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A new molecular subtyping approach was developed which is based on the amplification and sequencing of a repetitive region of the P1 gene of Mycoplasma pneumoniae. It allows the differentiation of all known subtypes and variants of M. pneumoniae as well as the identification of new subtypes directly in clinical samples to characterize endemic and epidemic M. pneumoniae infections.
The importance of epidemiological monitoring of yeasts involved in pathologic processes is unquestionable due to the increase of these infections over the last decade, the changes observed in species causing candidiasis, and empirical antifungal treatment. At the Mycology Center, 1006 isolates from a wide range of clinical samples were studied during 1999-2001. Candida albicans (40.3%) was the most isolated species, although, the Candida no albicans species with 54.9% showed the major prevalence. In blood cultures Candida parapsilosis (34.9%), C. albicans (30.2%) and C. tropicalis (25.6%) were recovered most frequently while C. glabrata represented only 2.3%. C. albicans with 60%-80% was the predominant specie in mucosal surface. We also detected Candida mediastinistis, which alert us over the importance at this location. Urinary tract infections caused by yeasts were more frequent in hospitalized patients, being C. albicans (47.7%), the most commonly isolated, followed by C. glabrata (24.8%) and C. tropicalis (20.0%). In the candidal onychomycoses, C. parapsilosis (37.7%) outplaced C. albicans (22.0%). Fluconazole susceptibility studies of Candida species allowed us to conclude that the majority of C. albicans islolates are susceptible, and that the highest resistance averages were observed in C. glabrata (21.41%) and C. krusei (69.23%).
The International Headache Society (IHS) diagnostic criteria for headache improved the accuracy of primary headache diagnoses, including migraine. However, many migraineurs receive an 'atypical migraine' diagnosis according to the IHS nosology (IHS 1.7), indicating that they approximate but do not fully meet all IHS criteria. This study characterized and sub-classified patients with atypical migraine. Within a clinical sample of 382 headache sufferers, 83 patients met IHS criteria for 'atypical migraine'. Patients receiving the IHS 1.7 designation did not converge to form a homogeneous group. Rather, distinct and clinically relevant subgroups were empirically derived (e.g. migraine with atypical pain parameters, brief migraine, chronic migraine). The results call for revisions of the IHS diagnostic criteria for migraine that would minimize the number of patients receiving an atypical diagnosis. Revisions would include decreasing the minimum headache duration criteria from 4 h to 2 h, and developing a classification for 'chronic migraine' for migraine greater than 15 days per month. The proposed revision provides a means of diagnosing the daily and near-daily headache commonly observed in clinical populations.
A single-tube, non-interrupted, one-step RT-PCR has been standardized to amplify the hypervariable region of the VP2 gene sequence of infectious bursal disease virus (IBDV). The technique standardized on purified viral RNA was successfully applied to the detection of the virus directly in clinical samples. The amplified products were confirmed to be IBDV specific by their size in ethidium bromide-stained agarose gel, nested PCR and restriction enzyme digestion. Digestion of the amplicons with StyI restriction enzyme also differentiated classical virus from six very virulent field isolates. The sensitivity of the one-step RT-PCR was found to be 0.2 pg of viral RNA.
We recently developed a simple new method which is designed to separate and concentrate bacteria from a sample by centrifugation in a gel system. Bacterial enzyme activity is then detected inside the gel without further manipulation using a colorimetric or fluorogenic substrate. The method provides a rapid, direct means of detecting bacteria in clinical samples, dispensing with the 24-h period normally required to isolate colonies on agar. Various applications of the method are described below, e.g. screening of negative urine samples, identification of Escherichia coli in urine samples, identification of Staphylococcus aureus in blood culture broths and detection of oxacillin-resistant S. aureus in blood culture broths. The advantages of the gel system and other applications are discussed.
