Maternity Centre, Fako Division, South West Province, Cameroon. METHODS: The malaria parasitaemia status of 770 umbilical cords, parturient women and placental impression smears were determined by light microscopy using blood samples collected between June 1999 and September 2001. The birthweights (BW) of the newborns were recorded soon after delivery. RESULTS: The results show that malaria parasites were present in the blood samples of 57 out of 730 (7.8%), 233/711 (32.8%) and 248/735 (33.7%) cord, maternal and placental biopsies respectively. Low birthweight (LBW) was recorded in 72 (9.6%) newborns, and the incidence was higher in primiparae. Newborns of mothers who had malaria parasites in their peripheral blood (12.4%) had a higher incidence (p=0.014) of LBW when compared with malaria parasite-free mothers (6.8%). Similarly, neonates born from malaria-positive placentas (13.5%) had a significantly higher incidence of LBW (p=0.006) than those from parasite-negative placentas (6.8%). Furthermore, newborns of malaria parasite-positive mothers, umbilical cords, placentas and primiparae had lower mean birthweight than malaria-negative mothers, placentas, umbilical cords and multiparae. CONCLUSION: We suggest that parity and maternal and placental malaria parasitaemia at delivery have an important negative impact on birthweight, especially in first pregnancies. This observation emphasizes the need for appropriate aggressive intervention strategies such as the use of insecticide-treated bed nets or intermittent preventive treatment to control malaria in pregnancy in the study area.

Malaria infection in pregnancy and its effects on haemoglobin levels in women from a malaria endemic area of Fako Division, South West Province, Cameroon.

Anaemia in pregnancy has been associated with maternal morbidity and mortality and is a risk factor for low birthweight. The importance of malaria as a major cause of anaemia in pregnancy in malaria endemic areas has not been fully elucidated. In two cross-sectional studies of pregnant women at antenatal enrolment and at delivery, we determined the prevalence of anaemia and assessed some risk factors associated with anaemia such as malaria parasitaemia and parity, in women from a malaria endemic area of south western Cameroon. Of the 1118 women whose Hb levels were analysed at first antenatal enrolment, 68.9% were anaemic (Hb<11.0 g/dL) although only 1.3% were severely anaemic (Hb<7 g/dl). At delivery, 69.9% (485/694) of the parturient women were anaemic with 4.3% having severe anaemia. The mean haemoglobin (Hb) level of the pregnant women at enrolment and at delivery was not significantly different. The mean Hb level of malaria parasite positive pregnant women (P=0.0001) and parturient women (P=0.0001) were significantly lower than those who were malaria parasite free. Similarly, the mean Hb level of primigravidae at antenatal enrolment (P=0.0001) and at delivery (primiparae; P=0.0001) was markedly lower than that of multigravidae or multiparae, respectively. Of the anaemic cases, 52.1% were malaria positive while 47.9% were malaria free at enrolment. By contrast, 36.9% (179/485) of the anaemic cases were associated with maternal malaria parasitaemia while 37.3% (174/466) were associated with placental malaria parasitisation. Thus at delivery, anaemia was more common in women without malaria parasitaemia (P=0.0003) or whose placentas were malaria free (63.1% vs 36.9%; P<0.05). The prevalence of anaemia was significantly higher (OR=2.399; P=0.001) in mothers whose peripheral blood and placental biopsy were free of malaria parasites (69.9%) than in those whose peripheral and placental samples had malaria parasites. The mean birthweight and placental weights of newborns of mothers with and without anaemia were similar. In addition, there was no association between maternal anaemia and the incidence of low birthweight. Our study demonstrates a high prevalence of mild to moderate anaemia amongst the study population with relatively low incidences of severe anaemia. Furthermore, at delivery >50% of the anaemic cases were not associated with maternal or placental malaria parasitaemia suggesting the existence of other causes of anaemia in this community. This observation is important in developing a strategy for controlling anaemia in the community.

In vivo efficacy study of quinine sulphate in the treatment of uncomplicated P. falciparum malaria in patients from Southwestern Cameroon.

