Umadevi S

The Isolation and the Biofilm Formation of Uropathogens in the Patients with Catheter Associated Urinary Tract Infections (UTIs)

BACKGROUND Urinary tract infections are the most commonly acquired bacterial infections and they account for an estimated 25-40% of the nosocomial infections. The microbial biofilms pose a public health problem for the persons who require indwelling medical devices, as the microorganisms in the biofilms are difficult to treat with antimicrobial agents. AIMS The present study included the isolation and the biofilm formation of the uropathogens in patients with catheter associated urinary tract infections. METHODS AND MATERIALS This prospective analysis which was carried out over a period of two months, included 50 urine samples from catheterized patients with symptoms of UTI. Following their isolation and identification, all the isolates were subjected to the biofilm detection by the tube adherence method and the Congo Red agar method. RESULTS E.coli was found to be the most frequently isolated uropathogen 35(70%), followed by Klebsiella pneumoniae 8(16%), Pseudomona aeruginosa 2(4%), Acinetobacter spp 1(2%), coagulase negative Staphylococci 3(6%) and Enterococci spp 1(2%). In the current study, 30 (60%) strains were positive in vitro for the biofilm production. CONCLUSION To conclude, there was significant bacteriuria in all the symptomatic catheterized patients and E.coli was the most frequent isolate. Diabetes (44%) was the most common factor which was associated with the UTIs in the catheterized patients.

Early dengue diagnosis by nonstructural protein 1 antigen detection: rapid immunochromotography versus two the enzyme-linked immunosorbent assay kits.

Dengue is known for its serious life-threatening complications. New rapid kits available recently in India target circulating non-structural protein (NS1) antigen from day one onwards. The sensitivity and specificity of a newly introduced rapid combo kit against two conventional ELISA kits is assessed. The performance of this kit is quite satisfactory since excellent agreement of 94.26% was observed with particular reference to NS1 antigen detection among all three kits namely Rapid SD Bioline dengue Duo (SD Korea), InBios DENV Detect NS1 ELISA, USA and dengue Early ELISA, Panbio, Australia. The false positivity of the rapid kit is very low since its specificity as for as NS1 antigen detection is concerned is 98.33%. The use of combination kit helps to detect additional cases of dengue, which are negative for NS1 antigen but positive for IgM and/or IgG antibodies, thus facilitating early diagnosis in remote areas and small laboratorie.

Laboratory evaluation of phenotypic detection methods of methicillin-resistant Staphylococcus aureus

Although conventional antibiotic susceptibility tests are most commonly performed for methicillin-resistant Staphylococcus aureus (MRSA), the results of these phenotypic tests are dependent on the standardization of the culture conditions. The aim of the study was to evaluate the conventional phenotypic screening tests in comparison to the mecA gene polymerase chain reaction (PCR). One hundred and two clinical isolates of MRSA identified by the oxacillin disk diffusion were subjected to PCR for the mecA gene and by the cefoxitin disk diffusion test and culture on oxacillin screen agar, mannitol salt agar, and methicillin-resistant Staphylococcus aureus Agar (MeReSA) selective medium, for MRSA. Although all 102 isolates were resistant in oxacillin and cefoxitin disk diffusion, 92 (90.1%) isolates were positive for the mecA gene. The sensitivities of the mannitol salt agar, MeReSA agar, and oxacillin screen agar were 89.13, 97.82, and 98.91%, respectively. The oxacillin screen agar may be recommended for confirming methicillin resistance in the disk diffusion test in resource-poor settings, where molecular methods are not available.

Determination of correlation between biofilm and extended spectrum β lactamases producers of Enterobacteriaceae

Background: Biofilm is a complex aggregate of microorganisms in which cells adhere to each other to a surface in a self-produced matrix of extra-cellular polymeric substance. Antibiotic resistance of bacteria in the biofilm mode of growth contributes to the chronicity of infections. Aims: This study was aimed to find out the prevalence of biofilm producers among the microorganisms isolated from our setup and to find out their antimicrobial susceptibility pattern with special reference to Extended Spectrum β-lactamase (ESBL) and AmpC producers. Settings and Design: It is a prospective, analytic study. Materials and Methods: The study included 169; non-repeat, clinical isolates of Enterobacteriaceae collected over a period of 6 months. All isolates were tested for biofilm production by tube adherence methods. Any bacterial species which showed resistance to any of third generation cephalosporin was tested for ESBL production and AmpC production. Statistical Analysis: Statistical analysis was carried out by taking percentage and simple ratios. Results: Among 169 isolates, 100 (59.2%) were biofilm producers. In our study, 44 (26%) and 43 (25%) isolates were ESBL and AmpC producers respectively. Of 44 ESBL producers, 42 (95.4%) and of 43 AmpC producers 39 (90.7%) were biofilm producers. Conclusions: Among 169 isolates, 26% and 25% were ESBL and AmpC producers. Of which, more than 90% were biofilm producers, which showed high resistance to commonly used antibiotics. Disabling biofilm resistance may enhance the ability of existing antibiotics to clear infections involving biofilms that are refractory to current treatments.

