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Background: Anaemia in pregnancy is typically due to iron deficiency (IDA) but remains a complex and pervasive problem, particularly in low resource settings. At clinics on the Myanmar–Thailand border, a protocol was developed to guide treatment by health workers in antenatal care (ANC). Objective: To evaluate the clinical use of a protocol to treat anaemia in pregnancy. Methods: The design was a descriptive retrospective analysis of antenatal data obtained during the use of a standard anaemia treatment protocol. Two consecutive haematocrits (HCT) ≺30% prompted a change from routine prophylaxis to treatment doses of haematinics. Endpoints were anaemia at delivery (most recent HCT before delivery ≺30%) and timeliness of treatment initiation. Women whose HCT failed to respond to the treatment were investigated. Results: From August 2007 to July 2012, a median [IQR] of five [4–11] HCT measurements per woman resulted in the treatment of anaemia in 20.7% (2,246/10,886) of pregnancies. Anaemia at delivery was present in 22.8% (511/2,246) of treated women and 1.4% (123/8,640) who remained on prophylaxis. Human error resulted in a failure to start treatment in 97 anaemic women (4.1%, denominator 2,343 (2,246 + 97)). Fluctuation of HCT around the cut-point of 30% was the major problem with the protocol accounting for half of the cases where treatment was delayed greater than 4 weeks. Delay in treatment was associated with a 1.5 fold higher odds of anaemia at delivery (95% CI 1.18, 1.97). Conclusion: There was high compliance to the protocol by the health workers. An important outcome of this evaluation was that the clinical definition of anaemia was changed to diminish missed opportunities for initiating treatment. Reduction of anaemia in pregnancy requires early ANC attendance, prompt treatment at the first HCT

Additional file 1: Table S2. Primers sequences, restriction enzymes and annealing temperatures of PCR-RFLP protocol used.

Summary Background Human genetic factors are important determinants of malaria risk. We investigated associations between multiple candidate polymorphisms—many related to the structure or function of red blood cells—and risk for severe Plasmodium falciparum malaria and its specific phenotypes, including cerebral malaria, severe malaria anaemia, and respiratory distress. Methods We did a case-control study in Kilifi County, Kenya. We recruited as cases children presenting with severe malaria to the high-dependency ward of Kilifi County Hospital. We included as controls infants born in the local community between Aug 1, 2006, and Sept 30, 2010, who were part of a genetics study. We tested for associations between a range of candidate malaria-protective genes and risk for severe malaria and its specific phenotypes. We used a permutation approach to account for multiple comparisons between polymorphisms and severe malaria. We judged p values less than 0·005 significant for the primary analysis of the association between candidate genes and severe malaria. Findings Between June 11, 1995, and June 12, 2008, 2244 children with severe malaria were recruited to the study, and 3949 infants were included as controls. Overall, 263 (12%) of 2244 children with severe malaria died in hospital, including 196 (16%) of 1233 with cerebral malaria. We investigated 121 polymorphisms in 70 candidate severe malaria-associated genes. We found significant associations between risk for severe malaria overall and polymorphisms in 15 genes or locations, of which most were related to red blood cells: ABO, ATP2B4, ARL14, CD40LG, FREM3, INPP4B, G6PD, HBA (both HBA1 and HBA2), HBB, IL10, LPHN2 (also known as ADGRL2), LOC727982, RPS6KL1, CAND1, and GNAS. Combined, these genetic associations accounted for 5·2% of the variance in risk for developing severe malaria among individuals in the general population. We confirmed established associations between severe malaria and sickle-cell trait (odds ratio [OR] 0·15, 95% CI 0·11–0·20; p=2·61 × 10−58), blood group O (0·74, 0·66–0·82; p=6·26 × 10−8), and –α3·7-thalassaemia (0·83, 0·76–0·90; p=2·06 × 10−6). We also found strong associations between overall risk of severe malaria and polymorphisms in both ATP2B4 (OR 0·76, 95% CI 0·63–0·92; p=0·001) and FREM3 (0·64, 0·53–0·79; p=3·18 × 10−14). The association with FREM3 could be accounted for by linkage disequilibrium with a complex structural mutation within the glycophorin gene region (comprising GYPA, GYPB, and GYPE) that encodes for the rare Dantu blood group antigen. Heterozygosity for Dantu was associated with risk for severe malaria (OR 0·57, 95% CI 0·49–0·68; p=3·22 × 10−11), as was homozygosity (0·26, 0·11–0·62; p=0·002). Interpretation Both ATP2B4 and the Dantu blood group antigen are associated with the structure and function of red blood cells. ATP2B4 codes for plasma membrane calcium-transporting ATPase 4 (the major calcium pump on red blood cells) and the glycophorins are ligands for parasites to invade red blood cells. Future work should aim at uncovering the mechanisms by which these polymorphisms can result in severe malaria protection and investigate the implications of these associations for wider health. Funding Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative.

