A bioassay method validation framework for laboratory and semi-field tests used to evaluate vector control tools.

Vector control interventions play a fundamental role in the control and elimination of vector-borne diseases. The evaluation of vector control products relies on bioassays, laboratory and semi-field tests using live insects to assess the product's effectiveness. Bioassay method development requires a rigorous validation process to ensure that relevant methods are used to capture appropriate entomological endpoints which accurately and precisely describe likely efficacy against disease vectors as well as product characteristics within the manufacturing tolerance ranges for insecticide content specified by the World Health Organization. Currently, there are no standardized guidelines for bioassay method validation in vector control. This report presents a framework for bioassay validation that draws on accepted validation processes from the chemical and healthcare fields and which can be applied for evaluating bioassays and semi-field tests in vector control. The validation process has been categorized into four stages: preliminary development; feasibility experiments; internal validation, and external validation. A properly validated method combined with an appropriate experimental design and data analyses that account for both the variability of the method and the product is needed to generate reliable estimates of product efficacy to ensure that at-risk communities have timely access to safe and reliable vector control products.

Human Infection Challenge with Serotype 3 Pneumococcus.

Rationale: Streptococcus pneumoniae serotype 3 (SPN3) is a cause of invasive pneumococcal disease and associated with low carriage rates. Following the introduction of pediatric 13-valent pneumococcal conjugate vaccine (PCV13) programs, SPN3 declines are less than other vaccine serotypes and incidence has increased in some populations coincident with a shift in predominant circulating SPN3 clade, from I to II. A human challenge model provides an effective means for assessing the impact of PCV13 on SPN3 in the upper airway. Objectives: To establish SPN3's ability to colonize the nasopharynx using different inoculum clades and doses, and the safety of an SPN3 challenge model. Methods: In a human challenge study involving three well-characterized and antibiotic-sensitive SPN3 isolates (PFESP306 [clade Ia], PFESP231 [no clade], and PFESP505 [clade II]), inoculum doses (10,000, 20,000, 80,000, and 160,000 cfu/100 µl) were escalated until maximal colonization rates were achieved, with concurrent acceptable safety. Measurement and Main Results: Presence and density of experimental SPN3 nasopharyngeal colonization in nasal wash samples, assessed using microbiological culture and molecular methods, on Days 2, 7, and 14 postinoculation. A total of 96 healthy participants (median age 21, interquartile range 19-25) were inoculated (n = 6-10 per dose group, 10 groups). Colonization rates ranged from 30.0-70.0% varying with dose and isolate. 30.0% (29/96) reported mild symptoms (82.8% [24/29] developed a sore throat); one developed otitis media requiring antibiotics. No serious adverse events occurred. Conclusions: An SPN3 human challenge model is feasible and safe with comparable carriage rates to an established Serotype 6B human challenge model. SPN3 carriage may cause mild upper respiratory symptoms.

A closer look at the WHO cone bioassay: video analysis of the hidden effects of a human host on mosquito behaviour and insecticide contact.

