Multi-insecticide resistant malaria vectors in the field remain susceptible to malathion, despite the presence of Ace1 point mutations.

Insecticide resistance in Anopheles mosquitoes is seriously threatening the success of insecticide-based malaria vector control. Surveillance of insecticide resistance in mosquito populations and identifying the underlying mechanisms enables optimisation of vector control strategies. Here, we investigated the molecular mechanisms of insecticide resistance in three Anopheles coluzzii field populations from southern Côte d'Ivoire, including Agboville, Dabou and Tiassalé. All three populations were resistant to bendiocarb, deltamethrin and DDT, but not or only very weakly resistant to malathion. The absence of malathion resistance is an unexpected result because we found the acetylcholinesterase mutation Ace1-G280S at high frequencies, which would typically confer cross-resistance to carbamates and organophosphates, including malathion. Notably, Tiassalé was the most susceptible population to malathion while being the most resistant one to the pyrethroid deltamethrin. The resistance ratio to deltamethrin between Tiassalé and the laboratory reference colony was 1,800 fold. By sequencing the transcriptome of individual mosquitoes, we found numerous cytochrome P450-dependent monooxygenases - including CYP6M2, CYP6P2, CYP6P3, CYP6P4 and CYP6P5 - overexpressed in all three field populations. This could be an indication for negative cross-resistance caused by overexpression of pyrethroid-detoxifying cytochrome P450s that may activate pro-insecticides, thereby increasing malathion susceptibility. In addition to the P450s, we found several overexpressed carboxylesterases, glutathione S-transferases and other candidates putatively involved in insecticide resistance.

Reduced proinsecticide activation by cytochrome P450 confers coumaphos resistance in the major bee parasite Varroa destructor.

Varroa destructor is one of the main problems in modern beekeeping. Highly selective acaricides with low toxicity to bees are used internationally to control this mite. One of the key acaricides is the organophosphorus (OP) proinsecticide coumaphos, that becomes toxic after enzymatic activation inside Varroa We show here that mites from the island Andros (AN-CR) exhibit high levels of coumaphos resistance. Resistance is not mediated by decreased coumaphos uptake, target-site resistance, or increased detoxification. Reduced proinsecticide activation by a cytochrome P450 enzyme was the main resistance mechanism, a powerful and rarely encountered evolutionary solution to insecticide selection pressure. After treatment with sublethal doses of [14C] coumaphos, susceptible mite extracts had substantial amounts of coroxon, the activated metabolite of coumaphos, while resistant mites had only trace amounts. This indicates a suppression of the P450 (CYP)-mediated activation step in the AN-CR mites. Bioassays with coroxon to bypass the activation step showed that resistance was dramatically reduced. There are 26 CYPs present in the V. destructor genome. Transcriptome analysis revealed overexpression in resistant mites of CYP4DP24 and underexpression of CYP3012A6 and CYP4EP4 RNA interference of CYP4EP4 in the susceptible population, to mimic underexpression seen in the resistant mites, prevented coumaphos activation and decreased coumaphos toxicity.

Species composition, infection rate and detection of resistant alleles in Anopheles funestus (Diptera: Culicidae) from Lare, a malaria hotspot district of Ethiopia.

