Chlorine inactivation of spores of Encephalitozoon spp.

This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of Encephalitozoon intestinalis and to investigate the effect of chlorine on two other species, E cuniculi and E. hellem, associated with human infection. The 50% tissue culture infective doses of these three species were also determined. On the basis of the results obtained, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic environment.

Alteration of anal sphincter morphology following vaginal delivery revealed by multiplanar anal endosonography.

OBJECTIVE: To assess morphologic change in the anal sphincters in the absence of endosonographic evidence of trauma after vaginal delivery. DESIGN: Prospective observational study. SETTING: District general hospital. POPULATION: Consecutively booked nulliparous pregnant women attending antenatal clinic. METHODS: All women were examined using three-dimensional anal endosonography, simple manometry and had questionnaire assessment of incontinence before and after delivery. MAIN OUTCOME MEASURES: Components of the anal canal were measured in the axial, sagittal and coronal planes and paired pre- and post-delivery examinations were compared. Any changes were related to changes in continence and anal canal manometry. RESULTS: Twenty-two women had a vaginal delivery and no endosonographic evidence of perineal trauma after delivery. After delivery, there was significant shortening of the length of the anterior external anal sphincter [EAS] (mean 21.7 vs 20.5 mm, P = 0.02) when measured in the sagittal plane, which increased in anterior angulation with respect to the axis of the anal canal (10 degrees vs 13.8 degrees, P = 0.03). In the axial plane, no change was seen in the thickness of any of the sphincter components after delivery. None of these morphologic changes correlated with changes in manometry or continence score. CONCLUSIONS: Anal sphincter morphology changes after an otherwise atraumatic vaginal delivery. This change does not correlate with any functional symptoms.

Assessment of portal venous system patency in the liver transplant candidate: a prospective study comparing ultrasound, microbubble-enhanced colour Doppler ultrasound, with arteriography and surgery.

AIM: To determine the role of microbubble-enhanced colour Doppler ultrasound (CDUS) in assessing portal venous patency prior to liver transplantation. MATERIALS AND METHODS: Over a 2-year period, all patients with chronic liver disease undergoing routine pre-transplant CDUS examination in whom the portal venous system was inadequately demonstrated were recruited to the study. CDUS was performed in 368 patients and 33 patients (9%) were recruited. A repeat CDUS examination following an intravenous bolus injection of the microbubble contrast agent Levovist (Schering Healthcare AG, Berlin, Germany) was performed. Diagnostic confidence was recorded on a free linear analogue scale for both examinations. Findings were compared with indirect portography and surgery. RESULTS: Of the 33 patients with sub-optimal baseline examinations, improvement in portal vein visualization was achieved in 31 patients (94%). Median diagnostic confidence increased from 50% (interquartile range 30-60) to 90% (interquartile range 75-98) (P < 0.001) following administration of Levovist. Overall accuracy of portal vein assessment using microbubble-enhanced CDUS in 15 patients in whom a definitive diagnosis was made within 2 months was 87%. CONCLUSION: Microbubble-enhanced CDUS is a simple, inexpensive adjunct to standard pre liver transplant screening of the portal vein. It is particularly helpful in patients with end-stage cirrhosis who are at high risk of portal vein thrombosis and in whom the conventional examination is sub-optimal.

Endosonographic anatomy of the normal anal canal compared with endocoil magnetic resonance imaging.

PURPOSE: This study was designed to clarify the sonographic anatomy of the normal anal canal by comparison with endoanal magnetic resonance imaging, to determine agreement between these imaging modalities and interobserver error in measuring layer thickness. METHODS: Three-dimensional endosonographic and endocoil magnetic resonance images of the anal canal were obtained in four males and five nulliparous females aged 22 to 34 years. Images were analyzed at similar levels throughout the canal using a graphics-overlay technique to compare sonographic with magnetic resonance images. Measurements were taken at one level for agreement analysis between modalities and for interobserver variability in the measurement of the thickness of the main anal canal layers. RESULTS: The muscularis submucosae ani, muscle bundles in the longitudinal muscle layer, and puboanalis were identified on sonography. The outer border of the external sphincter was demarcated by an interface reflection with ischioanal fat. Clarification of the external sphincter anatomy allowed excellent correlation (Ri = 0.96) for the assessment of thickness. There was excellent correlation for the interobserver measurement of the external and internal sphincters and the submucosal width on endosonography, but there was poor correlation for the longitudinal muscle (0.12). CONCLUSION: The overlay technique has improved endosonographic interpretation, and measurement of external sphincter thickness has been validated both by comparison with magnetic resonance and on interobserver agreement.

