A new malaria vector in Africa: Predicting the expansion range of Anopheles stephensi and identifying the urban populations at risk.

In 2012, an unusual outbreak of urban malaria was reported from Djibouti City in the Horn of Africa and increasingly severe outbreaks have been reported annually ever since. Subsequent investigations discovered the presence of an Asian mosquito species; Anopheles stephensi, a species known to thrive in urban environments. Since that first report, An. stephensi has been identified in Ethiopia and Sudan, and this worrying development has prompted the World Health Organization (WHO) to publish a vector alert calling for active mosquito surveillance in the region. Using an up-to-date database of published locational records for An. stephensi across its full range (Asia, Arabian Peninsula, Horn of Africa) and a set of spatial models that identify the environmental conditions that characterize a species' preferred habitat, we provide evidence-based maps predicting the possible locations across Africa where An. stephensi could establish if allowed to spread unchecked. Unsurprisingly, due to this species' close association with man-made habitats, our maps predict a high probability of presence within many urban cities across Africa where our estimates suggest that over 126 million people reside. Our results strongly support the WHO's call for surveillance and targeted vector control and provide a basis for the prioritization of surveillance.

Effects of the spatial repellent metofluthrin on landing rates of outdoor biting anophelines in Cambodia, Southeast Asia.

The emergence of artemisinin-resistant malaria in Southeast Asia is a major problem. The fact that many people become infected with malaria when they are outside has prompted the development of 'spatial' rather than topical repellents. The respective effects of one or four slow-release emanators of metofluthrin, a pyrethroid, were tested in Pailin, Pursat and Koh Kong, Cambodia. Numbers of mosquitoes counted in outdoor landing catches when one or four emanators were suspended close to the collector were compared with control collections. In Pailin, the effects of emanators on catches in Furvela tent traps and Centers for Disease Control (CDC) light traps suspended underneath houses were also investigated. Rate ratios were used to determine differences. A total of 29 255 mosquitoes were collected over 2934 h of landing collections, 87 nights of tent trapping and 81 nights of light trap capture. In Pailin, landing rates were reduced by 48% by a single emanator and by 67% by four emanators (P < 0.001). Similar reductions were observed in the number of mosquitoes collected in tent traps and the number of anophelines only collected in light traps. Results were similar in Pursat, but, for unknown reasons, those in Koh Kong showed no difference between control and metofluthrin collections (P > 0.05). These findings suggest that although the product can produce a significant effect, it requires further improvement.

Differential transcription profiles in Aedes aegypti detoxification genes after temephos selection.

The mosquito Aedes aegypti is the main vector of Dengue and Yellow Fever flaviviruses. The organophosphate insecticide temephos is a larvicide that is used globally to control Ae. aegypti populations; many of which have in turn evolved resistance. Target site alteration in the acetylcholine esterase of this species has not being identified. Instead, we tracked changes in transcription of metabolic detoxification genes using the Ae. aegypti 'Detox Chip' microarray during five generations of temephos selection. We selected for temephos resistance in three replicates in each of six collections, five from Mexico, and one from Peru. The response to selection was tracked in terms of lethal concentrations. Uniform upregulation was seen in the epsilon class glutathione-S-transferase (eGST) genes in strains from Mexico prior to laboratory selection, while eGSTs in the Iquitos Peru strain became upregulated after five generations of temephos selection. While expression of many carboxyl/cholinesterase esterase (CCE) genes increased with selection, no single esterase was consistently upregulated and this same pattern was noted in the cytochrome P450 monooxygenase (CYP) genes and in other genes involved in reduction or oxidation of xenobiotics. Bioassays using glutathione-S-transferase (GST), CCE and CYP inhibitors suggest that various CCEs instead of GSTs are the main metabolic mechanism conferring resistance to temephos. We show that temephos-selected strains show no cross resistance to permethrin and that genes associated with temephos selection are largely independent of those selected with permethrin in a previous study.

Transcription of detoxification genes after permethrin selection in the mosquito Aedes aegypti.

