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PubMed1994

Blockade of hippocampal M1 muscarinic receptors impairs working memory performance of rats.

In order to clarify the roles of hippocampal M1 and M2 muscarinic receptors in working and reference memory performance of rats, the effects of intrahippocampal injections of selective antagonists at both receptors on this behavior were examined with a three-panel runway task. In the working memory task, the M1 muscarinic receptor antagonist pirenzepine, injected bilaterally at 0.32 and 1.0 microgram/side into the dorsal hippocampus, significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). This effect of intrahippocampal pirenzepine (1.0 microgram/side) on working memory was attenuated by concurrent injection of 10 micrograms/side AF102B, the selective M1 muscarinic receptor agonist. Intrahippocampal injection of the M2 muscarinic receptor antagonist methoctramine at doses up to 1.0 microgram/side had no significant effect on the number of working memory errors. Intrahippocampal methoctramine injection at 3.2 micrograms/side produced a significant increase in working memory errors, an effect that was reversed by concurrent injection of 10 micrograms/side AF102B. Concurrent injection of 0.32 microgram/side methoctramine significantly reduced the increase in working memory errors induced by intrahippocampal pirenzepine (1.0 microgram/side). In the reference memory task, neither pirenzepine nor methoctramine affected the number of errors when injected into the hippocampus at doses up to 1.0 and 3.2 micrograms/side, respectively. These results suggest that processes mediated by M1 muscarinic receptors in the hippocampus are involved in working memory, but not in reference memory, and that blockade of hippocampal M2 muscarinic receptors ameliorates working memory deficits produced by M1 muscarinic blockade, possibly by increasing acetylcholine release.

Animals↗

PubMed1992

Histopathological classification of Hodgkin's lymphomas. Results from the reference pathology of the German Hodgkin Trial.

Diagnostic biopsies of 1,140 previously untreated patients with Hodgkin's lymphoma from the German Hodgkin Therapy Trial were evaluated by a panel of 4 histopathologists. Each case was classified by consensus of the four according to an extended Rye-System including the diversifying of NS.HD and the unclassifiable cases. The interobserver agreement rate was 92.1% among the panel members regarding the final classification. Primary Hodgkin diagnosis was approved in 91.7% (1045/1140 cases). The distribution of the 1,140 cases within this system was (in %) as follows: 2.7 LP.HD, 57.9 NS.HD, 17.3 MC.HD, 0.7 LD.HD, 13 HD of unclassifiable subtypes, 6.3 of not certain HD, 1.9 Non-Hodgkin Lymphoma, 1 case of non malignancy. Grading of NS.HD into 2 subgroups according to the British classification differed conspicuously by 84.5% versus 15.5% and 71.6% versus 28.4% in British results. This divergency is explained by interobserver disagreement ranking from 43% to 85%.

Biopsy↗

PubMed2005

Immunization against a low-frequency human platelet alloantigen in fetal alloimmune thrombocytopenia is not a single event: characterization by the combined use of reference DNA and novel allele-specific cell lines expressing recombinant antigens.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal immunization against a fetal platelet (PLT) alloantigen. In cases of FNAIT attributed to low-frequency PLT alloantigens, the laboratory diagnosis is often hampered by the lack of adequate PLTs. STUDY DESIGN AND METHODS: Three families with maternal immunization against fetal PLT antigens were analyzed. In Family 1, previous immunization of another female or woman has been observed. In Families 2 and 3, newborns presented with the typical clinical picture of FNAIT. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing with reference to DNA from Epstein-Barr virus-transformed B-lymphoblastoid cell lines. Antibodies were characterized by glycoprotein (GP)-specific immunoassay with a panel of stable Chinese hamster ovary cell lines expressing low-frequency alloantigens. RESULTS: In three families, maternal immunization associated with the low-frequency alloantigens human PLT antigen (HPA)-8bw (Sra), HPA-11bw (Groa), and HPA-13bw (Sita) was identified. Maternal serum samples showed positive reactions in an antigen capture assay with cell lines carrying recombinant GP IIb/IIIa (HPA-8bw and -11bw) or GPIa/IIa (HPA-13bw), respectively. These results could be confirmed by genotyping analysis of fathers and newborns. CONCLUSION: This study demonstrates that cases of FNAIT attributed to low-frequency PLT alloantigens cannot be regarded as single events. The availability of reference DNA and cell lines expressing recombinant PLT alloantigens can facilitate their identification.

