Deworming is recommended by the WHO in girls and pregnant and lactating women to reduce anaemia in areas where hookworm and anaemia are common. There is conflicting evidence on the harm and the benefits of intestinal geohelminth infections on the incidence and severity of malaria, and consequently on the risks and benefits of deworming in malaria affected populations. We examined the association between geohelminths and malaria in pregnancy on the Thai-Burmese border.Routine antenatal care (ANC) included active detection of malaria (weekly blood smear) and anaemia (second weekly haematocrit) and systematic reporting of birth outcomes. In 1996 stool samples were collected in cross sectional surveys from women attending the ANCs. This was repeated in 2007 when malaria incidence had reduced considerably. The relationship between geohelminth infection and the progress and outcome of pregnancy was assessed.A. lumbricoides (34.4% (285)) and T.trichuria (31.4% (250)) alone or in combination. A lower proportion of women (829) had mild (21.8% (181)) or severe (0.2% (2)) anaemia, or malaria 22.4% (186) (P.vivax monoinfection 53.3% (101/186)). A. lumbricoides infection was associated with a significantly decreased risk of malaria (any species)  and P.vivax malaria  whereas hookworm infection was associated with an increased risk of malaria (any species)  and anaemia . Hookworm was also associated with low birth weight .Stool sample examination  detected a high prevalence of geohelminths 70% (578/829), including hookworm (42.8% (355)), A. lumbricoides and hookworm appear to have contrary associations with malaria in pregnancy. Intestinal worms, particularly hookworm and whipworm, can cause anaemia, which is harmful for pregnant women. The WHO recommends deworming in pregnancy in areas where hookworm infections are frequent. Some studies indicate that coinfection with worms and malaria adversely affects pregnancy whereas other studies have shown that coinfection with worms might reduce the severity of malaria. On the Thai-Burmese border malaria in pregnancy has been an important cause of maternal death. We examined the relationship between intestinal helminth infections in pregnant women and their malaria risk in our antenatal care units. In total 70% of pregnant women had worm infections, mostly hookworm, but also roundworm and whipworm; hookworm was associated with mild anaemia although ova counts were not high. Women infected with hookworm had more malaria and their babies had a lower birth weight than women without hookworm. In contrast women with roundworm infections had the lowest rates of malaria in pregnancy. Deworming eliminates all worms. In this area it is unclear whether mass deworming would be beneficial. In 1994 and 2002 the World Health Organization (WHO) recommended anthelminthics be given to girls, pregnant and lactating women to reduce the burden of anaemia in areas where hookworm and anaemia are common P. falciparum malaria in presence of geohelminths Ascaris (A.) lumbricoides infections were linked to severe P. falciparum malaria in Senegal There are also conflicting and often confusing results regarding the impact of geohelminth infections on other infectious diseases, and in particular malaria P. falciparum malaria, was considered the main cause of anaemia in some P. falciparum and helminths resulted in a significantly lower mean birth weight than with P. falciparum infection alone in Nigeria and Ghana P.falciparum infection in women co-infected with A. lumbricoidesP.falciparum with any geohelminth and the risk remained significant for hookworm and A. lumbricoides alone Data from studies specific to pregnancy and helminths are also conflicting. Hookworm, not P. falciparum and P.vivax malaria species and where there has been no systematic deworming during pregnancy.Two cross-sectional helminth surveys (1996 and 2007) conducted among women attending antenatal clinics on the Thai-Burmese border were reviewed to determine whether there was any association between geohelminth infection and malaria in this area, endemic for both P. falciparumP. falciparum antimalarial drug that can be offered as intermittent presumptive therapy (IPT) or prophylaxis to pregnant women. Active weekly detection and early treatment of malaria has so far been the best method to prevent maternal death from malaria in this area The Shoklo Malaria Research Unit (SMRU) has five established clinics on the Thai-Burmese border. One is based in the largest of the refugee camps, Maela ; the others are stretched along 100 km of the border and serve a migrant population of circa 50,000 people. Antenatal clinics (ANC) have been operational since 1986 in the camp and 1998 in the migrant population. Malaria transmission is low and seasonal nd weekly haematocrit, provides routine iron and folate supplementation, and all necessary medical and obstetric care. A mother-to-child HIV transmission prevention program started in 2001 in the refugee camp and was introduced to the migrant population in 2008. HIV prevalence is low (<1.0%) and test uptake high (>90%) The ANC performs a weekly malaria smear for all women, 2P. falciparum in pregnant women was quinine or mefloquine mono-therapy in 1996, and quinine or artesunate with or without clindamycin in 2007. P.vivax episodes are treated with chloroquine alone. In 1996 women with non-severe (mild) anaemia  were treated with ferrous sulphate (200 mg three times daily) and folic acid (5 mg daily) until delivery. Women with severe symptomatic anaemia  were transfused. In 2007 all women received ferrous sulphate, 200 mg daily and folic acid, 5 mg weekly, from first consultation to delivery and treatment doses as stated previously if they became anaemic. Thailand has no deworming policy for pregnant women nor do the agencies working in the refugee camps.The antimalarial drug regimen for the treatment of Women were encouraged to deliver with trained midwives in the SMRU delivery rooms, those requiring Caesarean section were transferred to the nearest Thai Hospital. Gestational age was estimated by Dubowitz score Women participating in the surveys were of similarly deprived socioeconomic groups. Refugees in the camps receive food assistance and have access to medical care, but cannot work. Migrants work hard for low wages and lack access to medical care. Both groups are poor and economically weak Miscarriage was loss of products of conception or foetus before 28 completed weeks of gestation; stillbirth was delivery of a dead foetus aged 28 weeks or more; low birth weight (LBW) was a birth weight of <2500 grams measured in the first 5 days of life, and prematurity a delivery before 37.0 weeks of gestation; congenital abnormality was considered if a major defect was present at birth.The surveys in 1996 and 2007 were both conducted during the rainy season period (May–Oct) in order to allow comparison without having to take into account seasonality and because malaria peaks at that time of the year. Both surveys were exhaustive. The first survey, when SMRU only worked in the refugee camp, was to determine if worm infection was associated with anaemia. Every woman was asked to participate.The 2nd survey was done as a response to the preparation of a border wide medical guideline. The refugee camps on the border fall under the care of different NGOs (Non-Government Organisations) and there was debate on deworming in pregnancy. Since the last survey was old it was decided to resurvey pregnant women to determine if there was a need for deworming in pregnancy. At the time of this survey SMRU also provided antenatal care for migrants who have less access to health care than refugees. Hence refugee and migrant women were surveyed if they voluntarily gave a stool sample. Every 5th woman was asked to participate.Before each survey a general announcement was made to all pregnant women attending the ANC. Participation was voluntary. It was explained that if their stools were found positive for worms they would receive anthelminthic treatment. The importance of providing a fresh stool sample was explained.A. lumbricoides (roundworm) or Trichuris (T.) trichuria (whipworm) were treated with mebendazole 200 mg once daily for 3 days. In 1996 this treatment was given after delivery, in 2007 at the time of diagnosis or after the first trimester.Stool samples were examined on site. As Necator americanus and Ankylostoma duodenale ova cannot be differentiated by microscopy, the term hookworm was used. Women with a positive stool test result for hookworm, In this area the natural immunity to malaria is weak because the transmission is very low, so that most patients with malaria parasites become symptomatic. However because of the systematic weekly screening regardless of symptoms, many episodes are detected before symptoms arise. For this reason we cannot strictly speak of incidence or prevalence during the entire follow up period. Furthermore the number of infections of malaria relates not just to transmission but also to the poor response to antimalarial treatment. At the time of the 1996 survey quinine had an estimated failure rate of 23% and mefloquine 28% P. vivax group” or “ P. falciparum group” or “ mixed infection group”. Women in the P.vivax group only had one or more episodes of P.vivax, women in the P. falciparum group only had one or more episodes of P. falciparum and women in the mixed group may have had a single mixed infection of P.falciparum and P.vivax or on separate occasions a P.falciparum and a P.vivax. Results are given as proportions.In the analysis, women were categorized as “free of malaria” if all the malaria smears done at antenatal visits up to the day of the stool test were negative; women with a positive malaria smear up to the day of the stool test were categorized into one of 3 groups: “Following the ANC system as well as participation in the stool survey was voluntary. Providing a stool sample did not involve any risk for the pregnant woman. For these reasons no informed consent was obtained. Pregnancy records have been routinely entered to a data recording system since 1987. Ethical approval for analyzing these patient records was given by the Oxford Tropical Research Ethics Committee (reference: OXTREC 28–09).Stool samples were prepared using the formalin–ethyl acetate sedimentation technique Thick and thin malaria smears were stained with Giemsa and examined under oil immersion; the presence of any asexual blood stage parasite was declared as malaria positive. Smears were declared negative after reading 200 fields.Blood samples (finger prick) were centrifuged at 12000 rpm for 3 minutes and read using a standard haematocrit reader. The haematocrit value measured the day of the stool test was used to describe anaemia. If not available, the result the closest to the stool test day (but within 8 weeks prior to) was chosen.Data were entered using Microsoft Access, and analyzed using SPSS version 14 for Windows  and Epi Info (Centre for Disease Control and Prevention). Student's t-test and Mann-Whitney test were used for comparison of means and ranks respectively. Categorical data were compared using the chi-squared test or the Fisher's exact test, as appropriate. To assess independent predictors of malaria, anaemia and LBW, a multivariate unconditional logistic regression model was fitted using the variables that were significantly associated in univariate analysis.A total of 829 pregnant women provided a stool