Cases of Eastern equine encephalitis in humans associated with Aedes canadensis, Coquillettidia perturbans and Culiseta melanura mosquitoes with the virus in New York State from 1971 to 2012 by analysis of aggregated published data.

From 1971 to 2012, in New York State, years with human Eastern equine encephalitis (EEE) were more strongly associated with the presence of Aedes canadensis, Coquillettidia perturbans and Culiseta melanura mosquitoes infected with the EEE virus (Fisher's exact test, one-sided P = 0.005, 0.03, 0.03) than with Culiseta morsitans, Aedes vexans, Culex pipiens-restuans, Anopheles quadrimaculatus or Anopheles punctipennis (P = 0.05, 0.40, 0.33, 1.00, 1.00). The estimated relative risk of a case in a year in which the virus was detected vs. not detected was 14.67 for Ae. canadensis, 6.38 for Cq. perturbans and 5.50 for Cs. morsitans. In all 5 years with a case, Cs. melanura with the virus was detected. In no year was there a case in the absence of Cs. melanura with the virus. There were 18 years with no case in the presence of Cs. melanura with the virus. Such observations may identify the time of increased risk, and when the methods may be used to prevent or reduce exposure to vector mosquito species in this geographic region.

Knob-positive and knob-negative Plasmodium falciparum differ in expression of a strain-specific malarial antigen on the surface of infected erythrocytes.

We have investigated the expression of a strain-specific malarial antigen on the surface of erythrocytes infected with knobless (K-) variants of knob-positive (K+) strains of Plasmodium falciparum. Aotus blood infected with K+ or K- parasites derived from two independent geographical isolates (Malayan camp and Santa Lucia) was surface iodinated by the lactoperoxidase method. Infected and uninfected erythrocytes were then separated by a new procedure involving equilibrium density sedimentation on a Percoll gradient containing sorbitol. Strain-specific antigens were readily identified on the surface of erythrocytes infected with either of the K+ strains by their characteristic size and detergent solubility. These proteins were not detected on the surface of erythrocytes infected with either of the K- variants nor on uninfected erythrocytes isolated from K+- or K- -infected blood. These results are consistent with a role for the strain-specific surface antigen in cytoadherence of P. falciparum-infected erythrocytes. Our findings represent the second biochemical difference (with the knob-associated histidine-rich protein) between K+ and K- P. falciparum.

Platelet thrombospondin mediates attachment and spreading of human melanoma cells.

Human platelet thrombospondin adsorbed on plastic promotes attachment and spreading of human G361 melanoma cells. Attachment is rapid, and spreading is maximal by 90 min with 60-90% of the attached cells spread. In contrast, thrombospondin promotes attachment but not spreading of human C32 melanoma cells, which attach and spread only on laminin substrates. The specificity of these interactions and the regions of the thrombospondin molecule involved in attachment and spreading were examined using proteolytic fragments of thrombospondin and by inhibition studies. The sulfated fucan, fucoidan, and monoclonal antibody A2.5, which is directed against the heparin-binding domain of thrombospondin, selectively inhibit spreading but only weakly inhibit attachment. Monoclonal antibodies against some other domains of thrombospondin, however, are potent inhibitors of attachment. The amino-terminal heparin-binding domain of thrombospondin does not promote attachment. Large fragments lacking the heparin-binding domain support attachment but not spreading of G361 cells. Attachment activity is lost following removal of the 18-kD carboxyl-terminal domain. These results suggest that at least two melanoma ligands are involved in cell attachment and spreading on thrombospondin. The carboxyl-terminal region and perhaps other regions of the molecule bind to receptor(s) on the melanoma surface that promote initial attachment but not cell spreading. Interaction of the heparin-binding domain with sulfated glycoconjugates on melanoma surface proteoglycans and/or sulfated glycolipids mediates spreading. Monoclonal antibodies A2.5 and C6.7 also reverse spreading of G361 cells growing on glass culture substrates, suggesting that binding to thrombospondin mediates attachment of these melanoma cells in culture.

Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane.

The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage.

Parasite-infected-cell-agglutination and indirect immunofluorescence assays for detection of human serum antibodies bound to antigens on Plasmodium falciparum-infected erythrocytes.

Two methods are described for detecting the binding of serum antibodies from adults in an endemic malarious area (The Gambia) to surface antigens on Plasmodium falciparum-infected erythrocytes. An antibody-mediated parasite-infected-cell-agglutination assay (without secondary antibody) and an indirect immunofluorescence assay employing an anti-Fc secondary reagent were used to detect bound antibody. The surface of erythrocytes containing mature parasites bound antibody, but the surface of uninfected cells or cells containing early parasite stages did not react. Serum from 'non-immune' Europeans did not agglutinate infected erythrocytes, however, in the immunofluorescence test with anti-Ig and anti-F(ab')2 secondary reagents we could detect the binding of IgG antibody from 'non-immune' European serum to a small proportion of infected cells. In contrast to the results with freshly collected isolates, antibodies from sera of Gambian adults did not bind to the surface of infected cells from five different culture-adapted isolates of P. falciparum. These assays are suitable for studies on the antigenic diversity of erythrocyte antigens in natural infections and specific antibody responses to these antigens in infected patients.

Conservation of the Plasmodium falciparum sporozoite surface protein gene, STARP, in field isolates and distinct species of Plasmodium.

The extent of structural conservation of the Plasmodium falciparum sporozoite surface protein gene, STARP, recently characterized in the T9/96 clone, has been analyzed using the polymerase chain reaction. Results from Ivory Coast and Thai clones, field isolates originating from Brazil and Kenya and laboratory-maintained strains strongly suggest that this gene has a highly conserved structure throughout this species. This structure includes a complex repetitive central domain consisting of a mosaic region followed by tandem 45-amino acid-encoding (Rp45) and 10-amino acid-encoding (Rp10) repeat regions. Limited size variation in this domain appeared to result from highly localized duplication events in the Rp45 and Rp10 regions. No size variation was observed in the 5' and 3' coding non-repetitive regions, but minor size polymorphism was found in the single intron at the 5' end of the gene. No evidence was found of distinct families of polymorphic types, as has been observed with the blood-stage MSA-1, MSA-2 and S-antigens. The sequence of the STARP homologue in the phylogenetically close chimpanzee parasite, Plasmodium reichenowi, has also been elucidated and reveals high sequence conservation, although interesting differences were detected in the composition of the Rp10 region, known in P. falciparum to contain B- and T-cell epitopes. Finally, DNA hybridization reveals the presence in rodent malaria species of sequences containing homology to the STARP non-repetitive (though not the repetitive) regions, which would suggest that a similar, conserved gene may exist in these species.

Falciparum malaria parasitized erythrocytes bind to a carboxy-terminal thrombospondin fragment and not the amino-terminal heparin-binding region.

We investigated Plasmodium falciparum parasitized erythrocyte binding to proteolytic fragments of thrombospondin and the effects of anti-thrombospondin monoclonal antibodies on this binding. Purified human platelet thrombospondin was cleaved by trypsin, chymotrypsin or thrombin. Fragments were separated by heparin-agarose affinity chromatography, removing the amino-terminal heparin-binding region. Trypsin at 5.0 micrograms ml-1 of thrombospondin cleaved thrombospondin to reduced 140 and 120 kDa fragments plus a reduced 25-kDa heparin-binding fragment. Infected erythrocytes bound to intact thrombospondin (3420 +/- 460 infected erythrocytes mm-2) and the carboxy-terminal fragment, yielding 120-140-kDa fragments on sulfhydryl reduction, but not to the 25-kDa fragment (144 +/- 104 infected erythrocytes mm-2 (mean +/- s.d., N = 4). Similar results were obtained with chymotrypsin and thrombin cleavage. When the anti-thrombospondin monoclonal antibody MA-I was added to immobilized thrombospondin prior to infected erythrocytes, adherence was inhibited by 99%. At the same concentration, MA-I inhibited adherence to C32 melanoma cells by only 35%. MA-I binds to a calcium-dependent structure at the C-terminal globular region of thrombospondin. Monoclonal antibody MA-II inhibited adherence to thrombospondin by 46%, while MA-III had no effect. These antibodies bind to the N-terminal globular region which includes the heparin-binding site and the segment connecting the two globular regions, respectively. The site(s) for infected erythrocyte binding on thrombospondin reside in the large, 140- or 120-kDa, proteolytic cleavage fragments, and not in the N-terminal heparin-binding region.

