Detection of ESBL bacteria from clinical specimens: evaluation of a new selective medium.

Selective screening media for extended-spectrum beta-lactamase (ESBL)-producing bacteria are needed to guide antibiotic therapy and institute appropriate infection control measures. This study evaluates a selective cefpodoxime-incorporated chromogenic agar (CCA) medium for the detection of ESBLs from clinical specimens. The medium was formulated specifically for this study. For all culture-positive urine samples and wound swabs from intensive care unit (ICU) patients, CCA was compared with standard laboratory testing procedures and HPA/BSAC guidance on ESBL detection. The CCA medium was also evaluated for ESBL faecal carriage from patients on ICU and the haematology ward. These patients had no prior evidence of colonisation or infection with ESBL-producing bacteria. All ESBL isolates underwent minimum inhibitory concentration (MIC) testing to cefpodoxime. The Miles and Misra method and the ecometric methods were used to quality control the microbiological performance of the CCA medium, which proved satisfactory. A total of 750 specimens were examined (690 urines, 40 faeces, 20 wound swabs). From urine cultures, 92 suspect colonies were followed up. Eighteen were cefpodoxime-resistant on routine disc testing and all were confirmed subsequently as ESBL-positive. Conventional laboratory methods identified only two urinary ESBLs. Wound cultures revealed two suspect colonies, both of which were ESBL-positive and were also detected by routine methods. Faecal samples produced 10 suspect colonies, six of which were ESBL-positive. All ESBLs had cefpodoxime MICs >10 mg /L (75% were >256 mg/L). Thus, primary conventional culture methods cannot be relied upon to detect suspect ESBL-producing bacteria.

Impaired action of thyroid hormone associated with smoking in women with hypothyroidism.

BACKGROUND: The effect of smoking on thyroid function is controversial, and its effect on thyroid hormone action is unknown. We investigated the effects of cigarette smoking in women with various grades of hypothyroidism and in normal women. METHODS: We studied 138 normal women and 135 women with primary hypothyroidism, of whom 84 had subclinical hypothyroidism and 51 overt hypothyroidism. Sixty of the women with hypothyroidism were reevaluated during thyroxine therapy. The women were categorized as smokers or nonsmokers according to their responses to a questionnaire. Thyroid function was evaluated by measurements of serum thyrotropin, free thyroxine, and triiodothyronine. Peripheral thyroid hormone action was assessed by a clinical score and measurements of ankle-reflex time and serum lipids and creatine kinase. RESULTS: Among the women with subclinical hypothyroidism, the smokers had a higher mean (+/- SD) serum thyrotropin concentration (21.3 +/- 16.6 vs. 12.7 +/- 7.2 mU per liter, P = 0.004) and a higher ratio of serum triiodothyronine to serum free thyroxine (by 30 percent, P = 0.003) than the nonsmokers. Their serum concentrations of total cholesterol and low-density lipoprotein (LDL) cholesterol were higher (by 16 percent, P = 0.013; and 28 percent, P = 0.003, respectively). Among the women with overt hypothyroidism, the serum concentrations of thyrotropin, free thyroxine, and triiodothyronine were similar in the smokers and nonsmokers. As compared with the nonsmokers, the smokers had a clinical score indicating a greater degree of hypothyroidism (P < 0.001), higher serum concentrations of total and LDL cholesterol (by 25 percent, P < 0.001; and 24 percent, P = 0.002, respectively), longer ankle-reflex time (by 25 percent, P < 0.001), and higher serum concentrations of creatine kinase (by 236 percent, P < 0.001). There were dose-response relations between smoking and serum concentrations of total and LDL cholesterol, serum creatine kinase concentrations, and ankle-reflex time in the women with overt hypothyroidism, and between smoking and serum concentrations of total and LDL cholesterol in the women with subclinical hypothyroidism. CONCLUSIONS: Smoking increases the metabolic effects of hypothyroidism in a dose-dependent way. This may be explained by alteration of both thyroid function and hormone action.

Parathyroid hormone-related protein (PTHrP) in breast cancer and benign breast tissue.

