Role of fecal Clostridium difficile load in discrepancies between toxin tests and PCR: is quantitation the next step in C. difficile testing?

Direct tests for Clostridium difficile are 30-50 % more sensitive than tests for C. difficile toxins but the reasons for this discrepancy are incompletely understood. In addition to toxin degradation and strain differences, we hypothesized that C. difficile concentration could be important in determining whether toxins are detected in fecal samples. We performed standard curves on an FDA-approved real-time PCR test for the C. difficile tcdB gene (Xpert C. difficile/Epi, Cepheid) during a prospective comparison of a toxin immunoassay (Meridian Premier), PCR and toxigenic culture. Immunoassay-negative, PCR-positive samples were retested with a cell cytotoxin assay (TechLab). Among 107 PCR-positive samples, 46 (43.0 %) had toxins detected by immunoassay and an additional 18 (16.8 %) had toxin detected by the cytotoxin assay yielding 64 (59.8 %) toxin-positive and 43 (40.2 %) toxin-negative samples. Overall, toxin-negative samples with C. difficile had 10(1)-10(4) fewer DNA copies than toxin-positive samples and most discrepancies between toxin tests and PCR were associated with a significant difference in C. difficile quantity. Of the toxin-positive samples, 95 % had ≥ 4.1 log(10) C. difficile tcdB DNA copies/mL; 52 % of immunoassay-negative samples and 70 % of immunoassay and cytotoxin negative samples had <4.1 log(10) C. difficile tcdB DNA copies/mL. These findings suggest that fecal C. difficile concentration is a major determinant of toxin detection and C. difficile quantitation may add to the diagnostic value of existing test methods. Future studies are needed to validate the utility of quantitation and determine the significance of low concentrations of C. difficile in the absence of detectable toxin.

Decreases in unintegrated HIV DNA are associated with antiretroviral therapy in AIDS patients.

Better markers are needed to monitor the efficacy of antiretroviral drugs in persons infected with human immunodeficiency virus (HIV). We investigated the effects of zidovudine (ZDV) and dideoxycytidine (ddC) on the presence of unintegrated HIV-1 DNA in peripheral blood mononuclear cells (PBMCs) from AIDS patients. DNA was extracted from PBMCs and separated into low molecular weight (unintegrated) and high molecular weight (integrated) chromosomal fractions. These DNA fractions were then amplified by a quantitative polymerase chain reaction (PCR) and the amount and percentage of unintegrated HIV DNA were determined. Very high levels of unintegrated HIV DNA were found in AIDS patients not receiving treatment with ZDV or ddC (median = 95% unintegrated HIV DNA). In contrast, most patients who had received 4 or more weeks of antiretroviral therapy had lower levels of unintegrated HIV DNA (median = 30% unintegrated HIV DNA for patients receiving ZDV). Paired samples taken from five patients before and after therapy showed a striking reduction in the percentage of unintegrated HIV DNA. The decrease in the proportion of unintegrated HIV DNA in AIDS patients was due to both a reduction in the copy number of unintegrated HIV DNA and an increase in the copy number of integrated HIV DNA. Thus, measurements of unintegrated and integrated HIV DNA may be useful in providing objective assessments of the effectiveness of antiretroviral therapies.

A seven-year experience with methicillin-resistant Staphylococcus aureus.

From July 1983 through June 1990, 319 patients with methicillin-resistant Staphylococcus aureus (MRSA) were identified at the University of California, Davis Medical Center. Initially, our goal was eradication of MRSA from the hospital flora. Our approach was: (a) immediate notification of all MRSA isolates by the microbiology laboratory; (b) strict isolation; (c) cohorting; (d) bathing patients with an iodophor; (e) surveillance cultures of patients and staff; (f) treatment of all colonized persons; and (g) strict isolation on readmission. Control of spread was achieved but eradication was not. In 1987 our strategy was modified in order to establish the least restrictive methods to maintain control of the spread of MRSA. After notification by the microbiology laboratory, we now require: (a) contact isolation; (b) surveillance cultures of patients associated with each new case; and (c) contact isolation for all MRSA patients on readmission. Strict isolation and employee culturing are used only during major outbreaks. We have averaged four new cases of MRSA per month over the 7-year period, including four major outbreaks. Since 1987, we have averaged only three new cases per month with one major MRSA outbreak. Annual cost savings of greater than $50,000 have been realized through the policy modifications. We conclude that the use of contact isolation with some modifications has saved time and money and has successfully controlled the spread of MRSA in our university hospital.

Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction.

