A multicentre observational study on the epidemiology, risk factors, management and outcomes of mucormycosis in India.

OBJECTIVES: To describe the epidemiology, management and outcome of individuals with mucormycosis; and to evaluate the risk factors associated with mortality. METHODS: We conducted a prospective observational study involving consecutive individuals with proven mucormycosis across 12 centres from India. The demographic profile, microbiology, predisposing factors, management and 90-day mortality were recorded; risk factors for mortality were analysed. RESULTS: We included 465 patients. Rhino-orbital mucormycosis was the most common (315/465, 67.7%) presentation followed by pulmonary (62/465, 13.3%), cutaneous (49/465, 10.5%), and others. The predisposing factors included diabetes mellitus (342/465, 73.5%), malignancy (42/465, 9.0%), transplant (36/465, 7.7%), and others. Rhizopus species (231/290, 79.7%) were the most common followed by Apophysomyces variabilis (23/290, 7.9%), and several rare Mucorales. Surgical treatment was performed in 62.2% (289/465) of the participants. Amphotericin B was the primary therapy in 81.9% (381/465), and posaconazole was used as combination therapy in 53 (11.4%) individuals. Antifungal therapy was inappropriate in 7.6% (30/394) of the individuals. The 90-day mortality rate was 52% (242/465). On multivariate analysis, disseminated and rhino-orbital (with cerebral extension) mucormycosis, shorter duration of symptoms, shorter duration of antifungal therapy, and treatment with amphotericin B deoxycholate (versus liposomal) were independent risk factors of mortality. A combined medical and surgical management was associated with a better survival. CONCLUSIONS: Diabetes mellitus was the dominant predisposing factor in all forms of mucormycosis. Combined surgical and medical management was associated with better outcomes. Several gaps surfaced in the management of mucormycosis. The rarer Mucorales identified in the study warrant further evaluation.

Banana peel culture as an indigenous medium for easy identification of late-sporulation human fungal pathogens.

AIM: Fungi are increasing in incidence as human pathogens and newer and rarer species are continuously being encountered. Identifying these species from growth on regular culture media may be challenging due to the absence of typical features. An indigenous and cheap medium, similar to the natural substrate of these fungi, was standardised in our laboratory as an aid to species identification in a conventional laboratory setting. MATERIALS AND METHODS: Ripe banana peel pieces, sterilised in an autoclave at 121°C temperature and 15 lbs pressure for 15 min promoted good growth of hyphae and pycnidia or acervuli in coelomycetes, flabelliform and medusoid fruiting bodies of basidiomycetes and fruit bodies such as cleistothecium in ascomycetes. The growth from the primary isolation medium was taken and inoculated onto the pieces of double-autoclaved ripe banana peel pieces in a sterile glass Petri dish with some moisture (sprinkles of sterile distilled water). A few sterile coverslips were placed randomly inside the Petri dish for the growing fungus to stick on to it. The plates were kept at room temperature and left undisturbed for 15-20 days. At a time, one coverslip was taken out and placed on a slide with lactophenol cotton blue and focused under the microscope to look for fruit bodies. RESULTS: Lasiodiplodia theobromae, Macrophomina phaseolina, Nigrospora sphaerica, Chaetomium murorum, Nattrassia mangiferae and Schizophyllum commune were identified by characteristic features from growth on banana peel culture. CONCLUSIONS: Banana peel culture is a cheap and effective medium resembling the natural substrate of fungi and is useful for promoting characteristic reproductive structures that aid identification.

Application of PCR fingerprinting using (GACA)4 primer in the rapid discrimination of dermatophytes.

