Riad A. Bayoumi

Genetic diversity of Plasmodium falciparum in a village in eastern Sudan. 1. Diversity of enzymes, 2D-PAGE proteins and antigens

Twenty-nine plasmodium falciparum isolates from patients in Asar village, eastern Sudan, were characterized for variation in 18 different genetically controlled characters, including iso-enzymes, proteins detected by two-dimensional polyacrylamide gel electrophoresis and blood-stage antigens. Considerable allelic diversity in the genes determining these characters was detected. Each isolate contained genetically distinct parasites. Fifteen individuals were infected with more than one parasite genotype. The diversity of parasite types is most probably generated by recombination during mosquito transmission of mixed parasite clones.

Chloroquine-resistant Plasmodium falciparum in Eastern Sudan

In vivo testing of the sensitivity of Plasmodium falciparum to chloroquine was carried out in 61 falciparum malaria patients with acute symptoms, in Eastern Sudan. In 26 patients (42%), P. falciparum was resistant to chloroquine. Nine patients (15%) had RI resistance, seven (11%) had RII resistance while ten (16%) had RIII resistance. The persistance of parasitaemia and symptoms were highly correlated in patients with RIII responses. In 21 patients in vitro testing of chloroquine sensitivity was carried out simultaneously with the in vivo testing using the World Health Organization microtest. In vivo and in vitro testing were also highly correlated. Isolates from 12 patients with proven in vivo resistance, grew in vitro in the presence of chloroquine concentrations above 0.8 X 10(-6) mol/l blood. Resistant strains have either been spread by refugees across the borders from Ethiopia or have developed indigenously. Mounting drug pressure, mass movement of non-immune refugees and loss of immunity among local inhabitants, due to the drought, are in favour of development of an indigenous focus. Epidemics with intense transmission caused by heavy rains following the drought could have greatly enhanced the emergence and spread of resistant strains.

Severe autosomal recessive muscular dystrophy in an extended Sudanese kindred.

The clinical manifestations, biochemical, electrocardiographic, histological and histochemical features of a severe autosomal recessive muscular dystrophy (MD)‐as seen in 15 members of a large Sudanese kindred, both male and female‐are reported and discussed. The age of onset ranged from three to five years, the pattern of muscular weakness was similar to that of Duchenne MD and pseudohypertrophy was a remarkable feature. Both sexes became completely dependent by 16 years, and died at or before 20 years of age. The clinical picture in this family and the histological features are compared with those of Duchenne MD and with cases reported from Tunisia, Qatar and Libya. Certain clinical and histological features distinguish all of these types from each other and from the milder autosomal recessive MD of childhood which is more usual in the United Kingdom, Europe, Australia and North America.

Genetic diversity of Plasmodium falciparum in a village in eastern Sudan. 2. Drug resistance, molecular karyotypes and the mdr1 genotype of recent isolates.

Isolates of Plasmodium falciparum from a Sudanese village have been collected as part of a study of parasite genetic diversity during seasonal malaria epidemics. The sensitivity in vitro to chloroquine, pyrimethamine and mefloquine of these isolates has been determined. To assess the utility of pulse field gel chromosome separations in isolate characterization, 18 samples from individual patients in a single village were studied using this technique. Extensive variation in chromosome size was detected, no 2 isolates having identical molecular karyotypes. No multidrug resistance (mdr) gene amplification polymorphisms were detected in either chloroquine-resistant or chloroquine-sensitive isolates in this sample.

Cell-mediated immune responses to Plasmodium falciparum purified soluble antigens in sickle-cell trait subjects.

To determine the possible differences in the immune response to Plasmodium falciparum between sickle-cell trait (Hb AS) and normal haemoglobin (Hb AA) individuals, we examined 35 Hb AS and 24 Hb AA subjects matched for age and microenvironment. Their age was 2-55 years and all lived in a malaria endemic area 300 km south of Khartoum. Antibodies to ring-infected erythrocyte surface antigen (Pf155/RESA) and to circumsporozoite (CS) protein (anti-NANP40) indicated equal exposure to falciparum malaria. Peripheral blood mononuclear cells (BMNCs) from 20/35 (57%) Hb AS subjects compared with 10/24 (42%) Hb AA subjects, responded to affinity-purified P. falciparum soluble antigens (SPAg). Of those responding to SPAg, 9 (26%) Hb AS subjects and only two (8%) Hb AA subjects had high responses. The mean proliferative response to SPAg of BMNCs from Hb AS individuals was significantly higher than in Hb AA individuals (P less than 0.025). Responses of BMNCs to PPD and PHA were also higher among Hb AS individuals and correlated positively with responses to SPAg. These findings support the hypotheses that the sickle-cell trait protects individuals from P. falciparum infections, at least in part, by modulating the immune response.

