Comparison of agar-based media for primary isolation of glycopeptide-resistant enterococci

Objective: To compare four vancomycin-containing agar media for the isolation of glycopeptide-resistant enterococci (GRE) from clinical fecal specimens: kanamycin-aesculin-azide (KAA) agar; bile-aesculin-polymixin (BAP) agar; aztreonam-amphotericin blood (CBAA) agar; and neomycin blood (CBN) agar.
Methods: Fecal specimens from 125 patients were inoculated onto each medium. Media were examined for enterococci after incubation for up to 48 h. Enterococci were identified to species level, and glycopeptide phenotypes were determined by measuring minimum inhibitory concentrations of vancomycin and teicoplanin.
Results: GRE were isolated from 44/125 samples. Enterococcus faecalis and Enterococcus faecium isolates, expressing glycopeptide resistance of the VanA or VanB phenotypes, were recovered from 27/33 (82%) specimens on BAP medium, 26/33 (79%) on KAA medium, and 21/33 (64%) on CBN and CBAA media. Enterococcus gallinarum and Enterococcus casseliflavus isolates expressing low-level glycopeptide resistance (VanC phenotype) were recovered from 14/15 (93%) specimens on CBAA medium, 7/15 (47%) on KAA and CBN media, and 6/15 (40%) on BAP medium.
Conclusions: The media tested in this study, with the exception of CBN medium, detected at least 75% of patients colonized by GRE. Further development of BAP, CBAA and KAA media is warranted to improve growth and selectivity.     

A single amino acid substitution (D1441Y) in the carboxyl-terminal propeptide of the proalpha1(I) chain of type I collagen results in a lethal variant of osteogenesis imperfecta with features of dense bone diseases

Osteogenesis imperfecta (OI) is characterised by brittle bones and caused by mutations in the type I collagen genes, COL1A1 and COL1A2. We identified a mutation in the carboxyl-terminal propeptide coding region of one COL1A1 allele in an infant who died with an OI phenotype that differed from the usual lethal form and had regions of increased bone density. The newborn female had dysmorphic facial features, including loss of mandibular angle. Bilateral upper and lower limb contractures were present with multiple fractures in the long bones and ribs. The long bones were not compressed and their ends were radiographically dense. She died after a few hours and histopathological studies identified extramedullary haematopoiesis in the liver, little lamellar bone formation, decreased osteoclasts, abnormally thickened bony trabeculae with retained cartilage in long bones, and diminished marrow spaces similar to those seen in dense bone diseases such as osteopetrosis and pycnodysostosis. The child was heterozygous for a COL1A1 4321G→T transversion in exon 52 that changed a conserved aspartic acid to tyrosine (D1441Y). Abnormal proα1(I) chains were slow to assemble into dimers and trimers, and abnormal molecules were retained intracellularly for an extended period. The secreted type I procollagen molecules synthesised by cultured dermal fibroblasts were overmodified along the full length but had normal thermal stability. These findings suggest that the unusual phenotype reflected both a diminished amount of secreted type I procollagen and the presence of a population of stable and overmodified molecules that might support increased mineralisation or interfere with degradation of bone.     

Cerebro-osseous-digital syndrome: four new cases of a lethal skeletal dysplasia--distinct from Neu-Laxova Syndrome

