Modified technique for efficient detection of microsporidia

Changes in temperature (from room temperature to 50 degrees C) and staining time (from 90 to 10 min) were evaluated as a means of improving the detection of microsporidia from stool specimens. A blinded and independent comparison of 50 known positive matched-specimen pairs by three technologists resulted in consistently easier microscopic detection. The background is clearer, and spores stain more intensely. Staining time is reduced by 80 min.     

Involuntary disclosure of private medical records to the defence in criminal proceedings

The diagnosis of microsporidiosis by staining stools is known to be fast and cheap. To obtain a specific and sensitive result, two colorimetric methods must be used: staining by the fluorochrome Uvitex 2 B (VAN GOOL) and trichrome. Among the four staining methods of trichrome currently studied, the WEBER coloration could be considered as the most efficient. The density of microsporidia spores could be semi-quantitatively evaluated, because their distribution is homogeneous.     

Chaotic atrial rhythm

PURPOSE: To describe a case of microsporidial keratoconjunctivitis in a patient without human immunodeficiency virus (HIV) infection. METHODS: Case report. An epithelial corneal scraping from a woman with chronic bilateral keratoconjunctivitis was evaluated by Giemsa stain. RESULTS: Giemsa stain of an epithelial corneal scraping disclosed intracellular and extracellular spores characteristic of microsporidia. An HIV enzyme-linked immunosorbent assay (ELISA) test was negative. The signs and symptoms of the bilateral keratoconjunctivitis resolved after treatment with albendazole. CONCLUSION: Microsporidia may cause a chronic epithelial keratoconjunctivitis in the absence of HIV infection.     

Immunologic and molecular characteristics of Encephalitozoon-like microsporidia isolated from humans and rabbits indicate that Encephalitozoon cuniculi is a zoonotic parasite

To assess the zoonotic potential of Encephalitozoon-like microsporidia, we isolated and cultivated spores from specimens of urine, respiratory secretions, and stool from six patients infected with human immunodeficiency virus and from nine rabbits. Because spores of Encephalitozoon-like species are indistinguishable by microscopy, we characterized the isolates by western blot analysis and by restriction enzyme analysis of the small subunit (SSU) rDNA after amplification by the polymerase chain reaction. We identified Septata intestinalis in one patient and Encephalitozoon hellem in two symptomatic patients. Encephalitozoon cuniculi was found in all rabbits and in three patients. One of these patients had clinical manifestations of infection with this parasite (severe interstitial pneumonitis). We observed abatement of symptoms and cessation of parasite excretion when these patients were treated with albendazole. Our findings suggest that E. cuniculi may be pathogenic in humans and that it is a zoonotic parasite.     

Blinded, externally controlled multicenter evaluation of light microscopy and PCR for detection of microsporidia in stool specimens. The Diagnostic Multicenter Study Group on Microsporidia

The quality parameters for the detection of microsporidia in identical sets of 50 stool samples were determined for six laboratories where technicians used light microscopy and for six laboratories where technicians used PCR. The average overall sensitivities were 67% (89% for patient samples only) for the PCR laboratories and 54% (80% for patient samples only) for the light microscopy laboratories. Specificities were 98 and 95%, respectively. Differences in results were most apparent between the individual laboratories rather than between the two major methods used.     

Prevalence and toxigenicity of Clostridium difficile isolates in fecal microflora of preterm infants in the intensive care nursery

Fecal isolates of Clostridium difficile and its toxin B were followed prospectively in 50 preterm intensive care nursery (ICN) patients. The first stool specimen was obtained after 1 week of enteral feeding, at 15 +/- 1 days of life, and 2 more specimens were collected at 2-week intervals, 24 +/- 1 and 32 +/- 2 days of life. The stools were cultured for C. difficile, and tested for C. difficile toxin B. In the first specimen 15% of stools grew C. difficile. In the second specimen C. difficile isolation rates increased to 33% and plateaued. Toxin B was detected in 71, 93 and 100% of culture-positive stools in the first, second, and third specimens, respectively. C. difficile colonization was not associated with a higher incidence of necrotizing enterocolitis or diarrhea, and using precollected, frozen human milk did not protect from C. difficile colonization.     