sample for examination; this represented 85% (339/401) of the ANC attendees in 1996 and 33%  in 2007. There was no significant difference in the baseline demographics between women who provided a stool sample and those who did not (data not shown).In 1996 all women were from Maela refugee camp; in 2007, 42% (244/490) were from the camp, while the others attended the migrant antenatal clinics. Participants in the 2007 survey were older (+1.6 years), had their stool test at a later gestational age (+4 wks) and were less anaemic than those in the 1996 survey .vs. 20% (96/490), (P = 0.02), P. falciparum malaria, 14% (48/339) vs. 3% (13/490), (P<0.001) and anaemia, 27% (91/339) vs. 18% (90/490), (P = 0.005), but an increase in the proportion of women infected with P.vivax   P.vivax .A. lumbricoides (34% (285)) or T. trichuria (31% (250)) alone or in combination (vs. 62% (95% CI: 58–66) (305/490), P<0.001. The intensity of worm infections was low, with high hookworm ova counts (≥1000 ova/mL) found in <10% of the positive results, and a maximum count of 2900 ova/mL. Hookworm  of the 829 women were infected with at least one geohelminth, including hookworm (43% (355)), bination . PrevaleHookworm  and T.trThirty three pregnant women had their first malaria infection after stool testing and were excluded from further analysis related to geohelminths and malaria and anaemia.For the purpose of this analysis geohelminth infections were assumed to be present until mebendazole treatment was administered, as there was no routine deworming policy.P. falciparum infections only and 54% (83) P. vivax only. P. falciparum and P. vivax simultaneously or on separate occasions occurred in the remaining 11% (17).Overall 153/796 women (19%) had malaria detected at least once prior to, or at the day of the stool test. Most of the women presented with single species infections; 35% (53) had vs. 20% (109/554), P = 0.77  co-infections and the lowest with A. lumbricoides (±T. trichuria) co-infections. The protective effect of A. lumbricoides (±T. trichuria) remained significant for P.vivax malaria when stratifying by malaria species . There were only 3 women with hyper-parasitaemic malaria , 2 of them without worm infection, and 1 woman with hookworm infection. The relationship between malaria and stool ova counts for the hookworm (±T. trichuria) group and for ova count in the A. lumbricoides (±T. richuria) group was explored (A. lumbricoides (P = 0.92) stool ova counts. There was not sufficient data to study interaction between geohelminth single infections and their association with malaria, as nearly half of all infections were combinations of worms  P = 0.77 . There wP = 0.77 . The hig species . The oveexplored . There wof worms .vs. 17% (104/595) in multigravida, P = 0.029. The proportion of hookworm infection was higher in teenage women, although this was not significant.The proportions, by age and gravid groups, of women with malaria and those with geohelminths are presented . Age wasA. lumbricoides co-infection remained significant . Year of survey and ova counts for hookworm and A. lumbricoides were non-significant.In a multiple regression model, primigravida  and hookworm co-infection  remained the two independent factors associated with an increased risk of malaria while the protective effect of vs. 21% (150/699), (P = 0.011); in multigravida compared with primigravida, 24% (134/547) vs. 16% (30/183), (P = 0.024); those who were older than 25 years, 25% (100/394) vs. 19% (64/337), (P = 0.041); and those who had malaria, 30% (45/152) vs. 20% (119/581), (P = 0.021). In a logistic regression model high hookworm load , P = 0.049), malaria , being multigravid  and participating in the 1996 survey  remained independently associated with anaemia.Sixty five women (8%) did not have a haematocrit measurement at the time of or before the stool test and were not included in this part of the analysis. Mean haematocrit was similar whether geohelminth infection was present or not in the remaining 733 pregnant women. The proportion of women with anaemia was higher in women with high intensity hookworm infection compared to those with lower counts, 41% (14/34) Pregnancy outcome data were available for 94% (783/829) of women. There were 14 abortions (2%) and 8 stillbirths (1%). Eleven infants were born with congenital abnormalities (8 live-births and 3 stillbirths). Neither stillbirth nor congenital abnormalities were significantly associated with geohelminth infection. The mean gestational age at delivery was 38.9±1.7 [28.4–42.5] weeks. Mean gestational age and the proportion of premature infants were not significantly different in the presence or absence of geohelminth infection.vs. 10% (46/483), P<0.001), in women aged <25 years vs. older (19% (57/301) vs. 9% (30/345), P<0.001), with hookworm infection vs. none (17% (46/278) vs. 11% (41/370), P = 0.048), and in premature vs. term infants (58% (34/59) vs. 9% (53/589), P<0.001). Anaemia and malaria were not significant risk factors for LBW. In a logistic regression model excluding prematurity (the strongest risk factor for LBW (n = 59)), the presence of hookworm infection was independently but weakly associated with LBW  as was (more significantly so) being primigravid .Birth weight data were available for 87% (648/748) of live-born, normal, singletons. Mean birth weight was 2900±447 [1100–4400] g. The proportion of LBW newborns was significantly higher among primigravida compared to multigravida (25% (41/162) The proportion of intestinal geohelminth infections in pregnant women on the Thai-Burmese border is high and comparable to reports from other parts of South East Asia, including Thailand, Burma and Vietnam P.falciparum. Women are encouraged to attend ANC on a weekly basis. Attendance is high and most women average more than 10 consultations per pregnancy in both the refugee and migrant settings. Women with detectable parasitaemia on screening are treated regardless of symptoms as they are unlikely to clear parasitaemia without becoming symptomatic This site has a unique system of antenatal care established in 1986 in response to a high malaria related-maternal mortality rateA. lumbricoides infection has been associated with a reduced risk of P. vivax. The novelty of this finding might be influenced by the fact that many studies on interactions between worms and malaria were done in Africa, where P. falciparum is the predominant species. Other investigators in African settings have described higher prevalence of falciparum malaria in presence of A. lumbricoides in pregnant women Strongyloides stercoralis). The density of infection of hookworm and A. lumbricoides in these surveys was low, as is usually reported from Asian settings, and no relationship was found between malaria and geohelminth density.This is the first time, to our knowledge, that A. lumbricoides prevalence increased with gravidity, but whereas they observed the same trend with maternal age this was not observed on the Thai-Burmese borderSimilar to a study in Kenyan pregnant women P. falciparum in children P. falciparum but not P.vivax malaria P.vivax densities P. falciparum and to a much greater extent P. vivax prefer to invade young red cells. This would not account for the negative association seen between malaria and A. lumbricoides. However, an alternative hypothesis proposed by some authors is that residential location and spatial aspects of exposure may explain some of the associations between worms and malaria Co-infection with hookworm was associated in our setting with a significantly higher risk of malaria, but this did not reach statistical significance for the individual plasmodial species. Similar associations have been previously reported for P. falciparum observed on the Thai-Burmese border A. lumbricoides coinfection does indeed attenuate malaria, then mass deworming may reduce a potential protective benefit. On the other hand hookworm was associated with a higher proportion of malaria, low birth weight and anaemia suggesting that hookworm should be treated in pregnancy. As there is no specie selective antihelminth at hand, deworming policies should be based on local prevalence and intensity of geohelminths, malaria, and anaemia severity.Active weekly screening as part of routine antenatal care has made severe malaria a rare event. No association between disease severity and the prevalence of geohelminths could be demonstrated. The decreased proportion of women with mild anaemia between the two surveys could be related to the decrease of geohelminth infection; it also could be due to the reduction in A. lumbricoides and both P. falciparum and P. vivax malaria, during pregnancy. Our findings potentially have considerable practical and evolutionary implications. Future trials to confirm or deny the associations observed here require well designed longitudinal studies to account for the observed complex and conflicting interactions.The present paper has limitations: the surveys were cross sectional, pooling data from different periods and the sample size may not have been sufficient to detect quantitative effects of the different worm species on different plasmodial species. No plausible explanation has been provided for the observed associations. No socioeconomic, behavioral or environmental factors were available for analysis, however these tend to be uniformly similar across the population of refugees and migrant workers: These people live in poor conditions and all are economically deprived so that it is unlikely to be a confounder in the analysis. The assumption that worms observed in the stool sample were present at the time of malaria is plausible at the population level given the lifespan of worms but it is not possible to ascertain that was always the case in each individual. This may have reduced the precision. Nevertheless, the present paper presents for the first time in the same data set a range of complex interactions between hookworm,


