Scintigraphic evaluation of colon targeting pectin-HPMC tablets in healthy volunteers.

The in vivo evaluation of colon-targeting tablets was conducted in six healthy male volunteers. A pectin-hydroxypropyl methylcellulose coating was compressed onto core tablets labelled with 4MBq (99m)Tc-DTPA. The tablets released in the colon in all subjects; three in the ascending colon (AC) and three in the transverse colon (TC). Tablets that released in the TC had reached the AC before or just after food (Group A). The other three tablets released immediately upon AC entry at least 1.5h post-meal (Group B). Release onset for Group B was earlier than Group A (343min vs 448min). Group B tablets exhibited a clear residence period at the ileocaecal junction (ICJ) which was not observed in Group A. Prolonged residence at the ICJ is assumed to have increased hydration of the hydrogel layer surrounding the core tablet. Forces applied as the tablets progressed through the ICJ may have disrupted the hydrogel layer sufficiently to initiate radiolabel release. Conversely, Group A tablets moved rapidly through the AC to the TC, possibly minimising contact times with water pockets. Inadequate prior hydration of the hydrogel layer preventing access of pectinolytic enzymes and reduced fluid availability in the TC may have retarded tablet disintegration and radiolabel diffusion.

Emergence of vancomycin resistance in Staphylococcus aureus. Glycopeptide-Intermediate Staphylococcus aureus Working Group.

BACKGROUND: Since the emergence of methicillin-resistant Staphylococcus aureus, the glycopeptide vancomycin has been the only uniformly effective treatment for staphylococcal infections. In 1997, two infections due to S. aureus with reduced susceptibility to vancomycin were identified in the United States. METHODS: We investigated the two patients with infections due to S. aureus with intermediate resistance to glycopeptides, as defined by a minimal inhibitory concentration of vancomycin of 8 to 16 microg per milliliter. To assess the carriage and transmission of these strains of S. aureus, we cultured samples from the patients and their contacts and evaluated the isolates. RESULTS: The first patient was a 59-year-old man in Michigan with diabetes mellitus and chronic renal failure. Peritonitis due to S. aureus with intermediate resistance to glycopeptides developed after 18 weeks of vancomycin treatment for recurrent methicillin-resistant S. aureus peritonitis associated with dialysis. The removal of the peritoneal catheter plus treatment with rifampin and trimethoprim-sulfamethoxazole eradicated the infection. The second patient was a 66-year-old man with diabetes in New Jersey. A bloodstream infection due to S. aureus with intermediate resistance to glycopeptides developed after 18 weeks of vancomycin treatment for recurrent methicillin-resistant S. aureus bacteremia. This infection was eradicated with vancomycin, gentamicin, and rifampin. Both patients died. The glycopeptide-intermediate S. aureus isolates differed by two bands on pulsed-field gel electrophoresis. On electron microscopy, the isolates from the infected patients had thicker extracellular matrixes than control methicillin-resistant S. aureus isolates. No carriage was documented among 177 contacts of the two patients. CONCLUSIONS: The emergence of S. aureus with intermediate resistance to glycopeptides emphasizes the importance of the prudent use of antibiotics, the laboratory capacity to identify resistant strains, and the use of infection-control precautions to prevent transmission.

A special fluorescent in situ hybridization technique to study peripheral blood and assess the effectiveness of interferon therapy in chronic myeloid leukemia.

Using a highly sensitive fluorescence in situ hybridization method with probes for BCR and ABL1 (D-FISH), we studied 37 paired sets of bone marrow and blood specimens, collected within 24 to 96 hours of each other, from 10 patients before and during treatment for chronic myeloid leukemia (CML). The normal range for 500 interphase nuclei was

Pre-clinical evaluation of probes to detect t(8;21) AML minimal residual disease by fluorescence in situ hybridization.

