Prospective randomized placebo-controlled double-blind clinical study of adjuvant dexamethasone with surgery for chronic subdural haematoma with post-operative subdural drainage: Interim analysis.

Most chronic subdural haematomas (CSDH) are successfully treated neurosurgically. However, operative recurrences occur with a frequency 3-30%, consume resources and potentially prolong length-of stay (LOS). The only adjuvant factor proven to significantly decrease CSDH recurrence rate (RR) is post-operative subdural drainage. Corticosteroids have been used to conservatively manage CSDH. One non-randomised study also compared dexamethasone (DX) as an adjunct to surgery without post-operative drainage: whilst a null effect was observed, the 'surgery-alone' group consisted of only n = 13. We present an interim analysis of the first registered prospective randomised placebo-controlled trial (PRPCT) of adjuvant DX on RR and outcome after CSDH surgery with post-operative drainage. Participants were randomised to either placebo or a reducing DX regime over 2 weeks, with CSDH evacuation and post-operative drainage. Post-operative mortality (POMT) and RR were determined at 30 days and 6 months; modified Rankin Score (mRS) at discharge and 6 months. Post-operative morbidity (POMB) and adverse events (AEs) were determined at 30 days. Interim analysis at approximately 50% estimated sample size was performed (n = 47). Recurrences were not observed with DX: only with placebo (0/23 [0%] v 5/24 [20.83%], P = 0.049). There was no significant between-group differences in POMT, POMB, LOS, mRS or AEs. CONCLUSIONS: In this first registered PRPCT, interim analysis suggested that adjuvant DX with post-operative drainage is both safe and may significantly decrease recurrences. A 12.5% point between-groups difference may be reasonable to power a final sample size of approximately n = 89. Future studies could consider adjuvant DX for longer than the arbitrarily-chosen 2 weeks.

Epigenetic silencing of the intronic microRNA hsa-miR-342 and its host gene EVL in colorectal cancer.

MicroRNAs are small, non-coding RNAs that influence gene regulatory networks by post-transcriptional regulation of specific messenger RNA targets. MicroRNA expression is dysregulated in human malignancies, frequently leading to loss of expression of certain microRNAs. We report that expression of hsa-miR-342, a microRNA encoded in an intron of the gene EVL, is commonly suppressed in human colorectal cancer. The expression of hsa-miR-342 is coordinated with that of EVL and our results indicate that the mechanism of silencing is CpG island methylation upstream of EVL. We found methylation at the EVL/hsa-miR-342 locus in 86% of colorectal adenocarcinomas and in 67% of adenomas, indicating that it is an early event in colorectal carcinogenesis. In addition, we observed a higher frequency of methylation (56%) in histologically normal colorectal mucosa from individuals with concurrent cancer compared to mucosa from individuals without colorectal cancer (12%), suggesting the existence of a 'field defect' involving methylated EVL/hsa-miR-342. Furthermore, reconstitution of hsa-miR-342 in the colorectal cancer cell line HT-29 induced apoptosis, suggesting that this microRNA could function as a proapoptotic tumor suppressor. In aggregate, these results support a novel mechanism for silencing intronic microRNAs in cancer by epigenetic alterations of cognate host genes.

Diagnosis of herpes simplex virus infections in the clinical laboratory by LightCycler PCR.

Herpes simplex virus (HSV) causes several clinical manifestations in both normal and immunocompromised hosts; this agent is the most frequently detected virus in diagnostic laboratories. Recovery of the virus in cell culture is considered the "gold standard" for detection of this virus from sources other than cerebrospinal fluid. LightCycler is a newly developed, commercially available system designed to rapidly perform PCR, with real-time detection of PCR products by a fluorescence resonance energy transfer assay. We compared the detection of HSV for 200 specimens (number of genital specimens, 160; number of dermal specimens, 38; number of ocular specimens, 2) by shell vial cell cultures (MRC-5) and by LightCycler PCR. Of a total of 88 (44%) HSV strains detected, 69 (78%) were detected by both shell vial cell cultures and LightCycler PCR (DNA polymerase target). A total of 19 (22%) specimens were detected exclusively by LightCycler PCR. No specimens were positive by the shell vial assay only. All 19 discrepant samples had HSV DNA detected by an independent PCR directed to the thymidine kinase gene of the virus. The melting curve analysis feature of the LightCycler instrument identified identical genotype results for HSV type 1 (HSV-1) and HSV-2 from 84 of 88 (96%) positive samples. Specimens can be extracted, target HSV DNA can be amplified, and HSV PCR products can be identified by genotype within 2 h after receipt of specimen into the laboratory. The increased level of accurate identification (all 88 positive samples) compared with that of shell vial cell culture (69 of 88 samples identified as positive) and the agreement of LightCycler PCR results with all shell vial positive results indicate the potential for routine implementation of this technology for laboratory diagnosis of HSV infections.

