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Biomedical subjects

P Charache

Publications and source records attributed to P Charache.

At least 19 recordsLinked to original sources
PubMed1995

Clostridium difficile colitis: an efficient clinical approach to diagnosis.

OBJECTIVE: To define clinical and laboratory variables that suggest the presence of Clostridium difficile colitis and to establish the number of stool specimens needed to reasonably exclude the diagnosis of C. difficile colitis. DESIGN: Prospective study of consecutive inpatients whose stool specimens were sent to be evaluated for the presence of C. difficile toxin. SETTING: University teaching hospital. PATIENTS: 268 hospital inpatients in medical, surgical, and gynecology units. MEASUREMENTS: Structured history and physical examination; detection of C. difficile toxin by cytotoxin tissue-culture assay with anti-C. difficile antiserum neutralization and by enzyme-linked immunoassay (EIA) for C. difficile toxins A and B; and detection of fecal leukocytes by microscopic examination and by latex agglutination lactoferrin assay. RESULTS: 43 of 268 consecutive inpatients were positive for C. difficile toxin by EIA or tissue-culture assay. Although toxin was detected by EIA alone in 39 of the 43 patients, it was detected in an additional 4 patients (10%) by tissue-culture assay alone. Univariate and multivariate logistic regression analysis showed that the following clinical and laboratory features were associated with C. difficile toxin positivity: the onset of diarrhea 6 or more days after the administration of antibiotics (odds ratio, 1.38 [95% CI, 1.10 to 3.79]); hospital stay longer than 15 days (odds ratio, 1.33 [CI, 1.09 to 3.95]); the presence of fecal leukocytes determined by microscopy (odds ratio, 2.39 [CI, 1.05 to 5.42]) or lactoferrin assay (odds ratio, 3.74 [CI, 1.80 to 7.76]); the presence of semiformed (as opposed to watery) stools (odds ratio, 2.33 [CI, 1.10 to 4.90]); and cephalosporin use (odds ratio, 2.36 [CI, 1.10 to 5.09]). Toxin-positive patients were no more likely than controls to have had fever, abdominal pain or cramps, leukocytosis, green-colored diarrhea, or blood in the stool or to have received clindamycin or penicillin derivatives. Of the 43 patients with C. difficile toxin, 34 (79%) had positive results for the toxin on the first stool specimen, 5 (cumulative, 91%) had positive results on the second specimen, and 4 had positive results on the third specimen. Overall, the negative predictive value of the first stool specimen was 97%. All patients who had two or more clinical or laboratory predictors were diagnosed with C. difficile disease when either the first or the second stool specimen was positive for toxin. CONCLUSIONS: Clinicians at the bedside can use readily available clinical and laboratory information to decide which patients are likely to have C. difficile disease and when it is appropriate and useful to order specific diagnostic tests for C. difficile toxin. Such data are also useful in determining the number of stool samples that reasonably excludes the diagnosis of C. difficile colitis.

Aged↗
PubMed1995

Viral sensitivity testing in patients with cytomegalovirus retinitis clinically resistant to foscarnet or ganciclovir.

PURPOSE: Resistance to antiviral therapy is a potential cause of progression of cytomegalovirus retinitis in patients with the acquired immunodeficiency syndrome. We investigated the results of viral sensitivity testing in a series of patients with clinically resistant retinitis who had positive results of blood or urine cytomegalovirus cultures. METHODS: All patients with newly diagnosed cytomegalovirus retinitis between January 1990 and December 1991 were prospectively studied. Blood and urine cultures for cytomegalovirus were obtained in a nonrandomized subgroup of this group. The results of in vitro sensitivity to foscarnet and ganciclovir, determined by a DNA hybridization assay, were then analyzed in seven patients with clinically resistant cytomegalovirus retinitis and whose blood or urine culture results, or both, were positive for cytomegalovirus while on a treatment regimen. RESULTS: Foscarnet-resistant cytomegalovirus (ID50 > 300 microM) was isolated from two patients, one of whom was being treated with foscarnet. Ganciclovir-resistant cytomegalovirus (ID 50 > 6.0 microM) was isolated from four patients, three of whom were being treated with ganciclovir. Foscarnet- and ganciclovir-resistant cytomegalovirus occurred with previous ganciclovir therapy in one patient. Clinical improvement occurred in three patients whose change in therapy was based on viral sensitivity testing. In general, prolonged therapy with one drug was associated with a progressive increase in the ID 50 for that drug. CONCLUSIONS: Viral resistance to foscarnet or ganciclovir may explain refractory cytomegalovirus retinitis in some patients.

