[Show abstract][Hide abstract] ABSTRACT: Micronutrient fortified flour (MFF), supplementary food rations and micronutrient (MN) supplements may prevent deficiencies among pregnant women. Objectives of cross-sectional surveys in 2004 (n = 533) and 2006 (n = 515) were to assess the impact of new food rations (flour, oil) and supplements on MN status by trimester of pregnancy in the Maela refugee camp. Hemoglobin, iron status, zinc, retinol, β-carotene and tryptophan decreased, while α-/γ-tocopherol and 5-methyltetrahydrofolate (5-MTHF) increased from first to third trimester. In 2006, mean zinc and α-tocopherol for each trimester was significantly higher than in 2004. The weeks of supplemented thiamine and folic acid were positively correlated with thiamine diphosphate (TDP) and 5-MTHF, but not for ferrous sulfate as iron deficiency was observed in 38.5% of third-trimester women. Frequent consumption of fish paste and owning a garden or animal were associated with significantly higher iron status, retinol, β-carotene, and 5-MTHF. In conclusion, MFF and supplementary oil were most likely to explain improved zinc and α-tocopherol status, while thiamine and folate supplements ensured high TDP and 5-MTHF in late pregnancy. MN supplements, MN-rich staple food, small gardens, and programs to improve iron compliance are promising strategies to prevent MN deficiencies during pregnancy in vulnerable populations.
[Show abstract][Hide abstract] ABSTRACT: There are enormous numbers of microsatellites repeats (~1 microsatellite every 650bp) in the genome of the malaria parasite, Plasmodium falciparum. These markers are multiallelic and therefore more informative than SNPs for many research questions, yet they are problematic to score using next generation sequence data, and remain an untapped source of genetic variation for population genomic analyses. We used three approaches to validate scoring of microsatellite loci from next generation sequence data: (1) we sequenced the progeny of a genetic cross (n=14), and examined microsatellite calling in genome regions inherited from each parent; (2) we sequenced (in duplicate) parasites from a mutation accumulation experiment (n=38), and measured reproducibility and accuracy of scoring in >36K microsatellite loci throughout the parasite genome. (3) We compared microsatellite genotypes in genome sequence data from 6 single clone patient samples that had previously been scored for 335 microsatellites using traditional capillary electrophoresis. These analyses define the robustness with which microsatellites bearing different sequence motifs can be scored, and provide a map of callable microsatellites for large scale analysis of malaria parasite populations. To examine the utility of these markers for population genomic analyses, we determined genome-wide patterns of microsatellite variation in sequence data from 50 isolates obtained from a focus of emerging drug resistance in South-East Asia. We identify loci under strong, recent, positive selection in these samples, including known drug resistance loci, and we recapture the kelch locus underpinning artemisinin resistance.
[Show abstract][Hide abstract] ABSTRACT: Background:
Doxycycline is an antibiotic used in combination with quinine or artesunate for malaria treatment or alone for malaria chemoprophylaxis. Recently, one prophylactic failure has been reported, and several studies have highlighted in vitro doxycycline decreased susceptibility in Plasmodium falciparum isolates from different areas. The genetic markers that contribute to detecting and monitoring the susceptibility of P. falciparum to doxycycline, the pfmdt and pftetQ genes, have recently been identified. However, these markers are not sufficient to explain in vitro decreased susceptibility of P. falciparum to doxycycline. In this paper, the association between polymorphism of the small sub-unit ribosomal RNA apicoplastic gene pfssrRNA (PFC10_API0057) and in vitro susceptibilities of P. falciparum isolates to doxycycline were investigated.
Doxycycline IC50 determinations using the hypoxanthine uptake inhibition assay were performed on 178 African and Thai P. falciparum isolates. The polymorphism of pfssrRNA was investigated in these samples by standard PCR followed by sequencing.
No point mutations were found in pfssrRNA in the Thai or African isolates, regardless of the determined IC50 values.
The pfssrRNA gene is not associated with in vitro decreased susceptibility of P. falciparum to doxycycline. Identifying new in vitro molecular markers associated with reduced susceptibility is needed, to survey the emergence of doxycycline resistance.
[Show abstract][Hide abstract] ABSTRACT: Background
Plasmodium falciparum infections adversely affect pregnancy. Anti-malarial treatment failure is common. The objective of this study was to examine the duration of persistent parasite carriage following anti-malarial treatment in pregnancy.
