Screening of malaria infections in human blood samples with varying parasite densities and anaemic conditions using AI-Powered mid-infrared spectroscopy.

BACKGROUND: Effective testing for malaria, including the detection of infections at very low densities, is vital for the successful elimination of the disease. Unfortunately, existing methods are either inexpensive but poorly sensitive or sensitive but costly. Recent studies have shown that mid-infrared spectroscopy coupled with machine learning (MIRs-ML) has potential for rapidly detecting malaria infections but requires further evaluation on diverse samples representative of natural infections in endemic areas. The aim of this study was, therefore, to demonstrate a simple AI-powered, reagent-free, and user-friendly approach that uses mid-infrared spectra from dried blood spots to accurately detect malaria infections across varying parasite densities and anaemic conditions. METHODS: Plasmodium falciparum strains NF54 and FCR3 were cultured and mixed with blood from 70 malaria-free individuals to create various malaria parasitaemia and anaemic conditions. Blood dilutions produced three haematocrit ratios (50%, 25%, 12.5%) and five parasitaemia levels (6%, 0.1%, 0.002%, 0.00003%, 0%). Dried blood spots were prepared on Whatman™ filter papers and scanned using attenuated total reflection-Fourier Transform Infrared (ATR-FTIR) for machine-learning analysis. Three classifiers were trained on an 80%/20% split of 4655 spectra: (I) high contrast (6% parasitaemia vs. negative), (II) low contrast (0.00003% vs. negative) and (III) all concentrations (all positive levels vs. negative). The classifiers were validated with unseen datasets to detect malaria at various parasitaemia levels and anaemic conditions. Additionally, these classifiers were tested on samples from a population survey in malaria-endemic villages of southeastern Tanzania. RESULTS: The AI classifiers attained over 90% accuracy in detecting malaria infections as low as one parasite per microlitre of blood, a sensitivity unattainable by conventional RDTs and microscopy. These laboratory-developed classifiers seamlessly transitioned to field applicability, achieving over 80% accuracy in predicting natural P. falciparum infections in blood samples collected during the field survey. Crucially, the performance remained unaffected by various levels of anaemia, a common complication in malaria patients. CONCLUSION: These findings suggest that the AI-driven mid-infrared spectroscopy approach holds promise as a simplified, sensitive and cost-effective method for malaria screening, consistently performing well despite variations in parasite densities and anaemic conditions. The technique simply involves scanning dried blood spots with a desktop mid-infrared scanner and analysing the spectra using pre-trained AI classifiers, making it readily adaptable to field conditions in low-resource settings. In this study, the approach was successfully adapted to field use, effectively predicting natural malaria infections in blood samples from a population-level survey in Tanzania. With additional field trials and validation, this technique could significantly enhance malaria surveillance and contribute to accelerating malaria elimination efforts.

A semi-field evaluation in Thailand of the use of human landing catches (HLC) versus human-baited double net trap (HDN) for assessing the impact of a volatile pyrethroid spatial repellent and pyrethroid-treated clothing on Anopheles minimus landing.

BACKGROUND: The mosquito landing rate measured by human landing catches (HLC) is the conventional endpoint used to evaluate the impact of vector control interventions on human-vector exposure. Non-exposure based alternatives to the HLC are desirable to minimize the risk of accidental mosquito bites. One such alternative is the human-baited double net trap (HDN), but the estimated personal protection of interventions using the HDN has not been compared to the efficacy estimated using HLC. This semi-field study in Sai Yok District, Kanchanaburi Province, Thailand, evaluates the performance of the HLC and the HDN for estimating the effect on Anopheles minimus landing rates of two intervention types characterized by contrasting modes of action, a volatile pyrethroid spatial repellent (VSPR) and insecticide-treated clothing (ITC). METHODS: Two experiments to evaluate the protective efficacy of (1) a VPSR and (2) ITC, were performed. A block randomized cross-over design over 32 nights was carried out with both the HLC or HDN. Eight replicates per combination of collection method and intervention or control arm were conducted. For each replicate, 100 An. minimus were released and were collected for 6 h. The odds ratio (OR) of the released An. minimus mosquitoes landing in the intervention compared to the control arm was estimated using logistic regression, including collection method, treatment, and experimental day as fixed effects. RESULTS: For the VPSR, the protective efficacy was similar for the two methods: 99.3%, 95% CI (99.5-99.0) when measured by HLC, and 100% (100, Inf) when measured by HDN where no mosquitoes were caught (interaction test p = 0.99). For the ITC, the protective efficacy was 70% (60-77%) measured by HLC but there was no evidence of protection when measured by HDN [4% increase (15-27%)] (interaction test p < 0.001). CONCLUSIONS: Interactions between mosquitoes, bite prevention tools and the sampling method may impact the estimated intervention protective efficacy. Consequently, the sampling method must be considered when evaluating these interventions. The HDN is a valid alternative trapping method (relative to the HLC) for evaluating the impact of bite prevention methods that affect mosquito behaviour at a distance (e.g. VPSR), but not for interventions that operate through tarsal contact (e.g., ITC).

Multi-country evaluation of the durability of pyrethroid plus piperonyl-butoxide insecticide-treated nets: study protocol.