AIDS-Related Opportunistic Infections↗
PubMed1995

Association of BK virus with failure of prophylaxis against hemorrhagic cystitis following bone marrow transplantation.

PURPOSE: Hemorrhagic cystitis (HC) after bone marrow transplantation (BMT) has been ascribed to cyclophosphamide metabolites. HC has also been associated with excretion of the BK type of polyomavirus. The relative contributions of cyclophosphamide metabolites and BK virus in the development of HC following BMT are unknown. PATIENTS AND METHODS: We conducted a randomized trial to compare mesna with forced diuresis for prophylaxis against HC in 147 BMT recipients. We studied the association of BK virus with HC in 95 consecutive BMT recipients by prospectively monitoring urinary excretion of BK virus using polymerase chain reaction amplification of viral gene sequences. RESULTS: HC occurred in 37 of 147 (25.2%) transplant recipients. The incidence of HC was similar in patients given mesna (26.8%, 19 of 71) or forced diuresis (23.7%, 18 of 76), and in recipients of allogeneic (27.2%, 18 of 64) or autologous marrow (22.9%, 19 of 83). The incidence of HC was unrelated to primary disease, preparative regimen, or occurrence of graft-versus-host disease (GVHD). Excretion of BK virus was demonstrated in 50 of 95 patients (52.6%); 38 patients (40%) had persistent BK viruria (> or = two consecutive positive samples). HC occurred in 19 of 38 patients (50%) with persistent BK viruria, in one of 12 (8.3%) with only a single urine sample positive for BK virus, and in none of 45 who did not excrete BK virus (P < .0001). Shedding of BK virus also had a strong temporal correlation with onset of HC (r = .95). CONCLUSION: Mesna and forced diuresis are equally effective in abrogating the urothelial toxicity of preparative regimens for BMT. Since HC after BMT is virtually always associated with persistent BK viruria, strategies aimed at the prevention or elimination of viruria in BK seropositive recipients are warranted.

Adult↗
PubMed1992

New vancomycin disk diffusion breakpoints for enterococci. The National Committee for Clinical Laboratory Standards Working Group on Enterococci.

Since 1988, when the first vancomycin-resistant enterococcus was described, several descriptions of failures of disk diffusion breakpoints to detect low-level vancomycin resistance (MICs, 8 to 32 micrograms/ml) have been published. A four-laboratory collaborative study was undertaken to establish more accurate breakpoints for the disk test. Mueller-Hinton agar was used to perform dilution testing (in three laboratories) and disk diffusion testing (in all laboratories). Results were determined at 18, 24, and 48 h, and zones of inhibition were read using both transmitted and reflected light. One hundred organisms (35 Enterococcus faecalis, 55 E. faecium, and 10 E. gallinarum or E. casseliflavus isolates) were selected to represent vancomycin-susceptible and -resistant phenotypes. Interlaboratory agreement of agar dilution MICs was better at 24 h (91 to 94% within +/- 1 dilution) than at 18 h (76% within +/- 1 dilution). Therefore, 24-h agar dilution MIC results were used as the reference. For disk diffusion, it was critical to note the presence of a haze or colonies inside the zone when interpreting the test, since this correlated better with the results of the agar dilution test. The presence of a haze or inner colonies was best detected by reading the zones with transmitted light and incubating the plates for a full 24 h. When plotted against 24-h agar dilution MICs, breakpoints of </= 14 mm (resistant), 15 to 16 mm (intermediate), and >/= 17 mm (susceptible) resulted in 58 minor errors (14.5% of total values) and 5 very major errors (2.2% of resistant values or 1.3% of total values). No major errors were seen. Results of repeat testing using a common lot of Mueller-Hinton agar showed 52 minor errors (13.3%) and 4 major errors (4.2% of susceptible values of 1.0% pf total values) but no very major errors. It is recommended that any haze or colonies within the zone be taken into account when determining zones of inhibition and that an MIC test be performed for strains with intermediate zones if vancomycin is being considered for treatment.

