Changing patterns of referral into a family history clinic and detection of ovarian cancer: a retrospective 10-year review.

This study examines whether a change in the criteria for genetic testing for ovarian cancer risk changed the nature of referrals into our Familial Cancer service. This is a retrospective review of 273 women who underwent risk reducing surgery (RRS). The primary outcome was to establish whether there was an increase in women having RRS with a confirmed genetic mutation. Secondary outcomes included the incidence of occult cancer and of subsequent primary peritoneal cancer. The results showed an increase in women being offered RRS based on genetic diagnosis; 91% versus 32% before the criteria change. Four occult malignancies (1.5%) and two peritoneal cancers (0.7%) were noted.We have demonstrated a change in the nature of referrals to the familial cancer service from perceived risk to genetic diagnosis. We can now counsel women more accurately. With a defined risk we are enabling them to make an informed decision regarding risk reduction.

The roles of the pfcrt 76T and pfmdr1 86Y mutations, immunity and the initial level of parasitaemia, in predicting the outcome of chloroquine treatment in two areas with different transmission intensities.

The resistance of Plasmodium falciparum to chloroquine (CQ) is probably mediated by point mutations in two genes: pfcrt and pfmdr1. The aim of the present study was to investigate, in patients treated with CQ, the association between host factors, such as immunity and initial level of parasitaemia, and the ability to clear P. falciparum parasites carrying the key chloroquine-resistance (CQR) mutations, pfcrt 76T and pfmdr1 86Y. Identical CQ-efficacy trials were performed in 51 young children (aged <5 years) from Kibaha, in north-western Tanzania, and 44 patients (aged 3-57 years) from Darawish, in eastern Sudan. In both areas, all the CQ-treatment failures had infections with the 76T and 86Y alleles before treatment. Although the presence of these two alleles was significantly associated with treatment failure in Sudan (P=0.001), the corresponding association in Tanzania did not reach statistical significance (P=0.1). Of the 39 patients from Darawish and 44 from Kibaha who harboured parasites with the CQR mutations, 12 and 19, respectively, managed to clear their parasitaemias. The ability to clear CQR parasites was significantly associated with the initial level of parasitaemia (with P-values of 0.05 in Tanzania and 0.01 in Sudan) and with age-- the best surrogate for protective immunity in endemic areas (with P-values of 0.02 in Tanzania and 0.001 in Sudan). These results confirm previous observations that indicated that the 76T and 86Y alleles play a role in the mechanism of CQR, although other factors, such as level of parasitaemia when treated and age, are also important. The 76T and 86Y alleles could still be used as predictive markers for CQR, in non-immune individuals and low-transmission areas.

Antibodies to variable Plasmodium falciparum-infected erythrocyte surface antigens are associated with protection from novel malaria infections.

In areas of unstable transmission malaria affects all age groups, but the malaria incidence is lower in adults compared to children and teenagers. Under such conditions subclinical Plasmodium falciparum infections are common and some infections are controlled, because blood parasitaemia is maintained at low densities. Here, we test the hypothesis that the presence or absence of antibodies against variant antigens on the surface of P. falciparum-infected erythrocytes protect individuals against some infectious challenges and render them susceptible to others. Plasma collected in Daraweesh, eastern Sudan, before and after the malaria season from individuals who had (susceptible) or did not have malaria (protected) during the season, were tested for reactivity against variant antigens on the surface of nine parasite isolates by flow cytometry. Both protected and susceptible individuals acquired antibodies to variant antigens during the malaria season. The presence of antibody to a Ghanaian isolate before the season was statistically significantly associated with protection against malaria. When considering all nine isolates, the patterns of antibody acquisition differed between susceptible and protected individuals. Together, the results indicate that pre-existing anti-PfEMP1 antibodies can reduce the risk of contracting clinical malaria when challenged by novel parasite clones expressing homologous, but not heterologous variable surface antigens. The results also confirm that antibodies to variant antigens are induced by both clinical and subclinical infections, and that antibodies against several var sero-types are induced during an infection.

The epidemiology of febrile malaria episodes in an area of unstable and seasonal transmission.