BACKGROUND: Anopheles funestus, which is considered as secondary vector of malaria in Ethiopia, is known to have several morphologically indistinguishable (sibling) species. Accurate identification of sibling species is crucial to understand their biology, behaviour and vector competence. In this study, molecular identification was conducted on the Ethiopian An. funestus populations. Moreover, insecticide resistance mechanism markers were detected, including ace N485I, kdr L1014F, L1014S, and CYP6P9a TaqMan qPCR was used to detect the infective stage of the parasite from field collected adult female An. funestus populations. METHODS: Adult female mosquito collection was conducted from Lare, Gambella Regional State of Ethiopia between June 2018 to July 2020 using CDC light traps and HLC. Sub-samples of the morphologically identified An. funestus mosquitoes were molecularly identified using species-specific PCR, and the possible presence of insecticide resistance alleles was investigated using TaqMan qPCR (N485I-Ace-1), PCR-Sanger sequencing (L1014F-kdr), and PCR-RFLP (CYP6P9a resistance allele). Following head/thorax dissection, the TaqMan qPCR assay was used to investigate the presence of the infective stage Plasmodium parasite species. RESULTS: A total of 1086 adult female An. funestus mosquitoes were collected during the study period. All sub-samples (N = 20) that were morphologically identified as An. funestus sensu lato (s.l.) were identified as An. funestus sensu stricto (s.s.) using species- specific PCR assay. The PCR-RFLP assay that detects the CYP6P9a resistance allele that confers pyrethroid resistance in An. funestus was applied in N = 30 randomly selected An. funestus s.l. SPECIMENS: None of the specimens showed a digestion pattern consistent with the presence of the CYP6P9a resistance allele in contrast to what was observed in the positive control. Consequently, all samples were characterized as wild type. The qPCR TaqMan assay that detects the N485I acetylcholinesterase-1 mutation conferring resistance to organophosphates/carbamates in An. funestus was used in (N = 144) samples. All samples were characterized as wild type. The kdr L1014F and L1014S mutations in the VGSC gene that confer resistance to pyrethroids and DDT were analysed with direct Sanger sequencing after PCR and clean-up of the PCR products were also characterized as wild type. None of the samples (N = 169) were found positive for Plasmodium (P. falciparum/ovale/malariae/vivax) detection. CONCLUSION: All An. funestus s.l. samples from Lare were molecularly identified as An. funestus s.s. No CYP6P9, N485I acetylcholinesterase 1, kdr L1014F or L1014S mutations were detected in the An. funestus samples. None of the An. funestus samples were positive for Plasmodium. Although the current study did not detect any insecticide resistant mechanism, it provides a reference for future vector monitoring programmes. Regular monitoring of resistance mechanisms covering wider geographical areas of Ethiopia where this vector is distributed is important for improving the efficacy of vector control programs.

May EPH/Ephrin Targeting Revolutionize Lung Cancer Treatment?

Lung cancer (LC) is the leading cause of cancer death in the United States. Erythropoietin-producing hepatocellular receptors (EPHs) comprise the largest receptor tyrosine kinases (RTKs) family in mammals. EPHs along with their ligands, EPH-family receptor-interacting proteins (ephrins), have been found to be either up- or downregulated in LC cells, hence exhibiting a defining role in LC carcinogenesis and tumor progression. In their capacity as membrane-bound molecules, EPHs/ephrins may represent feasible targets in the context of precision cancer treatment. In order to investigate available therapeutics targeting the EPH/ephrin system in LC, a literature review was conducted, using the MEDLINE, LIVIVO, and Google Scholar databases. EPHA2 is the most well-studied EPH/ephrin target in LC treatment. The targeting of EPHA2, EPHA3, EPHA5, EPHA7, EPHB4, EPHB6, ephrin-A1, ephrin-A2, ephrin-B2, and ephrin-B3 in LC cells or xenograft models not only directly correlates with a profound LC suppression but also enriches the effects of well-established therapeutic regimens. However, the sole clinical trial incorporating a NSCLC patient could not describe objective anti-cancer effects after anti-EPHA2 antibody administration. Collectively, EPHs/ephrins seem to represent promising treatment targets in LC. However, large clinical trials still need to be performed, with a view to examining the effects of EPH/ephrin targeting in the clinical setting.

Pyrethroid Resistance Situation across Different Eco-Epidemiological Settings in Cameroon.

