Proteomics for the diagnosis of thyroid lesions: preliminary report.

OBJECTIVE: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a unique proteomic technology that explores the spatial distribution of biomolecules directly in situ, thus integrating molecular and morphological information. The possibility of correlating distribution maps of multiple analyses with cytological features makes it an ideal research tool for discovering new diagnostic markers. A previous study showed that MALDI-IMS could help discrimination between different types of thyroid lesions, especially papillary thyroid carcinoma (PTC); the present feasibility study on ex vivo fine needle aspiration (FNA) smears describes its potential in detecting new proteomic targets of other thyroid lesions (follicular lesions, medullary carcinoma). METHODS: MALDI-IMS was conducted on ex vivo FNAs obtained from surgical specimens and corresponding in vivo samples. Differences between proteomic profiles of different thyroid lesions were compared. RESULTS: Comparing the protein profiles of hyperplastic nodules obtained from three different patients with each other, and with a new PTC, showed a high degree of concordance, indicating good reproducibility of the IMS technology on cytological samples, suggesting its potential as a tool for biomarker discovery. Furthermore, comparison of the average proteomic profiles of hyperplastic nodules with a Hürthle cell adenoma revealed significant differences, underlying the capability of MALDI-IMS to distinguish between different thyroid lesions. Finally, the proteomic profile of medullary thyroid carcinoma was also characterized. CONCLUSIONS: Our results confirmed the possible role of MALDI-IMS in the search for diagnostic targets of PTC and follicular lesions, which could be applied in larger trials aimed at the identification of proteins, convertible to cost-effective diagnostic tools such as immunohistochemistry. These tests could be used to analyse in vivo cytological smears, improving the preoperative diagnosis of indeterminate thyroid nodules.

[Urinary NMP22 as a new marker in patients with transitional cell carcinoma]. / NMP22 urinario come nuovo marcatore tumorale in pazienti con carcinoma uroteliale.

To evaluate urinary NMP22 dosage as a marker of urothelial tumours, we have selected a group of 90 patients (85 males and 5 females, mean age 66 years) with clinical suspicion of transitional cell carcinoma (TCC), with microscopic or macroscopic hematuria, flank pain, urographic abnormalities and dysuria. All the patients have been evaluated by urinary cytology, renal and bladder ultrasound, cystoscopy. When a bladder tumour has been detected, bladder biopsies and, when required, I.V.P., CT or retrograde pyelography have been performed. A urine sample has been collected between midnight and noon; all samples from patients who were undergoing invasive procedures such as cystoscopy, were collected before or at least 5 days after the procedure. The test has been performed according to ELISA NMP22 (Matritech) technique; the test is specific for the nuclear matrix protein/nuclear mitotic apparatus protein expressed by cancer cells. When performing the test, 30 patients presented macroscopic hematuria. 22 patients resulted to have benign urinary tract conditions, 65 patients had TCC and 3 patients had a final evaluation suspicious for TCC. The NMP22 values were respectively 7.1 +/- 4.7 U/ml, 21.9 +/- 21.0 U/ml and 16 +/- 8.0 U/ml. From our study the sensitivity of the test is 67% (with a threshold value of 10 U/ml) and 55% (with a threshold value of 20 U/ml), while the urinary cytology resulted to have a sensitivity of 26% (p < 0.05). The sensitivity of the test in relation to staging was as follow: Tis 66% with a mean NMP22 value of 23.3 U/ml, Ta 26% with a mean NMP22 value of 13.2 U/ml, T1 100% with a mean NMP22 value of 40 U/ml, T2 73% with a mean NMP22 value of 36.4 U/ml. The specificity of the test was 100% with a threshold value of 20 U/ml. When considering a threshold value of 10 U/ml, the NMP22 test has a sensitivity higher than cytology, especially in low stage TCC. Macroscopic hematuria does not affect its sensitivity; the diagnostic efficacy of the test is not increased by the association of urinary cytology. It has an important role in the diagnosis of residual disease after TURB and in the follow-up evaluation of bladder cancer patients, since its dosage is not influenced by inflammatory conditions of the mucosa. We believe therefore that NMP22 is useful in clinical practice.

Determination of plasma metformin by a new cation-exchange HPLC technique.

