Virulence Markers and Antifungal Susceptibility Profile of Candida glabrata: An Emerging Pathogen.

Aims: To study virulence factors and antifungal susceptibility profile of C. glabrata isolated from various clinical specimens. Study Design: A total of 175 C. glabrata spp. isolated from various clinical specimens were included in the study. Place and Duration of Study: Department of Microbiology, Rural Medical College, Pravara Institute of Medical Sciences (PIMS), Loni, Maharashtra, India, between March 2008 to March 2013. Methodology: C. glabrata was identified by sugar assimilation and fermentation tests and colony color on Hichrome Candida agar. HiCandida identification kit supplemented the identification of the isolates. The virulence markers studied were production of extracellular hydrolytic enzymes (phospholipase, proteinase and coagulase), haemolytic activity and biofilm formation. The antifungal susceptibility profile of C. glabrata isolates was determined by Hicomb minimum inhibitory concentration (MIC) test. The antifungal agents used were amphotericin B (range 0.002-32 μg), fluconazole (range 0.016-256 μg), itraconazole (range 0.002-32 μg) and ketoconazole (range 0.002-32 μg). Results: Maximum number of isolates were obtained from blood culture (36%) followed by urine sample (29.7%). ICU stay followed by HIV infection were the main predisposing factors found to be associated with C. glabrata infection. A total of 53 (30.2%) C. glabrata isolates showed phospholipase activity. Proteinase production was seen in 56 (32%) isolates. 48 (27.4%) isolates were coagulase positive. Haemolytic activity was noted in 43 (24.5%) isolates. Most of C. glabrata produced β- type of haemolysis on sheep blood SDA agar. Biofilm forming ability was noted in 68 (38.8%) isolates. Maximum isolates were resistant to fluconazole (46.8%) and ketoconazole (46.8%) followed by itraconazole (45.7%). Amphotericin B resistance was seen in 58 (33.1%) isolates. Conclusion: Once considered as a non pathogenic human commensal, C. glabrata has emerged as an important pathogen in various clinical types of candidiasis. C. glabrata is innately resistant to antifungal drugs and various antifungal mechanisms of the body. Present research data available is not satisfactory to understand the pathogenic and other mechanisms involved in the transition of C. glabrata from nonpathogenic commensal to a potential pathogen. Therefore more research studies are needed to explain pathogenesis, host-pathogen interaction and other survival properties of this emerging pathogen.

Evaluation of Phospholipase Activity in Biofilm Forming Candida Species Isolated from Intensive Care Unit Patients.

Aims: To evaluate phospholipase activity in biofilm forming Candida spp. isolated from patients admitted in intensive care unit of rural tertiary care hospital. Study Design: A total of 135 biofilm forming Candida spp. isolated from various clinical specimens of patients admitted in ICU were included in the study. Place and Duration of Study: Department of Microbiology, Pravara Institute of Medical Science’s Rural Medical College India, between January 2010 and December 2012. Methodology: The Candida isolates were identified upto species level by conventional standard mycological techniques. The biofilm formation was assessed by inoculating the isolates in conical polystyrene test tube containing Sabouraud’s dextrose broth supplemented with glucose. Phospholipase activity of biofilm forming Candida isolates was detected by using egg yolk agar. Results: Out of 135 biofilm forming Candida spp. included in the study, 60 (44.4%) isolates were C. albicans. Among non-albicans Candida (NAC) spp. C. tropicalis was the major isolate followed by C. glabrata and C. parapsilosis. Phospholipase production was seen in 85 (62.9%) isolates. A total 49 (81.6%) isolates of C. albicans showed phospholipase activity. Among NAC spp. maximum phospholipase activity was seen in C. tropicalis and C. glabrata. Conclusion: Biofilm formed by the Candida spp. tend to be more resistant to antifungal drugs. Though C. albicans the most common species associated with Candida biofilms, the emergence of NAC spp. is of concern. NAC spp. shows varying degree of resistance either intrinsic or acquired or both to commonly used antifungal drugs. The isolation of NAC spp. from clinical specimens is no longer overlooked as these organisms are emerging pathogens. The virulence factors like biofilm formation and phospholipase activity is also noted in NAC spp. The study of these virulence factors would help in understanding the pathogenic role of NAC spp.

