Consensus statement on the interhospital transfer of patients with acute aortic syndrome: TRAVERSING Delphi study.

BACKGROUND: Standardisation of referral pathways and the transfer of patients with acute aortic syndromes (AAS) to regional centres are recommended by NHS England in the Acute Aortic Dissection Toolkit. The aim of the Transfer of Thoracic Aortic Vascular Emergencies to Regional Specialist INstitutes Group study was to establish an interdisciplinary consensus on the interhospital transfer of patients with AAS to specialist high-volume aortic centres. METHODS: Consensus on the key aspects of interhospital transfer of patients with AAS was established using the Delphi method, in line with Conducting and Reporting of Delphi Studies guidelines. A national patient charity for aortic dissection was involved in the design of the Delphi study. Vascular and cardiothoracic surgeons, emergency physicians, interventional radiologists, cardiologists, intensivists and anaesthetists in the United Kingdom were invited to participate via their respective professional societies. RESULTS: Three consecutive rounds of an electronic Delphi survey were completed by 212, 101 and 58 respondents, respectively. Using predefined consensus criteria, 60 out of 117 (51%) statements from the survey were included in the consensus statement. The study concluded that patients can be taken directly to a specialist aortic centre if they have typical symptoms of AAS on the background of known aortic disease or previous aortic intervention. Accepted patients should be transferred in a category 2 ambulance (response time <18 min), ideally accompanied by transfer-trained personnel or Adult Critical Care Transfer Services. A clear plan should be agreed in case of a cardiac arrest occurring during the transfer. Patients should reach the aortic centre within 4 hours of the initial referral from their local hospital. CONCLUSIONS: This consensus statement is the first set of national interdisciplinary recommendations on the interhospital transfer of patients with AAS. Its implementation is likely to contribute to safer and more standardised emergency referral pathways to regional high-volume specialist aortic units.

Interactions of Melanoma Cells with Distal Keratinocytes Trigger Metastasis via Notch Signaling Inhibition of MITF.

The most critical stage in initiation of melanoma metastasis is the radial to vertical growth transition, yet the triggers of this transition remain elusive. We suggest that the microenvironment drives melanoma metastasis independently of mutation acquisition. Here we examined the changes in microenvironment that occur during melanoma radial growth. We show that direct contact of melanoma cells with the remote epidermal layer triggers vertical invasion via Notch signaling activation, the latter serving to inhibit MITF function. Briefly, within the native Notch ligand-free microenvironment, MITF, the melanocyte lineage master regulator, binds and represses miR-222/221 promoter in an RBPJK-dependent manner. However, when radial growth brings melanoma cells into contact with distal differentiated keratinocytes that express Notch ligands, the activated Notch intracellular domain impairs MITF binding to miR-222/221 promoter. This de-repression of miR-222/221 expression triggers initiation of invasion. Our findings may direct melanoma prevention opportunities via targeting specific microenvironments.

Infection, nutritional status, and body composition: Associations at birth and 6 months postnatally in Soweto, South Africa.

INTRODUCTION: The impact of infection on infant nutritional status, body size, and growth is well documented. However, research into the impact of infection on infant body composition is limited. Greater understanding is, therefore, needed on the effects of infection in early life. METHODS: Associations between a composite morbidity index consisting of the sum of the cumulative tallies for a range of symptoms representing infection and morbidity in the infants and nutritional status (height-for-age (HAZ), and weight-for-height (WHZ)), and body composition (fat-free mass (FFM), fat mass (FM), fat-free mass index (FFMI), and fat mass index (FMI)) at 6 months of age were investigated using hierarchical regression analysis. RESULTS: The sample comprised data between birth and 6 months postnatally, of 156 infants who were a priori born healthy in Soweto, South Africa. Morbidity, over the cumulative period of birth to 6 months, was associated with lower FMI (ß = -1.77) and lower FM (ß = -0.61), and conversely with higher FFM (ß = 0.94), in infants at 6 months. No associations were found between the morbidity index and FFMI, HAZ, and WHZ. Increased birthweight was associated with a higher FFM (ß = 0.66), HAZ (ß = 1.14), and WHZ (ß = 0.87). Finally, safely managed sanitation facilities, representative of reduced environmental exposure to fecal-oral transmission pathways were associated with a higher HAZ (ß = 1.21). DISCUSSION: Reduction in FMI and FM and exposure to inflammatory cytokines associated with mounting an immune response could alter phenotypic trajectories during to this period of plasticity. From a public health perspective, these results imply that it is important to intensify efforts to prevent infection in infants in the first 6 months postnatally, and that these efforts should concentrate on access to safely managed sanitation facilities.

