A practical protocol to prepare paraffin-embedded whole tick histology sections.

Ticks are important ectoparasites that are capable of transmitting multiple classes of pathogens and are currently linked with many emerging tick-borne diseases worldwide. With increasing occurrences of tick-borne diseases in both humans and veterinary species, there is a continuous need to further our understanding of ticks and the pathogens they transmit. Whole tick histology provides a full scope of the tick internal anatomy, allowing researchers to examine multiple organs of interest in a single section. This is in contrast to other techniques that are more commonly utilized in tick-borne disease research, such as electron microscopy and light microscopy of individual organs. There is a lack of literature describing a practical technique to process whole tick histologic sections. Therefore, the current study aims to provide researchers with a workable protocol to prepare high quality paraffin-embedded whole tick histology sections. Amblyomma americanum adults were used as an example species for this study. After a series of pilot experiments using a combination of various fixatives, softening agents and processing techniques, we elected to compare two common fixatives, 10% neutral-buffered formalin (NBF) and Bouin's solution for whole ticks. Equal numbers of A. americanum unfed adults (n = 10/fixative) were processed identically and their whole tick histology coronal sections were individually scored. Higher scores were assigned to whole tick sections that contained more internal organs that are crucial for tick-borne disease research (e.g. salivary glands and midgut), high integrity of tissues and exoskeleton on the section, and good fixation and staining quality of the tissues. The mean total scores for Bouin's-fixed ticks were significantly higher compared to NBF-fixed ticks (p = 0.001). To further assess our preferred technique, we also demonstrated the feasibility of producing high quality whole tick sections for three other common tick species of medical importance (Rhipicephalus sanguineus, Ixodes scapularis, and Dermacentor variabilis) using Bouin's solution. While this technique may require further optimization for other tick species, we described a feasible protocol that uses commonly available tools, reagents and standard histologic equipment. This should allow any investigator to easily make adjustments to this protocol as needed based on their experimental goals.

Cytauxzoon felis in salivary glands of Amblyomma americanum.

Cytauxzoon felis is a tick-borne piroplasmid hemoparasite that causes life-threatening disease in cats. Despite the critical role that ticks play in pathogen transmission, our knowledge regarding the C. felis life cycle remains limited to the feline hosts. Specific life stages of C. felis within the tick host have never been visualized microscopically and previous investigations have been limited to molecular detection by polymerase chain reaction (PCR). Sporozoites are the infectious stage of piroplasmids that are transmitted by ticks. In other tick-borne piroplasmids, sporozoite-based vaccines play a key role in disease prevention and management. We believe sporozoites have similar potential for cytauxzoonosis. Therefore, the objective of this study was to use different molecular and microscopic techniques to detect and evaluate C. felis sporozoites in tick salivary glands (SG). A total of 140 Amblyomma americanum adults that were fed on C. felis-infected cats as nymphs were included for this study. Specifically, dissected SGs were quartered and subjected to C. felis RT-PCR, RNAscope® in situ hybridization (ISH), histology, direct azure staining, and transmission electron microscopy (TEM). Cytauxzoon felis RT-PCR was also performed on half tick (HT) carcasses after SG dissection. Cytauxzoon felis RNA was detected in SGs of 17/140 ticks. Of these, 7/17 ticks had microscopic visualization via ISH and/or TEM. The remaining 10/17 ticks had only molecular detection of C. felis in SGs via RT-PCR without visualization. Cytauxzoon felis RNA was detected solely in HT carcasses via RT-PCR in 9/140 ticks. In ISH-positive tick SGs, hybridization signals were present in cytoplasms of SG acinar cells. TEM captured rare C. felis organisms with characteristic ultrastructural features of sporozoites. This study describes the first direct visualization of any developing stage of C. felis in ticks. Forthcoming studies should employ a combination of molecular and microscopic techniques to investigate the C. felis life cycle in A. americanum.

Cochlear Enhancement May Precede Cochlear Obliteration After Vestibular Schwannoma Excision.