Metformin is an oral antihyperglycemic agent used in the therapy of noninsulin-dependent diabetic patients. This biguanide can induce dangerous complications such as lactic acidosis when its plasma concentration is too high. For this reason, the determination of plasma metformin should always be done during treatment. We developed a new HPLC method, for the routine determination of plasma metformin, with good reliability, rapid execution, and low costs. Sample preparation involved precipitation of the plasma proteins containing the internal standard buformin with a mixture of methanol, zinc sulfate, and ethylene glycol; the diluted supernatant was injected into a cation-exchange column. The mobile phase was potassium dihydrogenphosphate buffer-containing acetonitrile. The eluent was monitored at 236 nm. The calibration curve is linear within the range of 20-4000 ng/mL; the within-day coefficients of variation were less than 2.2% for metformin and 1.5% for buformin; the day-to-day coefficients of variation were less than 2.5% for metformin and 1.9% for buformin. The mean recoveries obtained from supplemented samples were included between 99.4 and 104.2% for metformin. Many characteristics make this method useful and easily accessible to all clinical laboratories equipped with HPLC instrumentation.

Comparative evaluation of the new gen-probe Mycobacterium tuberculosis amplified direct test and the semiautomated abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens.

Two commercial assays that detect Mycobacterium tuberculosis complex (MTB) in clinical specimens by rRNA target amplification (AMTDII) and ligase chain reaction (LCx) were evaluated. The tests were applied to 457 respiratory (n = 273) and extrapulmonary (n = 184) specimens collected from 357 patients. The results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered to be the "gold standard." Seventy specimens were from patients with pulmonary tuberculosis and 28 specimens were from patients with extrapulmonary tuberculosis. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values for respiratory specimens were 92.8, 99.4, 98.5, and 97%, respectively, for AMTDII and 75.7, 98.8, 96.4, and 90.5%, respectively, for LCx. With extrapulmonary specimens, the overall sensitivities, specificities, and positive and negative predictive values were 78.6, 99.3, 95.6, and 96.2%, respectively, for AMTDII and 53.6, 99.3, 93.7, and 92.1%, respectively, for LCx. The level of agreement between AMTDII and LCx assay results was 78.2%. We conclude that although both nucleic acid amplification methods are rapid and specific for the detection of MTB in clinical specimens, AMTDII is significantly more sensitive than LCx with both respiratory (P = 0.005) and extrapulmonary (P = 0.048) specimens.

Total carbon dioxide measured by the Vitros enzymatic method.

We evaluated the performance of an enzymatic method using dry chemistry for serum total carbon dioxide (tCO2) determination using a Vitros 500 analyser. Imprecision results were acceptable and the linearity was verified for concentrations within a range of 5.5-39.2 mmol/l, i.e. Y(measured) = 0.93 x(calculated) + 1.32, r = 0.99. The Vitros tCO2 method was unaffected by haemoglobin at all concentrations tested. Significant interference was caused by bilirubin at concentrations higher than 30 mumol/l; the addition of bilirubin lowered the apparent values for tCO2 dose-dependently. Serum tCO2 results were practically the same as those for plasma. The reference interval for venous tCO2 concentrations in a healthy population was: 22.4-34.2 mmol/l (mean: 28.3 mmol/l). Comparison of venous serum tCO2 results assayed using the Vitros method with bicarbonate (HCO3-) values calculated by blood gas determination of pCO2 and pH in arterial blood samples gave poor agreement, r = 0.58. The data revealed a mean difference of 5.48 +/- 3.09 mmol/l between the tCO2 measurements and calculated bicarbonate. This was statistically (p = 0.01) and clinically significant. We conclude that the Vitros method provides reliable tCO2 results in venous serum but this method must not be used as an interchangeable alternative to calculated arterial bicarbonate in order to avoid confusion, misinterpretation of results and erroneous therapeutic decisions.

Evaluation of a new method for rapid drug susceptibility testing of Mycobacterium avium complex isolates by using the mycobacteria growth indicator tube.

