Detection of 16S rRNA gene for rapid identification of bacterial pathogens causing peritonitis in patients on continuous ambulatory peritoneal dialysis.

PURPOSE: Peritonitis is the most important complication with high rate of morbidity and mortality in patients on continuous ambulatory peritoneal dialysis (CAPD) despite the success and advances. Rapid and accurate identification of pathogens causing peritonitis in a CAPD patient is essential for early targeted treatment. The aim of the study was to evaluate the role of 16S rRNA gene and ITS region PCR and sequencing in detecting bacterial and fungal pathogens from the dialysate of patients undergoing CAPD. METHODS: Fifty eight peritoneal dialysate from suspected cases of peritonitis on CAPD were subjected to conventional culture as per the ISPD guidelines and automated culture system. A conventional PCR was performed to detect the 16S rRNA gene and ITS region. Sequencing and analysis were performed to identify the etiological agent from the remaining dialysate. RESULTS: Among the 58 dialysate fluid, the etiological agents were identified in 8(14%) samples by conventional culture, 28(48%) by automated culture and 47(81%) by 16S rRNA sequencing and analysis. In 8 samples there was discordance in the results of the culture and 16S rRNA PCR. BLAST search of nine sequences obtained from 16S rRNA PCR revealed that these sequences matched best with uncultured bacterial clones. In eleven samples the sequence failed. CONCLUSION: The molecular tool 16S rRNA gene and ITS region PCR and sequencing cannot be used as a standalone test as it lacks sensitivity to identify some bacterial species due to high genetic similarity in some cases and inadequate database in GenBank. However, it could be used as a supplementary test to the culture method especially in the diagnosis of culture negative peritonitis.

Risk factors, bacteriological profile and outcome of surgical site infections following orthopaedic surgery.

BACKGROUND AND OBJECTIVES: Surgical site infection (SSI) is a challenge for the surgeon. Incidence of SSI reported in literature varies from 0.5% to 15%. Severity of SSI ranges from superficial skin infection to life-threatening condition like septicaemia. It is responsible for increased morbidity, mortality, and economic burden to the hospital in general, and the patient in particular. The aim of this study was to assess the risk factors, bacteriological profile, length of hospitalization, and cost due to orthopaedic SSI in patients admitted to a tertiary care hospital. MATERIALS AND METHODS: This was a prospective case control study. Cases were diagnosed based on CDC definition of nosocomial SSI. All cases were assessed preoperatively, intraoperatively and postoperatively, according to type of surgery, wound class, duration of operation, antimicrobial prophylaxis, use of drain, preoperative hospital stay, causative micro organism, total hospital stay, readmission rates and cost incurred. Age, sex and surgical procedure matched controls without SSI, were also assessed. Chi-square test and Fisher's exact test were used for analysis. P= <0.05 was considered significant. RESULTS: Out of 1023 patients, 47 cases had SSI, with a rate of 4.6%. Cigarette smoking was a risk factor for SSI (P = 0.0035). The most common etiologic agents were Acinetobacter baumannii and Staphylococcus aureus. Incidence of readmission among SSI cases was more compared to controls (P= 0.0001). Costs attributable to SSI (Indian Rupees) was Rs 32,542 (17,054 to 87,514) which was significantly more than those without SSI (P= <0.001). CONCLUSION: Despite latest surgical amenities, meticulous sterilization protocols and pre-operative antibiotic prophylaxis, SSI continues to be present in healthcare settings. The increase in duration of hospital stay due to SSI adds to additional burden to an already resource-constrained healthcare system.

Simple and economical method for identification and speciation of Staphylococcus epidermidis and other coagulase negative Staphylococci and its validation by molecular methods.

Coagulase-negative staphylococci (CoNS) have been increasingly recognized as a clinically important group of species that can cause several opportunistic nosocomial infections. There are at least 47 known species of Staphylococci and to differentiate all these species >40 biochemical tests need to be performed. The present study was able to refine the CoNS identification process by using only five tests to identify S. epidermidis from the rest and used six other tests to identify eleven other clinically significant CoNS species. A total of 242 CoNS isolates were collected from tertiary care hospitals and included in the study. The five-biochemical test scheme devised based on mathematical probability derived from a computer algorithm included fermentation of mannitol, maltose, mannose, trehalose and novobiocin susceptibility to differentiate S. epidermidis from other CoNS species. The remaining CoNS isolates other than S. epidermidis were further characterized with the help of six additional tests, which identified another eleven species. Species-specific PCR and 16SrDNA sequencing were used to confirm and validate the identification scheme. Species-specific PCR and 16SrDNA sequencing showed 100% agreement with non-divergent phenotypic test results, indicating that the five selected assays are highly specific for identifying S. epidermidis. In conclusion, this study used only 11 tests to identify most of the clinically significant CoNS that can reduce cost and time. This scheme is easy to perform in any laboratory with basic resources, the results of this study were validated using more accurate molecular methods such as PCR and 16S rDNA typing to confirm the utility of the proposed scheme.

Evaluation of Nuclear Morphometry and Ki-67 Index in Clear Cell Renal Cell Carcinomas: a Five-Year Study.

BACKGROUND AND OBJECTIVE: Clear Cell Renal Cell Carcinoma (CCRCC) is the most common adult renal neoplasm. Staging and grading of RCC are important predictors of survival. Fuhrman nuclear grading is widely used for CCRCC, the subjective nature of which has prompted more objective methods to evaluate nuclear features. Furthermore, Ki-67, a reliable marker of cellular proliferation may provide another variable for assessment of the biological behavior of RCC. The aim of this research was to study nuclear morphometry and Fuhrman nuclear grading of clear cell RCC, and to assess their relationship with the Ki-67 index. METHODS: Hematoxylin and eosin slides of forty cases of CCRCC were retrieved and studied for pathologic variables, including Fuhrman nuclear grade, pathological tumor and node stage. Nuclear morphometric analysis was performed using computer-assisted image analysis. The relationship between Fuhrman nuclear grading, pathologic stage, tumor size, nuclear morphometry and proliferative index were analyzed. RESULTS: According to Fuhrman grading, four (10%) cases were grade I, 23 (57.5%) were grade II, 12 (30%) were grade III, and one (2.5%) was grade IV. Moderate to high correlation was seen between Fuhrman nuclear grade and mean nuclear area, perimeter, diameter, length, nuclear roundness factor and Ki -67, with a P value of < 0.05. CONCLUSION: The CCRCC is an extremely heterogenous disease and clinical outcome is unpredictable despite several validated prognostic factors. The widely used Fuhrman nuclear grading is subjective, while nuclear morphometry, using computer assisted image analysis, can ensure more objective assessment. The Ki-67 index could provide reliable information and compliment the other prognostic parameters.

Morphologic heterogeneity in carcinosarcoma of the gallbladder: report of a rare cases.

Carcinosarcoma is a rare tumor composed of variable proportions of carcinomatous and sarcomatous elements and comprises less than one percent of all gallbladder malignancies. In most reported cases of carcinosarcoma of gallbladder, the epithelial component is adenocarcinoma. The mesenchymal component varies from homogenous sarcoma to more heterotopic elements like malignant bone, cartilage, and other mesenchymal tissues. We report a rare case of carcinosarcoma of the gallbladder in an 83-year-old male, with the carcinomatous component represented by undifferentiated carcinoma (spindle and giant cell type with osteoclastic giant cells) and the mesenchymal component seen as foci of chondrosarcoma.