Antifungal Susceptibility Profile of Clinical and Environmental Isolates of Aspergillus Species From a Tertiary Care Center in North India.

INTRODUCTION: Aspergillus species are ubiquitously found in the environment worldwide and are important causative agents for infection. Drug resistance among Aspergillus species is emerging, hence the present study was undertaken to look for antifungal susceptibility profiles of clinical and environmental isolates of Aspergillus species. MATERIALS AND METHODS: During the period from January 2018 to June 2019, a total of 102 Aspergillus isolates (40 clinical, 40 hospital, and 22 community environment) were tested for antifungal susceptibility testing for determination of minimum inhibitory concentration (MIC)/minimum effective concentration (MEC) as per Clinical and Laboratory Standards Institute (CLSI) M38-A3 method for itraconazole, voriconazole, amphotericin B, and caspofungin. RESULTS: Out of these 102 Aspergillus isolates, A. flavus was the most common species present. Aspergillus species were found to have low MIC values to azoles such as itraconazole and voriconazole except for one clinical isolate, which showed a MIC value of 2 µg/ml to voriconazole. Two isolates were non-wild-type for amphotericin B, but all isolates were wild-type for caspofungin. CONCLUSION: Antifungal susceptibility testing among clinical Aspergillus isolates and environmental surveillance studies in view of emerging drug resistance should be undertaken at a larger scale.

Phaeohyphomycosis: A study from tertiary health care center in North India.

Objectives: Phaeohyphomycosis refers to infections caused by phaeoid/dematiaceous or darkly pigmented fungi. This study was undertaken to further increase our knowledge about the incidence of phaeohyphomycosis and its causative agents. Materials and Methods: The present study was conducted over a period of one and a half years (January 2018-June 2019) on specimens received from patients with varied clinical manifestations ranging from superficial infections, subcutaneous cysts, pneumonia, brain abscess to a disseminated infection. These specimens were processed in the Department of Microbiology for potassium hydroxide (KOH) examination and culture and in Pathology for cytology/histopathological examination (HPE). All specimens positive on direct examination for dark grey, brown or black fungi were included in the study. Results: A total of 20 specimens were confirmed as phaeohyphomycosis. Most of the patients belonged to the age group of 41 to 50 years. Male: Female ratio was 2.3:1. Trauma was the most common risk factor. Spectra of the isolated fungal pathogens comprised of Bipolaris species, Exophiala species, Curvularia geniculata, Phialemonium species, Daldinia eschscholtzii, Hypoxylon anthochroum, Phaeoacremonium species, Leptosphaerulina australis, Medicopsis romeroi, Lasiodiplodia theobromae, Eutypella species, Chaetomium globosum, Alternaria species, Cladophialophora bantiana and 2 unidentified dematiaceous fungi. Recovery from phaeohyphomycosis was seen in 12 patients, 7 were lost to follow up and one patient succumbed to the illness. Conclusion: Infections caused by phaeoid fungi can no longer be viewed as rare. In fact, phaeohyphomycosis can have myriad of presentations spanning from mild cutaneous infections to fatal brain disease. Therefore, a high index of clinical suspicion is needed to diagnose such infections. The primary treatment modality remains surgical removal of the lesion in cutaneous or subcutaneous infections however disseminated disease with a guarded prognosis requires aggressive management.

Fatal Clostridium sporogenes Soft Tissue Polymicrobial Infections in Two Immunocompetent Cases: Case Report.

BACKGROUND: Clostridium sporogenes is reported rarely in literature. Reports from the skin and soft tissue infections are even less, more so in immunocompetent patients. CASE PRESENTATION: Two skin and soft tissue infections with C. sporogenes in immunocompetent patients have been presented in this study. One of the cases was following an electrical burn wound, and the other was following a bedsore. Both patients expired despite antibacterial treatment and debridement. DISCUSSION AND CONCLUSION: C. sporogenes had usually been reported after trauma particularly after penetrating and deep wound infection. More attention should be given to these patients so that the infection can be treated and diagnosed early in suspected anaerobic infections like Clostridium species.

Acrophialophora fusispora as an Agent of Mycotic Keratitis: A Case Report and Review of Literature.