Immunochemotherapy for visceral leishmaniasis: a controlled pilot trial of antimony versus antimony plus interferon-gamma.

Twenty-four Kenyan patients with visceral leishmaniasis were treated for 30 days with either conventional therapy (daily pentavalent antimony, n = 14) or experimental immunochemotherapy (daily antimony plus interferon-gamma [IFN-gamma] every other day, n = 10). All 24 patients responded clinically to treatment, and microscopic splenic aspirate scores rapidly decreased in both groups. As judged by splenic aspirate culture results, IFN-gamma-treated patients responded more quickly (50% versus 22% culture-negative after one week and 75% versus 58% culture-negative after two weeks). While not statistically significant, these differences raise the possibility that combination therapy using IFN-gamma, which was safe and well-tolerated, may accelerate the early parasitologic response in patients with visceral leishmaniasis.

Predicting outcome in malaria: correlation between rate of exposure to infected mosquitoes and level of Plasmodium falciparum parasitemia.

The level of Plasmodium falciparum parasitemia at clinical presentation has repeatedly been shown to correlate with severity of disease. Using data collected in western Kenya over 21 months, we examined associations between exposure variables, especially exposure to infective mosquitoes, and prevalence and density of P. falciparum parasitemia among 1,007 children six months to six years of age. The prevalence of P falciparum infection was similar at all exposure levels, but there was a correlation between exposure to sporozoite-infected mosquitoes over the previous 28-day period, and geometric mean parasite density of each cohort (Spearman rank coefficient = 0.724, P = 0.002). The relative odds of having a parasite density > or = 5,000/microliters was increased almost two-fold among individuals exposed to more than 10 infective bites during the prior 28-day period. Children enrolled during the highest incidence period were 80% more likely to have a density > or = 5,000/microliters relative to individuals enrolled during periods of lower incidence. The data suggest that measures, such as malaria vaccines, that reduce parasite densities by limiting numbers of sporozoites reaching the liver, or merozoites released from the liver, will reduce malaria-associated morbidity and mortality, even when they do not prevent all infections.

Studies on the prevalence of leishmanin skin test positivity in the Baringo District, Rift Valley, Kenya.

The leishmanin skin test (LST) was applied in 26 clusters of an average of 97 individuals in Baringo District, Kenya. These clusters were centered around recent cases of visceral leishmaniasis (VL). Of 2,411 individuals tested, 254 (10.5%, 155 males and 99 females) had a positive reaction. Among cured VL patients, the frequency was approximately 30% and no sex difference was observed. In the population as a whole, LST positivity increased with age to a stable level from approximately 15 years of age, reflecting an endemic situation. The level of LST positivity was 25-30% and 10-15% in males and females, respectively. Uninfected household contacts of VL cases had a higher frequency of LST reactivity than the rest of the population. This relationship was significant only in females and children, the prevalence ratio being 2.3 (95% confidence interval 1.3-4.1), 1.9 (1.1-3.5), and 1.4 (0.8-2.5) for females, children, and males, respectively. The frequency of LST positivity was higher individuals living in wood houses than in individuals living in house with mud or stone walls. Again, this difference was significant only in females and children (P = 0.02 and P = 0.04), but not in males (P = 0.7). The results suggest that children and women are exposed to the parasite in or around their houses, whereas adult males are, in addition, exposed elsewhere.

