Folate supplementation to prevent birth abnormalities: evaluating a community-based participatory action plan for refugees and migrant workers on the Thailand-Myanmar border.

OBJECTIVES: Preconception folic acid (PFA) taken at least 3 months before conception can decrease the incidence of neural tube defects (NTDs) by approximately 46%. NTDs contribute significantly to neonatal morbidity and mortality in migrant and refugee populations on the Thailand-Myanmar border (incidence 1.57/1000 live births). This audit aimed to assess uptake of PFA among migrant and refugee women, evaluate knowledge about PFA among local healthcare workers and implement a participatory community intervention to increase PFA uptake and decrease NTD incidence in this population. STUDY DESIGN: A mixed-methods baseline evaluation was followed by an intervention involving health worker education and a community outreach program. A follow-up audit was performed 18 months post-intervention. METHODS: Data were gathered via surveys, short interviews and focus group discussions. The intervention program included community-based workshops, production and distribution of printed flyers and posters, and outreach to various local organisations. RESULTS: Uptake of PFA was <2% both before and after the intervention. Despite a substantial increase in local healthcare worker knowledge of PFA, no significant improvement in PFA uptake after the intervention was detected. Most pregnancies in this local community sample were reported to be unplanned. CONCLUSIONS: High rates of NTDs with low PFA uptake remains a major public health challenge in this transient population. Results indicate that improved health worker knowledge alone is not sufficient to enhance PFA uptake in this population. Integration of PFA education within expanded family planning programs and broad-based food fortification may be more effective.

Post-treatment haemolysis in African children with hyperparasitaemic falciparum malaria; a randomized comparison of artesunate and quinine.

BACKGROUND: Parenteral artesunate is the treatment of choice for severe malaria. Recently, haemolytic anaemia occurring 1 to 3 weeks after artesunate treatment of falciparum malaria has been reported in returning travellers in temperate countries. METHODS: To assess these potential safety concerns in African children, in whom most deaths from malaria occur, an open-labelled, randomized controlled trial was conducted in Kinshasa, Democratic Republic of Congo. 217 children aged between 6 months and 14 years with acute uncomplicated falciparum malaria and parasite densities over 100,000/µL were randomly allocated to intravenous artesunate or quinine, hospitalized for 3 days and then followed for 42 days. RESULTS: The immediate reduction in haemoglobin was less with artesunate than with quinine: median (IQR) fall at 72 h 1.4 g/dL (0.90-1.95) vs. 1.7 g/dL (1.10-2.40) (p = 0.009). This was explained by greater pitting then recirculation of once infected erythrocytes. Only 5% of patients (in both groups) had a ≥ 10% reduction in haemoglobin after day 7 (p = 0.1). One artesunate treated patient with suspected concomitant sepsis had a protracted clinical course and required a blood transfusion on day 14. CONCLUSIONS: Clinically significant delayed haemolysis following parenteral artesunate is uncommon in African children hospitalised with acute falciparum malaria and high parasitaemias. TRIAL REGISTRATION: ClinicalTrials.gov ; Identifier: NCT02092766 (18/03/2014).

Depot medroxyprogesterone acetate and epithelial ovarian cancer: a multicentre case-control study.

OBJECTIVE: To evaluate the effect of depot medroxyprogesterone acetate (DMPA) in protecting against epithelial ovarian cancer (EOC) and to evaluate factors associated with the risk of EOC. DESIGN: A multicentre, case-control study. SETTING: Twelve hospitals located across Thailand. POPULATION: Three hundred and thirty patients with EOC ('cases') and 982 matched controls were recruited from the 12 hospitals. Cases were newly diagnosed patients with EOC, demonstrated pathologically. Controls were age-matched patients admitted to different wards in the same hospital. METHODS: Cases and controls were interviewed by trained interviewers using a standardised pre-tested questionnaire. The factors associated with EOC were evaluated using univariate and multivariate analyses. MAIN OUTCOME MEASURES: The odds ratio (OR) and 95% confidence interval (95% CI) were calculated to assess the relationship between DMPA and EOC. RESULTS: The use of DMPA was found to be associated with a 39% reduction in the risk of EOC with an OR of 0.61 and a 95% CI of 0.44-0.85 (P = 0.002). A significant risk reduction (83%) was observed when the duration of DMPA use was >3 years (OR 0.17; 95% CI 0.07-0.39; P < 0.001). Other factors associated with a reduced risk of EOC were the use of combined oral contraceptive pills and breastfeeding. A factor associated with an increased risk of EOC was a family history of gynaecological cancer. CONCLUSIONS: The results suggest that DMPA may have a protective effect against EOC. If this effect is real, then it represents an important non-contraceptive benefit of DMPA.