Parathyroid hormone-related protein (PTHrP) 1-86 was quantified by immunoassay in extracts of 132 breast cancers, 27 samples of normal breast tissue and four fibroadenomas. PTHrP 1-86, was detected in 68% of primary tumours (range 40-302,000 fmol/g), 33% of normal breast tissues (range 100-1800 fmol/g), and all four fibroadenomas (range 110-11,600 fmol/g). PTHrP displayed molecular heterogeneity on gel filtration chromatography, and 1-86, 1-34 and 37-67 immunoreactivity eluted as 25-27 kDa together with a peak of 19-21 kDA containing only 37-67 activity. Tumour PTHrP 1-86 levels correlated inversely with age (P < 0.05) and were higher in premenopausal women (P = 0.05). The proportion of tumours containing PTHrP was higher in axillary node positive premenopausal women (P < 0.05). These data suggest that oestrogen may regulate expression of PTHrP in breast cancer and that production of PTHrP may be linked to development of axillary node metastases.

The Zimbabwe External Quality Assessment Scheme (ZEQAS) in clinical chemistry: results of the pilot programme.

A pilot programme for assessing laboratory performance in clinical chemistry laboratories in Zimbabwe is described (ZEQAS). Twenty four laboratories providing patient care services participated. Eight lyphilised bovine sera were distributed over one year. Consensus values and the spread of interlaboratory agreement were calculated for each of 12 analytes and compared with results previously obtained in a large mature national EQA scheme in the UK (UK NEQAS). For all analytes except phosphate, the mean consensus value obtained in ZEQAS was between 94 and 108 pc of the UK target, although the spread of results in ZEQAS was generally two to threefold greater for individual analytes than in UK NEQAS. It is concluded the ZEQAS consensus values for the analytes surveyed provide a valid target against which individual laboratory performance can be assessed. The wide spread of results from individual laboratories suggests there is considerable scope for improving interlaboratory agreement. This is being addressed by the continuing programme, with increased interaction and production of local specimens.

Parathyroid hormone-related protein in human term placenta and membranes.

Parathyroid hormone-related protein (PTHrP), the hypercalcaemia of malignancy factor, is expressed in the tissues of the human uteroplacental unit, including the placenta, amnion and chorion. We have used three region-specific immunoassays to quantitate and compare the distribution of PTHrP in tissues obtained at term following spontaneous labour and vaginal delivery or elective Caesarean section. In non-labouring women highest PTHrP(1-86) and (37-67) immunoreactivity was found in amnion covering the placenta, rather than the decidua parietalis of the uterus (reflected amnion) (median 1020 vs 451 fmol/g; 2181 vs 1444 fmol/g respectively). In labouring women, the PTHrP(1-86) concentration in reflected amnion was inversely correlated with the interval between rupture of the membranes and delivery. Tissue PTHrP(1-86) concentrations were lower in placenta than in chorion and amnion (medians 12, 109 and 664 fmol/g respectively) and, in all tissues, PTHrP(1-34) and (37-67) concentrations were significantly higher than that of PTHrP(1-86). Bioactive PTHrP(1-34) was detected in placenta, chorion and amnion using the ROS cell bioassay. The PTHrP(1-86) concentration (mean +/- S.E.M. = 41.4 +/- 4.5 pmol/l) was high in amniotic fluid at term, although in maternal and cord plasma levels were only modestly increased. The molecular forms of PTHrP present in tissues and amniotic fluid were investigated by column chromatography which confirmed its molecular heterogeneity and suggested that processing is tissue-specific and occurs at both amino- and carboxy-terminals of the peptide.

The hypothalamo-pituitary-adrenal axis across the normal menstrual cycle and in polycystic ovary syndrome.