Analysis of candidate-host immunogenetic determinants in herpes simplex virus-associated Mollaret's meningitis.

Infection due to herpes simplex virus (HSV) is associated with recurrent aseptic meningitis (Mollaret's meningitis); however, the neuropathogenesis of this disease remains unknown. We collected 20 cerebrospinal fluid (CSF) specimens that were positive for HSV DNA by using polymerase chain reaction (PCR) assay from patients with a clinical diagnosis of Mollaret's meningitis. Patients were predominantly female (female:male, 22:1), with an average age of 32.8 years (range, 18-46 years). Using direct sequence analysis of HSV PCR products obtained from the CSF, we determined that all of the patients were infected with HSV type 2. In addition, we evaluated polymorphisms in 2 human genomic loci, which are associated with either severe or recurrent microbial infections (interferon-gamma receptor [IFN-gammaR] and mannose binding lectin [MBL]); these host genes were also amplified directly from the CSF specimens. No mutations were found in exons 2 or 3 of the IFN-gammaR gene (n=20). In contrast, there were 4 (20%), 4 (20%), and 0 mutations found in codons 52, 54, and 57, respectively, in exon 1 of MBL (n=20). A significantly higher frequency of codon 52 mutations (P=.04) was observed, compared with racially matched control patients.

Role of genotypic analysis of the thymidine kinase gene of herpes simplex virus for determination of neurovirulence and resistance to acyclovir.

Mutations in the thymidine kinase (TK) gene of herpes simplex virus (HSV) have been associated with resistance to acyclovir (ACY) and possible recognition of neurotropic strains. We sequenced a 335-bp segment of the TK gene to determine the frequency of mutations in HSV strains recovered from dermal, genital, and cerebrospinal fluid (CSF) specimens (n = 200; 102 HSV type 1 [HSV-1] 98 HSV-2 strains). Four polymorphic sites were detected in HSV-1 strains; C513T, A528G, C575T, and C672T. Among the polymorphisms, only C575T resulted in a change of amino acid sequence (residue 192, Ala-->Val). For HSV-2 strains, only one polymorphism (G420T) which resulted in an amino acid substitution (residue 139, Leu-->Phe) was detected. Phenotypic determination of resistance to ACY by a plaque reduction assay of 48 HSV isolates was not correlated with the sequence results of 11 strains in that 7 of these with genotypic polymorphisms were susceptible to the drug in vitro. In addition, of 32 ACY-resistant HSV strains, 28 (87.5%) had no polymorphisms detected in the 335-bp amplicon of the TK gene. There was no statistical difference in the frequency of polymorphisms according to the source of the specimens. We conclude that the detection of nucleic acid polymorphisms in a previously implicated 335-bp segment of the TK gene cannot be interpreted as indicative of either ACY resistance or neurotropism of HSV strains from dermal, genital, and CSF sources.

Ocular manifestations of Whipple disease: an atypical presentation.

A 62-year-old man developed bilateral granulomatous iridocyclitis after uncomplicated cataract surgery. On ophthalmic examination, we found moderate inflammation in the anterior chamber and vitreous, with granular crystalline deposits on the iris, intraocular lens, and capsular bag. Biopsy of the lens capsule and vitreous revealed periodic acid-Schiff-positive, diastase-resistant bacilli consistent with Tropheryma whippelii. Electron microscopy and polymerase chain reaction confirmed the diagnosis of Whipple disease. A jejunal biopsy specimen also revealed T whippelii. Treatment with trimethoprim-sulfamethoxazole, cefixime, rifampin, and doxycycline resulted in improvement of systemic symptoms, but intraocular inflammation persisted. Intraocular inflammation was eventually reduced with the intravenous administration of ceftriaxone sodium.