This study investigated the epidemiology of uncomplicated falciparum malaria in an area of unstable and seasonal transmission in eastern Sudan. About 90% of malaria morbidity in this region occurs in the months of September to November, and very few malaria cases occur during the intensely arid Sudanese dry season and during years of drought. The malaria situation in the study site, the village of Daraweesh, was analysed during 3 consecutive malaria seasons in 1993-95 during which the 457 inhabitants suffered at total of 436 episodes of falciparum malaria. Using an Andersen-Gill proportional hazard model for recurrent events stratified by family, we have calculated the relative hazard for clinical malaria episodes by age, sex, haemoglobin genotype, blood type and infection in the previous season. The malaria risk was significantly lower in individuals aged 20-88 years than in the 5-19 years age-group. The relative protection due to adulthood varied between seasons (relative risk, RR, 0x34 to 0x67). Serological data were not consistent with the hypothesis that the age difference in incidence was due to differences in exposure. During the 1993 season the malaria incidence in males was lower than in females (RR = 0x75), during the 1994 season the incidences were comparable, whereas males had an increased risk of malaria in 1995 (RR = 1x87). The relative risk in individuals carrying the haemoglobin AS genotype compared to homozygous AA individuals was 0x57.

Antiplasmodial activity of selected sudanese medicinal plants with emphasis on Acacia nilotica.

Twenty-two plant organs from eleven plants comprising five families were extracted and screened for antiplasmodial activity in vitro against Plasmodium falciparum 3D7 (chloroquine sensitive) and Dd2 (chloroquine resistant and pyrimethamine sensitive). Fifty nine percent of plant extracts from 22 extracts exerted activity on P. falciparum strain 3D7 with an IC(50) less than 50 microg/mL, whereas 43% of plant extracts showed an IC(50) value within 50 microg/mL on Dd2 strains. Plant extracts from Gardenia lutea, Haplophyllum tuberculatum, Cassia tora, Acacia nilotica and Aristolochia bracteolata possessed IC(50) values less than 5 microg/mL on both tested strains. Bioassay guided fractionation of A. nilotica revealed that the ethyl acetate extract possessed the highest activity (IC(50) = 1.5 microg/mL). Fraction 2 (R(f) = 0.75) prepared by preparative chromatography showed the highest activity on P. falciparum (IC(50) = 1.7 microg/mL). Phytochemical analysis indicated that the most active phase contained terpenoids and tannins and was devoid of alkaloids and saponins. The effect of plant extracts on lymphocyte proliferation showed low toxicity to the human cells. This plant has been subjected to long term clinical trials in folk medicine and is a promising plant.

Overlapping antigenic repertoires of variant antigens expressed on the surface of erythrocytes infected by Plasmodium falciparum.

Antibodies against variable antigens expressed on the surface of Plasmodium falciparum-infected erythrocytes are believed to be important for protection against malaria. A target for these antibodies is the P. falciparum erythrocyte membrane protein 1, PfEMP1, which is encoded by around 50 var genes and undergoes clonal variation. Using agglutination and mixed agglutination tests and flow cytometry to analyse the recognition of variant antigens on parasitized erythrocytes by plasma antibodies from individuals living in Daraweesh in eastern Sudan, an area of seasonal and unstable malaria transmission, we show that these antibodies recognize different variant antigens expressed by parasites of different genotype. Comparing the levels and acquisition of antibody to variant antigens in pairs of parasite isolates expressing different variant types, there is a correlation between the acquisition of antibodies to some combinations of variant antigens but not to others. These results indicate that (1) a single infection will induce the production of antibodies recognizing several variants of surface-expressed antigens, (2) the repertoire of variable antigens expressed by different parasites is overlapping and the degree of overlap differs between isolates, and (3) the expression of at least some variant antigens is genetically linked.

Nine-year longitudinal study of antibodies to variant antigens on the surface of Plasmodium falciparum-infected erythrocytes.