OBJECTIVE: We explored the hypothesis that activation of the hypothalamo-pituitary-adrenal axis is involved in the pathogenesis of hyperandrogenism in the polycystic ovary syndrome. PATIENTS: Seven women with polycystic ovary syndrome (mean age 27.6 +/- 1.6 (SEM) years) (hirsutism, oligo/amenorrhoea and elevated serum testosterone and dehydroepiandrosterone sulphate) and nine normal female controls (mean age 24.6 +/- 1.5 years) were studied. To exclude anovulation as a confounding factor, four of these normal women were studied in both the follicular and luteal phase of the menstrual cycle. DESIGN AND MEASUREMENTS: Plasma ACTH and cortisol levels were measured at 15-minute intervals between 0600 h and 1800 h. ACTH and cortisol mean levels, pulse number and amplitude were calculated using established computer software, programmed to identify ACTH and cortisol peaks. RESULTS: With the exception of mean plasma levels of ACTH over the 12-hour period, which were reduced in the luteal phase of the menstrual cycle (1.8 +/- 0.3 pmol/l) compared to the follicular phase (2.3 +/- 0.2 pmol/l, P < 0.05), there were no differences in the pattern of ACTH or cortisol secretion across the normal cycle. In polycystic ovary syndrome, 12-hour ACTH pulse frequency was reduced (3.6 +/- 0.7) compared with controls (5.9 +/- 0.6, P < 0.05), but cortisol pulsatility and ACTH and cortisol mean levels were similar in both groups. CONCLUSION: The hyperandrogenism of polycystic ovary syndrome cannot be explained by enhanced ACTH secretion. Normal circulating cortisol levels, yet elevated dehydroepiandrosterone sulphate levels, suggests that polycystic ovary syndrome is yet another example of discrepant adrenal glucocorticoid and androgen secretion, and provides further evidence for a putative adrenal androgen stimulating factor.

Immunochemical characterisation of parathyroid hormone-related protein from tumour and non-tumour cells.

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtration chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1-86 and 1-34) and bioactivity (1-34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19-22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10-16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1-34 or 37-67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24-25 kDa, consistently slightly larger than recombinant PTHRP(1-141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.

Hypothalamic abnormalities in patients with pituitary-dependent Cushing's syndrome.

OBJECTIVE: We aimed to investigate the pattern of 24-hour ACTH and cortisol secretion in pituitary-dependent Cushing's syndrome and to evaluate the pituitary and hypothalamic contributions to the disease. PATIENTS: Five women with Cushing's disease (mean age 35 +/- 5 (SEM) years) and five normal female controls (mean age 25 +/- 2 years) were studied. DESIGN AND MEASUREMENTS: Plasma ACTH and cortisol levels were measured every 15 minutes for 24 hours using established IRMA and RIA respectively. ACTH and cortisol mean and trough levels, pulse number and amplitude were calculated using established computer software, programmed to identify ACTH and cortisol peaks. RESULTS: Patients with Cushing's disease had a twofold increase in 24-hour mean cortisol levels and a threefold increase in 24-hour mean ACTH levels (Cushing's 5.9 +/- 1.0, controls 1.9 +/- 0.2 pmol/l, P less than 0.01). This was predominantly mediated by an increase in ACTH pulse amplitude. However, 24-hour ACTH pulse number was also increased (Cushing's 15.2 +/- 2.6, controls 10.6 +/- 1.7, P less than 0.05) due to an increase in pulse number between 1800 and 2400 h. ACTH trough levels were also higher in patients with Cushing's disease (Cushing's 5.3 +/- 1.3, controls 2.3 +/- 0.2 pmol/l, P less than 0.05). CONCLUSIONS: Twenty-four-hour mean plasma cortisol and ACTH levels are elevated two to three-fold in patients with Cushing's disease. The increase in ACTH pulse amplitude suggests a pituitary abnormality in patients with Cushing's disease. However, the increased ACTH pulse frequency together with elevated trough levels is interpreted as indicating coexisting hypothalamic stimulation (or loss of inhibition).

Casual urine concentrations of sodium and potassium and geographic blood pressure variations in Great Britain.

