Molecular typing of enteroviruses: comparing 5'UTR, VP1 and whole genome sequencing methods.

Enteroviruses (EV) commonly cause hand, foot and mouth disease (HFMD), and can also cause potentially fatal neurological and systemic complications. In our laboratory, sequencing 5' untranslated region (UTR) of the viral genome has been the routine method of genotyping EVs. During a recent localised outbreak of aseptic meningitis, sequencing the 5'UTR identified the causative virus as EV-A71, which did not fit with the clinical syndrome or illness severity. When genotyped using a different target gene, VP1, the result was different. This led us to evaluate the accuracy of the two different target genome regions and compare them against whole genome sequencing (WGS). We aimed to optimise the algorithm for detection and characterisation of EVs in the diagnostic laboratory. We hypothesised that VP1 and WGS genotyping would provide different results than 5'UTR in a subset of samples. Clinical samples from around New South Wales which were positive for EV by commercial polymerase chain reaction (PCR) assays were genotyped by targeting three different viral genome regions: the 5'UTR, VP1 and WGS. Sequencing was performed by Sanger and next generation sequencing. The subtyping results were compared. Of the 74/118 (63%) samples that were successfully typed using both the 5'UTR and the VP1 method, the EV typing result was identical for 46/74 (62%) samples compared to WGS as the gold standard. The same EV group but different EV types were found in 22/74 (30%) samples, and 6/74 (8%) samples belonged to different EV groups depending on typing method used. Genotyping with WGS and VP1 is more accurate than 5'UTR. Genotyping by the 5'UTR method is very sensitive, but less specific.

Angiographically Occult Subarachnoid Hemorrhage: Yield of Repeat Angiography, Influence of Initial CT Bleed Pattern, and Sources of Diagnostic Error in 242 Consecutive Patients.

BACKGROUND AND PURPOSE: Nearly 20% of patients with spontaneous SAH have no definitive source on initial DSA. The purpose of this study was to investigate the timing and yield of repeat DSA, to clarify the influence of initial CT bleed pattern, and to characterize sources of diagnostic error in this scenario. MATERIALS AND METHODS: We evaluated the yield of repeat DSA and clinical outcomes stratified by hemorrhage pattern on CT in consecutive patients with nontraumatic SAH with negative initial DSA findings at a referral center. Cases in which the culprit lesion was subsequently diagnosed were classified as physiologically occult (ie, undetectable) on the initial DSA, despite adequate technique and interpretation or misdiagnosed due to operator-dependent error. RESULTS: Two hundred forty-two of 1163 (20.8%) patients with spontaneous SAH had negative initial DSA findings between 2009 and 2018. The SAH CT pattern was nonperimesencephalic (41%), perimesencephalic (36%), sulcal (18%), and CT-negative (5%). Repeat DSA in 135/242 patients (55.8%) revealed a source in 10 patients (7.4%): 4 saccular aneurysms, 4 atypical aneurysms, and 2 arteriovenous shunts. The overall yield of repeat DSA was 11.3% with nonperimesencephalic and 2.2% for perimesencephalic patterns. The yield of the second and third DSAs with a nonperimesencephalic pattern was 7.7% and 12%, respectively. Physiologically occult lesions accounted for 6/242 (2.5%) and operator-dependent errors accounted for 7/242 (2.9%) of all angiographically occult lesions on the first DSA. CONCLUSIONS: Atypical aneurysms and small arteriovenous shunts are important causes of SAH negative on angiography. Improving DSAs technique can modestly reduce the need for repeat DSA; however, a small fraction of SAH sources remain occult despite adequate technique. These findings support the practice of repeating DSA in patients with a nonperimesencephalic SAH pattern.

Assessment of weight-based versus standard dosing of heparin in patients with unstable angina.

OBJECTIVE: To assess whether a weight-based dosing regimen (80 u/kg + 18 u/kg/h) or a standard-fixed dose regimen (5000 u + 1000 u/h) of heparin is more appropriate in patients with unstable angina (UA). METHOD: A drug use evaluation was conducted. Patient data for all patients weighing less than 100 kg who were in the coronary care unit of a Veterans Administration Hospital and who received heparin for UA (>24 h) over a 7-month period were included. For the first 4 months, patients were given standard heparin dosing and in the final 3 months patients were given heparin based on weight. The proportion of patients achieving activated partial thromboplastin times (aPTTs) that were at least therapeutic during therapy, the time to achieve the aPTT at a level that was at least therapeutic, and the number of patients with aPTTs over the therapeutic range were compared between groups. RESULTS: Patients in the group receiving weight-based heparin therapy (n = 23) were significantly more likely to achieve an aPTT that was at least therapeutic than patients receiving standard therapy (n = 42, 100% vs. 76%, respectively, P = 0.011). When all the patients in each group who achieved an aPTT that was at least therapeutic were compared, the weight-based group achieved the levels significantly faster than the standard-fixed dosing group (7.3 +/- 6.1 vs. 22.6 +/- 17.6 h, respectively, P = 0.0003). However, the use of weight-based dosing was associated with a higher incidence of achieving supertherapeutic aPTTs than standard therapy (78.3% vs. 50.0%, respectively, P = 0.049). CONCLUSION: Patients with UA may achieve therapeutic aPTTs faster than those on standard therapy but they also have a higher risk of achieving a supertherapeutic aPTT.

Validation of an in vitro RNA processing system for CT/CGRP precursor mRNA.

The pre-mRNA encoding calcitonin (CT) and calcitonin gene-related peptide (CGRP) is differentially processed in a tissue-specific fashion to include or exclude the calcitonin-specific exon 4. A minigene containing a viral first exon and exons 4, 5, and 6 from the human CT/CGRP gene was correctly processed in transfected HeLa or F9 teratocarcinoma cells to produce mRNA that included or excluded exon 4, respectively. This processing decision could be reproduced in vitro using nuclear extracts from these two cell lines and an RNA precursor from a similar minigene. Supplementation of extract from HeLa cells with extract from F9 cells resulted in the F9 splicing pattern in which exon 4 was excluded. This model system may be useful for the purification of splicing factors important in the regulation of this splice choice.

Calcitonin exon sequences influence alternative RNA processing.

The pre-mRNA encoding calcitonin (CT) and CT gene-related peptide (CGRP) is differentially processed in a tissue-specific fashion to include exon 4 (which encodes CT) or exclude this exon and splice to exon 5 (which encodes CGRP). We have used a CT-specific in vitro RNA-processing system to identify cis-acting sequences required to prevent splicing to exon 5. Deletion mapping demonstrated the presence of an element within the first 45 nucleotides of the CT-specific exon 4 that was required to suppress splicing to the CGRP-specific exon 5. This element was able to function in a completely heterologous system to suppress splicing when the CGRP exon was replaced with a constitutive viral exon. The element was unable to suppress splicing in the absence of a proximal CT-specific 3' splice site. Our results suggest that CT-specific splicing requires assisted recognition of its 3' splice site.