Diagnostic yield of duodenal biopsy and aspirate in AIDS-associated diarrhea

OBJECTIVES: To evaluate the diagnostic yield of performing duodenal biopsies and aspirates in AIDS patients with chronic diarrhea. METHODS: Retrospective review of esophagogastroduodenoscopy (EGD) records from January 1993 to March 1995 to identify those patients who underwent EGD for evaluation of AIDS associated diarrhea and had a duodenal biopsy and/or aspirate. Biopsies were examined for pathogens using routine histology and special stains, viral culture, and electron microscopy. Duodenal aspirates were evaluated for ova and parasites. All patients had previous negative stool studies. Pathology laboratory charges (hospital and professional fees) for each test and charges per positive test were determined. RESULTS: Of the 57 patients included in this study, 56 had a duodenal biopsy and 42 had a duodenal aspirate. An established pathogen was identified in only 15 (26%) patients. One patient had both Mycobacterium avium complex and microsporidia. Pathogens were identified in seven patients by hematoxylin and eosin stain, in three patients by acid-fast bacillus stain, and in six patients by electron microscopy. No pathogens were identified with Gomori's methenamine silver stain (44 patients), duodenal aspirate for ova and parasites (46 patients), immunoperoxidase stains (4 patients), or viral culture (4 patients). Cryptosporidia were identified in six, microsporidia in five, Mycobacterium avium complex in three, and Giardia lamblia and adenovirus each in one patient. CONCLUSIONS: In this series, the diagnostic yield of EGD with duodenal biopsy and aspirate in AIDS associated diarrhea was low. Pathogens were identified in 26% of patients; predominantly Cryptosporidium organisms and microsporidia. The routine performance of aspiration of duodenal contents for parasite examination and staining of duodenal tissue with Gomori's methenamine silver stain for fungal identification are not recommended. One should consider obtaining tissue for electron microscopy whenever duodenal biopsies are performed. The utility of EGD in AIDS associated diarrhea may improve as more effective therapies become available.     

Flow cytometric analysis of microsporidia belonging to the genus Encephalitozoon

Flow cytometry was used in the identification of human microsporidia belonging to the genus Encephalitozoon. Microsporidian spores of Encephalitozoon hellem, E. cuniculi, and E. intestinalis were propagated in axenic cultures of monkey kidney E6 cells, purified with Percoll, and exposed to homologous and heterologous rabbit antiserum and monoclonal antibody prepared against E. hellem spores. After reaction to goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG conjugated to fluorescein isothiocyanate, fluorescence histograms from gated data on light-scatter profiles showed that rabbit anti-E. hellem serum was reactive to E. hellem spores but also had cross-reactivity to spores of E. cuniculi and E. intestinalis. On the other hand, fluorescence histograms showed that rabbit anti-E. cuniculi and rabbit anti-E. intestinalis sera were reactive with homologous spores only. Monoclonal antibody prepared against E. hellem reacted only with spores of E. hellem. Neither the polyclonal antibodies nor the monoclonal antibodies reacted with Cryptosporidium parvum oocysts. Fluorescence histograms of spores treated with 10% formalin also showed reactivity, but the number of events in the most intense peaks of fluorescence was fewer (7 to 42%, depending on species) than the number of events in the most intense peaks of fluorescence for nontreated spores. By flow cytometry, formalin-treated and nontreated spores of Encephalitozoon were identified to the species level by using gated data on light-scatter profiles and analyzing the fluorescence histograms from the indirect immunofluorescence of the spores. Once a procedure is established for the isolation of Encephalitozoon spores from clinical specimens, identification of spores by flow cytometry may be useful not only for diagnosis but also for epidemiologic studies.     