BACKGROUND: Mass distributions of long-lasting insecticidal nets (LLINs) have contributed to large reductions in the malaria burden. However, this success is in jeopardy due in part to the increasing pyrethroid-resistant mosquito population as well as low LLINs coverage in various areas because the lifespan of LLINs is often shorter than the interval between replenishment campaigns. New insecticide-treated nets (ITNs) containing pyrethroid and piperonyl-butoxide (PBO) have shown a greater reduction in the incidence of malaria than pyrethroid LLINs in areas with pyrethroid-resistant mosquitoes. However, the durability (attrition, bio-efficacy, physical integrity and chemical retainment) of pyrethroid-PBO ITNs under operational settings has not been fully characterized. This study will measure the durability of pyrethroid-PBO ITNs to assess whether they meet the World Health Organization (WHO) three years of operational performance criteria required to be categorized as "long-lasting". METHODS: A prospective household randomized controlled trial will be conducted simultaneously in Tanzania, India and Côte d'Ivoire to estimate the field durability of three pyrethroid-PBO ITNs (Veeralin®, Tsara® Boost, and Olyset® Plus) compared to a pyrethroid LLIN: MAGNet®. Durability monitoring will be conducted up to 36 months post-distribution and median survival in months will be calculated. The proportion of ITNs: (1) lost (attrition), (2) physical integrity, (3) resistance to damage score, (4) meeting WHO bio-efficacy (≥ 95% knockdown after 1 h or ≥ 80% mortality after 24 h for WHO cone bioassay, or ≥ 90% blood-feeding inhibition or ≥ 80% mortality after 24 h for WHO Tunnel tests) criteria against laboratory-reared resistant and susceptible mosquitoes, and insecticidal persistence over time will be estimated. The non-inferiority of Veeralin® and Tsara® Boost to the first-in-class, Olyset® Plus will additionally be assessed for mortality, and the equivalence of 20 times washed ITNs compared to field aged ITNs will be assessed for mortality and blood-feeding inhibition endpoints in the Ifakara Ambient Chamber Test, Tanzania. CONCLUSION: This will be the first large-scale prospective household randomized controlled trial of pyrethroid-PBO ITNs in three different countries in East Africa, West Africa and South Asia, simultaneously. The study will generate information on the replenishment intervals for PBO nets.

Comparison of cone bioassay estimates at two laboratories with different Anopheles mosquitoes for quality assurance of pyrethroid insecticide-treated nets.

BACKGROUND: Quality assurance (QA) of insecticide-treated nets (ITNs) delivered to malaria-endemic countries is conducted by measuring physiochemical parameters, but not bioefficacy against malaria mosquitoes. This study explored utility of cone bioassays for pre-delivery QA of pyrethroid ITNs to test the assumption that cone bioassays are consistent across locations, mosquito strains, and laboratories. METHODS: Double-blinded bioassays were conducted on twenty unused pyrethroid ITNs of 4 brands (100 nets, 5 subsamples per net) that had been delivered for mass distribution in Papua New Guinea (PNG) having passed predelivery inspections. Cone bioassays were performed on the same net pieces following World Health Organization (WHO) guidelines at the PNG Institute of Medical Research (PNGIMR) using pyrethroid susceptible Anopheles farauti sensu stricto (s.s.) and at Ifakara Health Institute (IHI), Tanzania using pyrethroid susceptible Anopheles gambiae s.s. Additionally, WHO tunnel tests were conducted at IHI on ITNs that did not meet cone bioefficacy thresholds. Results from IHI and PNGIMR were compared using Spearman's Rank correlation, Bland-Altman (BA) analysis and analysis of agreement. Literature review on the use of cone bioassays for unused pyrethroid ITNs testing was conducted. RESULTS: In cone bioassays, 13/20 nets (65%) at IHI and 8/20 (40%) at PNGIMR met WHO bioefficacy criteria. All nets met WHO bioefficacy criteria on combined cone/tunnel tests at IHI. Results from IHI and PNGIMR correlated on 60-min knockdown (KD60) (rs = 0.6,p = 0.002,n = 20) and 24-h mortality (M24) (rs = 0.9,p < 0.0001,n = 20) but BA showed systematic bias between the results. Of the 5 nets with discrepant result between IHI and PNGIMR, three had confidence intervals overlapping the 80% mortality threshold, with averages within 1-3% of the threshold. Including these as a pass, the agreement between the results to predict ITN failure was good with kappa = 0.79 (0.53-1.00) and 90% accuracy. CONCLUSIONS: Based on these study findings, the WHO cone bioassay is a reproducible bioassay for ITNs with > 80% M24, and for all ITNs provided inherent stochastic variation and systematic bias are accounted for. The literature review confirms that WHO cone bioassay bioefficacy criteria have been previously achieved by all pyrethroid ITNs (unwashed), without the need for additional tunnel tests. The 80% M24 threshold remains the most reliable indicator of pyrethroid ITN quality using pyrethroid susceptible mosquitoes. In the absence of alternative tests, cone bioassays could be used as part of pre-delivery QA.

"In starvation, a bone can also be meat": a mixed methods evaluation of factors associated with discarding of long-lasting insecticidal nets in Bagamoyo, Tanzania.

BACKGROUND: Between 2000 and 2019, more than 1.8 billion long-lasting insecticidal nets (LLINs) were distributed in Africa. While the insecticidal durability of LLINs is around 3 years, nets are commonly discarded 2 years post distribution. This study investigated the factors associated with the decision of users to discard LLINs. METHODS: A mixed-method sequential explanatory approach using a structured questionnaire followed by focus group discussions (FGDs) to collect information on experiences, views, reasons, how and when LLINs are discarded. Out of 6,526 households that responded to the questionnaire of LLINs durability trial, 160 households were randomly selected from the households in four villages in Bagamoyo Tanzania for FGDs but only 155 households participated in the FGDs. Five of the household representatives couldn't participate due to unexpected circumstances. A total of sixteen FGDs each comprising of 8-10 adults were conducted; older women (40-60 years), older men (40-60 years), younger women (18-39 years), younger men (18-39 years). During the FGDs, participants visually inspected seven samples of LLINs that were "too-torn" based on Proportionate Hole Index recommended by the World Health Organization (WHO) guidelines on LLIN testing, the nets were brought to the discussion and participants had to determine if such LLINs were to be kept or discarded. The study assessed responses from the same participants that attended FGD and also responded to the structured questionnaire, 117 participants fulfilled the criteria, thus data from only 117 participants are analysed in this study. RESULTS: In FGDs, integrity of LLIN influenced the decision to discard or keep a net. Those of older age, women, and householders with lower income were more likely to classify a WHO "too-torn" net as "good". The common methods used to discard LLINs were burning and burying. The findings were seen in the quantitative analysis. For every additional hole, the odds of discarding a WHO "too-torn" LLIN increased [OR = 1.05 (95%CI (1.04-1.07)), p < 0.001]. Younger age group [OR = 4.97 (95%CI (3.25-7.32)), p < 0.001], male-headed households [OR = 6.85 (95%CI (4.44 -10.59)), p < 0.001], and wealthy households [OR = 3.88 (95%CI (2.33-6.46)), p < 0.001] were more likely to discard LLINs. CONCLUSION: Integrity of LLIN was the main determinant for discarding or keeping LLINs and the decision to discard the net is associated with socioeconomic status of the household, and the age and gender of respondents. WHO "too torn" nets are encouraged to be used instead of none until replacement, and disposal of nets should be based on recommendation.