OBJECTIVE: To evaluate clinical, parasitological and haematological responses to quinine sulphate therapy in patients with uncomplicated malaria using the 14-day WHO protocol. DESIGN: Longitudinal study. SETTING: The Buea Provincial hospital annex located in South Western Cameroon. SUBJECTS: The study participants consisted of children (> or = 8 months) and adults (< or = 550 years) with acute malaria attending the outpatient division of health institutions within Fako Division. RESULTS: Quinine sulphate failure was found in 42% of the patients. Of these 10% were resistant at the RI while 32% were at the RII level. Clinically, the overall success rate (ACR) was 94.2% while therapeutic failures (ETF and LTF) were observed in four patients (5.8%). 27.4% and 17.4% of the patients were anaemic at enrolment and day 14 respectively. The mean PCV levels of the patients increased during the follow-up period except on day three when mean PCV levels dropped. The difference in the mean PCV levels during the follow-up was significant (F = 60.29; P = 0.0001). CONCLUSION: The relatively high resistance of quinine sulphate observed in this study suggests the need to monitor the spread of resistance to this drug in the study region.

Plasmodium falciparum inhibitory capacities of paired maternal-cord sera from south-west province, Cameroon.

In malaria endemic areas, young children are protected against malaria attack during the first few weeks of life partially by transplacentally acquired antibodies. In this study, we show, using an in vitro assay, that part of these antibodies are involved with blocking the re-invasion of host red blood cells by erythrocytic merozoites. One hundred consecutive paired maternal-cord blood samples were collected at delivery and their plasma assayed for total IgG antibodies against crude blood stage antigens by the ELISA. The Ig fraction were precipitated from the plasma samples with (NH(4))(2)SO(4), purified on PD10 columns and used in vitro in determining the re-invasion inhibitory capacities. The mean (+/-SD) ELISA OD(405) IgG antibodies to crude blood stage antigens of maternal (0.476 +/- 0.48) and cord (0.421 +/- 0.39) plasma samples was not significantly different. However, the mean total protein concentration of the Ig fractions for maternal samples (15.82 +/- 3.85) was significantly higher (p=0.005) than that of paired cord samples (12.87 +/- 2.86 mg/ml). There was no correlation between anti-Plasmodium falciparum-specific IgG levels and total protein concentrations of the Ig fractions of both maternal and cord samples. The entire test Ig fractions were strongly inhibitory (>50 per cent) except for four paired maternal cord samples, which were moderately inhibitory (21--50 per cent) at the highest concentration tested (1:2 dilution). Furthermore, there was no correlation between maternal IgG levels and percentage re-invasion inhibition at the 1:2 dilution. The results suggest that mothers resident in malaria endemic areas possess naturally acquired re-invasion inhibitory antibodies and their foetuses can acquire these antibodies transplacentally, which may contribute to the relative protection observed in infants during their first few weeks of life.

In vivo efficacy study of quinine sulphate in the treatment of uncomplicated P. Falciparum malaria in patients from southwestern Cameroon

Objective: To evaluate clinical, parasitological and haematological responses to quinine sulphate therapy in patients with uncomplicated malaria using the 14-day WHO protocol. Design: Longitudinal study.Setting: The Buea Provincial hospital annex located in South Western Cameroon. Subjects: The study participants consisted of children (≥8 months) and adults (≤50 years) with acute malaria attending the outpatient division of health institutions within Fako Division.Results: Quinine sulphate failure was found in 42% of the patients. Of these 10% were resistant at the RI while 32% were at the RII level. Clinically, the overall success rate (ACR) was 94.2% while therapeutic failures (ETF and LTF) were observed in four patients (5.8%). 27.4% and 17.4% of the patients were anaemic at enrolment and day 14 respectively. The mean PCV levels of the patients increased during the follow-up period except on day three when mean PCV levels dropped. The difference in the mean PCV levels during the follow-up was significant (F=60.29; P=0.0001).Conclusion: The relatively high resistance of quinine sulphate observed in this study suggests the need to monitor the spread of resistance to this drug in the study region

An investigation of symptomatic malaria parasitaemia and anaemia in nursery and primary school children in Buea District Cameroon.

OBJECTIVES: To investigate the prevalence of asymptomatic malaria parasitaemia and anemia in nursery and primary school children and correlate parasite density with haemoglobin levels. DESIGN: Cross sectional study. SETTING: Samples were collected from children attending the Saint Theresa's bilingual school and the Government Primary school, Buea, South West Province, Cameroon. SUBJECTS: 297 nursery and primary school children two to 11 years old selected based on parental consent. MAIN OUTOME MEASURES: Relationship between asymptomatic malaria and anaemia. RESULTS: The prevalence of asymptomatic malaria in children was 30.3%. Parasite prevalence and density was independent of age and sex (p > 0.05). The mean haemoglobin level for parasitaemic children was 11.9 g/dl (+/- SD1.1) compared with 12.1 g/dl (+/- 1.2) for non-parasitaemic children. The difference was not significant (t = 1.918, p > 0.05). Anaemia when present was mild. No correlation was found between malaria parasite density and haemoglobin levels (r = -0.065; p > 0.05). CONCLUSION: Asymptomatic malaria was accompanied by low grade parasitaemia, which did not seem to have a significant effect on haemoglobin levels.