The sickle-cell trait modifies the intensity and specificity of the immune response against P. falciparum malaria and leads to acquired protective immunity

It is proposed that the in vivo mechanism of protection against falciparum malaria in individuals of the Hb AS genotype is not due solely to the adverse influence of Hb AS erythrocytes on the intraerythrocytic growth and development of P. falciparum. Instead, the simple physiological effect of Hb S on parasite growth appears to trigger an in vivo process of enhancement of the intensity and/or specificity of the host immune response, leading to acquired protective immunity, in a process simulating vaccination. Testing the hypothesis may lead to the identification of plasmodial antigens that induce protective responses in the human host and distinguish them from non-protective, immunosuppressive or decoy antigens that promote parasite survival. This may ultimately help in the selection of candidate antigens for a malaria blood-stage vaccine.

Seasonal changes in cell mediated immune responses to soluble Plasmodium falciparum antigens in children with haemoglobin AA and haemoglobin AS

In this longitudinal study peripheral blood mononuclear cells (PBMC) were obtained before and during the malaria season from healthy HbAA and HbAS children. Cells were compared for proliferation in response to stimulation by soluble Plasmodium falciparum antigens (SPAg) or purified derivative of tuberculin (PPD). The lymphoproliferative responses to SPAg of the paired PBMC samples showed 2 distinct seasonal changes in relation to the haemoglobin phenotype. In HbAA children, the lymphoproliferative responses to SPAg were suppressed during the malaria season. In contrast, they were enhanced in HbAS children during the malaria season. No distinct seasonal change in the response to PPD was found in relation to the haemoglobin phenotype. The study points to the role of the sickle cell trait in modulating the cellular immune responses to falciparum malaria.

Reduced cellular immune reactivity in healthy individuals during the malaria transmission season.

Antigen-induced cellular immune responses are suppressed during acute malaria. The present study engages the possibility that malaria-induced alterations in cellular immune reactivity extend beyond the clinical disease. Thus, lymphoproliferative responses of healthy individuals were diminished during the malaria transmission period in individuals living in an area of highly seasonal, unstable malaria transmission. This finding may have important implications for the design of studies of stimulatory properties of antigens using lymphocytes of endemic origin.

Lymphoproliferative Responses to Plasmodium falciparum Antigens in Children With and Without the Sickle Cell Trait

Blood mononuclear cells (BMNC) were isolated from sickle cell trait (HbAS) healthy donors and normal haemoglobin (HbAA) healthy donors resident in a P. falciparum endemic area of eastern Sudan. Blood samples were collected during the malaria season. BMNC were tested for their proliferative responses to phytohaemagglutinin (PHA), purified protein derivative of tuberculin (PPD) and to soluble P. falciparum antigens (SPAg). Higher responses to SPAg and PPD were observed in the HbAS children compared with the HbAA children, whereas no differences were observed among adults of the two phenotypes. Proliferative responses to PHA were comparable in all individuals tested.

Modulation of the cellular immune response during Plasmodium falciparum infections in sickle cell trait individuals

Plasma and peripheral blood mononuclear cells (PBMC) were obtained from P. falciparum‐infecled individuals with and without the sickle cell trait at diagnosis and 7 days after treatment. I lbAA and HbAS patients were compared for levels of plasma soluble IL‐2 receptors (IL‐2R) and the in vitro cellular reactivity to affinity‐purified soluble P. falciparum antigens (SPAg). PPD and phytohae‐magglutinin (PHA). At diagnosis. HbAS patients with clinical disease had lower plasma‐soluble IL‐2R levels and parasite counts than the corresponding HbAA patients, whereas HbAS and HbAA patients with asymptomatic infections had comparable soluble IL‐2R levels and parasite counts. PBMC from HbAS patients had higher proliferation and IFN‐γ production in response to SPAg than PBMC from HbAA patients. The difference in the lymphoproliferativc responses to SPAg between the two groups was evident in patients with asymptomatic infections. In all patients, the clinical severity, the soluble IL‐2R levels and the parasite counts were directly related. The former two were inversely related to the proliferative responses to SPAg. After treatment, all the studied parameters were comparable in the two groups. The study indicates that during P. falciparum infection, HbAS compared with HbAA patients had lower in vivo cellular activation and higher in vitro cellular reactivity in response to soluble malaria antigens.