The Centres for Disease Control light trap (CDC-LT) and the human decoy trap (HDT) compared to the human landing catch (HLC) for measuring Anopheles biting in rural Tanzania.

BACKGROUND: Vector mosquito biting intensity is an important measure to understand malaria transmission. Human landing catch (HLC) is an effective but labour-intensive, expensive, and potentially hazardous entomological surveillance tool. The Centres for Disease Control light trap (CDC-LT) and the human decoy trap (HDT) are exposure-free alternatives. This study compared the CDC-LT and HDT against HLC for measuring Anopheles biting in rural Tanzania and assessed their suitability as HLC proxies. METHODS: Indoor mosquito surveys using HLC and CDC-LT and outdoor surveys using HLC and HDT were conducted in 2017 and in 2019 in Ulanga, Tanzania in 19 villages, with one trap/house/night. Species composition, sporozoite rates and density/trap/night were compared. Aggregating the data by village and month, the Bland-Altman approach was used to assess agreement between trap types. RESULTS: Overall, 66,807 Anopheles funestus and 14,606 Anopheles arabiensis adult females were caught with 6,013 CDC-LT, 339 indoor-HLC, 136 HDT and 195 outdoor-HLC collections. Indoors, CDC-LT caught fewer An. arabiensis (Adjusted rate ratio [Adj.RR] = 0.35, 95% confidence interval [CI]: 0.27-0.46, p < 0.001) and An. funestus (Adj.RR = 0.63, 95%CI: 0.51-0.79, p < 0.001) than HLC per trap/night. Outdoors, HDT caught fewer An. arabiensis (Adj.RR = 0.04, 95%CI: 0.01-0.14, p < 0.001) and An. funestus (Adj.RR = 0.10, 95%CI: 0.07-0.15, p < 0.001) than HLC. The bias and variability in number of mosquitoes caught by the different traps were dependent on mosquito densities. The relative efficacies of both CDC-LT and HDT in comparison to HLC declined with increased mosquito abundance. The variability in the ratios was substantial for low HLC counts and decreased as mosquito abundance increased. The numbers of sporozoite positive mosquitoes were low for all traps. CONCLUSIONS: CDC-LT can be suitable for comparing mosquito populations between study arms or over time if accuracy in the absolute biting rate, compared to HLC, is not required. CDC-LT is useful for estimating sporozoite rates because large numbers of traps can be deployed to collect adequate mosquito samples. The present design of the HDT is not amenable for use in large-scale entomological surveys. Use of HLC remains important for estimating human exposure to mosquitoes as part of estimating the entomological inoculation rate (EIR).

Mosquito feeding behavior and how it influences residual malaria transmission across Africa.

The antimalarial efficacy of the most important vector control interventions-long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS)-primarily protect against mosquitoes' biting people when they are in bed and indoors. Mosquito bites taken outside of these times contribute to residual transmission which determines the maximum effectiveness of current malaria prevention. The likelihood mosquitoes feed outside the time of day when LLINs and IRS can protect people is poorly understood, and the proportion of bites received outdoors may be higher after prolonged vector control. A systematic review of mosquito and human behavior is used to quantify and estimate the public health impact of outdoor biting across Africa. On average 79% of bites by the major malaria vectors occur during the time when people are in bed. This estimate is substantially lower than previous predictions, with results suggesting a nearly 10% lower proportion of bites taken at the time when people are beneath LLINs since the year 2000. Across Africa, this higher outdoor transmission is predicted to result in an estimated 10.6 million additional malaria cases annually if universal LLIN and IRS coverage was achieved. Higher outdoor biting diminishes the cases of malaria averted by vector control. This reduction in LLIN effectiveness appears to be exacerbated in areas where mosquito populations are resistant to insecticides used in bed nets, but no association was found between physiological resistance and outdoor biting. Substantial spatial heterogeneity in mosquito biting behavior between communities could contribute to differences in effectiveness of malaria control across Africa.

From the factory to the field: considerations of product characteristics for insecticide-treated net (ITN) bioefficacy testing.