Aspergillosis is a disastrous cause of mortality and morbidity in patients in an immunocompromised state and old lung tuberculosis. However, the diagnosis of aspergillosis remains unsettled. In the present study, we developed a test to detect the Aspergillus fumigatus DNA using polymerase chain reaction (PCR) in clinical samples including sputum and bronchial aspirations from the patients with aspergillosis. We selected 2 pairs of oligonucleotide primers and the DNA for the small subunit ribosomal RNA of aspergillus fumigatus and they were significantly amplified. Although isolation of Aspergillus DNA may reflect contamination and colonization without infection, no aspergillus DNA was found in sputum or bronchial aspirations from the patients with lung cancer or interstitial lung diseases except a case of pulmonary fibrosis associated with rheumatoid arthritis chronically received 20 mg of corticosteroid. There was a 100% correlation between the culture results and the nested PCR results in the patients with pulmonary aspergillosis. These findings indicate that PCR detection of aspergillus fumigatus might be valuable for the diagnosis of aspergillosis.
A polymerase chain reaction (PCR) assay for the detection of Legionella spp. in clinical bronchial fluid samples was constructed. The assay could detect a 375 bp fragment of the 16S RNA gene, equivalent to 10 cfu in simulated clinical bronchial fluid and blood samples. Seven clinically relevant Legionella spp. involving 12 serogroups produced a positive signal, whereas three Legionella spp. and none of a panel of 26 bacterial strains produced a signal in the constructed assay. Investigation of bronchial fluid from 51 patients with clinically suspected Legionnaire's disease revealed the presence of Legionella DNA by PCR in seven patients. The results were verified by Southern blot analysis of the amplified DNA fragment. None of 37 control patients produced a positive signal. Only one of the seven bronchial fluid samples positive for Legionella by PCR was positive by conventional culture, thus indicating the increased sensitivity of PCR compared to culture.
Although peripheral blood and bone marrow are usually readily available from patients, present techniques of RNA extraction are tedious, require millilitres of starting material and removal of red blood cells before RNA purification. Further, successful reverse transcriptase polymerase chain reaction (RT-PCR) amplification requires the removal of haemoglobin derivatives which interfere with the PCR process. Recently, one step rapid use reagents have become available, claiming to be useful for obtaining high quality RNA from microlitre quantities of whole blood drawn directly from the patient. Their use to date in clinical samples appears limited with little information in the literature documented. In an attempt to overcome this, we tested the Trizol-LS, RNA-STAT-50 and Ultraspec-3 reagents upon a statistically significant number of clinical isolates of fresh and cryopreserved peripheral blood, bone marrow, blood apheresis products and a breast cancer cell line (MCF7) in order to evaluate whether these methods could be applied to routine laboratory use in an RT-PCR method capable of detecting rare gene expression. Our findings showed that there was some variation in the quality of RNA extracted which was indicated by absorbance spectrophotometry at 260 and 280 nm. 1% agarose gel electrophoresis showed that each of these methods could yield total RNA capable of generating the signature 18S and 28S rRNA bands. Using the Kruskal-Wallis non-parametric anova test combined with Dunn's multiple comparison test, the only statistically significant difference (p<0.05) indicated that Trizol-LS was more reliable than RNA-STAT-50-LS and Ultraspec-3 at extracting RNA from fresh peripheral blood. RNA extracted with the Trizol-LS and RNA STAT-50 reagents was successfully amplified in a multiplex RT-PCR reaction for detection of the multi-drug resistance related genes MDR1, the multi-drug resistance related protein (MRP) and topoisomerase IIalpha. Low level MDR1 gene expression could be detected in frozen whole blood. However, PCR products were only seen when the anti-coagulant heparin was removed from all samples prior to cDNA production. RT-PCR amplification was not 100% successful with RNA extracted with Ultraspec-3 reagent. In conclusion, we found that the RNA extracted from whole blood with the Trizol-LS and the RNA-STAT-50 are suitable for use in clinically relevant molecular biology protocols that analyze rare event genes without further purification. Our results indicated that the Trizol-LS reagent was generally more consistent in obtaining a pure and sufficient quantity of RNA from patient material as shown by the mean result of purity and quantity in comparison to either Ultraspec-3 or RNA-STAT-50-LS reagents. Ultraspec-3 is not easily suited for direct use with whole blood products.
