Focusing nucleic acid-based molecular diagnostics and xenomonitoring approaches for human helminthiases amenable to preventive chemotherapy

The current mainstay for control of the four major helminth diseases in humans (lymphatic filariasis, onchocerciasis, soil-transmitted helminthiases and schistosomiasis) is with preventive chemotherapy by mass administration of key anthelminthics. Following the London Declaration on Neglected Tropical Diseases in 2012, a roadmap for the elimination and control of these helminthiases by 2020 has been devised. With expected declines in prevalence and intensity of these infections, there is urgent need for implementing more sensitive, high-throughput and cost-effective diagnostic tools. Currently available diagnostic approaches for surveying, monitoring and evaluating helminth control programmes are based on microscopical observation of eggs/larvae, and/or detection of antibodies or parasite antigens in stool, urine or blood; all relatively low-throughput and of limited sensitivity and specificity. Newly proposed approaches for helminthiases diagnosis include the nucleic acid-based methods of (multiplex) real-time polymerase chain reaction assays, loop-mediated isothermal amplification and recombinase polymerase amplification. However, as well as sensitivity/specificity evaluation, their comparison to current ‘gold standard’ diagnostics and future application in individual-/community-based diagnosis, or in xenomonitoring requires consideration of relative costs, agreement of standard methods and strategic interpretation of resulting data before control/elimination programmes might best utilize molecular diagnostics to inform decision making. We review current nucleic-acid-based molecular diagnostic methods and highlight the needs and future research required to refine these tools for monitoring and evaluation of control and elimination programmes for four major human helminthiases

Insecticide resistance selection and reversal in two strains of Aedes aegypti

Background: Laboratory reared mosquito colonies are essential tools to understand insecticide action. However, they differ considerably from wild populations and from each other depending on their origin and rearing conditions, which makes studying the effects of specific resistance mechanisms difficult. This paper describes our methods for establishing multiple resistant strains of Aedes aegypti from two colonies as a new resource for further research on metabolic and target site resistance. Methods: Two resistant colonies of Ae. aegypti, from Cayman and Recife, were selected through 10 generations of exposure to insecticides including permethrin, malathion and temephos, to yield eight strains with different profiles of resistance due to either target site or metabolic resistance. Resistance ratios for each insecticide were calculated for the selected and unselected strains. The frequency of kdr alleles in the Cayman strains was determined using TaqMan assays. A comparative gene expression analysis among Recife strains was conducted using qPCR in larvae (CCae3A, CYP6N12, CYP6F3, CYP9M9) and adults (CCae3A, CYP6N12, CYP6BB2, CYP9J28a). Results: In the selected strain of Cayman, mortality against permethrin reduced almost to 0% and kdr became fixated by 5 generations. A similar phenotype was seen in the unselected homozygous resistant colony, whilst mortality in the susceptible homozygous colony rose to 82.9%. The Recife strains showed different responses between exposure to adulticide and larvicide, with detoxification genes in the temephos selected strain staying similar to the baseline, but a reduction in detoxification genes displayed in the other strains. Conclusions: These selected strains, with a range of insecticide resistance phenotypes and genotypes, will support further research on the effects of target-site and/or metabolic resistance mechanisms on various life-history traits, behaviours and vector competence of this important arbovirus vector

Filarial infection influences mosquito behaviour and fecundity

Understanding vector-parasite interactions is increasingly important as we move towards the endpoint goals set by the Global Programme for the Elimination of Lymphatic Filariasis (GPELF), as interaction dynamics may change with reduced transmission pressure. Elimination models used to predict programmatic endpoints include parameters for vector-specific transmission dynamics, despite the fact that our knowledge of the host-seeking behaviour of filariasis infected mosquitoes is lacking. We observed a dynamic, stage-specific and density dependent change in Aedes aegypti behaviour towards host cues when exposed to Brugia malayi filarial parasites. Infected mosquitoes exhibited reduced activation and flight towards a host during the period of larval development (L1/L2), transitioning to a 5 fold increase in activation and flight towards a host when infective stage larvae (L3) were present (p < 0.001). In uninfected control mosquitoes, we observed a reduction in convergence towards a host during the same period. Furthermore, this behaviour was density dependent with non-activated mosquitoes harbouring a greater burden of L1 and L2 larvae while activated mosquitoes harboured a greater number of L3 (p < 0.001). Reductions in fecundity were also density-dependent, and extended to mosquitoes that were exposed to microfilariae but did not support larval development

The impact of mosquito sampling strategies on molecular xenomonitoring prevalence for filariasis: a systematic review.