Background: Screening and monitoring serum bilirubin (SBR) in neonates is crucial to prevent neonatal hyperbilirubinemia (NH)-associated morbidity and mortality worldwide. A lack of resources is often a barrier for measuring SBR in developing countries. Reliable, cost-effective, easy to use point-of-care (POC) SBR tests are needed. This study aimed to evaluate the technical accuracy and usability of the Bilistick System (BS), a new bilirubin POC test, in a tropical setting. Methods: This was a mixed-methods study, including laboratory validation of the BS, direct observation of technical procedures as performed by the midwives and midwives’ assessment of the device’s easiness of use through focus group discussions (FGD) and a self-administered questionnaire. The study was conducted in a field clinic of the Shoklo Malaria Research Unit along the Thailand-Myanmar border between January and December 2017. Results: A total of 173 samples were tested at a median age of 4 days. BS generated an error message—providing no SBR readout—in 48.6% of the tests performed. For the tests that yielded a result, the correlation coefficient (95% CI) between BS and routine laboratory bilirubinometer SBR was 0.87 (0.77-0.93). The accuracy decreased with increasing haematocrit and at higher humidity (≥75%). Direct observation of the operators using the device and analysis of the focus group discussions and questionnaires indicated that the BS was considered easy to use and required limited training. Conclusions: This evaluation showed that the BS, in its current formulation, does not provide reliable results for measuring SBR in a tropical, low-resource setting but has acceptable usability features., SCOPUS: ar.j, info:eu-repo/semantics/published

Background Medicines that exert oxidative pressure on red blood cells (RBC) can cause severe hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Due to X-chromosome inactivation, females heterozygous for G6PD with one allele encoding a G6PD deficient protein and the other a normal protein produce two RBC populations each expressing exclusively one allele. The G6PD mosaic is not captured with routine G6PD tests. Methods An open-source software tool for G6PD cytofluorometric data interpretation is described. The tool interprets data in terms of % bright RBC, or cells with normal G6PD activity in specimens collected from two geographically and ethnically distinct populations, an African-American cohort (US), and a Karen and Burman ethnic cohort (Thailand) comprising 242 specimens including 89 heterozygous females. Results The tool allowed comparison of data across two laboratories and both populations. Hemizygous normal or deficient males and homozygous normal or deficient females cluster at narrow % bright cells with mean values of 96%, or 6% (males) and 97%, or 2% (females) respectively. Heterozygous females, show a distribution of 10-85% bright cells, and a mean of 50%. The distributions are associated to severity of the G6PD mutation. Conclusions Consistent cytoflourometric G6PD analysis facilitates inter-laboratory comparison of cellular G6PD profiles and contributes to understanding primaquine associated hemolytic risk.

Glucose-6-phosphate dehydrogenase (G6PD) activity is essential for redox equilibrium of red blood cells (RBCs) and, when compromised, the RBCs are more susceptible to haemolysis. 8-aminoquinolines (primaquine and tafenoquine) are used for the radical curative treatment of Plasmodium vivax malaria and can cause haemolysis in G6PD deficient subjects. Haemolytic risk is dependent on treatment dose and patient G6PD status but ultimately it correlates with the number of G6PD deficient RBCs. The G6PD spectrophotometric assay reliably identifies deficient subjects but is less reliable in heterozygous females, especially when other blood conditions are present. In this work we analysed samples with a range of G6PD phenotypes and haematologic conditions from 243 healthy volunteers of Asian or African-American heritage using both the spectrophotomeric assay and the G6PD flow-cytometric assay. Overall 18.5% of subjects (29.3% of Asian females) presented with anaemia, associated with decreased RBCs volume (MCV) and reticulocytosis; the flow-cytometric assay showed good correlation with the spectrophotometric assay (Pearson’s r 0.918–0.957) and was less influenced by haemoglobin concentration, number of RBCs and number of reticulocytes. This resulted in more precise quantification of the number of G6PD deficient RBCs and presumably higher predictive power of drug induced haemolytic risk.

Additional file 1. GeoDx. An online open source tool to guide G6PD diagnostics procurement.

Additional file 2: Table S3. Details regarding SNP and amino acid changes of the seven main G6PD variants along with sequences of the PCR primers, allele-specific primers and locus-speficic probes used to test samples collected from Cambodian males, Cambodia, National Malaria Survey 2013.