Neu-Laxova Syndrome (NLS) is a severe disorder with intrauterine growth retardation, edema, and characteristic face (including microcephaly with receding forehead, protuberant eyes, a flattened nose, deformed ears, cleft palate, and micrognathia). Ichthyosis is often present. Limb anomalies include hypoplastic fingers and syndactyly of fingers and toes. Patients are usually stillborn or die shortly after birth. We report five unrelated patients--four with atypical NLS and one with typical NLS. All five patients were stillbirths. Clinically, the atypical NLS patients showed a large skull; rhizo-, meso-, and acromelia; and hypoplasia of the metacarpals and phalanges. The feet were similarly affected. Radiographically, the atypical patients showed interpediculate narrowing and hypoplastic vertebral bodies. The long bones were stick-like, showing diaphyseal widening that spared the metaphyses and was more pronounced in the lower extremities. The ilia had a half-moon configuration with widening of the sacrosciatic notches. The ischia were vertical and the pubic bone was absent. The typical NLS patient showed microcephaly, normal vertebral body, and long bone ossification, but a pelvic configuration similar to that of the atypical NLS patients. The common and distinguishing clinical and radiographic features are reviewed. Scott et al. [1981: Am J Med Genet 9:165-175] described two patients with NLS with radiographic and clinical findings similar to patients 1-4 reported here. Patients 1-4 of this report lack the typical findings of NLS and likely represent a distinct lethal skeletal dysplasia.     

Cancer in the oldest old

Our previous work revealed that 88% of centenarians delay or escape the age-related lethal diseases cardiac disease, stroke and diabetes. In the cases of those having a history of cancer we have observed anecdotes of centenarians presenting with large primary tumors that would have otherwise been expected to have metastasized and to have been lethal. However, these tumors were removed without consequence. To better understand the relationship between cancer and exceptional longevity, we quantified age of cancer diagnoses, life-time clinically evident cancer prevalence, tobacco use and family histories through medical record review and interviews. One thousand one hundred and forty-three subjects were studied revealing 20% (N=152) of female and 22% (N=80) of male centenarians with a history of non-skin cancer. The most common cancers were prostate (11.7% of males), breast (8.2% of females), and colon (5.7%). The average age of diagnosis was 80.5 years compared to 63.2 years in the general population according to National Cancer Institute SEER data. Similar delays were noted when age of onset was examined according to specific type of cancer. In conclusion, the age of diagnosis of cancer is relatively delayed in those who live to 100 years. Some cancers are very rare among these individuals suggesting that there are certain cancers that may be incompatible with survival to extreme old age.     

Cytologic grade independently predicts survival of patients with pancreatic adenocarcinoma

Our objectives were to devise a cytologic grading system and determine whether it would predict survival of patients with solid-type pancreatic adenocarcinoma. We evaluated 116 consecutive patients from July 2000 to November 2002; they were followed up until September 2003. We scored the following features on rapid Romanowsky-stained endoscopic ultrasound-guided fine-needle aspiration smears: cell group architecture, single cells, nuclear grade, mucus, bizarre cells, and necrosis. A cytologic grade (low vs high) was assigned. The Kaplan-Meier estimate of 6-month survival was 76% (SE, 7%) for patients with low-grade tumors vs 50% (SE, 6%) for patients with high-grade carcinoma. The median survival for patients with low-grade vs high-grade tumors was 1 year vs 6 months, respectively (chi2 = 4.45; P = .035). Cox proportional hazards regression showed tumor stage, cancer-specific treatment, and cytologic grade to be independent predictors of survival (P = .001). No other factors (age, mass location, placement of stent, presence of concomitant chronic pancreatitis, race, sex) predicted survival. We devised a grading system that independently predicted survival in patients with pancreatic adenocarcinoma.     

Proximal urea cycle defects are challenging to detect with newborn screening: Results of a prospective pilot study using post-analytical tools

The purpose of this pilot project was to evaluate the efficacy of the Collaborative Integrated Laboratory Reports (CLIR) postanalytical tools from Mayo Clinic for detection of newborns with proximal urea cycle disorders (PUCD) in the Georgia newborn screening program that uses the underivatized Neobase2 kit (Perkin Elmer). We evaluated 138,560 newborn screening (NBS) samples (between 125,000 and 130,000 children) and used the CLIR result interpretation guidelines to stratify results. Children at higher risk of having a PUCD received follow-up services including confirmatory lab testing (ammonia, plasma amino acids, urine orotic acid) or a repeat NBS sample. We made multiple adjustments to our CLIR PUCD tool and to our follow-up algorithms in order to reduce false positives. Regardless, a high number of NBS samples resulted with false positives in part due to the glutamine peak also containing lysine. No children were diagnosed with a PUCD during our study period, and the Emory Genetics Metabolic Center is unaware of any children diagnosed outside of the NBS system during that time. Based on our experience, PUCD is not suitable for statewide NBS using Neobase2 and CLIR. Other methodologies that can separate glutamine from other amino acids may have better performance.     