Thrombospondin binding by parasitized erythrocyte isolates in falciparum malaria.

Toward understanding the pathogenesis of vascular sequestration in falciparum malaria, we investigated binding of Plasmodium falciparum parasitized erythrocyte isolates to thrombospondin and other adhesive proteins. Blood samples with rings from 12 patients with falciparum malaria were cultured 30 hr until parasites were mature trophozoites and schizonts. All parasitized erythrocyte isolates bound to thrombospondin, but not to fibronectin, laminin, vitronectin, or factor VIII/von Willebrand factor. Parasitized erythrocyte binding varied among isolates, ranging from 192 to 6,725 per mm2, average 2,953. There was good correlation between trophozoite plus schizont % parasitemia and thrombospondin binding (r = 0.884, P less than 0.001). In two patients with stupor, 3,642 and 2,864 parasitized erythrocytes bound per mm2, in proportion to parasitemia, suggesting cerebral malaria is not due to increased binding affinity. These results indicate there is a conserved function among isolates from this geographic region, known to be antigenically diverse at the parasitized erythrocyte membrane surface. These results support the hypothesis that specific binding to an endothelial receptor, possibly involving thrombospondin, plays a role in vascular sequestration in falciparum malaria.

Studies of the receptors on melanoma cells for Plasmodium falciparum infected erythrocytes.

We investigated whether thrombospondin plays a role in the binding of Plasmodium falciparum parasitized erythrocytes to C32 melanoma cells. Twelve patient isolates bound variably to melanoma cells, with good correlation between the degree of binding to cells and binding to thrombospondin. With a synchronous preparation of asexual parasites, acquisition of the capacity to bind to thrombospondin occurred at the same parasite stage as binding to melanoma cells. Development of parasites to trophozoites and schizonts correlated with binding of parasitized erythrocytes to thrombospondin and melanoma cells. The infected erythrocyte receptor for thrombospondin was destroyed by mild trypsinization, as was the receptor for melanoma cells. Although these results suggest similarity in the melanoma cell receptor and thrombospondin receptor for infected cells, other results showed that thrombospondin cannot alone be the melanoma cell receptor. Binding to other melanoma cell lines did not correlate with thrombospondin secretion: the RPMI 8252 and G361 cell lines bound few or no infected cells, yet secreted 50-100% as much thrombospondin as C32 cells. Iodinated thrombospondin bound in similar amounts to C32 cells and to noncytoadherent C361 melanoma cells. Binding and nonbinding melanoma cells did not differ in quantity of surface thrombospondin by radioimmunoassay. Thus, although purified, immobilized, thrombospondin binds parasitized erythrocytes, expression of thrombospondin alone on melanoma cells is not sufficient to mediate adherence.

Plasmodium falciparum incidence relative to entomologic inoculation rates at a site proposed for testing malaria vaccines in western Kenya.

Relationships between Plasmodium falciparum incidence and entomologic inoculation rates (EIRs) were determined for a 21-month period in Saradidi, western Kenya, in preparation for malaria vaccine field trials. Children, ranging in age from six months to six years and treated to clear malaria parasites, were monitored daily for up to 12 weeks to detect new malaria infections. Overall, new P. falciparum infections were detected in 77% of 809 children. The percentage of children that developed infections per two-week period averaged 34.7%, ranging from 7.3% to 90.9%. Transmission by vector populations was detected in 86.4% (38 of 44) of the two-week periods, with daily EIRs averaging 0.75 infective bites per person. Periods of intense transmission during April to August, and from November to January, coincided with seasonal rains. Relationships between daily malaria attack rates and EIRs indicated that an average of only 7.5% (1 in 13) of the sporozoite inoculations produced new infections in children. Regression analysis demonstrated that EIRs accounted for 74% of the variation in attack rates. One of the components of the EIR, the human-biting rate, alone accounted for 68% of the variation in attack rates. Thus, measurements of either the EIR or the human-biting rate can be used to predict corresponding attack rates in children. These baseline epidemiologic studies indicate that the intense transmission patterns of P. falciparum in Saradidi will provide excellent conditions for evaluating malaria vaccine efficacy.