The associations between blood pressure and urine sodium, potassium and creatinine have been studied among 1,240 men and 1,119 women living in nine British towns in order to assess the contribution of sodium and potassium intake to geographic BP variations within Great Britain. Significant positive associations were found between systolic BP and the urine sodium/potassium ratio for men (P less than 0.05) and for women (P less than 0.001), and for the sodium/creatinine ratio in men (P less than 0.01), after adjustment for age and body mass index. The findings for diastolic BP were similar, but non-significant for men. Associations between BP and sodium concentration were inconsistent and non-significant. The associations between BP and potassium concentration were consistently negative, and significant for diastolic in women (P less than 0.01). The correlations between the mean town systolic BPs and the sodium/potassium ratio were 0.65 (P = 0.058) for men, and 0.60 (P = 0.086) for women. Correlations for diastolic BP were such smaller. The association between BP and the sodium/potassium ratio in this study is consistent both within and between populations, although more so for women than for men. The results are also consistent with the results of other population studies using casual and 24 urine specimens. Although unable to quantify the effects of sodium and potassium with precision, the study suggests that the sodium/potassium ratio is of importance in geographic BP variations in Great Britain, at least for systolic blood pressure.

Modifying the request behaviour of clinicians.

AIM: To evaluate whether the feedback of laboratory use and cost data to clinicians modifies their request behaviour. METHODS: Over two years the effect of monthly feedback of clinical chemistry test use and revenue expenditure to three consultant physicians on their clinical chemistry and haematology requesting patterns was evaluated. Two physicians who received no information served as controls. RESULTS: Feedback over one year led to an immediate and sustained decrease of 15%, 27%, and 21% in clinical chemistry requests (p less than 0.01), tests (p less than 0.001), and revenue expenditure (p less than 0.001), respectively, and a 10% reduction in haematology tests (p less than 0.05) per outpatient visit. These changes persisted in the six months after the feedback was stopped. CONCLUSIONS: These results suggest that feedback of laboratory data to clinicians modifies their request behaviour and that supplying clinicians with information on what they do can influence the way they make decisions.

Production and characterisation of monoclonal antibodies to the mid-region 37-67 sequence of parathyroid hormone-related protein.

The production and characterisation of monoclonal antibodies (MAb) to the mid-region sequence 37-67 of human parathyroid hormone-related protein (PTHRP) is described. In spite of the poor immunogenicity of this sub-fragment of PTHRP, a high percentage of specific hybrids were produced by boosting with conjugate and free peptide prior to cell fusion. Seven of the MAbs produced cross-reacted with PTHRP37-67, PTHRP1-86 and native forms of PTHRP. Inhibition studies with peptide sub-fragments of PTHRP37-67 indicated that the majority recognised the 45-59 region. In a RIA for PTHRP1-86, detection limits ranged from 0.17 to 0.9 ng PTHRP1-86/tube, and no cross-reaction was found with PTH1-84. Two MAbs 1D11 and 4B10 were shown to be of potential use in measuring PTHRP1-86 in a two-site immunoradiometric assay in combination with either a solid phase consisting of a MAb to PTHRP1-34, or iodinated affinity purified rabbit antibodies to PTHRP1-34. MAb 1D11 coupled to Sepharose was suitable for immunoextraction of PTHRP, and successfully localised PTHRP on immunoblots. Two additional MAbs were produced which recognised an epitope unique to PTHRP37-67 located in the 37-46 region of the peptide.

Parathyroid hormone related protein and skeletal morbidity in breast cancer.

The presence of parathyroid hormone related protein (PTHRP) in human breast cancers has been assessed by immunohistochemistry using a polyclonal antiserum specific for the mid-region sequence 37-67 in an immunoperoxidase technique. The primary tumours from 155 normocalcaemic, consecutive women with early breast cancer who had been followed up for a minimum of 5 years were assessed. Dewaxed paraffin sections of formalin fixed tissue was used throughout. Positive PTHRP staining was detected in 56% of the cancers and was unrelated to standard prognostic factors, recurrence or survival. However, PTHRP positivity was related to the development of bone metastases (P less than or equal to 0.03) and hypercalcaemic episodes. PTHRP is implicated as the humoral factor responsible for hypercalcaemia associated with breast cancer and tumour positivity may be a useful predictor of which women will develop bone metastases.

Role of assays for parathyroid-hormone-related protein in investigation of hypercalcaemia.