A polyphasic taxonomic study (employing whole-cell protein and fatty acid analyses, 16S rDNA sequencing, DNA-DNA hybridization, determination of DNA G+C content, antibiotic susceptibility testing and extensive phenotypic characterization) was performed on 10 isolates that appeared to be related to Alcaligenes faecalis. The isolates were recovered from diverse environments that included human clinical samples. 16S rDNA sequence analysis indicated that these isolates belonged to the genus ACHROMOBACTER: Whole-cell protein analysis distinguished two groups, which were confirmed by DNA-DNA hybridization. Based on the results of this study, the organisms were classified as two novel Achromobacter species, Achromobacter insolitus sp. nov. (type strain, LMG 6003(T)) and Achromobacter spanius sp. nov. (type strain, LMG 5911(T)). Achromobacter insolitus can be distinguished from Achromobacter spanius by its ability to grow on acetamide and to assimilate mesaconate and aconitate, and by its inability to assimilate diaminobutane. Various tests allow the differentiation of both novel species from other Achromobacter species, including growth on acetamide, denitrification and assimilation of D-glucose, D-xylose, mesaconate, aconitate and diaminobutane.
During the last few years the direct diagnosis of Toxoplasma gondii infection has taken advantage of PCR. The present work tested the sensitivity and specificity of PCR for rDNA and p30 genes. Using ascitic fluid from infected mice rDNA PCR detected 0.5 tachyzoite/ml, while nested p30 PCR 1 tachyzoite/ml. The rDNA amplification was positive in all clinical samples from a single immuno compromised patient (blood, urine and bronchoalveolar fluid). In the same patient nested p30 PCR was positive only in urine and bronchoalveolar lavage (BAL) fluid. The rDNA and p30 amplicons were never found in any amniotic fluids tested. These results could prove the usefulness of rDNA amplification to detect T. gondii in blood.
BACKGROUND: The fluorescent probe Syto16 has been used successfully to measure P-glycoprotein (Pgp) function and, separately, early apoptosis and cell death. The present study was designed to evaluate whether the combined use of Syto16, the Pgp blocker PSC833, and 7-AAD allows simultaneous detection of all parameters, with emphasis on applications in acute myeloid leukemia (AML). METHODS: Pgp negative/positive KB cell lines treated with tumor necrosis factor alpha/hyperthermia and frozen-thawed AML samples were used as apoptosis/Pgp models. RESULTS: For the accurate assessment of apoptosis in samples with unknown Pgp status, it was essential to include a sample with PSC833: in such samples, viable cells always show a Syto16(high) and apoptotic cells a Syto16(low) fluorescence. Apoptotic cells loose their Pgp activity early on; in Pgp-positive cells, the Syto16(low) apoptotic cells then colocalize with the Syto16(low) viable cells in the situation minus PSC833. We have developed a gating strategy that, apart from quantifying apoptosis, allowed gating out these apoptotic cells for proper Pgp assessment. By using this strategy, no differences in Pgp activity were found in the treated versus the untreated samples (KB cells: P = 0.779, n = 10; AML cells: P = 0.525, n = 45). CONCLUSIONS: The use of the combination Syto16/PSC833/7-AAD provides a sensitive multiparameter flow cytometry method that enables accurate assessment of both apoptosis, cell death, and Pgp function in clinical samples.
