Development and validation of TaqMan real-time PCR for the detection of Burkholderia pseudomallei isolates from Malaysia

@#Rapid detection of Burkholderia pseudomallei, the etiologic agent of melioidosis, allows for timely initiation of appropriate treatment and better clinical outcomes. In the current gold standard, the culture method is time consuming and suffers from low sensitivity. Meanwhile, previously reported molecular assays are fast and sensitive, but their performance on isolates from Malaysia, an endemic region of melioidosis is under reported. This study designed oligonucleotides targeting orf2 of Type III secretion system (TTSS) genes cluster for the detection of Malaysian B. pseudomallei isolates and evaluated the assay on 95 local B. pseudomallei strains, 58 other microorganisms and 71 clinical specimens from patients. The developed assay exclusively detected all tested B. pseudomallei isolates with a detection limit of 20 fg per reaction (equivalent to ~2.5 copies). Subsequent testing on clinical samples showed that the assay detected all confirmed specimens with the growth of B. pseudomallei (n = 10/10). None of the negative specimens had a detectable signal of our TTSS-orf2 assay (n = 0/61). In conclusion, the present study provides crucial preliminary data for a subsequent study and should be considered as a potential alternative to current time-consuming culture method for the detection of B. pseudomallei.

A Recombinant Subunit HSABA392 as a potential Vaccine for Haemorrhagic septicaemia disease in livestock

@#Haemorrhagic septicaemia (HS) is a major disease in cattle and buffaloes, caused by certain serotypes of Pasteurella multocida, mainly B and E serotypes. Frequent HS outbreak has a major impact in many Asian countries, including Malaysia, where farmers encounter economic loss due to low milk production as well as death of their livestock. There are four types of vaccines available; broth bacterins, alum precipitated vaccine, aluminium hydroxide gel vaccine and oil adjuvant vaccine (OAV), but these vaccines can only provide short term immunity and therefore need to be administered annually. Hence, the development of a protein vaccine using recombinant antigen can be a potential candidate for the production of HS vaccine that would give longer immunity. We have successfully cloned the ABA392 gene fragment into a protein expression vector, pET-30a. The protein was expressed from our ABA392/pET30a clone and the immunogenicity of the protein has been tested on rats. This vaccine was able to trigger an immune response and therefore has the potential to be tested as suitable vaccine candidate in future studies. It is envisaged that this subunit vaccine will make a significant contribution in the management of HS among livestock in future.

Polycaprolactone Triol–Citrate Scaffolds Enriched with Human Platelet Releasates Promote Chondrogenic Phenotype and Cartilage Extracellular Matrix Formation

In this paper we report the differentiating properties of platelet-rich plasma releasates (PRPr) on human chondrocytes within elastomeric polycaprolactone triol–citrate (PCLT–CA) porous scaffold. Human-derived chondrocyte cellular content of glycosaminoglycans (GAGs) and total collagen were determined after seeding into PCLT–CA scaffold enriched with PRPr cells. Immunostaining and real time PCR was applied to evaluate the expression levels of chondrogenic and extracellular gene markers. Seeding of chondrocytes into PCLT–CA scaffold enriched with PRPr showed significant increase in total collagen and GAGs production compared with chondrocytes grown within control scaffold without PRPr cells. The mRNA levels of collagen II and SOX9 increased significantly while the upregulation in Cartilage Oligomeric Matrix Protein (COMP) expression was statistically insignificant. We also report the reduction of the expression levels of collagen I and III in chondrocytes as a consequence of proximity to PRPr cells within the scaffold. Interestingly, the pre-loading of PRPr caused an increase of expression levels of following extracellular matrix (ECM) proteins: fibronectin, laminin and integrin β over the period of 3 days. Overall, our results introduce the PCLT–CA elastomeric scaffold as a new system for cartilage tissue engineering. The method of PRPr cells loading prior to chondrocyte culture could be considered as a potential environment for cartilage tissue engineering as the differentiation and ECM formation is enhanced significantly.