The data presented here are a collation from previous studies carried out since 1994 in the Shoklo Malaria Research Unit (SMRU) on the Thailand-Myanmar border and performed using the same unique methodology detailed in the Materials and Methods section. Screening for malaria by microscopy is a routine part of weekly antenatal care (ANC) visits and therapeutic responses to anti-malarials were assessed in P. falciparum malaria cases. Women with microscopy confirmed P. falciparum malaria had a PCR blood spot from a finger-prick sample collected. Parasite DNA was extracted from the blood-spot samples using saponin lysis/Chelex extraction method and genotyped using polymorphic segments of MSP1, MSP2 and GLURP. Recurrent infections were classified by genotyping as novel, recrudescent or indeterminate. Factors associated with time to microscopy-detected recrudescence were analysed using multivariable regression techniques.
From December 1994 to November 2009, 700 women were treated for P. falciparum and there were 909 recurrent episodes (481 novel and 428 recrudescent) confirmed by PCR genotyping. Most of the recurrences, 85 % (770/909), occurred after treatment with quinine monotherapy, artesunate monotherapy or artesunate-clindamycin. The geometric mean number of days to recurrence was significantly shorter in women with recrudescent infection, 24.5 (95 %: 23.4-25.8), compared to re-infection, 49.7 (95 %: 46.9-52.7), P <0.001. The proportion of recrudescent P. falciparum infections that occurred after days 28, 42 and 63 from the start of treatment was 29.1 % (124/428), 13.3 % (57/428) and 5.6 % (24/428). Recrudescent infections ≥100 days after treatment occurred with quinine and mefloquine monotherapy, and quinine + clindamycin and artesunate + atovaquone-proguanil combination therapy. Treatments containing an artemisinin derivative or an intercalated Plasmodium vivax infection increased the geometric mean interval to recrudescence by 1.28-fold (95 % CI: 1.09-1.51) and 2.19-fold (1.77-2.72), respectively. Intervals to recrudescence were decreased 0.83-fold (0.73-0.95) if treatment was not fully supervised (suggesting incomplete adherence) and 0.98-fold (0.96-0.99) for each doubling in baseline parasitaemia.
Prolonged time to recrudescence may occur in pregnancy, regardless of anti-malarial treatment. Long intervals to recrudescence are more likely with the use of artemisinin-containing treatments and also observed with intercalated P. vivax infections treated with chloroquine. Accurate determination of drug efficacy in pregnancy requires longer duration of follow-up, preferably until delivery or day 63, whichever occurs last.
[Show abstract][Hide abstract] ABSTRACT: Objectives Methylene blue, once discarded due to its unsettling yet mild side effects, has now found a renewed place in the pharmacopoeia
of modern medicine. The continued spread of drug-resistant Plasmodium vivax and Plasmodium falciparum has also led to a recent re-examination of methylene blue's potent antimalarial properties. Here we examine the ex vivo susceptibility profile of Plasmodium spp. isolates to methylene blue; the isolates were from a region on the Thai–Myanmar border where there are increasing rates
of failure when treating vivax malaria with chloroquine.
Full-text · Article · Aug 2014 · Journal of Antimicrobial Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: The transition from enucleated reticulocytes to mature normocytes is marked by substantial remodeling of the erythrocytic cytoplasm and membrane. Despite conspicuous changes, most studies describe the maturing reticulocyte as a homogenous erythropoietic cell type. While reticulocyte staging based on fluorescent RNA stains such as thiazole orange have been useful in a clinical setting; these 'sub-vital' stains may confound delicate studies on reticulocyte biology and may preclude their use in heamoparasite invasion studies.
Here we use highly purified populations of reticulocytes isolated from cord blood, sorted by flow cytometry into four sequential subpopulations based on transferrin receptor (CD71) expression: CD71high, CD71medium, CD71low and CD71negative. Each of these subgroups was phenotyped in terms of their, morphology, membrane antigens, biomechanical properties and metabolomic profile.
Superficially CD71high and CD71medium reticulocytes share a similar gross morphology (large and multilobular) when compared to the smaller, smooth and increasingly concave reticulocytes as seen in the in the CD71low and CD71negativesamples. However, between each of the four sample sets we observe significant decreases in shear modulus, cytoadhesive capacity, erythroid receptor expression (CD44, CD55, CD147, CD235R, and CD242) and metabolite concentrations. Interestingly increasing amounts of boric acid was found in the mature reticulocytes.
Reticulocyte maturation is a dynamic and continuous process, confounding efforts to rigidly classify them. Certainly this study does not offer an alternative classification strategy; instead we used a nondestructive sampling method to examine key phenotypic changes of in reticulocytes. Our study emphasizes a need to focus greater attention on reticulocyte biology.