BACKGROUND: Superficial fungal infections have a major impact on cosmetic health, affecting more than 20-25% of the global population, which is predominantly caused by dermatophytes. As per literature search, molecular strain typing of dermatophytes has not been investigated in India. Therefore, the present study was carried out to characterise the dermatophyte species and strains by molecular methods. OBJECTIVE: To analyse the genotype variability by applying polymerase chain reaction (PCR) fingerprinting using a simple sequence repetitive oligonucleotide (GACA)4 primer to identify the species and strain variations among the dermatophytes isolated from a tertiary care centre in Chennai. MATERIALS AND METHODS: From January 2010 to December 2010, 81 dermatophytes were isolated and included for the present study. A simple sequence repetitive oligonucleotide (GACA)4 was used as a single primer in the amplification process. RESULTS: The (GACA)4-based PCR successfully amplified all the clinical isolates. Trichophyton rubrum and T. rubrum var. raubitschekii produced identical band profiles, where the latter could not be differentiated from the T. rubrum, which are being reported for the first time from south India. Epidermophyton floccosum produced species-specific band profiles. Intra-species variability was not observed among the T. rubrum and E. floccosum isolates. T. mentagrophytes produced three simple, distinct band patterns, which are surprisingly different from the earlier studies. CONCLUSION: The PCR-based genotype using the short primer is rapid and precise in direct identification of dermatophyte isolates by one-step PCR to the species level and strain discrimination of the T. mentagrophytes variants.

Improvement in the outcome of invasive fusariosis in the last decade.

Invasive fusariosis (IF) has been associated with a poor prognosis. Although recent series have reported improved outcomes, the definition of optimal treatments remains controversial. The objective of this study was to evaluate changes in the outcome of IF. We retrospectively analysed 233 cases of IF from 11 countries, comparing demographics, clinical findings, treatment and outcome in two periods: 1985-2000 (period 1) and 2001-2011 (period 2). Most patients (92%) had haematological disease. Primary treatment with deoxycholate amphotericin B was more frequent in period 1 (63% vs. 30%, p <0.001), whereas voriconazole (32% vs. 2%, p <0.001) and combination therapies (18% vs. 1%, p <0.001) were more frequent in period 2. The 90-day probabilities of survival in periods 1 and 2 were 22% and 43%, respectively (p <0.001). In period 2, the 90-day probabilities of survival were 60% with voriconazole, 53% with a lipid formulation of amphotericin B, and 28% with deoxycholate amphotericin B (p 0.04). Variables associated with poor prognosis (death 90 days after the diagnosis of fusariosis) by multivariable analysis were: receipt of corticosteroids (hazard ratio (HR) 2.11, 95% CI 1.18-3.76, p 0.01), neutropenia at end of treatment (HR 2.70, 95% CI 1.57-4.65, p <0.001), and receipt of deoxycholate amphotericin B (HR 1.83, 95% CI 1.06-3.16, p 0.03). Treatment practices have changed over the last decade, with an increased use of voriconazole and combination therapies. There has been a 21% increase in survival rate in the last decade.

Candidemia in intensive care unit patients: a one year study from a tertiary care center in South India.

BACKGROUND: The occurrence of candidemia is on a rise worldwide. Non-albicans Candida species have emerged as major causes of candidemia in many countries. Added to it is the problem of antifungal resistance in Candida isolates. OBJECTIVES: To find out the prevalence of candidemia in our intensive care unit (ICU) setup along with the antifungal susceptibility pattern of Candida isolates and various risk factors associated with candidemia. MATERIALS AND METHODS: All Candida isolates from blood stream infections of ICU patients were included in the 1 year study period (November 2008-October 2009). The isolates were speciated using various phenotypic tests. Antifungal susceptibility testing was done by disk diffusion methods according to Clinical and Laboratory Standards Institute guidelines and also using CANDIFAST. Various risk factors associated with the development of candidemia were looked into. RESULTS: A total of 39 Candida isolates were isolated during the study period of 1 year (prevalence of 0.65%). Candida tropicalis (74.35%) was the most common isolate followed by Candida albicans, Candida parapsilosis, Candida krusei and Candida glabrata. All the 39 Candida isolates (100%) were sensitive to amphotericin B while 12 isolates (30.8%) were resistant to fluconazole. The risk factors commonly associated with candidemia patients were long term antibiotic therapy (64.1%), use of central venous catheters (56.4%), urinary catheters (53.9%), steroid therapy (35.9%) and diabetes mellitus (33.3%). CONCLUSION: Candidemia is emerging as a significant problem in hospitalized patients, especially in ICU setups. Non-albicans Candida species are the major cause of candidemia as found in our study and few other studies in India. Multicentric studies involving many hospitals are required to know the true prevalence of candidemia and the status of antifungal drug resistance among Candida isolates in our country.

Unusual causes of fungal rhinosinusitis: a study from a tertiary care centre in South India.