BACKGROUND: The WHO cone test is one of three tests currently used to evaluate the efficacy of insecticide-treated bed nets (ITNs). It generates two test outputs, knockdown and 24-h mortality, both indicative of immediate toxicity but that reveal little about the nature of mosquito and ITN interaction or how results translate to real-world settings. METHODS: A human arm held 5 mm behind the net surface acted as a host attractant during cone tests and a smartphone was used to capture mosquito behaviour in the cone. Post-exposure blood feeding and survival for nine days were recorded; ingested blood meal size was determined by measuring excreted haematin. Four strains of Anopheles gambiae (insecticide susceptible: Kisumu and N'gousso; insecticide resistant: Banfora and VK7) were tested with and without the host attractant using untreated, Permanet 2.0 and Olyset nets. Video recordings were scan sampled every five seconds to record mosquito positions on either the net, in flight or in contact with the cone. Generalized estimating equations were used to analyse all data except survival within nine days which was analysed using Weighted Cox Regression. RESULTS: Net contact was the most frequently recorded behaviour in all Anopheles spp. strains on all nets. Adding the human host as attractant triggered excitatory behaviours: in all strains, the magnitude of net contact was significantly decreased compared to tests without a host. ITN exposure altered the observed behaviour of the two susceptible strains, which exhibited a decreased response to the host during ITN tests. The resistant strains did not alter their behaviour during ITN tests. Significantly less net contact was observed during Olyset Net tests compared to Permanet 2.0. The host presence affected survival after exposure: Banfora and VK7 mosquitoes exposed to Permanet 2.0 with a host lived longer compared to tests performed without a host. However, mosquitoes that blood-fed and survived long enough to digest the blood meal did not exhibit significantly reduced longevity regardless of the presence of the host attractant. CONCLUSIONS: Simple modifications to the WHO cone test and extension of post-test monitoring beyond the current 24 h enable detailed behavioural characterizations of individual ITNs to be compiled. The effects observed from testing with a host and including blood feeding suggest that more representative estimates of true of ITN efficacy are gained with these modifications than when using the current testing protocol.

Quantifying late-stage host-seeking behaviour of Anopheles gambiae at the insecticidal net interface using a baited-box bioassay.

BACKGROUND: Insecticide-treated nets (ITNs) are losing efficacy against pyrethroid-resistant malaria vector populations throughout Africa. Safeguarding bed net efficacy, vital for effective malaria control, requires greater knowledge of mosquito-ITN interactions and how this impacts on the mosquito. METHODS: A purpose-built benchtop apparatus with a closed 10 cm cubic chamber (the 'Baited-box') was used to video record behaviour of individual free-flying female Anopheles gambiae during approach and blood-feeding on a human hand through untreated nets and ITNs at close range. Time and duration of defined behavioural events, and knockdown and mortality at 1- and 24-h post-exposure respectively, were recorded for pyrethroid susceptible and resistant mosquitoes. RESULTS: Using three human volunteers differing in relative attractiveness to mosquitoes, 328 mosquitoes were individually tested. There were no significant differences between response rates to ITNs and untreated nets (P > 0.1) or between resistant (Tiassalé) and susceptible (Kisumu) mosquito strains, at untreated nets (P = 0.39) or PermaNet 2.0 (P = 1). The sequence of behavioural events from host-seeking to completion of blood-feeding was consistent in all tests but duration and start time of events involving net contact were reduced or delayed respectively with ITNs. Blood-feeding durations at untreated nets (means from 4.25 to 8.47 min (95% confidence interval (CI) = 3.39-9.89) at 3 human volunteers) were reduced by 37-50% at PermaNet 2.0, in susceptible (mean 2.59-4.72 min, 95% CI = 1.54-5.5, P = < 0.05) and resistant (mean 4.20 min, 95% CI = 3.42-4.97, P = 0.01) strains. Total accumulated net contact was approximately 50% lower at PermaNet and Olyset ITNs (P < 0.0001) in susceptible (two of the three volunteers) and resistant mosquitoes. Times prior to first net contact were similar at untreated nets and ITNs (P > 0.2), and neither ITN type showed detectable spatial repellency. After initial contact, blood-feeding commenced later at Olyset (mean 2.76 min, 95% CI = 1.74-3.76, P = 0.0009) and PermaNet (mean 2.4 min, 95% CI = 1.52-3.33, P = 0.0058) than untreated netting (mean 0.68 min, 95% CI = 0.42-0.94). CONCLUSIONS: The baited box offers a simple method for detailed characterization of mosquito behavioural responses to insecticidal nets, for comparing entomological modes of action between nets and for defining the behavioural responses of particular mosquito strains or populations. The device has potential as a screening assay in the search for novel net treatments and for investigations into behavioural resistance mechanisms.