Role of Appropriate Therapy in Combating Mortality among the Ventilated Patients.

CONTEXT Ventilator associated pneumonia (VAP) is a nosocomial infection prevalent among the intensive care unit (ICU) patients despite proper infection control practices. The diagnosis of VAP still remains controversial and hence the mortality rate is higher among this group of patients. AIM The aim of our study was to identify the antibiotic pattern and the appropriateness of treatment followed in the ICU in relation with the clinical pulmonary infection score (CPIS) as a tool to diagnose VAP. This was compared with patients who had an inappropriate treatment in comparison to the CPIS and the clinical outcome. RESULTS Out of the 18 VAP patients, 12 (66.7%) received appropriate therapy based on the antibiotic susceptibility pattern of the causative organism, while 1 (5.5%) received partially inappropriate therapy and 5 (27.8%) received totally inappropriate therapy. Nine of the 18 (50%) VAP patients died, while only 5 of the 58 (8.6%) patients without VAP died. 72.2% patients with VAP received appropriate treatment based on the sensitivity of the isolates. The mortality rate in VAP patients receiving inappropriate therapy was 80%, while in those receiving appropriate therapy the mortality rate was 38.5%. The mortality rate among VAP patients with blood culture positivity was 100%, while it was 43.75% among those with negative blood culture. CONCLUSION The mortality rate among the patients receiving inappropriate therapy is high compared to other group of patients. Hence, a proper evaluation and administration of appropriate antibiotics can curb mortality among the ventilated patients.

Multifocal keloids associated with Mycobacterium fortuitum following intralesional steroid therapy.

We report a case of subcutaneous abscess formation with Mycobacterium fortuitum following intralesional steroid injection into multifocal keloids. A high index of suspicion of atypical mycobacteria infection is needed in patients with a history of skin and soft tissue infections, in particular late-onset infections, which are negative for routine bacterial cultures and without a clinical response to antibiotics used for acute pyogenic infections.

Comparison of Microscopic Morphology of Fungi Using Lactophenol Cotton Blue (LPCB), Iodine Glycerol and Congo Red Formaldehyde Staining.