PfEMP1 is an antigenically variable molecule which mediates the adhesion of parasitized erythrocytes to a variety of cell types and which is believed to constitute an important target for naturally acquired protective immune responses in malaria. For 9 years we have monitored individuals living in an area of low-intensity, seasonal, and unstable malaria transmission in eastern Sudan, and we have used this database to study the acquisition, specificity, and duration of the antibody response to variant parasitized erythrocyte surface antigens. Both the levels and the spectrum of reactivity of these antibodies varied considerably among individuals, ranging from low levels of antibodies recognizing only few parasitized erythrocyte surface antigens to high levels of broad-specificity antibodies. In general, episodes of clinical malaria were associated with increases in the levels of parasitized erythrocyte surface-specific antibodies that subsided within months of the attack. This response was often, but not always, specific for the antigenic variants expressed by the parasite isolate causing disease. Our study provides evidence that Palciparum falciparum malaria is associated with a short-lived, variant-specific antibody response to PfEMP1-like antigens exposed on the surface of parasitized erythrocytes. Furthermore, our data suggest that the antigenic repertoires of variant antigens expressed by different parasite isolates show considerable overlapping, at least under Sahelian conditions of low-intensity, seasonal, and unstable malaria transmission. Finally, we demonstrate the existence of persistent differences among individuals in the capacity to mount antibody responses to variant surface antigens.

Antiplasmodial activity of selected Sudanese medicinal plants with emphasis on Maytenus senegalensis (Lam.) Exell.

The antiplasmodial activity of plant extracts related to four families was tested on chloroquine sensitive strain 3D7 and chloroquine resistant strain Dd2 of Plasmodium falciparum. The methanolic extract of Harrisonia abyssinica (Simaroubaceae) inhibited Dd2 with IC50 value of 4.7 microg/ml, while in 3D7, the IC50 value was 10 microg/ml. Most of the plants from the family Meliaceae showed highly potent antiplasmodial activity against the two tested strains. Khaya senegalensis, Azadirachta indica and Trichilia emetica showed IC50 values less than 5 microg/ml. The methanolic extract of Annona squamosa (Annonaceae) leaves showed high antiplasmodial activity with IC50 values of 2 and 30 microg/ml on 3D7 and Dd2, respectively. While stem bark showed moderate activity with IC50 values of 8.5 and 120 microg/ml on Dd2. Maytenus senegalensis (Celastraceae) possessed IC50 values of 3.9 on 3D7, 10 microg/ml on Dd2 and had no effect on lymphocyte proliferation even at the highest tested concentration; the IC50 was greater than 100 microg/ml. Liquid-liquid separation of the methanolic extract of M. senegalensis revealed that the dichloromethane extract possessed an IC50 value of only 2.1 microg/ml. Column fractionation of dichloromethane extract gave four fractions and fraction two showed an IC50 value of 0.5 microg/ml. Preliminary phytochemical analysis of dichloromethane fraction revealed terpenoids and traces of phenolic principles but no alkaloid, tannins or flavonoids were detected.

Characteristics of Plasmodium falciparum parasites that survive the lengthy dry season in eastern Sudan where malaria transmission is markedly seasonal.

We have examined 83 inhabitants of Asar village in eastern Sudan, where malaria transmission lasts approximately 2-3 months each year, for the presence of Plasmodium falciparum during the prolonged dry season. All patients were treated with a standard dose of chloroquine following the first diagnosis, then examined by microscopy and the polymerase chain reaction (PCR) every two weeks for the first two months and subsequently once each month for the next 15 months throughout the dry season until the following transmission season. The PCR primers used amplified polymorphic regions of the merozoite surface protein-1 (MSP-1), MSP-2, and glutamate-rich protein genes. Results show that subpatent and asymptomatic parasitemias persisted in some patients for several months throughout the dry season, often as genetically complex infections. Different genotypes could coexist together in a single infection and the proportions of each could fluctuate dramatically during this period. However, in some individuals, single genotypes appeared to persist for several months. Reappearance of clinical symptoms among patients with chronic infections was often associated with appearance of new alleles, indicating reinfections with parasites of novel genotypes.

Seasonal changes in the Plasmodium falciparum population in individuals and their relationship to clinical malaria: a longitudinal study in a Sudanese village.

Residents of Daraweesh village in Sudan were monitored for Plasmodium falciparum infection and malaria morbidity in 3 malaria seasons from 1993 to 1996. Malaria parasites were detected microscopically and by polymerase chain reaction (PCR) in a series of cross-sectional surveys. PCR revealed submicroscopical infections during the dry season, particularly among individuals who had recovered from a malaria episode following successful drug treatment. Clinical and subclinical infections were contrasted by assaying for allelic polymorphism at 2 gene loci, MSP-1 and GLURP and 2 hypotheses examined with reference to these data: that clinical malaria is associated with infection with novel parasite genotypes not previously detected in that host, or alternatively, that clinical malaria episodes are associated with an increased number of clones in an infection. We detected more mixed infections among clinical isolates, but people carrying parasites during the dry season were not found to have an increased risk of disease in the following malaria season. There was a clear association of disease with the appearance of novel parasite genotypes.