Changes in gene expression before, during and after five generations of permethrin laboratory selection were monitored in six strains of Aedes aegypti: five F(2)-F(3) collections from the Yucatán Peninsula of Mexico and one F(2) from Iquitos, Peru. Three biological replicate lines were generated for each strain. The response to selection was measured as changes in the lethal and knockdown permethrin concentrations (LC(50), KC(50)) and in the frequency of the Ile1,016 substitution in the voltage-gated sodium channel (para) gene. Changes in expression of 290 metabolic detoxification genes were measured using the 'Aedes Detox' microarray. Selection simultaneously increased the LC(50), KC(50) and Ile1,016 frequency. There was an inverse relationship between Ile1,016 frequency and the numbers of differentially transcribed genes. The Iquitos strain lacked the Ile1,016 allele and 51 genes were differentially transcribed after selection as compared with 10-18 genes in the Mexican strains. Very few of the same genes were differentially transcribed among field strains but 10 cytochrome P(450) genes were upregulated in more than one strain. Laboratory adaptation to permethrin in Ae. aegypti is genetically complex and largely conditioned by geographic origin and pre-existing target site insensitivity in the para gene. The lack of uniformity in the genes that responded to artificial selection as well as differences in the direction of their responses challenges the assumption that one or a few genes control permethrin metabolic resistance. Attempts to identify one or a few metabolic genes that are predictably associated with permethrin adaptation may be futile.

Malaria management: past, present, and future.

The prospect of malaria eradication has been raised recently by the Bill and Melinda Gates Foundation with support from the international community. There are significant lessons to be learned from the major successes and failures of the eradication campaign of the 1960s, but cessation of transmission in the malaria heartlands of Africa will depend on a vaccine and better drugs and insecticides. Insect control is an essential part of reducing transmission. To date, two operational scale interventions, indoor residual spraying and deployment of long-lasting insecticide-treated nets (LLINs), are effective at reducing transmission. Our ability to monitor and evaluate these interventions needs to be improved so that scarce resources can be sensibly deployed, and new interventions that reduce transmission in a cost-effective and efficient manner need to be developed. New interventions could include using transgenic mosquitoes, larviciding in urban areas, or utilizing cost-effective consumer products. Alongside this innovative development agenda, the potential negative impact of insecticide resistance, particularly on LLINs, for which only pyrethroids are available, needs to be monitored.

Effects of seasonality and irrigation on malaria transmission in two villages in Côte d'Ivoire.

The seasonality of malaria transmission was investigated in two villages in central Côte d'Ivoire: one usually with irrigated rice farming (Zatta) and one without (Tiémélékro). Adult mosquitoes were collected, from February 2002 to August 2005, inside and outside sentinel houses. In Tiémélékro, the biting rate of Anopheles gambiae s.s. showed a significant difference between the dry and rainy season only in 2003 (P<0.001). The corresponding rates for An. funestus s.s. showed significant seasonal differences in both 2002 and 2003 (P<0.001 for each year). In Zatta in 2003-2004, when irrigated rice farming was interrupted, there was no significant difference between the An. gambiae s.s. biting rates recorded in the dry and rainy seasons. In both 2002 and 2005, however, when irrigated rice farming was practised, the An. gambiae s.s. biting rate recorded in Zatta during the rainy season was significantly higher than that seen in the dry season (P<0.001 for each year). With just one exception (in Tiémélékro in 2005, the prevalence of Plasmodium infection in the An. funestus was significantly higher in the rainy season than in the dry season), no significant seasonal differences were seen in the prevalences of Plasmodium infection among the An. gambiae or An. funestus. In conclusion, although malaria transmission is quite stable in central Côte d'Ivoire throughout the year, it can be distinctly modified by irrigated rice farming.

Evolving Use of fMRI in Medicare Beneficiaries.

Using the Medicare Physician-Supplier Procedure Summary Master File, we evaluated the evolving use of fMRI in Medicare fee-for-service beneficiaries from 2007 through 2017. Annual use rates (per 1,000,000 enrollees) increased from 17.7 to 32.8 through 2014 and have remained static since. Radiologists have remained the dominant specialty group from 2007 to 2017 (86.4% and 88.6% of all services, respectively), and the outpatient setting has remained the dominant place of service (65.4% and 65.4%, respectively).

Propetamphos resistance in Rhipicephalus bursa (Acari, Ixodidae).