Comparative evaluation of colorimetric microtiter plate systems for detection of herpes simplex virus in cerebrospinal fluid.

In the past few years, application of the PCR to the detection of herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) from patients with encephalitis and meningitis has become standard laboratory practice. However, from an operational perspective, the true diagnostic value of PCR in this setting is yet to be realized because most laboratories subject the amplification products to lengthy probe hybridization procedures by Southern blotting. As alternatives to Southern blotting, we evaluated colorimetric microtiter plate (MTP) systems from ViroMed Laboratories, Inc. (PrimeCapture), CPG, Inc. (Quanti-PATH), and Incstar Corp. (GEN-ETI-K), in addition to a system developed at the Mayo Clinic with the PCR ELISA system (Boehringer Mannheim Corp.). We tested PCR products from 86 clinical CSF specimens submitted to our Molecular Microbiology Laboratory. The CSF specimens used had to have sufficient volume for comparative analysis. By conventional Southern blotting methods, 54 were positive and 32 were negative for HSV DNA. Compared with Southern blotting, the sensitivity and specificity were 63.0 and 100.0%, respectively, for the PrimeCapture system, 98. 2 and 96.9%, respectively, for the Quanti-PATH system, 98.2 and 100. 0%, respectively, for the GEN-ETI-K system, and 100.0 and 96.9%, respectively, for the Mayo system. All four MTP systems had turnaround times 12 to 24 h less than that for Southern blotting. There were no significant differences in costs or technologist time between the Mayo system and Southern blotting. Other features of the Mayo system include type-specific genotypic identification of HSV and the potential for determination of drug resistance by DNA sequencing. Overall, we found that colorimetric MTP systems were likely to improve test turnaround times and patient care at no additional cost.

Laboratory diagnosis of central nervous system infections with herpes simplex virus by PCR performed with cerebrospinal fluid specimens.

Until recently, the laboratory diagnosis of central nervous system (CNS) infections with herpes simplex virus (HSV) has been limited by poor sensitivity and/or specificity. We assessed the diagnostic utility of PCR for detection of HSV in over 2,100 specimens referred to the Mayo Clinic from August 1993 to May 1996. DNA extracted from cerebrospinal fluid (CSF) samples with IsoQuick was amplified by PCR with oligonucleotide primers directed to the DNA polymerase gene of HSV, yielding a 290-bp amplicon. HSV DNA was detected in 150 (135 by gel electrophoresis, 15 by Southern blotting only) of 2,106 (7.1%) specimens. PCR-positive CNS disease occurred in patients ranging in age from 13 days to 89 years; 59% of the cases occurred in patients between the ages of 30 and 69, and 21 (14%) of the patients were infants. Genotype analysis was not routinely performed; however, amplification of a 335-bp product within the thymidine kinase gene of HSV revealed 13 positions within a span of 80 nucleotides that accurately identified the two serotypes of the virus according to 14 reference strains. We conclude that PCR detection of HSV DNA in CSF specimens should be considered an emerging "gold standard" for the laboratory diagnosis of CNS infections with this virus.

Indeterminate results of the second-generation hepatitis C virus (HCV) recombinant immunoblot assay: significance of high-level c22-3 reactivity and influence of HCV genotypes.

A second-generation recombinant immunoblot assay (RIBA 2.0) is used in the United States to confirm infection with hepatitis C virus (HCV) in samples that are anti-HCV (enzyme immunoassay) positive. In some cases, indeterminate results of RIBA 2.0, which are defined as reactivity to a single antigen species or reactivity limited to two proteins derived from the same coding region of the HCV genome, are encountered. This study was performed to establish the significance of indeterminate RIBA 2.0 results in relation to HCV RNA detection, high positivity for the c22-3 band, and the HCV genotype as determined by direct DNA sequencing. Ninety-six samples with indeterminate RIBA 2.0 results were studied. HCV RNA was detected in 21 of 34 (62%) samples with high reactivity to c22-3 and in 8 of 62 (13%) samples with low reactivity to c22-3. The HCV genotype distribution in samples that were RIBA 2.0 indeterminate and HCV RNA positive was significantly different from that in samples of a control group with positive results for both the RIBA 2.0 and HCV PCR. These results suggest that highly positive c22-3 samples are likely to be associated with HCV viremia and that infection with less common HCV genotypes is more commonly associated with indeterminate RIBA 2.0 results.