Keloid tissue analysis discredits a role for myofibroblasts in disease pathogenesis.

Myofibroblasts, renowned for their contractility and extracellular matrix production, are widely considered the key effector cells for nearly all scars resulting from tissue repair processes, ranging from normal scars to extreme fibrosis. For example, it is often assumed that myofibroblasts underpin the characteristics of keloid scars, which are debilitating pathological skin scars lacking effective treatments because of a poor understanding of the disease mechanisms. Here, we present primary and published transcriptional and histological evidence that myofibroblasts are not consistently present in primary keloid lesions, and when alpha-smooth muscle actin (αSMA)-positive cells are detected, they are not greater in number or expressing more αSMA than in normal or hypertrophic scars. In conclusion, keloid scars do not appear to require αSMA-positive myofibroblasts; continuing to consider keloids on a quantitative spectrum with normal or hypertrophic scars, with αSMA serving as a biomarker of disease severity, is hindering advancement of understanding and therapy development.

Editor's Choice - A Comparison of Computed Tomography Angiography and Colour Duplex Ultrasound Surveillance Post Infrarenal Endovascular Aortic Aneurysm Repair: Financial Implications and Impact of Different International Surveillance Guidelines.

OBJECTIVE: Use of colour duplex ultrasound (CDUS) and computed tomography angiography (CTA) for infrarenal endovascular aortic aneurysm repair (EVAR) surveillance differs in internationally published guidelines. This study aimed firstly to compare CDUS detection of significant sac abnormalities with CTA. Secondly, a sensitivity analysis was conducted to compare financial estimates of the, predominantly CDUS based, local and Society of Vascular Surgery (SVS) protocols, the risk stratified European Society of Vascular Surgery (ESVS) protocol, and the CTA based National Institute of Health and Care Excellence (NICE) protocol. METHODS: Agreement between CDUS and CTA was assessed for detection of significant sac abnormalities. Surveillance protocols were extrapolated from published guidelines and applied to infrarenal EVAR patients active on local surveillance at a large, single centre. Surveillance intensity was dependent on presence of endoleak and subsequent risk of treatment failure in accordance with surveillance recommendations. Estimates for each surveillance protocol were inclusive of a range of published incidences of endoleak, contrast associated acute kidney injury (AKI), and excess hospital bed days, and estimated for a hypothetical five year surveillance period. RESULTS: The kappa coefficient between CDUS and CTA for detecting sac abnormalities was 0.68. Maximum five year surveillance cost estimates for the 289 active EVAR patients were £272 359 for SVS, £230 708 for ESVS, £643 802 for NICE, and £266 777 for local protocols, or £1 270, £1 076, £3 003, and £1 244 per patient. Differences in endoleak incidence accounted for a 1.1 to 1.4 fold increase in costs. AKI incidence accounted for a 3.3 to 6.2 fold increase in costs. CONCLUSION: A combined CTA and CDUS EVAR surveillance protocol, with CTA reserved for early seal assessment and confirmatory purposes, provides an economical approach without compromising detection of sac abnormalities. AKI, as opposed to direct imaging costs, accounted for the largest differences in surveillance cost estimates.

Alzheimer's pathology targets distinct memory networks in the ageing brain.

