Falciparum-infected Samples Research Articles

Detection and quantification of Babesia species intraerythrocytic parasites by flow cytometry.

Recent work has demonstrated that automated fluorescence flow cytometry (FLC) is a potential alternative for the detection and quantification of Plasmodium parasites. The objective of this study was to apply this novel FLC method to detect and quantify Babesia parasites in venous blood and compare results to light microscopy and polymerase chain reaction methods. An automated hematology/malaria analyzer (XN-31; Sysmex) was used to detect and quantify B microti-infected red blood cells from residual venous blood samples (n = 250: Babesia positive, n = 170; Babesia negative, n = 80). As no instrument software currently exists for Babesia, qualitative and quantitative machine learning (ML) algorithms were developed to facilitate analysis. Performance of the ML models was verified against the XN-31 software using P falciparum-infected samples. When applied to Babesia-infected samples, the qualitative ML model demonstrated an area under the curve (AUC) of 0.956 (sensitivity, 95.9%; specificity, 83.3%) relative to polymerase chain reaction. For valid scattergrams, the qualitive model achieved an AUC of 1.0 (sensitivity and specificity, 100%), while the quantitative model demonstrated an AUC of 0.986 (sensitivity, 94.4%; specificity, 100%). This investigation demonstrates that Babesia parasites can be detected and quantified directly from venous blood using FLC. Although promising, opportunities remain to improve the general applicability of the method.

Multilayer Thin-Film Optical Filters for Reflectance-Based Malaria Diagnostics.

Malaria diagnosis relies on optical microscopy and/or rapid diagnostic tests based on detecting specific malaria antigens. The clinical sensitivity of these methods is highly dependent on parasite density, with low levels of detection at low parasite density, challenging the worldwide malaria elimination efforts. Therefore, there is a need for diagnostic methods with higher sensitivity, demanding innovative diagnostics devices able to detect malaria at low parasite density and at early stages of the disease. We propose an innovative optical device for malaria diagnosis, based on optical reflectance spectrophotometry, for the detection of parasites through the quantification of haemozoin. For this purpose, a set of eight thin-film optical filters, based on multilayer stacks of MgO/TiO2 and SiO2/TiO2 thin-films, with high transmittance and low full width at half maximum (FWHM) at specific wavelengths, was designed and fully characterized (both numerically and experimentally). A preliminary assessment of its potential to reconstruct the original spectra of red blood cells was performed, both in uninfected and Plasmodium falciparum-infected samples. The obtained results show that, although the experimental filters have a non-ideal performance characteristic, they allow us to distinguish, based on only 8 discrete points in the optical spectrum, between healthy and malaria infected samples, up to a detection limit of 12 parasites/μL of red blood cells. Those results enhance the potential of using such a device for malaria diagnostics, aiming for non-invasiveness.

Decreased prevalence of the Plasmodium falciparum Pfcrt K76T and Pfmdr1 and N86Y mutations post-chloroquine treatment withdrawal in Katete District, Eastern Zambia

BackgroundIn 2002, Zambia withdrew chloroquine as first-line treatment for Plasmodium falciparum malaria due to increased treatment failure and worldwide spread of chloroquine resistance. The artemisinin combination regimen, artemether–lumefantrine, replaced chloroquine (CQ) as first choice malaria treatment. The present study determined the prevalence of CQ resistance molecular markers in the Pfcrt and Pfmdr1 genes in Eastern Zambia at 9 and 13 years after the removal of drug pressure.MethodsSamples collected from Katete District during the drug therapeutic efficacy assessments conducted in 2012 and 2016 were assayed by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) to determine the prevalence of genetic mutations, K76T on the Pfcrt gene and N86Y on the Pfmdr1 gene. A total of 204 P. falciparum-positive DBS samples collected at these two time points were further analysed.ResultsAmong the samples analysed for Pfcrt K76T and Pfmdr1 N86Y in the present study, 112 (82.4%) P. falciparum-infected samples collected in 2012 were successfully amplified for Pfcrt and 94 (69.1%) for Pfmdr1, while 69 (65.7%) and 72 (68.6%) samples from 2016 were successfully amplified for Pfcrt and Pfmdr1, respectively. In 2012, the prevalence of Pfcrt 76K (sensitive) was 97.3%, 76T (resistant) was 1.8%, and 0.8% had both 76K and 76T codons (mixed). Similarly in 2012, the prevalence of Pfmdr1 86N (sensitive) was 97.9% and 86Y (resistant) was 2.1%. In the 2016 samples, the prevalence of the respective samples was 100% Pfcrt 76K and Pfmdr1 86N.ConclusionThis study shows that there was a complete recovery of chloroquine-sensitive parasites by 2016 in Katete District, Eastern Zambia, 13 years following the withdrawal of CQ in the country. These findings add to the body of evidence for a fitness cost in CQ-resistant P. falciparum in Zambia and elsewhere. Further studies are recommended to monitor resistance countrywide and explore the feasibility of integration of the former best anti-malarial in combination therapy in the future.