BACKGROUND: Insecticide-treated nets (ITNs) undergo a series of tests to obtain listing by World Health Organization (WHO) Prequalification. These tests characterize the bioefficacy, physical and chemical properties of the ITN. ITN procurers assume that product specifications relate to product performance. Here, ITN test methods and their underlying assumptions are discussed from the perspective of the ITN manufacturing process and product characteristics. METHODS: Data were extracted from WHO Pesticide Evaluation Scheme (WHOPES) meeting reports from 2003 to 2017, supplemented with additional chemical analysis to critically evaluate ITNs bioassays with a focus on sampling, washing and wash resistance, and bioefficacy testing. Production methods for ITNs and their impact on testing outcomes are described. RESULTS AND RECOMMENDATIONS: ITNs are not homogenous products. They vary within panels and between the sides and the roof. Running tests of wash resistance using a before/after tests on the same sample or band within a net reduces test variability. As mosquitoes frequently interact with ITN roofs, additional sampling of the roof when evaluating ITNs is advisable because in nets where roof and sides are of the same material, the contribution of roof sample (20-25%) to the average is less than the tolerance for the specification (25%). Mosquito mortality data cannot be reliably used to evaluate net surface concentration to determine regeneration time (RT) and resistance to washing as nets may regenerate beyond the insecticide concentrations needed to kill 100% of susceptible mosquitoes. Chemical assays to quantify surface concentration are needed. The Wash Resistance Index (WRI) averaged over the first four washes is only informative if the product has a log linear loss rate of insecticide. Using a WRI that excludes the first wash off gives more reliable results. Storage conditions used for product specifications are lower than those encountered under product shipping and storage that may exceed 50 °C, and should be reconsidered. Operational monitoring of new ITNs and linking observed product performance, such as bioefficacy after 2 or 3 years of use, with product characteristics, such as WRI, will aid the development of more robust test methods and product specifications for new products coming to market.

Single blinded semi-field evaluation of MAÏA® topical repellent ointment compared to unformulated 20% DEET against Anopheles gambiae, Anopheles arabiensis and Aedes aegypti in Tanzania.

BACKGROUND: N,N-Diethyl-3-methylbenzamide (DEET) topical mosquito repellents are effective personal protection tools. However, DEET-based repellents tend to have low consumer acceptability because they are cosmetically unappealing. More attractive formulations are needed to encourage regular user compliance. This study evaluated the protective efficacy and protection duration of a new topical repellent ointment containing 15% DEET, MAÏA® compared to 20% DEET in ethanol using malaria and dengue mosquito vectors in Bagamoyo Tanzania. METHODS: Fully balanced 3 × 3 Latin square design studies were conducted in large semi-field chambers using laboratory strains of Anopheles gambiae sensu stricto, Anopheles arabiensis and Aedes aegypti. Human volunteers applied either MAÏA® ointment, 20% DEET or ethanol to their lower limbs 6 h before the start of tests. Approximately 100 mosquitoes per strain per replicate were released inside each chamber, with 25 mosquitoes released at regular intervals during the collection period to maintain adequate biting pressure throughout the test. Volunteers recaptured mosquitoes landing on their lower limbs for 6 h over a period of 6 to 12-h post-application of repellents. Data analysis was conducted using mixed-effects logistic regression. RESULTS: The protective efficacy of MAÏA® and 20% DEET was not statistically different for each of the mosquito strains: 95.9% vs. 97.4% against An. gambiae (OR = 1.53 [95% CI 0.93-2.51] p = 0.091); 96.8% vs 97.2% against An. arabiensis (OR = 1.08 [95% CI 0.66-1.77] p = 0.757); 93.1% vs 94.6% against Ae. aegypti (OR = 0.76 [95% CI 0.20-2.80] p = 0.675). Average complete protection time (CPT) in minutes of MAÏA® and that of DEET was similar for each of the mosquito strains: 571.6 min (95% CI 558.3-584.8) vs 575.0 min (95% CI 562.1-587.9) against An. gambiae; 585.6 min (95% CI 571.4-599.8) vs 580.9 min (95% CI 571.1-590.7) against An. arabiensis; 444.1 min (95% CI 401.8-486.5) vs 436.9 min (95% CI 405.2-468.5) against Ae. aegypti. CONCLUSIONS: MAÏA® repellent ointment provides complete protection for 9 h against both An. gambiae and An. arabiensis, and 7 h against Ae. aegypti similar to 20% DEET (in ethanol). MAÏA® repellent ointment can be recommended as a tool for prevention against outdoor biting mosquitoes in tropical locations where the majority of the people spend an ample time outdoor before going to bed.

The consequences of declining population access to insecticide-treated nets (ITNs) on net use patterns and physical degradation of nets after 22 months of ownership.

BACKGROUND: As insecticide-treated nets (ITNs) wear out and are disposed, some household members are prioritized to use remaining ITNs. This study assessed how nets are allocated within households to individuals of different age categories as ITNs are lost or damaged and as new ITNs are obtained. The study also explored how ITN allocation affects ITN durability. METHODS: A cross-sectional household survey and ITN durability study was conducted among 2,875 households across Tanzania to determine the proportion of nets that remain protective (serviceable) 22 months after net distribution aiming for universal coverage. Allocation of study nets within houses, and re-allocation of ITNs when new universal replacement campaign (URC) nets arrived in study households in Musoma District, was also assessed. RESULTS: Some 57.0% (95% CI 53.9-60.1%) of households had sufficient ITNs for every household member, while 84.4% (95% CI 82.4-86.4%) of the population had access to an ITN within their household (assuming 1 net covers every 2 members). In households with sufficient nets, 77.5% of members slept under ITNs. In households without sufficient nets, pregnant women (54.6%), children < 5 years (45.8%) and adults (42.1%) were prioritized, with fewer school-age children 5-14 years (35.9%), youths 15-24 years (28.1%) and seniors > 65 years (32.6%) sleeping under ITNs. Crowding ([Formula: see text] 3 people sleeping under nets) was twice as common among people residing in houses without sufficient nets for all age groups, apart from children < 5. Nets were less likely to be serviceable if: [Formula: see text] 3 people slept under them (OR 0.50 (95% CI 0.40-0.63)), or if nets were used by school-age children (OR 0.72 (95% CI 0.56-0.93)), or if the net product was Olyset®. One month after the URC, only 23.6% (95% CI 16.7-30.6%) of the population had access to a URC ITN in Musoma district. Householders in Musoma district continued the use of old ITNs even with the arrival of new URC nets. CONCLUSION: Users determined the useful life of ITNs and prioritized pregnant women and children < 5 to serviceable ITNs. When household net access declines, users adjust by crowding under remaining nets, which further reduces ITN lifespan. School-age children that commonly harbour gametocytes that mediate malaria transmission are compelled to sleep under unserviceable nets, crowd under nets or remain uncovered. However, they were accommodated by the arrival of new nets. More frequent ITN delivery through the school net programme in combination with mass distribution campaigns is essential to maximize ITN effectiveness.