Acute renal failure, disseminated intravascular coagulation (DIC), acute respiratory distress syndrome (ARDS), hypoglycemia, coma, or epileptic seizures are manifestations of severe Plasmodium falciparum malaria. On the other hand, Plasmodium vivax malaria seldom results in pulmonary damage, and pulmonary complications are exceedingly rare. We report the case of a 42-year-old male living in a malaria-endemic area who presented with ARDS and was diagnosed as having Plasmodium vivax malaria. A diagnosis of Plasmodium vivax malaria was established by a positive Plasmodium LDH immunochromatographic assay while a negative PfHRP2 based assay ruled out P. falciparum malaria. After specific anti-plasmodial therapy and intensive supportive care, the patient recovered and was discharged from hospital. The use of NIPPV in vivax-malaria related ARDS was associated with a good outcome. Plasmodium vivax is the most common of the four human malaria species. Symptoms of Plasmodium vivax malaria are similar to those of other types of malaria and include cyclical fever with chills, headache, weakness, vomiting, and diarrhoea. Other more severe manifestations like acute renal failure, DIC, ARDS, hypoglycemia, coma, or epileptic seizures are seen with Plasmodium falciparum malaria and are seldom associated with Plasmodium vivax. Non cardiogenic pulmonary edema, manifested as ARDS, is observed in 4–18% of the cases of uncomplicated Plasmodium falciparum malaria with a high rate of mortality while it is uncommonly seen with Plasmodium vivax. We report a case of ARDS complicating infection with Plasmodium vivax malaria.Malaria is a devastating parasitic disease transmitted through the bite of an infected Anopheles mosquitoes. A 42-year-old male patient was admitted with fever, chills, dry cough, headache, and body pain for 1 week. He was a known hypertensive taking losartan 50 mg daily.Plasmodium vivax. A Plasmodium LDH immunochromatographic assay was positive for Plasmodium vivax. Specific anti-plasmodial therapy was started with an injection of Artesunate 120 mg IV loading dose followed by 60 mg IV daily along with supportive therapy.On admission, he was conscious, oriented, and a general examination revealed a fever (99.6°F), mild hypertension (140/90 mm Hg), a pulse rate of 88 beats/min, and RR of 22/min with oxygen saturation of 99% on room air. Systemic examination was normal. Laboratory investigations were normal except for mild thrombocytopenia (platelet count = 130 × 109/L). The peripheral blood smear showed trophozoites and schizonts of 2-64.7 mmHg, pCO2-33.2 mmHg, HCO3-21.8 mmol/L, and a PaO2/FiO2 ratio of 161.7. A 2D Echo revealed normal cardiac function. A negative PfHRP2 assay ruled out P. falciparum infection. The patient's respiration was supported by non invasive positive pressure ventilation (NIPPV) with an IPAP of 12 cm H2O, an EPAP of 8 cmH2 O, and oxygen flow of 6 L/min to reduce the work of breathing. In view of a low PaO2/FiO2 ratio (<200), an injection of quinine 600 mg IV every 8 hours was added to the anti-plasmodium therapy. Central venous pressure was normal (8 cm H2O) and the patient was hemodynamically stable. Blood and urine cultures were sterile. An ABG done after 2 hours of NIPPV showed pH-7.38, pO2-120 mmHg, pCO2-38.4 mmHg, and HCO3-22.2 mmols/L.PaO2/FiO2 ratio increased to 240.On the third day of admission, the patient developed dyspnea and tachypnea with a respiratory rate of 45 to 50 /min and pulse oximetry showed decreased oxygen saturation (86% breathing on room air). He was shifted to the Intensive Care Unit. The chest X-ray showed bilateral non homogenous opacities consistent with ARDS  and arteOn Day 5 of intensive care, a significant improvement was seen on the chest X-ray while assisted ventilation was removed on Day 7. The patient was afebrile. Oxygen saturation was maintained using a face mask with an oxygen flow of 5 lit/min. The patient was discharged from the hospital after 14 days and Tab. Primaquine was given for 14 days for prevention of a relapse. On the follow-up visit after 15 days, the patient was asymptomatic with minimal residual findings seen on a chest X-ray.Plasmodium vivax malaria. The presence of trophozoites and schizonts of Plasmodium vivax on the peripheral smear and negative PfHRP2 assay along with the absence of cardiac factors for pulmonary edema establishes the diagnosis of ARDS due to Plasmodium vivax malaria. A PfHRP2 immunochromatic assay is proven to be a reliable method for diagnosis of Plasmodium falciparum malaria with a sensitivity of approx 0.001 % parasitaemia.[This case represents one of the rare occasions where respiratory complication was observed with Plasmodium vivax infected cases. Tanios, et al. had seen a case of Plasmodium vivax complicated by ARDS in 2001. Kochar reported 11 cases of severe vivax malaria in 2005.[2There are only a few well-documented reports of ARDS in in 2005.2Plasmodium vivax do not cytoadhere to endothelial cells; thus, the occurrence of ARDS in benign malaria suggests that lung injury in malaria cases is also determined by causes other than microvascular sequestration of parasitized red blood cells. Anstey, et al. suggest that lung monocyte accumulation occurs in vivax and ovale malaria, as well as in falciparum malaria, with intravascular inflammatory changes contributing to impaired gas transfer and respiratory manifestations.[The mechanisms underlying lung damage caused by Plasmodia are not well understood. Red blood cells parasitized by stations. In fact,in vitro but not in vivo data suggest that Plasmodium vivax-infected red cells may cytoadhere to the endothelial cell ligand chondroitin sulfate A (CSA).[Recent  A (CSA).6 Apart fNIPPV has revolutionized the management of acute respiratory failure and it can decrease the endotracheal intubation rates and even mortality. NIPPV has been successfully used in ARDS, which are rapidly reversible with treatment. In this P. falciparum or bacterial) or treat them simultaneously. The use of NIPPV in Plasmodium vivax related ARDS is associated with a good outcome.To conclude, it may be suggested that vivax malaria can cause ARDS and this point should be kept in mind while treating such patients. However, it is imperative to exclude associated mixed infections (


Reported incidences of prostate cancer and masculinization of animals indicate a release of compounds with androgenic properties into the environment. Large numbers of environmental pollutants have been screened to identify such compounds; however, not until recently was 1,2-dibromo-4-cyclohexane (TBECH) identified as the first potent activator of the human androgen receptor (hAR). TBECH has been found in beluga whales and bird eggs and has also been found to be maternally transferred in zebrafish.in vitro.In the present study we investigated interaction energies between TBECH diastereomers  and the hAR, and their ability to activate the receptor and induce prostate-specific antigen (PSA) expression in vitro competitive binding assays for AR by polarization fluorometry analysis. We used enzyme-linked immunosorbent assays to determine PSA activity in LNCaP and HepG2 cells.We performed computational modeling to determine interaction energies between the ligand and the AR ligand-binding site, and measured We found the γ and δ diastereomers to be more potent activators of hAR than the α and β diastereomers, which was confirmed in receptor binding studies. All TBECH diastereomers induced PSA expression in LNCaP cells even though the AR present in these cells is mutated (T877A). Modeling studies of LNCaP AR revealed that TBECH diastereomers bound to the receptor with a closer distance to the key amino acids in the ligand-binding domain, indicating stronger binding to the mutated receptor.The present study demonstrates the ability of TBECH to activate the hAR, indicating that it is a potential endocrine disruptor. The α and β forms are found in the commercial flame retardant Saytex BCL 462 , whereas the γ and δ forms are converted from α and β at temperatures > 120°C . In 2002on assay . In 1995on assay , and moron assay . In a reon assay .in silico and determined their potential to bind and activate the receptor and its downstream target, prostate-specific antigen (PSA) in vitro.In the present study, we analyzed the interaction energies between the different TBECH diastereomers and the hAR We synthesized TBECH diastereomers as previously described . DHT andS-value . Before calculation of interaction energies, the docked structures were subjected to relaxation, molecular dynamics simulations, and additional relaxation as previously described (We determined the interaction energies between the ligand and the AR ligand-binding site using the Molecular Operating Environment (MOE) program . The crystal structure of the hAR obtained from the Protein Data Bank entry 1e3g  was subjescribed . The AR escribed . The modWe performed competitive binding assays for AR using the PolarScreen AR competition assay  according to the manufacturer’s instructions, using polarization fluorometry analysis, with excitation at 485 nm and emission at 535 nm, on a GeniosPro instrument . The AR-LBD used in the PolarScreen AR competition assay is derived from rat but shows 100% sequence homology to the hAR: The amino acid sequences of the LBDs of rat and human AR . This was followed by the addition of 200 μL blocking buffer  and incubation for 1 hr at room temperature. The plates were then washed 3 times with 300 μL wash buffer and incubated with 100 μL primary antibody  for 1 hr at room temperature. The plates were washed and incubated with 100 μL secondary antibody  for 30 min at room temperature. Detection was performed by adding 100 μL 1-Step Turbo TMB-ELISA  for 30 min. The reaction was stopped with 50 μL 1 M Ht-test.Statistical significant differences were determined using two-tailed Student’s We determined ligand docking of TBECH in hAR and LNCaP AR LBD using molecular modeling techniques. The BFR TBECH can exist as four diastereomers , which differ in the manner in which the bromine atoms are oriented in their molecular structures . These dDocking simulations established that all four TBECH diastereomers occupied the same hAR LBP as did DHT . In the The substantial differences in distances between the amino acids in the LBP and ligand observed for the four TBECH diastereomers manifested as significant differences in interaction energies. The natural ligands DHT and testosterone bound to the hAR with interaction energies of −53.8 and −48.9 kcal/mol, respectively . Among t50) of 21.5 nM with a goodness of fit (R2) of 0.91. The relative affinity of the TBECH diastereomers was 655 nM (R2 = 0.96) for TBECH-β, 191 nM (R2 = 0.97) for a 50:50 mixture of TBECH-α and -β (TBECH-αβ), 47.4 nM (R2 = 0.98) for a 50:50 mixture of TBECH-γ and -δ (50:50 TBECH-γδ), and 35.9 nM (R2 = 0.99) for a 25:75 mixture of TBECH-γδ. This indicates that TBECH-γδ binds AR with an affinity very similar to that of DHT, whereas TBECH-β has the lowest affinity of the TBECH diastereomers . We used the slp-ARE-Luc vector because it contains four copies of an ARE that is highly specific for AR interactions. The optimal time and concentration of exposure was determined for DHT, testosterone, and different combination of the TBECH diastereomers β, 50:50 αβ, 50:50 γδ, and 25:75 γδ.We determined the activation capacity of the different TBECH diastereomers using transactivation studies in HepG2 and LNCaP cells. We used HepG2 cells to allow comparison with an earlier study , and we Stimulation of HepG2 cells with testosterone and DHT resulted in maximal induction after 8–12 hr of exposure , whereasAR activity decreased after further stimulation at 24 hr and 48 hr, suggesting early activation and fast cellular metabolism of DHT. We also assessed the time-dependent AR activation in response to different combinations of TBECH-γδ (50:50 and 25:75) using a final concentration of 1 μM. The two TBECH-γδ combinations activated the AR equally. Activation of AR reached statistical significance after 2 hr and maximal induction after 24 hr .50) of 14.9 nM (R2 = 0.96), whereas exposure to 25:75 TBECH-γδ resulted in an EC50 of 22.7 nM  and indicate that these TBECH diastereomers are highly potent androgen agonists. Determination of AR activation by 50:50 TBECH-αβ demonstrated that these diastereomers are less potent, with an EC50 of 174 nM (R2 = 0.94), one order of magnitude higher than DHT or TBECH-γδ. The weakest inducer of AR was TBECH-β, with an EC50 of 294 nM (R2 = 0.84). We also observed that TBECH-γ and -δ are complete agonists to DHT, whereas TBECH-α and -β are partial agonists because they conferred only partial induction. Determination of relative induction by the different compounds showed a 23.7 ± 5.0-fold induction after exposure to DHT, a 24.4 ± 5.1-fold induction with 50:50 TBECH-γδ, a 25.5 ± 1.3-fold induction with 25:75 TBECH-γδ, an 8.4 ± 1.2-fold induction with TBECH-αβ, and a 2.4 ± 0.3-fold induction with TBECH-β.To determine the optimal concentrations of DHT and TBECH isoforms for AR activation, we stimulated HepG2 cells with DHT in a dose-dependent manner for 8 hr and with TBECH for 40 hr. Exposure to 50:50 TBECH-γδ resulted in a half-maximal effective concentration  by TCDD  are listed in 50 of 21.5 nM, which correlates well with IC50 values reported in other studies , it retains the androgen binding and ligand specificity in the LBP of the AR . This muin vitro studies, this demonstrates that modeling is a powerful tool when identifying potential endocrine disruptors. However, it remains unknown how TBECH interacts with AR in other species, such as three-spined stickleback and zebrafish, that both have 11-ketotestoterone as their most potent activator of the AR (The present study provides important data on the ability of TBECH to bind and activate the hAR with high affinity. Combining the results from the molecular modeling, the competition assay, and the activation assay, the TBECH-δ diastereomer appears to be the most potent, followed by TBECH-γ and TBECH-α, with TBECH-β being the least potent activator of AR. Furthermore, as the modeling experiments are in agreement with results obtained from the f the AR . Therefo


