The influence of neonatal BCG vaccination on in vitro cytokine responses to Plasmodium falciparum.

BACKGROUND: Bacillus Calmette-Guérin (BCG) vaccination has off-target protective effects against infections unrelated to tuberculosis. Among these, murine and human studies suggest that BCG vaccination may protect against malaria. We investigated whether BCG vaccination influences neonatal in vitro cytokine responses to Plasmodium falciparum. Blood samples were collected from 108 participants in the Melbourne Infant Study BCG for Allergy and Infection Reduction (MIS BAIR) randomised controlled trial (Clinical trials registration NCT01906853, registered July 2013), seven days after randomisation to neonatal BCG (n = 66) or no BCG vaccination (BCG-naïve, n = 42). In vitro cytokine responses were measured following stimulation with P. falciparum-infected erythrocytes (PfIE) or E. coli. RESULTS: No difference in the measured cytokines were observed between BCG-vaccinated and BCG-naïve neonates following stimulation with PfIE or E. coli. However, age at which blood was sampled was independently associated with altered cytokine responses to PfIE. Being male was also independently associated with increased TNF-a responses to both PfIE and E. coli. CONCLUSION: These findings do not support a role for BCG vaccination in influencing in vitro neonatal cytokine responses to P. falciparum. Older neonates are more likely to develop P. falciparum-induced IFN-γ and IFN-γ-inducible chemokine responses implicated in early protection against malaria and malaria pathogenesis.

Prevalence and risk factors for Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis infection in pregnant women in Papua New Guinea.

OBJECTIVE: To determine the prevalence of, and risk factors associated with, Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis infection in pregnant women in Madang, Papua New Guinea (PNG). METHODS: A cross-sectional survey was conducted among 400 pregnant women presenting to antenatal clinics. Sociodemographic and behavioural data were collected and real-time PCR diagnostic methods were used to detect the presence of chlamydia, gonorrhoea and trichomonas in self-collected vaginal swabs. The relationships between symptoms, sociodemographic and behavioural factors and infection were assessed. RESULTS: The prevalence of C. trachomatis was 11.1%, N. gonorrhoeae was 9.7% and T. vaginalis was 21.3%. One-third of women (33.7%) had at least one infection. The most common symptom was abdominal pain (48.0%), but only abnormal vaginal discharge was consistently associated with infection (p<0.001). Women diagnosed with vaginal discharge syndrome were more likely to have at least one treatable infection (50.0% (47/94)  vs 26.8% (68/254), p<0.001), yet 59.1% of women with infection would have been missed by the current clinically-based syndromic diagnosis. Risk factors included having a partner at perceived risk of infection, maternal extramarital intercourse, early sexual debut, lack of formal education, urban residence and smoking. 78.8% of women reported never using condoms. CONCLUSIONS: The prevalences of T. vaginalis, C. trachomatis and N. gonorrhoeae were high among pregnant women in coastal PNG. The poor performance of clinically based syndromic diagnosis suggests that alternative strategies are urgently required to improve detection and reduce the burden of sexually transmitted infections and their associated adverse pregnancy outcomes in this population.

Adhesion of Plasmodium falciparum-infected erythrocytes to hyaluronic acid in placental malaria.

Infection with Plasmodium falciparum during pregnancy leads to the accumulation of parasite-infected erythrocytes in the placenta, and is associated with excess perinatal mortality, premature delivery and intrauterine growth retardation in the infant, as well as increased maternal mortality and morbidity. P. falciparum can adhere to specific receptors on host cells, an important virulence factor enabling parasites to accumulate in various organs. We report here that most P. falciparum isolates from infected placentae can bind to hyaluronic acid, a newly discovered receptor for parasite adhesion that is present on the placental lining. In laboratory isolates selected for specific high-level adhesion, binding to hyaluronic acid could be inhibited by dodecamer or larger oligosaccharide fragments or polysaccharides, treatment of immobilized receptor with hyaluronidase, or treatment of infected erythrocytes with trypsin. In vitro flow-based assays demonstrated that high levels of adhesion occurred at low wall shear stress, conditions thought to prevail in the placenta. Our findings indicate that adhesion to hyaluronic acid is involved in mediating placental parasite accumulation, thus changing the present understanding of the mechanisms of placental infection, with implications for the development of therapeutic and preventative interventions.

Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-infected erythrocytes.

Adherence of Plasmodium falciparum-infected erythrocytes to cerebral postcapillary venular endothelium is believed to be a critical step in the development of cerebral malaria. Some of the possible receptors mediating adherence have been identified, but the process of adherence in vivo is poorly understood. We investigated the role of carbohydrate ligands in adherence, and we identified chondroitin sulfate (CS) as a specific receptor for P. falciparum-infected erythrocytes. Parasitized cells bound to Chinese hamster ovary (CHO) cells and C32 melanoma cells in a chondroitin sulfate-dependent manner, whereas glycosylation mutants lacking chondroitin sulfate A (CSA) supported little or no binding. Chondroitinase treatment of wild-type CHO cells reduced binding by up to 90%. Soluble CSA inhibited binding to CHO cells by 99.2 +/- 0.2% at 10 mg/ml and by 72.5 +/- 3.8% at 1 mg/ml, whereas a range of other glycosaminoglycans such as heparan sulfate had no effect. Parasite lines selected for increased binding to CHO cells and most patient isolates bound specifically to immobilized CSA. We conclude that P. falciparum can express or expose proteins at the surface of the infected erythrocyte that mediate specific binding to CSA. This mechanism of adherence may contribute to the pathogenesis of P. falciparum malaria, but has wider implications as an example of an infectious agent with the capacity to bind specifically to cell-associated or immobilized CS.

The impact of maternal malaria on newborns.

BACKGROUND: Each year, malaria threatens 125 million pregnancies, and gestational malaria is responsible for up to 200,000 infant deaths in sub-Saharan Africa. With advancing knowledge of malaria in pregnancy and its impact on newborns, improved preventive and therapeutic interventions are possible. METHODS: We reviewed and, by consensus, evaluated published literature relevant to malaria and newborns. Important findings are summarised. RESULTS: Pregnant women are more likely than others to be inoculated with and infected by malaria parasites. Poor outcomes are particularly common in primigravid women and their offspring. The placenta is affected through cellular adhesion, cytokine production and mononuclear cell infiltrates. As a result, newborns may have low birthweight owing to intrauterine growth retardation or prematurity. Recent evidence suggests that a subset of these infants is also at higher risk of malaria infections later in life. Preventive strategies to improve maternal and fetal outcomes include intermittent preventive treatment and insecticide-treated bed nets. Asymptomatic malaria infection is not uncommon in newborns, and symptomatic disease occurs. Fever and death are possible during the early days of life, and presentation with a sepsis-like illness can occur during the 1st 2 months of life. Malaria-affected infants face higher than usual risks of infantile anaemia, subsequent malaria infection and death during the 1st year of life. CONCLUSIONS: Malaria is common during pregnancy and can have serious consequences for neonatal health. Neonatal morbidity and mortality can be significantly reduced by proper implementation of insecticide-treated nets and intermittent preventive treatment.

The effect of Plasmodium falciparum malaria on HIV-1 RNA blood plasma concentration.

