José Manuel González-Buitrago

Direct Identification of Urinary Tract Pathogens from Urine Samples by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

ABSTRACT Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been suggested as a reliable method for bacterial identification from cultures. Direct analysis of clinical samples might increase the usefulness of this method, shortening the time for microorganism identification. We compared conventional methods for the diagnosis of urinary tract infections (UTIs) and identification of the urinary tract pathogens (automated screening, plate cultures, and identification based on biochemical characteristics) and a fast method based on conventional screening and MALDI-TOF MS. For this latter method, 4 ml of urine was centrifuged at a low-revolution setting (2,000 × g) to remove leukocytes and then at high revolutions (15,500 × g) to collect bacteria. The pellet was washed and then applied directly to the MALDI-TOF MS plate. Two hundred sixty urine samples, detected as positive by the screening device (UF-1000i), were processed by culture and MALDI-TOF MS. Twenty samples were positive in the screening device but negative in culture, and all of them were also negative by MALDI-TOF MS. Two-hundred thirty-five samples displayed significant growth of a single morphological type in culture. Two-hundred twenty of them showed bacterial growth of >105 CFU/ml. Microorganism identifications in this group were coincident at the species level in 202 cases (91.8%) and at the genus level in 204 cases (92.7%). The most frequent microorganism was Escherichia coli (173 isolates). MALDI-TOF MS identified this microorganism directly from the urine sample in 163 cases (94.2%). Our results show that MALDI-TOF MS allows bacterial identification directly from infected urine in a short time, with high accuracy, and especially when Gram-negative bacteria with high bacterial counts are involved.

Microorganisms direct identification from blood culture by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows a fast and reliable bacterial identification from culture plates. Direct analysis of clinical samples may increase its usefulness in samples in which a fast identification of microorganisms can guide empirical treatment, such as blood cultures (BC). Three hundred and thirty BC, reported as positive by the automated BC incubation device, were processed by conventional methods for BC processing, and by a fast method based on direct MALDI-TOF MS. Three hundred and eighteen of them yield growth on culture plates, and 12 were false positive. The MALDI-TOF MS-based method reported that no peaks were found, or the absence of a reliable identification profile, in all these false positive BC. No mixed cultures were found. Among these 318 BC, we isolated 61 Gram-negatives (GN), 239 Gram-positives (GP) and 18 fungi. Microorganism identifications in GN were coincident with conventional identification, at the species level, in 83.3% of BC and, at the genus level, in 96.6%. In GP, identifications were coincident with conventional identification in 31.8% of BC at the species level, and in 64.8% at the genus level. Fungaemia was not reliably detected by MALDI-TOF. In 18 BC positive for Candida species (eight C. albicans, nine C. parapsilosis and one C. tropicalis), no microorganisms were identified at the species level, and only one (5.6%) was detected at the genus level. The results of the present study show that this fast, MALDI-TOF MS-based method allows bacterial identification directly from presumptively positive BC in a short time (<30 min), with a high accuracy, especially when GN bacteria are involved.

Rapid method for direct identification of bacteria in urine and blood culture samples by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: intact cell vs. extraction method

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable technology for the identification of microorganisms with proteomics approaches. Here, we compare an intact cell method and a protein extraction method before application on the MALDI plate for the direct identification of microorganisms in both urine and blood culture samples from clinical microbiology laboratories. The results show that the intact cell method provides excellent results for urine and is a good initial method for blood cultures. The extraction method complements the intact cell method, improving microorganism identification from blood culture. Thus, we consider that MALDI-TOF MS performed directly on urine and blood culture samples, with the protocols that we propose, is a suitable technique for microorganism identification, as compared with the routine methods used in the clinical microbiology laboratory.

MALDI-TOF mass spectrometry is a fast and reliable platform for identification and ecological studies of species from family Rhizobiaceae.