BACKGROUND: Acrophialophora species is an infrequent human opportunistic pathogen. It is widely distributed in temperate as well as tropical regions. Here, we present a rare case of fungal keratitis caused by A. fusispora. CASE PRESENTATION: A 26-year male driver presented with pain, watering, redness, whitish discoloration, and blurring of vision in the left eye for the last 3-4 days. Upon examination, he had a dry-looking corneal ulcer with infiltration and satellite lesions. Corneal scrapings were positive for septate fungal hyphae by Gram staining and KOH mount. After five days, the growth observed was presumptively identified as genus Acrophialophora and finally identified as Acrophialophora fusispora by genetic sequencing. The patient failed to respond medically and was planned for therapeutic keratoplasty. DISCUSSION: To date, four cases of ocular involvement due to Acrophialophora have been described. Amongst which one case was associated with an immunocompromised state. Three of the cases were resolved medically, while one required therapeutic keratoplasty, indicating possible strong pathogenicity to the eye. CONCLUSION: As Acrophialophora seems to have a predilection for eye infections, an early diagnosis with timely appropriate treatment is the best way to restore the normal vision of a patient.

Species Distribution and Antifungal Susceptibility Profile of Dermatophytes from a Tertiary Care Centre in North India.

Objective Dermatophytoses, one of the most ancient diseases, is becoming a menace in recent times. This has made the knowledge of antifungal susceptibility a priority in today's times. Material and Methods This is a prospective study conducted over 18 months including all dermatophytes isolated during the period. Dermatophytes were identified by routine phenotypic methods. Antifungal susceptibility testing was performed for griseofulvin, terbinafine, and itraconazole as per the Clinical Laboratory Standard Institute M38 A2, and minimum inhibitory concentrations (MICs) were read after 5 days. Results Patient details and associated risk factors were recorded. Fixed dose combinations with steroids were associated with 79.3% (46 out of 58) of patients with dermatophytosis of skin. Among the 72 dermatophytes isolated during the study period, 58 (80.5%) were isolated from skin scrapings and 14 (19.4%) from nail samples. Tinea corporis with cruris was the most common presentation. The most common dermatophyte isolated from skin scrapings was Trichophyton mentagrophytes complex (70.6%, 41 out of 58), while from nail samples it was Trichophyton rubrum complex (78.57%, 11 out of 14). Based on the MIC 50 and MIC 90 results, itraconazole showed the lowest MICs, followed by terbinafine and then griseofulvin. Conclusion With the changing epidemiology of species distribution and antifungal resistance, there is a need for continuous surveillance of these parameters of dermatophytes.

Epidemiological profile and antifungal susceptibility pattern of Trichosporon species in a tertiary care hospital in Chandigarh, India.

BACKGROUND AND PURPOSE: Trichosporon species are ubiquitous in nature which are associated with fatal opportunistic invasive infections, especially in immunocompromised patients. The present study aimed to evaluate the epidemiological and clinical details, as well as the antifungal susceptibility pattern of the patients with Trichosporon infections. MATERIALS AND METHODS: In total, 50 clinical isolates of Trichosporon species from various samples were included in this study. The samples were isolated over a period of 18 months from patients in a tertiary hospital in North India. The isolates were characterised phenotypically with Vitek MS (bioMérieux, France). Trichosporon spp. were isolated from urine (30%), nail (30%), tissue (16%), pleural fluid (14%), and sputum (5%). In total, majority of the isolates were of Trichosporon asahii (92%), followed by Trichosporon mucoides (6%), and Trichosporon ovoides (2%). It is noteworthy that most of the reported cases were from intensive care unit (34%). RESULTS: Intravenous catheters, antibiotics, and antifungal uptake were significantly associated risk factors with Trichosporon infection. All invasive isolates were observed to be resistant in vitro to caspofungin and exhibited high minimum inhibitory concentration (MIC) values against amphotericin B, fluconazole, and 5-flucytosine. The MICs for voriconazole and posaconazole were low. CONCLUSION: Trichosporonosis is being increasingly reported all around the world, including India. The results of this study highlighted the importance of early detection and treatment for this emerging yeast and also added to the ongoing surveillance for the antifungal susuceptibility pattern for this fungus.

A Comprehensive Review of Trichosporon spp.: An Invasive and Emerging Fungus.