OBJECTIVES: This study was undertaken to determine the relative effect of malaria infection on HIV concentration in blood plasma, and prospectively to monitor viral concentrations after antimalarial therapy. DESIGN: A prospective, double cohort study was designed to compare the blood HIV-1 RNA concentrations of HIV-positive individuals with and without acute malaria illness. Subjects were followed for 4 weeks after successful malaria therapy, or for 4 weeks from enrollment (controls). METHODS: Malawian adults with symptomatic Plasmodium falciparum parasitemia (malaria group) and asymptomatic, aparasitemic blood donors (control group) were tested for HIV-1 antibodies to identify appropriate study groups. The malaria group received antimalarial chemotherapy only and were followed with sequential blood films. In both groups, blood plasma HIV-1 RNA viral concentrations were determined at enrollment and again at 1, 2 and 4 weeks. RESULTS: Forty-seven malaria patients and 42 blood donors were enrolled. At enrollment blood plasma HIV-1 RNA concentrations were approximately sevenfold higher in patients with malaria than in blood donors (medians 15.1 x 10(4) and 2.24 x 10(4) copies/ml, respectively, P = 0.0001). No significant changes in median HIV-1 concentrations occurred in the 21 blood donors followed to week 4 (P = 0.68). In the 27 subjects successfully treated for malaria who were followed to week 4, a reduction in plasma HIV-1 RNA was observed from a median of 19.1 x 10(4) RNA copies/ml at enrollment, to 12.0 x 10(4) copies/ml at week 4, (P = 0.02). Plasma HIV-1 concentrations remained higher in malaria patients than controls (median 12.0 x 10(4) compared with 4.17 x 10(4) copies/ml, P = 0.086). CONCLUSIONS: HIV-1 blood viral burden is higher in patients with P. falciparum malaria than in controls and this viral burden can, in some patients, be partly reduced with antimalarial therapy.

Parasite adhesion and immune evasion in placental malaria.

Parasite sequestration in the placenta is a key feature of infection by Plasmodium falciparum during pregnancy and is associated with severe adverse outcomes for both mother and baby. Here, James Beeson and colleagues draw together the findings of recent studies on parasite mechanisms that mediate this process. They review evidence for novel parasite variants that appear able to evade pre-existing immunity, for the adhesion of P. falciparum-infected erythrocytes to placental glycosaminoglycans (and the molecular basis of these parasite properties) and for the expression of var genes encoding the variant antigen and adhesive ligand P. falciparum-erythrocyte membrane protein 1 (PfEMP1).

Cytoadherence characteristics of Plasmodium falciparum isolates from Thailand: evidence for chondroitin sulfate a as a cytoadherence receptor.

The association between cytoadherence of Plasmodium falciparum-infected erythrocytes and the severity of malaria has been evaluated. In this study, we investigate adherence to C32 melanoma cells, CD36, intracellular adhesion molecule-1 (ICAM-1), thrombospondin (TSP), E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and chondroitin sulfate A (CSA) of 36 P. falciparum isolates from patients suffering from acute falciparum malaria. Adherence to purified adhesion molecules varied greatly among different parasite isolates. All isolates but one adhered to CD36, but none bound to E-selectin and VCAM-1 beyond control levels. Some P. falciparum isolates adhered to ICAM-1 and to CSA, a newly identified receptor for adherence. There was no correlation between in vitro binding to any one receptor and the patients' conditions. In addition, we investigated the characteristics of adherence to CSA and to C32 melanoma cells. Infected erythrocytes continued to adhere after trypsin digestion and soluble CSA inhibited adherence to C32 melanoma cells in a dose-dependent manner. The results imply a role for CSA in the natural infection of P. falciparum.

Sulfated glycoconjugates as disrupters of Plasmodium falciparum erythrocyte rosettes.

Some strains of Plasmodium falciparum form erythrocyte rosettes that are believed to result from a lectin interaction between malaria-infected and uninfected erythrocytes. The sulfated glycoconjugate heparin and certain heparin derivatives have been observed to disrupt rosettes. To investigate this interaction further, we have studied the effects of four sulfated glycoconjugates on 15 fresh isolates of P. falciparum from Papua New Guinea. A broader range of sulfated glycoconjugates has been tested against a laboratory strain. A concentration of 1,000 micrograms/ml of dextran sulfate (molecular weight [MW] 500,000) was the most potent disrupter of rosettes. Fucoidan, heparin, and dextran sulfate (MW 5,000) were of decreasing effectiveness in 14 of 15 fresh isolates. The same relationship was true for the laboratory strain. Pentosan polysulfate and sulfatide also disrupted rosettes; chondroitin sulfates A, B, and C and keratan sulfate gave either minimal or no rosette disruption. Thus, some sulfated glycoconjugates are potent disrupters of P. falciparum erythrocyte rosettes. Sulfated glycoconjugates that are potent disrupters of P. falciparum rosettes may prove useful in identifying ligands involved in rosette formation.