Family Rhizobiaceae includes fast growing bacteria currently arranged into three genera, Rhizobium, Ensifer and Shinella, that contain pathogenic, symbiotic and saprophytic species. The identification of these species is not possible on the basis of physiological or biochemical traits and should be based on sequencing of several genes. Therefore alternative methods are necessary for rapid and reliable identification of members from family Rhizobiaceae. In this work we evaluated the suitability of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for this purpose. Firstly, we evaluated the capability of this methodology to differentiate among species of family Rhizobiaceae including those closely related and then we extended the database of MALDI Biotyper 2.0 including the type strains of 56 species from genera Rhizobium, Ensifer and Shinella. Secondly, we evaluated the identification potential of this methodology by using several strains isolated from different sources previously identified on the basis of their rrs, recA and atpD gene sequences. The 100% of these strains were correctly identified showing that MALDI-TOF MS is an excellent tool for identification of fast growing rhizobia applicable to large populations of isolates in ecological and taxonomic studies.

Identification of Brucella by MALDI-TOF Mass Spectrometry. Fast and Reliable Identification from Agar Plates and Blood Cultures

Background MALDI-TOF mass spectrometry (MS) is a reliable method for bacteria identification. Some databases used for this purpose lack reference profiles for Brucella species, which is still an important pathogen in wide areas around the world. We report the creation of profiles for MALDI-TOF Biotyper 2.0 database (Bruker Daltonics, Germany) and their usefulness for identifying brucellae from culture plates and blood cultures. Methodology/Principal Findings We created MALDI Biotyper 2.0 profiles for type strains belonging to B. melitensis biotypes 1, 2 and 3; B. abortus biotypes 1, 2, 5 and 9; B. suis, B. canis, B ceti and B. pinnipedialis. Then, 131 clinical isolates grown on plate cultures were used in triplicate to check identification. Identification at genus level was always correct, although in most cases the three replicates reported different identification at species level. Simulated blood cultures were performed with type strains belonging to the main human pathogenic species (B. melitensis, B. abortus, B. suis and B. canis), and studied by MALDI-TOF MS in triplicate. Identification at genus level was always correct. Conclusions/Significance MALDI-TOF MS is reliable for Brucella identification to the genus level from culture plates and directly from blood culture bottles.

Clinical evaluation of a new automated anti-dsDNA fluorescent immunoassay

Abstract The measurement of anti-double-stranded DNA (antidsDNA) antibodies is a useful tool for the diagnosis and the follow-up of systemic lupus erythematosus (SLE). Anti-dsDNA antibodies are involved in the pathogenesis of lupus nephritis and they are, specially the highavidity antibodies, the most specific antibodies associated with SLE nephritis and active SLE. The aim of the present study was to assess the clinical utility of an enzyme-linked immunosorbent assay (ELISA) that utilizes a circular double-stranded plasmid DNA as a nucleic acid source, adapted to an automated fluorescence immunoassay (EliA™ dsDNA, Pharmacia, Freiburg, Germany). Also, we compared this method with other immunoassays used in clinical laboratories. We have measured anti-dsDNA antibodies in the serum of 179 patients with a positive result for antinuclear antibodies (ANA). Seventy six sera were from SLE patients (14men and 62 women), and the other 103 sera (from 20 men and 83 women) constituted the control group. This latter group includes nine Sjögrens syndrome patients, six patients with rheumatoid arthritis and 88 with various other diseases, including connective tissue diseases (n=34), hepatopathies (n=17; 11 primary biliary cirrhosis and 6 autoimmune hepatitis), and 37 patients with nonautoimmune diseases (viral hepatitis, renal disease, diabetes, exanthema and hypertension). Methods used were “EliA™ dsDNA” (Pharmacia, Germany), “Varelisa® dsDNA” (Pharmacia, Germany), Farr (Amersham, UK) and Chritidia luciliae immunofluorescence test (Vitro-Immun, Germany). We assessed sensitivity, specificity, positive predictive value and negative predictive value in the clinical study, and kappa index and scatter plots in the comparative study. The results show a low concordance between methods (κ<0.6). The evaluated EliA method shows a very good specificity for SLE (93.2%) and a good sensitivity for active SLE (70.8%).

