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Fulltext Computerized Morphometric Analysis of Eryptosis

Jacob SS, Prasad K, Rao P, Kamath A, Hegde RB, Baby PM, et al.

Front Physiol, 2019;10:1230.
PMID: 31649550 DOI: 10.3389/fphys.2019.01230

Eryptosis is the suicidal destruction-process of erythrocytes, much like apoptosis of nucleated cells, in the course of which the stressed red cell undergoes cell-shrinkage, vesiculation and externalization of membrane phosphatidylserine. Currently, there exist numerous methods to detect eryptosis, both morphometrically and biochemically. This study aimed to design a simple but sensitive, automated computerized approach to instantaneously detect eryptotic red cells and quantify their hallmark morphological characteristics. Red cells from 17 healthy volunteers were exposed to normal Ringer and hyperosmotic stress with sodium chloride, following which morphometric comparisons were conducted from their photomicrographs. The proposed method was found to significantly detect and differentiate normal and eryptotic red cells, based on variations in their structural markers. The receiver operating characteristic curve analysis for each of the markers showed a significant discriminatory accuracy with high sensitivity, specificity and area under the curve values. The software-based technique was then validated with RBCs in malaria. This model, quantifies eryptosis morphometrically in real-time, with minimal manual intervention, providing a new window to explore eryptosis triggered by different stressors and diseases and can find wide application in laboratories of hematology, blood banks and medical research.
Fulltext Micro-Raman Spectroscopy Analysis of Optically Trapped Erythrocytes in Jaundice

Jacob SS, Bankapur A, Barkur S, Acharya M, Chidangil S, Rao P, et al.

Front Physiol, 2020;11:821.
PMID: 32754052 DOI: 10.3389/fphys.2020.00821

Derangements in bilirubin metabolism and/or dysfunctions in the hepato-biliary system lead to the unhealthy buildup of bilirubin in blood, resulting in jaundice. During the course of this disorder, circulating red cells are invariably subjected to toxic effects of serum bilirubin and an array of inflammatory compounds. This study aimed to investigate the vibrational spectroscopy of live red cells in jaundice using micro-Raman spectroscopy combined with optical-trap. Red cells from blood samples of healthy volunteers and patients with jaundice were optically immobilized and micro-Raman probed using a 785 nm diode laser. Raman signatures from red cells in jaundice exhibited significant variations from the normal and the spectral-markers were obtained from multivariate analytical methods. This research gives insightful views on how different pathologies can act as "stress-milieus" for red cells in circulation, possibly impeding their normal functions and also exasperating anemia. Raman spectroscopy, an emerging bio-analytical technique, is sensitive in detecting molecular-conformations in situ, at cellular-levels and in real-time. This study could pave way in understanding fundamental red cell behavior in different diseases by analyzing Raman markers.
Standardization of a bottle assay--an indigenous method for laboratory and field monitoring of insecticide resistance and comparison with WHO adult susceptibility test

Elamathi N, Barik TK, Verma V, Velamuri PS, Bhatt RM, Sharma SK, et al.

Parasitol Res, 2014 Oct;113(10):3859-66.
PMID: 25098343 DOI: 10.1007/s00436-014-4054-y

The WHO adult susceptibility test is in use for insecticide resistance monitoring. Presently, materials are being imported from the Universiti Sains Malaysia, Malaysia and sometimes it is cost prohibitive. As an alternative, we present here a method of bottle bioassay using indigenous material. Different aspects related to the assay were studied and validated in the field. Bottle assay was standardized in the laboratory by using locally sourced material and laboratory-maintained insecticide-susceptible Anopheles stephensi and Aedes aegypti strains against technical grade deltamethrin and cyfluthrin insecticides dissolved in ethanol in a range of different concentrations. The frequency of use of the deltamethrin-coated bottles and shelf-life were determined. Discriminating dose for deltamethrin and cyfluthrin was 10 μg against An. stephensi and 2 μg against Ae. aegypti females. Insecticide-coated bottles stored at 25 to 35 °C can be used for three exposures within 7 days of coating. The study carried out in the laboratory was validated on wild caught An. culicifacies in the states of Odisha and Chhattisgarh against deltamethrin-coated bottles in comparison to WHO adult susceptibility test. Results of the study indicated that deltamethrin-coated bottles were effective up to three exposures within 7 days of coating for field population and 100% mortality was recorded within 35 min as observed in laboratory studies for field collected susceptible population. Also in the WHO adult susceptibility test, 100% knock-down within 35 min and 100% mortality after 24 h holding period were observed in susceptible population, while in it was 50% knock-down in 1 h and 64% mortality after 24 h holding period for resistant population (50% mortality in bottle assay in 60 min). The bottle assay can be used as an alternative to the WHO adult susceptibility test both in the laboratory and field for monitoring insecticide resistance in mosquito vectors using locally sourced material.
Fulltext Design of a study to determine the impact of insecticide resistance on malaria vector control: a multi-country investigation

Kleinschmidt I, Mnzava AP, Kafy HT, Mbogo C, Bashir AI, Bigoga J, et al.