Comparative functional survival and equivalent annual cost of 3 long-lasting insecticidal net (LLIN) products in Tanzania: A randomised trial with 3-year follow up.

BACKGROUND: Two billion long-lasting insecticidal nets (LLINs) have been procured for malaria control. A functional LLIN is one that is present, is in good physical condition, and remains insecticidal, thereby providing protection against vector-borne diseases through preventing bites and killing disease vectors. The World Health Organization (WHO) prequalifies LLINs that remain adequately insecticidal 3 years after deployment. Therefore, institutional buyers often assume that prequalified LLINs are functionally identical with a 3-year lifespan. We measured the lifespans of 3 LLIN products, and calculated their cost per year of functional life, to demonstrate the economic and public health importance of procuring the most cost-effective LLIN product based on its lifespan. METHODS AND FINDINGS: A randomised double-blinded trial of 3 pyrethroid LLIN products (10,571 nets in total) was conducted at 3 follow-up points: 10 months (August-October 2014), 22 months (August-October 2015), and 36 months (October-December 2016) among 3,393 households in Tanzania using WHO-recommended methods. Primary outcome was LLIN functional survival (LLIN present and in serviceable condition). Secondary outcomes were (1) bioefficacy and chemical content (residual insecticidal activity) and (2) protective efficacy for volunteers sleeping under the LLINs (bite reduction and mosquitoes killed). Median LLIN functional survival was significantly different between the 3 net products (p = 0.001): 2.0 years (95% CI 1.7-2.3) for Olyset, 2.5 years (95% CI 2.2-2.8) for PermaNet 2.0 (hazard ratio [HR] 0.73 [95% CI 0.64-0.85], p = 0.001), and 2.6 years (95% CI 2.3-2.8) for NetProtect (HR = 0.70 [95% CI 0.62-0.77], p < 0.001). Functional survival was affected by accumulation of holes, leading to users discarding nets. Protective efficacy also significantly differed between products as they aged. Equivalent annual cost varied between US$1.2 (95% CI $1.1-$1.4) and US$1.5 (95% CI $1.3-$1.7), assuming that each net was priced identically at US$3. The 2 longer-lived nets (PermaNet and NetProtect) were 20% cheaper than the shorter-lived product (Olyset). The trial was limited to only the most widely sold LLINs in Tanzania. Functional survival varies by country, so the single country setting is a limitation. CONCLUSIONS: These results suggest that LLIN functional survival is less than 3 years and differs substantially between products, and these differences strongly influence LLIN value for money. LLIN tendering processes should consider local expectations of cost per year of functional life and not unit price. As new LLIN products come on the market, especially those with new insecticides, it will be imperative to monitor their comparative durability to ensure that the most cost-effective products are procured for malaria control.

Engineered Fn3 protein has targeted therapeutic effect on mesothelin-expressing cancer cells and increases tumor cell sensitivity to chemotherapy.

Mesothelin is a protein expressed at high levels on the cell surface in a variety of cancers, with limited expression in healthy tissues. The presence of mesothelin on tumor tissue correlates with increased invasion and metastasis, and resistance to traditional chemotherapies, through mechanisms that remain poorly understood. Molecules that specifically recognize mesothelin and interrupt its contribution to tumor progression have significant potential for targeted therapy and targeted drug delivery applications. A number of mesothelin-targeting therapies are in preclinical and clinical development, although none are currently approved for routine clinical use. In this work, we report the development of a mesothelin-targeting protein based on the fibronectin type-III non-antibody protein scaffold, which offers opportunities for applications where antibodies have limitations. We engineered protein variants that bind mesothelin with high affinity and selectively initiate apoptosis in tumor cells expressing mesothelin. Interestingly, apoptosis does not occur through a caspase-mediated pathway and does not require downregulation of cell-surface mesothelin, suggesting a currently unknown pathway through which mesothelin contributes to cancer progression. Importantly, simultaneous treatment with mesothelin-binding protein and chemotherapeutic mitomycin C had a greater cytotoxic effect on mesothelin-positive cells compared to either molecule alone, underscoring the potential for combination therapy including biologics targeting mesothelin.

Long-lasting insecticidal nets retain bio-efficacy after 5 years of storage: implications for malaria control programmes.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the most sustainable and effective malaria control tool currently available. Global targets are for 80% of the population living in malaria endemic areas to have access to (own) and use a LLIN. However, current access to LLINs in endemic areas is 56% due to system inefficiencies and budget limitations. Thus, cost-effective approaches to maximize access to effective LLINs in endemic areas are required. This study evaluated whether LLINs that had been stored for 5 years under manufacturer's recommended conditions may be optimally effective against Anopheles mosquitoes, to inform malaria control programmes and governments on the periods over which LLINs may be stored between distributions, in an effort to maximize use of available LLINs. METHODS: Standard World Health Organization (WHO) bioassays (cone and tunnel test) were used to evaluate the bio-efficacy and wash resistance of Olyset® and DawaPlus® 2.0 (rebranded Tsara® Soft) LLINs after 5 years of storage at 25 °C to 33.4 °C and 40% to 100% relative humidity. In addition, a small scale Ifakara Ambient Chamber test (I-ACT) was conducted to compare the bio-efficacy of one long stored LLINs to one new LLIN of the same brand, washed or unwashed. LLINs were evaluated using laboratory reared fully susceptible Anopheles gambiae sensu stricto (s.s.) (Ifakara strain) and pyrethroid resistant Anopheles arabiensis (Kingani strain). RESULTS: After 5 years of storage, both unwashed and washed, Olyset® and DawaPlus® 2.0 (Tsara® Soft) LLINs passed WHO bio-efficacy criteria on knockdown (KD60) ≥ 95%, 24-h mortality ≥ 80% and ≥ 90% blood-feeding inhibition in WHO assays against susceptible An. gambiae s.s. DawaPlus® 2.0 LLINs also passed combined WHO bioassay criteria against resistant An. arabiensis. Confirmatory I-ACT tests using whole nets demonstrated that long-stored LLINs showed higher efficacy than new LLINs on both feeding inhibition and mortality endpoints against resistant strains. CONCLUSIONS: Even after long-term storage of around 5 years, both Olyset® and DawaPlus® 2.0 LLINs remain efficacious against susceptible Anopheles mosquitoes at optimal storage range of 25 °C to 33.4 °C for temperature and 40% to 100% relative humidity measured by standard WHO methods. DawaPlus® 2.0 (Tsara® Soft) remained efficacious against resistant strain.