Alzheimer's disease researchers have been intrigued by the selective regional vulnerability of the brain to amyloid-ß plaques and tau neurofibrillary tangles. Post-mortem studies indicate that in ageing and Alzheimer's disease tau tangles deposit early in the transentorhinal cortex, a region located in the anterior-temporal lobe that is critical for object memory. In contrast, amyloid-ß pathology seems to target a posterior-medial network that subserves spatial memory. In the current study, we tested whether anterior-temporal and posterior-medial brain regions are selectively vulnerable to tau and amyloid-ß deposition in the progression from ageing to Alzheimer's disease and whether this is reflected in domain-specific behavioural deficits and neural dysfunction. 11C-PiB PET and 18F-flortaucipir uptake was quantified in a sample of 131 cognitively normal adults (age: 20-93 years; 47 amyloid-ß-positive) and 20 amyloid-ß-positive patients with mild cognitive impairment or Alzheimer's disease dementia (65-95 years). Tau burden was relatively higher in anterior-temporal regions in normal ageing and this difference was further pronounced in the presence of amyloid-ß and cognitive impairment, indicating exacerbation of ageing-related processes in Alzheimer's disease. In contrast, amyloid-ß deposition dominated in posterior-medial regions. A subsample of 50 cognitively normal older (26 amyloid-ß-positive) and 25 young adults performed an object and scene memory task while functional MRI data were acquired. Group comparisons showed that tau-positive (n = 18) compared to tau-negative (n = 32) older adults showed lower mnemonic discrimination of object relative to scene images [t(48) = -3.2, P = 0.002]. In a multiple regression model including regional measures of both pathologies, higher anterior-temporal flortaucipir (tau) was related to relatively worse object performance (P = 0.010, r = -0.376), whereas higher posterior-medial PiB (amyloid-ß) was related to worse scene performance (P = 0.037, r = 0.309). The functional MRI data revealed that tau burden (but not amyloid-ß) was associated with increased task activation in both systems and a loss of functional specificity, or dedifferentiation, in posterior-medial regions. The loss of functional specificity was related to worse memory. Our study shows a regional dissociation of Alzheimer's disease pathologies to distinct memory networks. While our data are cross-sectional, they indicate that with ageing, tau deposits mainly in the anterior-temporal system, which results in deficits in mnemonic object discrimination. As Alzheimer's disease develops, amyloid-ß deposits preferentially in posterior-medial regions additionally compromising scene discrimination and anterior-temporal tau deposition worsens further. Finally, our findings propose that the progression of tau pathology is linked to aberrant activation and dedifferentiation of specialized memory networks that is detrimental to memory function.

Subthreshold Amyloid Predicts Tau Deposition in Aging.

Current approaches to the early detection of Alzheimer's disease (AD) rely upon classifying individuals as "positive" or "negative" for biomarkers related to the core pathology of ß-amyloid (Aß). However, the accumulation of Aß begins slowly, years before biomarkers become abnormal. We used longitudinal [11C] Pittsburgh Compound B PET scanning and neuropsychological assessment to investigate the earliest changes in AD pathology and how it affects memory in cognitively normal older humans (N = 71; mean age 75 years; 35% male). We used [18F] AV-1451 PET scanning at the end of the observation period to measure subsequent tau deposition in a subset of our sample (N = 37). We found evidence for an inverted-U relationship between baseline Aß levels and Aß slope in asymptomatic older adults, suggesting a slowing of Aß accumulation even in cognitively normal adults. In participants who were nominally amyloid negative, both the rate of amyloid accumulation and the baseline levels of Aß predicted early tau deposition in cortical Braak regions associated with AD. Amyloid measures were only sensitive to memory decline as baseline levels of Aß increased, suggesting that pathological accumulation occurs before impacting memory. These findings support the necessity of early intervention with amyloid-lowering therapies even in those who are amyloid negative.SIGNIFICANCE STATEMENT The progressive nature of Alzheimer's disease (AD) necessitates the earliest possible detection of pathological or cognitive change if disease progression is to be slowed. We examined cognitively normal older adults in whom AD pathology is starting to develop, with the goal of early detection of AD pathology or cognitive changes. We found amyloid measures to be sensitive early on in predicting subsequent early tau deposition. Further, it appears that rates of amyloid accumulation already begin to slow in preclinical AD, suggesting that it is a relatively late stage of AD progression. Thus, it is crucial to examine older adults early, before amyloid levels have saturated, to intervene to slow disease progression.

Entorhinal Tau Pathology, Episodic Memory Decline, and Neurodegeneration in Aging.

