Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

BACKGROUND: The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. METHODS: The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. RESULTS: A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. CONCLUSIONS: The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.

Broiler response to growth promoter and potential to reserve meat nutrients

Three hundred day old Cobb commercial broilers were reared up to 32 days of age in a random distribution to 5 dietary treatments [A, B, C D and E], with 15 numbers having 4 replications in each fed with market available ready feed. Drinking water was supplemented with glucose and multiviuimin at onset, 4[th],11[th],23[rd] and 25[th] days. Besides, all the birds were immunized with ND clone-30 and D-78 at the age of 5[th] and II[th] day to prevent the Newcastle disease. Birds were medicated with four dietary additives that are being frequently used in commercial level, Chlortetracycline [10 gm/kg feed], Oxytetracycline [0.5 mg/liter water], Enrofloxacin [2.5 ml/ 10 liters water] and Doxicycline and Colistin sulphate 10.5 mg/liter] as per instruction of manufacturing company at the age from 7 to 24 days for dietary group A and from 19 to 24 days for B,C and D dietary group while E was controlled. At the end of the 3rd week [21 Day], representative medicated birds were slaughtered for inking samples to analyze the meat quality. In addition, at the end of trial, some antibiotic withdrawal birds were randomly selected and slaughtered for the same purpose. Live weight and dressed weight, breast, thigh, wings, heart, liver, spleen and gizzard weight were also taken using electric balance. Proximate components like DM, CP, and CF of meat were measured. The medication had no significant [P>0.05] effect on live weight gain followed by dressed weight and also for meat cuts in most cases. Only liver and gizzard showed the positive response in weight gain[gm] to medication [P<0.05]. In contrast, 7 days of withdrawal later, no antimicrobial [P>0.05] reply was found to live weight gain[g], dressed weight and different cut weight [g]. FCR was better in antibiotic groups in comparison with control group. Among medicated groups B was found best feed conversion efficiency at market age both in medication and withdrawal period. The essence is that proper management can recover the quality and quantity of chicken meat. Only oxytetracycline [B] may be used [if necessary] for increasing the breast meat quality [CP] up to the safe period [7 days of withdrawal] and to prevent the infection

Mutagenicity studies of cadmium chloride in mice

Cadmium chloride [CdCl2] was tested for its mutagenicity in Swiss albino mice by the dominant lethal mutation test and the micronuclei test singly or in combination with either nitrosoguanidine [NTG] or ascorbic acid. The results obtained showed that cadmium chloride is a weak mutagen when compared with NTG as a positive control. However, cadmium chloride [CdCl2] and NTG in combination showed a significant increase in the dominant lethal mutations when compared with the effect of either CdCl2 or NTG individually. A statistically significant increase of the frequency of micronucleated erythrocytes was also noticed when CdCl2 was combined with either NTG or ascorbic acid compared with CdCl2 individually

Intramedullary lipoma of the cervical and thoracic spinal cord

We report the case of a patient with quadriparesis due to a cervicothoracic intramedullary lipoma unassociated with spinal dysraphism. Intraspinal lipomas are usually associated with spinal dysraphism but in rare cases this association does not exist. Once the symptoms develop surgery does not usually result in any improvement in the neurological symptoms. Early Magnetic Resonance Imaging in patients with symptoms is recommended so that the tumour may he removed before permanent neurological deficit develops

specific gastric inhibitory polypeptide antiserum recognising a single molecular form

Gastric inhibitory polypeptide [GIP], a hormone of the proximal small intestine, potentiates glucose-induced insulin secretion in the presence of hyperglycaemia. It has been difficult to define the precise role of GIP due to the presence of a number of immunoreactive GIP components with differential immuno reactivity. We have raised an antiserum [S705] in sheep by multiple subcutaneous injections of natural porcine GIP after conjugating it to human albumin by the difluorodinitrobenzene [DFDNB] method. S705 recognizes only one component, immunoreactive GIP 5000, the major immunoreactive component of GIP which plays an important role in the enteroinsular axis

Regulatory peptides: a new concept

Recently, the new idea that the body has a signale system of chemical messenger peptides has developed. The same or similar clinical messenger may be utilized for endocrine, paracrine or neurocrine modes of transmission. Thus, the pavlovian theory of neural control and the circulating messenger concept of Bayliss and Starling are fused into a single concept of control by regulatory peptides