The rapid identification of Clostridium perfringens as the possible aetiology of a diarrhoeal outbreak using PCR.

A gastroenteritis outbreak occurred in a military camp where a laboratory and epidemiological investigation was carried out. The early onset of symptoms indicated probable food contamination with Clostridium perfringens. Stool samples collected from affected patients were tested within 4 h via real-time polymerase chain reaction (PCR) for the presence of the C. perfringens plc gene. Ten out of the 12 stool samples were positive. Confirmation of the molecular test results was carried out by enumeration of C. perfringens in stool by culture and shown to be in excess of 106 spores/g stool. The isolates obtained from culture were further analysed by PCR for the presence of the chromosomal enterotoxin (cpe) gene. Based on the clinical symptoms, epidemiological and laboratory investigations, C. perfringens was implicated as the aetiological agent. The ability to conduct real-time PCR analysis greatly shortens the time to diagnosis and allows for preventive and control measures to be effected quickly.

Molecular identification of coxsackievirus A24 variant, isolated from an outbreak of acute hemorrhagic conjunctivitis in Singapore in 2005.

An outbreak of acute hemorrhagic conjunctivitis (AHC) was reported in Singapore military camps in the year 2005. A total of 103 conjunctival swab specimens were collected from military personnel diagnosed clinically with AHC. PCR testing on these conjunctival specimens revealed the presence of an enterovirus, and this was confirmed by virus isolation. Molecular typing using a partial VP1 gene confirmed a variant of coxsackievirus A24 (CA24v) as the most likely etiological agent for the outbreak. Full-length genome sequencing was carried out on 2 selected virus strains, DSO-26SIN05 and DSO-52SIN05. Sequence comparison and phylogenetic analyses of the VP4, VP1 and 3Cpro gene regions were performed, clustering the Singapore CA24v strains with viruses originating from Asia in the post-2000 era. In addition, we report evolution rates of 4.2 x 10(-3) and 1.0 x 10(-3) nucleotide/year, respectively, for the VP4 capsid and 3Cpro gene regions. Our result shows a focal evolutionary point around 1965-1966, suggesting that the CA24v virus has been evolving constantly since its emergence in Singapore, nearly 40 years ago.

Oyster-associated outbreaks of Norovirus gastroenteritis in Singapore.

Outbreaks of gastroenteritis associated with the consumption of raw imported half-shelled frozen oysters occurred in Singapore between 16 Dec 2003 and 04 Jan 2004. A total of 305 cases were reported with clinical symptoms of diarrhoea (94%), abdominal cramps (72%), vomiting (69%) and fever (54%). The median incubation period was 30.8h and the duration of illness was 2-3 days. The overall relative risk of oyster consumption was 14.1 (95% CI: 8.3-24.0, P<0.001). Stool and oyster samples tested negative for common bacterial pathogens, including Vibrio parahaemolyticus. However, stool samples were positive for the presence of Norovirus group II RNA via RT PCR while oyster samples indicated the presence of Norovirus particles by electron microscopy. The clinical and epidemiological features were suggestive of Norovirus gastroenteritis and were subsequently confirmed by laboratory tests of stools and implicated oysters. Steps have been taken to ensure that food outlets do not thaw frozen oysters and serve them raw.

Histiocytosis presenting as a nasal mass.

The authors present a 1-year-old child who presented with an expansile enhancing mass of the nasal cavity that proved to be histiocytosis.

Amplified fragment length polymorphism fingerprinting of 16 banana cultivars (Musa cvs.).

Banana is one of the most important subtropical crops. The genetic system, however, is relatively unknown and is complicated by specific interhybridization, heterozygosity, and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, particularly when sterile. Amplified fragment length polymorphism (AFLP) analysis using eight primer combinations was carried out on 16 banana cultivars. Results showed that AFLP could be used to distinguish the different cultivars by their unique banding patterns. Unique AFLP molecular markers were detected for 12 banana cultivars, which can be used to develop specific probes for identification purposes. The cluster analysis also revealed the need for a link between genotype studies using molecular techniques and the current system of classification of Musa cultivars based purely on morphological traits.

