Effective High-Throughput Blood Pooling Strategy before DNA Extraction for Detection of Malaria in Low-Transmission Settings

In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 µl in 1 sample, was optimal, and the parasite density as low as 2 p/µl for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings.

The prognostic utility of bedside assessment of adults hospitalized.

Background: Data collected in clinical trials have been used to develop scoring systems that identify adults with malaria at greatest risk of death. One of these, the RCAM score, can be simply determined by measuring a patient’s Glasgow Coma Score and respiratory rate on admission to hospital. However the safety of using the RCAM score to define high-risk patients has not been assessed outside of the clinical trial setting. Methods: A retrospective audit of medical records of all adults admitted with a diagnosis of malaria to two tertiary referral hospitals in Lower Myanmar in 2013 was undertaken. An RCAM score was calculated in all patients and related to their subsequent clinical course. Results: The recent decline in malaria hospitalizations at both sites continued in 2013. During the year 90 adults were hospitalized with malaria; 62 (69%) had Plasmodium falciparum monoinfection, 11 (12%) had Plasmodium vivax mono-infection, 17 (19%) had mixed infection. All seven (7.7%) deaths occurred in patients infected with P. falciparum. An admission RCAM score < 2 identified all the patients that would survive to discharge (positive predictive value (95% confidence interval (CI)) 100% (94.9-100%) and also predicted a requirement for less supportive care: 9/70 (13%) patients with an admission RCAM score < 2 required supportive care (blood transfusion, vasopressor support or oxygen supplementation) during their hospitalization compared with 12/20 (60%) patients with an admission RCAM score ≥ 2 (p < 0.0001). No patient with P. vivax monoinfectionrequired supportive care during their hospitalization. Patients with an oxygen saturation ≤ 95% on room air on admission were more likely to die before discharge (odds ratio 17.3 (95% CI: 2.9-101.2) than patients with a higher oxygen saturation (p = 0.002). Conclusions: Even outside a clinical trial setting the RCAM score reliably identifies adults with malaria who are at greatest risk of death and can be safely used in the initial triage and management of these patients.

The use of parasite lactate dehydrogenase isoenzyme-based visual test for the speciation of Plasmodium Falciparum and Plasmodium vivax infections

Parasite specific lactate dehydrogenase (LDH) isoenzyme coated OptiMAL test strips were used to diagnose Plasmodium falciparum and p. vivax infections in one hundred clinically suspected malaria patients attending the Clinical Research Unit for Malaria, Defence Services General Hospital, Mingaladon during May to September 1999. Traditional microscopic examination was carried out in parallet to observe the sensitivity and specificity of the LDH-based OptiMAL test. the interpretation of the results by LDH-based test strips took only five minutes, whereas microscopic examination took thrity minutes. The sensitivities and specificities of pLDH-based OptiMAL test were 89.47 per cent and 100.0 per cent for P. falciparum; 100.0 per cent and 96.43 per cent for P.vivax respectively. Due to the rapid detection and speciation, prompt treatment was made without any delay.

Humoral immune response to falciparum malaria during pregnany

Humoral immune respone against Plasmodium Falciparum was studied ontwo hundred and thirty seven pregnant and non pregnant women in Tha-hton township during premonsoon season of 1998. Slide positivity rate among pregnant women was 7.3

(10/137), fifty percent of which were primigravide. Eighty percent of infection occured druing second trimester. Antimalarial antibiodies were determined by both Indirect Fluorescent Antibody Test (IFAT) and Enzyme Linked Immunosorbent Assay (ELISA). IFAT seropositivity rate was 63.33

(n=33) in primigravidae and 83.63

(n=11) in non pregnant healthy women. In the convalescent sera, the rates were 77.14

(n=7) and 87.33

(n=15) respectively. These IFAT results were also comparable with those of ELISA. Lower seropositivity rate and mean antibodytitre were observed in pregnant women compared with non-pregnants. These findings inply that there is suppression of antimalarial immunity druing pregnancy

The use of primaquine in malaria infected patients with red cell glucose-6-phosphate dehydrogenase (G6PD) deficiency in Myanmar.

32 subjects with Plasmodium falciparum gametocytes, and 31 cases with Plasmodium vivax infection from two military hospitals (Lashio, Mandalay) were treated with quinine 600 mg three times a day for 7 days followed by primaquine 45 mg single dose for gametocytes and 45 mg weekly x 8 weeks for vivax malaria. Although screening of red cell glucose-6-phosphate dehydrogenase (G6PD) was done prior to primaquine treatment, G6PD deficient subjects were not excluded from the trial. 20 patients hemizygous for mild G6PD deficiency (GdB- variant), 2 patients hemizygous for severe deficiency (Gd-Myanmar variant) completed the trial. No case of acute hemolysis was observed in all 22 patients with two genotypes of red cell G6PD deficiency status. Therefore, a single dose of primaquine 45 mg and/or weekly for 8 weeks is adequate for the treatment of patients with P. falciparum gametocytes and/or P. vivax malaria ignoring these red cell G6PD enzyme deficient variants in Myanmar.

A study of lipid profile in Myanmar smokers and nonsmokers

This study was carried out to see the levels of serum lipids in smokers and nonsmoders. A total of 155 healthy subjects from the staff of the Institute of Medicine II, Yangon were studied. Among the 155 subjects, 96 were nonsmokers and 59 were active current smokers, smoking 1-9 cigarettes a day. Over night fasting samples of serum total cholesterol, serum HDL cholesterol, serum trigly-cerides and serum phospholipids were measured. It was found that smokers had significantly higher level of serum total cholesterol than nonsmoders.

Plasmodium falciparum; merozoite invasion rate in erythrocytes with b-thalassaemia trait and glucose-6-phosphate dehydrogenase deficient genes

The malaria parasite invasion-erythrocyte membrane protein variation hypothesis was tested on red cells collected from subjects with G-6-PD deficiency and -thalassaemia trait genes. A series of in vitro competition assays were carried out by using serial dilution technique and the FITC-labelling method (1). The results showed that the invasion rates of P. falciparum merozoites into two G-6-PD hemizygotes deficient cell were Gd- 100 Percent of normal and Gd-Myanmar 95 Percent of normal. But in erythrocytes from two patients with double gene defects viz. G-6-PD mild deficient gene GdB-with -thalassaemia trait gene was 25 Percent and G-6-PD severely deficient gene Gd-Myanmar with -thalassaemia trait gene was 20 Percent susceptible to merozoite invasion. The invasion rate of malaria merozoites in these cells could be inhibited by the partial loss of vital attachement of the red cell membrane required for the process of invasion.

Some variation in membrane proteins of erythrocytes obtained from subjects with thalassaemia trait and G-6-PD deficient genes

A total of 350 erythrocyte samples were screened for haemoglobin variant, G-6-PD deficiency and thalassaemia trait genes. Of these 104 samples were subjected to membrane protein analysis using SDS-PAGE electrophoresis and double straining with silver and Coomassie Blue. Membrane protein patterns of erythrocytes from subjects with a single genetic defect (i.e. either G-6-PD deficient or thalassaemia trait) were found to be similar to that of normals but those from subjects with double genetic defects (i.e. combination of G-6-PD deficiency and thalassaemia trait) had a significantly reduced content of spectrin, band 3 and glycophorin A.