Advanced crystallisation methods for small organic molecules.

Molecular materials based on small organic molecules often require advanced structural analysis, beyond the capability of spectroscopic techniques, to fully characterise them. In such cases, diffraction methods such as single crystal X-ray diffraction (SCXRD), are one of the most powerful tools available to researchers, providing molecular and structural elucidation at atomic level resolution, including absolute stereochemistry. However SCXRD, and related diffraction methods, are heavily dependent on the availability of suitable, high-quality crystals, thus crystallisation often becomes the major bottleneck in preparing samples. Following a summary of classical methods for the crystallisation of small organic molecules, this review will focus on a number of recently developed advanced methods for crystalline material sample preparation for SCXRD. This review will cover two main areas of modern small organic molecule crystallisation, namely the inclusion of molecules within host complexes (e.g., "crystalline sponge" and tetraaryladamantane based inclusion chaperones) and the use of high-throughput crystallisation, employing "under-oil" approaches (e.g., microbatch under-oil and ENaCt). Representative examples have been included for each technique, together with a discussion of their relative advantages and limitations to aid the reader in selecting the most appropriate technique to overcome a specific analytical challenge.

Distinct genomic landscapes of gastroesophageal adenocarcinoma depending on PD-L1 expression identify mutations in RAS-MAPK pathway and TP53 as potential predictors of immunotherapy efficacy.

BACKGROUND: The impact of molecular alterations on programmed death-ligand 1 (PD-L1) combined positive score (CPS) is not well studied in gastroesophageal adenocarcinomas (GEAs). We aimed to characterize genomic features of tumors with different CPSs in GEAs. PATIENTS AND METHODS: Genomic alterations of 2518 GEAs were compared in three groups (PD-L1 CPS ≥ 10, high; CPS = 1-9, intermediate; CPS < 1, low) using next-generation sequencing. We assessed the impact of gene mutations on the efficacy of immune checkpoint inhibitors (ICIs) and tumor immune environment based on the Memorial Sloan Kettering Cancer Center and The Cancer Genome Atlas databases. RESULTS: High, intermediate, and low CPSs were seen in 18%, 54% and 28% of GEAs, respectively. PD-L1 positivity was less prevalent in women and in tissues derived from metastatic sites. PD-L1 CPS was positively associated with mismatch repair deficiency/microsatellite instability-high, but independent of tumor mutation burden distribution. Tumors with mutations in KRAS, TP53, and RAS-mitogen-activated protein kinase (MAPK) pathway were associated with higher PD-L1 CPSs in the mismatch repair proficiency and microsatellite stability (pMMR&MSS) subgroup. Patients with RAS-MAPK pathway alterations had longer overall survival (OS) from ICIs compared to wildtype (WT) patients [27 versus 13 months, hazard ratio (HR) = 0.36, 95% confidence interval (CI): 0.19-0.7, P = 0.016] and a similar trend was observed in the MSS subgroup (P = 0.11). In contrast, patients with TP53 mutations had worse OS from ICIs compared to TP53-WT patients in the MSS subgroup (5 versus 21 months, HR = 2.39, 95% CI: 1.24-4.61, P = 0.016). CONCLUSIONS: This is the largest study to investigate the distinct genomic landscapes of GEAs with different PD-L1 CPSs. Our data may provide novel insights for patient selection using mutations in TP53 and RAS-MAPK pathway and for the development of rational combination immunotherapies in GEAs.

Micro-CT imaging of Onchocerca infection of Simulium damnosum s.l. blackflies and comparison of the peritrophic membrane thickness of forest and savannah flies.

Onchocerciasis is a neglected tropical disease (NTD) caused by Onchocerca Diesing 1841 (Spirurida: Onchocercidae) nematodes transmitted by blackflies. It is associated with poverty and imposes a significant health, welfare and economic burden on many tropical countries. Current methods to visualize infections within the vectors rely on invasive methods. However, using micro-computed tomography techniques, without interference from physical tissue manipulation, we visualized in three dimensions for the first time an L1 larva of an Onchocerca species within the thoracic musculature of a blackfly, Simulium damnosum s.l. Theobald 1903 (Diptera: Simuliidae), naturally infected in Ghana. The possibility that thicker peritrophic membranes in savannah flies could account for their lower parasite loads was not supported, but there were limits to our analysis. While there were no statistically significant differences between the mean thicknesses of the peritrophic membranes, in the anterior, dorsal and ventral regions, of forest and savannah blackflies killed 34-48 min after a blood-meal, the thickness of the peritrophic membrane in the posterior region could not be measured. Micro-computed tomography has the potential to provide novel information on many other parasite/vector systems and impactful images for public engagement in health education.

Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro-CT.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro-computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio-opaque concretions. Moreover, the anatomy of O. ovis third-instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length.

Accidental Nasal Myiasis Caused by Megaselia rufipes (Diptera: Phoridae) in a Child.

A case of a nasal myiasis in a 3-yr-old Italian girl who was referred to Bambino Gesù Hospital in Rome, Italy, is reported. Larvae discharged with the nasal mucus were microscopically identified as Megaselia spp.; DNA barcoding analysis showed that they belonged to the 'scuttle fly' species Megaselia rufipes (Meigen). Based on the patient's history, she became infected when she played outside. This is the first report of myiasis in humans due to M. rufipes (Diptera: Phoridae).

Internal morphological changes during metamorphosis in the sheep nasal bot fly, Oestrus ovis.

During the larval stage, oestrid flies (Diptera: Oestridae) are obligate parasites, whereas during the adult stage they are free-living and do not feed. Like other cyclorrhaphous flies, oestrids undergo metamorphosis inside an opaque puparium, formed by the contracted and hardened cuticle of the third-instar larva. The present study documents the internal morphological changes taking place during metamorphosis of the sheep nasal bot fly, Oestrus ovis L., using non-invasive, micro-CT-based virtual histology and provides quantitative data of volumetric changes in specific organs. Virtual histological sections allowed visualisation of the progression and completion of the apolyses, which delimit the different intra-puparial stages, and the connection to the tracheal system of a large gas bubble, which plays an essential role during early metamorphosis. Overall, our results show that the sequence of morphological and volumetric changes in tissues and organs is similar to those found in other cyclorrhaphous flies, but they also reveal developmental differences that result in an adult vestigial digestive tract. Future studies could develop non-invasive, reliable methods for aging the intra-puparial forms of different oestrid species of veterinary importance, based on both qualitative and quantitative markers, thus improving our knowledge of their development and the efficiency of control strategies.

Health-related quality of life in patients with a germline BRCA mutation and metastatic pancreatic cancer receiving maintenance olaparib.

BACKGROUND: Patients with metastatic pancreatic cancer often have a detriment in health-related quality of life (HRQoL). In the randomized, double-blind, phase III POLO trial progression-free survival was significantly longer with maintenance olaparib, a poly(ADP-ribose) polymerase inhibitor, than placebo in patients with a germline BRCA1 and/or BRCA2 mutation (gBRCAm) and metastatic pancreatic cancer whose disease had not progressed during first-line platinum-based chemotherapy. The prespecified HRQoL evaluation is reported here. PATIENTS AND METHODS: Patients were randomized to receive maintenance olaparib (300 mg b.i.d.; tablets) or placebo. HRQoL was assessed using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30-item module at baseline, every 4 weeks until disease progression, at discontinuation, and 30 days after last dose. Scores ranged from 0 to 100; a ≥10-point change or difference between arms was considered clinically meaningful. Adjusted mean change from baseline was analysed using a mixed model for repeated measures. Time to sustained clinically meaningful deterioration (TSCMD) was analysed using a log-rank test. RESULTS: Of 154 randomized patients, 89 of 92 olaparib-arm and 58 of 62 placebo-arm patients were included in HRQoL analyses. The adjusted mean change in Global Health Status (GHS) score from baseline was <10 points in both arms and there was no significant between-group difference [-2.47; 95% confidence interval (CI) -7.27, 2.33; P = 0.31]. Analysis of physical functioning scores showed a significant between-group difference (-4.45 points; 95% CI -8.75, -0.16; P = 0.04). There was no difference in TSCMD for olaparib versus placebo for GHS [P = 0.25; hazard ratio (HR) 0.72; 95% CI 0.41, 1.27] or physical functioning (P = 0.32; HR 1.38; 95% CI 0.73, 2.63). CONCLUSIONS: HRQoL was preserved with maintenance olaparib treatment with no clinically meaningful difference compared with placebo. These results support the observed efficacy benefit of maintenance olaparib in patients with a gBRCAm and metastatic pancreatic cancer. CLINCALTRIALS.GOV NUMBER: NCT02184195.