The reliability of the Mycobacteria Growth Indicator Tube (MGIT [BBL]) for rapid drug susceptibility testing of Mycobacterium avium complex (MAC) isolates was evaluated. MICs of amikacin, clarithromycin, clofazimine, ethambutol, and rifabutin were determined by the MGIT system for 16 MAC strains. The results were compared with those obtained by the BACTEC broth macrodilution method. The turnaround times were 6 to 8 days (median, 7 days) for the MGIT and 5 to 7 days (median, 6 days) for the BACTEC system. Agreements with BACTEC system-determined MICs, within +/-1 log2 dilution, were 100, 100, 88, 63, and 44% for amikacin, clofazimine, rifabutin, clarithromycin, and ethambutol, respectively. Within +/-2 log2 dilutions, agreement with BACTEC system-determined MICs increased to 100% for all the tested drugs. In addition, if MGIT-determined MICs were evaluated according to the thresholds adopted for the interpretation of BACTEC system-determined ones, ethambutol was the only drug for which susceptible strains were frequently misclassified as resistant. It is concluded that the MGIT system is a promising, nonradiometric alternative to the BACTEC method for rapid susceptibility testing of MAC isolates; however, additional studies are required to confirm our results and to determine the optimal criteria for the interpretation of ethambutol MICs.

Serum markers for monitoring of prostatic carcinoma.

BACKGROUND: Serum TPS (tissue polypeptide-specific antigen) has been observed to be characteristic of carcinoma proliferation, and increased levels of TPS seem to be closely related to tumor progression. In this study we wanted to evaluate the importance of the tumor-marker TPS in the diagnosis and follow-up of patients with prostatic carcinoma, and to compare it with prostate-specific antigen (PSA). METHODS: We considered 39 patients with clinically confined disease, who underwent neoadjuvant hormonal therapy and thereafter radical prostatectomy, and 45 patients who did not undergo surgery and underwent hormonal adjuvant therapy alone. PSA and TPS were measured at the time of diagnosis and at regular intervals in the follow-up; TPS was measured in a control group of patients as well. RESULTS: We were able to observe that, in untreated patients, PSA correlates with clinical stage, increasing with increasing tumor stage; a similar correlation was not observed when considering TPS. After androgen ablation we observed a decrease in PSA, but the serum values of TPS remained higher, suggesting that activity still exists inside the tumor. The evaluation of TPS appeared to be of particular interest in the follow-up after radical prostatectomy, especially in patients undergoing hormonal therapy; in fact, we were able to observe that relapse of the disease can be suspected early by the increase of TPS in hormonally treated patients. CONCLUSIONS: We assert that TPS can add useful information on the state of neoplastic illness, especially in patients following adjuvant androgen-suppressive hormonal therapy, after radical prostatectomy; serial measurements of this marker could be useful in the early diagnosis of a relapse.

[Diagnostic efficacy of free SPA/total PSA ratio in the diagnosis of prostatic carcinoma]. / Efficacia diagnostica del rapporto PSA-free/PSA totale nella diagnosi del carcinoma prostatico.

Prostate specific antigen, specific organ and tissue marker, is a glycoprotein present in serum in different molecular forms, i.e. not protein bound and bound to proteins (PSA-ACT and PSA-AMG). The total PSA is expressed by the sum of the non protein bound value (free-PSA) and PSA-ACT. The aim of our study was to evaluate the hypothesis that measurement of free/total PSA ratio may be helpful in the differential diagnosis of prostatic pathology. Our study was conducted on 350 patients, to whom the total-PSA, free-PSA and f/t PSA had been performed; 250 patients showed a total PSA between 2.5 and 10 ng/ml and 185 of them had symptoms of bladder out-flow obstruction. In all of the 250 patients digital rectal examination, transrectal ultrasound and prostatic biopsy were performed. 100 patients were controls. The cut-off to differentiate between benign and malignant prostatic disease was 16%. The pathologic diagnosis was related to the f/t PSA ratio, and in particular those patients with a f/t PSA lower than 16% were expected to be prostatic carcinoma, while those with a f/t PSA higher than 16% were expected to be benign prostatic hypertrophy. The diagnostic accuracy of the ratio was calculated, and it was observed that it was 88.65% in the diagnosis of benign prostatic hypertrophy, while in the diagnosis of prostatic carcinoma it was 84.5%. We can therefore assume that f/t PSA can add useful information on prostatic pathology, eventually sparing unnecessary prostatic biopsies.