The medial temporal lobe (MTL) is an early site of tau accumulation and MTL dysfunction may underlie episodic-memory decline in aging and dementia. Postmortem data indicate that tau pathology in the transentorhinal cortex is common by age 60, whereas spread to neocortical regions and worsening of cognition is associated with ß-amyloid (Aß). We used [18F]AV-1451 and [11C]PiB positron emission tomography, structural MRI, and neuropsychological assessment to investigate how in vivo tau accumulation in temporal lobe regions, Aß, and MTL atrophy contribute to episodic memory in cognitively normal older adults (n = 83; age, 77 ± 6 years; 58% female). Stepwise regressions identified tau in MTL regions known to be affected in old age as the best predictor of episodic-memory performance independent of Aß status. There was no interactive effect of MTL tau with Aß on memory. Higher MTL tau was related to higher age in the subjects without evidence of Aß. Among temporal lobe subregions, episodic memory was most strongly related to tau-tracer uptake in the parahippocampal gyrus, particularly the posterior entorhinal cortex, which in our parcellation includes the transentorhinal cortex. In subjects with longitudinal MRI and cognitive data (n = 57), entorhinal atrophy mirrored patterns of tau pathology and their relationship with memory decline. Our data are consistent with neuropathological studies and further suggest that entorhinal tau pathology underlies memory decline in old age even without Aß.SIGNIFICANCE STATEMENT Tau tangles and ß-amyloid (Aß) plaques are key lesions in Alzheimer's disease (AD) but both pathologies also occur in cognitively normal older people. Neuropathological data indicate that tau tangles in the medial temporal lobe (MTL) underlie episodic-memory impairments in AD dementia. However, it remains unclear whether MTL tau pathology also accounts for memory impairments often seen in elderly people and how Aß affects this relationship. Using tau-specific and Aß-specific positron emission tomography tracers, we show that in vivo MTL tau pathology is associated with episodic-memory performance and MTL atrophy in cognitively normal adults, independent of Aß. Our data point to MTL tau pathology, particularly in the entorhinal cortex, as a substrate of age-related episodic-memory loss.

Enhancer methylation dynamics contribute to cancer plasticity and patient mortality.

During development, enhancers play pivotal roles in regulating gene expression programs; however, their involvement in cancer progression has not been fully characterized. We performed an integrative analysis of DNA methylation, RNA-seq, and small RNA-seq profiles from thousands of patients, including 25 diverse primary malignances and seven body sites of metastatic melanoma. We found that enhancers are consistently the most differentially methylated regions (DMR) as cancer progresses from normal to primary tumors and then to metastases, compared to other genomic features. Remarkably, identification of enhancer DMRs (eDMRs) enabled classification of primary tumors according to physiological organ systems, and in metastasis eDMRs are the most correlated with patient outcome. To further understand the eDMR role in cancer progression, we developed a model to predict genes and microRNAs that are regulated by enhancer and not promotor methylation, which shows high accuracy with chromatin architecture methods and was experimentally validated. Interestingly, among all metastatic melanoma eDMRs, the most correlated with patient survival were eDMRs that "switched" their methylation patterns back and forth between normal, primary, and metastases and target cancer drivers, e.g., KIT We further demonstrated that eDMR target genes were modulated in melanoma by the bone metastasis microenvironment, suggesting that eDMRs respond to microenvironmental cues in metastatic niches. Our findings that aberrant methylation in cancer cells mostly affects enhancers, which contribute to tumor progression and cancer cell plasticity, will facilitate development of epigenetic anticancer approaches.

Streptococcus pneumoniae as a Cause of Mycotic and Infected Aneurysms in Patients without Respiratory Features: Challenging Diagnoses Aided by 16S PCR.

BACKGROUND: Streptococcus pneumoniae is considered a rare cause of mycotic aneurysms. The microbiological diagnosis of mycotic aneurysms can be difficult, and many patients have negative blood culture results. METHODS: We describe a series of four consecutive cases of mycotic aneurysms caused by S. pneumoniae with no respiratory features or extravascular septic foci. In two patients with negative blood culture results, 16S PCR was used for the diagnosis of S. pneumoniae infection. RESULTS: Four men with mycotic aneurysms affecting the aorta, axillary, and popliteal arteries caused by S. pneumoniae presented to our center between 2015 and 2016. All were treated with at least one month of intravenous antibiotics, followed by at least 4 weeks of oral antibiotics. Two were additionally managed using endovascular surgical techniques, and one underwent an open surgical repair. The fourth patient presented with bilateral popliteal aneurysms, one of which ruptured and was managed using surgical ligation and bypass, whereas the other side subsequently ruptured and was repaired endovascularly. Three of the four patients are currently off antibiotics and considered cured, while one died of an unrelated cause. CONCLUSIONS: S. pneumoniae should be considered a potential causative agent of mycotic aneurysms. Diagnosis can be confirmed using 16S PCR, especially in patients where peripheral blood cultures are uninformative.