SEROPREVALENCE OF IMMUNOGLOBULIN IgG ANTIBODY RESPONSE TO PLASMODIUM FALCIPARUM MEROZOITE ANTIGENS AMONG CHILDREN IN MINNA, NORTH CENTRAL, NIGERIA

Objective: A cross-sectional study was carried out in a representative cohort of children in Minna aged 6 months–17 years to determine the correlation between immunoglobulin G (IgG) antibody responses to Plasmodium falciparum merozoite antigens.
 Methods: Plasma samples from 93 children were exposed to Enzyme-Linked Immunosorbent Assay for the measurement of IgG antibody production against P. falciparum. 
 Results: There was a high seroprevalence of IgG antibody against P. falciparum antigens tested with 74.20%. The seroprevalence for the male category was quite higher as compared with that of the female category, though, analysis using Mann–Whitney U-test revealed IgG antibody response to P. falciparum infection in the male was significantly different as compared to the female category (p<0.05). Furthermore, the prevalence of IgG antibody against P. falciparum antigen increased with age, with the lowest observed in 6 months–5 years 66.66%. Kruskal–Wallis H test showed a non-significant difference in the production of IgG antibody against P. falciparum antigen between different cohorts, and no correlation exists between them (p>0.05). An evidence of more than 50% was found for the production of IgG antibody by sub-microscopic parasite. On the other hand, microscopically positive P. falciparum samples recorded more seroprevalence of 68.81% as against negative samples, though significant difference between the negative and positive P. falciparum infected samples and the production of IgG antibody was not observed (p>0.05).
 Conclusion: This study has demonstrated a boosting immune responses by sub-microscopic parasite and also suggests a strong relationship between production of IgG antibody and malaria transmission, rather than protective immunity.

Pretreatment gametocyte carriage in symptomatic patients with Plasmodium falciparum and Plasmodium vivax infections on the Thai-Myanmar border.

One hundred and twenty-six blood samples were collected from patients with signs and symptoms of malaria who attended malaria clinics. Malaria positive cases detected by microscopic examination were confirmed by species-specific nested-PCR in 97 (29 and 68 samples for P. falciparum and P. vivax, respectively). The proportion of P. vivax and P. falciparum-infected samples was 70.1: 29.9%. The density in P. falciparum positive samples [median (95%CI): 10,340 (5280-19,200) μ/l] was significantly higher than P. vivax positive samples [4508 (3240-6120) μ/l]. Sixteen out of twenty-nine (55.2%) and 36 out of 68 (52.9%) P. falciparum- and P. vivax-infected samples, respectively, were gametocyte-positive. Gametocyte density in the P. falciparum-infected[124 (69-253) /μl] was significantly higher than that of the P. vivax-infected [54 (45-70)/μl] samples. A significant correlation between gametocyte density and pretreatment parasitemia was only detected in P. falciparum-infected, but not P. vivax-infected samples. The observed high prevalence rates of pretreatment gametocyte carriage of both malaria species, which serves as a large malaria reservoir, particularly in P. falciparum infection, could have a significant impact on malaria control in the endemic populations.

Stabilization of RDT target antigens present in dried Plasmodium falciparum-infected samples for validating malaria rapid diagnostic tests at the point of care

BackgroundMalaria rapid diagnostic tests (RDTs) are a great achievement in implementation of parasite based diagnosis as recommended by World Health Organization. A major drawback of RDTs is lack of positive controls to validate different batches/lots at the point of care. Dried Plasmodium falciparum-infected samples with the RDT target antigens have been suggested as possible positive control but their utility in resource limited settings is hampered by rapid loss of activity over time.MethodsThis study evaluated the effectiveness of chemical additives to improve long term storage stability of RDT target antigens (HRP2, pLDH and aldolase) in dried P. falciparum-infected samples using parasitized whole blood and culture samples. Samples were treated with ten selected chemical additives mainly sucrose, trehalose, LDH stabilizer and their combinations. After baseline activity was established, the samples were air dried in bio-safety cabinet and stored at room temperatures (~ 25 °C). Testing of the stabilized samples using SD Bioline, BinaxNOW, CareStart, and First Response was done at intervals for 53 weeks.ResultsStability of HRP2 at ambient temperature was reported at 21–24 weeks while that of PAN antigens (pLDH and aldolase) was 2–18 weeks of storage at all parasite densities. The ten chemical additives increased the percentage stability of HRP2 and PAN antigens. Sucrose alone and its combinations with Alsever’s solution or biostab significantly increased stability of HRP2 by 56% at 2000 p/µL (p < 0.001). Trehalose and its combinations with biostab, sucrose or glycerol significantly increased stability of HRP2 by 57% (p < 0.001). Unlike sucrose, the stability of the HRP2 was significantly retained by trehalose at lower concentrations (500, and 200 p/µL). Trehalose in combination biostab stabilizer increased the percentage stability of PAN antigens by 42, and 32% at 2000 and 500 p/µL respectively (p < 0.01). This was also the chemical combination with the shortest reconstitution time (~ < 20 min).ConclusionsThese findings confirm that stabilizing RDT target antigens in dried P. falciparum-infected samples using chemical additives provides field-stable positive controls for malaria RDTs.