Agar↗
PubMed1992

Detection of herpes simplex virus by a nonradiometric spin-amplified in situ hybridization assay.

An in situ hybridization kit (Diagnostic Hybrids, Inc., Athens, Ohio) was evaluated for use in the detection and identification of herpes simplex virus (HSV) from clinical specimens. For in situ hybridization, a 10-min spin amplification onto monolayers of African green monkey kidney cells (CV-1) in 24-well polystyrene dishes, 24-h culture amplification, and hybridization with an alkaline phosphatase-labeled DNA probe were used. A total of 648 specimens were tested, including 275 specimens from patients with symptomatic diseases sent specifically for HSV detection and 373 specimens from asymptomatic immunocompromised patients sent for detection of HSV shedding. Overall, the sensitivity of the hybridization assay was 97.8% (131 of 134 specimens), with 105 of 105 (100%) specimens from symptomatic patients and 26 of 29 (89.9%) specimens from asymptomatic patients being detected. The three specimens that were false negative by in situ hybridization had low virus titers, as determined by tissue culture. The specificity was 99.6% (512 of 514 specimens). The rapid, accurate results suggest that the in situ hybridization kit may be used as an alternative to conventional tissue culture for the detection of HSV.

DNA Probes↗
PubMed1992

In situ localization of Epstein-Barr virus in thymic carcinoma.

Epstein-Barr virus (EBV) genome is associated with a variety of lymphoid and epithelial malignancies. EBV DNA has been detected in some cases of thymic carcinoma, but the cellular locus of the virus has never been defined. Detection of EBV has also been reported in normal thymus, thymic lymphoid hyperplasia, and thymoma by some investigators but not by others. In order to better define the association of the virus with benign and malignant thymic tissues and to characterize its cellular locus, we applied a recently developed in situ hybridization technique using a very abundant EBV transcript (EBER1) as target to a variety of thymic tissues. We detected expression of this transcript only in the malignant epithelial cells in one case of thymic lymphoepithelioma-like carcinoma. EBV expression was not detected in six other cases of thymic carcinoma, nor in tissue from 16 normal thymuses, 14 thymomas, and 10 thymic lymphoid hyperplasias.

Adult↗
PubMed1991

Epstein-Barr virus in AIDS-related primary central nervous system lymphoma.

Primary central nervous system lymphoma occurs more often in patients with AIDS. Epstein-Barr virus (EBV) has been detected in these tumours, but the degree of association has not been defined because of both the highly restricted expression of EBV in malignant tissue and the lack of a technique that is reliable in formalin-fixed paraffin-embedded specimens. EBV-transformed lymphocytes contain short non-protein coding EBV transcripts (EBERs), which are expressed in much higher quantity than other EBV-latency transcripts. We describe a new strategy for detection of latent EBV with these transcripts as targets for in-situ hybridisation. 18 cases of AIDS-related primary CNS lymphoma from a consecutive necropsy series together with specimens from 3 further cases were studied. In each case, a strong positive signal over tumour cells indicated abundant expression of the EBV-EBER1 transcript. This 100% association suggests that the pathogenesis of these AIDS-associated lymphomas may differ from the systemic disease in which only 30-50% of tumours are associated with EBV. A pathogenetic role for EBV was further supported by showing expression of a viral protein (the latent membrane protein) that is implicated as an effector for EBV-associated lymphomagenesis. EBV might have a role as a tumour marker in the diagnosis and management of AIDS-related primary CNS lymphoma.

