BackgroundSuboptimal agreement between molecular assays for the detection of Acanthamoeba spp. in clinical specimens has been demonstrated, and poor assay sensitivity directly imperils the vision of those affected by amoebic keratitis (AK) through delayed diagnosis. We sought to develop and validate a single Taqman real time PCR assay targeting the Acanthamoeba 18S rRNA gene that could be used to enhance sensitivity and specificity when paired with reference assays.MethodsBiobanked DNA from surplus delinked AK clinical specimens and 10 ATCC strains of Acanthamoeba was extracted. Sequence alignment of 66 18S rRNA regions from 12 species of Acanthamoeba known to cause keratitis informed design of a new TaqMan primer set. Performance of the new assay was compared to the 2 assays used currently in our laboratory.ResultsAmong 24 Acanthamoeba-positive and 83 negative specimens by the CDC reference standard, performance characteristics of the newly designed primer set were as follows: sensitivity 100%, specificity 94%, PPV 82.8%, and NPV 100%. Compared to culture, sensitivity of the new primer set was 100%, and specificity 96%. No cross-reactivity of the primer set to non-acanthamoebae, including Balamuthia and Naegleria, was found.ConclusionsWe have validated a real time PCR assay for the diagnosis of AK, and in doing so, have overcome important barriers to rapid and sensitive detection of acanthamoebae, including limited sensitivity and specificity of commonly used assays.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-017-2666-x) contains supplementary material, which is available to authorized users.
Acanthamoeba spp. are the causative pathogens of several infections, including amoebic keratitis (AK), a vision-threatening infection. Acanthamoebae from corneal specimens of patients with AK harbor bacterial endosymbionts, which may increase virulence. We sought to understand the spectrum of bacterial endosymbionts present in clinical isolates of Acanthamoeba spp. identified in our reference parasitology laboratory. Isolates of Acanthamoeba spp. obtained from our biobank of anonymized corneal scrapings were screened for potential endosymbionts by PCR using primer pairs detecting bacteria belonging to orders Chlamydiales, Rickettsiales, or Legionellales and pan16S primers. Three primer pairs specific to the 18s rRNA gene of Acanthamoeba spp. were used for the amplification of Acanthamoeba DNA used for sequencing. Sanger sequencing of all PCR products was performed, followed by BLAST analysis for species identification. We screened 26 clinical isolates of Acanthamoeba spp. for potential endosymbionts. Five isolates (19%) were found to contain bacterial DNA belonging to Legionellales. Three (11%) contained members of the Rickettsiales and Pseudomonas genticulata was detected in a Rickettsia-positive sample. One strain (4%) contained Neochlamydia hartmannellae, a member of the Chlamydiales order. Bacterial endosymbionts are prevalent in clinical strains of Acanthamoeba causing AK isolated from corneal scrapings. The demonstration of these organisms in clinical Acanthamoeba isolates supports a potential exploration of anti-endosymbiont therapeutics as an adjuvant therapy in the treatment of AK.
Medical schools have been striving to equip students with the tools and skills needed to serve patients from the LGBTQ + community, also called the Sexual and Gender Minority (SGM) community. This study aims to assess student comfort with providing care, and faculty knowledge and preparedness in delivering SGM-centered education at our home institution. We conducted two mixed-methods surveys, one geared towards medical students across four years of study and one towards medicine faculty. Each survey collected first demographic information about participants, then used a validated tool to assess knowledge of the SGM community. The qualitative component of both surveys then consisted of a needs assessment to determine what students felt should be changed about their curriculum, and what faculty felt should change about their training to deliver this curriculum. We received 26 student responses from all 4 years of study and 35 faculty responses from a variety of medical specialties. Difference in knowledge assessment scores was not statistically significant across both cohorts. Most students felt overall comfortable providing care for sexual minority individuals, and faculty similarly felt comfortable teaching, but data showcases that perceived comfort is higher among the student cohort. We propose that students are acquiring knowledge and comfort with providing for SGM individuals from sources outside their curriculum, and that additional training of faculty is vital to ensure students not doing this independent learning do not fall through the cracks.
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