Applying the switch from presumptive treatment of malaria to new policies of anti-malarial prescriptions restricted to parasitologically-confirmed cases is a still unsolved challenge. Pragmatic studies can provide data on consequences of such a switch. In order to assess whether restricting anti-malarials to rapid diagnostic test (RDT)-confirmed cases in children of between five and 15 years of age is consistent with an adequate management of fevers, a school-based study was performed in Allada, Benin.Children in the index group (with fever and a negative RDT) and the matched control group (without fever and a negative RDT) were not prescribed anti-malarials and actively followed-up during 14 days. Blood smears were collected at each assessment. Self-medication with chloroquine and quinine was assessed with blood spots. Malaria attacks during the follow-up were defined by persistent or recurrent fever concomitant to a positive malaria test.484 children were followed-up (242 in each group). At day 3, fever had disappeared in 94% of children from the index group. The incidence of malaria was similar (five cases in the index group and seven cases in the control group) between groups. Self-medication with chloroquine and quinine in this cohort was uncommon.Applying a policy of restricting anti-malarials to RDT-confirmed cases is consistent with an adequate management of fevers in this population. Further studies on the management of fever in younger children are of upmost importance. Presumptive treatment of fevers with chloroquine has been formerly promoted in sub-Saharan malaria endemic areas. Treatment with anti-malarials restricted to parasitologically-confirmed cases is now promoted ,2. DiscrRestricting anti-malarials to parasitologically-confirmed cases has been recommended in many countries only for children of five years of age or above ; this discrepancy between age groups in terms of guidelines  on the mProviding that no diagnostic tool is perfect, collecting prospectively data in order to show that applying recent policies on the management of fever is safe and feasible  is now very important.The main objective of this school-based, prospective cohort study was to collect clinical, parasitological and pharmacological (self-medication) data in a pragmatic approach in order to see whether, regardless of cost-effectiveness issues, applying the algorithm on the management of fevers is consistent with adequate clinical care.Ethical clearance was obtained from the Faculty of Medicine ethics committee, Benin National University, Cotonou, and from the Deontology and Ethics Committee from the Institute of Research for Development. Written informed consent was obtained from all children or their parents/guardians before enrolment in the study.This study was designed to measure the incidence of malaria attacks during a two-week follow-up period in two populations of patients. First, an index group (with fever) constituted by schoolchildren attending the school nursery for fever and not diagnosed malaria, based on a negative HRP2-based RDT. Second, a control group (without fever) constituted of apparently healthy schoolchildren paired with index cases on gender, age, week of inclusion and malaria status (i.e. negative HRP2-based RDT).Hence, the objective of the study was to assess whether applying the algorithm of management of fevers in the school setting is consistent with adequate management of fevers, in the way it does not lead to a high number of undiagnosed (and thus untreated) malaria attacks, and by comparing the incidence of malaria attacks between the two groups (with and without fever).This study was performed from February through June 2008 (rains started in April) in four schools, located at Allada, southern Benin, where malaria transmission is intense and perennial . AltogetChildren attended school nurseries on their own, or led by their parents/guardians. Care was provided for any child from a school participating to the study. Children from the index group were included and actively followed only if individual informed consent was obtained  from a parent or guardian. Asymptomatic children were screened for the control group on gender, age, week of inclusion. The first child with a negative RDT and for whom an informed consent was obtained was included in the control group. Asymptomatic children (screened for inclusion into the control group) and with a positive RDT were advised to quickly attend at the school nursery as soon as symptoms of malaria would appear. Children treated for malaria within the prior month were not included in either group.®, Orchid Diagnostics) [All children were examined by a study nurse. Children with danger signs were referred to a health centre. Children attending the school nurseries were managed for fever only if fever was stated (tympanic temperature at 37.8°C or above ) or if anostics)  was perfFollow-up visits were on day 3, 7 and 14. A blood smear was collected at enrolment and at each scheduled visit. An additional blood spot for pharmacological data was collected for 100 children in each group on day 0 and day 14. When fever was reported and/or stated at a follow-up visit, a RDT was performed.RDTs were read by the study nurse, as recommended by manufacturer 15 minutes after it was performed. A Giemsa-stained thick blood film was prepared at inclusion and at each follow-up visit. Microscopy examination was done retrospectively  and blinded to the patient's identity. Parasite density was determined according to the number of parasites per 200 white blood cells (WBC), and assuming a total WBC count of 8,000/μL. Slide quality control was done by masked re-reading of 10% of slides, selected randomly.On day 0 and day 14 in the 100 first children from each group, whole blood was sampled on filter paper spots and dried at room temperature. Using these dried blood spot (DBS), chloroquine (and monodesethylchloroquine) and quinine were detected by high pressure liquid chromatography with UV detection to 254 nm using ammonium acetate /acetonitril (85/15% v/v) .pfhrp2 gene was amplified from genomic DNA in corresponding isolates, by PCR using the specific primer pairs Pfhrp2-F1 and Pfhrp2-R1 [In order to find an explanation to false negative RDT results, the exon 2 of fhrp2-R1 .Parametric tests  were positive. Among the 281 children who had a negative RDT, consent was obtained in 242 for their participation into the study. Most of them (62%) were prescribed an antibiotic and paracetamol was widely used.P. malariae parasitaemia). The exon 2 of pfhrp2 gene was amplified with success ruling out the hypothesis of pfhrp2 gene deletion to explain false negative results.The median delay between inclusions in the index group and in the control group was 0 day. In the index group, blood smears were positive in 12 children  in five index group children. No malaria severity or danger sign was observed. At enrolment, two  had a positive blood smear .Among the 12 children who had a positive blood smear at baseline, three had malaria attacks (two when taking into account solely cases of fever with a positive RDT) during the follow-up. The nine other children never presented with fever during the follow-up; parasitaemia spontaneously cleared in eight of these and remained persistently low (<100/μL) in one child.Children of the control group have been followed-up a total of 477 person-weeks Table . Three cWhen taking into account solely cases of fever with a positive RDT, 7 malaria attacks  were recorded in 7 children. At enrolment of these seven children, none had a positive blood smear. Overall, one malaria attack was not confirmed by microscopic examination. The clinical picture in this case was stomatitis. Rates of self-medication did not differ between groups  and no attack of severe malaria was observed during follow-up, as this was expected in this population of children. Since fever had disappeared in 94% of children from the index group, at day 3, educating mothers to bring back their child after two or three days in case of persistence of fever is, therefore, feasible: only a small minority of mothers would have to come back to the health care facility. Malaria attacks were not rare among children with persistent or recurrent fever (five out of 30) in the index group and in this case testing again with a RDT when fever persists, rather than referring patients directly, should be discussed.The baseline status of the two groups was the negativity of RDT. Interestingly, microscopically detectable parasitaemia at baseline, although rare in both groups, was more frequent in the index group. Differences in terms of self-medication can hardly explain these parasitological differences between groups. Because chloroquine and quinine were the most commonly drugs used for self-medication in the study area, they were chosen as markers of self-medication, though other anti-malarials like SP, were widely available at the time of the study.As in previous studies ,20, undiSelf-medication with quinine and chloroquine was uncommon in this cohort of children who had access to care, and it did not differ significantly between groups, though there was a trend to more self-medication with chloroquine at baseline in the index group.Applying fully a policy of treatment of malaria restricted to RDT-confirmed malaria cases appears to be consistent with adequate management of fevers in this population. Giving an anti-malarial treatment to a child with fever and a negative RDT or to an apparently healthy child with a negative RDT is equally useful. Further studies on the management of fever in younger children are of upmost importance.The authors declare that they have no competing interests.JFF designed the study, led field work interpreted the results and drafted the manuscript. PM, GA and TB participated in data collection. GA contributed with data collection in the field. SH performed molecular assays. EO designed the study. PH performed pharmacology assays. PD designed the study and critically read the manuscript, MC designed the study, performed the statistical analyses and critically read the manuscript. All authors read and approved the manuscript.