Hepatic artery pseudoaneurysms following liver transplantation: incidence, presenting features and management.

AIM: Hepatic artery pseudoaneurysm (PA) is an uncommon complication of liver transplantation. We report a series of 13 patients, the largest published review including outcome. The presenting features, risk factors and role of radiology in the diagnosis and management of this frequently fatal complication are discussed.

Survival and outcome after hepatic artery thrombosis complicating pediatric liver transplantation.

BACKGROUND/PURPOSE: Hepatic artery thrombosis (HAT) represents a significant cause of graft loss and mortality after pediatric orthotopic liver transplantation (OLT). The incidence and etiology of this complication have been investigated in detail but relatively little is known about outcome. METHODS: A review was conducted of all children with confirmed HAT complicating OLT during a 10-year period (1990 through 1999) in a single center. HAT was established by angiography or at operation in all cases. RESULTS: From a consecutive series of 400 pediatric OLTs, there were 31 (7.8%) instances of HAT in 29 children of median age 3.8 years (range, 8 days to 16 years). Twenty-four (83%) are alive after a median follow-up of 3.6 years. Fourteen cases occurred after transplantation of whole grafts and 17 after reduced or split livers. Of the 18 episodes resulting in retransplantation, there were 5 deaths and 2 second episodes of HAT; surviving children are alive with good graft function. Of the 13 episodes managed without retransplantation, 4 patients underwent attempted early revascularisation of the graft, which was successful in 2, and the remainder initially were treated conservatively. All 13 children are alive after a median follow-up of 4.1 years (range, 0.6 to 5.8), but 5 required radiologic or surgical intervention for biliary or septic complications; biochemical liver function is normal in 8, mildly abnormal in 3, and poor in 2. Retransplantation was less likely in those who had received reduced or split grafts (7 of 17) compared with those who had received whole grafts (11 of 14), but this difference just failed to reach statistical significance (chi(2) = 3.01, 0.1 > P > .05). CONCLUSIONS: Using a selective policy of retransplantation, revascularisation, and conservative treatment, 83% of children survived HAT complicating OLT. Approximately 40% of children with HAT survived without retransplantation. J Pediatr Surg 36:888-891.

Species-specific, nested PCR-restriction fragment length polymorphism detection of single Cryptosporidium parvum oocysts.

Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.

Multiplanar anal endosonography--normal anal canal anatomy.

INTRODUCTION: Volume acquisition during anal ultrasound enables multiplanar imaging of the anal canal. The normal ultrasonic multiplanar appearance of the anal canal is described and gender differences in canal anatomy are highlighted. METHODS: Ten male and 12 female normal volunteers (mean ages 31.5 years (s.d. 5.9) and 31.2 (s.d. 6.7)) had three-dimensional anal endosonography (3-D AES). Each volume dataset was seeded in the axial plane facilitating multiplanar identification of known anatomical structures. RESULTS: The anterior external anal sphincter (EAS) was significantly longer in men than women 30.1 mm (3.9) vs 16.9 mm (7.4) (P < 0.001). There was no difference in the length of the puborectalis 24.7 mm (6.4) vs 24 mm (5) (P=0.78) in men compared with women. The cranial extent of the anterior EAS was tilted forward in both sexes. The angle formed by the anterior EAS and the longitudinal axis of the anal canal was more acute in men than in women (11.1 degrees vs 18.6 degrees; P=0.007). Dataset volume seeding of familiar structures in the axial plane allowed the multiplanar endosonographic anatomy to be described. CONCLUSIONS: Multiplanar AES has enabled detailed longitudinal measurement of the components of the anal canal and has revealed important gender differences. The multiplanar ultrasonic appearance of the normal anal canal has been described for the first time.

Clinical use of Levovist, an ultrasound contrast agent, in the imaging of liver transplantation: assessment of the pre- and post-transplant patient.