Disseminated Encephalitozoon (Septata) intestinalis infection in a patient with AIDS: novel diagnostic approaches and autopsy-confirmed parasitological cure following treatment with albendazole

Encephalitozoon intestinalis is a recently described microsporidian which causes intestinal and disseminated infections in severely immunocompromised patients with AIDS. Preliminary data suggest that albendazole can be an effective therapy for patients with E. intestinalis infection. However, relapses have been reported following treatment in some cases. These results were based upon examination of cytologic, biopsy, or stool samples with an inherent sampling bias. This report documents the first postmortem evaluation of a patient with E. intestinalis infection treated with albendazole. Antemortem microsporidial diagnosis was performed on nasal mucosal smear and duodenal biopsy specimens by electron microscopy and a newly developed indirect fluorescent-antibody method based upon in vitro cultivation of the organism. This case represents the initial report of using nasal cytologic specimens for ultrastructural and antibody-based species-level diagnosis of microsporidiosis. Following successful treatment of this infection with albendazole, the patient died of other causes. A thorough autopsy examination failed to reveal the presence of E. intestinalis in any tissue, providing confirmatory evidence for a complete parasitological cure with albendazole.     

A likelihood approach to meta-analysis with random effects

In a meta-analysis of a set of clinical trials, a crucial but problematic component is providing an estimate and confidence interval for the overall treatment effect theta. Since in the presence of heterogeneity a fixed effect approach yields an artificially narrow confidence interval for theta, the random effects method of DerSimonian and Laird, which incorporates a moment estimator of the between-trial components of variance sigma B2, has been advocated. With the additional distributional assumptions of normality, a confidence interval for theta may be obtained. However, this method does not provide a confidence interval for sigma B2, nor a confidence interval for theta which takes account of the fact that sigma B2 has to be estimated from the data. We show how a likelihood based method can be used to overcome these problems, and use profile likelihoods to construct likelihood based confidence intervals. This approach yields an appropriately widened confidence interval compared with the standard random effects method. Examples of application to a published meta-analysis and a multicentre clinical trial are discussed. It is concluded that likelihood based methods are preferred to the standard method in undertaking random effects meta-analysis when the value of sigma B2 has an important effect on the overall estimated treatment effect.     

The current risk of retroviral infections transmitted by transfusion in patients who have undergone multiple transfusions. Cooleycare Cooperative Group

BACKGROUND: To conduct a multicenter, prospective survey within the program of the Cooleycare Cooperative Group to evaluate the rate of transfusion-transmitted infections with human immunodeficiency virus (HIV) and human T-lymphotropic virus (HTLV) in a cohort of patients who were homozygous for beta thalassemia and underwent multiple transfusions during the 6-year follow-up. PATIENTS AND METHODS: One thousand three hundred eighty-four patients with beta thalassemia from 36 centers were enrolled from December 1989 to March 1990. Serum samples were tested at regular intervals during the period from December 1989 to March 1996 for anti-HIV and anti-HTLV antibodies in 1 laboratory. Samples from 1073 and 1001 of the 1384 patients were available for evaluation also during the periods from December 1992 to March 1993 and December 1995 to March 1996, respectively. The risk of acquiring infection was calculated by the ratio between the number of patients who experienced seroconversion and the number of red blood cell units administered to the patients during the study period. RESULTS: The prevalence of HIV infection found in the period from December 1989 to March 1990 was 2.9% (40 of 1384 patients). During follow-up, 1 of 1001 patients showed anti-HIV seroconversion. The incidence of HIV infection was 1.7 per 10,000 person-years (upper boundary of the 95% confidence interval, 5 per 10,000). The risk of HIV infection was 1 in 190,000 U (upper boundary of the 95% confidence interval, 1 in 67,000). At baseline, 4 patients were infected with HTLV (3 with HTLV-1 and 1 with HTLV-2). No seroconversions were observed during follow-up; the risk of HTLV infection was less than 1 in 190,000 U. CONCLUSION: The application of reliable screening procedures for donor selection reduced the transmission of transfusion-associated HIV infection in 1989-1995 to fewer than 2 cases in 10,000 person-years or 1 case per 190,000 units of red blood cells.     