Isolation of Mycobacterium celatum from patients infected with human immunodeficiency virus.

Mycobacterium celatum is a recently described, slowly growing mycobacterium of still undefined clinical relevance. A retrospective study of seven patients was conducted to further elucidate the clinical presentation and prognosis of infection due to M. celatum in patients with AIDS. Three patients had an exclusively pulmonary infection and 3 had disseminated infection (including 2 patients with pulmonary and extrapulmonary involvement), and 1 patient had an exclusively extrapulmonary disease. Fever, weight loss, and productive cough lasting for >2 weeks were the most common symptoms. Chest radiographs showed diffuse or focal interstitial infiltrates without cavitation. The recovery of M. celatum from one patient was definitively determined to be clinically irrelevant. Our findings indicate that M. celatum may cause serious disease in patients with advanced human immunodeficiency virus-related immunosuppression. M. celatum infection appears to be responsive to antimycobacterial chemotherapy; however, further studies are needed to establish the optimal drug combination for this indication.

Determination of vanillylmandelic, 5-hydroxyindoleacetic and homovanillic acid in urine by isocratic liquid chromatography.

A new isocratic HPLC method, employing electrochemical detection, is described for the determination of urinary vanillylmandelic acid, 5-hydroxyindoleacetic acid and homovanillic acid. The main advantages of this technique are: simplicity, simultaneous determination of all analytes, the absence of an extraction procedure, isocratic elution and low cost. The diluted urine is injected onto a C18 reversed phase column. The mobile phase is potassium dihydrogenphosphate buffer containing 1-heptanesulphonic acid, methanol and acetonitrile. The calibration curves are linear from 0.1 to 50 mg/l; the precision data show CV less than 2.36% for within-day assay and less than 2.72% for day-to-day assays. The mean recoveries for supplemented samples are 98.2 to 102.0% for vanillylmandelic acid, 99.6 to 103.9% for 5-hydroxyindoleacetic acid and 98.7 to 102.0% for homovanillic acid. In comparisons of the present method with Radjaipur's extraction method (Radjaipur M. et al., Eur J Clin Chem Clin Biochem 1994; 32:609-13) the slopes for the three analytes were nearly 1, and the confidence region of the intercepts was close to 0. In conclusion the technique seems to be suitable for routine determination of the three analytes, especially for mass screening purposes.

Comparative evaluation of two commercial amplification assays for direct detection of Mycobacterium tuberculosis complex in respiratory specimens.

Two commercial assays detecting the presence of Mycobacterium tuberculosis complex in clinical specimens by rRNA target amplification (Gen-Probe Amplified M. tuberculosis Direct Test [AMTD]) and PCR (Amplicor) were evaluated. The tests were applied to 327 digested, decontaminated respiratory specimens collected from 236 patients. Results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered the "gold standard." A total of 60 specimens were collected from 27 patients with a diagnosis of pulmonary tuberculosis. Thirteen of these specimens were from patients receiving standard antituberculosis therapy and therefore were not included in the comparison. Of the remaining 47 specimens, 33 were smear positive, 40 were culture positive, 45 were AMTD positive, and 39 were Amplicor positive. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values were 77, 100, 100, and 95 for staining; 87, 100, 100, and 97.4 for culture; 95.9, 98.9, 94, and 99.2 for AMTD; and 85.4, 99.6, 97.9, and 97.1 for Amplicor, respectively. Agreement between AMTD and Amplicor assay results was 96.8%. It is concluded that although both nucleic acid amplification methods are rapid and specific for the detection of M. tuberculosis complex in respiratory specimens, AMTD appeared to be more sensitive than Amplicor.

An evaluation of the Ektachem serum lithium method and comparison with flame emission spectrometry.

