Implementation of a Clinical Decision Support Tool for Stool Cultures and Parasitological Studies in Hospitalized Patients.

There is substantial evidence that stool culture and parasitological examinations are of minimal to no value after 3 days of hospitalization. We implemented and studied the impact of a clinical decision support tool (CDST) to decrease the number of unnecessary stool cultures (STCUL), ova/parasite (O&P) examinations, and Giardia/Cryptosporidium enzyme immunoassay screens (GC-EIA) performed for patients hospitalized >3 days. We studied the frequency of stool studies ordered before or on day 3 and after day 3 of hospitalization (i.e., categorical orders/total number of orders) before and after this intervention and denoted the numbers and types of microorganisms detected within those time frames. This intervention, which corresponded to a custom-programmed hard-stop alert tool in the Epic hospital information system, allowed providers to override the intervention by calling the laboratory, if testing was deemed medically necessary. Comparative statistics were employed to determine significance, and cost savings were estimated based on our internal costs. Before the intervention, 129/670 (19.25%) O&P examinations, 47/204 (23.04%) GC-EIA, and 249/1,229 (20.26%) STCUL were ordered after 3 days of hospitalization. After the intervention, 46/521 (8.83%) O&P examinations, 27/157 (17.20%) GC-EIA, and 106/1,028 (10.31%) STCUL were ordered after 3 days of hospitalization. The proportions of reductions in the number of tests performed after 3 days and the associated P values were 54.1% for O&P examinations (P < 0.0001), 22.58% for GC-EIA (P = 0.2807), and 49.1% for STCUL (P < 0.0001). This was estimated to have resulted in $8,108.84 of cost savings. The electronic CDST resulted in a substantial reduction in the number of evaluations of stool cultures and the number of parasitological examinations for patients hospitalized for more than 3 days and in a cost savings while retaining the ability of the clinician to obtain these tests if clinically indicated.

Should varicella-zoster virus culture be eliminated? A comparison of direct immunofluorescence antigen detection, culture, and PCR, with a historical review.

A comparison of direct fluorescent-antibody assay (DFA), culture, and two PCR assays disclosed sensitivities of 87.8%, 46.3%, and 97.6% and 100%, respectively. We reviewed 1,150 results for clinical specimens submitted for DFA and culture and found that only 17 were culture positive/DFA negative. The incremental cost to detect these 17 positives was $3,078/specimen.

Two-week cast immobilization induced chronic widespread hyperalgesia in rats.

It has been postulated that physical immobilization is an essential factor in developing chronic pain after trauma or surgery in an extremity. However, the mechanisms of sustained immobilization-induced chronic pain remain poorly understood. The present study, therefore, aimed to develop a rat model for chronic post-cast pain (CPCP) and to clarify the mechanism(s) underlying CPCP. To investigate the effects of cast immobilization on pain behaviours in rats, one hindlimb was immobilized for 2 weeks with a cast and remobilization was conducted for 10 weeks. Cast immobilization induced muscle atrophy and inflammatory changes in the immobilized hindlimb that began 2 h after cast removal and continued for 1 week. Spontaneous pain-related behaviours (licking and reduction in weight bearing) in the immobilized hindlimb were observed for 2 weeks, and widespread mechanical hyperalgesia in bilateral calves, hindpaws and tail all continued for 5-10 weeks after cast removal. A sciatic nerve block with lidocaine 24 h after cast removal transitorily abolished bilateral mechanical hyperalgesia in CPCP rats, suggesting that sensory inputs originating in the immobilized hindlimb contribute to the mechanism of both ipsilateral and contralateral hyperalgesia. Intraperitoneal injection of the free radical scavengers 4-hydroxy-2,2,6,6-tetramethylpiperydine-1-oxy1 or N-acetylcysteine 24 h after cast removal clearly inhibited mechanical hyperalgesia in bilateral calves and hindpaws in CPCP rats. These results suggest that cast immobilization induces ischaemia/reperfusion injury in the hindlimb and consequent production of oxygen free radicals, which may be involved in the mechanism of widespread hyperalgesia in CPCP rats.

