Megh Singh Dhakad

Epidemiology and Virulence Determinants including Biofilm Profile of Candida Infections in an ICU in a Tertiary Hospital in India

The purpose of this prospective study was to isolate, speciate, and determine antifungal susceptibility and virulence patterns of Candida species recovered from the intensive care units (ICUs) in an Indian hospital. Study included 125 medical/postoperative patients admitted to ICU. Identification and speciation of yeast isolates were done by the biochemical methods. Antifungal susceptibility was done by broth microdilution method. Virulence testing of Candida species was done by phospholipase, proteinase, and adherence assay. A total of 103 Candida isolates were isolated; C. tropicalis was the predominant species (40.7%), followed by C. albicans (38.83 %), C. glabrata (11.65%), C. parapsilosis (3.88%), and 1.94% each of C. krusei, C. kefyr, and C. sphaerica. 60 Candida isolates (58.25%) showed resistance to fluconazole, while 7 (6.7%) isolates showed resistance to amphotericin B. Phospholipase and proteinase activities were seen in 73.8% and 55.3% Candida isolates with different species showing a wide range of activities, while 68.9% Candida isolates showed {4

Identification and Antifungal susceptibility testing of Candida species: A Comparison of Vitek-2 system with conventional and molecular methods

Background: Candida infection is a major cause of morbidity and mortality in immunocompromised patients; an accurate and early identification is a prerequisite need to be taken as an effective measure for the management of patients. The purpose of this study was to compare the conventional identification of Candida species with identification by Vitek-2 system and the antifungal susceptibility testing (AST) by broth microdilution method with Vitek-2 AST system. Materials and Methods: A total of 172 Candida isolates were subjected for identification by the conventional methods, Vitek-2 system, restriction fragment length polymorphism, and random amplified polymorphic DNA analysis. AST was carried out as per the Clinical and Laboratory Standards Institute M27-A3 document and by Vitek-2 system. Results: Candida albicans (82.51%) was the most common Candida species followed by Candida tropicalis (6.29%), Candida krusei (4.89%), Candida parapsilosis (3.49%), and Candida glabrata (2.79%). With Vitek-2 system, of the 172 isolates, 155 Candida isolates were correctly identified, 13 were misidentified, and four were with low discrimination. Whereas with conventional methods, 171 Candida isolates were correctly identified and only a single isolate of C. albicans was misidentified as C. tropicalis. The average measurement of agreement between the Vitek-2 system and conventional methods was >94%. Most of the isolates were susceptible to fluconazole (88.95%) and amphotericin B (97.67%). The measurement of agreement between the methods of AST was >94% for fluconazole and >99% for amphotericin B, which was statistically significant (P < 0.01). Conclusion: The study confirmed the importance and reliability of conventional and molecular methods, and the acceptable agreements suggest Vitek-2 system an alternative method for speciation and sensitivity testing of Candida species infections.

Spectrum of Opportunistic Fungal Infections in HIV/AIDS Patients in Tertiary Care Hospital in India

HIV related opportunistic fungal infections (OFIs) continue to cause morbidity and mortality in HIV infected patients. The objective for this prospective study is to elucidate the prevalence and spectrum of common OFIs in HIV/AIDS patients in north India. Relevant clinical samples were collected from symptomatic HIV positive patients (n = 280) of all age groups and both sexes and subjected to direct microscopy and fungal culture. Identification as well as speciation of the fungal isolates was done as per the standard recommended methods. CD4+T cell counts were determined by flow cytometry using Fluorescent Activated Cell Sorter Count system. 215 fungal isolates were isolated with the isolation rate of 41.1%. Candida species (86.5%) were the commonest followed by Aspergillus (6.5%), Cryptococcus (3.3%), Penicillium (1.9%), and Alternaria and Rhodotorula spp. (0.9% each). Among Candida species, Candida albicans (75.8%) was the most prevalent species followed by C. tropicalis (9.7%), C. krusei (6.4%), C. glabrata (4.3%), C. parapsilosis (2.7%), and C. kefyr (1.1%). Study demonstrates that the oropharyngeal candidiasis is the commonest among different OFIs and would help to increase the awareness of clinicians in diagnosis and early treatment of these infections helping in the proper management of the patients especially in resource limited countries like ours.

Pneumocystis pneumonia in HIV patients: a diagnostic challenge till date

HIV has become a major health problem in India, patients commonly succumb to opportunistic infections (OIs), respiratory infections being an important cause of morbidity and their accurate diagnosis is still a challenge. Our aim was to study the occurrence of Pneumocystis pneumonia (PCP) in HIV/AIDS patients with respiratory complaints attending ART clinic and to compare various diagnostic methodologies. One hundred and twenty five HIV/AIDS patients presenting with respiratory symptoms like cough, fever, breathlessness etc, were enrolled, and induced sputum samples were collected. Samples were homogenized using glass beads and Dithiothretol. Smears were prepared and examined by Immunoflourescent staining (IFAT), Gomori methanamine silver staining (GMSS), Toludine blue O staining (TBO) and Giemsa staining for Pneumocystis jiroveci. Among the 125 patients who presented with respiratory complaints, 34 cases (27.2%) were diagnosed as having PCP. All 34 cases were detected by IFAT followed by GMSS, Giemsa and Toludine blue O staining in decreasing order. The mean CD4 count was 67.27cells/μl. PCP has become an important health problem in HIV/AIDS patients with low CD4 counts in India. IFAT remains the most sensitive method for the detection of this uncultivable organism. In resource poor settings where an immunoflourecent microscope is not available, diagnosis of PCP still remains problematic.