Non albicans Candida species: its isolation pattern, species distribution, virulence factors and antifungal susceptibility profile.

Background: The change in epidemiology and antifungal susceptibility has generated interest of Clinical Microbiologists in identification of Candida up to species level along with antifungal susceptibility pattern. Non-albicans Candida (NAC) has emerged as an important opportunist pathogen. Extracellular hydrolytic enzymes are one of the important virulence attributes of Candida species. Aims & Objective: The present study aimed to determine the species distribution, virulence factors and antifungal susceptibility profile of NAC spp. isolated from various clinical specimens. Material and Methods: Speciation of Candida was done by assessing the germ tube formation, assimilation and fermentation of sugars and colony color on HICHROM Candida agar. In-vitro extracellular hydrolytic enzymes production in NAC spp. was assessed. Antifungal susceptibility testing of the isolates was performed by Hicomb minimum inhibitory concentration (MIC) test. Results: Majority of the isolates were obtained from urine sample (35.6%). C. tropicalis (29.4%) was the major isolate. Maximum extracellular hydrolytic enzymes activity was seen in C. tropicalis. A total of 79 (27.3%) isolates were resistant to fluconazole. Amphotericin B resistance was noted in 17 (5.8%) isolates. Conclusion: NAC spp. cannot be overlooked as mere containment or non-pathogenic commensals as most of them show reduced susceptibility to commonly used antifungal drugs. Extracellular hydrolytic enzymatic activity of NAC Spp. would be an important tool to prove the relation between the infective species of Candida and infection.

Species Identification And Antifungal Susceptibility Pattern Of Candida Isolates From Oropharyngeal Lesions Of HIV Infected Patients..

Background &Objective: Oropharyngeal candidiasis (OPC) is a common feature associated with HIV infection. Over the past decade, reports have documented a shift away from C. albicans as a major cause of infection to non albicans Candida (NAC) species. Several NAC spp are inherently resistant to commonly used antifungal drugs. The objective of the present study was to investigate the distribution pattern of Candida spp. from HIV infected patients with OPC and evaluate its antifungal susceptibility pattern. Methods: A total of 192 HIV infected patients with oropharyngeal lesions (OPL) suggestive of candidiasis and 60 non HIV infected healthy individuals presenting without any OPL were included in the study.Swabs collected from the site of lesions were used for the demonstration and isolation of Candida. Speciation of Candida isolates was done and antifungal susceptibility testing was performed by the disc diffusion method. Results: Out of 192 HIV-infected patients with OPL, 179(93.2%) showed growth of Candida. Isolation of NAC species was higher than C. albicans. Azole resistance was more in NAC species as compared to C. albicans.Conclusions: NAC species has emerged as an important cause of OPC in HIV infected patients. The increased isolation rates of NAC species and a gradual shift in the antifungal susceptibility profile underlines the need of early and accurate diagnosis of infecting Candida spp along with antifungal susceptibility testing for selecting the most appropriate antifungal agent for therapy.

Dental caries, gingivitis, periodontitis: a review

Microbial populations colonizing the teeth and periodontal tissues are a major source of pathogens responsible for oral and dental infections including dental caries, gingivitis, periodontitis etc. Dental caries is a multifactor and infectious disease resulting dueto interaction of three different aspects like dietary sugar, susceptible tooth enamel and oral microbial colonization. Plaques from caries active sites have significantly higher proportion of Streptococcus mutans (principle acid producer) with pH levels of 5.0 or lower. Dental decay occurs when normal demineralization remineralization is disturbed. On the other hand the most common form of gingivitis is chronic or long standing plaque induced gingivitis while acute necrotizing ulcerative gingivitis is most aggressive, developing gingivitis is associated with increasing numbers of Actinomyces israeliwhereas gingivitis with bleeding is associated with A. viscosus and pigmented Bacteroides. Periodontitis is defined as loss of alveolar support to the tooth and can be differentiated microbiologically and clinically into adult, localised juvenile and pre-pubertal periodontitis. Various species of Bacteroides, Actinomyces, Fusobacterium etc. have been isolated from cases of active periodontitis. Thus wherever possible both aerobic and anaerobic culture should be performed and appropriate antibiotic therapy should be prescribed instead of empirical treatment.