We developed a new method to amplify cell DNA in situ using the polymerase chain reaction (PCR). Proviral sequences of mouse mammary tumor virus (MMTV) contained in cultured cells and tissue sections were amplified intracellularly using a thermal cycler. Two techniques were employed to maintain the localization of the amplified DNA. First, complementary tails at the 5' ends of the oligonucleotide primers resulted in the synthesis of high molecular weight concatamers containing the target sequences. Second, the PCR was carried out in a thin film of agarose solidified over the tissue sections. The specifically amplified and localized DNA was then detected by in situ hybridization (ISH). Our results demonstrate that (a) DNA in tissue sections can serve as the target for the polymerase chain reaction in situ, (b) cell morphology is maintained, and (c) a target of 167 BP can be specifically detected in individual cells. This technique should be generally applicable to amplifying cellular DNA targets in tissue sections for detection in situ.

In situ detection of human immunodeficiency virus (HIV) nucleic acid in H9 cells using nonradioactive DNA probes and an image cytophotometry system.

Rapid and sensitive nonradioactive methods to detect human immunodeficiency virus (HIV)-infected cells are needed in clinical medicine. We developed an in situ hybridization test using 2-acetylaminofluorene (AAF)-labeled HIV DNA as a hybridization probe. Hybridized probe was detected using rabbit anti-AAF antibody, followed by alkaline phosphatase-conjugated goat anti-rabbit, and the bromochloroindolyl phosphate-nitroblue tetrazolium reaction. An image cytophotometry system was used to quantitate the percentage of HIV-infected cells. These methods were used to determine the percentage of H9 cells infected with HIV. HIV was detected in 0% of cells on day 1 post infection, 7% on day 4, 41% on day 8, and 5% on day 15. These results paralleled those of the reverse transcriptase assay and an antigen capture ELISA assay for HIV antigen. Thus the AAF modified HIV DNA probe detected HIV nucleic acid in infected H9 cells and the image cytophotometry system improved the sensitivity and objectivity of detection.

Sarcoidosis in hypersensitivity pneumonitis.

An abnormal chest x-ray film showing hilar adenopathy, diffuse interstitial pulmonary infiltrations, or both, combined with a tissue biopsy revealing noncaseating granuloma, are suggestive of sarcoidosis; however, non-caseating granuloma may also be found in other forms of pulmonary disease. Immunologic and environmental evaluation of three patients with the diagnosis of sarcoidosis made by the above criteria, revealed hypersensitivity pneumonitis in all. Since therapeutic considerations in these two diseases are different (avoidance being the mainstay in hypersensitivity pneumonitis), all methods to ensure a correct diagnosis should be employed.

Molecular typing methods for the epidemiological identification of Clostridium difficile strains.

Toxigenic Clostridium difficile is the etiologic agent of C. difficile-associated diarrhea (CDAD), the most common cause of nosocomial diarrhea. Cross-infection between patients and transmission through the environment and medical personnel are important factors in the acquisition of CDAD. In order to understand differences in epidemiology and pathogenesis, a number of typing schemes have been developed. We will review the typing methods used to study the epidemiology of C. difficile infections and how they have evolved from a phenotypic identification to state of the art molecular methods, detecting genetic polymorphisms among strains. These molecular methods include PCR-based methods (arbitrarily primed-PCR [AP-PCR] and PCR ribotyping), restriction endonuclease analysis (REA) and pulse field gel electrophoresis (PFGE). The application, usefulness and feasibility of these methods are compared and discussed. Finally, the role of genomics as a tool to investigate CDAD is introduced.

Risk factors associated with isolation of Stenotrophomonas (Xanthomonas) maltophilia in clinical specimens.

OBJECTIVE: To determine risk factors for patients whose cultures grew Stenotrophomonas maltophilia. DESIGN: Retrospective case-control study of 60 patients with cultures positive for S maltophilia, matched by specimen site to 120 controls whose cultures grew other gram-negative aerobic bacteria. SETTING: University medical center. RESULTS: S maltophilia was identified from the following sites: respiratory (36), wound (13), urinary (6), blood (4), and cerebral spinal fluid (1). By univariate analysis, cases had a higher risk of exposure than controls for ampicillin (P < .001), gentamicin (P < .001), vancomycin (P = .001), metronidazole (P = .003), piperacillin (P = .007), cefotaxime (P = .014), ceftazidime (P = .017), ciprofloxacin (P = .030), tobramycin (P = .040), and chronic respiratory disease (P = .024). Length of time foreign objects were in place prior to positive culture differed significantly between cases and controls only for endotracheal tubes in patients with respiratory isolates (median number of days: 12.5 for cases, 5 for controls; P = .007). For patients with urinary tract infections, having a urinary catheter increased the odds of infection 10 times over controls. Exposures found by multivariate analysis to be significantly more prevalent in cases than controls included ampicillin, cefotaxime, erythromycin, gentamicin, metronidazole, piperacillin, tobramycin, chronic respiratory disease, and female gender. Odds ratios were > 1 indicating higher risk for cases, except for erythromycin, which had an odds ratio < 1. CONCLUSIONS: The primary risk factor associated with isolation of S maltophilia was antibiotic use. For patients with pulmonary infections, chronic respiratory disease and length of time an endotracheal tube was in place also contributed to the risk. This suggests that judicious use of antibiotics may prevent some cases of S maltophilia infection.