Immunosuppression may lead to progression of hepatitis C virus-associated liver disease in hemophiliacs coinfected with HIV

OBJECTIVE: The aim of this study was to examine the interaction between HIV and hepatitis C virus (HCV) in hemophiliacs coinfected with the viruses and to investigate the possible relationship between immunosuppression and liver failure. METHODS: To identify risk factors for impending liver failure in hemophiliacs coinfected with HIV and HCV, we analyzed clinical and laboratory parameters, including CD4 count, aminotransferases (ALT, AST), cholinesterase, alkaline phosphatase, bilirubin, and gamma-glutamyltransferase, during 3 yr of follow-up (1990-1993) in four groups of patients: hemophiliacs with progressive immunodeficiency who were coinfected with HCV and HIV (group A, n = 49); hemophiliacs with stable immune function who were seropositive for HIV and HCV (group B, n = 95); hemophiliacs who were infected with HCV but not HIV (group C, n = 72); and homosexuals with progressive immunodeficiency who were infected with HIV but not HCV (group D, n = 24). RESULTS: Univariate analysis of data for group A showed a significant rise in gamma-glutamyltransferase and alkaline phosphatase (p < 0.01) that was not seen in groups B, C, and D. In a multivariate Cox regression analysis, age (odds ratio, 1.054 per yr; 95% confidence interval, 1.014-1.096 per yr), decline in CD4 count (odds ratio, 1.063 per cell/microl; 95% confidence interval, 1.037-1.091 per cell/microl), and alkaline phosphatase level (odds ratio, 1.012 per U/L; 95% confidence interval, 1.002-1.021 per U/L) emerged as independent determinants of death. CONCLUSIONS: Our data suggest that progressive immune dysfunction in hemophiliacs coinfected with HIV and HCV may influence progression of liver failure. In these patients cholestasis is an additional prognostic marker for survival that may reflect both exhausted immunity and impaired liver function.     

Justification for use of a single trichrome stain as the sole means for routine detection of intestinal parasites in concentrated stool specimens

Of 12,321 stool samples analyzed over a 6-year interval, 870 (7.1%) were positive for a total of 1,019 parasites, of which 1,011 (99.2%) were found in trichrome-stained smears of unconcentrated specimens while only 479 (47.0%) were detected in iodine-stained smears of concentrated samples. Stool specimens were next analyzed by trichrome staining of both unconcentrated and concentrated specimens preserved in either mercury-polyvinyl alcohol (PVA) or cupric PVA. Of 2,198 specimens, 171 (7.8%) were positive for a total of 208 parasites, 192 (92.3%) and 204 (98.1%) of which were found in the unconcentrated and concentrated specimens, respectively (P < 0.05). In our patient population, examination of a single trichrome-stained smear of a concentrated stool specimen is a cost-effective alternative to routinely analyzing both concentrated and unconcentrated specimens for parasites.     

Small volume bronchoalveolar lavage used in diagnosing Pneumocystis carinii pneumonia in HIV-infected patients

To determine the volume of bronchoalveolar lavage (BAL) fluid necessary to diagnose Pneumocystis carinii pneumonia (PCP) in immunocompromised patients, specimens from 25 patients were evaluated. Twenty-one patients were HIV infected. BAL was performed using three to four 60-mL aliquots of room temperature, sterile, saline solution. Each syringe of BAL effluent was numbered and its volume was measured. Immunofluorescent stains were performed on about 8-mL aliquots of the initial, final, and aggregate BAL specimens, and a modified Giemsa stain was also performed on a 0.4-mL aliquot of the aggregate specimen. Of 25 patients, Pneumocystis carinii organisms were identified in 9 with HIV infection, in whom all BAL specimens were positive with both immunofluorescence and Giemsa stains. In 16 patients, BAL specimens were negative for P carinii on both immunofluorescent and modified Giemsa testing. Both staining methods were 100% specific (95% confidence interval [CI], 83 to 100%) and 100% sensitive (95% CI, 72 to 100%). The volume of BAL effluent in the initial specimens positive for P carinii ranged from 15 to 25 mL. We conclude that in this small group of patients, PCP was accurately diagnosed from a single 60-mL BAL specimen stained with immunofluorescence methods.     