Increased risk of hepatocellular carcinoma in patients infected with hepatitis C genotype 1b.

UNLABELLED: Infection with hepatitis C virus (HCV) genotype 1b has been reported to be associated with more severe liver disease and an unfavorable outcome. Liver transplantation allows for a complete examination of the explanted liver for the detection of hepatocellular carcinoma (HCC). OBJECTIVE: To study the prevalence of HCC in patients with liver cirrhosis secondary to chronic infection with HCV genotype 1b compared with those infected with other genotypes. METHODS: Sera were collected from 48 consecutive patients undergoing liver transplantation for end stage liver disease secondary to HCV infection. RNA was extracted from serum using chaotropic lysis and isopropanol precipitation. Reverse transcriptase-polymerase chain reaction of the NS5 region was performed, followed by automated sequencing on desalted amplification products. Genotype assignment followed Simmonds's classification. All explanted livers were examined for the presence of HCC. RESULTS: HCV genotypes in our patients were as follows: subtype 1a, 20 patients (42%); 1b, 18 patients (37.5%); 2a, one patient (2%); 2b, six patients (12.5%); 3a, one patient (2%); and 4a, two patients (4%). Although five of 18 patients infected with HCV genotype 1b (28%) had HCC, only one of 30 patients (3%) infected with all other genotypes (1a, 2a, 2b, 3a, and 4a) had HCC (p = 0.02). CONCLUSION: Infection with HCV genotype 1b may carry a higher risk for the development of HCC than infection with other HCV genotypes.

Prevalence and outcome of hepatitis C infection among heart transplant recipients.

BACKGROUND: Hepatitis C virus infection is common in organ transplant recipients, and can be associated with significant morbidity and mortality. A unique feature of this infection among immunosuppressed patients is that it can progress without the development of hepatitis C virus antibodies. METHODS: To define the prevalence of hepatitis C virus infection in patients undergoing heart transplantation and identify clinical syndromes associated with hepatitis C virus infection in heart transplant recipients, we collected sera from 59 consecutive heart transplant recipients and their donors. Samples were tested before and after transplantation for hepatitis C virus antibodies with the use of a second-generation recombinant immunoblot assay and for hepatitis C virus RNA by means of reverse transcriptase polymerase chain reaction. RESULTS: Four of 59 patients (7%) had hepatitis C virus-RNA detected in posttransplantation serum samples; but only one of these was anti-hepatitis C virus antibody positive. Two of the four patients with hepatitis C virus RNA detected after transplantation received organs from donors who were positive for hepatitis C virus RNA/anti-hepatitis C virus. One of these two recipients tested positive for hepatitis C virus antibody and hepatitis C virus RNA before transplantation. The other two patients received organs from hepatitis C virus negative donors and possibly acquired infection after transplantation from blood or immunoglobulin preparations. One patient was anti-hepatitis C virus positive before transplantation but had no detectable hepatitis C virus RNA, and hepatitis C virus infection did not develop after transplantation. Progressive hepatitis C virus-induced cholestatic liver disease that led to hepatic failure and death after heart transplantation occurred in one of the four patients. CONCLUSION: Hepatitis C virus infection may occur after heart transplantation in the absence of anti-hepatitis C virus antibodies, and a syndrome of severe cholestatic liver disease may complicate heart transplantation in the presence of hepatitis C virus infection.

The morphological effects of two antimicrobial peptides, hecate-1 and melittin, on Escherichia coli.