Seasonal variation in agglutination of Plasmodium falciparum-infected erythrocytes.

Agglutination and rosette formation are in vitro characteristics of Plasmodium falciparum-infected erythrocytes, which have been associated with host protective immune responses and also with parasite virulence. The present study was carried out in an area of seasonal and unstable malaria transmission in eastern Sudan. Plasma samples were obtained before, during, and after the transmission season from a volunteer cohort of 64 individuals seven years of age and older. These plasmas were assayed for their ability to agglutinate cultured parasitized erythrocytes originally obtained from acute malaria infection samples taken from five of the cohort members. Our data show that the capacity of donor plasma samples to agglutinate parasitized cells depended largely on the time of sampling relative to the transmission season, at least within this epidemiologic setting. Thus, although less than half of the pretransmission season samples could agglutinate any of the five lines of cultured parasites, all post-transmission season samples could agglutinate at least one of the parasite lines, with 74% agglutinating two or more lines. This increase in the agglutination capacity of individual plasma samples after the transmission season occurred essentially regardless of whether an individual had experienced a clinical malaria attack during the transmission season. The study thus confirms the acquisition of agglutinating antibodies following episodes of clinical malaria, but also demonstrates that such acquisition can take place in the absence of disease, presumably as a consequence of subclinical infection. This is the first demonstration of marked seasonal fluctuations in the capacity of individuals' sera to agglutinate parasitized red blood cells. Possible explanations for this effect include a decrease in the levels of agglutinating antibodies between seasons, or shifts in the antigens being recognized by such antibodies from one transmission season to the next. Finally, we showed the existence of marked seasonal fluctuation in the levels of agglutinating antibodies, either because levels of such antibodies are not sustained between seasons or because the antigens recognized change from one season to the next.

Detection of very low level Plasmodium falciparum infections using the nested polymerase chain reaction and a reassessment of the epidemiology of unstable malaria in Sudan.

We have used the nested polymerase chain reaction (PCR) to assay for low level Plasmodium falciparum infections that were below the threshold of detection of blood film examination. This revealed a substantial group of asymptomatic, submicroscopically patent infections within the population of a Sudanese village present throughout the year although clinical malaria episodes were almost entirely confined to the transmission season. In our September, January, April, and June surveys, the PCR-detected prevalences were 13%, 19%, 24%, and 19%, respectively. These figures reveal a much higher prevalence of dry season infection than previous microscopic surveys have indicated. Furthermore, 20% of a cohort of 79 individuals were healthy throughout the September to November transmission season but were PCR-positive for P. falciparum in a least one of a series of samples taken in the ensuing months. Levels of exposure to P. falciparum infection were therefore higher than was previously believed in this region, highlighting the fact that many individuals were infected but healthy for most of the year. The reservoir parasite population was thus larger and more stable than previously thought, a finding that is consistent with the high levels of genetic variation at polymorphic loci reported from analysis of P. falciparum parasites in this area.

Genetic changes in the population of Plasmodium falciparum in a Sudanese village over a three-year period.

The prevalence of alleles of genes of the Plasmodium falciparum population of Asar village in eastern Sudan was monitored over three consecutive years. The characters studied were parasite surface antigens, proteins detected by two-dimensional polyacrylamide gel electrophoresis, enzymes, and drug response. Fluctuations in allele prevalences from one year to another were detected and are discussed in the context of seasonality of malaria transmission in the region studied.

High proportion of subclinical Plasmodium falciparum infections in an area of seasonal and unstable malaria in Sudan.

In the present longitudinal study, a cohort (n = 98) of children and adults 5-30 years of age living in an area of highly seasonal and unstable malaria transmission were followed for malaria morbidity during several successive transmission seasons. Based on morbidity surveillance during 1993 and measurements of antibody titers to the Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA), the cohort was divided into three groups: those who had at least one episode of clinical malaria (Group 1, n = 31), those who did not suffer from clinical malaria but had (Group 2, n = 63) or had not (Group 3, n = 4) a significant increase in antibody titers against the Pf155/RESA antigen. This increase was defined as equal to or greater than a four-fold increase in antibody titer in samples from same individuals taken at the beginning and the end of the malaria transmission season. Such increases in specific antibody levels suggested that the donors had been exposed to a P. falciparum blood-stage infection. Measurements of antibody titers to a peptide derived from the glutamate-rich protein exoantigen gave data parallel to those for Pf155/RESA. A surprisingly high fraction of individuals in the study cohort (approximately 66%) showed evidence of infection without ensuing clinical disease (Group 2).