Coverage outcomes (effects), costs, cost-effectiveness, and equity of two combinations of long-lasting insecticidal net (LLIN) distribution channels in Kenya: a two-arm study under operational conditions.

BACKGROUND: Malaria-endemic countries distribute long-lasting insecticidal nets (LLINs) through combined channels with ambitious, universal coverage (UC) targets. Kenya has used eight channels with variable results. To inform national decision-makers, this two-arm study compares coverage (effects), costs, cost-effectiveness, and equity of two combinations of LLIN distribution channels in Kenya. METHODS: Two combinations of five delivery channels were compared as 'intervention' and 'control' arms. The intervention arm comprised four channels: community health volunteer (CHV), antenatal and child health clinics (ANCC), social marketing (SM) and commercial outlets (CO). The control arm consisted of the intervention arm channels except mass campaign (MC) replaced CHV. Primary analysis used random sample household survey data, service-provider costs, and voucher or LLIN distribution data to compare between-arm effects, costs, cost-effectiveness, and equity. Secondary analyses compared costs and equity by channel. RESULTS: The multiple distribution channels used in both arms of the study achieved high LLIN ownership and use. The intervention arm had significantly lower reported LLIN use the night before the survey (84·8% [95% CI 83·0-86·4%] versus 89·2% [95% CI 87·8-90·5%], p < 0·0001), higher unit costs ($10·56 versus $7·17), was less cost-effective ($86·44, 95% range $75·77-$102·77 versus $69·20, 95% range $63·66-$77·23) and more inequitable (Concentration index [C.Ind] = 0·076 [95% CI 0·057 to 0·095 versus C.Ind = 0.049 [95% CI 0·030 to 0·067]) than the control arm. Unit cost per LLIN distributed was lowest for MC ($3·10) followed by CHV ($10·81) with both channels being moderately inequitable in favour of least-poor households. CONCLUSION: In line with best practices, the multiple distribution channel model achieved high LLIN ownership and use in this Kenyan study setting. The control-arm combination, which included MC, was the most cost-effective way to increase UC at household level. Mass campaigns, combined with continuous distribution channels, are an effective and cost-effective way to achieve UC in Kenya. The findings are relevant to other countries and donors seeking to optimise LLIN distribution. TRIAL REGISTRATION: The assignment of the intervention was not at the discretion of the investigators; therefore, this study did not require registration.

Mapping Aedes aegypti indoor resting behavior reveals a preference vulnerable to householder-led vector control.

Many mosquito vectors rest inside human habitations, a behavioral trait that is exploited for vector control by indoor residual spraying (IRS) of interior walls with insecticide. Although IRS and its refined version targeted IRS are very effective against Aedes aegypti, they are expensive and logistically challenging to deliver in densely populated urban areas where outbreaks of dengue and other arboviruses are the greatest challenge. In experiments in Recife, Brazil, we set out to quantify the indoor resting behavior of Ae. aegypti at a level beyond that previously reported. We found that significantly more Ae. aegypti males, unfed and fed females visited the base of walls (height 0-20 cm, corresponding to 12.3% of the total wall surface) more frequently than upper wall areas, with the difference more pronounced at higher temperatures. When the lowest 20 cm of the walls was treated with an appropriate insecticide and colored black, we recorded up to 85% cumulative mortality after 24-h exposure in the experimental room. The findings are significant because feasibly, householders could treat this small and accessible target zone manually, without the need for visits by costly IRS teams or equipment, reducing insecticide use and enabling communities to actively protect their own indoor environment.

Insecticidal roof barriers mounted on untreated bed nets can be as effective against Anopheles gambiae as regular insecticide-treated bed nets.