Hard tick infestation is an important problem in both traditional and industrial animal husbandry as well as in veterinary and public health. Several acaricides have been used to control tick infestation in Iran. Poor control has been attributed to acaricide resistance in tick populations although this has not been demonstrated experimentally. In this study, susceptibility status to propetamphos (Blotic) of 2-3-week-old larvae of Rhipicephalus bursa, one of the most common tick species in Iran, was evaluated using the FAO recommended larval packet test (LPT) method with some modifications. Log dosage probit analysis of propetamphos dose-response regression lines produced a resistance ratio based on LC(50) in the most resistant strain of approximately 103-fold. Biochemical assays suggested that the tick strains contained multiple insecticide resistance mechanisms involving elevation of esterases, GSTs, MFOs and alteration of acetylcholinesterase. In light of this data, practical implications for pest and pesticide resistance management strategies are discussed.

Characterization of inhibitors and substrates of Anopheles gambiae CYP6Z2.

Three CYP6Z genes are linked to a major pyrethroid resistance locus in the mosquito Anopheles gambiae. We have expressed CYP6Z2 in Escherichia coli and produced a structural model in order to examine its role in detoxification. E. coli membranes co-expressing CYP6Z2 and An. gambiae P450 reductase (AgCPR) catalysed the dealkylation of benzyloxyresorufin with kinetic parameters K(m) = 0.13 microM; K(cat) = 1.5 min(-1). The IC(50) values of a wide range of compounds were measured. Pyrethroids cypermethrin and permethrin produced low IC(50) values, but were not metabolized. Plant flavanoids were the most potent inhibitors. Several compounds were shown to be substrates, suggesting that CYP6Z2 has broad substrate specificity and plays an important chemo-protective role during the herbivorous phase of the life-cycle.

Operational impact of DDT reintroduction for malaria control on Anopheles arabiensis in Mozambique.

ABSTRACT With the increase in indoor residual spraying in many internationally and nationally funded malaria control programs, and affirmation by World Health Organization (WHO) that DDT is appropriate for use in the absence of longer lasting insecticide formulations in some malaria endemic settings, DDT has been reintroduced as a major malaria control intervention in Africa. Indoor residual spraying with DDT was reintroduced into Mozambique for malaria control in 2005, and it is increasingly becoming the main insecticide used for malaria vector control in Mozambique. The selection of DDT in Mozambique is evidence-based, taking account of the susceptibility of Anopheles arabiensis (Patton) and Anopheles gambiae (Giles) s.s. to all the available insecticide choices, as well as relative costs of the insecticide and the logistical costs of spraying. Before this time in Mozambique, DDT was replaced by h-cyhalothrin in 1993. Resistance occurred quickly to this insecticide, and in 2000 the pyrethroid was phased out and the carbamate bendiocarb was introduced. Low-level resistance was detected by biochemical assay to bendiocarb in 1999 in both Anopheles funestus (Giles) and An. arabiensis, although this was not evident in WHO bioassays of the same population. In the 2000-2006 surveys the levels of bendiocarb resistance had been selected to a higher level in An. arabiensis, with resistance detectable by both biochemical and WHO bioassay. The insecticide resistance monitoring program includes assessment of field populations by standard WHO insecticide susceptibility assays and biochemical assays. Monitoring was established in 1999, and it was maintained as part of an operational monitoring and evaluation program thereafter.

Electronic mosquito repellents for preventing mosquito bites and malaria infection.

BACKGROUND: Electronic mosquito repellents (EMRs) are marketed to prevent mosquitoes biting and to prevent malaria. OBJECTIVES: To assess whether EMRs prevent mosquito bites, and to assess any evidence of an effect on malaria infection. SEARCH STRATEGY: In August 2006, we searched the Cochrane Infectious Diseases Group Specialized Register, CENTRAL, MEDLINE, EMBASE, LILACS, Cambridge Scientific Abstracts, and the Science Citation Index. We also checked conference proceedings, contacted international specialist centres and EMR manufacturers, and checked reference lists. SELECTION CRITERIA: Field entomological studies, which controlled for geographic site, time, and attractiveness of human participants, of EMRs for preventing mosquito bites; and randomized and quasi-randomized controlled trials of EMRs to prevent malaria infection. DATA COLLECTION AND ANALYSIS: Two authors assessed trial quality, and extracted and analysed the data. MAIN RESULTS: Ten field entomological studies met the inclusion criteria. All 10 studies found that there was no difference in the number of mosquitoes caught from the bare body parts of the human participants with or without an EMR. No randomized or quasi-randomized controlled trials on the efficacy of EMR on malaria infection were found. AUTHORS' CONCLUSIONS: Field entomological studies confirm that EMRs have no effect on preventing mosquito bites. Therefore there is no justification for marketing them to prevent malaria infection.