PURPOSE: The frequency of mycotic infections of the nose and paranasal sinuses has been increasing over the past three decades. Apart from the common causes of fungal rhinosinusitis such as Aspergillus species and Penicillium species, there have been reports of rare and unusual fungi isolated from India and other countries. OBJECTIVE: The objective of this study is to find out the prevalence of fungal infections of the nose and paranasal sinuses caused by unusual fungal isolates at a tertiary care teaching hospital in South India. MATERIALS AND METHODS: Duration of the study period was from April 2009 to March 2010. Specimens were collected from the nose and paranasal sinuses of all clinically and radiologically diagnosed cases of rhinosinusitis. All the clinical specimens were processed by standard methods for fungal culture. This included initial screening by 10% potassium hydroxide, inoculation of the specimen onto Sabouraud dextrose agar and incubation at 25°C and 37°C, followed by slide culture and other special techniques wherever necessary. Histopathological examination was also performed for the specimens. RESULTS: A total of 60 specimens were received for fungal culture from cases of rhinosinusitis during the period, out of which 45 showed no growth. There were nine cases of Aspergillus flavus, 1 each of Aspergillus fumigatus and Penicillium species. The rest four specimens grew rare fungal isolates, i.e. Acremonium sp., Scedosporium apiospermun, Cladosporium cladosporioides and Lasiodiplodia theobromae. Histopathological findings were also positive for these four cases. CONCLUSION: Apart from the common causes, unusual fungal pathogens were isolated from cases of rhinosinusitis during the study period, which is in accordance with similar reports from other parts of India and some other countries.

A review of Candida species causing blood stream infection.

The incidence of candidemia has been on a rise worldwide. The epidemiology of invasive fungal infections in general and of candidemia in particular has changed in the past three decades because of a variety of factors like the AIDS epidemic, increased number of patients receiving immunosuppressive therapy for transplantation and the increasing use of antimicrobials in the hospital setups and even in the community. The important risk factors for candidemia include use of broad-spectrum antimicrobials, cancer chemotherapy, mucosal colonization by Candida species, indwelling vascular catheters like central venous catheters, etc. More than 90% of the invasive infections due to Candida species are attributed to five species-Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei. However, the list of new species of Candida isolated from clinical specimens continues to grow every year. Early diagnosis and proper treatment is the key for management of candidemia cases.

Trichosporon inkin, an unusual agent of fungal sinusitis: a report from south India.

The aetiology of fungal sinusitis is diverse and changing. Aspergillus species has been the most common cause for fungal sinusitis, especially in dry and hot regions like India. Trichosporon species as a cause for fungal sinusitis has been very rarely reported the world over. Here, we report a rare case of allergic fungal sinusitis caused by Trichosporon inkin in a 28-year-old immunocompetent woman. Bilateral nasal obstruction, nasal discharge and loss of smell were her presenting complaints. Diagnostic nasal endoscopy showed bilateral multiple polyps. Functional endoscopic sinus surgery was performed and many polyps were removed. Based on mycological and histopathological studies, the pathogen was identified as T. inkin.

Dentinal tubule disinfection with 2% chlorhexidine gel, propolis, morinda citrifolia juice, 2% povidone iodine, and calcium hydroxide.

AIM: To investigate the antimicrobial activity of 2% chlorhexidine gel, propolis, Morinda citrifolia juice (MCJ), 2% povidone Iodine (POV-I), and calcium hydroxide on Enterococcus faecalis-infected root canal dentine at two different depths (200 microm and 400 microm) and three time intervals (day 1, 3 & 5). METHODOLOGY: One hundred and eighty extracted human teeth were infected for 21 days with E. faecalis. Samples were divided into six groups. Group I (Saline) (Negative control), Group II (Propolis), Group III (MCJ), Group IV (2% Povidone Iodine), Group V (2% Chlorhexidine Gel), Group VI (Calcium hydroxide). At the end of 1, 3, and 5 days, the remaining vital bacterial population was assessed. Dentine shavings were collected at two depths (200 microm and 400 microm), and total numbers of colony forming units were determined. The values were analysed statistically with one-way analysis of variance followed by Tukey multiple comparison test. The paired t-test was used to check for differences in growth at different time intervals within groups and for differences at the two depths (P < 0.01) RESULTS: The number of colony-forming units was statistically significant in all groups compared to the control group (Saline). Group V (chlorhexidine gluconate) (100%) produced better antimicrobial efficacy followed by 2% POV-I (87%), propolis (71%), MCJ (69%), and calcium hydroxide (55%). There was no significant difference between propolis and MCJ and no significant difference between data at 200 microm and 400 microm. CONCLUSION: Propolis and MCJ were effective against E. faecalis in dentine of extracted teeth.