Challenges in the diagnosis and management of Nocardia infections in lung transplant recipients.

BACKGROUND: Nocardia infection occurs in 2.1-3.5% of lung transplant recipients, and may involve cavitary nodular pulmonary lesions, soft tissue infection, or other sites of dissemination. Nocardiosis can pose challenging clinical problems in the areas of diagnosis and treatment. Diagnostic delays may occur, and adverse reactions to therapy are common. This study reviews clinical and epidemiological aspects of nocardiosis in lung transplant recipients, with special attention to pitfalls in management. Clinicians should be alert for these possibilities in order to institute prompt therapy and to achieve successful outcomes. METHODS: A retrospective cohort study was conducted of 577 lung transplant recipients from January 1991 to May 2007. Demographics, reason for transplant, recent rejection, time from transplantation, site of infection, hypogammaglobulinemia, and/or neutropenia shortly before onset, Pneumocystis jiroveci prophylaxis, Nocardia species, radiographic findings, extrapulmonary lesions, nature and duration of treatment, adverse reactions, and outcomes were recorded. RESULT: Nocardia infection occurred in 1.9% (11/577). Mean onset was 14.3 months after transplant (range 1.5-39 months). N. asteroides was isolated in 55% (6/11). Emphysema was the most common reason for transplant (7/11, 64%). Six patients were receiving trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis at the time of diagnosis. Three patients had immune globulin G levels <400 mg/dL and 2 were neutropenic in the 3 months preceding diagnosis. Diagnosis was made by bronchoalveolar lavage (55%), skin abscess culture (18%), open lung biopsy (9%), pleural fluid (9%), and sputum culture (9%). Definitive diagnosis required a median of 9 days and a mean of 13.6 days (range 3-35 days) from the time of diagnostic sampling. Soft tissue lesions occurred in 3 and central nervous system involvement in 1 patient. Adverse reactions to therapy occurred in 9/10 (90%) of patients for whom information was available. Nocardia-related mortality occurred in 2/11 patients (18%). CONCLUSIONS: Nocardiosis occurred in 1.9% of lung transplant recipients and was associated with a mean of nearly 2 weeks to diagnosis and frequent adverse effects on therapy. TMP-SMX prophylaxis on a thrice weekly basis did not prevent all episodes of nocardiosis. Despite utilization of protocol bronchoscopies with cultures for Nocardia, this organism remains a source of clinical complexity in the lung transplant population.

Heterogeneous distribution of a gap junction protein, connexin43, in the gastroduodenal junction of the guinea pig.

The gastroduodenal junction differs in morphology and function from the stomach and the duodenum. We studied the immunohistochemical distribution of the gap junction protein, connexin43, and the nerve terminal proteins, SNAP-25 and synaptotagmin, in the musculature of the guinea pig gastroduodenal junction. Connexin43-immunopositive structures were distributed throughout the circular layer of the gastroduodenal junction, most densely in the duodenal circular layer. The difference in the distribution patterns of these structures between the stomach and the duodenum was readily observed in the gastroduodenal junction. In the inner part of the circular muscle layer of the gastroduodenal junction, the connexin43-immunopositive structures were relatively few or non-existent, whereas the SNAP-25-containing nerve fibers and synaptotagmin-containing nerve terminals, clearly observed, were numerous. These findings show a heterogeneous distribution of the gap junctions and nerves in the gastroduodenal junction. The results suggest that the gastroduodenal junction has heterogeneous electrical connections among smooth muscle cells via gap junctions, and specific nerve innervation, which regulates gastroduodenal motility.

Expression of granulocyte colony-stimulating factor receptor and platelet-derived endothelial cell growth factor in oral and oropharyngeal precancerous lesions.