To explore the impact of mosquito collection methods, sampling intensity and target genus on molecular xenomonitoring detection of parasites causing lymphatic filariasis. We systematically searched five databases for studies that used two or more collection strategies for sampling wild mosquitoes, and employed molecular methods to assess the molecular xenomonitoring prevalence of parasites responsible for lymphatic filariasis. We performed generic inverse variance meta-analyses and explored sources of heterogeneity using subgroup analyses. We assessed methodological quality and certainty of evidence. We identified 25 eligible studies, with 172 083 mosquitoes analysed. We observed significantly higher molecular xenomonitoring prevalence with collection methods that target bloodfed mosquitoes compared to methods that target unfed mosquitoes (prevalence ratio: 3.53; 95% confidence interval, CI: 1.52-8.24), but no significant difference compared with gravid collection methods (prevalence ratio: 1.54; 95% CI: 0.46-5.16). Regarding genus, we observed significantly higher molecular xenomonitoring prevalence for anopheline mosquitoes compared to culicine mosquitoes in areas where species are the primary vector (prevalence ratio: 6.91; 95% CI: 1.73-27.52). One study provided evidence that reducing the number of sampling sites did not significantly affect molecular xenomonitoring prevalence. Evidence of differences in molecular xenomonitoring prevalence between sampling strategies was considered to be of low certainty, due partly to inherent limitations of observational studies that were not explicitly designed for these comparisons. The choice of sampling strategy can significantly affect molecular xenomonitoring results. Further research is needed to inform the optimum strategy in light of logistical constraints and epidemiological contexts

A Superhydrophobic Cone to Facilitate the Xenomonitoring of Filarial Parasites, Malaria, and Trypanosomes Using Mosquito Excreta/Feces

Background: Molecular xenomonitoring (MX), the testing of insect vectors for the presence of human pathogens, has the potential to provide a non-invasive and cost-effective method for monitoring the prevalence of disease within a community. Current MX methods require the capture and processing of large numbers of mosquitoes, particularly in areas of low endemicity, increasing the time, cost and labour required. Screening the excreta/feces (E/F) released from mosquitoes, rather than whole carcasses, improves the throughput by removing the need to discriminate vector species since non-vectors release ingested pathogens in E/F. It also enables larger numbers of mosquitoes to be processed per pool. However, this new screening approach requires a method of efficiently collecting E/F. Methods: We developed a cone with a superhydrophobic surface to allow for the efficient collection of E/F. Using mosquitoes exposed to either Plasmodium falciparum, Brugia malayi or Trypanosoma brucei brucei, we tested the performance of the superhydrophobic cone alongside two other collection methods. Results: All collection methods enabled the detection of DNA from the three parasites. Using the superhydrophobic cone to deposit E/F into a small tube provided the highest number of positive samples (16 out of 18) and facilitated detection of parasite DNA in E/F from individual mosquitoes. Further tests showed that following a simple washing step, the cone can be reused multiple times, further improving its cost-effectiveness. Conclusions: Incorporating the superhydrophobic cone into mosquito traps or holding containers could provide a simple and efficient method for collecting E/F. Where this is not possible, swabbing the container or using the washing method facilitates the detection of the three parasites used in this study