Rapid emergence and spread of pyrethroid resistance in Anopheles gambiae populations is among the main factors affecting malaria vector control in Cameroon, but there is still not enough data on the exact pyrethroid resistance status across Cameroon. The present study assessed pyrethroid resistance profile in different eco-epidemiological settings in Cameroon. Susceptibility bioassay tests were performed with F0 An. gambiae females aged three to five days. Mosquito susceptibility to both permethrin and deltamethrin was assessed. Species of the An. gambiae s.l. complex were identified using molecular diagnostic tools. Target site mutations conferring resistance were detected using Taqman assays. Quantitative reverse transcription-real-time PCR (qRT-PCR) 3-plex TaqMan® assays were used for the quantification of detoxification genes implicated in pyrethroid resistance. An. gambiae, An. coluzzii and An. arabiensis were identified in the different settings. An. gambiae was dominant in Santchou, Kékem, Bélabo, Bertoua and Njombé, while An. coluzzii was abundant in Tibati and Kaélé. High frequencies of the kdr L1014F allele ranging from 43% to 100% were recorded in almost all sites. The L1014S kdr allele was detected at low frequency (4.10-10%) only in mosquito populations from Njombé and Tibati. The N1575Y mutation was recorded in Kaélé, Santchou, Tibati and Bertoua with a frequency varying from 2.10% to 11.70%. Six Cytochrome P450 genes (Cyp6p3, Cyp6m2, Cyp9k1, Cyp6p4, Cyp6z1, and Cyp4g16) were found to be overexpressed in at least one population. Analysis of cuticular hydrocarbon lipids indicated a significant increase in CHC content in mosquito populations from Kaélé and Njombé compared to Kékem, Bélabo and Bertoua populations. The study indicated high pyrethroid resistance across different ecological settings in Cameroon with different profile of resistance across the country. The present situation calls for further actions in order to mitigate the impact of insecticide resistance on vector control measures.

Pyrethroid and Etofenprox Resistance in Anopheles gambiae and Anopheles coluzzii from Vegetable Farms in Yaoundé, Cameroon: Dynamics, Intensity and Molecular Basis.

Previous studies have indicated widespread insecticide resistance in malaria vector populations from Cameroon. However, the intensity of this resistance and underlying mechanisms are poorly known. Therefore, we conducted three cross-sectional resistance surveys between April 2018 and October 2019, using the revised World Health Organization protocol, which includes resistance incidences and intensity assessments. Field-collected Anopheles gambiae s.l. populations from Nkolondom, Nkolbisson and Ekié vegetable farms in the city of Yaoundé were tested with deltamethrin, permethrin, alpha-cypermethrin and etofenprox, using 1× insecticide diagnostic concentrations for resistance incidence, then 5× and 10× concentrations for resistance intensity. Subsamples were analyzed for species identification and the detection of resistance-associated molecular markers using TaqMan® qPCR assays. In Nkolbisson, both An. coluzzii (96%) and An. gambiae s.s. (4%) were found together, whereas only An. gambiae s.s. was present in Nkolondom, and only An. coluzzii was present in Ekié. All three populations were resistant to the four insecticides (<75% mortality rates-MR1×), with intensity generally fluctuating over the time between mod-erate (<98%-MR5×; ≥98%-MR10×) and high (76-97%-MR10×). The kdr L995F, L995S, and N1570Y, and the Ace-1 G280S-resistant alleles were found in An. gambiae from Nkolondom, at 73%, 1%, 16% and 13% frequencies, respectively, whereas only the kdr L995F was found in An. gambiae s.s. from Nkolbisson at a 50% frequency. In An. coluzzii from Nkolbisson and Ekié, we detected only the kdr L995F allele at 65% and 60% frequencies, respectively. Furthermore, expression levels of Cyp6m2, Cyp9k1, and Gste2 metabolic genes were highly upregulated (over fivefold) in Nkolondom and Nkolbisson. Pyrethroid and etofenprox-based vector control interventions may be jeopardized in the prospected areas, due to high resistance intensity, with multiple mechanisms in An. gambiae s.s. and An. coluzzii.

miR-203 is an independent molecular predictor of prognosis and treatment outcome in ovarian cancer: a multi-institutional study.

Ovarian cancer (OC) accounts for the most gynecological cancer-related deaths in developed countries. Unfortunately, the lack of both evident early symptoms and effective asymptomatic population screening results in late diagnosis and inevitably poor prognosis. Hence, it is urgent to identify novel molecular markers to support personalized prognosis. In the present study, we have analyzed the clinical significance of miR-203 in OC using two institutionally independent cohorts. miR-203 levels were quantified in a screening (n = 125) and a validation cohort (n = 100, OVCAD multicenter study). Survival analysis was performed using progression and death as clinical endpoint events. Internal validation was conducted by bootstrap analysis, and decision curve analysis was used to evaluate the clinical benefit. Increased miR-203 levels in OC patients were correlated with unfavorable prognosis and higher risk for disease progression, independently of FIGO stage, tumor grade, residual tumor after surgery, chemotherapy response and age. The analysis of the institutionally independent validation cohort (OVCAD study) clearly confirmed the shorter survival outcome of the patients overexpressing miR-203. Additionally, integration of miR-203 levels with the established disease prognostic markers led to a superior stratification of OC patients that can ameliorate prognosis and benefit patient clinical management. In this regard, miR-203 expression constitutes a novel independent molecular marker to improve patients' prognosis in OC.

miR-181a overexpression predicts the poor treatment response and early-progression of serous ovarian cancer patients.