Epithelial hyperplasia and dysplasia have been diagnosed as precancerous lesions and have been discussed in relationship to carcinogenesis. We analyzed the immunohistochemical expression of granulocyte colony-stimulating factor receptor (G-CSFR) and platelet-derived endothelial cell growth factor (PD-ECGF) in oral and oropharynx; 33 samples of normal epithelium, 28 samples of hyperplasia, 16 samples of dysplasia and 58 samples of squamous cell carcinoma. Also, we examined mean vessel density (MVD) by using CD34 staining and proliferating cell nuclear antigen (PCNA) staining. Dysplasia and head and neck Squamous Cell Carcinoma (SCC) exhibited higher G-CSFR expression and MVD than normal or hyperplastic epithelium (p <0.01). In the PD-ECGF staining, significant differences were found between SCC and normal epithelium, hyperplasia and dysplasia (p<0.001). In dysplasia and hyperplasia, PD-ECGF expression was significantly correlated with PCNA expression (r=0.345, p=0.025), however it was not correlated with the MVD. G-CSFR expression was not correlated with either PCNA or MVD. These results suggest that G-CSFR and PD-ECGF might be concerned with different carcinogenesis pathways of the squamous cells in the oral region and that PD-ECGF may be concerned with epithelial proliferation rather than angiogenesis.

Distribution of preganglionic terminals in the cervical sympathetic ganglia detected by the expression of c-Fos like protein after electric stimulation of the ventral root.

To determine the segmental relationship between the upper thoracic spinal cord and cervical sympathetic ganglia, we observed the distribution pattern of postganglionic cells which expressed c-Fos like protein, one of the products of immediate early genes, after electrical stimulation of ventral roots at the T1-T3 spinal segments. We recognized a clear segmental arrangement of postganglionic cells in the stellate ganglion along its rostrocaudal direction corresponding to the segmental arrangement of preganglionic neurons in the spinal cord. That is, postganglionic neurons which expressed c-Fos like protein after stimulation of the T1 ventral root were distributed in the middle region of the stellate ganglion in the rostrocaudal direction. The c-Fos like protein-positive neurons after stimulation of the T2 ventral root were distributed in a more caudal region of the stellate ganglion than after T1 ventral root stimulation. C-Fos like protein-positive neurons after stimulation of the T3 ventral root were mainly situated in a more caudal region of the stellate ganglion than after T2 ventral root stimulation. There was, however, no segmental relationship between the upper thoracic levels of the spinal cord and superior cervical ganglion in the rostrocaudal direction. These results indicate that the segmental innervation of the upper thoracic spinal cord exists in the stellate ganglion, but not in the superior cervical ganglion.

Comparative histochemical study of Bowen's disease and actinic keratosis: preserved normal basal cells in Bowen's disease.

The degree of DNA-instability as revealed by immunohistochemical staining with anti-cytidine antibody after acid hydrolysis (DNA-instability test) has been recently used as a marker of malignancy. This technique was applied to examine 17 skin tissue samples of Bowen's disease, 47 of actinic keratosis, 15 of squamous cell carcinoma, 5 of seborrheic keratosis, and 10 of normal skin. All benign neoplastic cells of seborrheic keratosis and normal epidermal cells were negative. On the other hand, all cancer cells were positive with the DNA-instability test, indicating their malignancy, but all basal cells in Bowen's disease were completely negative. Compatible with this result, the basal cells in Bowen's disease were characteristically normal as evident in other histochemical examinations. Thus, they were negative with p53 immunohistochemistry, with normal signals of chromosome 17 in situ hybridisation and argyrophilic nucleolar organiser region, and showed slightly enhanced proliferative activity as revealed by proliferating cell nuclear antigen immunohistochemistry. Immunohistochemical staining with 34 beta E12 (monoclonal antibody against cytokeratins 1, 5, 10, and 14), which stains all normal epidermal keratinocytes including basal cells, showed that only the basal cells of Bowen's disease stained strongly and homogeneously, while all cancer cells in the upper layers of Bowen's disease and all layers of actinic keratosis were only sporadically or weakly stained. Staining with 34 beta B4 (monoclonal antibody against cytokeratin 1), which recognises the whole epidermis except for the basal layer in the normal epidermis, showed that the basal cells in the Bowen's disease were completely negative, and lower layer cells in the actinic keratosis and upper layer cells in Bowen's disease were only sporadically stained positive, although the superficial layer cells in actinic keratosis stained strongly and homogeneously. Our findings clearly indicate that the basal cells in Bowen's disease are normal. In support of this conclusion, the same cells showed normal morphology on electron microscopy with preserved basement membrane, although the latter was often damaged in actinic keratosis.