Malaria epidemics in highland areas of Kenya cause significant morbidity and mortality.To assess treatment-seeking behaviour for malaria in these areas, a questionnaire was administered to 117 randomly selected households in the highland area of Kipsamoite, Kenya. Self-reported episodes of malaria occurred in 100 adults and 66 children.P < 0.0001, and children, 79.6% v. 40.0%, P = 0.002, respectively). Individuals who attended medical facilities recalled receiving anti-malarial medication significantly more frequently than those who visited shops .The most frequent initial sources of treatment for malaria in adults and children were medical facilities (66.0% and 66.7%) and local shops (19.0% and 30.3%). Adults and children who initially visited a medical facility for treatment were significantly more likely to recover and require no further treatment than those who initially went to a local shop (adults, 84.9% v. 36.8%, A significant proportion of this highland population chooses local shops for initial malaria treatment and receives inappropriate medication at these localshops, reslting in delay of effective treatment. Shopkeeper education has the potential to be a component of prevention or containment strategies for malaria epidemics in highland areas. Malaria is a leading cause of death in children under the age of five years in sub-Saharan Africa . The RolA rapid treatment response is essential for effective home management. To this end, an understanding of treatment-seeking behaviour enables communities and the formal health care system to design interventions that cater to a specific population . Unlike One of the key strategic approaches outlined by the Kenyan National Malaria Strategy (KNMS) is the improvement of malaria epidemic preparedness and response . With meKipsamoite, Nandi District, is an epidemic-prone rural highland area with unstable malaria transmission. Altitude ranges from 1,910 m–2,154 m. The total population of the area is ~3,200 individuals. Study participants were recruited from all seven villages in the area. Subsistence farming is the principal occupation. More than 95% of the Kipsamoite population is of the Kalenjin Nandi tribe.dukas), offering over-the-counter medication, are frequently sought after alternatives to local health centres.There are several small hospitals within 10 kilometers of the study site, though most people tend to go to the local medical facilities for health care. Kipsamoite health centre is centrally located and offers free malaria microscopy testing and medication. However, traditional herbalists and shops  key informant interviews. The initial questionnaire used was adapted from a Knowledge, Attitudes and Practices questionnaire used by the Centers for Disease Control in an area of urban malaria transmission . It was The number of cows and the number of acres of land owned were weighted more heavily than other forms of property because cattle and land are the primary indicators of wealth in this community. Individuals were then ranked according to quartiles of wealth index. A number of individuals had the same wealth index number, resulting in quartiles with slightly greater or less than one quarter of the individuals.Study participants were recruited from a continually maintained census database of Kipsamoite. The study was conducted in August-September, 2002, just after a malaria epidemic had occurred in the area . A compuThis project was reviewed and approved by Case Western Reserve University/University Hospitals of Cleveland Institutional Review Board and the Ethics Committee of the Kenya Medical Research Institute, Nairobi, Kenya. The work was performed while the senior author (CCJ) was a faculty member at Case Western Reserve University. Informed consent was obtained from all participants.2) tests of association to test for significant differences. Adjusted odd ratios, by multivariate logistic regression, were used to compare the likelihood of seeking malaria treatment at a medical facility and within 1 day of symptom onset according to symptom severity, distance from medical facility, and socio-economic factors. Interaction terms were included in logistic regression models. No significant interaction was seen in any regression model.Frequencies and proportions were used for descriptive analysis. Since adults may behave differently when seeking treatment for themselves rather than their children, separate analysis was planned for adults vs. children as part of initial study design. Proportions were compared using Chi-square (χcheptigonit"). No household had more than two children with malaria reported, and only two households had two children with malaria – all other children with the only child in the household with reported malaria. Recent malaria episodes had occurred within six months of the interview more frequently in children than adults . Among symptomatic individuals, adults sought treatment within 1 day of symptom onset more often for themselves than for their children . Medical facilities were the most common initial treatment source for both adults (66.0%) and children (66.7%). Local shops were the most popular alternative, providing initial treatment for 19.0% of adults and 30.3% of children. Frequencies of treatment seeking behaviour were similar regardless of the time between questionnaire administration and the malaria episode. For example, among adults who had malaria episodes, health care was initially sought care at a medical facility 50%, 64% and 67.9% of the time for one, two and six month periods from the malaria episode to questionnaire administration, while in children it was sought at a medical facility 66.7%, 60% and 67.2% for the same time periods. A small number of symptomatic adults and parents of symptomatic children used traditional healers or herbalists as initial sources of treatment. The majority of adults and children who did not recover after initial treatment went to a medical facility as their second line of treatment . Thus, in the course of their illness, 88 of 100 adults (88.0%) and 65/66 children (98.5%) with self-diagnosed malaria went to a medical facility to seek treatment.Of the 120 households asked to participate, 117 agreed to administration of the questionnaire. Table P < 0.0001 for comparison of proportions for medical facilities vs. local shops, for both adults and children). Among the study participants who recalled receiving anti-malarial medications at the medical facility, chloroquine alone was received by only 4/51 adults (7.8%) and 3/32 children (9.4%); all others received medications recommended for malaria at the time of the study . Adults and children visiting local shops often received medicines without anti-malarial properties, such as acetaminophen, aspirin, anti-histamines, or antibiotics.Table P < 0.0001; children 79.6% v. 40.0%, P = 0.002, respectively, Table P > 0.99) to the adults and children who recalled receiving anti-malarial medication at a medical facility , suggesting that these individuals also received anti-malarial medication.Recovery rates of adults and children who attended a medical facility but could not recall what medication they received were essentially identical to those of the individuals who recalled receiving anti-malarial medication  might well have been due to other diseases in some cases. However, the high frequency of recalled response to medications with efficacy against malaria and the low frequency of recalled response to medications with no anti-malarial efficacy suggest that malaria was an accurate diagnosis in a majority of the cases. In this study, questions were asked of the household head or, for children, to the caretaker of the child. Since household heads may have greater access to care than other household members, this could have created a bias in the data obtained. Ideally, all household members should have been asked about malaria symptoms, but resource and time limitations precluded this preferred approach. Nonetheless, even if the data are not completely representative, the pattern of failure with treatment provided by shops is a significant and important finding, and likely reflects the experience of most in the community. Finally, malaria is infrequent in these areas in non-outbreak periods, so recall of exact malaria treatment could be difficult, particularly for individuals whose episode was >6 months prior to the questionnaire. Questions are ideally asked within weeks of the occurrence of the disease, but in this area this was not possible, as it would be very difficult to get adequate numbers of individuals with disease within 1 month of the questionnaire. This is demonstrated by the finding that although the questionnaire was administered shortly after an epidemic in the area, the majority of adults had still not had an episode of malaria during the prior 2-month epidemic period. Ideally, the survey should have been done in a much larger population to allow for a sufficient sample size to evaluate treatment seeking-behaviour within the prior 14 days. The limited resources available for conducting the present study precluded this more optimal testing. It seems likely that the data was accurate despite the variability in time from malaria episode to questionnaire administration for two reasons. First, the treatment options for malaria are limited, both in terms of places to seek care and types of treatment, so recalling what was done from this limited number of choices would not be particularly difficult. Second, the data on treatment-seeking in both children and adults was highly consistent over all time periods , supporting the idea that recall was accurate even over longer time periods.The present study in an epidemic-prone area of highland Kenya indicates that individuals in this area frequently sought treatment for malaria from local shopkeepers, and that treatment provided by these shopkeepers rarely had efficacy against malaria, leading to lack of recovery and delayed effective treatment for malaria. There may be an opportunity to improve the management and response to malaria epidemics by educating shopkeepers on the importance of prompt treatment of individuals with malaria symptoms with appropriate anti-malarial medication.The authors declare that they have no competing interests.POS was responsible for data analysis and interpretation and for the initial manuscript draft. LW and HK were involved in study design, data collection and revision of the manuscript. KJ was involved in data analysis, interpretation and critical revision of the manuscript. CCJ was responsible for study design and conception and involved in data analysis, interpretation and critical revision of the paper for intellectual content.