Protein-Polymer Conjugates Synthesized Using Water-Soluble Azlactone-Functionalized Polymers Enable Receptor-Specific Cellular Uptake toward Targeted Drug Delivery.

Conjugation of proteins to drug-loaded polymeric structures is an attractive strategy for facilitating target-specific drug delivery for a variety of clinical needs. Polymers currently available for conjugation to proteins generally have limited chemical versatility for subsequent drug loading. Many polymers that do have chemical functionality useful for drug loading are often insoluble in water, making it difficult to synthesize functional protein-polymer conjugates for targeted drug delivery. In this work, we demonstrate that reactive, azlactone-functionalized polymers can be grafted to proteins, conjugated to a small-molecule fluorophore, and subsequently internalized into cells in a receptor-specific manner. Poly(2-vinyl-4,4-dimethylazlactone), synthesized using reversible addition-fragmentation chain transfer polymerization, was modified post-polymerization with substoichiometric equivalents of triethylene glycol monomethyl ether to yield reactive water-soluble, azlactone-functionalized copolymers. These reactive polymers were then conjugated to proteins holo-transferrin and ovotransferrin. Protein gel analysis verified successful conjugation of proteins to polymer, and protein-polymer conjugates were subsequently purified from unreacted proteins and polymers using size exclusion chromatography. Internalization experiments using a breast cancer cell line that overexpresses the transferrin receptor on its surface showed that the holo-transferrin-polymer conjugate was successfully internalized by cells in a mechanism consistent with receptor-mediated endocytosis. Internalization of protein-polymer conjugate demonstrated that the protein ligand maintained its overall structure and function following conjugation to polymer. Our approach to protein-polymer conjugate synthesis offers a simple, tailorable strategy for preparing bioconjugates of interest for a broad range of biomedical applications.

The development and evaluation of a self-marking unit to estimate malaria vector survival and dispersal distance.

BACKGROUND: A clear understanding of mosquito biology is fundamental to the control efforts of mosquito-borne diseases such as malaria. Mosquito mark-release-recapture (MMRR) experiments are a popular method of measuring the survival and dispersal of disease vectors; however, examples with African malaria vectors are limited. Ethical and technical difficulties involved in carrying out MMRR studies may have held back research in this area and, therefore, a device that marks mosquitoes as they emerge from breeding sites was developed and evaluated to overcome the problems of MMRR. METHODS: A modified self-marking unit that marks mosquitoes with fluorescent pigment as they emerge from their breeding site was developed based on a previous design for Culex mosquitoes. The self-marking unit was first evaluated under semi-field conditions with laboratory-reared Anopheles arabiensis to determine the marking success and impact on mosquito survival. Subsequently, a field evaluation of MMRR was conducted in Yombo village, Tanzania, to examine the feasibility of the system. RESULTS: During the semi-field evaluation the self-marking units successfully marked 86% of emerging mosquitoes and there was no effect of fluorescent marker on mosquito survival. The unit successfully marked wild male and female Anopheles gambiae sensu lato (s.l.) in sufficiently large numbers to justify its use in MMRR studies. The estimated daily survival probability of An. gambiae s.l. was 0.87 (95% CI 0.69-1.10) and mean dispersal distance was 579 m (95% CI 521-636 m). CONCLUSIONS: This study demonstrates the successful use of a self-marking device in an MMRR study with African malaria vectors. This method may be useful in investigating population structure and dispersal of mosquitoes for deployment and evaluation of future vector control tools, such as gene drive, and to better parameterize mathematical models.

Protecting migratory farmers in rural Tanzania using eave ribbons treated with the spatial mosquito repellent, transfluthrin.

BACKGROUND: Many subsistence farmers in rural southeastern Tanzania regularly relocate to distant farms in river valleys to tend to crops for several weeks or months each year. While there, they live in makeshift semi-open structures, usually far from organized health systems and where insecticide-treated nets (ITNs) do not provide adequate protection. This study evaluated the potential of a recently developed technology, eave ribbons treated with the spatial repellent transfluthrin, for protecting migratory rice farmers in rural southeastern Tanzania against indoor-biting and outdoor-biting mosquitoes. METHODS: In the first test, eave ribbons (0.1 m × 24 m each) treated with 1.5% transfluthrin solution were compared to untreated ribbons in 24 randomly selected huts in three migratory communities over 48 nights. Host-seeking mosquitoes indoors and outdoors were monitored nightly (18.00-07.00 h) using CDC light traps and CO2-baited BG malaria traps, respectively. The second test compared efficacies of eave ribbons treated with 1.5% or 2.5% transfluthrin in 12 huts over 21 nights. Finally, 286 farmers were interviewed to assess perceptions about eave ribbons, and their willingness to pay for them. RESULTS: In the two experiments, when treated eave ribbons were applied, the reduction in indoor densities ranged from 56 to 77% for Anopheles arabiensis, 36 to 60% for Anopheles funestus, 72 to 84% for Culex, and 80 to 98% for Mansonia compared to untreated ribbons. Reduction in outdoor densities was 38 to 77% against An. arabiensis, 36 to 64% against An. funestus, 63 to 88% against Culex, and 47 to 98% against Mansonia. There was no difference in protection between the two transfluthrin doses. In the survey, 58% of participants perceived the ribbons to be effective in reducing mosquito bites. Ninety per cent were willing to pay for the ribbons, the majority of whom were willing to pay but less than US$2.17 (5000 TZS), one-third of the current prototype cost. CONCLUSIONS: Transfluthrin-treated eave ribbons can protect migratory rice farmers, living in semi-open makeshift houses in remote farms, against indoor-biting and outdoor-biting mosquitoes. The technology is acceptable to users and could potentially complement ITNs. Further studies should investigate durability and epidemiological impact of eave ribbons, and the opportunities for improving affordability to users.