Neurons in Golgi-stain-like images revealed by GFP-adenovirus infection in vivo.

Neurons in the adult brain have a very complex morphology with many processes, including tremendously long axons. Since dendrites and axons play key roles in the input and output of neural information, respectively, the visualization of complete images of these processes is necessary to reveal the mechanism of neural information processing. Here we made a recombinant adenovirus vector which encodes green fluorescent protein (GFP) tagged with a palmitoylation site, a membrane-targeting signal, produced specific antibodies to GFP, and used them as probes for staining the nervous system. In the neocortex, after injection of the recombinant virus and immunoperoxidase staining with the antibodies, many different types of cells were labeled in a Golgi stain-like fashion. Although the number of labeled cells varied depending on the amount of virus injected, the recombinant virus was considered to be infectious to cortical neurons of all cell types without selectivity. In contrast, the viral infection in the cerebellar cortex and superior cervical ganglion showed some selectivity toward the cell type. It is expected that this recombinant virus will be a useful tool for the morphological analysis of neuronal connections, especially the analysis of microcircuitry in the cerebral cortex.

The developmental fate of the rostral/caudal half of a somite for vertebra and rib formation: experimental confirmation of the resegmentation theory using chick-quail chimeras.

To determine whether resegmentation of somites forms the axial skeleton, we traced the development of the rostral and the caudal half of a somite during skeletogenesis in chick-quail chimeras by replacing the rostral or caudal half of a newly formed chick somite with that of a quail somite. The rostral half-somite transplant formed the caudal half of the vertebral body, the entire spinous process and the distal rib, while the caudal half-somite transplant formed the rostral half of vertebral body, the rostral half of spinous process, the vertebral arch, the transverse process and the entire rib. These findings confirm the resegmentation theory except the spinous process and the distal rib.

Femoral intercondylar notch measurements in osteoarthritic knees.

METHODS: We measured the dimensions of the intercondylar notch of the femur in 32 patients with primary severe osteoarthrosis (OA) of the knee and 54 embalmed cadaveric knees. RESULTS: There were 56 knees with morphologically normal anterior cruciate ligament (ACL), 11 knees with lax or partially ruptured ACL and 19 knees with missing ACL. The average width of the intercondylar notch in knees with lax and missing ACL was significantly narrower than that of knees with normal ACL. In addition, knees with missing ACL had a significantly smaller notch depth than knees with normal ACL. In medial compartment OA (56 knees), the notch width and depth in knees with severe OA (37 knees) were significantly smaller than those in normal (19 knees) and mild to moderate OA groups (19 knees). CONCLUSION: Our results indicate that osteophyte growth in the femoral intercondylar notch seems to correlate with the progression of medial compartment OA of the knee.

Arborization pattern of sympathetic preganglionic axons in the rat superior cervical and stellate ganglia.