Assessing fluctuating odontometric asymmetry among fossil hominin taxa through alternative measures of central tendency: effect of outliers and directional components on reported results.

Preliminary inquires into the distribution and expression of fluctuating odontometric asymmetry (FOA), among selected fossil hominins, have revealed results that may be serviceable within studies that assess, among others, palaeobiological, evolutionary processes and events. Though several intricate statistical applications have aided in the advancement of FOA to the hominin fossil record, little is known regarding the influence of outliers and directional components on reported results. Moreover, most methods employed to test homogeneity among FOA datasets are sensitive to the assumption that underlying samples reflect Gaussian distributions. Because this assumption is often violated, alternative formulations of Levene's test statistic, which have been shown to be robust under non-normality, have been suggested. Unfortunately, previous FOA studies have failed to address their potential. Given this, we considered two areas that may influence interpretations of FOA among fossil hominin studies. Firstly, we assessed distributions of signed data (d(u)) among samples of Australopithecus africanus, Paranthropus robustus and Homo habilis for outliers and directional asymmetry to evaluate their influence on reported heterogeneity. Secondly, in an attempt to decrease the probability of falsely rejecting H(0) due to non-normality, we considered alternative estimates of central tendency for comparisons of FOA. Our study confirms the need for intrinsic scrutiny of data, as the removal of one extreme value within the buccolingual H. habilis sample produced statistically significant outcomes at the sample level, while directional asymmetry was exposed within an expanded buccolingual P. robustus sample. However, though servicing alternative measures of central tendency remains informative, except for the buccolingual P. robustus sample before the correction of directional asymmetry, replacement of the mean was not required herein. Consistent with previous investigations, significant differences between buccolingual values in apposing arcades were unique among A. africanus and P. robustus, with the latter expressing greater FOA overall. Finally, our results strengthen the assertion that the individuals sampled among H. habilis may indicate an episode of developmental compromise where external and/or internal noises are lessened through internal homeostasis.

Insecticide resistance in Anopheles arabiensis and Anopheles gambiae from Mozambique.

Malaria control in the southern part of Mozambique is currently by indoor residual spraying with a carbamate insecticide and by pyrethroid-treated bed-nets distributed to pregnant women and children under five in northern Mozambique. The susceptibility of Anopheles gambiae s.s. and Anopheles arabiensis Patton to pyrethroid, carbamate, organochlorine, and organophosphorus insecticides was determined by World Health Organization adult mosquito susceptibility tests at 17 localities in Mozambique, from March 2000 to July 2002. Biochemical assays were carried out on mosquitoes from the same families to detect shifts in the quantity or activity of enzyme families involved in insecticide detoxification. An. gambiae s.s. from all localities remained fully susceptible to DDT and the organophosphorus insecticide malathion. A low level of pyrethroid resistance was detected in populations in southern Mozambique. Populations outside Maputo province were still susceptible to pyrethroids. Low level resistance to the carbamate propoxur also was detected in An. arabiensis from two localities. Mosquitoes from five of the localities had elevated p450 estimates, compared with the insecticide susceptible Durban strain. The lack of cross-resistance between pyrethroids and DDT in Mozambican populations suggests that a kdr-type target site resistance mechanism has not been selected. Increased frequencies of insecticide insensitive acetylcholinesterase, the target site for carbamates and organophosphates, were found in 16 of the populations tested. Although vector control with bendiocarb is not being compromised by the presence of the acetylcholinesterase mechanism alone, the high level of insensitive acetylcholinesterase unless sensibly managed may have long-term implications for malaria control programs in Mozambique.

Insecticide resistance in Anopheles funestus (Diptera: Culicidae) from Mozambique.