Towards the automated identification of Chrysomya blow flies from wing images.

The Old World screwworm fly (OWSF), Chrysomya bezziana (Diptera: Calliphoridae), is an important agent of traumatic myiasis and, as such, a major human and animal health problem. In the implementation of OWSF control operations, it is important to determine the geographical origins of such disease-causing species in order to establish whether they derive from endemic or invading populations. Gross morphological and molecular studies have demonstrated the existence of two distinct lineages of this species, one African and the other Asian. Wing morphometry is known to be of substantial assistance in identifying the geographical origin of individuals because it provides diagnostic markers that complement molecular diagnostics. However, placement of the landmarks used in traditional geometric morphometric analysis can be time-consuming and subject to error caused by operator subjectivity. Here we report results of an image-based approach to geometric morphometric analysis for delivering wing-based identifications. Our results indicate that this approach can produce identifications that are practically indistinguishable from more traditional landmark-based results. In addition, we demonstrate that the direct analysis of digital wing images can be used to discriminate between three Chrysomya species of veterinary and forensic importance and between C. bezziana genders.

Micro-computed tomography visualization of the vestigial alimentary canal in adult oestrid flies.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage as they acquire all necessary nutrients during the parasitic larval stage. The adult mouthparts and digestive tract are therefore frequently vestigial; however, morphological data on the alimentary canal in adult oestrid flies are scarce and a proper visualization of this organ system within the adult body is lacking. The present work visualizes the morphology of the alimentary canal in adults of two oestrid species, Oestrus ovis L. and Hypoderma lineatum (de Villiers), with the use of non-invasive micro-computed tomography (micro-CT) and compares it with the highly developed alimentary canal of the blow fly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). Both O. ovis and H. lineatum adults showed significant reductions of the cardia and the diameter of the digestive tract, an absence of the helicoidal portion of the midgut typical of other cyclorrhaphous flies, and a lack of crop and salivary glands. Given the current interest in the alimentary canal in adult dipterans in biomedical and developmental biology studies, further understanding of the morphology and development of this organ system in adult oestrids may provide valuable new insights in several areas of research.

Component alignment and clinical outcome following total knee arthroplasty: a randomised controlled trial comparing an intramedullary alignment system with patient-specific instrumentation.

AIMS: We conducted a randomised controlled trial to assess the accuracy of positioning and alignment of the components in total knee arthroplasty (TKA), comparing those undertaken using standard intramedullary cutting jigs and those with patient-specific instruments (PSI). PATIENTS AND METHODS: There were 64 TKAs in the standard group and 69 in the PSI group. The post-operative hip-knee-ankle (HKA) angle and positioning was investigated using CT scans. Deviation of > 3° from the planned position was regarded as an outlier. The operating time, Oxford Knee Scores (OKS) and Short Form-12 (SF-12) scores were recorded. RESULTS: There were 14 HKA-angle outliers (22%) in the standard group and nine (13%) in the PSI group (p = 0.251). The mean HKA-angle was 0.5° varus in the standard group and 0.2° varus in the PSI group (p = 0.492). The accuracy of alignment in the coronal and axial planes and the proportion of outliers was not different in the two groups. The femoral component was more flexed (p = 0.035) and there were significantly more tibial slope outliers (29% versus 13%) in the PSI group (p = 0.032). Operating time and the median three-month OKS were similar (p = 0.218 and p = 0.472, respectively). Physical and mental SF-12 scores were not significantly different at three months (p = 0.418 and p = 0.267, respectively) or at one year post-operatively (p = 0.114 and p = 0.569). The median one-year Oxford knee score was two points higher in the PSI group (p = 0.049). CONCLUSION: Compared with standard intramedullary jigs, the use of PSI did not significantly reduce the number of outliers or the mean operating time, nor did it clinically improve the accuracy of alignment or the median Oxford Knee Scores. Our data do not support the routine use of PSI when undertaking TKA. Cite this article: Bone Joint J 2016;98-B:1043-9.