Parathyroid-hormone-related protein (PTHrP) has been implicated as a humoral mediator of hypercalcaemia in malignant disease. We have investigated the contributions of PTHrP and parathyroid hormone (PTH) to the hypercalcaemia seen in routine clinical practice by means of highly sensitive immunoradiometric assays. PTHrP concentrations in plasma and PTH concentrations in serum were measured in 121 consecutive patients with hypercalcaemia (corrected serum calcium above 2.65 mmol/l) identified from routine biochemical profiles in a district general hospital. Hypercalcaemia was due to primary hyperparathyroidism in 63 (52%) patients and to malignant disease in 40 (49%). Plasma PTHrP was detectable in 35 (88%) of 40 patients with solid tumours and 3 of 9 patients with haematological malignant disease; it was undetectable in 92% of patients with primary hyperparathyroidism. 7 patients with malignant disease had PTH concentrations above 4.0 pmol/l, consistent with coexisting primary hyperparathyroidism. Measurement of both PTH and PTHrP in all patients led to a change in the diagnosis in 7% of patients. This study provides direct evidence for a humoral role of tumour-derived PTHrP in hypercalcaemia, and shows how PTHrP assays can be used appropriately, in conjunction with PTH assays, to investigate hypercalcaemia in routine clinical practice.

Parathyroid hormone related protein and hypercalcaemia in breast cancer.

OBJECTIVE: To see whether parathyroid hormone related protein has a humoral role in breast cancer. DESIGN: Plasma concentrations and tumour expression of parathyroid hormone related protein were determined (by two site immunoradiometric assay and immunohistochemistry respectively) in women with breast cancer and related to the presence of bone metastases and serum calcium concentrations. SUBJECTS: Plasma concentrations of parathyroid hormone related protein were measured in 57 women with early breast cancer without apparent bone metastases, 28 women with bone metastases, and 13 women with bone metastases and hypercalcaemia. Tissue positivity for parathyroid hormone related protein was determined retrospectively in 106 primary breast tumours from women without apparent bone metastases and 72 tumours from women with bone metastases, 25 of whom subsequently developed hypercalcaemia. RESULTS: Plasma parathyroid hormone related protein concentrations were detectable (greater than 0.23 pmol/l) in 12 (92%) of the 13 hypercalcaemic patients with bone metastases compared with 10 (36%) of the 28 normocalcaemic patients with bone metastases and five (9%) of the 57 normocalcaemic patients without bone metastases. Parathyroid hormone related protein concentrations were significantly higher in hypercalcaemic than normocalcaemic patients with bone metastases. Tumour staining was positive for parathyroid hormone related protein in 22 (88%) of the 25 primary breast cancers from patients with bone metastases. Tumour staining was positive for parathyroid hormone related protein in 22 (88%) of the 25 primary breast cancers from patients with bone metastases who later developed hypercalcaemia compared with 25 (53%) of the 47 from women in this group who remained normocalcaemic and 55 (52%) of the 106 early breast cancers from women without known metastases. CONCLUSION: Tumour derived parathyroid hormone related protein may have an important humoral role in hypercalcaemia associated with metastatic breast cancer.

Immunoreactivity of plasma parathyrin-related peptide: three region-specific radioimmunoassays and a two-site immunoradiometric assay compared.

We measured parathyrin (parathyroid hormone)-related peptide (PTHRP) in plasma by three region-specific RIAs and compared them with an established two-site immunoradiometric assay (IRMA) of PTHRP1-86 in samples from control subjects and from patients with primary hyperparathyroidism (PH) and humoral hypercalcemia of malignancy (HHM). The two direct RIAs of PTHRP1-34 and PTHRP37-67 were specific for regions 9-18 and 52-61, respectively. In the extraction RIA of PTHRP1-34 we used an affinity gel containing a monoclonal antibody specific for the 17-27 sequence; cross-reacting PTHRP species eluted from the gel were assayed by the RIA of PTHRP1-34. PTHRP1-86 plasma concentrations by IRMA were less than 0.23 pmol/L in control subjects and patients with PH, and were significantly increased in patients with HHM (mean 6.1 pmol/L, P less than 0.001). In contrast, plasma PTHRP1-34 concentrations were not significantly different in the three groups; in HHM patients, the mean was 190 pmol/L. Plasma PTHRP37-67 concentrations were similar in control and PH groups and, although significantly increased in HHM patients (mean 440 pmol/L, P less than 0.002), showed poor diagnostic discrimination. PTHRP1-34 concentrations after affinity extraction of plasma were also significantly higher in HHM patients (mean 10.7 pmol/L, P less than 0.001), but showed incomplete diagnostic discrimination. We conclude that the diagnostic utility of the direct RIAs for quantifying PTHRP is markedly inferior to the IRMA of PTHRP1-86.