Central Nervous System Neoplasms↗
PubMed1991

The effect of tetracycline on levels of oral contraceptives.

Despite the widespread use of tetracycline for treatment of dermatologic disorders and sexually transmitted diseases, the pharmacodynamics of oral contraceptive use in the presence of tetracycline has not been studied. Seven normal women ingested an oral contraceptive containing ethinyl estradiol 35 micrograms and norethindrone 1 mg in the follicular phase of the menstrual cycle. On day 0 baseline ethinyl estradiol and norethindrone levels were obtained at 0, 1/2, 3/4, 1, 2, 4, 12, and 24 hours after oral contraceptive administration. On day 1 tetracycline 500 mg was given orally every 6 hours while the oral contraceptive was continued. Tetracycline, ethinyl estradiol, and norethindrone levels were determined at the same time intervals as on day 0. Oral contraceptive and tetracycline were continued for up to 10 days, and additional concentrations of ethinyl estradiol, norethindrone, and tetracycline were determined between days 5 and 10. Four additional normal women ingested tetracycline for 5 to 10 days. Tetracycline levels were determined at the time intervals noted above on day 1 and days 5 to 10. No significant decrease in plasma ethinyl estradiol or norethindrone concentration was seen with either short-term (24 hours) or long-term (5 to 10 days) ingestion of tetracycline. Similarly, levels of tetracycline do not significantly decrease with ingestion of a low-dose oral contraceptive.

Adolescent↗
PubMed1991

Culture confirmation of cytomegalovirus and herpes simplex virus by direct enzyme-labeled DNA probes and in situ hybridization.

Using probes consisting of horseradish peroxidase (HRP) directly attached to DNA, scrapings or trypsinized cells from 217 adequate clinical samples were cultured and analyzed in 3 blind studies by in situ hybridization for the presence of cytomegalovirus (CMV) and herpes simplex virus (HSV). Sixty samples were judged inadequate due to insufficient cell numbers; however, this problem was significantly decreased during the course of the study. One hundred and eighteen samples were found positive and 70 samples were found negative for CMV. Scrapings of cultured cells from 29 clinical samples revealed 9 samples which were positive and 20 samples which were negative for HSV. Forty-two additional samples, containing either uninfected cells or cells infected with various strains of CMV, were analyzed for the ability of the HRP-DNA CMV probe to detect such isolates. Twenty samples were positive and 22 negative for CMV. No false-negatives or false-positives were observed for either CMV or HSV. In addition to the specificity noted above neither the CMV nor the HSV DNA probe hybridized to potential contaminants found in clinical specimens.

Cell Line↗
PubMed1991

Prevalence of infection with human immunodeficiency virus in elective surgery patients.

An anonymous survey of elective surgery patients was performed to assess prevalence of antibody to human immunodeficiency virus (HIV) in a large urban hospital. Of 4087 patients evaluated, 18 (0.4%) were found to be infected with HIV as confirmed by a positive Western blot antibody test. Assessment of risk factors demonstrated that patients with a history of a blood transfusion did not differ in demographics or rate of infection from the population as a whole. Of the 18 HIV infected patients, 13 gave an admission history of one or more risk factors, including 10 with a history of a prior positive test. Only five, or 0.12% of the patients, provided no history of a risk factor or a history of transfusion only. The authors conclude that the prevalence of HIV infection among elective surgery patients is low, and that there would not be any substantial benefit from screening such patients for antibody against HIV.

AIDS Serodiagnosis↗
PubMed1991

Abundant expression of EBER1 small nuclear RNA in nasopharyngeal carcinoma. A morphologically distinctive target for detection of Epstein-Barr virus in formalin-fixed paraffin-embedded carcinoma specimens.