Cerebral infarction in pediatric acquired immunodeficiency syndrome.

Cerebral infarction is an uncommon complication of AIDS in pediatric patients. We have seen three HIV-infected children who developed acute neurological deficits due to stroke. Cerebral infarction must be considered in the work-up of a child with AIDS who presents with focal neurological deficit, seizure or mental status change. Stroke is a complication of HIV infection that occurs in approximately 1% of affected children [1]. At autopsy, evidence of cerebral infarction was documented in 10-30% of children with HIV infection [2]. We have seen three children with focal infarction who are HIV positive.

Whole-body autoradiographic disposition, elimination and placental transport of [14C]tri-o-cresyl phosphate in mice.

Tri-o-cresyl phosphate (TOCP) is used commercially as a plasticizer and flame retardant. The disposition, metabolism, elimination and transplacental uptake of [phenyl-U-14C]TOCP and/or its metabolites, in pregnant and non-pregnant mice, were examined. Pregnant (18th-day gestation) and non-pregnant, ICR mice were given an i.v. dose of [14C]TOCP (557 microCi kg-1; Specified activity 4.83 microCi mumol-1). At various time intervals (1, 24, 48 and 72 h) the animals were processed for whole-body autoradiography (WBA). Over 72 h the non-pregnant mice excreted 55% of the 14C in the urine and 9% in the feces, while excretion in the urine and feces by the pregnant mice was 50% and 9% of the total dose, respectively. The WBA and its computer-assisted image analysis indicated extensive distribution of the 14C label originally dosed as [14C]TOCP in pregnant mice and their fetuses. The retention of radioactivity in organs such as lung, spleen, gall-bladder and liver of mother and its fetuses suggest that these are the target sites of TOCP toxicity. The distribution in non-pregnant and pregnant mice and in the fetal tissues followed a similar pattern in uptake and retention until 72 h. Brain and spinal cord had the least amount of [14C]TOCP. This finding may support reports that explain the insensitivity of the mice towards organophosphate-induced delayed neurotoxicity (OPIDN) of TOCP.

Effect of glutathione modulation on molecular interaction of [14C]-chloroacetonitrile with maternal and fetal DNA in mice.

Binding of haloacetonitriles or their reactive metabolites to macromolecules of fetal tissue may be responsible for reproductive toxicity. To investigate the role of glutathione (GSH) in the metabolism and reproductive toxicity of haloacetonitriles, irreversible interaction of chloroacetonitrile (CAN) with maternal uterine and fetal DNA was assessed in a time course study among normal and among glutathione-depleted mice treated with [2-14C]-CAN. GSH was depleted in maternal and fetal tissues by treating of animals with diethylmaleate (DEM) 1 h before [2-14C]-CAN administration. Maternal urinary excretion of thiocyanate was 5 times higher in glutathione-depleted mice than in controls. At 8 and 24 h following [2-14C]-CAN administration, total radioactivity uptake in maternal uterine tissue, amniotic fluid, and fetal tissue was higher in glutathione-depleted mice than in control. Also the interaction of CAN or its reactive metabolites with maternal uterine DNA was enhanced following glutathione depletion. At 24 h after treatment, the covalent binding to DNA in fetal tissue was significantly increased in glutathione depleted mice (205% of control). The magnitude of interaction of CAN in fetal DNA was about 4 times higher than that in uterine DNA. The time course study in either maternal uterine or fetal DNA revealed elevated and persistent levels of covalent binding of [ C]-CAN to DNA at 72 h after treatment. Enhancement of the molecular interaction of CAN in maternal and fetal DNA following GSH depletion indicates an important role for GSH in CAN metabolism.

Studies on the mechanism of acetonitrile toxicity. I: Whole body autoradiographic distribution and macromolecular interaction of 2-14C-acetonitrile in mice.