[Show abstract][Hide abstract] ABSTRACT: Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC50s) were determined by a modified sigmoid Emax model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC50s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.
[Show abstract][Hide abstract] ABSTRACT: The quantity of circulating reticulocytes is an important indicator of erythropoietic activity in response to a wide range of haematological pathologies. While most modern laboratories use flow cytometry to quantify reticulocytes, most field laboratories still rely on 'subvital' staining. The specialist 'subvital' stains, New Methylene Blue (NMB) and Brilliant Crésyl Blue are often difficult to procure, toxic, and show inconsistencies between batches. Here we demonstrate the utility of Giemsa's stain (commonly used microbiology and parasitology) in a 'subvital' manner to provide an accurate method to visualize and count reticulocytes in blood samples from normal and malaria-infected individuals.
[Show abstract][Hide abstract] ABSTRACT: The Shoklo Malaria Research Unit has been working on the Thai-Myanmar border for 25 y providing early diagnosis and treatment (EDT) of malaria. Transmission of has declined, but resistance to artesunate has emerged. We expanded malaria activities through EDT and evaluated the impact over a 12-y period.
Between 1 October 1999 and 30 September 2011, the Shoklo Malaria Research Unit increased the number of cross-border (Myanmar side) health facilities from two to 11 and recorded the number of malaria consultations. Changes in malaria incidence were estimated from a cohort of pregnant women, and prevalence from cross-sectional surveys. In vivo and in vitro antimalarial drug efficacy were monitored. Over this period, the number of malaria cases detected increased initially, but then declined rapidly. In children under 5 y, the percentage of consultations due to malaria declined from 78% (95% CI 76-80) (1,048/1,344 consultations) to 7% (95% CI 6.2-7.1) (767/11,542 consultations), 0.001. The ratio of declined from 1.4 (95% CI 1.3-1.4) to 0.7 (95% CI 0.7-0.8). The case fatality rate was low (39/75,126; 0.05% [95% CI 0.04-0.07]). The incidence of malaria declined from 1.1 to 0.1 episodes per pregnant women-year. The cumulative proportion of decreased significantly from 24.3% (95% CI 21.0-28.0) (143/588 pregnant women) to 3.4% (95% CI 2.8-4.3) (76/2,207 pregnant women), 0.001. The in vivo efficacy of mefloquine-artesunate declined steadily, with a sharp drop in 2011 (day-42 PCR-adjusted cure rate 42% [95% CI 20-62]). The proportion of patients still slide positive for malaria at day 3 rose from 0% in 2000 to reach 28% (95% CI 13-45) (8/29 patients) in 2011.
Despite the emergence of resistance to artesunate in , the strategy of EDT with artemisinin-based combination treatments has been associated with a reduction in malaria in the migrant population living on the Thai-Myanmar border. Although limited by its observational nature, this study provides useful data on malaria burden in a strategically crucial geographical area. Alternative fixed combination treatments are needed urgently to replace the failing first-line regimen of mefloquine and artesunate. Please see later in the article for the Editors' Summary.
[Show abstract][Hide abstract] ABSTRACT: Whole genome sequencing (WGS) of Plasmodium vivax is problematic due to the reliance on clinical isolates which are generally low in parasitaemia and sample volume. Furthermore, clinical isolates contain a significant contaminating background of host DNA which confounds efforts to map short read sequence of the target P. vivax DNA. Here, we discuss a methodology to significantly improve the success of P. vivax WGS on natural (non-adapted) patient isolates. Using 37 patient isolates from Indonesia, Thailand, and travellers, we assessed the application of CF11-based white blood cell filtration alone and in combination with short term ex vivo schizont maturation. Although CF11 filtration reduced human DNA contamination in 8 Indonesian isolates tested, additional short-term culture increased the P. vivax DNA yield from a median of 0.15 to 6.2 ng µl(-1) packed red blood cells (pRBCs) (p = 0.001) and reduced the human DNA percentage from a median of 33.9% to 6.22% (p = 0.008). Furthermore, post-CF11 and culture samples from Thailand gave a median P. vivax DNA yield of 2.34 ng µl(-1) pRBCs, and 2.65% human DNA. In 22 P. vivax patient isolates prepared with the 2-step method, we demonstrate high depth (median 654X coverage) and breadth (≥89%) of coverage on the Illumina GAII and HiSeq platforms. In contrast to the A+T-rich P. falciparum genome, negligible bias was observed in coverage depth between coding and non-coding regions of the P. vivax genome. This uniform coverage will greatly facilitate the detection of SNPs and copy number variants across the genome, enabling unbiased exploration of the natural diversity in P. vivax populations.