Multilumen catheter sepsis and an educational program to combat it.

Catheter-related sepsis is a problem with many variables. A process of elimination may eventually identify the actual cause(s) of this phenomenon. We began our problem-solving approach by observing personnel inserting and caring for central lines, which showed a lack of compliance with existing protocols. A program was designed to provide the correct procedural activities. This study plans to test whether ongoing educational programs and an infection control department that maintains a high degree of visibility are effective in reducing nosocomial line-related infections.

Ovine pulmonary transit of tetracycline and minocycline.

Following cannulation of the right external jugular vein and the efferent duct of the right caudal mediastinal lymph node (the caudal end of this node was ligated to cut off the inflow of systemic lymph, i.e., 90%-95% of the efferent lymph was of pulmonary origin), sheep were given either tetracycline or minocycline as single doses of 5 mg/kg body weight infused intravenously over 30 min. Venous blood plasma and pulmonary lymph collected contemporaneously before infusion and from 5 min to 24 hr postinfusion were assayed by a well-agar diffusion method using Bacillus cereus. Peak concentrations of both drugs were observed in both plasma and lymph at 5 min postinfusion. Tetracycline penetrated into the lymph better than minocycline (percent penetration 67.3% of cf. 38.2%). The concentration of tetracycline was significantly higher in lymph during and 5 min postinfusion (p less than 0.01), a factor that may be of importance when selecting a tetracycline for the treatment of a pulmonary infection.

Isolation of Clostridium innocuum from cases of recurrent diarrhea in patients with prior Clostridium difficile associated diarrhea.

Clostridium innocuum isolates resistant to vancomycin (MIC values of 16-24 microg/mL) were isolated from three patients with recurrent Clostridium difficile -associated diarrhea (CDAD). We discuss the clinical significance and problems associated with the identification and differentiation of these two clostridial species, which may result in misdiagnosis of patients.

Genotyping of Bacteroides fragilis isolates from stool specimens by arbitrarily-primed-PCR.

In order to determine genetic relatedness of Bacteroides fragilis isolates from different clinical sources, arbitrarily primed polymerase chain reaction (PCR) (AP-PCR) was used to compare 17 strains isolated from patients with inflammatory bowel disease (IBD) and 20 strains isolated from foals with diarrhea. Three reference ATCC strains were also analyzed. Eighteen unique types were identified with a 22-mer arbitrary primer (ERIC-2) among the 20 patient isolates. Types 1 (enterotoxigenic) and 9 (nonenterotoxigenic), were each found in the stools of two patients. All other isolates showed a distinct and unique DNA banding pattern indicating a high degree of genotypic variability. Eleven types were identified among the foal isolates. Type 20, a nonenterotoxigenic type, was present in 30% of the foals. No correlation was found between the human and horse isolates. No clear relationship between a disease state (diarrhea or IBD) and specific types was observed. AP-PCR will be useful as a rapid method to determine genetic relatedness and in future epidemiologic studies of diarrheal diseases due to B. fragilis.

Antecedent use of fluoroquinolones is associated with resistance to moxifloxacin in Clostridium difficile.

OBJECTIVE: Moxifloxacin is characterized by high activity against Gram-positive cocci and some Gram-positive and -negative anaerobes, including Clostridium difficile. This study investigates the role of prior quinolone use in relation to patterns of susceptibility of C. difficile to moxifloxacin. METHODS: Sixty-three clinical isolates of C. difficile were investigated for toxigenicity, susceptibility to moxifloxacin, and mutations in the DNA gyrase gene. The medical histories for 50 of these patients were available and used to identify previous fluoroquinolone use. RESULTS: Thirty-three (52.4%) strains showed resistance to moxifloxacin (MICs > or = 16 mg/L). All moxifloxacin-resistant strains harbored a mutation at amino acid codon Ser-83 of gyrA. Forty-five isolates (71.4%) were toxigenic; all moxifloxacin-resistant strains were in this group. Resistance to moxifloxacin was associated with prior use of fluoroquinolones (P-value 0.009, chi-square). CONCLUSIONS: Although the use of moxifloxacin to treat C. difficile-associated diarrhea is not likely to be common, these data show a relationship between antecedent fluoroquinolone use and resistance to moxifloxacin in C. difficile isolates, and raise questions regarding selection pressure for resistance placed on colonizing bacteria exposed to fluoroquinolones. Mutations in gyrA are involved in moxifloxacin resistance.