Acetonitrile, a commonly used solvent is known to cause central nervous system dysfunctions. In order to gain an insight onto the mechanism of acetonitrile toxicity, we studied the kinetics of acetonitrile distribution in mice. Male ICR mice were given a tracer dose of 2-14C-acetonitrile intravenously (60 mu mol/kg or 684 mu Ci/kg, spec. act. 11.4 mCi/mmol). At various time intervals (5 min., 0.5, 1, 4, 8, 24 and 48 hr) after treatment, mice were anaesthetized and frozen by immersion in a dry ice/hexane mixture, or they were dissected for collection of organs and tissues. Frozen mice were processed for whole body autoradiography, which allows the detection of non-volatile metabolites of acetonitrile at their sites of accumulation. Covalent binding of acetonitrile metabolites in tissues was determined using trichloroacetic acid followed by ethanol/ether extraction techniques. Whole body autoradiography revealed heavy localization of acetonitrile metabolites in the gastrointestinal tissues and bile. At 5 min., the highest levels of radioactivity occurred in the liver and kidney; levels declined over time. At 24 and 48 hr, acetonitrile derived radioactivity were detected in the gastrointestine, thymus, liver and male reproductive organs. Covalent binding studies at 24 and 48 hr after treatment indicated that 40-50% of the total radioactivity present in the liver was bound to the macromolecular fractions of the tissues. The radioactivity contents of other organs were, in large part (40-50% of total), present in the lipid fraction of the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic interaction of recombinant interferon-beta and zidovudine in AIDS patients.

Zidovudine (AZT), the only currently approved drug for treatment of human immunodeficiency virus (HIV) in AIDS, is known to be metabolized by mammalian systems to a variety of metabolites including 3'-azido-3'-deoxy-5'-O-glucuronide (GAZT). Interferons (IFNs) are known to alter the microsomal enzyme system responsible for the metabolism of some compounds. The aim of the present study was to investigate the effect of combination therapy of recombinant (r) IFN-beta and AZT on the rates of metabolism of AZT in AIDS patients. AZT was given orally (200 mg every 4 h) for 8 weeks prior to initiation of rIFN-beta therapy (45 X 10(6) U/day, s.c.). Serum samples from 8 patients were obtained prior to and at days 3 and 15 following initiation of rIFN-beta therapy. Serum was analyzed by high-performance liquid chromatography (HPLC) for both AZT and GAZT. The serum data were analyzed by a computer-assisted pharmacokinetics program that calculates rates of AZT metabolism. The half life for AZT was increased approximately two-fold by day 15. The rate of metabolism of AZT was diminished from 1.43 h-1 prior to IFN-beta therapy, to 0.4 h-1 and 0.05 h-1 at days 3 and 15, respectively. The volume of distribution of AZT was 2 l/kg at day 0 and increased to 3.2 and 3.5 l/kg on days 3 and 15, respectively. In conclusion, the results indicate that rIFN-beta inhibits the rate of AZT metabolism in AIDS patients.

Studies on the mechanism of haloacetonitriles toxicity: quantitative whole body autoradiographic distribution of [2-14C]chloroacetonitrile in rats.

Chloroacetonitrile (CAN), a drinking water disinfectant by-product, possesses mutagenic and carcinogenic properties. The objective of this study was to investigate the biologic fate of CAN, using whole body autoradiographic (WBA) techniques. Male Sprague-Dawley rats were treated with a tracer dose of [2-14C]CAN (i.v., 88 muCi/kg, spec. act 4.07 mCi/mmol). At various time intervals (0.08, 1, 3, 6, 12, 24, and 48 h) after treatment, rats were processed for WBA. Over 12 h after administration, the radioactivity excreted in urine, feces, and exhaled as 14CO2 accounted for 51%, 2.7%, and 12% of the dose, respectively. Only 0.8% of the administered dose was exhaled as unchanged CAN. At an early time interval (5 min) extensive accumulation of radioactivity was observed in liver, kidney, and gastrointestinal (G.I.) walls. In addition, high levels of 14C were detected in the thyroid gland, lung bronchioles, adrenal cortex, salivary gland, and testes. At 1 h following administration, the olfactory bulb, olfactory receptor area of the brain and lumbar cistern showed high accumulations of radioactive CAN or its equivalents. At 3, 6, and 12 h after treatment, the radioactivity diffused homogeneously in all tissues and reconcentrated in several organs at later time periods (24 and 48 h). Our studies indicate extensive metabolic biotransformation of CAN in rats. The retention of radioactivity in the tissues of the thyroid gland, G.I., testes, brain and eye suggest that those organs are potential target sites of CAN toxicity.