Ovarian cancer (OC) remains a leading cause of gynecological cancer-related death worldwide, characterized by poor 5-year survival. Molecular markers could serve as crucial tools of personalized prognosis and therapy. Herein, we present miR-181a as novel predictor of OC prognosis, using five independent OC cohorts. In particular, a screening (n = 81) and an institutionally independent validation (n = 100, OVCAD multicenter study) serous OC (SOC) cohorts were analyzed. Bagnoli et al (2016) OC179 (n = 124) to OC133 (n = 100) and TCGA (n = 489) served as external validation cohorts. Patients' survival and disease progression were assessed as clinical endpoint events. Bootstrap analysis was performed for internal validation and decision curve analysis was utilized to evaluate clinical benefit. miR-181a overexpression was unveiled as powerful and independent molecular predictor of patients' poor survival and higher risk for disease progression after debulking surgery and platinum-based chemotherapy. Analysis of the OVCAD institutionally independent cohort, as well as of Bagnoli et al. and TCGA external cohorts further confirmed the unfavorable prognostic nature of miR-181a overexpression in SOC. Strikingly, multivariate prognostic models incorporating miR-181a with established disease markers clearly improved patients' risk-stratification and offered superior clinical benefit in OC prognostication. Conclusively, miR-181a evaluation could augment prognostic accuracy and support precision medicine decisions in OC.

Cytochrome P450-based metabolic insecticide resistance in Anopheles and Aedes mosquito vectors: Muddying the waters.

Despite the substantial progress achieved in the characterization of cytochrome P450 (CYP) -based resistance mechanisms in mosquitoes, a number of questions remain unanswered. These include: (i) the regulation and physiology of resistance conferring CYPs; (ii) the actual contribution of CYPs in resistance alone or in combination with other detoxification partners or other resistance mechanisms; (iii) the association between overexpression levels and allelic variation, with the catalytic activity and the intensity of resistance and (iv) the true value of molecular diagnostics targeting CYP markers, for driving decision making in the frame of Insecticide Resistance Management applications. Furthermore, the translation of CYP - based insecticide resistance research in mosquitoes into practical applications, is being developed, but it is not fully exploited, as yet. Examples include the production of high throughput platforms for screening the liability (stability) or inhibition potential of novel insecticidal leads and synergists (add-ons), as well as the exploration of the negative cross resistance concept (i.e. detoxification of certain insecticides, but activation of others pro-insecticides). The goal of this review is to critically summarise the current knowledge and the gaps of the CYP-based metabolic insecticide resistance in Anopheles and Aedes mosquito vectors. The progress and limitations of the protein and the reverse/forward genetic approaches, the understanding and importance of molecular and physiological aspects, as well as the current and future exploitation routes of CYP research are discussed.

Vector population monitoring tools for insecticide resistance management: Myth or fact?

Insecticide resistance is a large and growing problem for the control of mosquito disease vectors. The World Health Organization (WHO) established the Global Plan for Insecticide Resistance Management (GPIRM) in 2012. In that context, both classical and molecular tools, as well as entomological databases and decision support platforms have been developed and used for IRM. Despite major advances in the molecular elucidation of resistance mechanisms and the development of diagnostic tools, their impact on disease control programs has been limited. In most cases diagnostic tools provide a retrospective examination of changes imposed by insecticides rather than a prospective analysis to guide vector control strategies. The uncertainty of the predictive value of markers, the assay robustness and the common misconceptions in resistance diagnosis terminology are continuing challenges in monitoring vector resistance. Furthermore, an often logistics, as opposed to systematic scientific evidence, based approach to decision for the use of the very few alternative chemicals in vector control, has reduced the value of resistance monitoring in practice. The current deployment of new insecticidal active ingredients should necessitate the application of companion diagnostics (CDx) and the development of modern ways for interpretation and management of the data by trained programme managers. This will establish their real value for use in decision-making, in line with evidence based choice of chemicals in agriculture and medical applications.