Plasmodium falciparum infection rates for some Anopheles spp. from Guinea-Bissau, West Africa.

Presence of Plasmodium falciparum circumsporozoite protein (CSP) was detected by enzyme linked immunosorbent assay (ELISA) in a sample of Anopheles gambiae s.s., A. melas and A. pharoensis collected in Guinea-Bissau during October and November 2009. The percentage of P. falciparum infected samples (10.2% overall; confidence interval (CI): 7.45-13.6%) was comparable to earlier studies from other sites in Guinea-Bissau (9.6-12.4%). The majority of the specimens collected were identified as A. gambiae which had an individual infection rate of 12.6 % (CI: 8.88-17.6) across collection sites. A small number of specimens of A. coluzzii, A. coluzzii x A. gambiae hybrids, A. melas and A. pharoensis were collected and had infection rates of 4.3% (CI:0.98-12.4), 4.1% (CI:0.35-14.5), 11.1% (CI:1.86-34.1) and 33.3% (CI:9.25-70.4) respectively. Despite being present in low numbers in indoor collections, the exophilic feeding behaviors of A. melas (N=18) and A. pharoensis (N=6) and high infection rates observed in this survey suggest falciparum-malaria transmission potential outside of the protection of bed nets.

Plasmodium falciparum infection rates for some Anopheles spp. from Guinea-Bissau, West Africa

Presence of Plasmodium falciparum circumsporozoite protein (CSP) was detected by enzyme linked immunosorbent assay (ELISA) in a sample of Anopheles gambiae s.s., A. melas and A. pharoensis collected in Guinea-Bissau during October and November 2009. The percentage of P. falciparum infected samples (10.2% overall) was comparable to earlier studies from other sites in Guinea-Bissau (9.6-12.4%). The majority of the specimens collected were identified as A. gambiae which had an individual infection rate of 12.6 % across collection sites. A small number of specimens of A. coluzzii, A. coluzzii x A. gambiae hybrids, A. melas and A. pharoensis were collected and had infection rates of 4.3%, 4.1%, 11.1% and 33.3% respectively. Despite being present in low numbers in indoor collections, the exophilic feeding behaviors of A. melas (N=18) and A. pharoensis (N=6) and high infection rates observed in this survey suggest falciparum-malaria transmission potential outside of the protection of bed nets.

Detection of a substantial number of sub-microscopic Plasmodium falciparum infections by polymerase chain reaction: a potential threat to malaria control and diagnosis in Ethiopia

BackgroundPrompt and effective malaria diagnosis not only alleviates individual suffering, but also decreases malaria transmission at the community level. The commonly used diagnostic methods, microscopy and rapid diagnostic tests, are usually insensitive at very low-density parasitaemia. Molecular techniques, on the other hand, allow the detection of low-level, sub-microscopic parasitaemia. This study aimed to explore the presence of sub-microscopic Plasmodium falciparum infections using polymerase chain reaction (PCR). The PCR-based parasite prevalence was compared against microscopy and rapid diagnostic test (RDT).MethodsThis study used 1,453 blood samples collected from clinical patients and sub-clinical subjects to determine the prevalence of sub-microscopic P. falciparum carriages. Subsets of RDT and microscopy negative blood samples were tested by PCR while all RDT and microscopically confirmed P. falciparum-infected samples were subjected to PCR. Finger-prick blood samples spotted on filter paper were used for parasite genomic DNA extraction.ResultsThe prevalence of sub-microscopic P. falciparum carriage was 19.2% (77/400) (95% CI = 15. 4–23.1). Microscopy-based prevalence of P. falciparum infection was 3.7% (54/1,453) while the prevalence was 6.9% (100/1,453) using RDT alone. Using microscopy and PCR, the estimated parasite prevalence was 20.6% if PCR were performed in 1,453 blood samples. The prevalence was estimated to be 22.7% if RDT and PCR were used. Of 54 microscopically confirmed P. falciparum-infected subjects, PCR detected 90.7% (49/54). Out of 100 RDT-confirmed P. falciparum infections; PCR detected 80.0% (80/100). The sensitivity of PCR relative to microscopy and RDT was, therefore, 90.7% and 80%, respectively. The sensitivity of microscopy and RDT relative to PCR was 16.5 (49/299) and 24.2% (80/330), respectively. The overall PCR-based prevalence of P. falciparum infection was 5.6- and 3.3 fold higher than that determined by microscopy and RDT, respectively. None of the sub-microscopic subjects had severe anaemia, though 29.4% had mild anaemia (10–11.9 g/dl).ConclusionsAsymptomatic, low-density malaria infection was common in the study area and PCR may be a better tool for measuring Plasmodium prevalence than microscopy and RDT. The inadequate sensitivity of the diagnostic methods to detect substantial number of sub-microscopic parasitaemia would undoubtedly affect malaria control efforts, making reduction of transmission more difficult. RDT and microscopy-based prevalence studies and subsequent reports of reduction in malaria incidence underestimate the true pictures of P. falciparum infections in the community. PCR, on the other hand, seems to have reasonable sensitivity to detect a higher number of infected subjects with low and sub-microscopic parasite densities than RDTs or microscopy.