Bacteriological study of paediatric and adult chronic suppurative otitis media.

Chronic suppurative otitis media (CSOM) is one of the commonest illnesses in ENT practice which requires medical attention all the more in children of poor socio-economic status having in past inadequate treatment and negligent medical care. The present study was conducted to find out the various aerobic and anaerobic microorganisms associated with CSOM in paediatric and adult cases and their current antimicrobial susceptibility pattern as a guide to therapy. Samples were collected from 109 clinically diagnosed cases of CSOM and processed according to standard protocols. Out of 74 paediatric CSOM cases, 72 (97.2%) were bacterial culture positive while out of 35 adult CSOM cases, 28 (80%) were culture positive. Bilateral CSOM was slightly more common in adults (25%) than paediatric (21.4%) age group. Polymicrobial nature of CSOM was noted in both paediatric (70.8%) and adult (71.4%) cases while number of organisms isolated per lesion was slightly higher in adults (2.5) as compared to paediatric (1.95) cases. In paediatric CSOM, Staphylococcus aureus was the commonest aerobic isolate while in adult CSOM, Pseudomonas aeruginosa was the commonest one. Among anaerobes Peptostreptococcus spp. was commonest in CSOM where as Prevotella melaninogenica in adult CSOM. Sensitivity of S. aureus to cefuroxime was 72.2% while that of gram negative bacilli was higher to cefotaxime (90 to 100%). Among anaerobes higher sensitivity was seen to metronidazole (98.6%), clindamycin (95.7%) and chloramphenicol (98.6%). In view of the polymicrobial etiology of CSOM, prompt appropriate antimicrobial therapy can effectively reverse the disease process thereby preventing longterm sequelae.

Surgical infections: a microbiological study

Surgical infections are mostly polymicrobial, involving both aerobes and anaerobes. One hundred seventeen cases comprised of abscesses (n=51), secondary peritonitis (n=25), necrotizing fascitis (n=22) and wounds with devitalized tissues (n=19) were studied. The number of microorganisms isolated per lesion was highest in secondary peritonitis (2.32). The aerobe/ anaerobe ratio was 0.81 in secondary peritonitis and 1.8 in necrotizing fascitis. Most secondary peritonitis (80 percent), necrotizing fascitis (75 percent) and wounds with devitalized tissues (66.7 percent) were polymicrobial. Common microorganisms isolated in our study were E. coli, Staphylococcus aureus, Klebsiella spp., Pseudomonas aeruginosa, Bacteroides fragilis and Peptostreptococcus spp. The most effective antibiotics for S. aureus were clindamycin (79.1 percent) and cefuroxime (70.8 percent). For Gram-negatives (Klebsiella spp., E. coli and Proteus spp.), the most effective antibiotics were cefotaxime, ceftizoxime, amikacin and ciprofloxacin. Pseudomonas aeruginosa was maximally sensitive to amikacin (35.2 percent) and ciprofloxacin (35.2 percent). The greatest degree of multidrug resistance to all the drugs was found in P. aeruginosa (52.9 percent), followed by Klebsiella spp. (33.3 percent), Proteus spp. (33.3 percent), E. coli (22.2 percent), and S. aureus (12.5 percent). All the anaerobes that we isolated were 100 percent sensitive to metronidazole and chloramphenicol, followed by clindamycin (95 percent to 100 percent). Apart from antibiotic therapy, non-antimicrobial methods, such as hyperbaric oxygen therapy and debridement also play an important role in the treatment of surgical infections.

C-reactive proteins as an indicator of sub-clinical infection in cases of premature rupture of membranes.

The present study was conducted on 25 cases of preterm, premature rupture of membranes and 25 patients preterm with intact membranes, between 28-36 weeks of gestation, admitted in the maternity ward of Obstetrics and Gynecology, PGIMS, Rohtak. The sensitivity and specificity of CRP determination was found to be 80% each as an early predictor of subclinical chorioamnionitis. TLC had a low sensitivity of 20% and specificity of 60% in detecting histopathological chorioamnionitis. So CRP estimation is a reliable marker for detection of early choriamnionitis, at the same time, it is quite affordable and reasonably simple.