Colour Doppler US is well established for imaging of hepatic vessels in the assessment of pre- and post-liver transplant patients. Unfortunately, a full colour Doppler US examination of the portal or hepatic venous and hepatic arterial systems is frequently precluded by technical factors. Ultrasound contrast agents are useful in enhancing vascular Doppler signal and play an important role in liver transplantation assessment. A series of patients with vascular problems illustrates the role of US contrast in the pre-transplant candidate, where portal vein patency and direction of flow is assessed, presence of portal vein thrombus is confirmed and cavernous transformation demonstrated. Occlusion of hepatic veins in Budd-Chiari syndrome is confidently confirmed. Following liver transplantation, US contrast allows a comprehensive assessment of hepatic artery thrombosis, hepatic artery stenosis and pseudoaneurysm formation. The need for further imaging is reduced or confidently deferred in many instances. Ultrasound contrast agents play an important role in the liver transplant candidate.

Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts.

Several in vitro surrogates have been developed as convenient, user-friendly alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts. Such viability assays have been used increasingly to determine oocyst inactivation following treatment with chemical, physical, or environmental stresses. Defining the relationship between in vitro viability assays and oocyst infectivity in susceptible hosts is critical for determining the significance of existing oocyst inactivation data for these in vitro assays and their suitability in future studies. In this study, four viability assays were compared with mouse infectivity assays, using neonatal CD-1 mice. Studies were conducted in the United States and United Kingdom using fresh (<1 month) or environmentally aged (3 months at 4 degrees C) oocysts, which were partially inactivated by ozonation before viability and/or infectivity analyses. High levels of variability were noted within and between the viability and infectivity assays in the U.S. and United Kingdom studies despite rigorous control over oocyst conditions and disinfection experiments. Based on the viability analysis of oocyst subsamples from each ozonation experiment, SYTO-59 assays demonstrated minimal change in oocyst viability, whereas 4',6'-diamidino-2-phenylindole-propidium iodide assays, in vitro excystation, and SYTO-9 assays showed a marginal reduction in oocyst viability. In contrast, the neonatal mouse infectivity assay demonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in vitro viability assays cannot be used to reliably predict oocyst inactivation following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a "gold standard" until suitable alternative viability surrogates are identified for disinfection studies.

Inter-laboratory comparison of the CD-1 neonatal mouse logistic dose-response model for Cryptosporidium parvum oocysts.

cryptosporidium parvum oocyst viability can be determined by vital dyes, in vitro excystation, and cell culture; however, neonatal mouse infectivity assays are the reference method. Unfortunately, there have been few efforts to standardize methods for infectivity assays thus casting a veil of uncertainty over the significance and comparability of results. In order to address this issue, two laboratories proficient in measuring oocyst infectivity conducted independent dose titration studies with neonatal CD-1 mice using standardized protocols and a well-characterized isolate of Cryptosporidium parvum. The resulting independent logistic dose-response models derived by regression analysis were compared with each other and with a published model. The comparisons showed these dose-response functions to be reproducible under standardized conditions. It is important to standardize mouse strain, age of mice at inoculation and necropsy, oocyst isolate, and age of oocysts. However, other factors, including methods used to detect infectivity and to count oocyst doses, appear less critical. Adopting a standardized assay for oocyst infectivity will provide both a basis for comparing data from various oocyst disinfection studies and a suitable platform for evaluating new or existing in vitro viability surrogates such as excystation, vital dyes or cell culture.

A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis.

The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID(50)) and a minimal infective dose (MID) for E. intestinalis. The TCID(50) is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID(50) have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25 degrees C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log(10) reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.

The effects of complex wrist and forearm posture on wrist range of motion.

Previous research on wrist functionally has focused almost entirely on range of motion (ROM) in 2 or 3 isolated planes (flexion/extension, radial/ulnar deviation, and forearm pronation/supination), without investigating the potential effects of complex wrist/forearm posture on ROM. A quantitative analysis of these effects on wrist ROM was performed. ROM was measured in one plane using both a manual method and an electrogoniometer while the participant maintained a fixed, secondary wrist and forearm posture. The study revealed that combinations of wrist/forearm postures have significant effects on wrist ROM; the largest effects are those of wrist flexion/extension on radial deviation ROM. The study also found that, consistent with previous research, wrist deviation measurements obtained with an electrogoniometer were significantly different from those obtained manually. Biomechanical theories for the results obtained are discussed. This research could be used to enhance ergonomic evaluation techniques by providing a more accurate risk assessment of certain complex wrist postures, particularly those in which wrist flexion/extension is combined with radial deviation.

Waterborne protozoan pathogens.