Accurate malaria diagnosis is mandatory for the treatment and management of severe cases. Moreover, individuals with asymptomatic malaria are not usually screened by health care facilities, which further complicates disease control efforts. The present study compared the performances of a malaria rapid diagnosis test (RDT), the thick blood smear method and nested PCR for the diagnosis of symptomatic malaria in the Brazilian Amazon. In addition, an innovative computational approach was tested for the diagnosis of asymptomatic malaria.The study was divided in two parts. For the first part, passive case detection was performed in 311 individuals with malaria-related symptoms from a recently urbanized community in the Brazilian Amazon. A cross-sectional investigation compared the diagnostic performance of the RDT Optimal-IT, nested PCR and light microscopy. The second part of the study involved active case detection of asymptomatic malaria in 380 individuals from riverine communities in Rondônia, Brazil. The performances of microscopy, nested PCR and an expert computational system based on artificial neural networks  using epidemiological data were compared.Plasmodium species in 12 cases of mixed infections . Moreover, the microscopy presented low performance in the detection of asymptomatic cases (61.25% of correct diagnoses). The MalDANN system using epidemiological data was worse that the light microscopy (56% of correct diagnoses). However, when information regarding plasma levels of interleukin-10 and interferon-gamma were inputted, the MalDANN performance sensibly increased (80% correct diagnoses).Nested PCR was shown to be the gold standard for diagnosis of both symptomatic and asymptomatic malaria because it detected the major number of cases and presented the maximum specificity. Surprisingly, the RDT was superior to microscopy in the diagnosis of cases with low parasitaemia. Nevertheless, RDT could not discriminate the An RDT for malaria diagnosis may find a promising use in the Brazilian Amazon integrating a rational diagnostic approach. Despite the low performance of the MalDANN test using solely epidemiological data, an approach based on neural networks may be feasible in cases where simpler methods for discriminating individuals below and above threshold cytokine levels are available. Plasmodium spp. in Giemsa-stained blood smears from individuals presenting with malaria-like symptoms persists as the gold standard for the diagnosis of malaria and is mandatory to obtain access to the anti-parasitic treatment. Microscopic parasitological diagnosis requires continued personnel training and supervision of users in addition to a minimum laboratory structure, which is difficult to maintain in remote areas of the rainforest. Additionally, such a test is prone to large observer-related variation [Despite global efforts, the malaria burden is increasing worldwide, with almost two million estimated deaths annually . The lacariation ,3 and laariation .Other diagnostic methodologies have arisen to overcome the inefficient malaria diagnosis, such as PCR-based genetic tests. Nested PCR and real time PCR present higher sensitivity and specificity to malaria diagnosis compared to light microscopy ,6. NeverPlasmodium infection is very high in the Brazilian Amazon [Plasmodium to uninfected Anopheles mosquitoes [Within the Rondônia State in the Brazilian Amazon, the incidence of malaria and the occurrence of drug resistant cases are increasing . In Burin Amazon , furthersquitoes  and may Plasmodium infection, and it was compared to field microscopy and nested PCR.Herein, the diagnostic effectiveness between an RDT , field microscopy and nested PCR was compared in individuals with malaria-related symptoms from an Amazonian region, which presents an increasing incidence of malaria . FurtherThis study was approved by the Ethical Committee of the São Lucas University, Rondônia, Brazil, for the human subject protocol. The clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. All participants or legal guardians gave written informed consent before patients entered the study.P. vivax. The prevalence of P. falciparum infection in the Brazilian Amazon is 23.7% [Plasmodium malariae is about 10% in Rondônia [This study was performed in Rondônia State in the southwestern Brazilian Amazon. Within this region, the malaria transmission is unstable, with an increasing number of cases being detected annually from April to September . Most mais 23.7% , and theRondônia .For the first part of the study, a cross-sectional investigation was performed between May 2006 and September 2007 in Buritis, Rondônia, Brazil , a recently urbanized municipality with high prevalence of symptomatic malaria [Plasmodium infection, riverine communities close to Demarcação, Rondônia, Brazil , in which a high prevalence of asymptomatic Plasmodium infection has been reported [To test the efficacy regarding the diagnosis of asymptomatic reported , were stThe individuals were examined and interviewed by a trained physician. In the first part of the study, the thick blood smear and the Optimal-IT RDT  were run at the same time. The Optimal RDT was performed according to the manufacturer's instructions. For estimation of parasitaemia, experienced malaria field microscopists from the FUNASA malaria diagnostic center counted parasitaemia on slides using the thick film method. All the slides were stained using Giemsa pH 7.2. The results were reported as parasites/μL. In addition, 300 μL of total blood were collected in EDTA-treated tubes and stored for the nested PCR.Plasmodium infections, they were followed for 30 days. Only the individuals who remained without malaria-related symptoms and positive nested PCR test after this period were classified as asymptomatic malaria cases.The molecular diagnosis of malaria infection was performed in all subjects using the nested PCR technique described previously . To contPlasmodium infection, an expert system based on Artificial Neural Networks (ANN) [To identify asymptomatic ks (ANN)  was deveP. vivax and/or P. falciparum) according to the nested PCR and clinical evaluation described above. The objective was to develop a helpful method for discriminating asymptomatic plasmodial infections from uninfected cases.The database provided by the survey contained 380 records with information from non-infected individuals (n = 178) and those with asymptomatic malaria (n = 202)  and interferon-gamma (IFN-gamma) were measured using the Cytometric Bead Array - CBA®  according to the manufacturer's protocol, with all samples run in a single assay in the main laboratory at the Centro de Pesquisas Gonçalo Moniz, Bahia, Brazil. The flow cytometric assay was performed and analyzed by a single operator, and standard curves were derived from cytokine standards. The cytokine levels were used for other studies addressing pathogenic aspects of malaria in this region [Plasmodium infections. The MalDANN software used this additional information together with clinical and epidemiological data to enhance the power of prediction of asymptomatic malaria cases.During the active search for subjects with asymptomatic s region , and thePlasmodium species were compared between the diagnostic methods using Fisher's exact test. Within all comparisons, differences in which p < 0.05 were considered statistically significant. The statistical analyses were made using the Graphpad Prism 5.0b .The overall performances of diagnostic methods were compared using Fisher's exact test (when two methods were compared) or a chi-square test (when three methods were compared). The results for the sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) obtained for each Plasmodium infection . Concerning the overall performance for the malaria diagnosis independently of the parasite species, the nested PCR resulted in the highest number of positive exams , significantly superior to the RDT  and the field microscopy . As expected, the general positivity of the RDT was equivalent to the microscopy . Given that the nested PCR test presented higher positivity, it was considered as the gold standard to calculate the power of the two other tests. Therefore, for the diagnosis of symptomatic Plasmodium sp. infection, the RDT presented a sensitivity of 89.02% (95% CI: 83.38%-93.26%), a specificity of 100% (95% CI: 97.36%-100%), a positive predictive value (PPV) of 100% (95% CI: 97.36%-100%) and a negative predictive value (NPV) of 87.90% (95% CI: 81.75%-92.55%). Surprisingly, the light microscopy presented a lower sensitivity , an equivalent specificity  and PPV , and a lower NPV  than the RDT. Within the subjects evaluated in this study, no P. malariae case was detected. Under this circumstance, we decided to consider P. non-falciparum infection as P. vivax malaria cases for the RDT results.Of the 311 individuals presenting with malaria-related symptoms screened by FUNASA diagnostic centers, 188 (60.45%) were male, and the median age was 33.5 (Table Plasmodium species was assessed (Table P. vivax (61.84% of the positive cases), 53 individuals infected solely with P. falciparum (30.63% of the positive cases) and 13 cases of mixed infection . The light microscopy detected 84 cases of vivax malaria (23 cases fewer than nested PCR), 45 cases of falciparum malaria (eight cases fewer than nested PCR), and 12 mixed malaria cases (one fewer than nested PCR). The RDT detected 56 falciparum malaria cases and 98 vivax malaria cases. Importantly, the RDT used had limitations in the species identification because it intrinsically cannot discriminate mixed infections [P. falciparum infection and four cases of vivax malaria. In a larger sample, this important issue can bring problems concerning the adequate management of the patients with mixed infections. Concerning the discrimination of Plasmodium species, both RDT and light microscopy presented similar performances , while the RDT detected 13 cases . It was then possible that these patients had a parasitaemia below 100 parasites/μL because they were positive in two qualitative tests and negative by the parasite quantification using microscopy. On the other hand, the concordance of the results among the tests was similar when the patients presented with higher parasitaemia, except for the known absence of mixed infections detected by the RDT , P. falciparum  and P. vivax  infections, with similar specificities and PPV . These findings indicate that for this endemic area, the RDT is superior to field light microscopy to identify individuals with low parasitaemia, albeit not detecting a few cases of mixed infections.The Table DT Table . FurtherPlasmodium-infected individuals who are common in the Amazonian riverine communities [The next step was to assess the diagnosis of symptomless munities  and may munities . Previoumunities . In the et al. unpublished data). Initially, the information provided by the data bank included test results of several immunological parameters, socioeconomic, environmental, clinical and epidemiological data . The ANN pre-processing of