Comparative disposition and whole-body autoradiographic distribution of [2-14C]azidothymidine and [2-14C]thymidine in mice.

Azidothymidine (AZT) is the only approved drug for treatment of acquired immunodeficiency syndrome caused by human immunodeficiency virus. The drug is known to be metabolized by mammalian systems. The objectives of this study were: 1) to investigate the biologic fate of AZT using whole-body autoradiography; and 2) to compare the biologic fate of AZT with that of the parent molecule thymidine (dThd). Male Sprague-Dawley mice were given (intravenously) a tracer dose of [2-14C]AZT (273 microCi/kg) or [2-14C]dThd (218 microCi/kg). Treated animals were sacrificed at various time periods (2 min, 5 min, 4 hr and 24 hr) and processed for whole-body autoradiography. Tissue distribution of radioactivity in the autoradiographs was quantitated using computer-aided image analysis. The elimination of AZT and dThd was also examined by radiochemical analyses of urine, feces and expired air of treated animals over a 24-hr period. Twenty-four hr following AZT treatment, the radioactivity excreted in urine, feces and in exhaled air (as 14CO2) accounted for 86, 4.6 and 3.7% of the dose, respectively. Within 2 min after administration of AZT, maximum radioactivity was detected in the kidney. The brain, spinal cord and testes were conspicuous because of virtual lack of radioactivity. All other parenchymatous organs (liver, lung, heart and spleen) had apparent similar levels of radioactivity that were higher than those in the connective tissues. At a later time period (4 hr), the radioactivity in most organs was eliminated except in the renal medulla, contents of gastrointestinal tract, urinary bladder and mouth cavity.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mechanism of haloacetonitrile-induced gastrointestinal toxicity: interaction of dibromoacetonitrile with glutathione and glutathione-S-transferase in rats.

The haloacetonitrile, dibromoacetonitrile (DBAN), is a direct-acting genotoxic agent that has been detected in drinking water. In a time course study, male Sprague-Dawley rats were treated with DBAN (75 mg/kg PO), and killed at 0.5, 1, 2, and 4 hr after treatment. In a dose response study, animals were treated orally with various doses of DBAN (25, 50, 75, and 100 mg/kg) and killed at one-half hour after treatment. Control animals received 1 ml/kg PO of the vehicle dimethyl sulfoxide (DMSO). In both experiments blood and organs were collected and stored at -80 degrees C until the time of analysis. At 0.5 hr after treatment, a single oral dose of DBAN caused a significant decrease of glutathione (GSH) concentrations in liver (54% of control) and stomach (6% of control). Hepatic GSH depletion was maximal at 0.5 hr and rebound to the control levels by 4 hr. In contrast, gastric GSH concentrations remained low at all time points. DBAN caused an insignificant change in both kidney and blood GSH levels. DBAN significantly inhibited glutathione-S-transferase (GST) activity in liver and stomach. Hepatic GST inhibition was maximal (34% of control) at 2 hr and minimal (80% of control) at 4 hr. Meanwhile, in the stomach GST activity was inhibited at 1 hr (60% of control) and remained low at all times after treatment. Both GSH depletion and GST inhibition were dose-dependent. This study indicates that GSH and GST play an important role in the metabolism and detoxification of DBAN in rats. The prolonged depletion of GSH and inhibition of GST in the gastrointestinal (GI) tissues suggest that the GI tract is a major target for DBAN toxicity.

Determination of camptothecin in biological fluids using reversed-phase high-performance liquid chromatography with fluorescence detection.