Malaria parasitaemia and immunoglobulin levels in paired maternal-cord sera from south western Nigeria.

Blood samples were collected from one hundred and sixteen parturient women and one hundred and seventeen umbilical cords at delivery for the detection of malaria parasitaemia and determination of total serum immunoglobulins (IgG, IgM and IgA). Immunoglobulin levels were measured by the single radial immunodiffusion method and the enzyme-linked immunosorbent assay for cord blood IgM. Malaria parasites were found in 2.6% (3/117) of cord blood and 22.4% (26/116) of maternal samples. Primiparae had the highest incidence and density of parasites compared with multiparae. A negative correlation was obtained between parasite density and parity of the parturient women (r = -0.54, P < 0.005). Mean cord blood IgG (P < 0.001) and IgM (P < 0.0001) were significantly lower than the mean maternal IgG and IgM. Maternal IgG (r = 0.65, P < 0.001) but not IgM (r = 0.09, P < 0.50) correlated with those of cord blood. Mean IgM (P < 0.001) but not IgG (P > 0.50) and IgA (P < 0.40) was significantly higher in malaria positive parturient women compared with malaria negative women. These data confirms the transplacental transfer of IgG across the placenta and the higher incidence of malaria parasitaemia in primiparae. The presence of IgM in cord blood samples suggest intrauterine sensitization of the foetus to common infections.

Studies on Plasmodium falciparum parasitemia and development of anemia in Nigerian infants during their first year of life.

Bimonthly surveys were carried out for 12 months to investigate the dynamics of the acquisition of malaria parasitemia in relation to hemoglobin genotype, development of anemia, and body weight in infants during their first year of life. Thick blood smears for malaria, a capillary blood sample for measurement of packed cell volume (PCV) levels, and body weights were obtained at each survey. Generally, parasite rates (P < 0.001) and mean parasite densities (P < 0.025) increased with age. With a few exceptions, parasite rates and densities were similar in infants with hemoglobin AA and AS during the first year of life. Malaria parasitemia significantly lowered the PCV levels of the study infants only at four (P < 0.001), six (P < 0.025), eight (P < 0.001), and 10 (P < 0.01) months of age. No significant difference was observed in the mean body weight of malaria-positive and -negative infants during the first year of life except in infants two months of age (P < 0.05). The fairly rapid increase in parasite rate and density after two months of age is indicative of the decrease in protection after the first 2-3 months of life.

Lack of association between levels of transplacentally acquired Plasmodium falciparum-specific antibodies and age of onset of clinical malaria in infants in a malaria endemic area of Nigeria.

A cohort of 117 newborns was followed longitudinally for 12 months to determine the age of onset of clinical malaria and the subsequent episodes of malaria, and to investigate the possible existence of a correlation between level of transplacentally acquired Plasmodium falciparum-specific antibodies and age of onset of malaria in the infant. The mean age of onset of malaria in 49 infants was 4.48 +/- 1.54 months. Mean (+/- S.D.) age of onset of clinical malaria in haemoglobin AA infants (4.38 +/- 1.14) was significantly (P < 0.05) lower compared with haemoglobin AS (5.58 +/- 2.43) infants. No correlation was obtained between the age of onset of malaria and the level of cord serum total IgG, IgM and antibodies to P. falciparum antigens. Cord blood seropositivity for antibodies to the blood stage antigen Pf155/RESA and its C-terminal repeat sequence (EENV)6 or to the (NANP)6 peptide representing repeats of the circumsporozoite protein (CSP) did not influence the age of onset of clinical malaria. However, infants with haemoglobin AS whose cord blood was seropositive for antibodies to the (EENV)6 or (NANP)6 peptide showed delayed onset (P < 0.001) of malaria compared with AA seropositive infants. Although our results indicate that transplacentally acquired antibodies to the studied antigens alone offer no significant protection against malaria during the first few months of life, antibodies in concert with other factors such as haemoglobin genotype may contribute to the protection of the newborn.