the same data displayed better prediction results when all of the cited variables were used, including gender and the use of a bed net.One artificial neural networks-based test  was developed, and fed with a data bank built during another study . It was noticed that among the training data were data from patients with very similar values but with different diagnoses. Hence, there was a need for greater number of neurons to process and better differentiate the pattern of input data. A new network was created using ten neurons in the hidden layers, keeping the other settings Figure . Thus thFurther, the diagnostic performances of the MalDANN and light microscopy were compared, considering nested PCR as the gold standard. Using exclusively the epidemiological data, the software diagnosed correctly only 56% of the samples , exhibiting lower performance than microscopic diagnosis due to increased number of false positive exams  were negative by light field microscopy. Although these individuals probably had low parasitaemia, they were symptomatic and did not receive anti-malarial treatment because of the negative microscopy exam. This finding reinforces that the field microscopists from this area need continued refinement, and investments are necessary to improve the quality of the malaria screening. It also indicates that the large demand for microscopic tests in this area, including those from patients with other infections, could contribute to the reduced quality of the tests. Recent evidence indicates that there is a large inter-rater reliability of the parasite counts for the malaria diagnosis [The use of nested PCR as the gold standard was done when it was noted that it presented the highest sensitivity. Considering the principle of this molecular assay, in which small fragments of iagnosis .In addition, the RDT presented higher effectiveness in the identification of malaria cases with low parasitaemia than the light microscopic test. Many other studies worldwide have indicated diverse findings ,21,22. TP. vivax and P. falciparum infections is critical because the drug therapies and the treatment durations are different. Actually, there are other RDT that can discriminate mixed infections [Optimal-IT, the RDT used in the present study, cannot discriminate mixed infections. The discrimination between fections , and thePlasmodium infection is a major problem in many regions worldwide [Plasmodium parasites after repeated infections [Plasmodium-infected individuals.The asymptomatic orldwide ,26, inclorldwide . Symptomfections , which lfections . While ufections , which iPlasmodium infection [Plasmodium infection. This idea was confirmed when the software performance significantly improved after the addition of information regarding IL10 - and IFN-gamma plasma measurements. In addition, other routine biochemical laboratory exams did not improved the MalDANN performance. These cytokines were chosen in the light of evidences that individually or as ratios they are associated with the malaria severity [Asymptomatic individuals frequently refuse to give blood for tests, which hampers the detection of symptomless plasmodial infections. Additionally, the routinely used thick blood smear exam displays a low performance in individuals with low parasite burdens, as is the case in asymptomatic nfection . Herein,nfection . Thus, tnfection . The disseverity ,30. BesiThis study shows that the chosen RDT  performed superiorly in discriminating symptomatic malaria cases with low parasitaemia than field microscopy, although it did not discriminate mixed infections. An RDT for malaria diagnosis may find a promising use in the Brazilian Amazon integrating a rational diagnostic approach. Despite the low performance of the MalDANN test using solely epidemiological data, an approach basedon neural networks may be feasible in cases where simpler methods for discriminating individuals below and above threshold cytokine levels are available.The authors declare that they have no competing interests.Wrote the paper: BBA; Performed data analysis: BBA and ARF; Performed the field study and clinical examinations: BBA, SMSN and LMAC; Performed molecular experiments: LLN, KFF and BBA; Designed and validated the Expert Based System Networks for malaria diagnosis: AMB and AD; Participated in the design of the study and helped with the manuscript: LMAC, EC and AB; Coordinated the study and helped to draft the manuscript: MBN. All authors have read and approved the final version of the manuscript.BBA, ARF, SMSN and LLN received fellowships from the Brazilian National Research Council (CNPq). EC, AB and MBN are senior investigators from CNPq. This work was supported by FINEP (010409605)/FNDCT-CT Amazônia. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.


Cerebral malaria is the most severe neurological complication of falciparum malaria and a leading cause of death and neuro-disability in sub-Saharan Africa. This study aimed to describe functional deficits and behaviour problems in children who survived cerebral malaria with severe neurological sequelae and identify patterns of brain injury.Records of children attending a specialist child neurology clinic in Uganda with severe neurological sequelae following cerebral malaria between January 2007 and December 2008 were examined to describe deficits in gross motor function, speech, vision and hearing, behaviour problems or epilepsy. Deficits were classified according to the time of development and whether their distribution suggested a focal or generalized injury. Any resolution during the observation period was also documented.Thirty children with probable exposure to cerebral malaria attended the clinic. Referral information was inadequate to exclude other diagnoses in 7 children and these were excluded. In the remaining 23 patients, the commonest severe deficits were spastic motor weakness (14), loss of speech (14), hearing deficit (9), behaviour problems (11), epilepsy (12), blindness (12) and severe cognitive impairment (9). Behaviour problems included hyperactivity, impulsiveness and inattentiveness as in attention deficit hyperactivity disorder (ADHD) and conduct disorders with aggressive, self injurious or destructive behaviour. Two patterns were observed; a) immediate onset deficits present on discharge and b) late onset deficits. Some deficits e.g. blindness, resolved within 6 months while others e.g. speech, showed little improvement over the 6-months follow-up.In addition to previously described neurological and cognitive sequelae, severe behaviour problems may follow cerebral malaria in children. The observed differences in patterns of sequelae may be due to different pathogenic mechanisms, brain regions affected or extent of injury. Cerebral malaria may be used as a new model to study the pathogenesis of ADHD. Cerebral malaria is the most severe neurological complication of falciparum malaria. Children with cerebral malaria are admitted with fever, seizures, coma and brainstem signs and despite adequate treatment using current guidelines, about 20% die. EarlierA wide range of functional deficits are described -7, the iThis was a case series of children exposed to cerebral malaria that developed severe neurological deficits and attended specialist neurology care.The weekly Child Neurology Clinic in Mulago  in Kampala, Uganda, is the only specialist care centre for children with neurological disorders in the country. This ouStudy subjects were children ages 12-79 months, who attended the clinic from January 2007 to December 2008;Plasmodium falciparum parasites on a blood smear, a normal cerebrospinal fluid and no other cause to explain the coma)[a) following hospitalization for World Health Organization defined cerebral malaria (unarousable coma with the coma),b) were discharged with or had severe neurological sequelae developing after discharge andc) had detailed referral information, a discharge summary or both.All had been exposed to cerebral malaria between the ages of 5-46 months and first attended the clinic 1-48 months later. Subsequent re-attendances were at intervals determined by the attending clinician but mostly at 1-3 month intervals and each subject was followed up for a minimum of six months. Children with incomplete records or for whom the reported diagnosis of cerebral malaria could not be ascertained were excluded.Records of incident patients who attended the clinic during the study period and fulfilled the inclusion criteria were examined. Parental reports of acquired deficits in vision, hearing or speech, regression in milestones and severe cognitive impairment, gross motor deficits and severe behavioural problems were documented. Motor assessment was based on a system for classifying function in children with cerebral palsy, the gross motor function classification system (GMCFS). This isOf the 1,136 incident patients registered in the clinic over the two-year period, 835 were attended to and had records available for review. The majority of missing records were for patients who were registered in the clinic, had an appointment booked but did not attend. Thirty patients out of the 835 reported exposure to cerebral malaria of whom, 23 fulfilled the inclusion criteria. Original hospitalisation notes were obtained for 11 of these patients. Seven patients did not have sufficient information to exclude other central nervous system infections. The median age on exposure was 30 (5-72) months and 13 were males.The majority of patients had been discharged with multiple deficits. Severe neurological sequelae included loss of speech 14), hearing impairment (9), motor deficits (14), behaviour problems (11), epilepsy (12), blindness (12) and cognitive impairment (9). Figure 4, hearinBased on the time of onset, severe neurological sequelae could be categorized into two;i. Immediate onset sequelae - evident on discharge from hospital. The majority of these patients had prolonged coma and status epilepticus during the acute illness  or months to years (epilepsy) after the insult.Cortical blindness has been described in earlier studies and because it is reversible, its pathogenesis has been attributed to transient ischaemia. In this series, blindness was observed in 12/23 subjects and often, was one of many deficits on discharge. The shortest time to resolution was one month. Visual acuity in ten of the remaining patients improved over a 2-6 months period and in all cases, almost normal vision was restored by 7 months.Hearing impairment has rarely been reported in previous series. In this series, severe hearing impairment was reported in 9/23 children. Two other children had milder degrees of hearing impairment. Five of the nine children with severe impairment had formal audiometric testing: 3 had profound impairment and 2 had severe impairment with a hearing threshold of 80 decibels. The parents of only one child could afford hearing aids.Aphasia was reported in 14 children. Three other children had dysarthria. All three had oro-motor problems. Although speech therapy was offered at weekly or fortnightly