Antigens, Human Platelet↗

PubMedDate not supplied

Chiropractic technique procedures for specific low back conditions: characterizing the literature.

BACKGROUND: Many original clinical trials and several review papers have come to the conclusion that manipulation is safe and effective for the treatment of low back pain. However, it is necessary to determine which specific types of manipulation and nonmanipulative types of chiropractic adjustive care are most effective for particular types of low back pain across both tissue-specific and functional classifications. OBJECTIVE: To characterize the quantity and quality of literature gathered for an Expert Panel that was convened to rate various specific chiropractic adjustive procedures for the treatment of common types of low back pain, drawing on the clinical expertise of the panel members and the relevant literature. STUDY DESIGN: Systematic review of treatment-specific, condition-specific trials, studies, and case reports of chiropractic care for low back pain. METHODS: Computerized searching and hand searching were used to identify references in the medical and chiropractic literature pertaining to the chiropractic treatment of low back pain in which both the condition and specific treatment procedures were adequately described. This literature was then categorized according to a variety of characteristics and used by a panel to evaluate the specific procedures. RESULTS: The 3 most studied adjustive procedures are side-posture high-velocity, low-amplitude; distraction (mostly flexion distraction); and mobilization, respectively. The clinical condition most commonly addressed by the included studies is low back pain. The procedure with the widest base of evidence support is side posture manipulation for low back pain.

Chiropractic↗

PubMed2005

Feasibility study of the use of frozen human sera in split-sample comparison of immunoassays with candidate reference measurement procedures for total thyroxine and total triiodothyronine measurements.

BACKGROUND: Diagnostic manufacturers must ensure/document metrologically traceable assays. We report on a feasibility study of a split-sample comparison for that purpose. Processed, frozen single-donation sera, assigned target values by candidate reference measurement procedures (cRMPs), were used with immunoassays for total thyroxine (TT(4)) and triiodothyronine (TT(3)) as models. METHODS: Two serum panels were quantified for TT(4) and TT(3) with validated cRMPs and measured in parallel with at least 14 immunoassays. The results were interpreted in terms of traceability of calibration (trueness) and of the individual measurement result (accuracy) by linear regression analysis and graphical representation against specifications. The commutability of the sera was investigated by parallel analysis of TT(4) in freshly collected but nonfiltered specimens. RESULTS: The TT(4) (TT(3)) concentrations in the sera (according to the cRMPs) were 64-269 nmol/L (0.88-13.7 nmol/L). The method comparison showed that for TT(4), on average, the immunoassays produced results in agreement with the cRMPs, whereas for TT(3), results were typically higher. It also demonstrated a considerable between-assay divergence in traceability of calibration and accuracy. The evidence of noncommutability of the sera attributable to processing, however, indicates that the interpretation should be treated with caution. CONCLUSIONS: Frozen sera can be used for documenting/validating traceability of total thyroid measurements. The way in which the sera are processed may jeopardize commutability, however, and therefore requires in-depth investigation.

Cryopreservation↗

PubMedDate not supplied

Clinical management of primary hyperparathyroidism and thresholds for surgical referral: a national study examining concordance between practice patterns and consensus panel recommendations.

OBJECTIVE: To determine whether 1990 guidelines established by the National Institutes of Health (NIH) for the optimal management (surgical versus nonsurgical) of patients with asymptomatic primary hyperparathyroidism (PHPT) are known and followed by endocrinologists. METHODS: We surveyed endocrinologists in the United States who were randomly selected in 1998 from two endocrine societies. Endocrinologists were asked about management of asymptomatic patients with PHPT, awareness of NIH recommendations, practice demographics, and annual PHPT case volume. We classified endocrinologists into either low-volume (<12 cases of PHPT per year) or high-volume (> or = 12 cases per year) physicians. RESULTS: Of 374 eligible physicians, 146 (39%) responded to our survey. In comparison with nonrespondents, respondents were of similar age, sex, years in practice, and geographic location profiles; this finding suggested minimal nonresponse bias. More high-volume physicians than low-volume physicians were aware of the NIH guidelines (75% versus 50%; P<0.01). Management of asymptomatic patients was similar between volume groups; overall, 39% of patients were referred for surgical treatment. Nevertheless, considerable variation in management existed; 7% of all physicians referred > or = 90% of their asymptomatic patients for surgical treatment, whereas 31% referred < or = 10%. Adherence to monitoring guidelines for nonsurgically managed patients ranged widely, depending on the specific recommendation (from 6% of physicians obtaining creatinine clearance assessments every 6 months to 78% of physicians ordering serum calcium measurements every 6 months). Surgical referral practices also varied substantially, with 25% of all physicians referring a 40-year-old patient with PHPT when hypercalcemia was mild (< or = 1 mg/dL above normal), 39% when hypercalcemia was moderate, and 31% when hypercalcemia was severe (>1.5 mg/dL above normal). Of the responding physicians, 4% reported that hypercalcemia alone was not sufficient justification to refer a patient for surgical intervention. Higher PHPT case volume was not associated with differences in surgical referral. CONCLUSION: Suboptimal awareness of the 1990 NIH panel recommendations and the substantial variation in clinical management of PHPT indicate that newer NIH guidelines developed in 2002 must be more widely disseminated and strongly recommended if practice patterns are to be influenced and clinical outcomes improved.

