Biodegradation of organophosphate pesticide chloropyrifos by Egyptian bacterial isolates.

Sixteen microbial isolates capable of growing on Dursban as a secondary substrate were isolated from three soil and sewage water samples collected from different localities polluted with pesticides. Six developed isolates only were capable of biodegrading Dursban and utilizing it as only sole source of carbon, energy and phosphorus. The six bacterial isolates were managed to grow on enrichment medium containing Dursban up to 40 ml/liter, for seven days at 25 degrees C. Each isolate exhibited growth and degradation of Dursban concentrations that best bacteria were identified as Pseudomonas stutzeri S7B4 and Flavobacterium balustinum S8B6. These two bacterial isolates were subjected to some environmental and nutritional parameters that affect the biodegradation process of Dursban. The optimum conditions includes :incubation period, 7 days; Dursban concentrations, 10 ml/l; inoculum size, 4 ml/l; incubation temperature, 35 degrees C; optimum pH value, 7; carbon source, fructose and ribose, respectively; nitrogen source, urea and peptone, respectively; amino acid, histidine; and vitamin, yeast extract, under shaking condition (200 rpm). Only the most potent microbial isolate Pseudomonas stutzeri was grown on their own mineral salts medium which contained 40 mlM/l in case of Dursban in the absence and presence of fructose as the best carbon source for two time intervals i.e. 7 and 15 days. Absence of phosphorus and the presence of many oxidized compounds revealed that the ability of P. stutzeri to biodegrade and detoxify Dursban using it as the sole phosphorus, carbon and energy sources. GC-MS analysis of all three treatments of Dursban-bioremediation process showed no detection of any phosphorus compounds especially Dursban in the three treatments, indicated that both bacterial strains i.e. P. stutzeri S7-B4 and F. balustinum S8B6 were able to utilize Dursban pesticide as carbon and phosphorus sources. Thus, it is possible to use both bacterial strains in the bioremediation of pesticides especially Dursban-contaminated sites.

Polymorphic N-acetyltransferase (NAT2) genotyping of Emiratis.

Polymorphic N-acetyltransferase (NAT2) genotypes were determined in 106 unrelated Emiratis by PCR-RFLP analysis to obtain estimates of allele frequencies. Thirteen different genotypes were found, four associated with the rapid acetylator phenotype and nine with the slow acetylator phenotype. Among 67 phenotypically slow acetylators, there was 100% concordance between phenotype and genotype. Among 39 phenotypically rapid acetylators, 37 possessed at least one wild type allele; a 95% concordance with genotype. Seven different NAT2 alleles associated with slow acetylation were found. The commonest was a NAT2*5 type (C481T) allele which occurred with a frequency of 0.53, a significantly higher frequency than has been reported for other ethnic groups. A second slow allele, a NAT2*6 type (G590A), occurred with a frequency of 0.21. The most common genotypes found were NAT2*5/*5 homozygotes, NAT2*5/*6 heterozygotes and NAT2*4/*5 heterozygotes with frequencies of 0.25, 0.25 and 0.22 respectively. The high overall prevalence of alleles associated with slow acetylation (173/212; 81.6%) among Emiratis is consistent with previously reported high frequency of the slow acetylator phenotype in Arabs. Two apparently new slow alleles were identified but have not yet been fully characterized. One appears to be a NAT2*5 variant allele. The other uncharacterized allele appears likely to contain an entirely new mutation associated with slow acetylation.

Digital codes from hypervariable tandemly repeated DNA sequences in the Plasmodium falciparum circumsporozoite gene can genetically barcode isolates.

DNA typing systems currently used in parasitology involve either hybridising Southern blots with repetitive sequence probes or amplifying genomic sequences using the polymerase chain reaction (PCR). Both such approaches assay allelic length variation, usually in unexpressed tandemly repeated DNA sequences. Where an appropriate target locus exists, an alternative PCR-based strategy which reveals allelic sequence variation in tandemly repeated DNA offers a more accurate and internally controlled assay. We describe such a strategy for the rapid extraction of information on tandem repeat sequence variation from hypervariable alleles, and apply it to the Plasmodium falciparum CS gene. The extreme variability of such DNA 'barcodes' can be used to identify parasite stocks and lineages. This system is also potentially useful for population genetic and epidemiological studies since it offers the possibility of following the spread of distinctively marked parasite genotypes in samples taken from infected individuals.