[Show abstract][Hide abstract] ABSTRACT: The establishment of a Plasmodium vivaxin vitro culture system is critical for the development of new vaccine, drugs and diagnostic tests. Although short-term cultures have been successfully set up, their reproducibility in laboratories without direct access to P. vivax-infected patients has been limited by the need for fresh parasite isolates. We explored the possibility of using parasite isolates and reticulocytes, both cryopreserved, to perform invasion and initiate short-term culture. Invasion results obtained with both cryopreserved isolates and reticulocytes were similar to those obtained with fresh samples. This method should be easily replicated in laboratories outside endemic areas and will substantially contribute to the development of a continuous P. vivax culture. In addition, this model could be used for testing vaccine candidates as well as for studying invasion-specific molecular mechanisms.
Full-text · Article · Feb 2012 · International journal for parasitology
[Show abstract][Hide abstract] ABSTRACT: Chloroquine (CQ) remains the treatment of choice for Plasmodium vivax malaria. Initially confined to parts of Indonesia and Papua, resistance of P. vivax to CQ seems to be spreading, and alternative treatments are required.
We conducted a randomized controlled study to compare the efficacy and the tolerability of CQ and dihydroartemisinin-piperaquine (DP) in 500 adults and children with acute vivax malaria on the Northwestern border of Thailand.
Both drugs were well tolerated. Fever and parasite clearance times were slower in the CQ than in the DP group (P < .001). By day 28, recurrent infections had emerged in 18 of 207 CQ recipients compared with 5 of 230 treated with DP (relative risk, 4.0; 95% confidence interval [CI], 1.51-10.58; P = .0046). The cumulative risk of recurrence with P. vivax at 9 weeks was 79.1% (95% CI, 73.5%-84.8%) in patients treated with CQ compared with 54.9% (95% CI, 48.2%-61.6%) in those receiving DP (hazard ratio [HR], 2.27; 95% CI, 1.8-2.9; P < .001). Children <5 years old were at greater risk of recurrent P. vivax infection (74.4%; 95% CI, 63.2%-85.6%) than older patients (55.3% [95% CI, 50.2%-60.4%]; HR, 1.58 [95% CI, 1.1-2.2]; P = .005). In vitro susceptibility testing showed that 13% of the tested isolates had a CQ median inhibitory concentration >100 nmol/L, suggesting reduced susceptibility.
The efficacy of CQ in the treatment of P. vivax infections is declining on the Thai-Myanmar border. DP is an effective alternative treatment.
Full-text · Article · Nov 2011 · Clinical Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing.
Full-text · Article · Oct 2011 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: Currently, there are no reliable RBC invasion assays to guide the discovery of vaccines against Plasmodium vivax, the most prevalent malaria parasite in Asia and South America. Here we describe a protocol for an ex vivo P vivax invasion assay that can be easily deployed in laboratories located in endemic countries. The assay is based on mixing enriched cord blood reticulocytes with matured, trypsin-treated P vivax schizonts concentrated from clinical isolates. The reliability of this assay was demonstrated using a large panel of P vivax isolates freshly collected from patients in Thailand.
[Show abstract][Hide abstract] ABSTRACT: Resistance of vivax malaria to treatment with antifolates, such as pyrimethamine (Pyr), is spreading as mutations in the dihydrofolatereductase
(dhfr) genes are selected and disseminated. We tested the antitumor drug methotrexate (MTX), a potent competitive inhibitor of
dhfr, against 11 Plasmodium vivax isolates ex vivo, 10 of which had multiple dhfr mutations associated with Pyr resistance. Despite high-grade resistance to Pyr (median 50% inhibitory concentration [IC50], 13,345 nM), these parasites were all highly susceptible to MTX (median IC50, 2.6 nM). Given its potency against Pyr-resistant P. vivax, the antimalarial potential of MTX deserves further investigation.
Full-text · Article · Jan 2011 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Plasmodium species ex vivo sensitivity assay protocols differ in the requirement for leukocyte removal before culturing. This study shows that the presence
of leukocytes significantly increases the 50% inhibitory concentration (IC50) of P. vivax and P. falciparum to artesunate and chloroquine relative to results with the paired leukocyte-free treatment. Although leukocyte removal is
not an essential requirement for the conduct of ex vivo assays, its use has important implications for the interpretation of temporal and spatial antimalarial sensitivity data.
Full-text · Article · Dec 2010 · Antimicrobial Agents and Chemotherapy