With the explosion in interest in cytokines and an increasing understanding of their possible involvement in the pathogenesis of many diseases, the number of studies measuring cytokines in body fluids has increased markedly over the last 5 to 10 years. However, it is readily apparent from the literature that the level of awareness of the many factors that can potentially influence the results obtained is very low. Such factors are likely to contribute considerably to the disparities seen among similar types of study. These include assay properties such as standardization and specificity, type of sample and sample handling, and many biological influences such as age, sex, ethnic background, and diurnal rhythmicity. Clearly, it is not possible to control for all these factors, but a minimum evaluation of assays should be carried out as described, including recovery and parallelity studies using relevant samples and the establishment of reproducibility and normal healthy population levels. An awareness of these factors, which is the purpose of this review, may hopefully lead to an improvement in the quality of studies incorporating the measurement of cytokines in clinical samples and should aid in the interpretation of the data.
(E)-4-Hydroxy-2-nonenal (HNE) is a highly reactive product of the free radical-stimulated lipid peroxidation of phospholipid-bound arachidonic acid in cellular membranes. We describe a sensitive and specific method for the determination of HNE in clinical samples. The method is based on the formation of the O-pentafluorobenzyl (O-PFB) oxime derivative of HNE, which is then extracted and cleaned up by solid-phase extraction. The HNE O-PFB oxime is then analysed without further derivatisation by capillary column gas chromatography-negative ion chemical ionisation mass spectrometry (GC-NICI-MS) using selected-ion monitoring. Concentrations down to the pmol range were achieved using deuterated HNE as an internal standard. The method was used to determine HNE in the cerebrospinal fluid and plasma of patients with Parkinson's disease, the plasma of patients with HIV-1 infection and AIDS and in inflamed mucosal biopsy specimens from patients with inflammatory bowel disease.
A high-performance liquid chromatographic (HPLC) method with electrochemical detection and solid-phase extraction (SPE) using cartridges of weak cation-exchange capacity as the primary retention mechanism is described for the separation and determination of methylnaltrexone (MNTX) in small clinical samples of plasma or urine. The procedure was performed using a Phenomenex Prodigy ODS-2, 5 microm, 150x3.2 mm analytical column and 50 mM potassium acetate buffer, with 11% methanol as organic modifier at pH* 4.5 at a flow-rate of 0.5 ml/min. The detection potential was 700 mV. The six-point standard calibration curves were linear over three consecutive days in the range from 2 to 100 ng/ml. The average goodness of fit (r) was 0.9993. The lower limit of detection (LOD) and limit of quantification (LOQ) were found to be 2.0 and 5.0 ng/ml, respectively. At the LOQ, the coefficient of variation for the entire method was 8.0% and the accuracy was 10.0% (n = 10). Recovery of the drug from plasma was in the region of 94%. The method was applied to a pharmacokinetics study of methylnaltrexone after subcutaneous administration and in numerous assays of analytes in blood plasma and urine. The pharmacokinetics parameters for a single dose of 0.1 or 0.3 mg/kg in plasma were C(max) = 110 (+/-55) and 287 (+/-101) ng/ml and t(max) = 16.7 (+/-10.8) and 20.0 (+/-9.5) min, respectively. The method is simple, yet sensitive for the detection and determination of methylnaltrexone in biological samples at the level of the physiological response.
The aim of this work was to compare data obtained using PCR assay for detection of Mycobacterium tuberculosis with culture. We report a test for detection of tubercle bacilli by PCR and identification at species level by capture plate hybridisation and enzyme-linked immunoassay. 222 clinical samples obtained from patients with confirmed and suspected tuberculosis were analysed. These specimens were tested parallelly by conventional culture on Löwenstein-Jensen slants and PCR test. For 205 samples a complete agreement between these methods was observed.