Malaria control in southern Mozambique is currently by indoor residual carbamate insecticide treatment, with pyrethroid-treated bed-nets distributed to pregnant women and children under five in northern Mozambique. The susceptibility of Anopheles funestus s.s. to pyrethroid, carbamate, organochlorine, and organophosphorus insecticides was determined by World Health Organization adult mosquito susceptibility tests at 19 localities in Mozambique, from March 2000 to July 2002. Biochemical assays were carried out on mosquitoes from the same families to detect shifts in the quantity or activity of enzyme families involved in insecticide detoxification. An. funestus from all localities remained fully susceptible to DDT and the organophosphorus insecticide malathion. A high level of pyrethroid resistance was detected in An. funestus populations in southern Mozambique. An. funestus outside Maputo province were still susceptible to pyrethroids. An. funestus from six localities also were resistant to carbamate insecticides propoxur and bendiocarb. Both pyrethroid and carbamate resistance occurred in five of these six localities. Mosquitoes from five of the localities with elevated p450 estimates, compared with the insecticide-susceptible Durban strain, were pyrethroid-resistant. The only exception to this trend was Mozal, which had elevated p450 estimates but full pyrethroid susceptibility by bioassay. The lack of cross-resistance between pyrethroids and DDT in Mozambican An. funestus suggests that a kdr-type target site resistance mechanism has not been selected. Low levels of insecticide-insensitive acetylcholinesterase, the target site for carbamates and organophosphates, were found in all populations tested. The high level of metabolically based pyrethroid resistance has implications for current malaria control programs in Mozambique.

Same-Day Sinus and Brain CT Imaging in the Medicare Population: Are Practice Patterns Changing in Association with Medicare Policy Initiatives?

BACKGROUND AND PURPOSE: Monitoring the frequency of same-day sinus and brain CT (Outpatient Measure 14, "OP-14") is part of a recent large Centers for Medicare and Medicaid Services hospital outpatient quality initiative to improve imaging efficiency. This study investigates patient-level claims data in the Medicare population focusing on where same-day sinus and brain CT imaging is performed and how the frequency of same-day studies changed with time before and during OP-14 measure program implementation. MATERIALS AND METHODS: Research Identifiable Files were used to identify all sinus and brain CT examinations from 2004 through 2012 for a 5% random patient sample of Medicare fee-for-service beneficiaries. Overall and site of service use rates were calculated for same- and non-same-day examinations. Changes were mapped to policy initiative timetables. RESULTS: The number of same-day sinus and brain CT studies from 2004 to 2012 increased 67% from 1.85 (95% CI, 1.78-1.91) per 1000 Medicare beneficiaries in 2004 to 3.08 (95% CI, 3.00-3.15) in 2012. The biggest driver of increased same-day studies was the emergency department setting, from 0.56 (95% CI, 0.53-0.60) per 1000 to 1.78 (95% CI, 1.72-1.84; +215.7%). Overall use of brain CT from 146.0 (95% CI, 145.1-146.9) per 1000 to 176.3 (95% CI, 175.4-177.2; +21%) and sinus CT from 12.6 (95% CI, 12.4-12.8) per 1000 to 15.4 (95% CI, 15.2-15.6; +22%) increased until 2009 and remained stable through 2012. CONCLUSIONS: Previously increasing same-day sinus and brain CT in Medicare beneficiaries plateaued in 2009, coinciding with the implementation of targeted measures by the Centers for Medicare and Medicaid Services. Same-day imaging continues to increase in the emergency department setting.

Spatiotemporal distribution of insecticide resistance in Anopheles culicifacies and Anopheles subpictus in Sri Lanka.

The malaria situation in Sri Lanka worsened during the 1990s with the emergence and spread of resistance to the drugs and insecticides used for control. Chloroquine resistance has increased rapidly over this period, but adverse changes in malaria transmission are more closely associated with insecticide use rather than drug resistance. Insecticide susceptibility tests were routinely carried out in key anopheline vectors across the country for more than a decade. These sentinel data were combined with data collected by other research programmes and used to map the spatial and temporal trends of insecticide resistance in the main vectors, Anopheles culicifacies and A. subpictus, and to examine the relationship between insecticide resistance, changes in national spraying regimens and malaria prevalence. Both species had widespread resistance to malathion, the insecticide of choice in the early 1990s. Both species were initially susceptible to the organophosphate and pyrethroid insecticides used operationally from 1993, but some resistance has now been selected. The levels of malathion and fenitrothion resistance in A. subpictus were higher in some ecological regions than others, which may be related to the distribution of sibling species, agricultural pesticide exposure and/or environmental factors. The study highlights that the emergence and spread of insecticide resistance is a constant threat and that active surveillance systems are vital in identifying key vectors and evidence of resistance.