Genetic testing for hereditary cancer predisposition: BRCA1/2, Lynch syndrome, and beyond.

Obstetrician/gynecologists and gynecologic oncologists serve an integral role in the care of women at increased hereditary risk of cancer. Their contribution includes initial identification of high risk patients, screening procedures like bimanual exam, trans-vaginal ultrasound and endometrial biopsy, prophylaxis via TAH and/or BSO, and chemoprevention. Further, gynecologists also serve a central role in the management of the secondary repercussions of efforts to mitigate increased cancer risks, including vasomotor symptoms, sexual function, bone health, cardiovascular disease, and mental health. The past several years has seen multiple new high and moderate penetrance genes introduced into the clinical care of women at increased risk of gynecologic malignancy. Awareness of these new genes and the availability of new multi-gene panel tests is critical for providers on the front-line of women's health.

Morphology successfully separates third instar larvae of Muscina.

Three representatives of the muscid genus Muscina Robineau-Desvoidy (Diptera: Musicidae), Muscina levida (Harris), Muscina prolapsa (Harris) and Muscina stabulans (Fallén), are well known for their medical, veterinary and forensic importance. However, data in the literature provide contradictory information for the identification of third instar larvae of these species. This hinders easy species differentiation because it requires the rearing of material to adult stages in order to facilitate reliable identification. Third instar larvae of these three Muscina species were studied in detail using light and scanning electron microscopy to provide thorough redescriptions of their morphology. Existing information concerning third instar morphology, with particular reference to its value for taxonomy and identification, is revised and discussed. Emden's spiracular distance factor (SDF) is considered here as inappropriate for identification purposes in third instar larvae of Muscina spp. because its values are not constant ratios but increase during larval maturation and overlap in the examined species. These species were, instead, discriminated here by differences in the spinulation patterns of their abdominal segments.

A randomized controlled trial of postoperative analgesia following total knee replacement: transdermal Fentanyl patches versus patient controlled analgesia (PCA).

BACKGROUND: This randomized controlled trial compared a standard patient controlled analgesic (PCA) regime with a transdermal and oral Fentanyl regime for post-operative pain management in patients undergoing total knee replacement. METHODS: One hundred and ninety-six patients undergoing total knee replacement were recruited. Pre- and post-operatively Visual Analogue Score (VAS), Oxford Knee Score, Health Anxiety and Depression Score and Brief Pain Inventory Score were completed. According to the day 1, VAS score patients were randomly allocated to either a PCA regime or a Fentanyl transdermal/oral regime. Patient reported outcomes were measured until the patients were discharged. RESULTS: The results demonstrate that in terms of analgesic effect, day of discharge and side effect profile the two regimes are comparable. CONCLUSIONS: We conclude that a Fentanyl transdermal regime provides adequate analgesic effect comparable to a standard PCA regime in conjunction with a low side effect profile. Using a transdermal analgesic system provides efficient continuous delivery enabling a smooth transition from hospital to home within the first week. Transdermal Fentanyl provides an alternative analgesic regime that can provide an equivalent analgesic effect so as to enable a satisfactory outcome for the patient in terms of function and pain. LEVEL OF EVIDENCE: II.

Pharmacology of the glucagon-like peptide-1 analog exenatide extended-release in healthy cats.

Exenatide extended-release (ER) is a microencapsulated formulation of the glucagon-like peptide 1-receptor agonist exenatide. It has a protracted pharmacokinetic profile that allows a once-weekly injection with comparable efficacy to insulin with an improved safety profile in type II diabetic people. Here, we studied the pharmacology of exenatide ER in 6 healthy cats. A single subcutaneous injection of exenatide ER (0.13 mg/kg) was administered on day 0. Exenatide concentrations were measured for 12 wk. A hyperglycemic clamp (target = 225 mg/dL) was performed on days -7 (clamp I) and 21 (clamp II) with measurements of insulin and glucagon concentrations. Glucose tolerance was defined as the amount of glucose required to maintain hyperglycemia during the clamp. Continuous glucose monitoring was performed on weeks 0, 2, and 6 after injection. Plasma concentrations of exenatide peaked at 1 h and 4 wk after injection. Comparing clamp I with clamp II, fasting blood glucose decreased (mean ± standard deviation = -11 ± 8 mg/dL, P = 0.02), glucose tolerance improved (median [range] +33% [4%-138%], P = 0.04), insulin concentrations increased (+36.5% [-9.9% to 274.1%], P = 0.02), and glucagon concentrations decreased (-4.7% [0%-12.1%], P = 0.005). Compared with preinjection values on continuous glucose monitoring, glucose concentrations decreased and the frequency of readings <50 mg/dL increased at 2 and 6 wk after injection of exenatide ER. This did not correspond to clinical hypoglycemia. No other side effects were observed throughout the study. Exenatide ER was safe and effective in improving glucose tolerance 3 wk after a single injection. Further evaluation is needed to determine its safety, efficacy, and duration of action in diabetic cats.