The Effect of Age and Gender on Visual Search During Lane Changing

This study examined visual search behavior relative to three regions of interest (ROI) (side mirror, rear view mirror, and blind spot) for self-initiated lane changes in a sample of 108 drivers under actual highway conditions. As has been observed previously, few drivers scan all three of the ROI prior to executing a lane change, with turning around to inspect the blind spot being the lowest frequency behavior. Age, gender and direction (left or right lane change) were found to influence visual search behaviors. For lane changes to the right, blind spot checking occurred less than 32% of the time in females and less than 15% of the time in males. This low level of blind spot checking to the right was consistent across younger and older age groupings. Interestingly, the most notable age discrepancy was in checking the left blind spot. Younger drivers checked their left blind spot 53.3% of the time compared to a rate of 23.9% for drivers in their 60s. Implications of these findings for both driver remediation programs and the increasing availability of blind spot identification systems are considered

Measuring quality of life in opioid dependent people : a systematic review of assessment instruments

Purpose Opioid dependence is a chronic relapsing disorder. Despite increasing research on quality of life (QOL) in people with opioid dependence, little attention has been paid to the instruments used. This systematic review examines the suitability of QOL instruments for use in opioid-dependent populations and the instruments’ quality. Methods A systematic search was performed in the databases Medline, PsycInfo, The Cochrane Library, and CINAHL. Articles were eligible if they assessed QOL of opioid-dependent populations using a validated QOL instrument. Item content relevance to opioid-dependent people was evaluated by means of content analysis, and instrument properties were assessed using minimum standards for patient-reported outcome measures. Results Eighty-nine articles were retrieved, yielding sixteen QOL instruments, of which ten were assessed in this review. Of the ten instruments, six were disease specific, but none for opioid dependence. Two instruments had good item content relevance. The conceptual and measurement model were described in seven instruments. Four instruments were developed with input from the respective target population. Eight instruments had low respondent and administrator burden. Psychometric properties were either not assessed in opioid-dependent populations or were inconclusive or moderate. Conclusions No instrument scored perfectly on both the content and properties. The limited suitability of instruments for opioid-dependent people hinders accurate and sensitive measurement of QOL in this population. Future research is in need of an opioid dependence-specific QOL instrument to measure the true impact of the disease on people’s lives and to evaluate treatment-related services

Evaluating the diagnostic test accuracy of molecular xenomonitoring methods for characterising the community burden of Onchocerciasis

Background: Molecular xenomonitoring (MX), the detection of parasite nucleic acid in the vector population, is recommended for onchocerciasis surveillance in elimination settings. However, the sensitivity of MX for detecting onchocerciasis-positive communities has not previously been evaluated. MX may have additional applications for control programmes but its utility is restricted by a limited understanding of the relationship between MX results and human prevalence. Methods: We conducted a systematic review of studies reporting the prevalence of Onchocerca volvulus DNA in wild-caught Simulium spp. flies (MX rate) and corresponding prevalence of microfilaria (mf) in humans. We evaluated the sensitivity of MX for detecting onchocerciasis-positive communities and describe the characteristics of studies with reduced sensitivity. We conducted a linear regression to evaluate the relationship between mf prevalence and MX rate. Results: We identified 15 relevant studies, with 13 studies comprising 34 study communities included in the quantitative analyses. Most communities were at advanced stages towards elimination and had no or extremely low human prevalence. MX detected positive flies in every study area with >1% mf prevalence, with the exception of one study conducted in the Venezuelan Amazonian focus. We identified a significant relationship between the two measurements, with mf prevalence accounting for half of the variation in MX rate (R2 0.50, p<0.001). Conclusion: MX is sensitive to communities with ongoing onchocerciasis transmission. It has potential to predict human mf prevalence, but further data is required to understand this relationship, particularly from MX surveys conducted earlier in control programmes before transmission has been interrupted

Evaluating the Evidence for Lymphatic Filariasis Elimination

In the global drive for elimination of lymphatic filariasis (LF), 15 countries have achieved validation of elimination as a public health problem (EPHP). Recent empirical evidence has demonstrated that EPHP does not always lead to elimination of transmission (EOT). Here we show how the probability of elimination explicitly depends on key biological parameters, many of which have been poorly characterized, leading to a poor evidence base for the elimination threshold. As more countries progress towards EPHP it is essential that this process is well-informed, as prematurely halting treatment and surveillance programs could pose a serious threat to global progress. We highlight that refinement of the weak empirical evidence base is vital to understand drivers of elimination and inform long-term polic