Optimizing the culture of Plasmodium falciparum in hollow fiber bioreactors.

The hollow fiber bioreactor (HFBR) is a cell culturing system allowing continuous perfusion of medium. It was designed to grow microorganisms in a dynamically altering medium mimicking change in the in vivo intravascular and extravascular compartments. The cell compartment (extra capillary space) and medium compartment (intra capillary space) are connected through pores of semipermeable fiber membranes. These membranes allow exchange of gas and nutrients. We have adapted this system for the ex vivo culture of Plasmodiumfalciparum at high parasite densities. A Thai P. falciparum isolate (TM036) cultured in RPMI, supplemented with 0.5% Albumax II, could be maintained continuously in the system by daily changes of a small volumes of medium. Under optimized conditions the HFBR cultures attained 8% parasitemia in 40% hematocrit, thereby providing a total parasite biomass of 6.0 x 10(9) parasitized erythrocytes. The main problem encountered was clogging of micropores in the hollow fiber system by cellular debris over time. Although 'reverse flushing' partly prevented this, a larger pore size might be needed to overcome this problem. The system opens new possibilities for the study of in vitro drug sensitivity under conditions mimicking in vivo pharmacokinetics, and the selection of anti-malarial drug resistance and associated parasite biological and genomic changes.

Plasmodium malariae in Bangladesh.

We describe a 32-year-old Bangladeshi male presenting with severe malaria caused by a mono-infection with Plasmodium malariae. Rosetting of infected and uninfected erythrocytes, a putative virulence factor in falciparum malaria, was observed in the blood slide. Severe disease caused by P. malariae is extremely rare. The patient made a rapid recovery with intravenous quinine treatment.

Immune response to Plasmodium vivax has a potential to reduce malaria severity.

Plasmodium falciparum infection causes transient immunosuppression during the parasitaemic stage. However, the immune response during simultaneous infections with both P. vivax and P. falciparum has been investigated rarely. In particular, it is not clear whether the host's immune response to malaria will be different when infected with a single or mixed malaria species. Phenotypes of T cells from mixed P. vivax-P. falciparum (PV-PF) infection were characterized by flow cytometry, and anti-malarial antibodies in the plasma were determined by an enzyme-linked immunosorbent assay. We found the percentage of CD3+delta2+-T cell receptor (TCR) T cells in the acute-mixed PV-PF infection and single P. vivax infection three times higher than in the single P. falciparum infection. This implied that P. vivax might lead to the host immune response to the production of effector T killer cells. During the parasitaemic stage, the mixed PV-PF infection had the highest number of plasma antibodies against both P. vivax and P. falciparum. Interestingly, plasma from the group of single P. vivax or P. falciparum malaria infections had both anti-P. vivax and anti-P. falciparum antibodies. In addition, antigenic cross-reactivity of P. vivax or P. falciparum resulting in antibodies against both malaria species was shown in the supernatant of lymphocyte cultures cross-stimulated with either antigen of P. vivax or P. falciparum. The role of delta2 +/- TCR T cells and the antibodies against both species during acute mixed malaria infection could have an impact on the immunity to malaria infection.

Oxidative stress and rheology in severe malaria.

There is mounting evidence that the release of haemozoin (beta-haematin), which is produced in large amounts during malaria infection and is released into the circulation during schizont rupture, is associated with damage to cell membranes through an oxidative mechanism. The red blood cell membrane is thus oxidised, causing rigidity of the cell. This can contribute to the pathophysiology of severe malaria, since red blood cells will have to deform considerably in order to squeeze through the microcirculation, the patency of which is disturbed by sequestered red blood cells containing the mature forms of the parasite. Rigidity of red blood cells forms a new target for intervention. Since this seems to be caused by oxidative damage to the red blood cell membrane, the anti-oxidant N-acetylcysteine is a promising candidate for adjunctive treatment in severe malaria, which still has a mortality rate as high as 20%.

Identification of cryptic coinfection with Plasmodium falciparum in patients presenting with vivax malaria.