We evaluated the performance of a new colorimetric method in dry chemistry for serum lithium (Li) assay using an Ektachem E500 analyser. Imprecision results were acceptable and the linearity was verified for concentrations within the range of 0.2-3.9 mmol l-1, i.e. y(measured) = 1.02x(calculated) + 0.07, r = 0.99. The Ektachem Li assay was unaffected by potassium (K), calcium (Ca) and magnesium (Mg) at all concentrations tested. Significant interference was caused by sodium (Na) and haemoglobin. Statistically and clinically significant interference was caused by high concentrations of Na (213 mmol l-1) with a bias of 0.20 mmol l-1 (p = 0.02) and by high levels of haemoglobin (280 mumol l-1) with a bias of 0.20 mmol l-1 (p = 0.01). Comparison of serum Li results from 80 patient samples assayed using the Ektachem method with those obtained using the IL943, a flame atomic emission spectrometry (FAES)-based method, gave a regression line equation: Ektachem = 0.95xFAES + 0.13, r = 0.96. The data revealed a mean difference of 0.10 mmol l-1 between the Ektachem and FAES results that was statistically significant (p = 0.01), but clinically negligible. We conclude that the Kodak method provides reliable Li serum results and represents a valid alternative to the FAES method.

The effect of different glucose endovenous administrations on amylin and insulin blood concentration in healthy subjects.

Islet amyloid polypeptide (IAPP) or amylin is a 37 amino-acid peptide involved in carbohydrate metabolism. It is a hormone secreted from the pancreatic cells which is reported to be co-secreted with insulin. Its function and secretion is not well known. A paracrine inhibition of insulin secretion could be the main function of this hormone. Different hypotheses are considered regarding IAPP co-localization with insulin in the secretory granules and its subsequent secretion. In the attempt to clarify these controversial findings we evaluated the plasma concentrations of IAPP and insulin during different intravenous glucose administrations in healthy patients. Twenty normoglycemic patients (10 females, 10 males, age +/- SD 37 +/- 9 years) underwent two different endovenous glucose administrations: a) "long duration" infusion, in which 500 ml of 33% glucose solution was administered in 60 minutes, b) a standard intra-venous glucose tolerance test (IVGTT) characterized by an infusion of 0.33 g/Kg of glucose in 4 minutes. Blood samples were collected at fixed times (from 0 to 180 minutes) to assay IAPP, insulin and glucose concentration. The IAPP secretion seemed to be related more to the glycemic variation than to the insulin secretion. A proportional behavior of IAPP and insulin was observed only in the first 90 minutes of the long duration infusion. The highest increases of IAPP concentrations were found when there was a rapid glucose decrease in the blood. These findings suggest a significant role of IAPP in regulating the rapid decreases of glycemia.

Activity of seven antimicrobial agents, alone and in combination, against AIDS-associated isolates of Mycobacterium avium complex.

The activity of seven antimicrobial agents (and five two-drug combinations and five three-drug combinations) was investigated against 37 clinical isolates of Mycobacterium avium recovered from blood cultures of AIDS patients. The susceptibility tests were performed in Middlebrook 7H12 broth using a radiometric method. MICs of amikacin, ciprofloxacin, clarithromycin, clofazimine, ethambutol, rifabutin and sparfloxacin were determined. Five antimicrobial agents were tested in combination with clarithromycin and also with clarithromycin plus amikacin to look for possible synergic activity. Synergic activity in combination with clarithromycin and with clarithromycin plus amikacin, was detected for rifabutin (54% and 51% of isolates, respectively), clofazimine (38% and 35%), ethambutol (16% and 32%), ciprofloxacin (8% and 14%) and sparfloxacin (3% and 8%). No antagonism was observed. We conclude that clarithromycin is an essential component in the chemotherapy of M. avium complex disease.

Comparison of two selective media and a membrane filter technique for isolation of Campylobacter species from diarrhoeal stools.

Diarrhoeal stool specimens from 415 patients were examined for Campylobacter spp. by culture on charcoal cefoperazone deoxycholate agar (CCDA), Skirrow medium and Columbia blood agar overlaid with a 0.65 micron pore size membrane filter. Forty-eight Campylobacter strains were isolated from 45 (10.8%) specimens by all media; 44 were Campylobacter jejuni (91.7%), three were Campylobacter coli (6.3%) and one was Campylobacter hyointestinalis (2.0%). The percentages of Campylobacter-positive specimens isolated on Skirrow medium, CCDA and the membrane filter were 62, 82 and 95%, respectively. The recovery of more Campylobacter spp. from the same stool sample was achieved by the membrane filter method only. The highest isolation rate (100%) was observed when culture on CCDA and the membrane filter method were combined.