Adult↗

PubMed2001

Reference values for peripheral blood B-lymphocyte subpopulations: a basis for multiparametric immunophenotyping of abnormal lymphocytes.

BACKGROUND AND OBJECTIVES: Immunophenotyping has become a useful tool for the differential diagnosis of chronic B-cell lymphoproliferative disorders. The aim of this work was to determine reference values of normal B-cell subpopulations. MATERIAL AND METHODS: Blood samples from 38 healthy volunteers were analyzed by multidimensional flow cytometry, using a panel of directly conjugated antibodies. Results were expressed as percent of positive B cells and as median fluorescence intensity, an indirect assessment of the expression level. RESULTS: CD20, CD22, CD24, CD40, CD79a, CD79b, FMC7, CD11a, CD18, CD44 were positive in the whole B cell population, whereas CD10, CD86, CD103, CD154 and FasL were almost absent from the B-lymphocyte population. 75% were IgD positive. The kappa/lambda ratio was 1.5. CD5, CD23, CD25, CD38, CD43, CD54, CD62L, CD80 and CD95 were positive in different B-cell subpopulations. The utility of all these markers in the differential diagnosis of chronic B-cell lymphoproliferative disorders is discussed. CONCLUSION: In order to interpret a pathological immunophenotype, it is necessary to refer to quantitative and qualitative values of normal B-cell subpopulations.

Adult↗

PubMed2003

Genetic heterogeneity in lung and colorectal carcinoma as revealed by microsatellite analysis in plasma or tumor tissue DNA.

BACKGROUND: Determination of tumor clonality has implications for molecular characterization and the optimal treatment of cancer. Allelotyping allows detection of the two alleles, maternal and paternal, and provides additional information regarding clonal genetic defects. The presence of allelic imbalances (AI) in tumors is a general event, but is not necessary at the same allele (alternative AI). The authors' goal was to determine whether the presence of alternative AI (AA) was a marker of heterogeneity and prognosis. METHODS: To further analyze the heterogeneity of lung tumors, tumor DNA released in the plasma was compared with primary tumor DNA from 24 lung carcinoma patients. The comparison was performed by allelotyping using 12 microsatellites targeting 9 chromosomal regions, taking in each case leukocyte DNA as reference. To extend and confirm these observations, 26 primary colorectal carcinomas with paired synchronous liver metastasis were analyzed using an enlarged panel of 33 microsatellites. RESULTS: AA were observed in 40% (20 of 50) of all patients, in 25% (6 of 24) of lung carcinoma patients but at a higher level, and in 54% (14 of 26) of colorectal carcinoma patients. They affected different chromosome localizations and each tumor stage. In both types of cancer, patients with AA had a higher AI mean frequency in their primary tumor. CONCLUSIONS: Detection of AA is an original marker of heterogeneous tumors, demonstrating that independent events occurred on specific genetic sites required for cancer progression.

Adenocarcinoma↗

PubMed2003

Antigenic and genetic diversity of early European isolates of Infectious bursal disease virus prior to the emergence of the very virulent viruses: early European epidemiology of Infectious bursal disease virus revisited?