Trichosporon species are basidiomycetous yeast-like organisms found ubiquitous in nature. They are increasingly been recognized as opportunistic pathogens capable of causing life-threatening invasive diseases (trichosporonosis), especially in immuno-suppressed patients and rarely in immuno-competent patients too. Earlier multiple members of the genus Trichosporon were clubbed together as T. beigelli but after the advent of molecular techniques, more than 50 different subspecies and around 16 different strains causing human diseases are reported. It is known to cause a wide range of diseases, from superficial to probable and proven invasive diseases to summer hypersensitivity. The ability of Trichosporon strains to form biofilms on implanted devices, glucuronoxylomannan in their cell walls, and production of proteases and lipases lead to the virulence of this genus. This ubiquitous fungus exhibits intrinsic resistance to echinocandins, variable minimum inhibitory concentrations (MIC) for amphotericin B, and moderate susceptibility to fluconazole and Itraconazole, which are the commonly used anti-fungal agents for any invasive fungal infections which lead to the re-emergence of this notorious yet neglected pathogen and hence the reports of breakthrough infections among patients receiving these antifungals. This review is to understand the epidemiological, clinical details, and antifungal susceptibility pattern of various Trichosporon infections and it highlights the importance of early detection and treatment for this emerging yeast and also will add to the ongoing surveillance for the anti-fungal susceptibility pattern for this fungus.

Aspergillus candidus eumycetoma with review of literature.

OBJECTIVE: Mycetoma is a slowly progressive, chronic granulomatous infection of skin and subcutaneous tissues with involvement of underlying fasciae and bones, usually affecting extremities. In India, among mycetoma, the eumycetoma constitutes about 35% of cases. Hereby, we report the first case of eumycetoma caused by uncommon fungus, Aspergillus candidus. CASE REPORT: A 61 year old female presented to the Department of Dermatology with history of swelling of right foot associated with multiple firm nodules of approx. 1.5 × 1.5 cm each over dorsum of foot with discharging sinus containing white color granules. Biopsy of lesion on right foot on direct KOH examination revealed septate hyphae. Histopathology examination showed a histiocytic granuloma with fungal elements. Culture on Sabouraud Dextrose Agar grew white mycelial colonies which were identified to be Aspergillus species phenotypically. Genetic sequencing using Internal transcribed spacer gene, beta tubulin gene and Calmodulin gene was done and the isolate was identified to be Aspergillus candidus. Lesion was excised and patient was started on itraconazole. CONCLUSION: Timely identification and starting of antifungal treatment can help in reducing morbidity due to eumycetoma to a large extent. As newer and newer species of fungi are emerging as significant causative agents of human infections, it is pertinent to report such findings from epidemiological point of view.

Nocardia Infections: Ten Years Experience from a Tertiary Health Care Center in North India (2007-2016).

BACKGROUND: Nocardia species are important cause of infections in humans but are underreported due to missed diagnosis as well as misdiagnosis. Majority of the literature on these infections consists of case reports or series with few articles describing high number of cases. OBJECTIVE: To study the epidemiology of Nocardia infections in a tertiary care center. MATERIALS AND METHODS: This retrospective observational study was done in a tertiary care centre of North India over a period of 10 years (2007-2016). The detection of Nocardia spp. from clinical specimens was done by conventional methods viz. direct microscopy (Gram's stain, modified Ziehl -Neelsen stain [1%], KOH examination) and culture. RESULTS: A total of 25 cases of nocardiosis were diagnosed during the study period. The mean age of the patients was 50.9 years (range 30-72 years) with a male:female ratio of 3:2. The site of disease in these patients included pulmonary (n=18), cutaneous (n=4), perinephric abscess (n=1), ocular (n=1) and bone (n=1). Risk factors associated were underlying lung disease (n=11), smoking (n=7), diabetes (n=5) and steroid therapy (n=4) in pulmonary nocardiosis, iatrogenic (n=1) and leprosy (n=1) in cutaneous nocardiosis, diabetes in perinephric abscess and cataract surgery in ocular nocardiosis. Culture was positive in 12/25 (48%) patients for Nocardia spp. Direct microscopy was positive in 22 patients. We wish to highlight that meticulous observation of KOH wet mount examination helped in clinching the diagnosis of Nocardiosis in 3 cases which were earlier missed by other methods. CONCLUSION: Good communication with the clinician alongside a meticulous effort in the laboratory is essential for appropriate diagnosis and management of these cases.