Multianalyte quantitative competitive PCR on optically encoded microspheres for an eight-gene panel related to prostate cancer.

Nucleic acid-based tests have a profound impact in every medical discipline. Because multigene tests offer higher diagnostic accuracy and lower overall cost than single assays, they are especially useful for diseases, like prostate cancer, that present variability at the molecular level and diversity of available therapeutic interventions. We have developed a quantitative competitive PCR for an eight-gene panel, related to prostate cancer, that includes five genes of the human tissue kallikrein family (KLKs), prostate-specific membrane antigen (PSMA), prostate cancer antigen 3 (PCA3), and HPRT1 as a reference gene. Using PCR as a synthetic tool, a competitor was prepared for each target sequence containing the same primer binding sites as the target but differing in a short segment to enable discrimination by hybridization. The assay involves multiplex amplification of targets and competitors followed by a multiplex hybridization assay for the 16 amplification products. The assay was performed on optically encoded microspheres with oligonucleotide probes attached to their surface. The microspheres were analyzed rapidly (1 min) by flow cytometry. The signal ratio of the target and cognate competitor is a function of the target copy number in the sample prior to amplification. The multiplexing potential of the proposed method is much higher than real-time PCR and other end-point methods since there are 100 sets of commercially available microspheres.

Uncovering the clinical utility of miR-143, miR-145 and miR-224 for predicting the survival of bladder cancer patients following treatment.

Accurate prognosis is a key factor in establishing optimal therapeutic decisions; yet in the case of bladder cancer (BlCa) current prognostic indicators cannot ensure optimal disease management. Here, we aimed to evaluate the previously unexplored clinical potential of the urological cancer-related miR-145, miR-143 and miR-224 in BlCa. A total of 279 bladder tissue specimens were included in this study (133 BlCa, 107 adjacent normal and 39 healthy samples). Total RNA was extracted from tissues, it was polyadenylated and reverse transcribed to cDNA. The expression of target molecules was measured via quantitative real-time PCR. The expression levels of both miR-143 and miR-145 were significantly decreased, whereas those of miR-224 were increased in BlCa. Receiver operating characteristic curve analysis indicated a significant discriminatory capacity for miR-143/miR-145 levels. Important associations with disease aggressiveness were observed for all three microRNAs; elevated levels were observed in tumors of higher stage and grade, as well as in 'high-risk' TaT1 patients. More importantly, high miR-143/145 levels could effectively prognose inferior overall survival for muscle-invasive patients and could independently predict the progression of superficial tumors. Finally, the combination of miR-143/145 overexpression with the widely used prognostic markers of European Organization for Research and Treatment of Cancer-risk groups or recurrence at the first follow-up cystoscopy resulted to a superior positive prediction of non-muscle-invasive bladder cancer short-term progression compared with the use of the abovementioned markers alone. The cancer-related miR-143, miR-145 and miR-224 were investigated for the first time in the clinical setting of BlCa, and miR-143/145 cluster constitutes a novel marker helpful for providing an enhanced prediction of oncologic outcome for BlCa patients.

Kallikrein-related peptidase 13: an independent indicator of favorable prognosis for patients with nonsmall cell lung cancer.

The KLK13 gene is dysregulated in several carcinomas, and its expression levels seem to be associated with disease prognosis. The aim of our study was to investigate the prognostic potential of KLK13 mRNA expression for patients with nonsmall cell lung cancer (NSCLC). Total RNA was isolated from cancerous and normal tissues from a cohort of 128 NSCLC patients. The KLK13 mRNA transcription levels were measured using a sensitive quantitative RT-PCR method. The results were normalized by dividing the KLK13 mRNA values with the geometric mean of mRNA expression from four reference genes: beta-actin, TATA-binding protein, hypoxanthine phosphoribosyltransferase 1, and acidic ribosomal phosphoprotein P0. The malignant tissues from the majority of patients (59.3 %) contained significantly more KLK13 mRNA transcripts than did the paired nonmalignant tissues (median difference 11.1-fold, P = 0.008). KLK13 was expressed at higher levels in females than that in males (P = 0.021). No other statistically significant association with clinicopathological data was observed. Kaplan-Meier survival analyses demonstrated that patients with KLK13-positive tumors survived significantly longer than those with KLK13-negative ones (P = 0.009). KLK13 expression was also shown to be able to stratify high-risk individuals among patients with early disease stages (P = 0.030). Multivariate Cox regression analysis showed that KLK13 expression is a favorable, independent prognostic indicator of overall survival (OS) (P = 0.024). Our results suggest that KLK13 mRNA expression constitutes a novel biomarker for the prediction of overall survival in NSCLC and that its quantitative assessment in tumor tissues can aid in treatment decision making.