Accuracy of estimated creatinine clearance in obese patients with stable renal function in the intensive care unit

We compared agreement between creatinine clearance values in obese, critically ill patients calculated using three common empirically derived formulas and modifications thereof, with creatinine clearance obtained by conventional 24-hour urine collection. We selected the charts of 22 patients in intensive care units (86% medical, 14% surgical) according to the following criteria: actual body weight greater than 150% of ideal body weight; serum creatinine variation of less than 15% from the day of starting 24-hour urine collection to the day before or after the collection; presence of a urinary bladder catheter; no history of renal dialysis; and clinical indication for renal function assessment. Mean measured 24-hour urinary creatinine clearance for all patients was 72 +/- 64 ml/minute (range 8-248 ml/min). The method of estimating creatinine clearance that showed the least mean bias was the equation of Salazar and Corcoran using a corrected serum creatinine concentration (mean bias -2 ml/min); however, the corresponding 95% confidence intervals were wide (-133-129 ml/min). The narrowest range of 95% confidence intervals were seen with Jelliffe's equation (mean bias 25 ml/min, 95% confidence intervals -41-90 ml/min). In this sample, estimated creatinine clearances did not agree acceptably with measured values. Despite low mean bias values, none of the empirically derived equations that we studied had clinically acceptable 95% confidence intervals. We recommend using the 24-hour urine collection method when assessing creatinine clearance in obese, critically ill patients.     

Synthesis of a potent wide-spectrum serotonin-, norepinephrine-, dopamine-reuptake inhibitor (SNDRI) and a species-selective dopamine-reuptake inhibitor based on the gamma-amino alcohol functional group

Mortality data for B6CF1 mice exposed to 60Co gamma rays for the duration of life were used to make quantitative predictions of age-specific mortality observed in comparably exposed beagles. Simple Kaplan-Meier survival curves for the beagles and their 95% confidence intervals were computed for each dose-rate group observed. A dose-response equation was estimated from the mortality data for mice using a proportional hazard model. The dose-response model for mice was then used to generate predicted survivorship curves at dose rates that would recreate the dose burdens observed in the beagle at comparable points within the life span of the two organisms. When these predicted survivorship curves were scaled to adjust for species differences in the life span of control animals, the predictions for the mouse fell within the confidence intervals observed for the beagle. The successful interspecies extrapolation of age-specific mortality risks for species as different as the mouse and dog enhances both the value of studies involving laboratory animals and the potential relevance of the animal studies to the prediction of health effects in humans.     

Adolescent drinking behaviour and the role of family life: a Scottish perspective

We report on a simplified method of cytomorphological in vitro confirmation of newly established lung cancer cell lines by using multicellular tumor spheroids (MTS) and flow cytometry (FCM). Eleven cell lines were established from 11 patients with lung cancer. The MTS were produced by culturing cells in agar-coated dish. Cytomorphological studies were made using smears of crushed MTS and frozen sections of MTS. The MTS were fixed doubly with paraformaldehyde and osmic acid for scanning and transmission electron microscopy. Bivariate fluorescence of fluorescein isothyocyanate (FITC, tumor associated antigen, TAA) and propidium iodide (DNA) were measured by FCM. The MTS grew anchorage-independently. Cytopathological and electron microscopic findings of MTS were similar to those of the original clinical specimens. The DNA index and TAA were useful in evaluating the presence or absence of contamination by cells of non-tumor origin. The new cell lines satisfied a minimum of four conditions to confirm their establishment: (a) they originated from humans, (b) they were cytomorphologically identified with specimens from primary lesions, (c) they showed tumorigenicity, and (d) they were free from contamination by cells of different origin. From these findings, the establishment of new cell lines can be confirmed in vitro by using MTS and FCM.     