Barrier bednets (BBnets), regular bednets with a vertical insecticidal panel to target mosquitoes above the bednet roof, where they are most active, have the potential to improve existing Insecticidal Treated Bednets (ITNs), by reducing the quantity of insecticide required per net, reducing the toxic risks to those using the net, potentially increasing insecticide choice. We evaluated the performance of PermaNet 3.0 (P3) and untreated (Ut) bed nets with and without pyrethroid and piperonyl butoxide roof barriers in killing pyrethroid-resistant and susceptible Anopheles gambiae, simultaneously video-recording mosquito flight tracks. Bioassay results showed that treated roof barriers, particularly the longitudinal P3 barrier (P3L) could be an effective addition to a bed net: P3 + P3L were consistently significantly more effective than the reference P3 bednet while performance of untreated nets could be raised to equal that of the reference P3 following the addition of a P3 barrier. The BBnet's potential to augment existing bednets and enhance their performance is considered.

Video augmentation of the WHO cone assay to quantify mosquito behavioural responses to insecticide-treated nets.

BACKGROUND: Insecticide-treated nets (ITNs) using pyrethroids have been the main vector control tools deployed in malaria endemic countries and are responsible for the dramatic reduction in African malaria cases in the early 2000s. The World Health Organization (WHO) cone test was designed to assess the rapid toxicity effects of pyrethroid exposure on mosquito vectors but has yielded no insights beyond 60-min knockdown and 24-h mortality. As dual-active-ingredient (AI) ITNs become more widespread, bioassays that can provide realistic assessment of single- and dual-treated ITNs (i.e. nets with more than one active ingredient) are urgently needed. METHODS: We present an augmentation of the cone test that enables accurate quantification of vector behavioural responses (specifically movement, spatial and temporal occupancy) to ITNs using video recording and bespoke software that uses background segmentation methods to detect spatial changes in the movement of mosquitoes within the cone. Four strains of Anopheles gambiae sensu lato (s.l.) were exposed to four ITNs (PermaNet 2.0, PermaNet 3.0, Olyset Net, Interceptor G2) and untreated nets in these modified cone tests. Life history data (post-exposure blood-feeding, blood meal weight, longevity) for individual mosquitoes were recorded. RESULTS: All mosquitoes responded to the presence of ITNs, spending from 1.48 to 3.67 times more time in the upper region of the cone, depending on the ITN type. Of all ITNs, PermaNet 2.0 provoked the smallest change in behavioural response. Activity in the cone influenced observed post-exposure longevity, and in resistant strains exposed to Interceptor G2, the higher the activity, the greater the risk of dying, as long as the proportion of activity at the net surface was less than 50%. All ITNs inhibited blood-feeding, and smaller blood meals were taken when mosquitoes fed. CONCLUSIONS: The additional mosquito behaviour data obtained by using this modification to the WHO cone test provides unique insight into the innate responses of different mosquito strains on untreated nets and the entomological mode of action of ITNs, important evidence when evaluating ITN characteristics.

Participant perceptions and experiences of a novel community-based respiratory longitudinal sampling method in Liverpool, UK: A mixed methods feasibility study.

Longitudinal, community-based sampling is important for understanding prevalence and transmission of respiratory pathogens. Using a minimally invasive sampling method, the FAMILY Micro study monitored the oral, nasal and hand microbiota of families for 6 months. Here, we explore participant experiences and opinions. A mixed methods approach was utilised. A quantitative questionnaire was completed after every sampling timepoint to report levels of discomfort and pain, as well as time taken to collect samples. Participants were also invited to discuss their experiences in a qualitative structured exit interview. We received questionnaires from 36 families. Most adults and children >5y experienced no pain (94% and 70%) and little discomfort (73% and 47% no discomfort) regardless of sample type, whereas children ≤5y experienced variable levels of pain and discomfort (48% no pain but 14% hurts even more, whole lot or worst; 38% no discomfort but 33% moderate, severe, or extreme discomfort). The time taken for saliva and hand sampling decreased over the study. We conducted interviews with 24 families. Families found the sampling method straightforward, and adults and children >5y preferred nasal sampling using a synthetic absorptive matrix over nasopharyngeal swabs. It remained challenging for families to fit sampling into their busy schedules. Adequate fridge/freezer space and regular sample pick-ups were found to be important factors for feasibility. Messaging apps proved extremely effective for engaging with participants. Our findings provide key information to inform the design of future studies, specifically that self-sampling at home using minimally invasive procedures is feasible in a family context.