Soluble haemoglobin is a marker of recent Plasmodium falciparum infections.

Monoclonal antibodies (Mab) were raised against haemoglobin (Hb) associated with Plasmodium falciparum protein and used to develop an ELISA, measuring circulating levels of released Hb. This assay was evaluated in different malaria patients in parallel with ELISA assays for C-reactive protein (CRP) and haptoglobin. Levels of Hb were negatively associated with levels of haptoglobin. Increased levels of serum Hb and CRP and decreased levels of haptoglobin were seen in Danish malaria patients. Consecutive studies showed that increased Hb levels were detectable 3-7 days after initiation of treatment probably because of drug induced destruction of infected erythrocytes. Increased levels of CRP were measured 0-3 days after initiation of treatment. The Hb assay was used in an epidemiological study of malaria in an area of Sudan with unstable malaria transmission. The proportion of Sudanese adults with detectable soluble Hb was higher in the rainy season with malaria transmission compared to the dry season. Hb levels in the rainy season were negatively associated with levels of haptoglobin. Most adults had increased levels of soluble Hb and decreased levels of haptoglobin 7 and 30 days after their treatment of P. falciparum malaria compared to the levels during acute disease. Thus, both soluble Hb and haptoglobin appear to be markers of recent P. falciparum infections. Very high levels of CRP protein were measured in some of the malaria patients at the day of treatment while lower levels were recorded 7 and 30 days after treatment. Soluble Hb levels were associated with malariometric parameters in a similar fashion to haptoglobin. The new Mab-based assay for measuring soluble Hb in the peripheral blood of malaria patients may be useful for future epidemiological studies of 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.

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.

HLA genotypes and variant alleles in Sudanese families of Arab-Negroid tribal origin.

The indigenous population of the Blue Nile Province, Sudan, is an Arab-Negroid admixture, although some isolates of west African origin (Fallata) have begun to intermix with the indigenous population. Consanguineous marriages are common in these Muslim families. Members of 22 nuclear families were typed for HLA class I and II antigens using complement-dependent microcytotoxicity with Ninth International Workshop (9th WS) and local reagents. Considerable polymorphism was observed at each locus with a majority of the World Health Organization (WHO)-recognized alleles represented in the parental sample, albeit at low frequency. Seven parental haplotypes carried A locus alleles which were not identical to WHO-recognized specificities. All appeared to be Aw19-related specificities. Five B locus serologic variants were observed; all appeared distinct from WHO-recognized specificities. In one family we observed a new DR-DQ association; DR4 segregated with DQw2, rather than with DQw3. As has been observed for several other genetic systems in these tribes, considerable polymorphism was found for all class I and class II gene products in spite of a high level of consanguinity. Tribal admixture and/or a selective advantage in protecting the population against disease may account for this unexpectedly high level of heterozygosity.

Cell-mediated immune responses to soluble Plasmodium falciparum antigens in residents from an area of unstable malaria transmission in the Sudan.

This paper describes immune responses to P. falciparum infection in individuals living in an area of highly seasonal, unstable malaria transmission. The in vitro cellular immune responses of peripheral blood mononuclear cells (PBMC) from 36 Sudanese donors to a complex of affinity purified soluble P. falciparum antigens (SPag) and two components thereof (Ag1 and Ag7) were examined and compared to humoral immune parameters. In 29/36 Sudanese donors, SPag induced a significant lymphoproliferative response in vitro. In contrast only 3/27 Danish donors never exposed to malaria responded to SPag. Ag1 and Ag7 induced significant lymphoproliferation in 9/34 and 13/36 Sudanese donors respectively, whereas no Danish donors responded. Significant interferon-gamma production was observed in 16/27, 1/5 and 3/12 Sudanese donors when stimulated by SPag, Ag1 and Ag7 respectively. Lymphoproliferative responses to SPag correlated with proliferative responses to Ag1 and Ag7, and with Spag-induced interferon-gamma production. These results indicate that T-cell clones recognizing epitopes on Ag1 and Ag7 have been expanded in the studied population as a result of exposure to P. falciparum infection. The T-cell parameters did not correlate with the presence of antibodies to SPag, Pf155/RESA or a crude parasite sonicate or with the schizont IFA titers of the plasma. This indicates that parameters outside the degree of exposure to P. falciparum influence the cellular immune responses to malaria.