Malar J, 2015 Jul 22;14:282.
PMID: 26194648 DOI: 10.1186/s12936-015-0782-4

BACKGROUND: Progress in reducing the malaria disease burden through the substantial scale up of insecticide-based vector control in recent years could be reversed by the widespread emergence of insecticide resistance. The impact of insecticide resistance on the protective effectiveness of insecticide-treated nets (ITN) and indoor residual spraying (IRS) is not known. A multi-country study was undertaken in Sudan, Kenya, India, Cameroon and Benin to quantify the potential loss of epidemiological effectiveness of ITNs and IRS due to decreased susceptibility of malaria vectors to insecticides. The design of the study is described in this paper.
METHODS: Malaria disease incidence rates by active case detection in cohorts of children, and indicators of insecticide resistance in local vectors were monitored in each of approximately 300 separate locations (clusters) with high coverage of malaria vector control over multiple malaria seasons. Phenotypic and genotypic resistance was assessed annually. In two countries, Sudan and India, clusters were randomly assigned to receive universal coverage of ITNs only, or universal coverage of ITNs combined with high coverage of IRS. Association between malaria incidence and insecticide resistance, and protective effectiveness of vector control methods and insecticide resistance were estimated, respectively.
RESULTS: Cohorts have been set up in all five countries, and phenotypic resistance data have been collected in all clusters. In Sudan, Kenya, Cameroon and Benin data collection is due to be completed in 2015. In India data collection will be completed in 2016.
DISCUSSION: The paper discusses challenges faced in the design and execution of the study, the analysis plan, the strengths and weaknesses, and the possible alternatives to the chosen study design.
Fulltext A new WHO bottle bioassay method to assess the susceptibility of mosquito vectors to public health insecticides: results from a WHO-coordinated multi-centre study

Corbel V, Kont MD, Ahumada ML, Andréo L, Bayili B, Bayili K, et al.

Parasit Vectors, 2023 Jan 20;16(1):21.
PMID: 36670470 DOI: 10.1186/s13071-022-05554-7

BACKGROUND: The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both.
METHODS: A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species (Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids (metofluthrin, prallethrin and transfluthrin), neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration-response curves for each insecticide-species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population (LC50 and LC99, respectively) and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively.
RESULTS: Overall, about 200,000 mosquitoes were tested with the new bottle bioassay, and LC50/LC99 or OI50/OI99 values were determined for all insecticides. Variation was seen between laboratories in estimates for some mosquito species-insecticide combinations, while other test results were consistent. The variation was generally greater with transfluthrin and flupyradifurone than with the other compounds tested, especially against Anopheles species. Overall, the mean within-bioassay variability in mortality and oviposition inhibition were
Fulltext Implications of insecticide resistance for malaria vector control with long-lasting insecticidal nets: a WHO-coordinated, prospective, international, observational cohort study

Kleinschmidt I, Bradley J, Knox TB, Mnzava AP, Kafy HT, Mbogo C, et al.

Lancet Infect Dis, 2018 Jun;18(6):640-649.
PMID: 29650424 DOI: 10.1016/S1473-3099(18)30172-5

BACKGROUND: Scale-up of insecticide-based interventions has averted more than 500 million malaria cases since 2000. Increasing insecticide resistance could herald a rebound in disease and mortality. We aimed to investigate whether insecticide resistance was associated with loss of effectiveness of long-lasting insecticidal nets and increased malaria disease burden.
METHODS: This WHO-coordinated, prospective, observational cohort study was done at 279 clusters (villages or groups of villages in which phenotypic resistance was measurable) in Benin, Cameroon, India, Kenya, and Sudan. Pyrethroid long-lasting insecticidal nets were the principal form of malaria vector control in all study areas; in Sudan this approach was supplemented by indoor residual spraying. Cohorts of children from randomly selected households in each cluster were recruited and followed up by community health workers to measure incidence of clinical malaria and prevalence of infection. Mosquitoes were assessed for susceptibility to pyrethroids using the standard WHO bioassay test. Country-specific results were combined using meta-analysis.
FINDINGS: Between June 2, 2012, and Nov 4, 2016, 40 000 children were enrolled and assessed for clinical incidence during 1·4 million follow-up visits. 80 000 mosquitoes were assessed for insecticide resistance. Long-lasting insecticidal net users had lower infection prevalence (adjusted odds ratio [OR] 0·63, 95% CI 0·51-0·78) and disease incidence (adjusted rate ratio [RR] 0·62, 0·41-0·94) than did non-users across a range of resistance levels. We found no evidence of an association between insecticide resistance and infection prevalence (adjusted OR 0·86, 0·70-1·06) or incidence (adjusted RR 0·89, 0·72-1·10). Users of nets, although significantly better protected than non-users, were nevertheless subject to high malaria infection risk (ranging from an average incidence in net users of 0·023, [95% CI 0·016-0·033] per person-year in India, to 0·80 [0·65-0·97] per person year in Kenya; and an average infection prevalence in net users of 0·8% [0·5-1·3] in India to an average infection prevalence of 50·8% [43·4-58·2] in Benin).
INTERPRETATION: Irrespective of resistance, populations in malaria endemic areas should continue to use long-lasting insecticidal nets to reduce their risk of infection. As nets provide only partial protection, the development of additional vector control tools should be prioritised to reduce the unacceptably high malaria burden.
FUNDING: Bill & Melinda Gates Foundation, UK Medical Research Council, and UK Department for International Development.