Asymptomatic malaria parasitaemia and seroreactivities to Plasmodium falciparum antigens in blood donors from Ibadan, south-western Nigeria.

Malaria parasite rates, parasite densities and seroreactivities to two Plasmodium falciparum antigens (Pf155/RESA and circumsporozoite protein) were investigated in a random sample of 416 blood donors attending the Blood Transfusion Unit of the University College Hospital in Ibadan, south-western Nigeria: 224 in October-November 1991 and 192 in March 1992. The incidence of malaria parasitaeia observed in 1991 was significantly higher than that seen in 1992 (41% v. 19%; P < 0.001). In contrast, the geometric mean parasite density in 1992 was significantly higher than in 1991 (440 v. 191) parasites/microliters blood; P < 0.001). Although parasite rates were highest in the group aged 25-31 years in both surveys, there was no apparent correlation between age of donor and parasite density in either survey. Parasite density was significantly higher in AA- than in AS-haemoglobin individuals only in the 1992 survey (P = 0.050). All the blood donors were seropositive for antibodies to crude parasite antigens, indicating heavy exposure to malaria infection. Seroreactivity to Pf155/RESA was similar in the two surveys but that to circumsporozoite protein (CSP) was significantly higher in 1991 than in 1992 (P < 0.001). The seropositivity rates were generally similar to malaria-positive and -negative blood donors. In 1992, however, all the blood donors with high reactivities to Pf155/RESA, as detected by erythrocyte membrane immunofluorscence, were negative for malaria parasites, indicating that this group was relatively protected against malaria parasitaemia. It is recommended that blood samples from prospective blood donors be examined for malaria parasites and that recipients of malaria-infected blood samples be given a curative regimen of antimalarials.

Antibodies to Pf155/RESA and circumsporozoite protein of Plasmodium falciparum in paired maternal-cord sera from Nigeria.

Paired maternal-cord serum samples were analysed for antibodies to the Pf155/RESA and circumsporozoite protein (CSP) antigens of Plasmodium falciparum. Malaria parasites were found in 2.6% (3/117) of cord blood and 22.4% (26/116) of maternal samples. Immunofluorescence assays detected P. falciparum-specific IgG antibodies in all paired samples while P. falciparum-specific IgM was detected in 5.8% (7/121) of cord samples. The positivity rates for antibodies to Pf155/RESA and (NANP)6 but not (EENV)6, a C-terminal repeat sequence of Pf155/RESA, were significantly higher in maternal as compared with cord samples. Seropositivity rates to Pf155/RESA and (EENV)6 were not related to maternal parity group while positivity rates to the (NANP)6 peptide were higher in primiparae and multiparae of > or = 4 parity. These data confirm the transplacental transfer of P. falciparum-specific antibodies and the higher incidence of malaria parasitaemia in primiparae. The presence of P. falciparum-specific IgM in some cord samples suggests intrauterine sensitization of the foetus to malarial antigens.

A longitudinal study of seroreactivities to Plasmodium falciparum antigens in Nigerian infants during their first year of life.

The kinetics of passively transferred maternal antibodies to antigens of Plasmodium falciparum and the dynamics of acquisition of these antibodies during the first year of life was investigated in infants born in a malaria endemic area of south-western Nigeria. Blood samples were collected from the infants at bi-monthly follow-up visits for the analysis of total serum immunoglobulin G, IgM, IgA and antibodies to the antigen Pf155/RESA and against synthetic peptides representing antigenic sequences of the blood stage antigen Pf155/RESA and Ag332 or the circumsporozoite protein (CSP). IgG levels fell from birth till 4 months and a steady rise was observed thereafter till ten months of life. On the contrary mean IgM and IgA levels increased throughout the first year of life. Generally the number of infants positive for antibodies to the antigens under investigation fell from birth and between 4-6 months of age was either low or absent. None of the infants were positive for antibodies to the peptide representing Ag332 during the first year of life. The earliest seroconversion was detected at 6 months of age involving the Pf155/RESA and (NANP)6 antigens. The results indicate a high level of exposure in this study area to malaria infection early in life. The finding of an active antibody response to malarial antigens in infancy encourages the hope that a malaria vaccine administered early in life may accelerate the development of naturally acquired immunity and thus protect the population most at risk.