Comparing the new Ifakara Ambient Chamber Test with WHO cone and tunnel tests for bioefficacy and non-inferiority testing of insecticide-treated nets.

BACKGROUND: Insecticide-treated net (ITN) durability, measured through physical integrity and bioefficacy, must be accurately assessed in order to plan the timely replacement of worn out nets and guide procurement of longer-lasting, cost-effective nets. World Health Organization (WHO) guidance advises that new intervention class ITNs be assessed 3 years after distribution, in experimental huts. In order to obtain information on whole-net efficacy cost-effectively and with adequate replication, a new bioassay, the Ifakara Ambient Chamber Test (I-ACT), a semi-field whole net assay baited with human host, was compared to established WHO durability testing methods. METHODS: Two experiments were conducted using pyrethroid-susceptible female adult Anopheles gambiae sensu stricto comparing bioefficacy of Olyset®, PermaNet® 2.0 and NetProtect® evaluated by I-ACT and WHO cone and tunnel tests. In total, 432 nets (144/brand) were evaluated using I-ACT and cone test. Olyset® nets (132/144) that did not meet the WHO cone test threshold criteria (≥ 80% mortality or ≥ 95% knockdown) were evaluated using tunnel tests with threshold criteria of ≥ 80% mortality or ≥ 90% feeding inhibition for WHO tunnel and I-ACT. Pass rate of nets tested by WHO combined standard WHO bioassays (cone/tunnel tests) was compared to pass in I-ACT only by net brand and time after distribution. RESULTS: Overall, more nets passed WHO threshold criteria when tested with I-ACT than with standard WHO bioassays 92% vs 69%, (OR: 4.1, 95% CI 3.5-4.7, p < 0.0001). The proportion of Olyset® nets that passed differed if WHO 2005 or WHO 2013 LN testing guidelines were followed: 77% vs 71%, respectively. Based on I-ACT results, PermaNet® 2.0 and NetProtect® demonstrated superior mortality and non-inferior feeding inhibition to Olyset® over 3 years of field use in Tanzania. CONCLUSION: Ifakara Ambient Chamber Test may have use for durability studies and non-inferiority testing of new ITN products. It measures composite bioefficacy and physical integrity with both mortality and feeding inhibition endpoints, using fewer mosquitoes than standard WHO bioassays (cone and tunnel tests). The I-ACT is a high-throughput assay to evaluate ITN products that work through either contact toxicity or feeding inhibition. I-ACT allows mosquitoes to interact with a host sleeping underneath a net as encountered in the field, without risk to human participants.

Mosquito repellents for malaria prevention.

BACKGROUND: Malaria is an important cause of illness and death across endemic regions. Considerable success against malaria has been achieved within the past decade mainly through long-lasting insecticide-treated nets (LLINs). However, elimination of the disease is proving difficult as current control methods do not protect against mosquitoes biting outdoors and when people are active. Repellents may provide a personal protection solution during these times. OBJECTIVES: To assess the impact of topical repellents, insecticide-treated clothing, and spatial repellents on malaria transmission. SEARCH METHODS: We searched the following databases up to 26 June 2017: the Cochrane Infectious Diseases Group Specialized Register; the Central Register of Controlled Trials (CENTRAL), published in the Cochrane Library; MEDLINE; Embase; US AFPMB; CAB Abstracts; and LILACS. We also searched trial registration platforms and conference proceedings; and contacted organizations and companies for ongoing and unpublished trials. SELECTION CRITERIA: We included randomized controlled trials (RCTs) and cluster-randomized controlled trials of topical repellents proven to repel mosquitoes; permethrin-treated clothing; and spatial repellents such as mosquito coils. We included trials that investigated the use of repellents with or without LLINs, referred to as insecticide-treated nets. DATA COLLECTION AND ANALYSIS: Two review authors independently reviewed trials for inclusion, extracted the data, and assessed the risk of bias. A third review author resolved any discrepancies. We analysed data by conducting meta-analysis and stratified by whether the trials had included LLINs. We combined results from cRCTs with individually RCTs by adjusting for clustering and presented results using forest plots. We used GRADE to assess the certainty of the evidence. MAIN RESULTS: Eight cRCTs and two RCTs met the inclusion criteria. Six trials investigated topical repellents, two trials investigated insecticide-treated clothing, and two trials investigated spatial repellents.Topical repellentsSix RCTS, five of them cluster-randomized, investigated topical repellents involving residents of malaria-endemic regions. Four trials used topical repellents in combination with nets, but two trials undertaken in displaced populations used topical repellents alone. It is unclear if topical repellents can prevent clinical malaria (RR 0.65, 95% CI 0.4 to 1.07, very low certainty evidence) or malaria infection (RR 0.84, 95% CI 0.64 to 1.12, low-certainty evidence) caused by P. falciparum. It is also unclear if there is any protection against clinical cases of P. vivax (RR 1.32, 95% CI 0.99 to 1.76, low-certainty evidence) or incidence of infections (RR 1.07, 95% CI 0.80 to 1.41, low-certainty evidence). Subgroup analysis of trials including insecticide-treated nets did not show a protective effect of topical repellents against malaria. Only two studies did not include insecticide-treated nets, and they measured different outcomes; one reported a protective effect against clinical cases of P. falciparum (RR 0.40, 95% CI 0.23 to 0.71); but the other study measured no protective effect against malaria infection incidence caused by either P. falciparum or P. vivax.Insecticide-treated clothingInsecticide-treated clothing were investigated in trials conducted in refugee camps in Pakistan and amongst military based in the Colombian Amazon. Neither study provided participants with insecticide-treated nets. In the absence of nets, treated clothing may reduce the incidence of clinical malaria caused by P. falciparum by approximately 50% (RR 0.49, 95% CI 0.29 to 0.83, low-certainty evidence) and P. vivax (RR 0.64, 95% CI 0.40 to 1.01, low-certainty evidence).Spatial repellentsTwo cluster-randomized RCTs investigated mosquito coils for malaria prevention. We do not know the effect of spatial repellents on malaria prevention (RR 0.24, 95% CI 0.03 to 1.72, very low certainty evidence). There was large heterogeneity between studies and one study had high risk of bias. AUTHORS' CONCLUSIONS: There is insufficient evidence to conclude topical or spatial repellents can prevent malaria. There is a need for better designed trials to generate higher certainty of evidence before well-informed recommendations can be made. Adherence to daily compliance remains a major limitation. Insecticide-treated clothing may reduce risk of malaria infection in the absence of insecticide-treated nets; further studies on insecticide-treated clothing in the general population should be done to broaden the applicability of the results.