Study of TH1/TH2 Cytokine Profiles in HIV/AIDS Patients in a TertiaryCare Hospital in India

Background and Objectives: Switch of cytokines from a T helper 1 (Th1) to a Th2 is an important factor in the progression of HIV infection to AIDS. Hence, our objectives were to analyze the levels of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytokines and their correlation with clinical and immunological profiles in HIV/AIDS patients. Methods: We studied 234 symptomatic HIV positive patients (case group) attending OPDs, ART clinic and medical wards of the Hospital. CD4+T-cell count was determined by flow cytometry using Fluorescent Activated Cell Sorter Count system. Quantitative determination of cytokines (Th1 subtype: IL-2, IFN-γ and Th2 subtype: IL-4, IL-10) was done by ELISA. Results: Patients (08-68 years) had CD4+T-cell counts ranged from 16-1033 cells/μl. The median CD4+T cell count was 204.50 cells/μl and the mean was 265.48 cells/μl. The concentration of IL-2 and IFN-γ were significantly lower in case group compared to asymptomatic HIV patients (P<0.001) while the IL-4 in symptomatic HIV negative was higher than healthy controls and case group compared to asymptomatic HIV patients. Concentration of IL-10 was also higher in case group compared to asymptomatic HIV patients. A positive correlation was found between IL-4, IFN-γ, IL-2 with CD4+T-cells and a negative correlation between IL-10 and CD4+T-cells among the case group. Conclusions: Hence a decline in type-1 cytokines (IFN-γ, IL-2) and rise in type-2 cytokines (IL-4, IL-10) was observed in symptomatic patients with HIV infection in comparison to asymptomatic HIV patients suggesting a shift from Th1 to Th2 type cytokine response.

Allergic Fungal Rhinosinusitis: A Study in a Tertiary Care Hospital in India

The study was conducted to study the occurrence and clinical presentation of allergic fungal rhinosinusitis (AFRS), characterize the same, and correlate with the microbiological profile. Clinically suspected cases of fungal rhinosinusitis (FRS) depending upon their clinical presentation, nasal endoscopy, and radiological evidences were included. Relevant clinical samples were collected and subjected to direct microscopy and culture and histopathological examination. 35 patients were diagnosed to have AFRS. The average age was 28.4 years with a range of 18–48 years. Allergic mucin was seen in all the AFRS patients but fungal hyphae were detected in only 20%. 80% of cases were positive for IgE. All the patients had nasal obstruction followed by nasal discharge (62.8%). Polyps were seen in 95% (unilateral (48.57%) and bilateral (45.71%)), deviated nasal septum was seen in 28.57%, and greenish yellow secretion was seen in 17.14%. Direct microscopy and septate hyphae were positive in 71.42% of cases. 91.4% of cases were positive by culture. 5.7% yielded mixed growth of A. flavus and A. niger. Prompt clinical suspicion with specific signs and symptoms along with timely sampling of the adequate patient specimens and the optimal and timely processing by microscopy and culture and histopathological examination is a must for early diagnosis and management.

An Unusual Case of Cystic Fibrosis Associated Pneumocystis jiroveci Pneumonia in an Infant.

Pneumocystis jiroveci pneumonia (PJP) is one of the major infections in patients with impaired immunity. The entity is common in HIV-seropositive individuals but quite very rare in HIV-seronegative individuals especially children. We report here a case of 16-week-old HIV-seronegative infant with chief complaint of chronic cough of one month of evolution. Sweat chloride test for diagnosis of cystic fibrosis was positive. Bronchoalveolar lavage (BAL) fluid was collected and Pseudomonas aeruginosa was isolated on culture. Empirical antibiotic regimen comprising ceftriaxone and azithromycin was initiated that was switched to meropenem as per antimicrobial susceptibility report, but the patient did not improve. Subsequently, an immunofluorescence staining of BAL fluid was performed and P. jiroveci cysts were detected. Following a laboratory confirmation of Pneumocystis pneumonia, cotrimoxazole was added and the clinical condition of the patient significantly improved. This is an unusual case wherein unsuspected PJP occurred and since signs and symptoms of the patient persisted even after the initiation of antimicrobial therapy for Pseudomonas infection and resolved only after treatment for PJP was started, it suggests a causative role of P. jiroveci rather than colonization/contamination.

Invasive Fungal Rhinosinusitis: An Observational Study in an Indian Tertiary Care Hospital

Invasive fungal rhinosinusitis (IFRS) is a challenging condition and the mortality of IFRS in immuno compromised patients is very high. 75 patients of suspected FRS were included to study the burden and the clinicopathological and mycological profile of invasive fungal rhinosinusitis (FRS) in these patients. The samples collected were exudate from nasal debri, discharge and intraoperative tissue sample and were subjected to direct microscopy to histopathological examination and direct microscopy (KOH and calcofluor white) and culture on Sabouraud dextrose agar. Identification of molds and yeasts were done by conventional methods. 25 cases were suspected to have IFRS were confirmed by microbiological and histo pathological examination, comprising 56% of AIFRS (acute IFRS), 36% CIFRS (chronic IFRS) and 8% CGFRS (chronic granulomatous FRS). Rhizopus arrhizus (64.2%) was the most common isolate followed by Aspergillus flavus (35.7%). Mucor species were solely isolated from AIFRS (14.3%). In CIFRS, Aspergillus flavus (44.4%) seemed to be the major isolate with Aspergillus niger, Alternaria spp., Penicillium spp. and Candida albicans. Necrosis of submucosa, bone, vascular tissue was seen in 96% of cases with aseptate hyphae in 78.5% AIFRS and 77.7% in CIFRS. Radiological features help in presumptive diagnosis of FRS. Direct microscopy along with culture conformation is important for diagnosis and early initiation of treatment.