Evidence of endothelial inflammation, T cell activation, and T cell reallocation in uncomplicated Plasmodium falciparum malaria.

To explain the observation that acute Plasmodium falciparum malaria is associated with a transient inability of peripheral blood cells to respond to antigenic stimulation in vitro, we have postulated the disease-induced reallocation of peripheral lymphocytes, possibly by adhesion to inflamed endothelium. We measured plasma levels of soluble markers of endothelial inflammation and T cell activation in 32 patients suffering from acute, uncomplication P. falciparum malaria, as well as in 10 healthy, aparasitemic control donors. All donors were residents of a malaria-endemic area of Eastern State Sudan. In addition, we measured the T cell surface expression of the interleukin-2 receptor (CD25) and the lymphocyte function-associated antigen (LFA-1; CD11a/CD18). We found that the plasma levels of all inflammation and activation markers were significantly increased in the malaria patients compared with the control donors. In addition, we found a disease-induced depletion of T cells with high expression of the LFA-1 antigen, particularly in the CD4+ subset. The results obtained provide further support for the hypothesis of T cell reallocation to inflamed endothelium in acute P. falciparum malaria.

Genetic evidence that RI chloroquine resistance of Plasmodium falciparum is caused by recrudescence of resistant parasites.

Isolates of Plasmodium falciparum from patients in a Sudanese village exhibiting RI resistance to chloroquine have been typed for allelic variants of 2 merozoite surface antigens, MSP1 and MSP2. Blood forms were taken from each patient before chloroquine was administered, and after parasites had reappeared following treatment. Each patient was found to be infected with genetically different parasites. However, in each patient the parasites of the recrudescent infections possessed the same alleles of each gene as those of the primary infection. The results show that the parasites which reappeared after chloroquine were a genuine recrudescence of the primary forms, and not derived from a new infection.

Differential T-cell expression of LFA-1 in residents from Africa and Denmark. Description of the phenomenon and its possible basis.

All circulating T cells constitutively express the adhesion molecule leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18) at either low or high surface density. In the present paper we have compared the expression of the LFA-1 alpha-chain CD11a on peripheral T cells obtained from indigenous Africans with permanent residence in Africa to T cells from indigenous Danes with permanent residence in Denmark. The Africans had a higher percentage of T cells with high CD11a expression than did Danish donors. The difference was evident in both the CD3-, CD4+, and CD8+ subsets. The difference did not appear to reflect a higher degree of peripheral T-cell activation in the African donors, as T-cell expression of the activation marker IL-2 receptor (CD25) was similar in the two groups. Furthermore, we observed no apparent correlation between CD3+ CD11a(hi) and CD3+ CD25+ values in individual donors. LFA-1 expression on T cells obtained from expatriate Africans with long-term residence in Denmark resembled that of Danish permanent residents more than that of Africans with permanent residence in Africa. In addition, T cells obtained from two expatriate Danes with long-term residence in rural Africa were phenotypically similar to those from African permanent residents. The data suggest that the observed difference is environmental rather than ethnic and may reflect the degree of exposure to infectious agents.

The efficacy of artemether in the treatment of Plasmodium falciparum malaria in Sudan.

The efficacy of artemether (a qinghaosu derivative) administered intramuscularly for the treatment of Plasmodium falciparum malaria was compared to quinine in an open randomized trial including 54 patients in eastern Sudan, where chloroquine resistance is common. The artemether treatment (5 d intramuscular regimen) was effective and the drug was well tolerated. All patients had cleared the parasitaemia and were free of symptoms 48 h after initiation of treatment. The parasite clearance time was comparable in patients receiving artemether and quinine. No side effect was reported by patients receiving artemether. No recrudescence was seen in 21 patients treated with artemether who completed 28 d follow-up. In the quinine group 3 of 18 patients had recrudescences, or possibly reinfections, on days 14, 21 and 28.

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.