OBJECTIVE: Cochlear obliteration after vestibular schwannoma excision has been noted, with implications on cochlear implantation. Early postoperative cochlear enhancement with gadolinium on magnetic resonance imaging (MRI) has also been observed. Timing of enhancement and association with obliteration is described here. STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral center, ambulatory. PATIENTS: Patients receiving vestibular schwannoma excision surgery by the senior author performed at one institution between January 2015 and July 2017 with postoperative MRIs INTERVENTION:: Diagnostic. MAIN OUTCOME MEASURE(S): The imaging characteristics on postoperative MRIs examined were loss of fluid signal on postoperative T2 images and cochlear enhancement on gadolinium enhanced T1 images. In the patients receiving labyrinthine sparing procedures, presence of postoperative hearing was evaluated. RESULTS: Of the 42 patients evaluated, 24 received the translabyrinthine approach and 18 received a labyrinth sparing surgery. Twenty-nine had evidence of cochlear enhancement on T1 with gadolinium contrast, and 27 had evidence of cochlear obliteration on T2 images. The odds ratio of patients with cochlear enhancement having obliteration was 30.0:1 (p < 0.0001). Intense cochlear enhancement (n = 21) appeared a median of 163 days after surgery, and complete or near complete obliteration (n = 18) appeared a median of 480 days after surgery, a statistically significant difference (p < 0.001). Within the labyrinth sparing group, there was no statistically significant association between hearing loss and cochlear obliteration or enhancement. CONCLUSIONS: Cochlear enhancement is correlated with cochlear obliteration and may precede it.

Is MRI Equal to CT in the Evaluation of Thin and Dehiscent Superior Semicircular Canals?

OBJECTIVE: Can magnetic resonance imaging (MRI) diagnose abnormally thin and dehiscent superior semicircular canals (SSCs) that traditionally rely on evaluation by computed tomography (CT) imaging? STUDY DESIGN: Retrospective clinical study. SETTING: Tertiary referral center. PATIENTS: Adults who underwent both MRI and CT of the temporal bones over the past 3 years. INTERVENTIONS: CT and MR images of SSCs were separately reviewed, in a blinded fashion by three neuroradiologists at our institution. CT diagnosis of abnormally thin or dehiscent SSC was used as the "gold" standard. MAIN OUTCOME MEASURES: 1) Dehiscent SSC. 2) Abnormally thin SSC. 3) Normal SSC. RESULTS: One hundred temporal bones with evaluable superior semicircular canals from 51 patients were eligible for review on CT and MR imaging. There were 26 patients of thin SSC and 17 patients of SSC dehiscence on CT imaging, of which 13 and 15 respectively were also found on MRI. There were nine false-positive dehiscent SSC patients and four thin SSC patients observed on MR imaging while not observed on CT. For thin SSCs, MRI sensitivity was 61.9% and specificity of 94.3% with a positive predictive value of 81.3% and a negative predictive value of 86.2%. For dehiscent SSCs, sensitivity was 88.2% and specificity of 89.2% with a positive predictive value of 62.5% and a negative predictive value of 97.4%. CONCLUSION: In this series, MRI in the axial and coronal plane had a high negative predicative value for thin SSC (86%) and dehiscent SSC (97%). However, MRI cannot conclusively diagnose thin or dehiscent SSCs.

Does subspecialty referral affect the pretest probability for an abnormal finding on knee MRI of pediatric patients?

OBJECTIVE: Pediatricians and orthopedists comprise the largest referral basis for knee MR examinations at our institution. In an era of cost optimization, the purpose of this study was to compare differences in pretest probability for an abnormal finding on knee MRI based on referral subspecialty. MATERIALS AND METHODS: A retrospective review of 501 consecutive knee MR examinations of pediatric patients (56% male; mean age, 14 years; age range, 1-18 years) referred by nononcology orthopedic surgeons and 93 consecutive knee MR examinations of patients (47% male; mean age, 14 years; age range, 2-18 years) referred by general pediatricians from 2005 to 2009 were reviewed. Two patient groups based on the MR report were established: patients with entirely normal MRI findings and those who had a clinically significant MRI finding or findings. The latter group included children with any internal derangement, a discoid meniscus, a neoplasm, or evidence of infection or inflammation. RESULTS: The incidence of an entirely normal knee MRI from pediatrician referral and orthopedic referral was 24% (22/93) and 17% (87/501) (p = 0.15), respectively. The respective incidence of selected specific injuries identified from pediatrician and orthopedic referral included any internal derangement, 39.8% and 48.3% (p = 0.13); neoplasm, 2.2% and 1.4% (p = 0.64); and inflammation, 6.5% and 2.0% (p = 0.03). CONCLUSION: Despite differences in subspecialty training, we found no significant differences in the proportion of normal knee MRI examinations and no statistical difference in the proportion of patients with internal derangement. Further investigation is necessary to determine whether these findings translate into overall cost-savings or differences in patient outcome.