Urinary hCG patterns during the week following implantation

BACKGROUND: Human chorionic gonadotrophin (hCG) is used to monitor pregnancy status. Yet the pattern of hCG excretion in the first week following implantation has not been adequately described.Therefore the aim of this study was to describe the average profile of hCG and its variability during the 7 days following estimated implantation in a population of naturally conceived pregnancies. METHODS: We measured daily hCG concentrations in first-morning urine for 142 clinical pregnancies from women with no known fertility problems. Mixed-effects regression models were used to estimate the hCG trajectory and its variability in relation to pregnancy outcomes. RESULTS: hCG rose 3-fold between the day of detection and the next day (95% CI = 2.7-3.4). The relative rate of rise decreased thereafter, reaching 1.6-fold (95% CI = 1.5-1.8) between days 6 and 7. HCG levels followed a log-quadratic trajectory, and the patterns of rise were unrelated to number of fetuses, risk of spontaneous abortion or sex of the baby. Later implantations (after 10 luteal days) produced slower rates of increase. CONCLUSIONS: Although mean hCG follows a log-quadratic trajectory during the first week of detectability, there is high variability across pregnancies. Later implantation may reflect characteristics of the uterus or conceptus that slow hCG production.     

Comparison of Nine Commercially Available Clostridium difficile Toxin Detection Assays,a Real-Time PCR Assay for C. difficile tcdB,and a Glutamate Dehydrogenase Detection Assay to Cytotoxin Testing and Cytotoxigenic Culture Methods