intervals, over the follow up period, no full recovery was reported suggesting that loss of speech after childhood cerebral malaria has a poorer prognosis.Gross motor deficits following cerebral malaria include hemiplegia, diplegia, quadriparesis or quadriplegia. In thisBehaviour problems observed in 11 children were some of the most striking difficulties. Using items in the DSM IV-TR criteria, 3 types of behaviour problems could be recognized:a) Hyperactivity, impulsiveness and inattentiveness as in ADHD;b) Conduct disorders ;c) Autistic spectrum and pervasive developmental disorder-like behaviours with or without other neuro-psychiatric problems .th child were presumed to be an adverse reaction to phenobarbitone and the symptoms improved when this drug was withdrawn.The earliest behavioural symptoms developed within two weeks of discharge. The most common were ADHD-like symptoms. These often were associated with poor night-time sleep. Symptoms improved over 1-3 weeks with haloperidol and/or small doses of methylphenidate. Worsening hyperactivity developed in a child with self injurious behaviour following the initiation of phenobarbitone to treat epilepsy. Behaviour problems in the 12Aggressive behaviour included beating up peers, throwing stones at people and cars with no or minimal provocation and in two cases, uncontrollable anger. One child with gelastic seizures and a history of febrile seizures in multiple family relations would in addition to anger and aggressive behaviour get prolonged bouts of laughter.Parents of two other children with labile moods reported bouts of excessive crying following minimal scolding. In the first of the two children, these crying episodes started two weeks after discharge from hospital; the child would cry continuously for over two hours. The electroencephalogram was normal.Three other children were said to wander off and would sometimes get lost unless closely watched. Parents of one would tie him up in the house. The strain on the family was so immense that one parent described life at home almost like, "hell" and grandparents were called in to help care for two of the other children. These symptoms appeared to improve with haloperidol: in 2/3 cases (including the child who was being tied up), the wandering stopped within a month.Other than socialization, the effect of the behaviour problems on school attendance in previously school-going children was detrimental; teachers could not cope, school performance declined significantly and all dropped out of school. A brain CT scan was performed in two children - one, a child with intractable epilepsy and aggressive behaviour who was initially discharged with blindness, loss of hearing and speech. The scans showed no focal lesions in the brain parenchyma but bilateral atrophy of the temporal lobes. The second CT scan was in a child discharged with hemiplegia, blindness, feeding difficulties, loss of speech and who later developed ADHD-like symptoms. The acute CT showed brain swelling and meningeal enhancement over the temporo-parietal area and the convalescent CT, cortical atrophy in the same area.EEG was performed in 8 of the 12 patients with epilepsy. In patients with generalized seizures, diffuse epileptiform discharges were observed on the electroencephalogram. Localized discharges over the temporal region were seen in two patients. Two children had multiple seizure types with more than 10 seizures a day. Seizures in these two were unresponsive to therapy with phenobarbitone (later withdrawn) and carbamazepine. Although seizures in one eventually improved on sodium valproate, in the second child who in addition had severe behavioural problems, sodium valproate did not reduce the seizure frequency. Instead, the frequency reduced and the behaviour problems improved upon the initiation of clonazepam.Several patients with multiple sequelae had severe cognitive impairment. Child neglect and malnutrition were evident especially among those who presented >6 months after exposure. Malnutrition was compounded by feeding difficulties in the spastic children.This study aimed to describe functional and behaviour deficits in children with severe impairments following cerebral malaria and the patterns of these deficits. We found that deficits in motor function, behaviour, vision, speech and hearing or epilepsy were major long-term sequelae. Two main patterns were observed: a) immediate and, b) late-onset deficits. Some deficits (e.g. blindness) resolved, others (e.g. loss of speech) showed little improvement over the follow-up period, while some (e.g. behaviour problems) developed long after exposure.Earlier studies of cerebral malaria mostly focused on reducing the high mortality. Although the long-term outcome has been described in recent times,6,7,12 aThis was a retrospective study of children with severe impairments in a specialist clinic but not a representative sample of children surviving cerebral malaria, a selection bias that may have over-estimated the frequency of sequelae. Secondly, the World Health Organization definition for cerebral malaria is not specific and so, Despite the above weaknesses and the small sample size, this is one of the first papers to provide a comprehensive description of impairments following recovery from cerebral malaria by using domain specific assessments in individual children. The inclusion of behaviour assessments has for the first time highlighted behaviour difficulties as major sequelae of cerebral malaria in children. In addition, the two patterns of deficits described here may help guide pathogenesis studies although the observed differences may be due to different brain regions affected or extent of injury. Cerebral malaria may also be used as a new model to study the pathogenesis of ADHD in children.The authors declare that they have no competing interests.RI and TJ conceived the study. RI, AK, SB, GM, CM and JB saw the patients. RI performed the analysis and wrote the first draft while AK SB, JB and TJ critically read through the manuscript.Supplementary Table S1: Severe Neurological and Behavioural Sequelae following Cerebral Malaria in Ugandan children. This table summarises the demographic and clinical characteristics of the individual study subjects at the time of exposure, the documented risk factors for neurological sequelae, the types of sequelae observed and changes over the follow up period.Click here for file


To the Editor: A 30-year-old African man, without any specific medical history, came to the emergency department of Brugmann University Hospital in Brussels, Belgium, on March 18, 2008, because of malaise, profuse transpiration, and dizziness that day. He also reported a 3-day history of muscle pain and pain while urinating, for which his general practitioner prescribed ciprofloxacin. Originally from Guinea-Conakry, the patient reported no travel outside Belgium after his arrival >9 years earlier. No recurrent fever episodes were noted during this period. Two weeks before becoming ill, a friend visiting from Guinea-Conakry stayed at his home for 7 days.Plasmodium falciparum with a parasite density of 0.1%. Serologic tests showed an antibody titer to Plasmodium spp. of 3,200. A blood sample was sent to the national reference laboratory at the Institute of Tropical Medicine in Antwerp. The diagnosis of P. falciparum malaria was confirmed by microscopy and real-time PCR. Follow-up was uneventful because the patient responded to a 7-day course of oral quinine and doxycycline.Results of a physical examination were normal, except for a temperature of 37.5°C. Blood analysis showed moderate anemia (hemoglobin 12.9 g/dL) and thrombocytopenia  with total bilirubin and C-reactive protein levels of 1.5 mg/dL and 7.0 mg/dL, respectively. Because we suspected subfebrile malaria in this patient, a blood smear was prepared. It showed ring-shaped trophozoites of P. falciparum, usually occurs within 2 months after the bite of an infective mosquito. A few reports mention a delay of >1 year between exposure and initial clinical symptoms, probably related to the disappearance of residual protective immunity in immigrants (P. falciparum infection that becomes clinically evident in an immunocompromised person (Anopheles spp. mosquito, referred to as airport, luggage, or container malaria (P. falciparum malaria without any evidence of a mosquito bite have been reported and related to transfusion of parasitized erythrocytes, intravenous drug use, or accidental needlestick injuries (Malaria, a potential life-threatening disease caused by P. falciparum malaria that may have been contracted by the bite of an anopheline mosquito captured in the luggage of the patient’s visiting friend (Anopheles spp. in Belgium is difficult. The patient did not receive any recent blood transfusions and denied being an intravenous drug user, although this possibility cannot be excluded.We report a clinically atypical case of late P. falciparum. Another possible reason is residual immunity to malaria, which was no longer protective but still capable of attenuating symptoms or signs of malaria. This finding implicates recrudescence of disease after a long period of asymptomatic infection with P. falciparum. Serologic analysis detected high levels of antibodies to Plasmodium spp., which suggests a chronic infection rather than a new one.This type of malaria, also known as luggage or suitcase malaria, makes adequate and timely diagnosis difficult because a history of exposure to a possibly malaria-infected mosquito is apparently absent. Moreover, our patient had few classic symptoms or signs, such as high-grade fever, chills, or headaches; this pattern complicates diagnosis. This lack of typical malaria symptoms may be related to the fact that before coming to the hospital, the patient took ciprofloxacin, which has in vitro activity against P. falciparum in the area visited, which would enable the parasite to evade the host’s immune response (P. falciparum has been suggested, which would link persistent immunity with late recrudescence or with less severe or complicated disease in immigrants (P. falciparum has been transmitted through blood transfusions from donors from malaria-endemic regions several years after exposure, which suggests long subpatent periods (It is generally accepted that protective immunity wanes after several months of nonexposure. Support for this thesis is the frequency of clinical malaria in African adults who visit their families after a long stay in a country where the disease is not endemic. However, these cases might be caused by antigenic variation of P. falciparum malaria in patients without a recent travel history to malaria-endemic areas. In such cases, autochthonous malaria, whether transmitted by an imported or an indigenous mosquito or by infected blood cells or needles, should be excluded. Residual protective immunity, even after several years of nonexposure to P. falciparum, may explain persistent asymptomatic infection and late recrudescence of disease.This case highlights the problem of diagnosing