The highly restricted expression of the Epstein-Barr virus (EBV) genome in malignancy has limited the use of EBV detection methods applicable to formalin-fixed paraffin-embedded carcinoma specimens. In EBV-transformed lymphocytes very short nonprotein coding EBV transcripts (EBERs) are expressed in much higher abundance (10(7) copies per cell) than other EBV latency transcripts. Using a 3H riboprobe, the authors demonstrated EBER1 expression in NASOPHARYNGEAL CARCINOMAS (NPCs) as well as in parotid salivary gland. Recognition of EBER1 expression was facilitated by the intensity of hybridization and its characteristic morphology (nuclear with nucleolar sparing). EBER1 expression was not demonstrated in other epithelial malignancies arising from mucosal surfaces (oropharynx, uterine cervix) from which EBV shedding has been detected. Repeat study of the NPC specimens with digoxigenin-labeled probe yielded hybridization signal with subcellular morphologic detail and without background in a 12-hour procedure. Thus the EBER1 transcript is an appropriate target for in situ hybridization detection of EBV in formalin-fixed paraffin-embedded carcinoma specimens.

Carcinoma↗
PubMed1990

Detection of EBV gene expression in Reed-Sternberg cells of Hodgkin's disease.

EBV DNA has been detected by Southern blot hybridization in 20-25% of Hodgkin's disease tumor specimens and localized to the Reed-Sternberg cells by in situ hybridization. In the present investigation we used a 3H-labelled EBER I anti-sense RNA for in situ hybridization of archival formalin-fixed paraffin-embedded Hodgkin's disease specimens previously shown by Southern Blot hybridization to be EBV-positive. In 6 of 8 specimens neoplastic cells showed an intense signal in virtually all of the tumor cells. The background lymphocytes, eosinophils, plasma cells and histiocytes did not demonstrate significant hybridization. In each case hybridization tended to spare the nucleoli. Hybridization was detected in specimens with histologies of mixed cellularity and nodular sclerosis. The intensity of signal relative to background is better than that in previous studies utilizing 35S-labelled large internal repeat probes for EBV in Reed-Sternberg cells. The exposure time is one week in contrast to the 4-5 weeks reported by others for detection of EBV in Hodgkin's disease. Both increased relative intensity and shorter exposure requirements may be attributed to the very high number of EBER transcripts in the target cells. The demonstration that EBER I is expressed is consistent with a role for EBV in growth regulation of Reed-Sternberg cells and suggests that the virus is not merely a silent passenger in Hodgkin's disease.

Blotting, Southern↗
PubMed1990

Oligonucleotides for polymerase chain reaction amplification and hybridization detection of Epstein-Barr virus DNA in clinical specimens.

We designed synthetic oligonucleotide primers and hybridization probe for use in polymerase chain reaction (PCR) amplification and hybridization detection of Epstein-Barr virus (EBV) nucleic acid sequences. Primer sequences were chosen from the coding region for the Epstein-Barr virus nuclear antigen-1 (EBNA-1). PCR amplification and hybridization with these oligonucleotides was carried out on standard laboratory cell lines including African Burkitt's lymphoma and infectious mononucleosis derived cell lines, as well as cell lines recently established from clinical EBV isolates from bone marrow transplant recipients. All EBV cell lines tested were positive. No false-positives were detected with uninfected cell lines, human placental DNA or with other viruses. The sensitivity of the detection procedure was such that four copies of the EBV genome could consistently be detected in a background of 1 microgram of placental DNA. EBV was detected in DNA extracts from the peripheral blood mononuclear cells of two patients with infectious mononucleosis and one patient with viral-associated hemophagocytic syndrome. Three of 18 EBV seropositive patients without known ongoing EBV-associated illness undergoing ambulatory surgery also had EBV detected in DNA extracts from their peripheral blood mononuclear cells. EBV was detected in DNA extracts from lymphoma tissue from two patients with post-transplant lymphomas and two AIDS patients with primary CNS lymphomas. EBV was not detected in 12 B-cell lymphoma specimens from patients without history of immunocompromise. DNA extracts from formalin-fixed paraffin-embedded Hodgkin's tissues previously shown to be EBV positive by Southern blot were also demonstrated to be EBV positive by PCR. Thus, with the oligonucleotides described, PCR is applicable to the detection of EBV in a spectrum of clinical isolates. The broad specificity of these oligonucleotides for all strains of EBV tested is probably a function of the highly conserved sequence of the EBNA-1 DNA binding domain.