Development, application and evaluation of three novel TaqMan qPCR assays for phosphine resistance monitoring in major stored product pests Tribolium castaneum and Rhyzopertha dominica.

BACKROUND: Stored product protection from insect pests relies heavily on the use of phosphine. The most serious drawback of phosphine is the development of resistance in major stored product insects worldwide, including the red flour beetle, Tribolium castaneum (Herbst) and the lesser grain borer, Rhyzopertha dominica (F.). Two genetic loci are responsible for phosphine resistance: the rph1 (S349G mutation in the cyt-b5-r homolog) in T. castaneum and the rph2 (P45/49S mutation in the dihydrolipoamide dehydrogenase (dld) gene) in T. castaneum and R. dominica. RESULTS: In this study, we have developed and applied high-throughput, practical and specific molecular diagnostics (TaqMan qPCR) for monitoring mutations S349G, P45S and P49S. In our pilot monitoring application, we have included phosphine-resistant and susceptible populations from different parts of the world (USA, Australia, Brazil) and European strains from Greece and Serbia. Our results for the resistant T. castaneum showed a P45S mutant allele frequency (MAF) of 100% and 75.0% in the populations from Serbia and Brazil, respectively. Regarding the susceptible T. castaneum, P45S was detected in Greece (MAF = 62.5%) and was absent in Australia (MAF = 0.0%). Additionally, the S349G mutation was found to be fixed in all resistant populations, while it was also detected in susceptible ones (frequencies: 65.0% and 100.0%). The only case where both mutations were fixed (100%) was a resistant population from Serbia. In R. dominica, the P49S mutation was found only in the two resistant R. dominica populations from Serbia and Greece (50.0% and 100%) and was absent from the susceptible one from Greece; thus, P49S seems to be a satisfactory indicator for monitoring phosphine resistance. CONCLUSIONS: Our P49S detection assay in R. dominica seems to be a viable option in this direction, yet its utilization needs additional large-scale confirmatory work. The identification of additional resistance markers also should be prioritized. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Molecular monitoring of insecticide resistance in major disease vectors in Armenia.

BACKGROUND: Armenia is considered particularly vulnerable to life-threatening vector-borne diseases (VBDs) including malaria, West Nile virus disease and leishmaniasis. However, information relevant for the control of the vectors of these diseases, such as their insecticide resistance profile, is scarce. The present study was conducted to provide the first evidence on insecticide resistance mechanisms circulating in major mosquito and sand fly populations in Armenia. METHODS: Sampling sites were targeted based mainly on previous historical records of VBD occurrences in humans and vertebrate hosts. Initially, molecular species identification on the collected vector samples was performed. Subsequently, molecular diagnostic assays [polymerase chain reaction (PCR), Sanger sequencing, PCR-restriction fragment length polymorphism (RFLP), quantitative PCR (qPCR)] were performed to profile for major insecticide resistance mechanisms, i.e. target site insensitivity in voltage-gated sodium channel (vgsc) associated with pyrethroid resistance, acetylcholinesterase (ace-1) target site mutations linked to organophosphate (OP) and carbamate (CRB) resistance, chitin synthase (chs-1) target site mutations associated with diflubenzuron (DFB) resistance and gene amplification of carboxylesterases (CCEs) associated with resistance to the OP temephos. RESULTS: Anopheles mosquitoes were principally represented by Anopheles sacharovi, a well-known malaria vector in Armenia, which showed no signs of resistance mechanisms. Contrarily, the knockdown resistance (kdr) mutations V1016G and L1014F/C in the vgsc gene were detected in the arboviral mosquito vectors Aedes albopictus and Culex pipiens, respectively. The kdr mutation L1014S was also detected in the sand fly, vectors of leishmaniasis, Phlebotomus papatasi and P. tobbi, whereas no mutations were found in the remaining collected sand fly species, P. sergenti, P. perfiliewi and P. caucasicus. CONCLUSIONS: This is the first study to report on molecular mechanisms of insecticide resistance circulating in major mosquito and sand fly disease vectors in Armenia and highlights the need for the establishment of systematic resistance monitoring practices for the implementation of evidence-based control applications.