Development and validation of an immunoradiometric assay of parathyrin-related protein in unextracted plasma.

This two-site immunoradiometric assay for human parathyrin-related protein 1-86 (PTHRP1-86) in plasma uses a mouse monoclonal antibody to PTHRP1-34 coupled to cellulose particles for immunoextraction of N-terminal immunoreactivity, and a rabbit antiserum to PTHRP37-67 that is indirectly labeled with 125I-labeled PTHRP37-67 for quantifying the bound analyte. The detection limit of the assay is 0.23 pmol/L, corresponding to 0.4 pg (0.04 fmol) per tube, for a sample volume of 200 microL. Recovery of PTHRP1-86 added to serum is essentially quantitative, and within- and between-batch precision is 4.4% and 11.1%, respectively. PTH1-84, PTHRP18-34, PTHRP9-34, PTHRP1-34, and PTHRP37-67 do not cross-react in the assay at concentrations up to 2 nmol/L. Plasma concentrations of PTHRP1-86 were below or close to the detection limit of the assay in normal subjects and in patients with primary hyperparathyroidism, hypoparathyroidism, chronic renal failure, and normocalcemic malignancy. In 37 hypercalcemic patients with various malignancies, we found detectable PTHRP1-86 concentrations in 35 (95%, mean 7.4 pmol/L, range 0.46-24.7). The data support the proposed humoral role of PTHRP in cancer-associated hypercalcemia and suggest that the assay has clinical utility in the differential diagnosis of hypercalcemia.

Feedback of laboratory usage and cost data to clinicians: does it alter requesting behaviour?

In a 1 year prospective study we evaluated the effect of feedback of laboratory data on the requesting behaviour of physicians in general medicine. Data on within-hours and out-of-hours clinical chemistry laboratory usage and revenue expenditure for inpatients and outpatients, expressed in terms of clinical workload, were supplied monthly to a group of three consultant physicians in general medicine. With these data the physician could monitor his performance over a period of time and compare it with that of his peers. Two consultants in general medicine who received no information served as controls. Over a period of 6 months, there was a 25%, 13% and 18% decrease in tests (P less than 0.01), requests (P less than 0.05) and revenue expenditure (P less than 0.01) per outpatient visit, respectively, in the intervention group of physicians following the introduction of feedback when compared to their baseline period and to the control group. The decrease (P less than 0.01) was in the commonly requested and 'seemingly cheap' tests. There was no significant change in laboratory use and expenditure on inpatients. The feedback of laboratory data was acceptable to the physicians, raised their awareness of laboratory usage and costs and decreased laboratory workload and expenditure.

UK external quality assessment scheme for immunoassays in endocrinology.

UK EQAS provide the UK with a comprehensive system for EQA in endocrinology, as well as in other aspects of clinical chemistry and laboratory medicine. UK EQAS in endocrinology are scientifically designed to yield an objective assessment of participants' performance and stimulate improvements in between-laboratory agreement. The design uses appropriate specimens, based on liquid human serum and prepared with minimal processing and additives in the organising centres to enable detailed study of recovery and other important factors. Target values are validated by reproducibility on repeated distribution and by recovery and parallelism studies. Reports are presented informatively, and emphasise the cumulative scoring system (bias and variance) for performance assessment. Computerised data processing and data presentation form an integral part of these schemes, and a common core computing system is in use throughout these UK EQAS. Participants receive advice and assistance in the interpretation of performance data and, when appropriate, in the resolution of problems.