Fungal infections in the soft tissue: a study from a tertiary care center.

Isolated fungal soft-tissue infections are uncommon but may cause severe morbidity or mortality, especially among immunosuppressed patients. In this study, a total of 56 soft-tissue specimens from patients with clinical suspicion of fungal infection collected at a tertiary care centre in Chennai during the period December 2005 to May 2007 were evaluated. Among the culture positives, majority were from diabetic patients. Among the 34 culture positives, the isolates consisted of Candida 12, Fusarium 4, Rhizopus 1 and Aspergillus 3 one each of Absidia corymbifera and Apophysomyces elegans. Treatment of fungal soft-tissue infection requires a team approach of surgeons, pathologists and microbiologists.

Maxillary sinusitis caused by Lasiodiplodia theobromae.

Lasiodiplodia (monotypic) comprises a very small proportion of the fungal biota. It is a common plant pathogen in tropical and subtropical regions. Clinical reports on its association with onychomycosis, corneal ulcer and phaeohyphomycosis are available. However, Lasiodiplodia theobromae causing fungal sinusitis has not been reported. We present here a case of fungal sinusitis in a 30-year-old woman, who came to the ENT OPD (out patient department) with complaints of intermittent bleeding and nasal discharge from the left side for a week. The patient complained of headache, predominantly on the left side and heaviness on and off since two months. Diagnosis was based on radiological and mycological evidence; the patient underwent endoscopic surgery and was started on antifungal treatment.

Nattrassia mangiferae causing fungal keratitis.

We report a case of fungal keratitis caused by the coelomycetous fungus Nattrassia mangiferae in a 70 year old gentleman, agriculturist by occupation, with a history of injury to his right eye. The scraping showed narrow septate fungal hyphae on a KOH mount, isolation of a fast growing black mould, which demonstrated hyphae and arthroconidia of varying widths typical of the Scytalidium synanamorph (S. dimidiatum). The formation of the pycnidia, which at maturity, expressed conidia. The patient was started on topical itraconazole one hourly and topical atropine thrice a day. The patient was lost to follow up hence we are not able to comment on the final outcome of the patient.

Forty-one recent cases of invasive zygomycosis from a global clinical registry.

BACKGROUND: Invasive zygomycosis accounts for a significant proportion of all invasive fungal diseases (IFD), but clinical data on the clinical course and treatment response are limited. PATIENTS AND METHODS: Fungiscope-A Global Rare Fungal Infection Registry is an international university-based case registry that collects data of patients with rare IFD, using a web-based electronic case form at www.fungiscope.net. RESULTS: Forty-one patients with invasive zygomycosis from central Europe and Asia were registered. The most common underlying conditions were malignancies (n = 26; 63.4%), diabetes mellitus (n = 7; 17.1%) and solid organ transplantation (n = 4; 9.8%). Diagnosis was made by culture in 28 patients (68.3%) and by histology in 26 patients (63.4%). The main sites of infection were the lungs (n = 24; 58.5%), soft tissues (n = 8; 19.5%), rhino-sinu-orbital region (n = 8; 19.5%) and brain (n = 6; 14.6%). Disseminated infection of more than one non-contiguous site was seen in six patients (14.6%). Mycocladus corymbifer was the most frequently identified species (n = 10, 24.4%). A favourable response was observed in 23 patients (56.1%). Overall survival was 51.2% (n = 21). At diagnosis, four patients (9.8%) were on continuous antifungal prophylaxis with itraconazole (n = 1; 2.4%) or posaconazole (n = 3; 7.3%). Initial targeted treatment with activity against zygomycetes was administered to 34 patients (82.9%). Liposomal amphotericin B was associated with improved response (P = 0.012) and survival rates (P = 0.004). CONCLUSIONS: Pathogen distribution and, consequently, drug susceptibility seem to vary across different geographic regions. Furthermore, protection from invasive zygomycosis for patients on posaconazole prophylaxis is not absolute. Our findings indicate that the use of liposomal amphotericin B as first-line treatment for patients diagnosed with zygomycoses merits further investigation, preferably in the form of a clinical trial.