Eleven Polish and Hungarian isolates of Infectious bursal disease virus (IBDVs) obtained in the 70/80s (early IBDV) and in the 90s (recent IBDV) were characterized in an Antigen-Capture-ELISA with a panel of neutralizing monoclonal antibodies (Mabs), and by nucleotide sequencing of the VP2 variable domain (vVP2). The viruses were compared with reference IBDV strains, among others with Faragher 52/70 (F52/70, classical, isolated 1970), 89163 (typical very virulent-vvIBDV, isolated 1989) and 91168 (antigenically modified vvIBDV, isolated 1991). Only one of the early isolates (Hungarian strain P1) proved antigenically and genetically similar to F52/70. Other early isolates exhibited no reactivity versus Mabs 3, 4, 5 and/or 8 and had a common previously unrecognized combination of amino acid changes in vVP2. The recent isolates all proved antigenically and genetically related to typical vvIBDV strain 89163, except the Polish isolate 93/35 which proved related to the 91168 strain although no epidemiological relationship had been documented between these viruses in the field. Phylogenetic analysis confirmed that the non-P1 early IBDVs represent a previously unrecognized group among serotype 1 IBDVs. It is discussed whether these early isolates are derivatives of the F52/70-like viruses that might still be present in the field, or whether they represent early IBDV strains that might have been present prior to and progressively replaced by the F52/70-like viruses, as the latter have been replaced by vvIBDVs in the late eighties.

Amino Acid Sequence↗

PubMed2006

The four Es of problem gambling: a psychological measure of risk.

A focus group of Reno area Gamblers Anonymous members identified four psychological traits contributing to risk for problem gambling, including: Escape, Esteem, Excess and Excitement. A panel of four experts authored 240 Likert-type items to measure these traits. By design, none of the items explicitly referred to gambling activities. Study 1 narrowed the field of useful items by employing a quasi-experimental design which compared the answers of Reno area Gamblers Anonymous members (N = 39) to a control sample (N = 34). Study 2 submitted successful items, plus new items authored with the knowledge gained from Study 1, to validation in a random sample telephone survey across Queensland, Australia (N=2577). The final 40 item Four Es scale (4Es) was reliable (alpha=.90); predicted gambling problems as measured by the Canadian Problem Gambling Index of Severity (PGSI, Ferris & Wynne (2001). The Canadian Problem Gambling Index: Final Report: Canadian Centre on Substance Abuse); and distinguished problem gamblers from persons with alcohol abuse problems. The new scale can provide a basis for further study in harm minimization, treatment, and theory development.

Adult↗

PubMed1988

Chromal focus of acquired chromatic discrimination loss and solvent exposure among printshop workers.

Acquired dyschromatopsia has been associated with exposure to organic solvents. However, the chromal focus of the loss may be indicative of its gravity. According to Kollner's rule, blue-yellow loss reflects changes in external retinal layers, while red-green loss appears to be indicative of internal retinal or optic nerve damage. The objective of the present study was to examine chromatic discrimination capacity of 30 printshop workers exposed to organic solvent mixtures, and of a non-exposed reference group. Colour vision was assessed with a colour arrangement test designed to detect acquired dyschromatopsia, the Lanthony D-15 desaturated panel. Quantitative analysis, using Bowman's colour confusion index, revealed significantly higher scores indicative of colour vision loss among the exposed workers as compared to the non-exposed. Analysis of covariance, with age as co-variate, showed colour confusion index to be significantly associated with job category. Similarly, qualitative analysis showed that the exposed workers presented a significantly higher prevalence of acquired dyschromatopsia as compared to the non-exposed group. However, analysis of the type of chromatic discrimination loss showed that among the nonexposed persons, dyschromatopsia was localized only in the blue-yellow range, while for 35% of the dyschromatopic-exposed persons, red-green loss as well as blue-yellow loss were present. Three-dimensional chi 2-analysis showed that the complex pattern of dyschromatopsia was not related to age, but to job category. These findings suggest that the type of dyschromatopsia, reflecting the gravity of neural alterations, may be a function of exposure level and/or the ophthalmotoxic properties of the particular solvents used.

Adult↗

PubMed1995

In vitro bioassay with enhanced sensitivity for human granulocyte colony-stimulating factor.

A method for the determination of human granulocyte colony-stimulating factor (hG-CSF) activity, based on stimulation of cellular proliferation, was developed using a subclone of the murine myeloid leukemia cell line NFS-60, with an improved sensitivity for hG-CSF, as indicator. The optimal range for quantitative analysis of hG-CSF was about 4-60 pg ml-1. The stimulatory effect was measured by a colorimetric microassay: the optical density of formazan, which is produced by viable cells from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), was obtained by reading plates in a multi-channel photometer. The assay was designed as a five-dose parallel line test, employing three or four doses for potency determinations, which fulfil pharmacopoeial requirements for assay validity. Inter-assay relative standard deviation (RSD) varied between 5.2 and 12.0%. Most assay experiments revealed potencies within limits of error of 90-110% and the mean index of precision value was 0.057. The recently developed yeast cell-derived International Standard (88/502) served as a reference for activity of rhG-CSF. Specificity of the assay was demonstrated by absence of response upon exposure to a panel of biomolecules, including recombinant human interleukin-3, and by the suppression of growth stimulation in the presence of neutralizing anti hG-CSF antibodies. Potency readings of unglycosylated rhG-CSF were dependent on pH of assay medium with higher relative activities observed at pH 6.6 than at 7.4. Moreover, SDS-PAGE analysis of the carbohydrate-deficient preparation, following incubation at physiological pH, revealed several high molecular weight rhG-CSF bands and decreased monomeric form. The method described was found suitable for potency assessments of pharmaceutical formulations of hG-CSF.