In Thailand, approximately 8% of patients treated for vivax malaria are found subsequently to have coinfection with Plasmodium falciparum. A P. falciparum histidine rich protein 2 (PfHRP-2) dipstick test was evaluated as a predictor of mixed infections with subpatent P. falciparum in a prospective study of 238 patients admitted to the hospital with acute vivax malaria. Of these, 23 (10%) had subsequent development of falciparum malaria without reexposure. Patients with cryptic P. falciparum infection had a significantly lower mean (standard deviation) hematocrit than those with P. vivax alone: 29.6 (7.6%) versus 37.2 (6.4%) (P < 0.0001). Using microscopic appearance of P. falciparum after the start of treatment as the reference standard, the PfHRP-2 test was 74% sensitive and 99% specific in predicting mixed infections with subpatent P. falciparum parasitemia at presentation. The PfHRP-2 dipstick test may be a useful adjunct to microscopy in areas where mixed infections are common.

A comparison of the in vivo kinetics of Plasmodium falciparum ring-infected erythrocyte surface antigen-positive and -negative erythrocytes.

Ring-infected erythrocyte surface antigen (RESA)-positive, Plasmodium falciparum-negative red blood cells (RBCs) are cells from which the malaria parasite has been removed by the host without the destruction of the erythrocyte ("pitting"). The survival of RESA-RBCs in vivo was assessed in 14 severe and 6 uncomplicated falciparum malaria patients. The mean RESA-RBC life of 183 hours (95% confidence interval [CI], 136-246) was longer than the median parasite clearance time of 66 hours (range, 30-108 hours) but shorter than the mean red cell life of 1027 hours (95% CI, 840-1213) (P =.0004), with a median ratio of 0.2:1.0 (range, 0.1-0.7). The estimated median percentage of parasites pitted/body transit was 0.003% (range, 0.001%-0.05%). The rate of rise of the RESA-RBC count during the first 24 hours after antimalarial treatment was significantly faster (P =.036) and the subsequent RESA-RBC survival significantly shorter (P =.017) after treatment with an artemisinin derivative than after treatment with quinine. Parasitization of red cells leads to changes in the erythrocyte that shorten their survival even if the parasite is removed subsequently.

Fake artesunate in southeast Asia.

Artesunate is a key antimalarial drug in the treatment of multidrug-resistant Plasmodium falciparum malaria in southeast Asia. We investigated the distribution of counterfeit artesunate tablets by use of the validated, simple, and inexpensive Fast Red TR dye technique. We also aimed to identify distinguishing characteristics of the fake drugs. Of 104 shop-bought "artesunate" samples from Cambodia, Laos, Myanmar (Burma), Thailand, and Vietnam, 38% did not contain artesunate. Characteristics such as cost and physical appearance of the tablets and packaging reliably predicted authenticity. The illicit trade in counterfeit antimalarials is a great threat to the lives of patients with malaria. The dye test will assist national malaria control authorities in urgently needed campaigns to stop this murderous trade.

Persistence of Plasmodium falciparum HRP-2 in successfully treated acute falciparum malaria.

The potential for Plasmodium falciparum histidine-rich protein-2 (PfHRP-2) dipstick tests to predict antimalarial treatment failure was investigated in a prospective study in Thailand of 38 patients admitted with severe malaria and 54 hospitalized with uncomplicated P. falciparum infections. Of these, 40 had subsequent recrudescence of their infections. Overall, 89% of patients with severe malaria and 61% of patients with uncomplicated malaria had positive PfHRP-2 dipstick tests for > 2 weeks following the start of treatment. Persistence was correlated positively with admission parasite counts, PfHRP-2 intensity scores and disease severity. PfHRP-2 tests which remained positive for > 2 weeks and PfHRP-2 reactive intensity scores on admission, at day 7 and day 14 did not predict treatment failure independent of admission parasitaemia. Freezing and thawing the blood samples did not significantly affect PfHRP-2 results tested by the dipstick technique. The PfHRP-2 dipstick test provides a useful indicator of recent severe malaria, but does not predict the therapeutic response.

Ex-vivo short-term culture and developmental assessment of Plasmodium vivax.

A simple reproducible method for short-term ex-vivo Plasmodium vivax culture is presented in which glucose, ascorbic acid, thiamine, hypoxanthine, and 50% human AB+ serum are added to the standard P. falciparum in-vitro culture medium. Culture of freshly obtained blood samples from patients with acute vivax malaria with > 0.5% parasitaemia resulted in > 95% complete schizogony. Culture could be continued for 5-6 cycles without the addition of red cells. Criteria for staging the erythrocytic development of P. vivax in the first schizogonic cycle based on synchronous ex-vivo culture are presented. The asexual cycle was divided into 7 morphological stages: tiny ring (0-6 h), small ring (6-12 h), large ring (12-18 h), early trophozoite (18-28 h), late trophozoite (28-36 h), early schizont (36-42 h) and mature schizont (42-48 h). This simple method of culturing P. vivax ex vivo is suitable for antimalarial susceptibility and immunoparasitology studies.