Combined H1 and H2 antihistamine therapy in chronic urticaria.

Chronic urticaria is a frustrating problem for the patient and the physician. The cause is usually undetermined, and the therapy is directed toward controlling symptoms. Recent evidence that human skin blood vessels possess H2 receptors, as well as the commonly recognized H1 receptors, suggests a possible reason for the frequent failure of H1 antihistamines in controlling this disorder. Eighteen patients with refractory chronic idiopathic urticaria participated in a double-blind, cross-over study to evaluate the efficacy of combined H1 (hydroxyzine hydrochloride) and H2 (cimetidine) antihistamines vs H1 antihistamines alone. This study indicates that combined H1 and H2 antihistamine therapy is statistically more effective than H1 antihistamines alone in controlling the symptoms of chronic urticaria.

The emergency department presentation of patients with active pulmonary tuberculosis.

OBJECTIVE: To determine the clinical presentation of emergency department (ED) patients with active pulmonary tuberculosis (TB). METHODS: This was a retrospective medical record review of adult patients, identified through infection control records, diagnosed as having active pulmonary TB by sputum culture over a 30-month period at an urban teaching hospital. The ED visits by these patients from one year before to one year after the initial positive sputum culture were categorized as contagious or noncontagious, using defined clinical and radiographic criteria. The medical records of patients with contagious visits to the ED were reviewed to determine chief complaint, presence of TB risk factors and symptoms, and physical examination and chest radiograph findings. RESULTS: During the study period, 44 patients with active pulmonary TB made 66 contagious ED visits. Multiple contagious ED visits were made by 12 patients (27%; 95% CI = 15% to 43%). Chief complaints were pulmonary 33% (95% CI = 22% to 46%), medical but nonpulmonary 41% (95% CI = 29% to 54%), infectious but nonpulmonary 14% (95% CI = 6% to 24%), and traumatic/orthopedic 12% (95% CI = 5% to 22%). At least one TB risk factor was identified in 57 (86%; 95% CI% = 76 to 94%) patient visits and at least one TB symptom in 51 (77%; 95% CI = 65% to 87%) patient visits. Cough was present during only 64% (95% CI = 51% to 75%) of the patient visits and hemoptysis during 8% (95% CI = 3% to 17%). Risk factors and symptoms that, if present, were likely to be detected at triage were foreign birth, homelessness, HIV positivity, hemoptysis, and chest pain. CONCLUSIONS: Patients with active pulmonary TB may have multiple ED visits, and often have nonpulmonary complaints. Tuberculosis risk factors and symptoms are usually present in these patients but often missed at ED triage. The diversity of clinical presentations among ED patients with pulmonary TB will likely make it difficult to develop and implement high-yield triage screening criteria.

Microcratering within the lunar regolith--a theory and observation.

Since the Apollo 11 mission to the moon, there has been substantial analysis of the lunar rocks and soil grains, utilizing more recent advances in electron probe technologies. It is the objective of this research to revisit the theories concerning the microcratering within the lunar regolith. Recent theories have included the idea that the microcratering phenomenon was caused by meteoric impacting onto the lunar surface during early lunar history. Other theories have suggested that the microcratering was a result of secondary ejector associated with micrometeoric and meteoric impact. This research team suggests that microcratering may have been associated with primordial dust during and before the formation of our solar system.

Character and frequency of discomfort immediately following restorative procedures.

Within 24 hours after dental treatment, discomfort was experienced by 78% of the patients questioned. Sensitivity to cold was the most frequent type of discomfort experienced (reported by 50% of the patients). Postoperative discomfort resulting from cold stimulus was most often mild (in 78%), occasionally moderate (in 22%), and never severe. The duration of postoperative discomfort resulting from cold stimulus was most often fleeting (reported by 94%) and seldom prolonged (reported by 6%). The second most frequent type of postoperative discomfort reported was associated with the administration of the local anesthetic. Sensitivity to sweets does not seem to be a source of postoperative discomfort following routine operative dental procedures.

Experimental evaluation of public policy: the case of state legislation for child passenger safety.

Observations of children in automobiles were made in seven states before and after implementation of legislation requiring use of child passenger safety devices. Increases in safe seating for children covered by state laws and children under 1 year old were observed in three of the five states implementing legislation during this study. Decreases in safe seating for these age groups were observed in two states, however. Increases in safe seating for children from 1 to 5 years old were observed in four of these five states. Although methodological limitations require cautious interpretation, these data suggest the impact child safety seat laws may have on compliance. Implications of this research for policies on child passenger safety and the importance of exploiting naturally occurring public experiments are discussed.