Linezolid Resistance in Staphylococcus haemolyticus - Case Series and Review of Literature.

BACKGROUND: Coagulase negative Staphylococci (CoNS) are important. The common antibiotics used for the treatment of the infections caused by CoNS are penicillin, oxacillin, ciprofloxacin, clindamycin, erythromycin, gentamicin, and vancomycin. Linezolid is an oxazolidinone group of antibiotic with activity against Gram-positive bacteria. It is used for the treatment of serious infections caused by Gram-positive bacteria resistant to other antibiotics, including streptococci, vancomycin-resistant enterococci (VRE). AIMS AND OBJECTIVE: This study emphasizes on the judicious use of newer antibiotics to contain the spread of resistance. METHOD: We are discussing five cases of Linezolid resistant Staphylococcus Haemolyticus which were reported in our laboratory during one year from patients with device related infections and also review of literature is being presented for an update. RESULT: In our study, the isolates were resistant to other groups of antimicrobials but susceptible to glycopeptides. All the isolates were methicillin-resistant. CONCLUSION: Linezolid is approved as an alternative drug to be given for catheter-related bloodstream infections. In earlier studies, linezolid-resistant staphylococci have been reported increasingly all over the world. This study is to create awareness amongst clinicians that improper and excessive use of linezolid will make this antibiotic-resistant and thus will be of no help in future, so judicious and relevant use of antibiotics needs to be emphasized.

Assessment of tumor growth in pancreatic neuroendocrine tumors in von Hippel Lindau syndrome.

BACKGROUND: The incidence of pancreatic neuroendocrine tumors (PNETs) is increasing, but only a subset of these heterogeneous tumors will progress to malignant disease, which is associated with a poor prognosis. Currently, there are limited data on the natural history of these tumors and it is difficult to determine which patients require surgical intervention because the risk of metastatic disease cannot be accurately determined. STUDY DESIGN: We conducted a prospective study of 87 patients with von Hippel Lindau syndrome-associated solid pancreatic lesions to determine the natural history of these tumors with biochemical testing, follow-up anatomic and functional imaging, and advanced imaging analysis, with a median follow-up of 4 years. RESULTS: Approximately 20% of consecutive tumor measurements during follow-up were decreased in size and 20% showed no change. This included 2 of 4 surgically proven malignant tumors, which had a net decrease in tumor size over time. Tumor volume, as derived from greatest diameter and volumetric measurements, showed good correlation to pathology tumor measurement of surgically resected tumors (Spearman rank correlation ρ = 0.72, p = 0.0011, and ρ = 0.83, p < 0.0001, respectively). Tumor density measurement had an inverse relationship with tumor size (Spearman rank correlation -0.22, p = 0.0047). A tumor density cutoff of 200 was 75% specific for malignant tumors. CONCLUSIONS: Pancreatic neuroendocrine tumors demonstrate a nonlinear growth pattern, which includes periods of no growth and apparent decrease in size by imaging. These growth patterns are variable and are not associated with tumor grade and malignancy. Tumor density, as measured in this cohort, may offer a specific diagnostic tool for malignant disease.

MiR-34a and miR-483-5p are candidate serum biomarkers for adrenocortical tumors.

BACKGROUND: Nonfunctioning adrenal incidentalomas are common and many patients undergo adrenalectomy to exclude adrenocortical carcinoma (ACC). Recent studies have shown dysregulated microRNA (miRNA) expression in ACC. The objective of this study was to determine the feasibility and diagnostic accuracy of measuring serum miRNAs in patients with benign and malignant adrenocortical neoplasms. METHOD: Five miRNAs were selected from miRNA profiling studies in ACC (miR-let-7d, -34a, -195, -214, and 483-5p). Total miRNA was extracted from serum samples in patients with malignant and benign adrenal neoplasms. miRNAs levels were measured by quantitative reverse transcript polymerase chain reaction and normalized to miR-16. To determine if miRNAs were secreted from ACC cells, we measured miRNA levels in culture. RESULTS: Serum samples from 22 patients with cortical adenomas and 17 patients with ACC were analyzed, and all 5 miRNAs were detected. We found greater levels of miR-34a (P = .001) and miR-483-5p (P = .011) in patients with ACC. The area under the receiver operating characteristic curve was 0.81 for miR-34a and 0.74 for miR-438-5p. MiR-34a and miR-483-5p levels in ACC cells were greater in the supernatant at 48 hours compared with intracellular levels. CONCLUSION: We show that dysregulated miRNAs in ACC are detectable in human serum samples. MiR-34a and miR-483-5p are candidate serum biomarkers for distinguishing between benign and malignant adrenocortical tumors.