Pharmacokinetics and pharmacodynamics of the glucagon-like peptide-1 analog liraglutide in healthy cats.

Glucagon-like peptide-1 (GLP-1) is an intestinal hormone that induces glucose-dependent stimulation of insulin secretion while suppressing glucagon secretion. Glucagon-like peptide-1 also increases beta cell mass and satiation while decelerating gastric emptying. Liraglutide is a fatty-acid derivative of GLP-1 with a protracted pharmacokinetic profile that is used in people for treatment of type II diabetes mellitus and obesity. The aim of this study was to determine the pharmacokinetics and pharmacodynamics of liraglutide in healthy cats. Hyperglycemic clamps were performed on days 0 (HGC) and 14 (LgHGC) in 7 healthy cats. Liraglutide was administered subcutaneously (0.6 mg/cat) once daily on days 8 through 14. Compared with the HGC (mean ± standard deviation; 455.5 ± 115.8 ng/L), insulin concentrations during LgHGC were increased (760.8 ± 350.7 ng/L; P = 0.0022), glucagon concentrations decreased (0.66 ± 0.4 pmol/L during HGC vs 0.5 ± 0.4 pmol/L during LgHGC; P = 0.0089), and there was a trend toward an increased total glucose infused (median [range] = 1.61 (1.11-2.54) g/kg and 2.25 (1.64-3.10) g/kg, respectively; P = 0.087). Appetite reduction and decreased body weight (9% ± 3%; P = 0.006) were observed in all cats. Liraglutide has similar effects and pharmacokinetics profile in cats to those reported in people. With a half-life of approximately 12 h, once daily dosing might be feasible; however, significant effects on appetite and weight loss may necessitate dosage or dosing frequency reductions. Further investigation of liraglutide in diabetic cats and overweight cats is warranted.

Genetic counselors' (GC) knowledge, awareness, understanding of clinical next-generation sequencing (NGS) genomic testing.

Genomic tests are increasingly complex, less expensive, and more widely available with the advent of next-generation sequencing (NGS). We assessed knowledge and perceptions among genetic counselors pertaining to NGS genomic testing via an online survey. Associations between selected characteristics and perceptions were examined. Recent education on NGS testing was common, but practical experience limited. Perceived understanding of clinical NGS was modest, specifically concerning tumor testing. Greater perceived understanding of clinical NGS testing correlated with more time spent in cancer-related counseling, exposure to NGS testing, and NGS-focused education. Substantial disagreement about the role of counseling for tumor-based testing was seen. Finally, a majority of counselors agreed with the need for more education about clinical NGS testing, supporting this approach to optimizing implementation.

Changes in the frequency of benign focal spikes accompany changes in central information processing speed: a prospective 2-year follow-up study.