Rapid and reliable testing for clinically actionable EGFR mutations in non-small cell lung cancer using the IdyllaTM platform: a real-world two-center experience in Greece.

BACKGROUND: Limited information exists on epidermal growth factor receptor (EGFR) molecular epidemiology in Greece. Next-generation sequencing (NGS) is the recommended method for EGFR genotyping in NSCLC. The Idylla Biocartis platform is a fully automated system for actionable EGFR mutation detection. RESEARCH DESIGN AND METHODS: We describe the prevalence of EGFR mutations in NSCLC patients in two high-volume clinical centers in Greece and compare key methods used for their determination. Eight hundred and fifty-seven FFPE samples from NSCLC patients were tested for EGFR mutations at University of Crete (UoC; n = 324) and at Evangelismos Hospital, Athens (Evangelismos; n = 503). RESULTS: The prevalence of EGFR mutations was 11.1% in the whole cohort (11.5% in non-squamous). The detection rate was 11.0% by NGS, 9.8% by Sanger and 11.3% by Idylla for the whole cohort (12.0% in non-squamous). The agreement between Idylla and Sanger was 93.2%. A targetable EGFR mutation was detected in 10.0% using tissue NGS alone, and in 16.0% using concurrent Idylla ctEGFR testing. CONCLUSION: The frequency of EGFR mutations was as expected for a Caucasian population. The Idylla EGFR test performance is comparable to reference methods and with a shorter TAT. Adding a concurrent plasma Idylla test to tissue NGS testing increases the detection rate of EGFR mutations in NSCLC.

Quantified KLK15 gene expression levels discriminate prostate cancer from benign tumors and constitute a novel independent predictor of disease progression.

BACKGROUND: Several transcript variants of the kallikrein-related peptidase 15 gene (KLK15) have been identified up to now. The classical KLK15 mRNA isoform encodes for a non-truncated, functional protein. Aberrant KLK15 expression is found in breast, ovarian, and prostate cancers. Our aim in this present study was the specific quantitative expression analysis of the classical KLK15 mRNA transcript in prostate tumors and the examination of its clinical significance in prostate cancer. METHODS: We isolated total RNA from 150 prostate tissue specimens and, following cDNA synthesis, the expression of KLK15 classical mRNA transcript was measured via quantitative Real-Time PCR using the TaqMan® chemistry. HPRT1 was used as a reference gene, and the absolute quantification approach, through the incorporation of standard curves, was applied for the calculation of normalized KLK15 expression. RESULTS: KLK15 expression levels were significantly upregulated in malignant compared to benign samples (P < 0.001). The discriminatory value of KLK15 was confirmed by ROC curve and logistic regression analysis (both P < 0.001). KLK15 was also associated with advanced pathological stage (P = 0.023). KLK15-positive prostate cancer patients presented significantly shorter progression-free survival intervals, determined by biochemical relapse (P = 0.006), compared to KLK15-negative ones. Multivariate Cox regression analysis revealed that KLK15 expression is an independent predictor of biochemical recurrence (HR = 3.36, P = 0.038). CONCLUSIONS: The present study unravels the important role of quantified KLK15 classical mRNA expression levels as a novel biomarker helpful for the differential diagnosis and prognosis of prostate cancer patients.

Downregulation and prognostic performance of microRNA 224 expression in prostate cancer.