[Treatment of hyperlipidemia in obese patients: monotherapy versus bi-therapy]. / Traitement de l'hyperlipidémie chez les sujets obèses: monothérapie versus bithérapie.

OBJECTIVES: Severe hyperlipoproteinaemia (increased LDL, light density lipoproteins, and VLDL, very light density lipoproteins) in patients with high body mass index (BMI) is positively associated with the occurrence of coronary heart disease. This condition requires combined drug regimen because high lipid levels frequently remain after monotherapy and diet. The aim of our study was to investigate the efficacy of combined therapy utilizing the following association: HMG CoA reductase inhibitors plus fibrates. METHODS: We examined 50 patients, males, affected by obesity (BMI > 30) and hyperlipoproteinaemia (phenotype IIB, Fredrickson). The first group, 20 obese subjects with severe dislipidaemia, and the second group, 10 mildly hyperlipidaemic obese patients received bezafibrate 600 mg/d and pravastatin 40 mg/d. The other subjects, all obese and highly dyslipidaemic patients, received monotherapy: 10 patients, bezafibrate 600 mg/d and the rest pravastatin 40 mg/d. Weekly, for ten weeks, we evaluated the following serum parameters: total cholesterol and HDL-cholesterol, triglycerides and apolipoprotein A1 and B. RESULTS: We observed no significant changes in HDL-cholesterol and apolipoprotein levels, while an important reduction in total cholesterol and triglycerides, induced by combined therapy, was particularly evident in those patients with the higher lipidic alterations, compared with the additive effects of single drugs. CONCLUSIONS: The data show that this combined treatment could be proposed for these subjects to reduce hyperlipidaemia and the risk of premature atherosclerosis.

Isolation of the newly described species Mycobacterium celatum from AIDS patients.

Mycobacterium celatum is a recently described species which, on the basis of conventional tests, may be misidentified as Mycobacterium xenopi or as belonging to the Mycobacterium avium complex. Only genomic sequencing or high-performance liquid chromatography of cell wall mycolic acids can presently allow a correct identification of this mycobacterium. Two cases of infection due to M. celatum, in AIDS patients, are described here. The quantitative susceptibility pattern of the isolates to a wide spectrum of drugs is also reported.

Bacteriostatic and bactericidal activities of paromomycin against Mycobacterium avium complex isolates.

The MICs and MBCs of paromomycin for 32 Mycobacterium avium complex (MAC) strains isolated from patients with the acquired immunodeficiency syndrome were determined by a radiometric broth dilution method. The MICs for the majority of strains were either 8 or 16 mg/L and the MBCs were four- to eight-fold higher. Paromomycin merits further evaluation as oral prophylaxis against disseminated MAC infection.

Value of antigen and antibody detection in the serological diagnosis of invasive aspergillosis in patients with hematological malignancies.

In a study to determine the value of antigen and antibody detection in the serological diagnosis of invasive aspergillosis in patients with hematological malignancies, 436 sequential serum samples from 79 neutropenic patients were tested. Circulating galactomannan antigen was detected with a latex agglutination method (Pastorex Aspergillus) and antibody to Aspergillus fumigatus with an immunodiffusion test on agar gel. Among the 79 patients 18 cases of invasive aspergillosis were detected (4 proven, 6 highly probable, 3 probable, 5 suspected). Antigen was detected in 7 of these 18 patients. The sensitivity of the antigen test was 38.8% and the specificity 95%. The antibody test showed 55.5% sensitivity and 100% specificity. In this study the antigen test was less sensitive than previously reported but highly specific and might serve as a supplementary diagnostic test.

Blood catecholamine levels and lymphocyte beta-adrenoceptors following acute noise stress.

Blood hormonal levels and lymphocyte beta-adrenoceptor characteristics have been studied in subjects exposed to acute noise stress. Cortisol and DHEAS show a peak at 15 min after the beginning of the stimulus. Catecholamine levels show an increase in 4 out of 8 subjects. However, no statistically significant changes have been observed in beta-adrenoceptor characteristics.