Cytoadherence characteristics of Plasmodium falciparum-infected erythrocytes from Malawian children with severe and uncomplicated malaria.

Cytoadherence of Plasmodium falciparum-infected erythrocytes to the microvascular endothelium is believed to be a key factor in the development of cerebral malaria. Erythrocyte rosette formation has been correlated with malaria severity in studies from east and west Africa. We cultured fresh isolates from Malawian children with severe (n = 76) or uncomplicated (n = 79) malaria to pigmented trophozoite stage and examined rosette formation and adherence to CD36, intercellular adhesion molecule-1 (ICAM-1), chondroitin sulfate A (CSA), and thrombomodulin (TM). Most (126 of 148) isolates bound to CD36, and 76 of 136 bound to ICAM-1. Fewer bound to CSA (40 of 148) or TM (23 of 148). After controlling for parasitemia, there was an inverse association between binding to CD36 (P = 0.004) or ICAM-1 (P = 0.001) and disease severity. Parasites from children with severe malaria anemia bound least to CD36, whereas ICAM-1 binding was lowest in children with cerebral malaria. There was no difference in rosette formation between any of the groups. In Malawian children, there was no evidence of a positive association between adherence to any of the receptors examined and disease severity. The negative association found raises the possibility that adherence to certain receptors could instead be an indicator of a less pathogenic infection.

Diversity of agglutinating phenotype, cytoadherence, and rosette-forming characteristics of Plasmodium falciparum isolates from Papua New Guinean children.

The relationship between antigenic variation, cytoadherence, rosette formation, and the pathogenesis of malaria has led to great interest in the diversity of these properties in Plasmodium falciparum isolates from different communities. In this study, we extend previous investigations by delineating the spectrum of agglutinating phenotypes, adherence to C32 melanoma cells, human umbilical vein endothelial cells (HUVEC), CD36, and intracellular adhesion molecule-1 (ICAM-1), and rosette-forming ability of a group of 20 P. falciparum isolates from Papua New Guinean children. Agglutination phenotypes were determined by using both the children's convalescent serum and a panel of adult immune sera. The wide range of variant antigenic types in the community was demonstrated by the failure of the agglutination assays to identify any two isolates with the same agglutinating phenotype in this, the largest study of its kind. Comparison of agglutination profiles from fresh and cryopreserved isolates demonstrated the general acceptability of cryopreservation before testing, but cautioned that some isolates may undergo selection and phenotypic change during the process. Nineteen isolates were able to bind to at least one of the four ligands studied and showed marked variation in both avidity and specificity of binding. The purified proteins ICAM-1 and CD36 proved to be the most useful assay ligands for investigating field isolates, with 18 isolates binding to at least one protein and 14 to both. No correlation was found between the binding of isolates to any two ligands nor between the binding of a standardized inoculum and the level of the patient's presenting parasitemia. All isolates from the study group were found to form rosettes (at a mean rate of 14.6% of cultured trophozoites involved in rosettes). A lack of correlation between rosette formation and CD36 binding suggests that the previously reported role of CD36 as a rosette formation receptor may not be important for isolates from Papua New Guinea.

Malaria and anemia in antenatal women in Blantyre, Malawi: a twelve-month survey.