Comparative performance of three experimental hut designs for measuring malaria vector responses to insecticides in Tanzania.

BACKGROUND: Experimental huts are simplified, standardized representations of human habitations that provide model systems to evaluate insecticides used in indoor residual spray (IRS) and long-lasting insecticidal nets (LLINs) to kill disease vectors. Hut volume, construction materials and size of entry points impact mosquito entry and exposure to insecticides. The performance of three standard experimental hut designs was compared to evaluate insecticide used in LLINs. METHODS: Field studies were conducted at the World Health Organization Pesticide Evaluation Scheme (WHOPES) testing site in Muheza, Tanzania. Three East African huts, three West African huts, and three Ifakara huts were compared using Olyset(®) and Permanet 2.0(®) versus untreated nets as a control. Outcomes measured were mortality, induced exophily (exit rate), blood feeding inhibition and deterrence (entry rate). Data were analysed using linear mixed effect regression and Bland-Altman comparison of paired differences. RESULTS: A total of 613 mosquitoes were collected in 36 nights, of which 13.5% were Anopheles gambiae sensu lato, 21% Anopheles funestus sensu stricto, 38% Mansonia species and 28% Culex species. Ifakara huts caught three times more mosquitoes than the East African and West African huts, while the West African huts caught significantly fewer mosquitoes than the other hut types. Mosquito densities were low, very little mosquito exit was measured in any of the huts with no measurable exophily caused by the use of either Olyset or Permanet. When the huts were directly compared, the West African huts measured greater exophily than other huts. As unholed nets were used in the experiments and few mosquitoes were captured, it was not possible to measure difference in feeding success either between treatments or hut types. In each of the hut types there was increased mortality when Permanet or Olyset were present inside the huts compared to the control, however this did not vary between the hut types. CONCLUSIONS: Both East African and Ifakara huts performed in a similar way although Ifakara huts allowed more mosquitoes to enter, increasing data power. The work convincingly demonstrates that the East African huts and Ifakara huts collect substantially more mosquitoes than the West African huts.

Durability of Olyset campaign nets distributed between 2009 and 2011 in eight districts of Tanzania.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the first line choice for malaria vector control in sub-Saharan Africa, with most countries adopting universal coverage campaigns. However, there is only limited information on LLIN durability under user conditions. Therefore, this study aimed to assess the durability of Olyset(®) LLINs distributed during campaigns between 2009 and 2011 in Tanzania. METHODS: A retrospective field survey was conducted in eight districts in Tanzania mainland to assess the durability of Olyset campaign nets. Household questionnaires were used to assess attrition, i.e. net loss. All nets remaining in households were collected. A sub-sample of 198 Olyset campaign nets was examined for bio-efficacy against Anopheles gambiae s.s. mosquitoes, permethrin content and physical integrity following standard World Health Organization (WHO) methods. RESULTS: Of 6067 campaign nets reported to have been received between 2009 and 2011, 35% (2145 nets) were no longer present. Most of those nets had been discarded (84%) mainly because they were too torn (94%). Of the 198 sub-sampled Olyset LLINs, 61% were still in serviceable physical condition sufficient to provide personal protection while 39% were in unserviceable physical condition according to WHO proportionate Hole Index (pHI). More than 96% (116/120) of nets in serviceable condition passed WHO bioefficacy criteria while all nets in unserviceable condition passed WHO bioefficacy criteria. Overall mean permethrin content was 16.5 g/kg (95% CI 16.2-16.9) with 78% of the sub-sampled nets retaining recommended permethrin content regardless of their age or physical condition. Nets aged 4 years and above had a mean permethrin content of 14 g/kg (95% CI 12.0-16.0). The only statistically significant predictor of reduced physical net integrity was rats in the house. CONCLUSIONS: Two-to-four years after a mass campaign, only 39% of distributed nets remain both present and in serviceable physical condition, a functional survival considerably below WHO assumptions of 50% survival of a 'three-year' net. However, the majority of nets still retained substantial levels of permethrin and could still be bio-chemically useful against mosquitoes if their holes were repaired, adding evidence to the value of net care and repair campaigns.