While most bacteria involved in dentoalveolar infection are highly susceptible to penicillin, some Prevotella strains exhibit resistance to this agent through the production of beta-lactamase. The production of beta-lactamase by Prevotella spp. is in turn associated with the expression of the genes cfxA and cfxA2. The aim of the present study was to determine the prevalence of cfxA and cfxA2 in Prevotella strains by use of real-time PCR and to assess the performance of this molecular method for the direct detection of the genes in 87 clinical samples (pus and root canal exudates) from dentoalveolar infection. Production of beta-lactamase by each isolate was determined using a nitrocefin disk. beta-Lactamase production was seen in 31% of Prevotella isolates, while all isolates of other species were beta-lactamase negative. The penicillin resistance of isolates strongly correlated with the production of beta-lactamase. Real-time PCR was found to detect the cfxA and cfxA2 genes from at least five cells per reaction mixture (5 x 10(3) CFU/ml of pus). Using real-time PCR, the presence of cfxA and cfxA2 was evident for all 48 beta-lactamase-positive Prevotella strains. In contrast, neither beta-lactamase-negative Prevotella (n = 91) or non-Prevotella (n = 31) strains were positive for the genes. In this study, 31 of the 87 samples yielded beta-lactamase-positive Prevotella results, and cfxA and cfxA2 were detected in all 31 samples. Of the 56 culture-negative samples, 8 (14%) were positive for cfxA and cfxA2 by the real-time PCR. This sensitive and specific molecular method offers a rapid clinical test for aiding in the selection of an appropriate antibiotic for treatment of dentoalveolar infection. Although penicillin remains largely effective in the treatment of dentoalveolar infection, beta-lactamase-stable antibiotics should be considered in cases in which beta-lactamase-positive Prevotella strains are involved.
In this study, real-time PCR with pathogen-specific molecular beacons (MB) and primers was evaluated for prediction of community-acquired pneumonia (CAP) causative agents, detecting six main CAP agents, Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Streptococcus pyogenes, simultaneously. The PCR assay was evaluated for fresh clinical specimens from infants and children (n = 389) and from adults (n = 40). The MB probes and primers are both pathogen specific, namely, the lytA gene for S. pneumoniae, the mip gene for L. pneumophila, and 16S rRNA genes for the remaining four organisms. DNA extraction of clinical specimens was performed with a commercially available EXTRAGEN II kit, and amplification was performed with Stratagene Mx3000P. The limit of detection for these pathogens ranged from 2 copies to 18 copies. The whole process from DNA extraction to the analysis was finished in less than 2 h. The obtained sensitivity and specificity of this real-time PCR study relative to those of conventional cultures were as follows: 96.2% and 93.2% for S. pneumoniae, 95.8% and 95.4% for H. influenzae, 100% and 100% for S. pyogenes, and 100% and 95.4% for M. pneumoniae, respectively. The sensitivity and specificity for M. pneumoniae relative to those of a serologic assay were 90.2% and 97.9%, respectively. In six clinical samples of C. pneumoniae, the real-time PCR gave positive predictable values, and in those cases, elevation of the titer value was also observed. In conclusion, we demonstrated that a real-time PCR assay with pathogen-specific MB is useful in identifying CAP causative agents rapidly and in examining the clinical course of empirical chemotherapy in a timely manner, supporting conventional culture methods.
Strains of Chlamydophila pneumoniae may be associated with respiratory disease or atherosclerosis. Two real-time quantitative PCR assays targeting the species-specific genes Cpn0278 and ArgR were developed to compare the in vitro growth of respiratory strains AR39 and K6 with that of atherosclerotic strain A03 and to quantify C. pneumoniae in clinical samples. A third real-time PCR assay was designed to assess contamination with Mycoplasma spp. The assays targeting C. pneumoniae detected DNA concentrations corresponding to 10(4) to 10(-4) inclusion-forming units (IFU)/reaction and were highly specific. AR39 exhibited the longest lag phase and period of exponential growth; K6 augmented growth rates at higher inocula; and A03 grew at highest rates. Contamination with Mycoplasma spp. of AR39 and A03 unlikely accounted for growth differences between them. Numbers of IFU in C. pneumoniae-positive respiratory secretions varied within 4 to 5 orders of magnitude. The assays described may prove valuable for pathogenicity studies.