We prospectively examined whether changes in the frequency of benign focal spikes accompany changes in cognition. Twenty-six children with benign focal spikes (19 with Rolandic epilepsy) and learning difficulties were examined with repeated 24-hour EEG recordings, three cognitive tests on central information processing speed (CIPS), and questionnaires on cognition and behavior at baseline, 6months, and 2years. Antiepileptic drug changes were allowed when estimated necessary by the treating physician. At baseline, a lower CIPS was correlated with a higher frequency of diurnal interictal epileptiform discharges (IEDs) and with worse academic achievement. At follow-up, there was a significant correlation between changes in CIPS and EEG changes in wakefulness (in the same direction) when the EEG outcome was dichotomized in IED frequency "increased" or "not increased". Behavioral problems were more often observed in patients with higher frequency of IEDs in sleep at baseline and in those with ongoing IEDs compared with those with EEG remission (without or with sporadic IEDs in the recording) at the end of the study period. No changes were observed in the results of the questionnaires. A lower diurnal IED frequency at baseline, lack of serial IEDs, and occurrence of only unilateral IEDs were correlated with a higher chance of EEG remission at 2-year follow-up. Electroencephalography remission could not be predicted from other epilepsy variables except from seizure freedom in the last six months. Our results confirm the nonbenign character of 'benign' focal spikes. Whether an early and stable EEG remission can be achieved through antiepileptic treatment and whether this is of benefit for cognitive development should be examined in prospective placebo-controlled randomized trials.

Improved method for screening mitochondrial cytochrome b markers to identify regional populations of the Old World screwworm fly and other myiasis agents.

A new protocol was developed to overcome obstacles to the high-throughput sequence analysis of the 716-717 nucleotides at the carboxyl terminal of the mitochondrial gene cytochrome b (cyt b) of the myiasis flies Chrysomya bezziana and Wohlfahrtia magnifica. For both of these obligate parasites, cyt b haplotypes provide diagnostic markers for phylogeographic populations, markers that identify the origins of emerging populations causing economically important myiasis in livestock and, in the case of C. bezziana (Old World screwworm fly), could help select reproductively-compatible populations for use in the Sterile insect technique as part of area wide integrated pest management. High sequence quality is important for unambiguously detecting the few mutations that are diagnostic for regional cyt b haplotypes and their lineages. A key innovation is the design of a new forward primer for the specific PCR amplification and high-quality sequencing of cyt b. The improved protocol will facilitate the use of this established comparative cyt b sequence analysis, not only by teams lacking the resources for whole genome sequencing (WGS) but also by those requiring reference sequences for developing comparative mitogenomics based on WGS.

Environmental and phylogeographical determinants of the distribution of the Old World screwworm fly in Indonesia.

The Old World screwworm (OWS) fly, Chrysomya bezziana, is an obligate parasite of livestock, and the myiasis caused by its larval infestations is economically important in Indonesia. The current spatial distribution of such a pest depends on two main factors: the current environmental conditions in which it can survive; and, its ability to occupy those environments by dispersal, which can be inferred from phylogeography and population genetics. These indicate that all OWS flies in Indonesia have mitochondrial cytochrome b (cyt b) haplotypes of the Asian lineage, and the regional separation of its four sub-lineages is the result of infrequent long-distance dispersal. We report the first investigation to associate regional cyt b sub-lineages of the OWS fly with environmental variables. Principal Components Analysis was used to demonstrate that these sub-lineages are associated with very similar macro-climates throughout Indonesia. Then, a species distribution model for the OWS fly in Indonesia was obtained by using the Maxent program. This indicated that elevation captured information not given by other environmental variables, and cattle density provided the most useful information by itself. The results of our study provide some important leads for future research, which will require better, stratified sampling.

A retrospective and geographical epidemiological survey of traumatic myiasis in southern Italy.

A survey on the prevalence and geographical distribution of traumatic myiasis in sheep, and the risk factors for the disease, was carried out in a region of southern Italy. A total of 138 sheep flocks were selected and visited to acquire data on the presence or absence of traumatic myiasis using both a questionnaire for retrospective analysis and animal inspection. Prevalences registered at the farm and animal levels, respectively, were 8.7% and 6.3% in 2010, and 5.8% and 5.0% in 2011. Records of the occurrence of the parasitic disease in this region are recent: a statistically significant (P < 0.01) progressive increase in the number of farms affected (from 0.7% to 8.7%) has been registered since 2007. Wohlfahrtiosis was found in 11 of 95 (11.6%) geographical units sampled and three significant (P < 0.05) clusters of spatial farm aggregation were identified in the southern part of the study area. A total of 158 presently uninfested farms were considered to be at high risk for transmission as a result of their proximity to infested farms. The spreading of Wohlfahrtia magnifica (Diptera: Sarcophagidae) in southern regions of Italy represents a warning that the risk for infestation may become more significant in other Italian regions, as well as other European countries.