Camptothecin, a plant alkaloid with antitumor activity, is a potent and rapidly acting inhibitor of DNA synthesis. The objective of this study was to develop a sensitive high-performance liquid chromatographic (HPLC) method for the detection and estimation of the camptothecin concentration in biological fluids. Using HPLC coupled with fluorescence detection, at an excitation wavelength of 370 nm and an emission wavelength of 434 nm, we found that the lower limits of detection for camptothecin in aqueous, plasma and urine samples were 0.5, 1 and 10 ng/ml, respectively. The ideal mobile phase used was methanol-10 mM potassium phosphate (75:25, v/v, pH 4.0). To determine the utilization of the method in a biological system, we studied the pharmacokinetics of camptothecin in mice. Elimination of camptothecin from mice blood was triphasic and followed first-order kinetics. The half-life of camptothecin in mouse blood was 25.7 min. Our studies indicate that HPLC with fluorescence detection for the determination of camptothecin in different media is a simple, rapid, sensitive and reproducible method.

Studies on the mechanism of haloacetonitriles toxicity: inhibition of rat hepatic glutathione S-transferases in vitro.

Acetonitrile (AN) and seven of its halogenated derivatives known to be water disinfectant by-products were evaluated for their action on hepatic cytosolic glutathione S-transferase (GST) activity using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Increasing concentrations of acetonitrile, monofluoroacetonitrile (MFAN), monochloroacetonitrile (MCAN), and monobromoacetonitrile (MBAN) up to 10 mM failed to produced 50% inhibition of the activity of GST enzyme. However, dichloroacetonitrile (DCAN), trichloroacetonitrile (TCAN), dibromoacetonitrile (DBAN), and monoiodoacetonitrile (MIAN) were potent inhibitors with 150 values of 2.49, 0.34, 0.82, and 4.44 mM, respectively. At concentrations equivalent to their 150, MIAN, DCAN, and DBAN decrease both apparent Km and Vmax of the enzyme activity toward glutathione (GSH) to 20-50% of control. TCAN significantly increases both apparent Km and Vmax for GSH to 650 and 120% of control values, respectively. The inhibitory effect of haloacetonitriles (HAN) on hepatic GST activity toward CDNB was found to be a mixed type. The inhibitory effect of DCAN, DBAN, and TCAN on the hepatic GST activity was found to be reversible and the activity was completely recovered after dialysis of the inhibited enzyme. MIAN, however, inhibited GST activity in an irreversible manner. Haloacetonitriles' induced inhibition of hepatic GST activity in vitro is consistent with that observed in vivo. The data presented in this study show that haloacetonitriles induced reversible inhibition of hepatic GST activities, and this effect may lead to decreased detoxification of other electrophilic chemicals.

Pontine calcification following radiotherapy: CT demonstration.

Development of pontine calcifications following radiation therapy for suprasellar tumors is described in two patients, 5 and 9 years old. Post-radiotherapy brain calcifications are rare in the brain stem.

Pediatric AIDS. Neuroradiologic and neurodevelopmental findings.

A group of 23 pediatric patients seropositive for HIV antibody were studied by computed tomography and evaluated neurodevelopmentally. Significant neurodevelopmental delays were found in over 95% of the patients studied. CT findings in six patients were normal and thirteen of 23 (57%) had prominence of the CSF spaces. Less frequent findings included calcifications in the basal ganglia and white matter. Cerebral mass lesions included one case of lymphoma and one case of hemorrhage. The CT findings in the pediatric age group differs from the adult population in that contrast enhancing inflammatory mass lesions are uncommon.

Dominantly-inherited polycystic kidneys in infants: association with hypertrophic pyloric stenosis.

Newborn male fraternal twins presented at 10 days of age will bilateral flank masses; intravenous urograms showed polycystic kidney disease. Both babies also had hypertrophic pyloric stenosis (HPS). Their father has radiographic and sonographi findings of previously unsuspected polycystic kidneys and has a history of HPS in infancy. The association of dominantly-inherited polycystic kidneys (DPK) and HPS in this family is probably due to chance. However the authors speculate that the autosomal gene for DPK may occur at one of several loci that carry the genetic liability for HPS, A DISORDER TRANSMITTED BY POLYGENIC INHERITANCE.