Antigens, Viral↗
PubMed1990

Rapid detection of Pneumocystis carinii using a direct fluorescent monoclonal antibody stain.

A collaborative study was undertaken at two institutions to assess the performance of a direct fluorescent-antibody stain for the detection of Pneumocystis carinii in respiratory specimens from patients with known or suspected human immunodeficiency virus type 1 infections. A total of 163 specimens (125 induced sputa, 37 bronchoalveolar lavage fluids, and 1 tracheal aspirate) from 124 patients were examined by using modified Giemsa (Diff-Quik; Baxter American Scientific Products, Chicago, Ill.) and direct fluorescent-antibody stains. A total of 73 specimens contained P. carinii, which was detected in 66 (92%) of the specimens by using the modified Giemsa and in 71 (97%) of the specimens by using the fluorescent-antibody stain. One bronchoalveolar lavage fluid specimen in which P. carinii was detected only with the fluorescent-antibody stain was determined to be a false-positive based on subsequent clinical evaluation of the patient. Although the overall time for processing and examining specimens stained with either stain was not significantly different for those specimens containing P. carinii, considerably less time was required for microscopic examination of those fluorescent-antibody-stained specimens lacking P. carinii.

Antibodies, Monoclonal↗
PubMed1990

Clinical application of novel sample processing technology for the identification of salmonellae by using DNA probes.

Two hundred and fifty clinical fecal specimens collected over a 7-month period were analyzed for the presence of salmonellae by a rapid DNA hybridization procedure. Hybridizations were performed by using a novel specimen processing protocol called wicking and a previously unreported 1,600-base-pair probe cloned from Salmonella enteritidis DNA. The probe was shown to be reactive with all 70 Salmonella serotypes tested and not reactive with 101 stock strains of other enteric bacteria. Southern analysis of 30 Salmonella isolates representing 22 serotypes suggested that the probe sequence was highly conserved, appearing as a 1,600-base-pair band in a BglII digest of isolate DNA in 29 of 30 isolates and as a 2,300-base-pair fragment in 1 of the isolates. The probe correctly identified all salmonellae (nine isolates) among 47 H2S-producing colonies tested from among 250 clinical specimens cultured on xylose-lysine-desoxycholate medium. Salmonellae grown on xylose-lysine-desoxycholate medium gave consistently higher hybridization values than did those grown on either MacConkey or Hektoen enteric agar. In addition, of eight gram-negative broth enrichments in which salmonellae were identified by conventional means, seven were probe positive. The use of this nucleic acid probe and hybridization technique provides a simple and rapid identification of Salmonella species.

Bacteriological Techniques↗
PubMed1990

Detection of surgical pathogens by in vitro DNA amplification. Part I. Rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene.

The management of candidemia and disseminated candidiasis depends on rapid, unambiguous identification of Candida. Such identification is retarded by the slow growth of Candida from clinical specimens. Administration of effective but potentially toxic antifungal therapy is often withheld pending identification. To circumvent this slow growth and thus to expedite diagnosis and therapy, the polymerase chain reaction (PCR) was used to amplify a segment of fungal DNA coding for the cytochrome P450L1A1 (lanosterol-14 alpha-demethylase) in vitro. The technique provides unambiguous evidence of C. albicans in as few as 6 hours with a detection threshold of 10 organisms in a 100 mu specimen. Clinical specimens of urine (n = 4), sputum (n = 6), wound fluid (n = 1, and blood (n = 2) were collected from patients, and C. albicans was conventionally documented at these sites; in each case, PCR was confirmed. Of 17 additional specimens that were culture negative, PCR suggested the presence of yeast in two of the specimens. PCR-based detection of surgical pathogens may have broad application in rapid screening for the presence of organisms either indigenous to a particular surgical intensive care unit or peculiar to selected patient populations.

Candida albicans↗