Estimating confidence intervals for cost-effectiveness ratios: an example from a randomized trial

Cost-effectiveness ratios usually appear as point estimates without confidence intervals, since the numerator and denominator are both stochastic and one cannot estimate the variance of the estimator exactly. The recent literature, however, stresses the importance of presenting confidence intervals for cost-effectiveness ratios in the analysis of health care programmes. This paper compares the use of several methods to obtain confidence intervals for the cost-effectiveness of a randomized intervention to increase the use of Medicaid's Early and Periodic Screening, Diagnosis and Treatment (EPSDT) programme. Comparisons of the intervals show that methods that account for skewness in the distribution of the ratio estimator may be substantially preferable in practice to methods that assume the cost-effectiveness ratio estimator is normally distributed. We show that non-parametric bootstrap methods that are mathematically less complex but computationally more rigorous result in confidence intervals that are similar to the intervals from a parametric method that adjusts for skewness in the distribution of the ratio. The analyses also show that the modest sample sizes needed to detect statistically significant effects in a randomized trial may result in confidence intervals for estimates of cost-effectiveness that are much wider than the boundaries obtained from deterministic sensitivity analyses.     

A score method of constructing asymmetric confidence intervals for the mean of a rating scale item.

This article presents a generalization of the Score method of constructing confidence intervals for the population proportion (E. B. Wilson, 1927) to the case of the population mean of a rating scale item. A simulation study was conducted to assess the properties of the Score confidence interval in relation to the traditional Wald (A. Wald, 1943) confidence interval under a variety of conditions, including sample size, number of response options, extremeness of the population mean, and kurtosis of the response distribution. The results of the simulation study indicated that the Score interval usually outperformed the Wald interval, suggesting that the Score interval is a viable method of constructing confidence intervals for the population mean of a rating scale item. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A comparison of hemodynamic parameters derived from transthoracic electrical bioimpedance with those parameters obtained by thermodilution and ventricular angiography

OBJECTIVE: To determine the limits of agreement between the cardiac output and volumetric data estimated by impedance cardiography with the cardiac output determined by thermodilution and the left ventricular ejection fraction and end-diastolic volume estimated from left ventriculography. DESIGN: A prospective study. SETTING: The cardiac catheterization laboratory of a university-affiliated teaching hospital. PATIENTS: Twenty-four patients with coronary artery disease undergoing elective left- and right heart catheterization. INTERVENTIONS: Cardiac output was measured by the thermodilution method and the ejection fraction and left ventricular volumetric data were determined by ventriculography. These same measurements were obtained by simultaneously performed impedance cardiography using a commercially available bioimpedance device. MEASUREMENTS AND MAIN RESULTS: The patients' mean cardiac output was 4.6 +/- 1.7 L/min by bioimpedance and 5.0 +/- 1.1 L/min by thermodilution. The limits of agreement between the two methods was -4.1 to 3.5 L/min. The 95% confidence intervals for the lower and upper limits of agreement were -2.7 to -5.5 L/min and 2.1 to 4.9 L/min, respectively. The mean ejection fraction was 63 +/- 8% by bioimpedance and 53 +/- 15% by ventriculography. The limits of agreement between the ejection fraction estimated by bioimpedance and ventriculography was -35% to 37%. The 95% confidence intervals for the lower and upper limits of agreement were -22% to -48% and 24% to 50%, respectively. The mean left ventricular end-diastolic volume was 108 +/- 47 mL, as estimated by bioimpedance, and 121 +/- 35 mL, as estimated by ventriculography. The limits of agreement between the left ventricular end-diastolic volume as estimated by bioimpedance and ventriculography was -139 to 113 mL. The 95% confidence intervals for the lower and upper limits of agreement were -184 to -94 mL and 68 to 158 mL, respectively. CONCLUSIONS: The 95% confidence range defining the limits of agreement between cardiac output and volumetric data estimated by bioimpedance, with the cardiac output measurement by thermodilution and the volumetric data estimated from left ventriculography, were wide, making the degree of agreement clinically unacceptable. In the opinion of the authors, impedance cardiography should not replace invasive hemodynamic monitoring at this time.