Development and evaluation of a two-step multiplex TaqMan real-time PCR assay for detection/quantification of different genospecies of Borrelia burgdorferi sensu lato

Nowadays, at least four clinically important B. burgdorferi sensu lato (s.l.) genospecies (B. afzelii, B. garinii, B. burgdorferi sensu stricto (s.s.) and B. lusitaniae) circulate in Portugal. Each genospecies has a different tropism that resuls in a diverse array of clinical manifestations. The standard diagnostic procedure used is normally simple, nevertheless, during the “window-period” phase, in which specific antibodies cannot yet be detected, diagnosis becomes difficult, and calls for reliable, sensitive and specific laboratory methods, such as molecular tests. The aim of this study was to develop and evaluate a multiplex TaqMan real-time PCR assay to infer the presence of B. burgdorferi s.l. genospecies in clinical and vector-derived samples. The assay consists of two steps: (i) a first duplex real-time PCR targeting both flaB of B. burgdorferi s.l., and an internal control (18S rDNA for tick samples or the mammal β-actin gene for clinical samples); and (ii) a second tetraplex real-time PCR targeting the flaB gene of B. afzelii, B. garinii, B. burgdorferi s.s. and B. lusitaniae. The first step revealed a high specificity and sensitivity, allowing the detection of as low as 20 genome equivalents (GE) of B. burgdorferi s.l. from isolated cultures, clinical samples and ticks. The second step revealed high specificity, but a slightly lower sensitivity (2×102 GE) for detection of B. afzelii, B. garinii, B. burgdorferi s.s. and B. lusitaniae in purified DNA extracts, and particularly when testing cerebrospinal fluid (CSF) samples. Nonetheless, both real-time PCR protocols were developed to be applied at the beginning of the infection, to improve early diagnosis of Lyme borreliosis (LB), where detection of Borrelia should not rely on the use of CSF samples. The assay here described is of special interest for the analysis of both environmental and clinical samples, being advantageous in the former phase screening of Lyme borreliosis, when the efficiency of serologically based diagnoses may be seriously compromised

Master of Pharmacy students’ knowledge and awareness of antibiotic use, resistance and stewardship

Background. Antibiotic resistance has become a global public health concern. In this study we investigated the knowledge and awareness of antibiotic use, resistance and stewardship, held by the pharmacy students currently studying at the University of Brighton.Study design. This was a cross-sectional, online survey, and email invitations to participate were sent to all students attending our Master of Pharmacy (MPharm) course (n = 583). Students’ knowledge was assessed with 29 items; responses for these were totaled before comparison among students.Comparison of scores between groups of students was performed using the Kruskal-Wallis or the MannWhitney test, as appropriate.Results. The response rate was 32%. The overall median knowledge score was 7.9. There was a statistically significant difference in knowledge scores between years of study (p = 0.02), particularly between year of study 1 (7.6) and 4 (8.3). A statistically significant difference was found between the knowledge scores of male (8.4) and female (7.9) students (p = 0.03). Most students believed a strong knowledge of antibiotics, and microbiology and infection control is important for their pharmacy careers and more than 90% agreed that antibiotic resistance will be a greater clinical problem in thefuture.Conclusions. Although the MPharm students studied achieved good overall knowledge scores, a significant proportion showed a lack of understanding with regards to some important aspects of antibiotic resistance mechanisms, factors promoting the emergence and spread of antibioticresistance, and antibiotic stewardship policies

A field evaluation of an isothermal DNA amplification assay for the detection of Theileria annulata infection in cattle

A loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Theileria annulata infection in cattle. The results were compared with a real-time PCR used for the quantification of T. annulata parasitaemia. One hundred bovine blood samples from 16 cattle farms were tested with LAMP and real-time PCR, with T. annulata DNA being detected in 66% and 67% of the samples, respec- tively. The results showed that the LAMP assay detects a parasitaemia as low as 0.00025%, indicating ahigh analytical sensitivity of LAMP for clinical diagnosis of bovine theileriosis

Kerion caused by Microsporum audouinii in a child

Kerion celsi is rarely associated with Microsporum audouinii infection. We report the case of a 3-year-old girl with a kerion celsi caused by M. audouinii and successfully treated with oral terbinafine. Fungi identification was made by macro and microscopical colony morphology analyses and molecular (genotypic) studies

Environmental distribution of Cryptococcus neoformans and Cryptococcus gattii around the Mediterranean basin

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-15. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected, and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus, and two Olea trees. C. neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy, and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent animportant niche for these yeasts

Fundamental niche prediction of the pathogenic yeasts cryptococcus neoformans and cryptococcus gattii in europe

Fundamental niche prediction of Cryptococcus neoformans and Cryptococcus gattii in Europe is an important tool to understand where these pathogenic yeasts have a high probability to survive in the environment and therefore to identify the areas with high risk of infection. In this study, occurrence data for C. neoformans and C. gattii were compared by MaxEnt software with several bioclimatic conditions as well as with soil characteristics and land use. The results showed that C. gattii distribution can be predicted with high probability along the Mediterranean coast. The analysis of variables showed that its distribution is limited by low temperatures during the coldest season, and by heavy precipitations in the driest season. C. neoformans var. grubii is able to colonize the same areas of C. gattii but is more tolerant to cold winter temperatures and summer precipitations. In contrast, the C. neoformans var. neoformans map was completely different. The best conditions for its survival were displayed in sub-continental areas and not along the Mediterranean coasts. In conclusion, we produced for the first time detailed prediction maps of the species and varieties of the C. neoformans and C. gattii species complex in Europe andMediterranean area