Tracing the origins and signatures of selection of antifolate resistance in island populations of Plasmodium falciparum

BackgroundResistance of the malaria parasite Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) has evolved worldwide. In the archipelago of São Tomé and Principe (STP), West Africa, although SP resistance is highly prevalent the drug is still in use in particular circumstances. To address the evolutionary origins of SP resistance in these islands, we genotyped point mutations at P. falciparum dhfr and dhps genes and analysed microsatellites flanking those genes.MethodsBlood samples were collected in July and December 2004 in three localities of São Tomé Island and one in Principe Island. Species-specific nested-PCR was used to identify P. falciparum infected samples. Subsequently, SNPs at the dhfr and dhps genes were identified through PCR-RFLP. Isolates were also analysed for three microsatellite loci flanking the dhfr gene, three loci flanking dhps and four loci located at putative neutral genomic regions.ResultsAn increase of resistance-associated mutations at dhfr and dhps was observed, in particular for the dhfr/dhps quintuple mutant, associated with clinical SP failure. Analysis of flanking microsatellites suggests multiple independent introductions for dhfr and dhps mutant haplotypes, possibly from West Africa. A reduced genetic diversity and increased differentiation at flanking microsatellites when compared to neutral loci is consistent with a selective sweep for resistant alleles at both loci.ConclusionsThis study provides additional evidence for the crucial role of gene flow and drug selective pressures in the rapid spread of SP resistance in P. falciparum populations, from only a few mutation events giving rise to resistance-associated mutants. It also highlights the importance of human migration in the spread of drug resistant malaria parasites, as the distance between the islands and mainland is not consistent with mosquito-mediated parasite dispersal.

A Multiplex Ligase Detection Reaction-Fluorescent Microsphere Assay for Simultaneous Detection of Single Nucleotide Polymorphisms Associated with Plasmodium falciparum Drug Resistance

Incomplete malaria control efforts have resulted in a worldwide increase in resistance to drugs used to treat the disease. A complex array of mutations underlying antimalarial drug resistance complicates efficient monitoring of parasite populations and limits the success of malaria control efforts in regions of endemicity. To improve the surveillance of Plasmodium falciparum drug resistance, we developed a multiplex ligase detection reaction-fluorescent-microsphere-based assay (LDR-FMA) that identifies single nucleotide polymorphisms (SNPs) in the P. falciparum dhfr (9 alleles), dhps (10 alleles), and pfcrt (3 alleles) genes associated with resistance to Fansidar and chloroquine. We evaluated 1,121 blood samples from study participants in the Wosera region of Papua New Guinea, where malaria is endemic. Results showed that 468 samples were P. falciparum negative and 453 samples were P. falciparum positive by a Plasmodium species assay and all three gene assays (concordance, 82.2%). For P. falciparum infections where the assay for each gene was positive, 2 samples carried resistance alleles for all three genes, 299 carried resistance alleles for dhfr and pfcrt, 131 carried resistance alleles for only one gene (dhfr [n = 40], dhps [n = 1], or pfcrt [n = 90]), and 21 carried only sensitive alleles at all three genes. Mixed-strain infections characterized 100 samples. Overall, 95.4% (432/453) of P. falciparum-infected samples carried at least one allele associated with resistance to Fansidar or chloroquine. In view of the fact that 86.3% (391/453) of P. falciparum-infected samples carried pfcrt mutations, chloroquine is largely ineffective against P. falciparum in Papua New Guinea. Surveillance of additional dhfr and dhps polymorphisms in order to monitor the continued effectiveness of Fansidar is recommended.