Anterograde labeling technique with Phaseolus Vulgaris leucoagglutinin (PHA-L) was employed to observe how a single preganglionic axon arborizes in the superior cervical ganglion (SCG) and stellate ganglion (STG) of rats. PHA-L was injected into the intermediolateral nucleus of the spinal cord at the middle point between segments T1 and T2, and labeled axons were detected immunohistochemically in serial sections. We traced and drew three preganglionic axons over their full length in the SCG and STG. In SCG, the labeled axons bifurcated repeatedly and extended to a length of 600-700 microns in the rostrocaudal direction, and about 200 microns in the transverse direction. These three preganglionic axons made 11, 14 and 11 dense terminal plexus regions along their trajectory. The pattern of the most dense terminal plexus corresponded to the pericellular type dendritic plexus, one of the plexus patterns of dendritic collaterals of SCG neurons. In the STG, the extent of axonal arborization was more variable than that in the SCG, ranging from 400 to 800 microns in the rostrocaudal direction and about 400 microns in the transverse direction. The three analyzed axons made 21, 19 and 20 dense terminal plexus regions along their trajectory, with a similar pattern to those in SCG. These results indicated that there might be a columnar or ellipsoidal organization of postganglionic neurons which are innervated by single preganglionic axons.

NOS-positive preganglionic neurons innervate a subpopulation of postganglionic neurons in superior cervical ganglion in rats.

To determine the postganglionic targets of NOS-containing preganglionic neurons, we studied the association of NADPH-diaphorase positive preganglionic fibers and retrogradely labeled postganglionic neurons in the superior cervical ganglion (SCG) in rats. Wheat germ agglutinin-horseradish peroxidase solution was applied to the anterior chamber of the eye, middle cerebral artery, subcutaneous layer of the facial skin, or submucosal layer of the inside of the lip. Two days after tracer application, the rats were perfused with fixative solution. Serial sections of the SCG were stained histochemically for NADPH-diaphorase followed by diaminobenzidine reaction. More than 80% of the labeled postganglionic neurons innervating the structures in the subcutaneous or submucosal layer showed close association with NADPH-diaphorase positive preganglionic nerve terminals; approximately one-third of these labeled neurons were encircled by dense baskets of pericellular terminals. On the other hand, most of the postganglionic neurons innervating the iris (69%) or the cerebral artery (90%) did not show a distinct association with NADPH-diaphorase positive terminals. These results suggest that one of the principal roles of the NOS-containing preganglionic neurons may be in controlling the postganglionic neurons which innervate the structures in the subcutaneous or submucosal layer.

Restriction of the fate of early migrating trunk neural crest in gangliogenesis of avian embryos.

Trunk neural crest is the source of peripheral nervous tissue, the adrenal medulla and pigment cells. To quantitatively assay the potency of neural crest to form each derivative tissue, we isolated fragments of neural crest from quail embryos and transplanted them into the migration pathways of chicken embryos. In the resultant chimeras, we counted the quail cells derived from grafts distributed in the dorsal root ganglia, the sympathetic tissues around the aorta and the spinal nerves. Descendant cells of quail neural crest derived from the brachial or lumbosacral and lower levels were more abundant in the dorsal root ganglia than in the sympathetic tissue, while those derived from adreno-medullary levels were more abundant in the sympathetic tissue than in the dorsal root ganglia. No correlation was seen between the distribution pattern of quail cells and the rostrocaudal levels of graft site in chick embryos. These findings suggest that the developmental potency of truncal neural crest in gangliogenesis is restricted in the early phase of their migration and differs along the rostrocaudal axis, although it is not clear whether this restriction reflects determination of each crest cell. The size of the rudiments of the dorsal root ganglia in the normal embryo differed along the rostrocaudal axis, these differences being consistent with those in the fate of the neural crest at a given somite level.

[Study on the distribution of muscle spindles in the rat extensor digitorum longus muscle].