Malaria and anemia are common in pregnant African women. We screened 4,764 Malawian women at first antenatal visits for malaria and anemia. A total of 42.7% had a malaria infection, which was more common and of higher density in primigravidae (prevalence = 47.3%, geometric mean = 332 parasites/microl) and teenagers (49.8%, 390/microl) than in multigravidae (40.4%, 214/microl) or older women (40.6%, 227/microl). However, 35% of gravida 3+ women were parasitemic. A total of 57.2% of the women was anemic (hemoglobin < 11 g/dl), with moderate anemia (7.0-8.9 g/dl) in 14.9% and severe anemia (< 7 g/dl) in 3.2%. Prevalences of malaria and anemia were highest in the rainy season. Women with moderate/severe anemia had higher parasite prevalences and densities than women with mild/no anemia. Logistic regression showed that age, season, and trimester of presentation were significantly associated with the prevalence of malaria, but gravidity was not. In this urban setting, age and season are more important than gravidity as predictors of malaria at first antenatal visit, and parasitemia is frequent in women of all gravidities.

An alternative agglutination assay to measure antibodies to variant surface antigens of Plasmodium falciparum-infected erythrocytes.

Agglutination assays detect antibodies to variant parasite antigens expressed on the surface of Plasmodium falciparum-infected erythrocytes. Standard techniques require immediate analysis limiting the number of samples that can be processed simultaneously and preclude re-examination of slides. Fixed Giemsa-stained smears allow long-term storage and re-examination, without fluorescence microscopy.

Hydrocortisone in chloramphenicol-treated severe typhoid fever in Papua New Guinea.

We investigated the effect of hydrocortisone on mortality and complications in chloramphenicol-treated severe typhoid fever (STF) in Goroka, Papua New Guinea. Of 374 culture-positive patients, 146 formed a retrospective comparison group, of whom 41 had STF. Of 228 patients in the intervention group, 58 had STF. Patients without STF had low mortality (2.5%) with standard treatment. In the intervention group, hydrocortisone was used in two dosage schedules, 100 mg for 12 doses (23 patients) and 400 mg for 12 doses (23 patients). There was no difference in mortality between steroid-treated and comparison STF patients (44.8% versus 43.9%) or in complications, and we conclude that moderate doses of steroids are not beneficial in severe typhoid fever.

Intermittent sulfadoxine-pyrimethamine in pregnancy: effectiveness against malaria morbidity in Blantyre, Malawi, in 1997-99.

Plasmodium falciparum malaria in pregnancy predisposes to maternal and foetal morbidity. In 1993 Malawi adopted intermittent presumptive therapy with sulfadoxine-pyrimethamine (SP) as malaria prophylaxis for all pregnant women. To assess operational effectiveness of SP, we examined (in 1997-99) the relationship between number of doses of SP prescribed in antenatal clinic and indicators of malaria infection and morbidity at delivery, including peripheral and placental parasitaemia, maternal and neonatal anaemia, and birthweight. Among Malawian women delivering in a large urban hospital, SP prescription was associated with a decrease in placental malaria prevalence (from 31.9% with no SP prescription to 22.8% with > or = 2 doses SP) and density, decreased prevalence of low birthweight (from 23% in women not receiving SP to 10.3% in women given > or = 2 doses), and higher maternal haemoglobin concentrations. These effects were most marked in first and second pregnancies, in which malaria prevalence was highest. Maternal and cord blood malaria prevalence and mean cord blood haemoglobin concentrations did not differ with SP usage. Implementation of the SP administration policy was incomplete: 24% of women were not prescribed any SP, and only 30% were prescribed at least 2 doses as recommended. Intermittent presumptive treatment with SP is having a positive impact on some, but not all indicators of malaria infection and morbidity in Malawi. Improved implementation and continued surveillance are essential.

Disruption of erythrocyte rosettes and agglutination of erythrocytes infected with Plasmodium falciparum by the sera of Papua New Guineans.