Amyloid and tau PET demonstrate region-specific associations in normal older people.

ß-amyloid (Aß) and tau pathology become increasingly prevalent with age, however, the spatial relationship between the two pathologies remains unknown. We examined local (same region) and non-local (different region) associations between these 2 aggregated proteins in 46 normal older adults using [18F]AV-1451 (for tau) and [11C]PiB (for Aß) positron emission tomography (PET) and 1.5T magnetic resonance imaging (MRI) images. While local voxelwise analyses showed associations between PiB and AV-1451 tracer largely in the temporal lobes, k-means clustering revealed that some of these associations were driven by regions with low tracer retention. We followed this up with a whole-brain region-by-region (local and non-local) partial correlational analysis. We calculated each participant's mean AV-1451 and PiB uptake values within 87 regions of interest (ROI). Pairwise ROI analysis demonstrated many positive PiB-AV-1451 associations. Importantly, strong positive partial correlations (controlling for age, sex, and global gray matter fraction, p<.01) were identified between PiB in multiple regions of association cortex and AV-1451 in temporal cortical ROIs. There were also less frequent and weaker positive associations of regional PiB with frontoparietal AV-1451 uptake. Particularly in temporal lobe ROIs, AV-1451 uptake was strongly predicted by PiB across multiple ROI locations. These data indicate that Aß and tau pathology show significant local and non-local regional associations among cognitively normal elderly, with increased PiB uptake throughout the cortex correlating with increased temporal lobe AV-1451 uptake. The spatial relationship between Aß and tau accumulation does not appear to be specific to Aß location, suggesting a regional vulnerability of temporal brain regions to tau accumulation regardless of where Aß accumulates.

Mycotic Aneurysm following a Dog Bite: The Value of the Clinical History and Molecular Diagnostics.

A 63-year-old Caucasian taxi driver presented with a 3-week history of malaise, night sweats, 7 kg weight loss, generalized arthralgia, and persistent mid-lower abdominal pain. Blood inflammatory markers were raised, and a computed tomography scan demonstrated an irregular degeneration of the infrarenal aorta, with a differential diagnosis including aortic infection. An urgent type IV thoracoabdominal aneurysm repair was performed with a rifampicin-soaked aortic tube graft during an open procedure. No organisms were grown from multiple peripheral blood cultures or culture of the affected aorta. However, subsequent 16S ribosomal polymerase chain reaction analysis of the resected aorta identified Capnocytophaga canimorsus as the causative organism-a commensal that lives in the mouth of dogs and cats. The patient subsequently gave a history of multiple bites from his pet dog over recent months-the likely source of infection. He was treated with 8 weeks of intravenous antibiotics before switching to oral antibiotics for an additional 6 weeks.

Pax6 regulates gene expression in the vertebrate lens through miR-204.

During development, tissue-specific transcription factors regulate both protein-coding and non-coding genes to control differentiation. Recent studies have established a dual role for the transcription factor Pax6 as both an activator and repressor of gene expression in the eye, central nervous system, and pancreas. However, the molecular mechanism underlying the inhibitory activity of Pax6 is not fully understood. Here, we reveal that Trpm3 and the intronic microRNA gene miR-204 are co-regulated by Pax6 during eye development. miR-204 is probably the best known microRNA to function as a negative modulator of gene expression during eye development in vertebrates. Analysis of genes altered in mouse Pax6 mutants during lens development revealed significant over-representation of miR-204 targets among the genes up-regulated in the Pax6 mutant lens. A number of new targets of miR-204 were revealed, among them Sox11, a member of the SoxC family of pro-neuronal transcription factors, and an important regulator of eye development. Expression of Trpm/miR-204 and a few of its targets are also Pax6-dependent in medaka fish eyes. Collectively, this study identifies a novel evolutionarily conserved mechanism by which Pax6 controls the down-regulation of multiple genes through direct up-regulation of miR-204.

The prevalence and impact of undiagnosed cognitive impairment in older vascular surgical patients.