Contribution of humoral immunity to the therapeutic response in falciparum malaria.

The contribution of humoral immunity to the therapeutic response in acute falciparum malaria was assessed in a case-control study. Forty adult Thai patients with acute falciparum malaria who had subsequent recrudescent infections and 40 patients matched for age, therapeutic regimen, and disease severity who were cured by Day 28 were studied. All cured patients had positive immunoglobulin (Ig) G to ring-infected erythrocyte surface antigen (RESA) in their admission plasma, compared with only 60% of patients who failed to respond to treatment (P < 0.001). The proportion of IgM-positive cases at admission was also higher in the successfully treated group than in the group with failure (70% versus 30%) (P < 0.001). The geometric mean (95% confidence interval) reciprocal IgG titer at admission was significantly higher in cured patients (187.0 [83.5-418.3]) compared with those who experienced treatment failure (11.6 [5.1-26.5]) (P < 0.001). The patients with uncomplicated malaria who were both IgG and IgM positive at admission had significantly shorter fever clearance times and lower admission parasitemia levels compared with those who were negative (P = 0.01 and P = 0.02, respectively). The median (range) in vitro parasite multiplication rate was significantly lower in cultures containing positive anti-RESA antibody plasma compared with those containing normal plasma (0.7 [0.1-3.5] versus 2.6 [0.1-12.1]; P < 0.001). These results suggest that antimalarial antibodies may play an important supportive role in the therapeutic response to antimalarial drugs during acute falciparum malaria.

The resistance to physiological shear stresses of the erythrocytic rosettes formed by cells infected with Plasmodium falciparum.

Rosetting forces are believed to be an important contributor to the microcirculatory obstruction that occurs in malaria caused by Plasmodium falciparum. In this study, rosettes of erythrocytes from cultures of this parasite were suspended in different media and exposed to shear stresses corresponding to those encountered on the arterial and venous sides of the human circulation. The rosettes formed by infected erythrocytes in malaria culture medium containing 10% AB serum were disrupted easily (approximately 50% being broken) when exposed to very low shear stresses of < 0.5 Pa. However, use of higher concentrations of serum strengthened the rosetting binding forces considerably. Suspension of rosettes in a viscous colloid (e.g. dextran) increased the adherence forces between infected and uninfected red cells. The results indicate that rosettes do resist the physiological shear forces that are encountered in the venular side of the circulation and could thus contribute to microvascular obstruction in falciparum malaria.

The mechanisms of parasite clearance after antimalarial treatment of Plasmodium falciparum malaria.

Studies were conducted to determine how malaria parasites are cleared from the blood after antimalarial treatment. Neither artesunate nor quinine decreased parasitized red cell deformability or increased antibody binding. In acute falciparum malaria, ring-infected erythrocyte surface antigen (RESA) was observed in erythrocytes without malaria parasites (RESA-red blood cell [RBC]), indicating prior parasitization. In uncomplicated malaria, RESA-RBC numbers increased significantly (P=.002) within 24 h of starting artesunate but rose much more slowly (7 days) after quinine treatment. In severe malaria, RESA-RBC increased significantly (P=. 001) within hours of starting artesunate but not with quinine treatment (P=.43). RESA-RBCs were not produced after drug treatment of malaria parasite cultures in vitro. Rapid malaria parasite clearance after treatment with artemisinin derivatives results mainly from the extraction of drug-affected parasites from host erythrocytes-presumably by the spleen. This explains why the fall in hematocrit after treatment of hyperparasitemia is often less than that predicted from loss of parasitized cells.

Parasite multiplication potential and the severity of Falciparum malaria.

The multiplication rates and invasiveness of Plasmodium falciparum parasites isolated from adult Thai patients hospitalized with uncomplicated malaria (n=34) were compared with those from persons with severe malaria (n=42). To simulate severe malaria and control for host effects, the in vitro cultures were adjusted to 1% parasitemia and used the same red blood cell donor. P. falciparum isolates from persons with severe malaria had initial cycle multiplication rates in vitro that were 3-fold higher than those from uncomplicated malaria (median [95% confidence interval], 8.3 [7. 1-10.5] vs. 2.8 [1.7-3.9]; P=.001). Parasites causing severe malaria exhibited unrestricted red blood cell invasion, whereas those from uncomplicated malaria were restricted to a geometric mean of 40 (31%-53%) of red blood cells. P. falciparum parasites causing severe malaria were less selective and multiplied more at high parasitemias than those causing uncomplicated malaria.