Sir, Although fungi were considered as innocuous environmental saprophyte, the significance of fungi as human pathogen is on the raise [1] . Hence, laboratory diagnosis of fungal infections has critical role in patient care. In this prospective study, which was carried out from March to September, 2013, we have evaluated the performance of iodine-glycerol in comparison to lactophenol cotton blue (LPCB) and Congo-red formaldehyde as an alternative method for identification of filamentous fungi. A total of 180 clinical samples, obtained from skin, hair, nail, sputum, broncho-alveolar lavage, corneal scrapings and tissue of various inpatient and outpatient departments of Mahatma Gandhi Medical College & Research Institute, Pondicherry, India were processed for fungi and included in this study. Direct microscopic examination was performed using potassium hydroxide (KOH) iodine glycerol and Congo red formaldehyde wet mounts [Table/Fig-1,​,22and​and3].3]. Culture was done in duplicate in SDA with and without cycloheximide at 25oC and 37oC incubation. [Table/Fig-1]: Tease mount preparation of fungal culture using LPCB showing penicillium sp [Table/Fig-2]: Formalin-Congo red-stained tease mount preparations of zygomyces showing aseptate hyphae [Table/Fig-3]: Wet preparation using iodine glycerol showing macroconidia of Microsporum spp Tease mount and slide cultures of 20 standard fungal strains (belonging to the genus Aspergillus, Penicillium, Fusarium, Curvuleria, Mucor, Rhizopus, Syncephalastrum, Trichophyton, Epidermophyton and Microsporum) and fungal clinical isolates grown on culture were stained by Congo red formaldehyde, LPCB and iodine-glycerol in an attempt to compare their staining characteristics. Iodine glycerol reagent was prepared in-house by addition of glycerol (0.25%) in iodine solution as described by Vignesh et al., [2] . Whereas for Congo red formaldehyde stain, 10% formaldehyde solution was diluted to 1% and Congo red was added to achieve 0.1% final concentration at pH 7.1 [3] . Wet preparations of Congo red formaldehyde were examined under fluorescence microscope. Fungal culture was considered as the gold standard test and sensitivity, specificity, positive predictive value and negative predictive value were calculated. Total 32 fungal isolates were recovered in culture [Table/Fig-4]. The performance of KOH, iodine glycerol and Congo red formaldehyde wet mounts in comparison to culture are expressed in terms of sensitivity, specificity, positive predictive value and negative predictive value and detailed in [Table/Fig-5]. Direct microscopic detection of fungal pathogens has immense value in routine mycology workup. Especially in case of opportunistic mycoses, isolates grown on culture often require direct microscopic confirmation to rule out the possibility of laboratory contamination [4] . KOH, in various concentrations, has been employed as a keratolytic agent to demonstrate fungi in tissue [5] . However, it does not stain fungal elements. Consequently, delicate semi-transparent fungal elements escape detection [6-8] . On the other hand, collagen fibers, tissue matrix, lipid vesicles, air bubbles, and textile fibers are often difficult to distinguish from fungi [6] . Several authors had reported low sensitivity and specificity of KOH mounts [6-8] . Saxena et al., reported 68% sensitivity and 40% specificity [6] . In our study, the sensitivity and specificity of KOH was 65.6% and 93.9% respectively. [Table/Fig-4]: Fungal isolate recovered in culture from clinical samples [Table/Fig-5]: Comparison of three methods for microscopic detection of fungi in clinical samples Fluorescent dyes which bind specifically to fungal cell wall have essential role in mycology. Calcofluor white was traditionally used as a fluorescent brightener in paper and textile industries [3,5] , has been extensively used to study cell walls of fungi and bacteria. The use of other fluorescent agents in mycology have been reported infrequently. Congo red is a diazo compound which is commonly used to stain amyloid in tissue sections [3] . Pertaining to its high affinity for cellulose or chitin, Congo red also permits the detection of fungal elements in tissue. Although Congo red is cost-effective compared to Calcofluor white, its use as fungal stain is still unconventional [3,9] . Congo red in conjunction with glutaraldehyde or formaldehyde, as fungicidal agent, had been reported to be useful for rapid detection fungi in both clinical specimens and fungal culture preparations [3] . We found that yeast cells and both septate and asepate hyphae in clinical specimens had bright orange fluorescence. In case of culture preparations of filamentous fungi, the cell walls, cross-walls of hyphae, as well as the hyphal tips and rhizoids were visible. However, the conidia of molds, Aspergillus, Penicillium and Fusarium species in particular, were indistinct and often not visible under fluorescent microscope. Slifkin et al., found good staining result of Congo red for a variety of yeasts and molds except the cell wall of Phialophora species and the capsule of Cryptococcus neoformans which were poorly stained [3] . In another study, Candida and Histoplasma isolates were found Congo red negative unlike Pityrosporum and Blastomyces isolates [9] . Although KOH and Congo red are easily available and inexpensive reagent, both may have potential health hazard. As per material safety data sheets, formaldehyde used in Congo red preparations is cytotoxic and carcinogenic [10] . Whereas, KOH solutions with concentrations higher than 2% are corrosive [11] . In a recent study, better microscopic morphology of fungi was obtained using iodineglycerol in comparison to LPCB [2] . In iodine-glycerol method, iodine acts as fungicide as well as an agent for staining fungal structures. Glycerol prevents rapid drying of the wet preparation. In contrast to phenol, KOH and formaldehyde, iodine has no potential biohazards [2] . Furthermore, while comparing microscopic morphology of fungi, best results were achieved with LPCB and iodine glycerol. Both preparations had more transparency and high clarity. Thin delicate hyphae and semitransparent spores were visualized clearly which allowed rapid identification. However, we observed that iodine-glycerol and Congo red formaldehyde preparations were unsatisfactory for detection of fungi in clinical samples. The sensitivity of iodine-glycerol and Congo red formaldehyde methods were 59.3% and 43.8% [Table/Fig-5]. This may be due to the inability of these two methods to visualize fungal elements embedded deeply in tissue as keratin and tissue materials were not digested by a keratolytic agent. Furthermore, Congo red formaldehyde method also had limitations of rapid fading of fluorescence on light exposure and drying. As a result, Congo red formaldehyde preparations were not suitable for long term preservation. In conclusion, Iodine-glycerol preparation was found to be a promising technique for identification fungal isolates which may be employed as a non-hazardous and safer alternative to LPCB for fungal identification.

Septic Arthritis caused by Group A Streptococcus in Newborn: An Unusual Presentation.

Streptococcal sepsis in neonates is a potentially lethal condition. A wide spectrum of clinical presentations has been often reported in Group B Streptococcal infections in neonates. Bone and joint infections which are caused by Group B Streptococcus are also encountered frequently, but they have not yet been reported in case of Group A Streptococcal infection in neonates. Here, we are reporting a case of septic arthritis and late onset neonatal sepsis which were caused by Group A Streptococcus in a full term, healthy baby.