The 8;21 translocation in acute myeloid leukemia (AML) results in a consistent fusion transcript, AML1/ETO. Long-term clinical remission occurs in some patients despite incomplete eradication of AML1/ETO as demonstrated by RT-PCR, thus limiting the usefulness of this assay. An important future goal will be to determine if there is a level of minimal residual disease (MRD) in patients below which relapse is unlikely. For the detection of MRD, we have developed reagents for fluorescence in situ hybridization (FISH) that identify both derivative 8 and 21 chromosomes with a high analytical sensitivity. In t(8;21) AML cells, two fused signals were detected in addition to the normal 8 and 21 alleles. The sensitivity and specificity of this probe mixture were analyzed in cell lines and patient bone marrows. One and two randomly juxtaposed signals were observed in 2.4 and 0.04% of normal cells, respectively. However, these were easily differentiated from t(8;21) cells by the absence of signals from the normal alleles. Using as criteria the presence of two fused signals plus the normal alleles, we observed no false positives among 5,000 normal cells. The probe correctly identified 20/20 patients with t(8;21) AML and 10/10 non-t(8;21) patients. In cell dilution experiments, the analytical sensitivity of this reagent was equal to that of the X chromosome and Y chromosome alpha-satellite probes. These optimized probes should facilitate the quantitative assessment and study of MRD in t(8;21) AML.

A 500-kb physical map and contig from the Harvey ras-1 gene to the 11p telomere.

A contiguous physical map was constructed from the Harvey ras-1 (HRAS1) gene to the 11p telomere. The contig spans approximately 500 kb and is minimally composed of a telomere-containing YAC and P1 and cosmid clones. Included in the contig are 11 sequence-tagged sites derived from P1 and cosmid ends. Three genes were placed on the contig in the following order: telomere-ribonuclease/angiogenin inhibitor (RNH)-Harvey ras-1 (HRAS1)-HRAS1-related complex (HRC). Two novel tetranucleotide repeats (heterozygosity of 66 and 68%) and a complex CA repeat (heterozygosity of 78%) were isolated and characterized.

Physical mapping of the holoprosencephaly critical region in 21q22.3, exclusion of SIM2 as a candidate gene for holoprosencephaly, and mapping of SIM2 to a region of chromosome 21 important for Down syndrome.

We set out to define the holoprosencephaly (HPE) critical region on chromosome 21 and also to determine whether there were human homologues of the Drosophila single-minded (sim) gene that might be involved in HPE. Analysis of somatic cell hybrid clones that contained rearranged chromosomes 21 from HPE patients defined the HPE minimal critical region in 21q22.3 as D21S113 to qter. We used established somatic cell hybrid mapping panels to map SIM2 to chromosome 21 within subbands q22.2-q22.3. Analysis of the HPE patient-derived somatic cell hybrids showed that SIM2 is not deleted in two of three patients and thus is not a likely candidate for HPE1, the HPE gene on chromosome 21. However, SIM2 does map within the Down syndrome critical region and thus is a candidate gene that might contribute to the Down syndrome phenotype.

Amplification and overexpression of HER-2/neu in carcinomas of the salivary gland: correlation with poor prognosis.

There are few reliable prognostic markers of biological aggressiveness for head and neck carcinomas in general. For salivary gland carcinomas, anatomic location, tumor size, histological grade, and extent of disease involvement are considered to be clinically important risk factors for recurrent disease. Molecular genetic alterations in salivary gland carcinomas have not been characterized, and tumor cell proteins have not been shown to be prognostically significant. Here a cohort of mucoepidermoid carcinomas of the major (parotid and submandibular) salivary glands are analyzed for a molecular genetic alteration, HER-2/neu gene amplification, and gene amplification and expression results are compared with long-term clinical follow-up information. Archival tissues resected from 58 patients with mucoepidermoid carcinoma of salivary glands were evaluated for HER-2/neu gene amplification by fluorescence in situ hybridization and for gene expression by immunohistochemistry in a blinded fashion. Clinical follow-up information was compared with the results of these analyses to determine whether there were significant associations. Overexpression, identified as membrane immunostaining by immunohistochemistry, was observed in 22 of 58 (38%) mucoepidermoid carcinomas. Gene amplification, characterized by fluorescence in situ hybridization, was observed in 12 (21%) cases. Eleven of the 12 cases with gene amplification were also immunostained for HER-2/neu. Both gene amplification (P = 0.0001, P < 0.0001) and immunostaining (P < 0.0001, P < 0.0001) were correlated with shorter disease-free interval and poorer overall patient survival, respectively. Multivariate analysis showed that HER-2/neu immunostaining and amplification were markers of poor prognosis independent of histopathological grade, tumor size, and involvement of regional lymph nodes. HER-2/neu is amplified and/or overexpressed in approximately one-third of mucoepidermoid carcinomas of salivary glands. Amplification and/or overexpression appears to be an independent marker of poor prognosis in mucoepidermoid carcinomas of the salivary glands as it is in carcinomas of the breast, ovary, and endometrium.