Animals↗

PubMed2001

Sex differences in the dynamics of cue utilization and exploratory behavior.

The purpose of the present study was to investigate the effects of sex and estrous cycle on exploratory behavior, as well as the degree to which reliance on environmental cues changes with training. Fischer 344 rats were placed three times in an open field box that contained three objects (two identical bottles and a cylinder). During the initial exposure to the environment all females showed higher activity levels and explored a larger region of the environment compared to males. However, upon subsequent exposure to the same environment, these sex differences disappeared. During the third and final session, the locations of the bottle and the cylinder were switched. The estrous females and to a lesser degree male rats, responded to the relocation of objects with a renewal of exploration and activity; proestrous females did not show this response. The rats were then trained on a four-arm radial maze reference memory task. The correct arm could be located by its relation to extra-room cues, a large distal white panel, or to local inserts on the maze. Once the animals consistently chose the goal arm, a probe session was conducted to determine which cues the animals were using to solve the task. During the probe trial both the white panel and the local inserts were rotated 90 degrees clockwise and counterclockwise respectively and the animals' choice of arm recorded. During the first probe, females tended to rely on all three types of cues in solving the task. With additional training there was a shift towards predominantly using the distal visual information. In contrast, male rats did not show this shift; by the first probe session the males were predominantly using the distal visual information to solve the task. The findings indicate: (1) sex differences in the initial use of environmental cues; (2) the usage of environmental information is dynamic and changes with additional exposures to the environment. The results are related to previous findings on sex differences and estrous cycle effects, with an emphasis on the implications for hippocampal processing.

Animals↗

PubMed1999

Incidence and aetiology of heart failure; a population-based study.

AIMS: To determine the incidence and aetiology of heart failure in the general population. METHODS AND RESULTS: New cases of heart failure were identified from a population of 151 000 served by 82 general practitioners in Hillingdon, West London through surveillance of acute hospital admissions and through a rapid access clinic to which general practitioners referred all new cases of suspected heart failure. On the basis of clinical assessment, electrocardiography, chest radiography and transthoracic echocardiography, a panel of three cardiologists decided that 220 patients met the case definition of new heart failure over a 20 month period (crude incidence rate of 1.3 cases per 1000 population per year for those aged 25 years or over). The incidence rate increased from 0.02 cases per 1000 population per year in those aged 25-34 years to 11.6 in those aged 85 years and over. The incidence was higher in males than females (age-adjusted incidence ratio 1.75 [95% confidence interval 1.34-2.29, P<0.0001]). The median age at presentation was 76 years. The primary aetiologies were coronary heart disease (36%), unknown (34%), hypertension (14%), valve disease (7%), atrial fibrillation alone (5%), and other (5%). CONCLUSIONS: Within the general population, new cases of heart failure largely occur in the elderly, and the incidence is higher in men than women. The single most common aetiology is coronary heart disease, but in a third of cases the aetiology cannot be determined on the basis of non-invasive investigation alone. To be relevant to clinical practice, future clinical trials in heart failure should not exclude the elderly.

Adult↗

PubMed1991

Maintaining competence for first-line nurse managers: an evaluation of the use of the literature.

The use of nursing literature is frequently advocated as a means of maintaining competence. The purpose of this study was to compare the frequency of references to competencies in the literature with those retained for the role of first-line nurse managers by an expert panel involved in the Delphi technique. In this way, the use of literature in maintaining managerial competence for first-line nurse managers was evaluated. When the 20 most frequently mentioned competencies in the literature were compared with the highest mean scores and those ranked as most important by the expert panel, only two competencies appeared on all three lists. Fifteen competencies were frequently mentioned in the literature but were not identified by the expert panel as important to the role of first-line nurse managers. A further three competencies identified in the literature were excluded by the expert panel, indicating they were not perceived to be a function of first-line nurse managers.