People living in areas endemic for Plasmodium falciparum develop humoral responses which may contribute to protection against clinical disease but the specificity of such protective antibody responses remains to be defined. Antibodies disrupting erythrocyte rosettes have been associated with protection against cerebral malaria, and antibodies agglutinating infected erythrocytes with reduced episodes of clinical disease. We have studied the capacity of serum from Papua New Guinean adults and children with a spectrum of malaria exposure, including children and adults at the time of clinical disease, to disrupt erythrocyte rosettes and cause agglutination of infected erythrocytes. Using a single parasite isolate, almost all sera from adults from highly endemic areas agglutinated infected erythrocytes, and the majority disrupted rosettes, in some cases at greater titres than hitherto described. There was a correlation between rosette disruption and agglutination in highly exposed adults. Rosette disrupting antibodies were equally frequent in children with cerebral and uncomplicated malaria. Antibodies causing rosette disruption were frequent only in adults with a long history of malarial exposure. Rosette disrupting antibodies do not appear to protect Papua New Guinean children or adults against cerebral malaria.

The age-specific prevalence of Plasmodium falciparum in migrants to Irian Jaya is not attributable to agglutinating antibody repertoire.

Previous observations have shown that individuals migrating from a malaria free area to a malaria endemic region in North Eastern Irian Jaya quickly acquire anti-parasite immunity, in an age-dependent manner. Sera from migrants and long-term residents in this area were examined for their ability to agglutinate a range of Plasmodium falciparum isolates and to disrupt erythrocyte rosettes. Antibody responses to merozoite surface protein 2 (MSP2) and ring-infected erythrocyte surface antigen (RESA) were also determined. The range of isolates agglutinated by sera from the migrants approached that seen in long-term residents. No difference was found between migrant adults and children in the range of agglutinating antibody, size of agglutinates, nor disruption of rosettes. Anti-MSP2 and anti-RESA antibodies were the only factors examined which showed a correlation with age. We conclude that although antibody to parasite neoantigens expressed on the surface of infected erythrocytes may play a role in the acquisition of immunity, the humoral response to other P. falciparum antigens is more likely to account for the age-dependent prevalence of parasitaemia observed.

Reactive arthritis complicating group G streptococcal septicaemia.

We describe a case of group G streptococcal septicaemia complicated by sterile polyarthritis and pericarditis. We suggest that this organism may cause a 'reactive arthritis' type of illness, and that this is in keeping with known properties of the organism. We note that our patient's clinical features fit the modified Jones criteria for the diagnosis of rheumatic fever.

Autonomic neuropathy is common in human immunodeficiency virus infection.

We prospectively evaluated autonomic nervous system function in 17 consecutive ambulant patients with HIV infection [mean age (S.D.) 34.5 (5.5) years] and 17 controls matched for sex and age [31.2 (7.4) years]. A questionnaire was administered, and neurological examination and standard bedside autonomic function tests were performed. Eleven of 17 HIV-infected individuals (64.7%) had symptoms suggestive of autonomic dysfunction, particularly urogenital problems. Thirteen of 17 (76.5%) had autonomic neuropathy (one or more abnormal tests), including nine of 11 symptomatic individuals. Only two controls had any autonomic symptoms, and all had normal tests. Autonomic dysfunction is common in those with HIV infection and AIDS.

The platelet count as a simple measure to distinguish inflammatory bowel disease from infective diarrhoea.

A total of 212 adult patients with infective diarrhoea and 27 with inflammatory bowel disease (IBD), admitted consecutively to an infectious disease unit, were studied in order to determine whether clinical features and laboratory measurements performed on admission identified cases of IBD. Long-standing diarrhoea, blood in the faeces, anaemia, leucocytosis, thrombocytosis, raised ESR and a reduced concentration of serum albumin were more common in patients with IBD (P less than 0.05). The most striking difference was in the platelet count with 59% patients with IBD and 1.6% patients with infective diarrhoea having platelet counts greater than 450 x 10(9)/l. A raised platelet count in a patient admitted to hospital with 'acute gastro-enteritis' suggests IBD.