The molecular basis of two contrasting metabolic mechanisms of insecticide resistance.

The esterase-based insecticide resistance mechanisms characterised to date predominantly involve elevation of activity through gene amplification allowing increased levels of insecticide sequestration, or point mutations within the esterase structural genes which change their substrate specificity. The amplified esterases are subject to various types of gene regulation in different insect species. In contrast, elevation of glutathione S-transferase activity involves upregulation of multiple enzymes belonging to one or more glutathione S-transferase classes or more rarely upregulation of a single enzyme. There is no evidence of insecticide resistance associated with gene amplification in this enzyme class. The biochemical and molecular basis of these two metabolically-based insecticide resistance mechanisms is reviewed.

Differential glycosylation produces heterogeneity in elevated esterases associated with insecticide resistance in the brown planthopper Nilaparvata lugens Stål.

The major insecticide resistance mechanism in the brown planthopper Nilaparvata lugens involves overproduction of esterases. Esterases purified from a resistant strain appeared as a ladder of bands on isoelectric focussing (IEF) gels from pI 4.7 to 5.0. Two-dimensional electrophoresis showed that isozymes ranged in size from 66 to 68 kDa with those of lower pI being apparently smaller. All isozymes detected by two-dimensional electrophoresis were glycosylated. N-glycosidase A reduced the number of isozymes on IEF to two, with increased pI and an increased molecular weight of 69 kDa. No O-linked glycans were detected. Deglycosylation had no effect on esterase activity, hence glycosylation is not involved in active site conformation. As N-glycosidase F completely deglycosylated the esterases, none of the glycans has an alpha1,3-bound core fucose. Reactivity with the lectins GNA, MAA and DSA, combined with differential cleavage of N-linked glycans with endoglycosidases F1 and F2, indicated that terminally linked mannose is present in high mannose and/or hybrid type glycans and that terminally linked sialic acid and galactose-beta(1-4)-N-acetylglucosamine are present in biantennary complexes. Neuraminidase treatment had the same effect on pI of isozymes as complete deglycosylation. Therefore, the majority of the heterogeneity of elevated esterases on IEF is due to differential attachment of sialic acid to glycans of the two proteins.

Cloning, expression and characterization of an insect class I glutathione S-transferase from Anopheles dirus species B.

Insect class I glutathione S-transferases (GSTs) were expressed from cDNA obtained from larvae of the Thai malaria vector. Anopheles dirus in a PCR RACE (rapid amplification of cDNA ends) reaction using a primer to the conserved N-terminal region of An. gambiae class I GSTs and a synthetic oligo d(T)-adaptor primer. Seven different plasmids, resulting from sub-cloning of an original single 0.7 Kb PCR band, were picked at random and sequenced. Four of these were clearly GSTs on the basis of putative amino acid sequence conservation. All the sequences had a conserved N-terminal region, but were highly divergent at the C-terminus. The variability in the PCR products suggests that there is a high level of GST class I isoenzyme variability in larval An. dirus. One of the subclones from the PCR reaction contained a full coding region of the cDNA for GST. This had a putative amino acid sequence which was 76 and 91% identity to the An. gambiae GST class I, agGST 1-5 and agGST 1-6 respectively, but only 48% identity to agGST 1-2. The catalytically active enzyme, expressed in Escherichia coli, was strongly immuno-cross reactive with antisera raised against the two An. gambiae class I GSTs. The expressed enzyme was purified to homogeneity from an E. coli cell lysate by S-hexylglutathione agarose affinity chromatography. The enzyme had a high specific activity with CDNB, and also used DCNB and ethacrynic acid as substrates. In addition, it had peroxidase and DDTase activity and its activity with CDNB, was strongly inhibited by a range of organophosphorus and pyrethroid insecticides. This is consistent with the predicted role of this GST class in insecticide resistance.