The continuing rise in the incidence of Clostridium difficile infection is a cause for concern, with implications for patients and health care systems. Laboratory diagnosis largely relies on rapid toxin detection kits, although assays detecting alternative targets, including glutamate dehydrogenase (GDH) and toxin genes, are now available. Six hundred routine diagnostic diarrheal samples were tested prospectively using nine commercial toxin detection assays, cytotoxin assay (CYT), and cytotoxigenic culture (CYTGC) and retrospectively using a GDH detection assay and PCR for the toxin B gene. The mean sensitivity and specificity for toxin detection assays were 82.8% (range, 66.7 to 91.7%) and 95.4% (range, 90.9 to 98.8%), respectively, in comparison with CYT and 75.0% (range, 60.0 to 86.4%) and 96.1% (91.4 to 99.4%), respectively, in comparison with CYTGC. The sensitivity and specificity of the GDH assay were 90.1% and 92.9%, respectively, compared to CYT and 87.6% and 94.3%, respectively, compared to CYTGC. The PCR assay had the highest sensitivity of all the tests in comparison with CYT (92.2%) and CYTGC (88.5%), and the specificities of the PCR assay were 94.0% and 95.4% compared to CYT and CYTGC, respectively. All kits had low positive predictive values (range, 48.6 to 86.8%) compared with CYT, assuming a positive sample prevalence of 10% (representing the hospital setting), which compromises the clinical utility of single tests for the laboratory diagnosis of C. difficile infection. The optimum rapid single test was PCR for toxin B gene, as this had the highest negative predictive value. Diagnostic algorithms that optimize test combinations for the laboratory diagnosis of C. difficile infection need to be defined.Clostridium difficile is a major nosocomial pathogen causing a range of symptoms from mild to severe diarrhea and is the etiological agent of pseudomembranous colitis. The incidence of C. difficile infection has increased markedly in many countries, notably associated with the epidemic spread of PCR ribotype 027 (NAP1) since its recognition in the United States and Canada (6, 7, 13). It is essential to have accurate laboratory diagnosis of C. difficile infection to ensure patients receive appropriate treatment and that correct infection control measures are put in place. Also, inaccurate testing will potentially lead to poor quality surveillance data that may lead to inappropriate infection prevention measures.The cytotoxin assay (CYT), first described by Chang et al., detects the toxins produced by C. difficile in the supernatants of patient feces, using both antitoxin-protected and nonprotected cell monolayers (2). This assay is commonly used as the gold standard method for comparison in toxin kit evaluations, although its use in routine microbiology laboratories has largely been superseded. Cytotoxogenic culture (CYTGC) has been used as an alternative gold standard method to CYT testing, i.e., where CYT testing is performed using culture supernatants instead of directly from the fecal sample (1). These are lengthy assays, however, with results delayed for 24 to 48 h for the CYT and for more than 72 h for the CYTGC assay.Rapid, commercially available, toxin detection kits removed the need for laboratories to maintain the cell lines necessary for CYT testing. Although originally designed to detect either toxin A or toxin B, the kits currently available detect both toxins to enable detection of toxin A-negative, toxin B-positive strains. Alternative detection methods have now been developed, including an assay that detects a surface-associated enzyme of C. difficile, glutamate dehydrogenase (GDH). Zheng et al. reported that the Techlab C. diff Chek-60 GDH assay had good sensitivity compared to CYT testing of 92%, but it had a low specificity of 89.1% and poor positive predictive value (PPV) of 57.7% (21). Commercial molecular diagnostic tests, such as the BD GeneOhm C. difficile PCR assay, which detects the tcdB toxin gene of C. difficile, are now available. A recent study compared this assay to CYT testing and found a sensitivity and specificity of 90.9% and 95.2%, respectively (15). The PPVs of the BD GeneOhm C. difficile PCR assay were only 70.2% compared with CYT testing and 89.5% compared with CYTGC (15), with a prevalence of toxin-positive fecal samples of 15.2%.Despite numerous evaluations of C. difficile testing methods, no evaluation has compared all methods on the same sample set. This study compared six commercially available enzyme immunoassays (EIAs) and three lateral-flow assays for detection of C. difficile toxins A and B, a PCR assay for detection of the tcdB gene of C. difficile, and an assay for detection of C. difficile-specific GDH, with CYT testing and CYTGC.     

Folate and one-carbon metabolism gene polymorphisms and their associations with oral facial clefts

Folate metabolism plays a critical role in embryonic development. Prenatal folate supplementation reduces the risk of neural tube defects and probably oral facial clefts. Previous studies of related metabolic genes have associated polymorphisms in cystathionine-beta-synthase (CBS) and 5,10-methylenetetrahydrofolate reductase (MTHFR) with cleft risk. We explored associations between genes related to one-carbon metabolism and clefts in a Norwegian population-based study that included 362 families with cleft lip with or without cleft palate (CL/P) and 191 families with cleft palate only (CPO). We previously showed a 39% reduction in risk of CL/P with folic acid supplementation in this population. In the present study we genotyped 12 polymorphisms in nine genes related to one-carbon metabolism and looked for associations of clefting risk with fetal polymorphisms, maternal polymorphisms, as well as parent-of-origin effects, using combined likelihood-ratio tests (LRT). We also stratified by maternal periconceptional intake of folic acid (>400 microg) to explore gene-exposure interactions. We found a reduced risk of CL/P with mothers who carried the CBS C699T variant (rs234706); relative risk was 0.94 with one copy of the T allele (95% CI 0.63-1.4) and 0.50 (95% CI 0.26-0.96) with two copies (P = 0.008). We found no evidence of interaction of this variant with folate status. We saw no evidence of risk from the MTHFR C677T variant (rs1801133) either overall or after stratifying by maternal folate intake. No associations were found between any of the polymorphisms and CPO. Genetic variations in the nine metabolic genes examined here do not confer a substantial degree of risk for clefts.