Antibody reactivities to glutamate-rich peptides of Plasmodium falciparum parasites in humans from areas of different malaria endemicity.

Synthetic P. falciparum peptides were evaluated as tools in epidemiological investigations of malaria. Plasma IgM and IgG antibody reactivities against synthetic peptides covering sequences of glutamate-rich protein (GLURP) and acidic-basic repeat antigen (ABRA) were measured by ELISA in individuals from malaria-endemic areas of Sudan, Indonesia and The Gambia to study antibody responses to these peptides in donors living in areas of different malaria endemicity. IgG and IgM reactivities to the peptides increased with malaria endemicity, although there were no differences in reactivities to the GLURP peptide between non-exposed donors and donors living in areas of low malaria endemicity. IgG reactivities to the GLURP peptide in Sudanese adults were high one month after treatment in all adults tested, while IgG reactivities to the ABRA peptide were infrequent. IgM responses to the peptides tested were shortlived in most patients. In Gambian children with malaria, IgM reactivities but not IgG antibody reactivities against the ABRA peptide were higher in those with mild malaria than in those with severe malaria. The peptides may be useful in future epidemiological studies, especially in areas of low malaria endemicity.

Resistance to falciparum malaria among adults in central Sudan.

Adults claiming resistance to malaria were identified in the Sennar region of central Sudan, where P. falciparum is hyperendemic but seasonal in transmission. Indirect fluorescent antibody (IFA) titers of sera from these individuals were comparable to those of malaria patients with positive blood films, indicating equal exposure, while in vitro antiparasitic activity of their sera tended to be higher, indicating an effective immunological response to falciparum malaria. Hemoglobin S (Hb S) was significantly more prevalent in adults resistant to malaria. This trait offers protection at the erythrocyte level and it is also possible that it could enhance the ability of carrier adults to acquire protective immunity. Erythrocyte 6-phosphogluconate dehydrogenase A (PGDA) and phosphoglucomutase 1 (PGM1), phenotypes of unknown relevance to protection against falciparum malaria, were also significantly more prevalent in those claiming resistance to malaria. A trend of higher prevalence for erythrocyte glucose-6-phosphate dehydrogenase deficiency (G6PD-), Kell (+) and transferrin D (TfD) was detected among resistant individuals and higher KP(a+) and P2 among malaria patients, but the numbers evaluated in this study did not allow determination of statistical significance. No association was found with erythrocyte glyoxalases, ABO and Duffy blood groups and serum haptoglobins.

Sickle cell disease in Sudan.

The clinical, haematological and biochemical features of 50 Sudanese patients with sickle cell disease (SCD) were determined. Of 23 patients with complete family data, 21 had sickle cell anaemia (homozygous HbSS), 2 had sickle-cell/beta+thalassaemia but none had sickle cell/beta Othalassaemia. The remaining 27 patients had HbSS phenotype. 84% of patients were from the Baggara tribe in western Sudan, where HbS is a natural extension of the west African HbS belt. 21 patients were children under 2 years old; 19 were 3-10 years old; and the remaining 10 were over 10 years old. Young patients presented mainly with painful vaso-occlusive crisis, severe anaemia, hand and foot syndrome, fever, underweight, malnutrition and various infectious diseases. All patients had mild to moderate cardiac enlargment; 42% had a moderately enlarged spleen but only 10% had an enlarged liver; 20% had infarctive lesions of long bones and another 8% had Salmonella osteomyelitis. Leg ulcers, priapism, enuresis and cholelithiasis were not observed. Patients had a mean haemoglobin concentration of 7.3 g/dl; reticulocyte count of 15.1%; serum bilirubin of 2.1 mg/dl; HbA2 level of 2.8% and HbF of 7%. Thus, the observed pattern of SCD in Sudan is comparable to the severe type described for Africans and not comparable to the benign form found in Shiite Moslem Arabs of Saudi Arabia. 6 adults with mild SCD had HbF levels below 5%. Amelioration of the disease, therefore, does not seem to be related to HbF levels; nor was it possible to relate it to high levels of erythrocyte 2,3-diphosphoglycerate.