Fatal cellulitis caused by Apophysomyces elegans.

A case of cellulitis of the left lateral side of the face caused by the zygomycete Apophysomyces elegans in a healthy male following a road traffic accident is reported. The contaminated soil was the source of fungus. Broad aseptate fungal hyphae were seen in the necrosed tissues. Extensive tissue debridement and treatment with amphotericin B were not successful in controlling the rapid invasion of the tissues by the fungus. Patient developed angioinvasion, severe cellulitis and finally succumbed to the infection three weeks after admission.

Multiple discharging sinuses: an unusual presentation caused by Absidia corymbifera.

A case of zygomycosis presenting with non-healing multiple discharging sinuses in a diabetic patient is reported here. The debrided tissue on histopathological examination revealed dense infiltration with aseptate fungal hyphae. Potassium hydroxide mount showed hyaline aseptate hyphae suggestive of zygomycosis. On culture, Absidia corymbifera was isolated. The patient responded to surgical debridement and therapy with amphotericin B followed by itraconazole.

Identification of Malassezia species.

Malassezia spp. are lipophilic unipolar yeasts recognized as commensals of skin that may be pathogenic under certain conditions. The genus Malassezia now comprises of seven species. This study was aimed at using a simple practical approach to speciate Malassezia yeasts from clinical material. Seventy skin scrapings from patients with pityriasis versicolor infection, positive in 10% potassium hydroxide (KOH), were cultured onto modified Dixon's agar (mDixon's agar) and Sabouraud dextrose agar (SDA) and incubated at 32 degrees C. Speciation was done on the basis of Gram stain morphology, catalase test, and utilization of Tweens. Out of 70 scrapings 48 (68.75%) showed growth on mDixon's agar. The commonest isolate was M. sympodialis (28, 58%) followed by M. globosa (19, 40%) and one isolate was (2%) of M. restricta. M. sympodialis was the commonest species affecting our population and there was no isolation of M. obtusa, M. slooffiae, M. pachydermatis and M. furfur.

Rare co-existence of Salmonella typhi and mycobacteria tuberculosis in a psoas abscess--a case report.

We report a rare case of dual infection in a psoas abscess. Pus from the abscess grew Salmonella typhi and the abscess wall showed epitheloid granulomas giant cells, which we confirmed as tuberculosis by PCR. Such dual infection cases may be missed unless looked for since both these infections are common in our country.

Role of surface swab, core swab and fine needle aspiration in isolating the core bacteria in inflammed tonsils.

The microbial flora of 75 patients with chronic tonsillitis were sudied to identify the commonest organism causing chronic tonsillitis and to compare surface swab, fine needle aspiration (FNA) and core swab in isolating the pathogens. Staphylococcus aureus was the predominant isolate from surface core swab FNA and surface swab. In our study the correlation between surface swab with core swab was 47.9% and between surface swab with FNA 57.3%. The correlation between FNA with core swab of 69.3% showed that FNA for culture was superior to surface swab in predicting the core culture. Hence FNA of the tonsil has more advantage over the routinely performed surface swab in representing the core pathogen. Thus the rationale of treating chronic tonsillitis medically should be based on the knowledge of the common core pathogen.

Legionella as a lower respiratory pathogen in north India.

One hundred patients of lower respiratory tract infection (LRTI) were prospectively studied over 2 years to find out if Legionella is a causative agent in these patients. In addition, 50 environmental samples and 50 age and sex matched controls were studied. Culture and direct fluorescent antibody testing (DFA) of respiratory tract secretions, and serodiagnosis by indirect immunofluorescence (IIF) and ELISA, were employed to detect Legionella. Respiratory tract secretions from all patients were negative for Legionella on culture and DFA. Low antibody titters to Legionella were observed in 21 patients and these could be attributed to cross reaction with other gram-negative bacteria. All environmental samples and controls tested negative for Legionella. Legionella does not seem to be an important lower respiratory tract pathogen in this part of the country and empirical addition of erythromycin to treatment regimens for pneumonia is not warranted in our setting.