Protozoan parasites were the most frequently identified etiologic agents in waterborne disease outbreak from 1991 to 1994. The waterborne parasites Giardia lamblia, Naegleria fowleri, Acanthamoeba spp., Entamoeba histolytica, Cryptosporidium parvum, Cyclospora cayetanesis, Isospora belli, and the microsporidia are reviewed. For each parasite, the review includes history, life cycle, incidence, symptoms, and therapy. Clinical detection methods are compared, and emerging technologies are discussed. Information on the association of these parasites with waterborne outbreaks is reviewed. Current information on protozoan parasites identified as etiological agents in waterborne outbreaks is discussed. Water industry issues related to recent disease outbreaks are examined in the context of water quality testing regulations for G. lamblia and those proposed for C. parvum. The review identifies the limitations of the American Society of Testing and Materials water-testing method for these parasites. An overview of federal regulations affecting the water industry and laboratories that test for water quality is also provided. The article highlights the importance of the clinical laboratory as a frontline defense for the detection of infectious organisms. The review points to the need for clinical laboratories, physicians, and public health personnel to cooperatively plan and assess the challenge of meeting this potential public health threat.

Identification and healing of leg ulcers with impaired circulation.

Leg ulcers may fail to heal because inaccurate assessment has resulted in inappropriate treatment. This article suggests a more accurate method of assessment and describes the use of zinc oxide paste bandage to heal leg ulcers where there is impaired arterial circulation.

Biochemical and genetic characterization of Corynebacterium pseudotuberculosis.

Isolates (n = 94) of Corynebacterium pseudotuberculosis were obtained from sheep, goats, horses, and cattle from various parts of the world. The isolates were characterized biochemically and by restriction endonuclease analysis of DNA. We found near homogeneity in the ability of isolates to ferment carbohydrates and to produce urease. All isolates produced phospholipase D and catalase. The ability of isolates from horses to reduce nitrate, the inability of isolates from sheep and goats to do so, and the correlation of this characteristic with results of restriction endonuclease analyses confirmed the existence of 2 biovars of C pseudotuberculosis. We propose that these biovars be referred to as biovar equi for isolates that reduce nitrate and biovar ovis for isolates that fail to do so.

A modified direct phosphate assay for studying ATPases.

A simple, rapid assay for purified ATPases is presented, based upon the formation of phosphomolybdate and its extraction into butyl acetate. The inclusion of imidazole makes the assay more sensitive and reproducible apparently because of the formation of an imidazole-phosphomolybdate complex. Protein (100 micrograms), Hepes buffer [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (0.1 M) and nucleotides (1 mM) were all shown to cause interference. The interference by nucleotides could be counteracted by using more molybdate. Butyl acetate was shown to extract virtually all of the phosphomolybdate almost instantaneously upon vortex mixing.

Hypothesis--a chemical mechanism for the biosynthesis of ATP involving ion-exchange reactions.

Dissociation constants for Mg . ATP were determined by displacing ATP from Dowex-1 resin with magnesium. These constants were then used to analyze the kinetics of yeast mitochondrial ATPase, in terms of the concentrations of free magnesium and free ATP, at a series of pH values. Both Mg . ATP and hydroxide ions were found to compete with the binding of ATP to the enzyme. These results were interpreted, in terms of an ion-exchange model, to mean that the synthesis of ATP may require the utilization of both magnesium and hydroxide ions for the dissociation of ATP from the enzyme as Mg . ATP. The concentrations of Mg and hydroxide required to compete with ATP were both found to be about three orders of magnitude greater than those required to form products, indicating that magnesium and hydroxide ions can contribute about 8 kcal of energy when ATP is synthesized.

Serologic survey for leptospirosis in Arizona beef cattle in 1981.

Sera from 1,215 beef cattle in Arizona were evaluated by leptospiral microscopic agglutination test in 1981. Over 25% had agglutinins to greater than or equal to 1 of 5 serovars of Leptospira interrogans used as antigens (canicola, grippotyphosa, hardjo, icterohaemorrhagiae, and pomona) at a titer of greater than or equal to 1:100, and 8.2% had titers of greater than or equal to 1:400 to greater than or equal to 1 serovars. The most common serovar to which reactions were detected was hardjo; agglutinins were detected at titers of greater than or equal to 1:100 in 14.3% and of greater than or equal to 1:400 in 5.5%. Cross reactions were rare at serum dilutions greater than or equal to 1:100 (2%) and extremely rare at greater than or equal to 1:400 (0.7%). Because vaccination with leptospiral bacterins is seldom practiced in Arizona beef cattle, a titer of greater than or equal to 1:100 may be useful in estimating incidence and prevalence of the disease and as an aid to diagnosis of leptospirosis.