Prospective screening in familial nonmedullary thyroid cancer.

BACKGROUND: Approximately 8% of nonmedullary thyroid cancers are familial. The optimal age for screening in familial nonmedullary thyroid cancer (FNMTC) is unknown. METHODS: Kindreds with FNMTC (2 or more first-degree relatives affected) were screened prospectively with thyroid ultrasonography. RESULTS: Fifteen kindreds showed an overall prevalence of thyroid nodule(s) ≥5 mm of 44% at screening; 19% in the second generation, and 90% in the generation anterior to the index case. The youngest age of detection was 10 years for thyroid nodules and 18 years for thyroid cancer. Microcalcification of thyroid nodules at screening was associated with a greater risk of cancer (P < .05). Family members diagnosed with thyroid cancer by ultrasonographic screening were diagnosed at a younger age and had a lower rate of extra thyroidal invasion (P < .05). CONCLUSION: In FNMTC, first-degree relatives 10 years or older, including the generation anterior to the index case, should have thyroid screening by ultrasonography, which may result in earlier diagnosis.

Telomere length is shorter in affected members of families with familial nonmedullary thyroid cancer.

BACKGROUND: The theory that short telomere length and genetic defects in maintaining telomere length are associated with familial nonmedullary thyroid cancer (FNMTC) is controversial. Thus, the aim of this study was to determine whether telomere length and genes involved in maintaining telomere length are altered in FNMTC. METHODS: Blood samples were collected from 44 members (13 affected and 31 unaffected) of six families with FNMTC and from 60 controls. Quantitative polymerase chain reaction (Q-PCR) and reverse transcription PCR were performed to analyze relative telomere length (RTL), gene copy number, and mRNA expression of telomerase reverse transcriptase (hTERT), telomere repeat binding factor 1 (TRF1), telomere repeat binding factor 2 (TRF2), repressor activator protein 1 (RAP1), TRF1 interacting nuclear factor 2 (TIN2), tripeptidyl peptidase 1 (TPP1), and protection of telomere 1 (POT1). RESULTS: Affected members had shorter RTL, as compared with unaffected members (0.98 vs. 1.23, p<0.01). There was no significant difference in hTERT, TRF1, TRF2, RAP1, TIN2, TPP1, and POT1 gene copy number or mRNA expression between affected and unaffected members. CONCLUSIONS: RTL is shorter in affected members with FNMTC but is not associated with altered copy number or expression in hTERT, TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. The small differences in RTL preclude the utility of RTL as a marker for FNMTC in at-risk individuals.

AmpC ß-lactamases in nosocomial isolates of Klebsiella pneumoniae from India.