OBJECTIVE: The objectives of this observational cohort study were to investigate the prevalence of undiagnosed cognitive impairment in older patients presenting for vascular surgery, to examine its association with adverse postoperative outcomes, and to test the feasibility of a preoperative cognitive assessment tool. METHODS: Patients aged 60 years or older were recruited by consent on admission to the vascular surgical ward of an inner-city teaching hospital with a large tertiary referral practice for proposed elective or emergency aortic or lower limb arterial intervention. Cognition was assessed preoperatively by the Montreal Cognitive Assessment (MoCA), and a score below 24/30 indicated cognitive impairment or dementia. The mean length of time taken to complete the assessment was recorded. Baseline characteristics (medical multimorbidity, frailty, and laboratory tests), hospital length of stay (LOS), and postoperative complications were documented. RESULTS: Preoperative MoCA was completed in 114 patients with a mean age of 76.3 years (standard deviation, 7.36 years); 67.5% were men, and 55.3% of procedures were elective. The MoCA was completed in 100% of patients and was quick and acceptable to patients in this setting. Cognitive impairment or dementia was found in 68% of patients (77 of 114) and was previously unrecognized in 88.3% of patients (68 of 77). Therefore, 60.5% of patients (68 of 114) aged 60 years or older presenting for vascular surgery had previously undiagnosed cognitive impairment. MoCA <24 was univariately associated with pre-existing frailty (Edmonton Frail Scale [EFS] score ≥6.5) and longer LOS (≥12 days). In logistic regression modeling, MoCA <24 was strongly independently associated with frailty EFS score ≥6.5 (odds ratio, 12.55; P < .001). By use of the area under the receiver operating characteristic curve (AUC), MoCA <24 was predictive of longer LOS of ≥12 days (AUC, 0.621; P = .049). The strength of predictive power increased with the addition of frailty (EFS score ≥6.5) to the models (AUC, 0.695; P = .002). CONCLUSIONS: The prevalence of cognitive impairment among older patients presenting for vascular surgery is high and frequently undiagnosed before admission. It is feasible to use the MoCA to identify cognitive impairment in this high-risk surgical group preoperatively. The combined assessment of frailty and cognition is predictive of adverse postoperative outcomes and longer LOS.

Deletion of Hsd11b1 suppresses caloric restriction-induced bone marrow adiposity in male but not female mice.

Bone marrow adipose tissue (BMAT) comprises >10% of total adipose mass in healthy humans. It increases in diverse conditions, including ageing, obesity, osteoporosis, glucocorticoid therapy, and notably, during caloric restriction (CR). BMAT potentially influences skeletal, metabolic, and immune functions, but the mechanisms of BMAT expansion remain poorly understood. Our hypothesis is that, during CR, excessive glucocorticoid activity drives BMAT expansion. The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) amplifies glucocorticoid activity by catalysing intracellular regeneration of active glucocorticoids from inert 11-keto forms. Mice lacking 11ß-HSD1 resist metabolic dysregulation and bone loss during exogenous glucocorticoid excess; thus, we hypothesised that 11ß-HSD1 knockout mice would also resist excessive glucocorticoid action during CR, thereby restrining BMAT expansion and bone loss. To test this, we first confirmed that 11ß-HSD1 is expressed in mouse and human bone marrow. We then investigated the effects of CR in male and female control and 11ß-HSD1 knockout mice from 9 to 15 weeks of age. CR increased Hsd11b1 mRNA in adipose tissue and bone marrow. Deletion of Hsd11b1 did not alter bone or BMAT characteristics in mice fed a control diet and had little effect on tibial bone microarchitecture during CR. Notably, Hsd11b1 deletion attenuated the CR-induced increases in BMAT and prevented increases in bone marrow corticosterone in males but not females. This was not associated with suppression of glucocorticoid target genes in bone marrow. Instead, knockout males had increased progesterone in plasma and bone marrow. Together, our findings show that knockout of 11ß-HSD1 prevents CR-induced BMAT expansion in a sex-specific manner and highlights progesterone as a potential new regulator of bone marrow adiposity.

Extra-aortic mycotic aneurysm due to group A Streptococcus after pharyngitis.

Mycotic aneurysms, especially outside the aorta, are uncommon, with group A Streptococcus a particularly rare cause. We report a case of extra-aortic mycotic aneurysm following a sore throat without demonstrable bacteremia where identification of the pathological organism was made by molecular diagnostic techniques after a standard laboratory culture was negative.