Molecular genetic analysis of the 3p- syndrome.

Molecular genetic analysis of five cases of 3p- syndrome (del(3)(qter-->p25:)) was performed to investigate the relationship between the molecular pathology and clinical phenotype. Fluorescence in situ hybridization studies and analysis of polymorphic DNA markers from chromosome 3p25-p26 demonstrated that all four informative cases had distal deletions. However, the extent of the deletion was variable: in two patients with the most extensive deletions the deletion breakpoint mapped between RAF1 and D3S1250, in one patient the deletion breakpoint was between D3S1250 and D3S601, and in two patients the deletion commenced telomeric to D3S601 (and telomeric to D3S1317 in one of these). All five patients displayed the classical features of 3p- syndrome (mental retardation, growth retardation, microcephaly, ptosis and micrognathia) demonstrating that loss of sequences centromeric to D3S1317 is not required for expression of the characteristic 3p- syndrome phenotype. The three patients with the most extensive deletions had cardiac septal defects suggesting that a gene involved in normal cardiac development is contained in the interval D3S1250 and D3S18. The PMCA2 gene is contained within this region and deletion of this gene may cause congenital heart defects. At least three patients were deleted for the von Hippel-Lindau (VHL) disease gene although none had yet developed evidence of VHL disease. We conclude that molecular analysis of 3p- syndrome patients enhances the management of affected patients by identifying those at risk for VHL disease, and can be used to elucidate the critical regions for the 3p- syndrome phenotype.

Norman-Roberts syndrome: clinical and molecular studies.

We report on a 7-year-old boy with microcephaly, bitemporal hollowing, low sloping forehead, slightly prominent occiput, widely set eyes, broad and prominent nasal bridge, and severe postnatal growth deficiency. Hypertonia, hyperreflexia, seizures, and profound mental retardation were also present. Brain MRI documented partial agyric cortex with patchy pachygyria, colpocephaly, and hypoplasia of corpus callosum and brain stem, which is consistent with the diagnosis of lissencephaly type I grade 2. On the basis of his phenotypic appearance the patient is considered to have the Norman-Roberts syndrome. Molecular studies, performed by means of in situ hybridization and DNA probe analysis, did not demonstrate deletion in the Miller-Dieker/isolated Lissencephaly critical region on the short arm of chromosome 17.

Physical mapping of the holoprosencephaly critical region on chromosome 7q36.

Holoprosencephaly (HPE) is a developmental field defect involving the brain and face. Cytogenetic deletions in patients with HPE have localized one of the HPE genes to chromosomal region 7q36. We have characterized the 7q deletions in thirteen HPE patients. The result is the construction of a high resolution physical map of 7q32-qter. As a first step towards cloning an HPE gene crucial for normal brain development, we have defined the HPE minimal critical region in 7q36 between D7S292 and D7S392.

A cluster of pseudofungemia associated with hospital renovation adjacent to the microbiology laboratory.

OBJECTIVE: To determine the clinical significance and source of fungemia following a cluster of positive blood cultures during a 3-day period. DESIGN: Chart review was used to determine the clinical significance of positive blood cultures. Microbiologic sampling of the laboratory environment was used to determine potential sources of fungal contamination. SETTING: A large, tertiary care, community teaching hospital. PATIENTS: All patients with blood cultures positive for Aspergillus species, Penicillium species, or both during the outbreak period. RESULTS: Thirteen patients, all children, were reported to have positive blood cultures for fungus during a 3-day period in early 1990. None had clinical features consistent with fungemia. Investigation of specimen processing procedures revealed that microbiologic plates were not processed--as per protocol--under the biologic hood but inadvertently were left open to air on the work bench by laboratory technicians. Settling plates left at the workbench, at door entry sites, and at sites of renovation immediately adjacent to the laboratory were positive for Aspergillus and/or Penicillium; control plates placed elsewhere were negative. Airflow patterns suggested spread into the microbiologic laboratory through an open door located near the implicated workbench station and a false ceiling above the workbench area. CONCLUSIONS: Our investigation demonstrates that faulty technique in the laboratory coupled with a change in environmental conditions can result in false-positive cultures and an outbreak of pseudofungemia.