Plasmodium falciparum gametocytemia in Kenyan children: associations among age, intensity of exposure to transmission, and prevalence and density of subsequent gametocytemia.

Recently, an association was described between the density of Plasmodium falciparum asexual parasitemia in Kenyan children and the entomologic inoculation rate (EIR) measured prior to measurement of asexual parasitemia. This study examined whether transmission pressure, as represented by the EIR, was associated with the prevalence or density of gametocytemia in Kenyan children. Each month for 19 months, a cohort of approximately 50 children was given a radical cure and enrolled in the study. Blood films were taken on days 0, 7, and 14. The EIR was calculated for the 28-day period ending 14 days prior to enrollment: the relationship between blood film data from day 7 and exposure variables was explored. We found that younger children were more likely to be gametocytemic than older children and, if gametocytemic, were more likely to have a dense gametocytemia. There was an inverse relationship between the number of infective bites per night received and prevalence but not density of gametocytemia, even after age adjustment. Concordance of gametocytemia prevalence on days 0 (64%), 7 (66%), and 14 (52%) was poor; 84% of the children were positive on at least one day. This indicates that in many subjects the detectable gametocytemia varied over the 14 days. Under these holoendemic transmission conditions, the EIR is inversely correlated with prevalence of gametocytemia, and point measurements of gametocytemia by conventional microscopy underestimate the number of infective donor hosts.

Agglutination of Plasmodium falciparum-infected erythrocytes from east and west African isolates by human sera from distant geographic regions.

Plasmodium falciparum-infected erythrocytes (PfE) were collected from acutely infected children in The Gambia and Tanzania and cultured for more than 30 hr until the parasites were mature trophozoites. Sera collected from these countries, other African countries, Asia, and South America were used in the PfE microagglutination test to determine whether PfE from East and West Africa share surface antigens. From the patterns of agglutination reactivity, we identified extensive antigenic diversity in surface antigens, but obtained no evidence for greater differences between isolates from East or West Africa and those within one region. The majority of sera from immune adults from The Gambia, Tanzania, Sudan, Nigeria, or Ghana were pan-agglutinating, and agglutinated all PfE isolates from The Gambia and Tanzania. Some sera from immune adults of Irian Jaya also agglutinated each of the seven African isolates, while others agglutinated many but not all of the isolates, similar to sera from immune adults of Flores, Indonesia. In contrast, sera from nonimmune adults from Colombia agglutinated few of the African isolates. It was remarkable, however, that sera from nonimmune Colombians agglutinated any African isolates. Our results are consistent with the following conclusions: some PfE surface antigen(s) are very diverse; this diversity is a feature of the parasite worldwide; the repertoire of isolate-specific surface antigens, although large, includes antigens that are either identical or antigenically cross-reactive in geographically very distant parasite populations; and African adults have pan-agglutinating antibodies that may contribute to protective immunity. Such pan-agglutinating antibodies could reflect the accumulation of a large repertoire of isolate-specific antibodies. The contribution of antibody against any shared PfE surface antigen to the pan-agglutinating reactivities is unknown and awaits development of the appropriate reagents.

A new rural focus of cutaneous leishmaniasis caused by Leishmania tropica in Kenya.