Epidemiology of dengue fever in Guatemala.

Dengue fever occurs worldwide and about 1% of cases progress to severe haemorrhage and shock. Dengue is endemic in Guatemala and its surveillance system could document long term trends. We analysed 17 years of country-wide dengue surveillance data in Guatemala to describe epidemiological trends from 2000 to 2016.Data from the national dengue surveillance database were analysed to describe dengue serotype frequency, seasonality, and outbreaks. We used Poisson regression models to compare the number of cases each year with subsequent years and to estimate incidence ratios within serotype adjusted by age and gender. 91,554 samples were tested. Dengue was confirmed by RT-qPCR, culture or NS1-ELISA in 7097 (7.8%) cases and was IgM ELISA-positive in 19,290 (21.1%) cases. DENV1, DENV2, DENV3, and DENV4 were detected in 2218 (39.5%), 2580 (45.9%), 591 (10.5%), and 230 (4.1%) cases. DENV1 and DENV2 were the predominant serotypes, but all serotypes caused epidemics. The largest outbreak occurred in 2010 with 1080 DENV2 cases reported. The incidence was higher among adults during epidemic years, with significant increases in 2005, 2007, and 2013 DENV1 outbreaks, the 2010 DENV2 and 2003 DENV3 outbreaks. Adults had a lower incidence immediately after epidemics, which is likely linked to increased immunity.

A "One-Stop" Screening Protocol for Haemoglobinopathy Traits and Iron Deficiency in Sri Lanka.

Introduction: The high frequencies of carriers of severe haemoglobinopathies and of iron deficiency in Southeast Asia require reliable and affordable tests to improve on current screening procedures. Objectives: We evaluate a "one stop" approach using the THALCON dichlorophenolindophenol (DCIP) and one-tube osmotic fragility (OF) tests and measurement of Zinc Protoporphyrin (ZPP) to detect and distinguish HbE and ß-thalassaemia traits from iron deficiency. We compare findings with current screening practice in Sri Lanka that relies on the identification of low mean red cell volume and/or mean red cell hemoglobin for this purpose. Methods: Between November 2017 and May 2018, we undertook a cross-sectional survey of secondary school students in Gampaha district, Sri Lanka. The THALCON-DCIP and OF tests were compared to capillary electrophoresis (CE), used as a gold standard to detect haemoglobinopathies. ZPP was measured in whole blood. Plasma ferritin and C-reactive protein (CRP) were measured in students with a raised ZPP concentration. Results: We collected venous blood samples from 1,324/1,332 (99.4%) students. The median age of the students was 17 (IQR 16-18) years, all were Sinhalese and 814/1,324 (61.5%) were female. CE identified 3 students with HbE trait and 26 students with ß-thalassaemia trait. The THALCON-DCIP test was positive only in the 3 students with HbE (sensitivity 100%, 95% CI 29.2-100.0; specificity 100%, 95% CI 99.7-100.0). The THALCON-OF test identified all 26 students with ß-thalassaemia trait (sensitivity = 100%, 95% CI 86.8-100.0) and 287 students with a normal CE result (specificity = 77.9%; 95% CI 75.5-80.1). It was also positive in 2/3 (66.7%) students with HbE trait. Iron deficiency (ZPP > 70 µmol/mol heme) was present in 118/1,240 (9.5%) students with a normal hemoglobin genotype, all of whom had plasma ferritin <15 ng/ml and CRP <5 mg/L. Conclusion: This one-stop approach offers reliable and affordable population screening for both haemoglobinopathy traits and iron deficiency in resource-limited settings where these conditions are common and ensures that iron supplements are targeted only to those who require them. Further work is warranted to refine the OF test to reduce the number of false positive results.