The distribution of muscle spindles (MS) in the extensor digitorum longus (EDL) muscle of the rat hind limb was morphologically studied on a series of longitudinal cryostat sections. The intrafusal muscle fibers were brownishly stained with the acetylcholinesterase reaction, and the mucopolysaccharide contained in the equatorial periaxial space was stained with alcian blue. This double-staining method made it easy for us to find the MS and to decide the equatorial portion and the whole extension of MS. From a series of camera lucida drawings the distribution of all MS was reconstructed on a sheet of paper and three-dimensionally imaged on a personal computer using image reconstructing software. The MS were distributed mainly in the superficial and lateral part of EDL muscle. Additionally, an ATPase reaction was employed to detect the red muscle fibers, and it was confirmed that their distribution of them is similar to that of MS.

Monoclonal antibodies specific to quail embryo tissues: their epitopes in the developing quail embryo and their application to identification of quail cells in quail-chick chimeras.

Quail-chick chimeras have been used extensively in the field of developmental biology. To detect quail cells more easily and to detect cellular processes of quail cells in quail-chick chimeras, we generated four monoclonal antibodies (MAb) specific to some quail tissues. MAb QCR1 recognizes blood vessels, blood cells, and cartilage cells, MAb QB1 recognizes quail blood vessels and blood cells, and MAb QB2 recognizes quail blood vessels, blood cells, and mesenchymal tissues. These antibodies bound to those tissues in 3-9-day quail embryos and did not bind to any tissues of 3-9-day chick embryos. MAb QSC1 is specific to the ventral half of spinal cord and thymus in 9-day quail embryo. No tissue in 9-day chick embryo reacted with this MAb. This antibody binds transiently to a small number of brain vesicle cells in developing chick embryo as well as in quail embryo. A preliminary application of two of these MAb, QCR1 and QSC1, on quail-chick chimeras of neural tube and somites is reported here.

Determination of somite cells: independence of cell differentiation and morphogenesis.

Somites are mesodermal structures which appear transiently in vertebrates in the course of their development. Cells situated ventromedially in a somite differentiate into the sclerotome, which gives rise to cartilage, while the other part of the somite differentiates into dermomyotome which gives rise to muscle and dermis. The sclerotome is further divided into a rostral half, where neural crest cells settle and motor nerves grow, and a caudal half. To find out when these axes are determined and how they rule later development, especially the morphogenesis of cartilage derived from the somites, we transplanted the newly formed three caudal somites of 2.5-day-old quail embryos into chick embryos of about the same age, with reversal of some axes. The results were summarized as follows. (1) When transplantation reversed only the dorsoventral axis, one day after the operation the two caudal somites gave rise to normal dermomyotomes and sclerotomes, while the most rostral somite gave rise to a sclerotome abnormally situated just beneath ectoderm. These results suggest that the dorsoventral axis was not determined when the somites were formed, but began to be determined about three hours after their formation. (2) When the transplantation reversed only the rostrocaudal axis, two days after the operation the rudiments of dorsal root ganglia were formed at the caudal (originally rostral) halves of the transplanted sclerotomes. The rostrocaudal axis of the somites had therefore been determined when the somites were formed. (3) When the transplantation reversed both the dorsoventral and the rostrocaudal axes, two days after the operation, sclerotomes derived from the prospective dermomyotomal region of the somites were shown to keep their original rostrocaudal axis, judging from the position of the rudiments of ganglia. Combined with results 1 and 2, this suggested that the fate of the sclerotomal cells along the rostrocaudal axis was determined previously and independently of the determination of somite cell differentiation into dermomyotome and sclerotome. (4) In the 9.5-day-old chimeric embryos with rostrocaudally reversed somites, the morphology of vertebrae and ribs derived from the explanted somites were reversed along the rostrocaudal axis. The morphology of cartilage derived from the somites was shown to be determined intrinsically in the somites by the time these were formed from the segmental plate. The rostrocaudal pattern of the vertebral column is therefore controlled by factors intrinsic to the somitic mesoderm, and not by interactions between this mesoderm and the notochord and/or neural tube, arising after segmentation.