BACKGROUND & OBJECTIVES: AmpC ß-lactamases are clinically significant since these confer resistance to cephalosporins in the oxyimino group, 7-α methoxycephalosporins and are not affected by available ß-lactamase inhibitors. In this study we looked for both extended spectrum ß-lactamases (ESBL) and AmpC ß-lactamases in Klebsiella pneumoniae clinical isolates. METHODS: One hundred consecutive, non-duplicate clinical isolates of K. pneumoniae collected over a period of one year (June 2008 - June 2009) were included in the study. An antibiotic susceptibility method was used with 10 antibiotics for Gram-negative infections which helped in screening for ESBL and AmpC ß-lactamases and also in confirmation of ESBL production. The detection of AmpC ß-lactamases was done based on screening and confirmatory tests. For screening, disc diffusion zones of cefoxitin <18 mm was taken as cefoxitin resistant. All cefoxitin resistant isolates were tested further by AmpC disk test and modified three dimensional test. Multiplex-PCR was performed for screening the presence of plasmid-mediated AmpC genes. RESULTS: Of the 100 isolates of K. pneumoniae studied, 48 were resistant to cefoxitin on screening. AmpC disk test was positive in 32 (32%) isolates. This was also confirmed with modified three dimensional test. Indentation indicating strong AmpC producer was observed in 25 isolates whereas little distortion (weak AmpC) was observed in 7 isolates. ESBL detection was confirmed by a modification of double disk synergy test in 56 isolates. Cefepime was the best cephalosporin in synergy with tazobactam for detecting ESBL production in isolates co-producing AmpC ß-lactamases. The subsets of isolates phenotypically AmpC ß-lactamase positive were subjected to amplification of six different families of AmpC gene using multiplex PCR. The sequence analysis revealed 12 CMY-2 and eight DHA-1 types. INTERPRETATION & CONCLUSIONS: Tazobactam was the best ß-lactamase inhibitor for detecting ESBL in presence of AmpC ß-lactamase as this is a very poor inducer of AmpC gene. Amongst cephalosporins, cefepime was the best cephalosporin in detecting ESBL in presence of AmpC ß-lactamase as it is least hydrolyzed by AmpC enzymes. Cefepime-tazobactam combination disk test would be a simple and best method in detection of ESBLs in Enterobacteriaceae co-producing AmpC ß-lactamase in the routine diagnostic microbiology laboratories.

Candida colonization in urine samples of ICU patients: determination of etiology, antifungal susceptibility testing and evaluation of associated risk factors.

The presence of Candida in urine presents a therapeutic challenge for the physician as it is often asymptomatic, and management guidelines have not been clearly laid down on this issue. The presence of Candida in urine may represent contamination of clinical sample, actual colonization of the lower urinary tract or may be a true indicator of invasive infection of lower and/or upper urinary tract. In a clinical setting like the ICU, multiple risk factors for Candida colonization may be present in the same patient, thereby increasing the chances of candiduria, manifold. In the present study on 80 patients in ICU, high rate of Candida colonization (57.5%) was found in urine samples of ICU patients with C. tropicalis (57.3%) being the predominant species. We also isolated 8 strains of Trichosporon species, all of these presented as a mixed infection along with Candida species. Among the various risk factors studied, urinary catheterization and previous antibiotic therapy were identified as statistically significant (P value <0.05). The minimum inhibitory concentration of the isolates was determined for amphotericin B, fluconazole and itraconazole by E-test. Most of the isolates were susceptible to amphotericin B. The C. parapsilosis strains did not show any drug resistance; however, resistance to fluconazole was observed 18.6, 27.27, 50 and 25% in C. tropicalis, C. albicans, C. glabrata and Trichosporon species, respectively.

Evaluation of various methods for the detection of meticillin-resistant Staphylococcus aureus strains and susceptibility patterns.

Meticillin-resistant Staphylococcus aureus (MRSA) has been recognized as one of the major pathogens in hospital as well as community settings. In India, the mean isolation rate of MRSA is 20-40 % and many studies have suggested an escalating rate of infections caused by this organism. Despite pharmaceutical and technological advancement, infections caused by MRSA still remain difficult to diagnose. The present study was undertaken to compare five phenotypic methods for the detection of MRSA. This involved examining 200 isolates of S. aureus by oxacillin disc diffusion, cefoxitin disc diffusion, oxacillin screen agar test, the latex agglutination test and growth on CHROMagar. PCR for mecA gene detection was taken as the gold standard. It was found that 35 % of all S. aureus infections were caused by MRSA. The cefoxitin disc diffusion method, as recommended by the Clinical and Laboratory Standards Institute, was found to be a reliable method for MRSA detection but it should be supplemented with some other method like latex agglutination, CHROMagar or oxacillin screen agar testing so that no MRSA is missed. We recommend that along with cefoxitin disc diffusion, another method, preferably latex agglutination, should be routinely used in all hospitals to detect MRSA.

Fusarium sacchari: a cause of exogenous fungal endophthalmitis: first case report and review of literature.

A case of exogenous fungal endophthalmitis due to Fusarium sacchari is being reported. It has never been reported in this clinical entity before; hence, to the best of our knowledge, this is first report of F. sacchari as a cause of exogenous fungal endophthalmitis and also the first time when this fungus has been isolated from an Indian patient.