The effects of the 26 amino acid, cationic, amphipathic, antibacterial peptide melittin and hecate-1, a 23 amino acid analog of it, on the gram negative bacterium Escherichia coli were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and freeze-fracture. Both peptides killed virtually all bacteria at the peptide concentration and cell density used. TEM and SEM revealed aggregates of bacteria entangled with material extruded from the bacterial surfaces. SEM revealed irregular bacterial surfaces with bleb-like projections. TEM and freeze-fracture indicate that the bacterial inner and outer membranes, as well as the peptidoglycan layer between, were extensively damaged. The cytoplasmic contents of the cells, however, did not appear radically disturbed, providing little evidence for osmotically induced cytolysis.

Early detection of human cytomegalovirus viremia in bone marrow transplant recipients by DNA amplification.

Surveillance blood cultures for human cytomegalovirus (HCMV) are commonly used to identify the bone marrow transplant (BMT) recipients with the highest risk of serious HCMV disease and for whom early interventional ganciclovir therapy would be beneficial. We monitored 36 allogeneic BMT recipients weekly for the presence of HCMV in the blood from 0 to 100 days posttransplantation. Viable HCMV in leukocytes (WBC) was detected by shell vial and tube culture methods. HCMV DNA in WBC and plasma was detected by PCR and DNA hybridization using primers and a probe from the EcoRI fragment D region of HCMV AD169. A uracil-N-glycosylase-dUTP PCR protocol was used to prevent false-positive results due to amplicon carryover. Seventeen patients had multiple consecutive positive samples containing HCMV DNA in plasma or WBC. In 14 of 17 patients, HCMV was also detected by blood culture. HCMV DNA was detected sporadically in six patients, none of whom had positive cultures. One patient had HCMV viremia detected by WBC culture only. The remaining 12 patients had no positive PCR assays or blood cultures. For the patients with positive blood cultures, PCR detection of HCMV DNA in plasma preceded detection of HCMV in culture by a mean of 8 days and detection in WBC preceded detection in culture by 6 days. HCMV disease (interstitial pneumonia) was documented for two patients with viremia (blood culture and PCR positive) and one patient without viremia (blood culture and PCR negative). The earlier recognition of high-risk patients provided by detection of HCMV DNA in plasma or WBC may improve the efficacy of early interventional antiviral therapy.

Isolation of hepatitis C virus RNA from serum for reverse transcription-PCR.

Standard multistep extraction and isolation of RNA for hepatitis C virus (HCV) reverse transcription (RT)-PCR are impractical for routine use in clinical laboratories. We compared three simple commercially available methods for RNA isolation (RNAzol B, TRISOLV, and ULTRASPEC; Biotecx Laboratories, Houston, Tex.) and a total nucleic acid isolation method (IsoQuick; MicroProbe Corp., Garden Grove, Calif.) for the recovery of HCV RNA from sera obtained from 12 viremic patients for RT-PCR. RNAzol B, TRISOLV, ULTRASPEC, and IsoQuick extraction methods detected 87.5, 79.2, 33.3, and 58.3% of the paired positive samples, respectively. The method used for isolation of RNA is an important concern when optimizing HCV RT-PCR.

Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization.

A polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary tuberculosis was developed by using oligonucleotide primers to amplify a fragment of IS6110, an insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis. Sediment obtained from sputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simple lysis buffer and was heated at 100 degrees C for 30 min prior to amplification. A dUTP-uracil N-glycosylase PCR protocol was used to prevent false-positive test results because of the carryover of products from previous amplification reactions. The 317-bp amplicon was detected by direct gel analysis and Southern blotting and then hybridization with a biotin-labeled internal probe. Hybrid molecules were detected by using a commercially available avidin-alkaline phosphatase-chemiluminescent substrate system (Tropix, Inc., Bedford, Mass.). The analytical sensitivity of the assay was 10 fg of purified mycobacterial DNA. The limits of detection by culture (Middlebrook 7H11 agar and Lowenstein-Jensen medium) and by PCR were equivalent in terminal dilution experiments for organism suspensions and positive sputa. An internal control was used to detect the presence of amplification inhibitors in each negative reaction mixture. DNA was purified from inhibitory specimens by phenol-chloroform extraction and ethanol precipitation. PCR results were compared with results of microscopy and conventional culture for the detection of M. tuberculosis in 313 sputum specimens. There were 124 specimens that were positive for M. tuberculosis by conventional methods and 113 (91%) that were positive by PCR. PCR detected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%). This PCR assay innovations that make application of this new technology feasible in clinical microbiology laboratories.