High-density genetic and physical mapping of DNA markers near the X-linked Alport syndrome locus: definition and use of flanking polymorphic markers.

To refine the genetic and physical mapping of the locus for Alport syndrome (ATS), 22 X-chromosome restriction fragment length polymorphism (RFLP) markers that fall between Xq21.3 and Xq25 were tested for genetic linkage with the disease and also mapped with respect to a series of physical breakpoints in this region. The location of the COL4A5 gene, which has recently been shown to be mutated in at least some families with Alport syndrome, was determined with respect to the same physical breakpoints. Two large Utah kindreds were included in the genetic studies, kindreds P and C, with 125 and 63 potentially informative meioses, respectively. Both kindreds have essentially identical nephritis; however, kindred P has sensorineural hearing loss associated with the nephritis, while kindred C does not. A mutation in COL4A5 has been demonstrated for kindred P, but no change in this gene has yet been detected for kindred C. Twelve informative probes did not recombine with the disease locus in either kindred (theta = 0.0, with combined lod scores for the two kindreds ranging from 7.7 to 30.0). The closest markers that could be demonstrated to flank the disease locus were the same for each kindred and thus the locations of the mutations causing the two disease phenotypes are not distinguishable at the current level of genetic resolution. The flanking markers are also useful for the resolution of questionable diagnoses and allow accurate estimates for these families of the rate of sporadic hematuria in noncarrier females (7%) and the penetrance of hematuria for carrier females (93%).

Complications associated with central venous catheters inserted in critically ill neonates.

OBJECTIVE: To assess the incidence and spectrum of complications associated with central venous catheter (CVC) placement in the critically ill infant. DESIGN: A prospective study of all babies hospitalized in a neonatal intensive care unit (NICU) from January 1989 to December 1989. Potential risk factors associated with infection were evaluated by a case-control comparison. SETTING: Conducted at a university-affiliated, tertiary care community hospital. PATIENTS: Neonates requiring intensive care and a central venous catheter. Controls consisted of noninfected babies. RESULTS: Of 263 critically ill neonates, only 13 (4.9%) required a CVC insertion. Seventeen CVCs were placed in these 13 neonates for a total duration of 600 days (median, 32 days/cannula). Fifteen (88%) of these cannulas had one or more complications during its catheter life including dislodgement or leakage (53%), occlusion or thrombosis (47%), infections (29%), or minor bleeding (12%). Five babies (29%) developed 6 episodes of bloodstream infection including 3 sporadic cases due to Staphylococcus epidermidis and a cluster of fungemia due to Malassezia furfur associated with lipid emulsion therapy. Infants with a CVC-associated infection were a younger gestational age (24 weeks versus 32 weeks, p = .04) and weighed less at birth (580 g versus 1285 g, p = .02). The overall rate of bloodstream infection was one episode per 100 days of catheter use. CONCLUSIONS: CVCs may be lifesaving to a critically ill neonate, but complications occur frequently. Use must be restricted to infants in whom alternate delivery routes of intravenous therapy or support are otherwise unavailable.

Outbreak of Brucella melitensis among microbiology laboratory workers in a community hospital.