Phytoestrogen (Daidzein) Promotes Chondrogenic Phenotype of Human Chondrocytes in 2D and 3D Culture Systems

Clinical investigations have shown a significant relationship between osteoarthritis (OA) and estrogens levels in menopausalwomen. Therefore, treatment with exogenous estrogens has been shownto decrease the risk ofOA.However, the effect estrogen has not been clearly demonstrated in the chondrocytes using phytoestrogens, which lack the specific side-effects of estrogens, may provide an alternative therapy. This study was designed to examine the possible effects of phytoestrogen (daidzein) on human chondrocyte phenotype and extracellular matrix formation. Phytoestrogens which lack the specific side-effects of estrogens may provide beneficial effect without causing hormone based side effect. Human chondrocytes cells were cultured in 2D (flask) and 3D (PCL-CA scaffold) systems. Daidzein cytotoxic effect was determined by MTT assay. Chondrocyte cellular content of glycosaminoglycans (GAGs), total collagen and chondrogenic gene expression were determined in both culture systems after treatment with daidzein.Daidzein showedtime-dependent and dose-independent effects on chondrocyte bioactivity.Thecompound at low doses showed significant (p0.05). The expression levels of Fibronectin, Laminin and Integrin b1were significantly increased especially in3Dculture system. This studywas illustrated the potential positive effects of daidzein onmaintenance of human chondrocyte phenotype and extracellular matrix formation suggesting an attractive and viable alternative therapy for OA.

The timing and sequence of emergence of permanent teeth in Malay schoolchildren in Kota Bharu, Malaysia

The purpose of this study was to determine the timing and sequence of emergence of the first 28 permanent teeth in a cross-section of Malay children in the district of Kota Bharu, in northeastern Peninsula Malaysia. The sample consists of 478 boys and 908 girls of Malay descent aged between 5-19 years. The criterion for tooth emergence was the appearance of the tooth through the gingivae. Descriptive statistics were calculated and probit regression analysis performed to determine the mean age of emergence of the permanent dentition. The mean age at emergence was found to conform to general trends, with emergence seen earlier in girls than in boys. Comparisons were made with the Chinese (Hong Kong) and the Punjabi (Chandigarh) profiles, which showed earlier emergence timings in those ethnic groups. The emergence timing in Malays, however, was earlier than in Thais (Central Thailand). The sequence of emergence was determined by referring to the mean age of tooth emergence of individual teeth and conforms to the general trend seen in other studies.

Mini laparoscopic cholecystectomy: a better outcome

The outcomes after traditional laparoscopic cholecystectomy [LC; one 10 mm port, one 12 mm port and two 5 mm ports] and mini laparoscopic cholecystectomy [MLC; three 3mm ports and one 12mm port] for gall stone disease were compared. The study was a randomized, single blind trial comparing LC with MLC. The randomization period was from February 2003 to December 2004. Two groups of patients underwent the surgery, 48 underwent LC and 47 underwent MLC. Patients were randomized by means of a blind envelop system just before surgery. The groups were matched for age, sex and preoperative characteristics. Median [range] operating times for LC and MLC were similar 45 [20- 120] and 50[20-170] mm. respectively. Intraoperative and postoperative complication rates, the time for the patient to resume walking, eating and passing stools, and median hospital stay were the same in the two groups. The level of post operative pain was lower in the MLC group at 1 h, 3h, 6h, 12h and 24h. Patients who had MLC received fewer injections of analgesic [p=0.036] and more patients in this group expressed satisfaction with the cosmetic result [p= 0.001]. MLC took a similar time to perform and caused less postoperative pain than the standard laparoscopic procedure. Reducing the port size further enhanced the advantages of laparoscopic over open cholecystectomy

Role of surgery for chronic groin pain by inguinal neurectomy

Inguinal nerve entrapment is a debilitating post operative problem. Fifty patients were treated for inguinal nerve entrapment including 26 men and 24 women with an average age of 45 +/- 14 years Most patients had inguinal hernia repairs or Pfannenstiel incisions. Mesh was found in 27% of patients. Symptoms included pain [100%] radiation of pain to the thigh and/or genital area [59%], and postural pain [59%]. Diagnosis was made by physical examination, postural maneuvering, and inguinal nerve block. Proximal nerve resection was followed by polyester ligature and absolute alcohol application to prevent neuromas. Eight percent of patients had minor complications. Total pain relief was attained in 72% of patients, partial relief in 25%, and no relief in 3%. Two patients complained of numbness postoperatively. Multifactorial analysis showed recurrent hernia repair as a significant predictive factor