Red cell selectivity in malaria: a study of multiple-infected erythrocytes.

To characterize red cell susceptibility to invasion in malaria, a selectivity index (SI) was calculated as the ratio of observed number of multiple-infected red cells to that expected from a random process (Poisson distribution). In patients with falciparum malaria (n = 100) SI decreased with increasing parasitaemia (P < 0.001), and correlated inversely with plasma lactate concentrations, chosen prospectively as a measure of disease severity (r = -0.36, P < 0.001). For parasitaemias < 5%, the SI was lower in patients with severe malaria (geometric mean 1.35; 95% confidence interval 1.01-1.80) than in uncomplicated malaria (2.31; 1.89-2.81; P = 0.003), despite similar parasite counts. The geometric mean (range) SI in vivax malaria (n = 20), 7.69 (1.67, 29.75), was significantly greater than that in falciparum malaria at comparable parasitaemias (< or = 2%), 2.44 (0.45, 14.05), P < 0.001, suggesting that about 13% of circulating erythrocytes were susceptible to invasion by Plasmodium vivax. This translates into susceptibility for about 2 weeks after emergence from the bone marrow, if age is the sole determinant of this process. In falciparum malaria selectivity was inversely proportional to severity; lack of selectivity could reflect either a 'favourable' host red cell phenotype, or an indiscriminate parasite population. Both are dangerous for the host.

Red cell deformability, splenic function and anaemia in thalassaemia.

Red cell deformability (RCD) was measured in 38 patients with alpha-thalassaemia and 48 patients with beta-thalassaemia, of whom 13 had undergone splenectomy. All splenectomized patients, but none of those with intact spleens, had very rigid erythrocytes with an elongation index <0.45 at a high shear stress of 30 Pa suggesting a splenic recognition threshold for removal of rigid red cells. At this shear stress RCD correlated strongly with the degree of anaemia in both the splenectomized (r = 0.81, P < 0.001) and non-splenectomized beta-thalassaemic patients (all patients r = 0.81, P < 0.001; homozygous beta-thalassaemic patients r = 0.51, P = 0. 01). These data suggest that reduced RCD is a major determinant of anaemia in thalassaemia.

Red blood cell deformability as a predictor of anemia in severe falciparum malaria.

Decreased erythropoiesis and increased clearance of both parasitized and noninfected erythrocytes both contribute to the pathogenesis of anemia in falciparum malaria. Erythrocytes with reduced deformability are more likely to be cleared from the circulation by the spleen, a process that is augmented in acute malaria. Using a laser diffraction technique, we measured red blood cell (RBC) deformability over a range of shear stresses and related this to the severity of anemia in 36 adults with severe falciparum malaria. The RBC deformability at a high shear stress of 30 Pa, similar to that encountered in the splenic sinusoids, showed a significant positive correlation with the nadir in hemoglobin concentration during hospitalization (r = 0.49, P < 0.002). Exclusion of five patients with microcytic anemia strengthened this relationship (r = 0.64, P < 0.001). Reduction in RBC deformability resulted mainly from changes in unparasitized erythrocytes. Reduced deformability of uninfected erythrocytes at high shear stresses and subsequent splenic removal of these cells may be an important contributor to the anemia of severe malaria.

The effects of quinine and artesunate treatment on plasma tumor necrosis factor levels in malaria-infected patients.

Tumor necrosis factor-alpha (TNF-alpha) is an endogenous mediator of shock and inflammation including malaria. Many lines of evidence suggest that cytoadherence, the life-threatening pathology associated with complicated and cerebral malaria, results from the overproduction of TNF in response to malarial parasite. Quinine has been shown to inhibit TNF synthesis and cytoadherence in vitro suggesting an additional beneficial effect of quinine on its anti-TNF action. On the other hand, artesunate inhibits cytoadherence better than quinine does not suppress TNF production in vitro. The present study compares the effect of artesunate and quinine on TNF levels of malaria-infected patients. Surprisingly, plasma TNF levels increased dramatically after quinine administration but did not increase after artesunate administration. This difference may be explained by previous observations showing that artesunate kills parasites in vitro and clears parasitemias in vivo for more rapidly than quinine. The rapid clearance of plasma TNF in quinine treated patients might be due to the drug's TNF-suppressive activity.