Clinical Competence↗

PubMed2000

Characterisation of isolates of Haemophilus paragallinarum from Indonesia.

OBJECTIVE: To characterise 18 isolates of Haemophilus paragallinarum isolated from chickens in Indonesia. PROCEDURE: The isolates were identified to species level by traditional phenotypic methods. Six of the isolates were also identified by a species-specific polymerase chain reaction. Fourteen of the isolates were examined for resistance to a panel of seven antimicrobial agents using a disc diffusion method. All 18 isolates were serotyped according to the Page scheme using reference antisera in a haemagglutination inhibition test. RESULTS: Four of the 18 isolates were obtained from indigenous (kampung) chickens, with the remainder being from typical intensive poultry production systems. The 18 isolates were obtained from 11 outbreaks that showed the typical clinical signs of infectious coryza and 11 of the isolates were obtained from chickens that had been vaccinated with infectious coryza vaccines. All 18 isolates were confirmed as H paragallinarum by biochemical testing and six isolates were also identified as H paragallinarum by the polymerase chain reaction test. Eleven isolates were resistant to erythromycin and streptomycin, 10 to neomycin, eight to oxytetracycline, five isolates to doxycycline, three to sulphamethoxazoltrimethoprim but only one to ampicillin. Seven isolates were Page serovar A, four were Page serovar B and seven were Page serovar C. CONCLUSION: The presence of all three Page serovars (A, B and C) has been confirmed for the first time in Indonesian chickens. As the majority of the infectious coryza vaccines in use in Indonesia contain only serovar A and C, the presence of serovar B in chickens indicates that the protection by these bivalent vaccines would be reduced. The use of trivalent infectious coryza vaccines that contain serovars A, B and C is recommended for use in Indonesia.

Animals↗

PubMed2005

Sensitivity and specificity of a monoclonal antibody-based fluorescence assay for detecting Enterocytozoon bieneusi spores in feces of simian immunodeficiency virus-infected macaques.

Enterocytozoon bieneusi is clinically the most significant among the microsporidia causing chronic diarrhea, wasting, and cholangitis in individuals with human immunodeficiency virus/AIDS. Microscopy with either calcofluor or modified trichrome stains is the standard diagnostic test for microsporidiosis and does not allow species identification. Detection of E. bieneusi infection based on PCR is limited to a few reference laboratories, and thus it is not the standard diagnostic assay. We have recently reported the development and characterization of a panel of monoclonal antibodies against E. bieneusi, and in this publication we evaluated the specificity and sensitivity of an immunofluorescence assay (IFA), compared with PCR, in simian immunodeficiency virus-infected macaques. The IFA, which correlated with the primary PCR method, with a detection limit of 1.5 x 10(5) spores per gram of feces, will simplify considerably the detection of E. bieneusi spores in clinical and environmental specimens and in laboratory and epidemiological investigations.

Animals↗

PubMed2005

Multilocus sequence typing of Neisseria meningitidis directly from clinical samples and application of the method to the investigation of meningococcal disease case clusters.

Infections associated with Neisseria meningitidis are a major public health problem in England, Wales, and Northern Ireland. Currently, over 40% of cases are confirmed directly from clinical specimens using PCR-based methodologies without an organism being isolated. A nested/seminested multilocus sequence typing (MLST) system was developed at the Health Protection Agency Meningococcal Reference Unit to allow strain characterization beyond the serogroup for cases confirmed by PCR only. This system was evaluated on a panel of 20 meningococcus-positive clinical specimens (3 cerebrospinal fluid and 17 blood samples) from different patients containing various concentrations of meningococcal DNA that had corresponding N. meningitidis isolates. In each case, the sequence type generated from the clinical specimens matched that produced from the corresponding N. meningitidis isolate; the sensitivity of the MLST system was determined to be less than 12 genome copies per PCR. The MLST system was then applied to 15 PCR meningococcus-positive specimens (2 cerebrospinal fluid and 13 blood samples), each from a different patient, involved in three case clusters (two serogroup B and one serogroup W135) for which no corresponding N. meningitidis organisms had been isolated. In each case, an MLST sequence type was generated, allowing the accurate assignment of individual cases within each of the case clusters. In summary, the adaptation of the N. meningitidis MLST to a sensitive nested/seminested format for strain characterization directly from clinical specimens provides an important tool for surveillance and management of meningococcal infection.

Bacterial Proteins↗