We have identified a new rural focus of cutaneous leishmaniasis caused by Leishmania tropica in Muruku sublocation, Salama location, Laikipia district, Rift Valley province, Kenya. Based on a few available case histories, previous reports of L. tropica in Kenya indicated a tentative geographical distribution. Recently 6 indigenous Kenyans from the new focus, who had never travelled outside Kenya, developed cutaneous lesions on the face and/or extremities found to contain Leishmania by culture and smear. Most of the patients manifested the typical 'urban' dry sore which grew slowly into a nodule measuring 2 x 1 cm to 9.5 x 3 cm, and after some months formed a central crust surrounded by small satellite papules. After treatment with Pentostam (sodium stibogluconate), about 40% of the sores failed to heal completely, either scarring centrally with fulminating papules at the edges and spreading peripherally, or healing but then recrudescing at the edge of the scar. Stationary-phase promastigotes from culture isolates were analysed by cellulose acetate electrophoresis. Isoenzyme profiles of 6 isolates were compared with those of World Health Organization reference strains using 12 enzyme loci; they were indistinguishable from those of 2 L. tropica reference strains. All 6 case sites lay within a radius of 4 km. Several other suspected cases from the same area are being investigated.

Safety and immunogenicity of a Plasmodium falciparum sporozoite vaccine: boosting of antibody response in a population with prior natural exposure to malaria.

Recombinant sporozoite vaccine or placebo were administered once to 25 volunteers from an area endemic for malaria. Antibody to R32tet32 rose in 9 of 15 receiving vaccine and remained elevated in 6 for 6 months. Mean absorbance increase was 0.43 +/- 0.40 with vaccine, 0.01 +/- 0.23 with placebo, and 0.72 +/- 0.19 in responders. Six non-responders had significantly lower pre-immunization levels (0.07 +/- 0.05) than responders (0.39 +/- 0.25). There was an association between an increase in immunofluorescence (n = 4) and an increase in absorbance (n = 9) among vaccine recipients (n = 15). Vaccine-induced increase in antibody to natural circumsporozoite antigen was indicated by increases in immunofluorescence and by increases in circumsporozoite precipitation score in 2 of the 5 responders with highest antibody increase measured by enzyme-linked immunosorbent assay. Response to subunit sporozoite vaccine paralleled response to prior natural sporozoite exposure and was significant and prolonged in a population with prior natural exposure to malaria.

Paecilomyces pyelonephritis complicating nephrolithiasis and review of Paecilomyces infections.

We report a case of Paecilomyces variotii isolated from the renal pelvis at ureterolithotomy. The patient presented with nephrolithiasis, acute flank pain, fever and pyuria, which resolved postoperatively. Paecilomyces has infected the cornea, prosthetic lens implants, lacrimal sac, maxillary sinuses, prosthetic mitral and aortic valves, skin and a ventriculoperitoneal shunt.

Identification of isolate-specific proteins on sorbitol-enriched Plasmodium falciparum infected erythrocytes from Gambian patients.

We have compared the surface radio-iodinated proteins of uninfected and Plasmodium falciparum-infected erythrocytes from natural infections of human patients. Cryopreserved infected blood from Gambian children with falciparum malaria was thawed, cultured to the middle trophozoite stage, and surface radio-iodinated. Trophozoite-infected cells were enriched about 10-fold on a Percoll gradient newly designed to separate cells based on their differential permeability to sorbitol. Infected blood was radio-iodinated and erythrocytes from the fraction enriched in parasitized cells and uninfected erythrocytes from the same sample obtained from the gradient and compared by SDS-PAGE and autoradiography. In each sample, parasitized erythrocytes contained one or more polypeptides of very high molecular weight (Mr 250 000-300 000) that were not found on uninfected erythrocytes from the same patient. These proteins were isolate-specific in size and number, suggesting that natural isolates contain a variable number of different P. falciparum phenotypes for this surface protein. In addition, these radio-iodinated surface proteins could not be extracted from the host cell membrane by the non-ionic detergent Triton X-100, but were extracted by SDS. The properties of these proteins suggest they are the equivalent for natural infections of the strain-dependent antigen previously described (Leech, Barnwell, Miller & Howard, 1984) on the surface of P. falciparum-infected Aotus erythrocytes. In addition, we observed a second parasite-dependent modification of labelled proteins on infected erythrocytes with the appearance of a new band of Mr 30 000. There were also variations in the pattern of radio-isotope labelled proteins on uninfected erythrocytes from different patients.