Biological Variation of Interleukin-1β, Interleukin-8 and Tumor Necrosis Factor-α in Serum of Healthy Individuals

Abstract Components of biological variation can be used to assess the usefulness of reference values, to evaluate the significance of changes in serial results from an individual and to define objective analytical goals. The aim of the study was to assess, in 15 healthy subjects studied at regular monthly intervals over a period of 6 consecutive months, the biological variation of interleukin-1β (IL-1β), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α). Biological variation data (within-subject and between-subject coefficient of variation (CV)) were determined using a simple nested analysis of variance. Derived parameters (index of individuality, reliability coefficient and critical diferences) were calculated from within-subject and between-subject CV. The mean and standard deviation (SD), within-subject CV, between-subject CV, index of individuality and reliability coefficient were as follows: for IL-1β, 0.67 (0.32) pg/ml, 30%, 36%, 0.85, and 0.76; for IL-8, 3.68 (1.45) pg/ml, 24%, 31%, 0.85 and 0.75; and for TNF-α, 3.14 (1.87) pg/ml, 43%, 29%, 1.56 and 0.50, respectively. We conclude that between-subject variation and within-subject variation are quite similar for IL-1β and IL-8 and are relatively high for the three cytokines studied. Index of individuality is less than 1.4 for IL-1β and IL-8, and thus reference intervals based on population studies are of limited value. On the contrary, the index of individuality for TNF-α is greater than 1.4 and reference values can be used for diagnosis. Quality goals for imprecision are easily achieved for the three cytokines with current methodology.

Urinary levels of regenerating islet-derived protein III β and gelsolin differentiate gentamicin from cisplatin-induced acute kidney injury in rats

A key aspect for the clinical handling of acute kidney injury is an early diagnosis, for which a new generation of urine biomarkers is currently under development including kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin. A further diagnostic refinement is needed where one specific cause among several potentially nephrotoxic insults can be identified during the administration of multidrug therapies. In this study we identified increases in regenerating islet-derived protein III beta (reg IIIb) and gelsolin as potential differential urinary markers of gentamicins nephrotoxicity. Indeed, urinary levels of both reg IIIb and gelsolin distinguish between the nephrotoxicity caused by gentamicin from that caused by cisplatin where these markers were not increased by the latter. Reg IIIb was found to be overexpressed in the kidneys of gentamicin-treated rats and excreted into the urine, whereas urinary gelsolin originated from the blood by glomerular filtration. Our results illustrate an etiological diagnosis of acute kidney injury through analysis of urine. Thus, our results raise the possibility of identifying the actual nephrotoxin in critically ill patients who are often treated with several nephrotoxic agents at the same time, thereby providing the potential for tailoring therapy to an individual patient, which is the aim of personalized medicine.

Dose reduction of efavirenz: an observational study describing cost–effectiveness, pharmacokinetics and pharmacogenetics

AIM Antiretroviral treatment implies a high cost to the healthcare system. The aim of this study was to evaluate the clinical and economic impact of efavirenz (EFV) dose adjustment by monitoring plasma concentrations and pharmacogenetic analysis of the 516G>T CYP2B6 polymorphism. MATERIALS & METHODS One hundred and ninety HIV patients treated with EFV were studied. Plasma EFV concentrations were measured by HPLC with ultraviolet detection, and pharmacogenetic analysis was performed by Real Time (RT)-PCR. RESULTS One hundred and ninety patients initially treated with a standard dose of EFV (600 mg/day) were studied. In 31 (16.3%) patients, EFV dose was reduced. A total of 87.1% of patients were heterozygous/homozygous carriers (GT/TT). CD4(+) count increased while the minimum steady-state plasma concentration and adverse effects decreased significantly after dose adjustment. Considering only the dose reduction, the adjustments accounted for a saving of 43,539 €/year. CONCLUSION The individualization of EFV dosage guided by genotyping 516G>T CYP2B6 and therapeutic drug monitoring could increase the efficiency of EFV use in antiretroviral treatment.

Multiplexed testing in the autoimmunity laboratory.

Abstract A variety of technologies for autoantibody profiling have been developed. The main techniques are line-blot immunoassays, bar-coded nanoparticle immunoassays, and bead-based assays with flow cytometry detection and antigen microarrays. Some of these technologies are only able to measure a limited number of autoantibodies, while others can detect elevated numbers. Assays for antinuclear antibody specificities using line-blot immunoassays and bead-based assays with flow cytometry detection are already commercialised. Antigen microarrays for autoantibody measurement are only in the development phase, although in the not too distant future these assays will probably appear on the market. Multiplexed testing in the autoimmunity laboratory appears to have a promising future, since this technique permits a reduction in analytical time, with a shorter turnaround time. Clin Chem Lab Med 2006;44:1169–74.