From May to September 1988, eight employees of a microbiology laboratory developed acute brucellosis (attack rate, 31%). Seven of the eight affected employees had clinical illness ranging from a nonspecific, flulike illness to severe hepatitis. Blood cultures obtained from five of the affected employees (63%) were positive for Brucella melitensis, biotype 3. Comparison of cases and controls showed that there were no risk factors besides employment in the laboratory. Based on work locations, assignments, and interviews, it was found that person-to-person, droplet, food-borne, and waterborne spread were unlikely. Our investigation disclosed that 6 weeks before the outbreak began, a frozen brucella isolate from a patient hospitalized 3 years earlier had been thawed and subcultured without the use of a biologic safety cabinet. This clinical isolate was subsequently identified as B. melitensis, biotype 3, identical to the employee isolates. It is presumed that transmission occurred via the airborne route. This outbreak reemphasized that all work on Brucella species, an established biosafety level 3 organism, must be conducted under a biologic safety hood. Furthermore, it might be prudent to perform all clinical "setups" under a safety hood since aerosolization commonly occurs during the initial processing of specimens and the majority of these specimens are from patients with uncertain diagnoses.

Person-to-person transmission of Brucella melitensis.

Human brucellosis is primarily an occupational hazard in the USA; in the Middle East and Africa ingestion of contaminated dairy products is an important route of infection. Whether human beings can become infected via person-to-person spread is uncertain. During an investigation of a commonsource, laboratory-associated outbreak due to Brucella melitensis, biotype 3, the wife of a microbiologist with serologically proven brucellosis became infected. Her blood isolate was indistinguishable from the epidemic strain. In the absence of other risk factors, we suggest that sexual intercourse is a possible means of transmission.

Characterization of polymorphic loci on a telomeric fragment of DNA from the long arm of human chromosome 7.

The 240-kb yeast artificial chromosome (YAC) HTY146 (D7S427) containing the telomere from the q arm of human chromosome 7 was subcloned into the cosmid vector sCOS-1. Cosmid subclones were screened for DNA polymorphisms by Southern blot analysis of restriction digests of DNA from random individuals. Four distinct polymorphisms were characterized. These markers provide a resource for defining the end of the genetic map for the long arm of human chromosome 7.

Noncompliance with Universal Precautions Policy: why do physicians and nurses recap needles?

In 1987 the Centers for Disease Control published a Universal Precautions Policy establishing blood and body fluid procedures to be used consistently with all patients. An important and unequivocal Universal Precautions Policy recommendation with regard to avoidance of needlestick injuries is that needles should never be recapped. We examined the recapping-related attitudes and behaviors of physicians and nurses at four large teaching hospitals with patients with acquired immunodeficiency syndrome and with Universal Precautions Policy in-service training programs. Compliance was found to be considerably less than optimal. According to unannounced needle counts in disposal boxes, the percentage of recapped needles was always greater than 25% and exceeded 50% in four instances. Recapping was related to inadequate knowledge, concerns about personal risk, forgetfulness, being "too busy" to follow the Universal Precautions Policy, and the misperception that recapping is a way to avoid needlestick injury. Strategies are suggested to improve and supplement traditional in-service education.

The clinical spectrum of endophthalmitis: incidence, predisposing factors, and features influencing outcome.

Endophthalmitis is a virulent ocular inflammation typically developing suddenly and progressing rapidly. To better understand the incidence and factors predisposing to infection and influencing outcome, records of 114 patients with endophthalmitis hospitalized at one institution from 1980 to 1986 were reviewed. An infectious origin was confirmed in 79 patients (69%). The most common pathogens included staphylococcal species (Staphylococcus epidermidis, 33 cases; Staphylococcus aureus, 8 cases), streptococci (18 cases), gram-negative organisms (10 cases), and fungi (7 cases). Predisposing factors for infective endophthalmitis included preceding ocular surgery (67%), penetrating trauma (13%), systemic infection (11%), and periocular infection (5%). Despite vitrectomies and aggressive use of antibiotics, 42 patients (53%) with infective endophthalmitis suffered major visual loss. Morbidity was less pronounced with infection caused by S. epidermidis (23%; P less than .05). Patients with infective endophthalmitis more likely required repeated vitrectomies, were hospitalized longer, and had a worse outcome (as measured by complete enucleation) than those with "sterile" endophthalmitis. On the basis of these data, empiric vancomycingentamicin might be initiated in patients with endophthalmitis. Studies to define optimal management are needed, because the morbidity associated with this entity remains pessimistically high despite state-of-the-art treatment.