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.

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.

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.

Drug response and genetic characterization of Plasmodium falciparum clones recently isolated from a Sudanese village.

We have isolated 20 clones of Plasmodium falciparum from isolates from patients attending a village clinic in Sudan during 10 d in October-November 1989. The clones were genetically diverse, having highly variable molecular karyotypes and a wide range of drug responses. Chloroquine-sensitive (50% inhibitory concentration [IC50] in the 4-15 nM range) and chloroquine-resistant clones (IC50 in the 40-95 nM range) co-existed in the population, but no obvious amplification of the P-glycoprotein homologue gene, Pgh1 (previously known as the multi-drug resistance gene, mdr1) marked the chloroquine-resistant clones. Chloroquine resistance was reversible by verapamil in these clones, although they varied in their susceptibility to verapamil alone. These observations indicate that the biochemical characteristics of the Sudanese chloroquine-resistant P. falciparum are similar to those reported from south-east Asian and Latin American isolates, which is consistent with there being a similar molecular basis for this phenomenon.

Activation by pyridoxal 5-phosphate of aspartate transaminase in serum of patients with heart and liver disease.

When aspartate transaminase activity in serum is increased, pyridoxal 5-phosphate addition produces more pronounced activation of post-myocardial infarct sera than of sera from patients with chronic liver disease. Possible explanations for this are considered. Routine pre-incubation of sera with pyridoxal phosphate prior to aspartate transaminase determination is recommended.

Uptake and efflux of chloroquine by chloroquine-resistant Plasmodium falciparum clones recently isolated in Africa.

In recently isolated African Plasmodium falciparum clones, the intracellular chloroquine concentration at steady-state, under standard culture conditions, could not differentiate chloroquine-sensitive from resistant parasites. However, under an atmosphere of air the chloroquine-resistant P. falciparum clones released pre-accumulated [3H]chloroquine more rapidly than sensitive clones. The very fast efflux of the pre-accumulated drug from chloroquine-resistant (CQR) parasites resulted in a differential in the drug retained by resistant and sensitive parasites. The chloroquine-sensitive parasites retained 2-3 times more chloroquine than resistant parasites. The steady-state uptake of [3H]chloroquine appeared to be enhanced by verapamil and desipramine in the chloroquine-resistant clones, while the opposite was observed with sensitive clones. This confirmed the suggestion that verapamil inhibits the rapid efflux in CQR parasites resulting in a readily detectable increase in chloroquine accumulation. These observations indicate that the biochemical phenotypes of African chloroquine-resistant P. falciparum are similar to those reported from S.E. Asia and Latin America and are consistent with a common molecular basis for the phenomenon.

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.

A study of chloroquine and desethylchloroquine plasma levels in patients infected with sensitive and resistant malaria parasites.

This study was carried out on 63 patients in the town of Gadaref in eastern Sudan; each patient was given the standard therapeutic dose of chloroquine (CQ). Plasma levels of chloroquine and its major metabolite desethylchloroquine (DCQ) were measured by means of a high-performance liquid chromatographic method (HPLC) in patients infected with sensitive (sensitive group) and resistant (resistant groups) strains of Plasmodium falciparum. The ratios of chloroquine to desethylchloroquine (CQ/DCQ) in different groups were calculated and the results obtained were compared and correlated with the degree of parasitaemia. The statistical analysis of the results showed that the plasma content of CQ and the CQ/DCQ ratio in the majority of the patients fall within the normal mode of distribution. A small group of patients showed a deviation from the normal mode by having a rather high CQ plasma level and a high ratio of CQ/DCQ. The mean plasma levels of CQ and the CQ/DCQ ratio in the sensitive group was found to be higher than that in the resistant groups. However, these differences were found to be not significant. Correlation tests showed that the levels of CQ and the CQ/DCQ ratios increase with the increase of the degree of parasitaemia in the sensitive group but decrease with the increase of parasitaemia in resistant groups.

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.