Stomatococcus mucilaginosus bacteremias. Typical case presentations, simplified diagnostic criteria, and a literature review.

Even though Stomatococcus mucilaginosus is considered indigenous oral-pharyngeal flora, cited literature and case reports indicate that it can be the cause of infectious conditions. Tested strains were isolated from blood, the oral region, and wound sources. The organism was routinely misidentified or not identified by conventional or commercial systems (Vitek, STAPH-Trac). Four antimicrobial diagnostic disks for example, bacitracin (0.04 units; Taxo A), furazolidone (100 micrograms), novobiocin (5 micrograms), and polymyxin B (300 units), were evaluated as possible addition to previously applied biochemical characteristics that differentiate between S. mucilaginosus, Micrococcus sp., and coagulase-negative staphylococci. Consistent antimicrobial susceptibility patterns among our isolates to the diagnostic disks produced applicable characteristics for discriminating S. mucilaginosus from similar microorganisms. However, therapeutic choices of antimicrobial agents should be guided by individual organism susceptibility test results because of variable, often resistant patterns to beta-lactams, aminoglycosides, macrolides, new fluoroquinolones, and sulfonamides.

Effect of active immunization against estrogen on gonadotropin response to testosterone propionate treatment in ovariectomized pony mares.

An experiment was conducted to determine whether partial neutralization of estrogens via active immunization alters testosterone propionate (TP)-induced increases in FSH secretion after GnRH administration in ovariectomized pony mares. Twenty mares were used in a 2 X 2 factorial arrangement of treatments (n = 5/group). Factor 1 was long-term active immunization against either bovine serum albumin (BSA) or estrone-17-oxime-BSA. Factor 2 was 11-d administration of either vehicle (vegetable oil) or TP (175 micrograms/kg BW). Plasma concentrations of FSH were not affected (P greater than .1) by either factor. As expected, the FSH response to exogenous GnRH was threefold greater (P less than .05) in BSA-immunized mares treated with TP than in BSA-immunized mares receiving oil. However, immunization against estrogens reduced (P less than .05) this TP-induced increase in FSH response by 52%. Plasma concentrations of LH were decreased (P less than .08) by TP; this effect was not altered (P greater than .1) by immunization against estrogen. The LH response to exogenous GnRH was not affected (P greater than .1) by either factor. We conclude that aromatization of testosterone to estrogen is partially responsible for the increased FSH response to exogenous GnRH in TP-treated mares. In contrast, suppression of LH concentrations by TP appears to involve only the androgenic effect of TP.

Effects of active immunization against gonadotropin releasing hormone on gonadotropin secretion after ovariectomy and testosterone propionate administration to mares.

Five lighthorse mares were actively immunized against gonadotropin releasing hormone (GnRH) conjugated to bovine serum albumin (BSA) to study the involvement of GnRH in luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion following ovariectomy (OVX) and after administration of testosterone propionate (TP). Five mares immunized against BSA served as controls. Immunizations were started on November 1, and OVX was performed in June (d 1). All mares were treated with TP from d 50 to 59 after OVX. On the day of OVX, concentrations of LH were lower (P less than .05) in GnRH-immunized mares than in BSA-immunized mares and were generally nondetectable; FSH concentrations were reduced (P less than .05) by 50% in GnRH-immunized mares relative to BSA-immunized mares. In contrast to BSA-immunized mares, plasma concentrations of LH or FSH did not increase after OVX in GnRH-immunized mares. The LH response to GnRH analog (less than .1% cross-reactive with GnRH antibodies) on d 50 was reduced (P less than .05) by 97% in GnRH-immunized mares relative to BSA-immunized mares, whereas the FSH response was similar for both groups. Treatment with TP for 10 d reduced (P less than .01) the LH response and increased (P less than .01) the FSH response to GnRH analog in BSA-immunized mares, but it had no effect (P greater than .1) on the response of either gonadotropin in GnRH-immunized mares.(ABSTRACT TRUNCATED AT 250 WORDS)