INTRODUCTION: The extensive use of prostate-specific antigen as a general prostate cancer biomarker has introduced the hazards of overdiagnosis and overtreatment. Recent studies have revealed the immense biomarker capacity of microRNAs (miRNAs) in prostate cancer. The aim of this study was to analyze the expression pattern of miR-224, a cancer-related miRNA, in prostate tumors and investigate its clinical utility. METHODS: Total RNA was isolated from 139 prostate tissue samples. After the polyadenylation of total RNA by poly(A) polymerase, cDNA was synthesized with a suitable poly(T) adapter. miR-224 expression was assessed by quantitative real-time PCR and analyzed with the comparative quantification cycle method, C(q)(2(-ΔΔCq)). We performed comprehensive biostatistical analyses to explore the clinical value of miR-224 in prostate cancer. RESULTS: miR-224 expression was significantly downregulated in malignant samples compared with benign samples (P < 0.001). Higher miR-224 expression levels were found in prostate tumors that were less aggressive (P = 0.017) and in an earlier disease stage (P = 0.018). Patients with prostate cancer who were positive for miR-224 had significantly enhanced progression-free survival intervals compared with miR-224-negative patients (P = 0.021). Univariate bootstrap Cox regression confirmed that miR-224 was associated with favorable prognosis (hazard ratio 0.314, P = 0.013); nonetheless, multivariate analysis, adjusted for conventional markers, did not identify miR-224 as an independent prognostic indicator. CONCLUSIONS: miR-224 is aberrantly expressed in prostate cancer. Its assessment by cost-effective quantitative molecular methodologies could provide a useful biomarker for prostate cancer.

Monitoring of Insecticide Resistance Mutations and Pathogen Circulation in Sand Flies from Emilia-Romagna, a Leishmaniasis Endemic Region of Northern Italy.

The continuously expanding distribution of sand flies, proven vectors of Leishmania and of several phleboviruses, is a growing public health issue in Europe. Especially in Italy, visceral leishmaniasis (VL) is occurring with increasing incidence northward, in previously non-endemic provinces. Around the globe, disease elimination efforts largely focus on sand fly vector insecticidal control, often leading to the development of resistance. In Emilia-Romagna (ER), northern Italy, insecticides are heavily applied for agricultural and mosquito control, but not specifically against sand flies. Here, we investigated the sand fly species composition in certain environmental settings in ER provinces and monitored the presence of pyrethroid resistance mutations and pathogen circulation. Phlebotomus perfiliewi, a dominant vector of Leishmania infantum, was detected almost exclusively in the region. No mutations in the voltage-gated sodium channel gene, e.g., knock-down resistance mutations I1011M, L1014F/S, V1016G, or F1020S, were recorded. Pathogen monitoring revealed that almost 40% of the tested sand fly pools were positive for Leishmania, while the presence of Toscana and Fermo phleboviruses was also observed in much lower frequencies (≤3% positive pools). Regular epidemiological and entomological monitoring, alongside resistance surveillance, is highly recommended to ensure the sustainability and efficiency of vector control interventions.

Molecular diagnostics for monitoring insecticide resistance in the western flower thrips Frankliniella occidentalis.

BACKGROUND: Insecticide resistance has emerged in various western flower thrips (WFT) populations across the world, threatening the efficiency of chemical control applications. Elucidation of insecticide resistance mechanisms at the molecular level provides markers for the development of diagnostics to monitor the trait and support evidence-based resistance management. RESULTS: TaqMan and Droplet Digital polymerase chain reaction (ddPCR) diagnostics were developed and validated, against Sanger sequencing, in individual and pooled WFT samples respectively, for the G275E mutation (nicotinic acetylcholine receptor α6 gene, nAChR α6) associated with resistance to nAChR allosteric modulators, site I (spinosyns); L1014F, T929I, T929C and T292V mutations (voltage-gated sodium channel gene, vgsc) linked with pyrethroid resistance; and I1017M (chitin synthase 1 gene, chs1) conferring resistance to growth inhibitors affecting CHS1 (benzoylureas). The detection limits of ddPCR assays for mutant allelic frequencies (MAF) were in the range of 0.1%-0.2%. The assays were applied in nine WFT field populations from Crete, Greece. The G275E (MAF = 29.66%-100.0%), T929I and T929V (combined MAF = 100%), L1014F (MAF = 11.01%-37.29%), and I1017M (MAF = 17.74%-51.07%) mutations were present in all populations. CONCLUSION: The molecular diagnostics panel that was developed in this study can facilitate the quick and sensitive resistance monitoring of WFT populations at the molecular level, to support evidence-based insecticide resistance management strategies. © 2022 Society of Chemical Industry.