Additional file 1: S1 Table. Definition of primary efficacy endpoint by initial species. S2 Table. Dose of each compound given to the participants. S3 Table. The baseline characteristics of women with Plasmodium falciparum mono-infection. S4 Table. The baseline characteristics of women with Plasmodium vivax mono-infection. S5 Table. The baseline characteristics of patients with Plasmodium falciparum infection, including both P. falciparum mono-infection and co-infection of P. falciparum and Plasmodium vivax. S6 Table. Cumulative proportion of fever and parasite clearance of falciparum and vivax for each treatment arm among pregnant women with history of fever or documented fever at enrolment. S7 Table. Description of gametocyte carriage, congenital and placental malaria. S8 Table. Univariable and multivariable analyses of the risk of PCR-corrected treatment failure in pregnant women with uncomplicated falciparum malaria using a Cox proportional hazard model. S9 Table. Univariable and multivariable analyses on the risk of PCR-corrected treatment failure in pregnant women with uncomplicated falciparum malaria using a Cox proportional hazard model with Pfkelch13 mutations. S10 Table. Prevalence of symptoms before and after treatment. S11 Table. Description of pregnancy outcomes. S12 Table. Details of pregnant women who had miscarriage or stillbirth. S13 Table. Details of congenital abnormality and ICD-10 coding. S14 Table. Details of neonatal mortality. S15 Table. Univariable and multivariable linear regression analyses of the characteristics associated with gestational week at birth among those who had malaria in pregnancy. S16 Table. Univariable and multivariable linear regression analyses of the characteristics associated with SGA z-score (birthweight for gestational age and newborn sex at birth). S17 Table. Birth outcomes in pregnant women who had malaria and were enrolled in the first trimester. S1 Figure. Monthly number of pregnant women with malaria for each malaria species over the study period (from February 2010 to August 2016). S2 Figure. Kaplan-Meier survival curves for antimalarials in pregnancy: Panel a. recurrence of any malaria species, Panel b. Plasmodium falciparum PCR-corrected efficacy censoring intercalated Plasmodium vivax malaria, Panel c. P. falciparum PCR-uncorrected efficacy , and Panel d. P. vivax parasitaemia after P. falciparum mono-infection. S3 Figure. Kaplan-Meier survival curves for parasite clearance of Plasmodium falciparum (Panel a) or Plasmodium vivax (Panel b) for each treatment arm. S4 Figure. Fractional change in haematocrit from baseline during the follow-up for each species (Panel a) or treatment stratified by malaria species (Panel b).

Additional file 2: Table S3. Details regarding SNP and amino acid changes of the seven main G6PD variants along with sequences of the PCR primers, allele-specific primers and locus-speficic probes used to test samples collected from Cambodian males, Cambodia, National Malaria Survey 2013.

Additional file 1: S1 Table. Definition of primary efficacy endpoint by initial species. S2 Table. Dose of each compound given to the participants. S3 Table. The baseline characteristics of women with Plasmodium falciparum mono-infection. S4 Table. The baseline characteristics of women with Plasmodium vivax mono-infection. S5 Table. The baseline characteristics of patients with Plasmodium falciparum infection, including both P. falciparum mono-infection and co-infection of P. falciparum and Plasmodium vivax. S6 Table. Cumulative proportion of fever and parasite clearance of falciparum and vivax for each treatment arm among pregnant women with history of fever or documented fever at enrolment. S7 Table. Description of gametocyte carriage, congenital and placental malaria. S8 Table. Univariable and multivariable analyses of the risk of PCR-corrected treatment failure in pregnant women with uncomplicated falciparum malaria using a Cox proportional hazard model. S9 Table. Univariable and multivariable analyses on the risk of PCR-corrected treatment failure in pregnant women with uncomplicated falciparum malaria using a Cox proportional hazard model with Pfkelch13 mutations. S10 Table. Prevalence of symptoms before and after treatment. S11 Table. Description of pregnancy outcomes. S12 Table. Details of pregnant women who had miscarriage or stillbirth. S13 Table. Details of congenital abnormality and ICD-10 coding. S14 Table. Details of neonatal mortality. S15 Table. Univariable and multivariable linear regression analyses of the characteristics associated with gestational week at birth among those who had malaria in pregnancy. S16 Table. Univariable and multivariable linear regression analyses of the characteristics associated with SGA z-score (birthweight for gestational age and newborn sex at birth). S17 Table. Birth outcomes in pregnant women who had malaria and were enrolled in the first trimester. S1 Figure. Monthly number of pregnant women with malaria for each malaria species over the study period (from February 2010 to August 2016). S2 Figure. Kaplan-Meier survival curves for antimalarials in pregnancy: Panel a. recurrence of any malaria species, Panel b. Plasmodium falciparum PCR-corrected efficacy censoring intercalated Plasmodium vivax malaria, Panel c. P. falciparum PCR-uncorrected efficacy , and Panel d. P. vivax parasitaemia after P. falciparum mono-infection. S3 Figure. Kaplan-Meier survival curves for parasite clearance of Plasmodium falciparum (Panel a) or Plasmodium vivax (Panel b) for each treatment arm. S4 Figure. Fractional change in haematocrit from baseline during the follow-up for each species (Panel a) or treatment stratified by malaria species (Panel b).

Additional file 3. PCR-based ligase detection reaction-fluorescent microsphere assay (PCR-LDR-FMA) protocol.