Thangam Menon

Phospholipase and Proteinase Activities of Clinical Isolates of Candida from Immunocompromised Patients

Sixty-one isolates of Candida recovered from HIV seropositive and cancer patients were studied for elaboration of putative virulence determinants – phospholipase (PL) and secreted aspartyl proteinase (Sap). Forty two (68.85%) isolates examined were PL producers and 51 (83.6%) were positive for Sap. 57.37% (35/61) isolates produced both enzymes. Enzymatic activity was more pronounced in Candida albicans with 100% PL and 94.1% Sap activity. In contrast, non-C. albicans species demonstrated only 29.6% PL and 70.3% Sap activity, indicating interplay of other virulence determinants in these yeasts in colonization and disease.

Comparison of double disc and three dimensional methods to screen for ESBL producers in a tertiary care hospital.

Extended spectrum beta lactamases (ESBLs) continue to be a major problem in clinical setups world over, conferring resistance to the expanded spectrum cephalosporins. An attempt was made to study ESBL production among Enterobacteriaceae members from a tertiary care center in Chennai. A total of seventy randomly collected isolates of the family Enterobacteriaceae from a tertiary care center was studied for their susceptibility patterns to various antibiotics and detection of ESBL producers by double disc synergy (DDS) test and three dimensional test (TDT). Eighty percent of the isolates were multidrug resistant (MDR) and 20% were ESBL producers. TDT detected 85.7% whereas only 14.2% were detected by DDS. In the present study, a large number of isolates were found to be MDR and ESBL producers. TDTs were found to be better than DDS in the detection of ESBLs. Continued monitoring of drug resistance is necessary in clinical settings for proper disease management.

Actividad esterasa en especies de Candida procedentes de pacientes inmunodeficientes

A total of 149 clinical isolates of Candida species isolated from immunocompromised patients were examined to ascertain their esterase activity by the Tween 80 opacity test, which is a biochemical test used mainly to differentiate between Candida albicans and Candida dubliniensis. Our results showed that C. albicans (92.3%), Candida tropicalis (92.3%), Candida parapsilosis (25%), C. dubliniensis (16.6%), Candida inconspicua (100%), and Candida lipolytica (100%) produced opacity halos through the 10-day post-inoculation period. The remaining Candida species did not produce a positive test response. These findings indicate that Tween 80 opacity test cannot be used as the sole phenotypic trait in the differentiation of C. albicans and C. dubliniensis.

Anticandidal activity of Azadirachta indica

OBJECTIVE: To study the antifungal activity of 10 different extracts of seed kernels of Azadirachta indica A. Juss (Meliaceae) on Candida sps. isolated from immunocompromised patients. MATERIALS AND METHODS: The extractants used were hexane, methanol, chloroform, water, petroleum ether, dichloromethane, acetone and absolute alcohol. The products of a successive extraction procedure involving hexane, chloroform and methanol were also tested for anticandidal activity. The minimum inhibitory concentration was tested by broth dilution method at concentrations ranging from 1 to 0.0625 mg/ml. RESULTS: The ethanol extract of commercial neem seed oil, ethanol extract of neem seed kernels and the hexane extract showed best results. All strains were resistant to methanol: chloroform: water extracts and chloroform extracts of the successive extraction procedure. CONCLUSION: The hexane and alcoholic extracts of neem seed seem to be promising anticandidal agents.

Role ofCandida in indirect pathogenesis of antibiotic associated diarrhoea in infants

One hundred and thirty seven isolates ofCandida species were isolated from antiobiotic associated diarrhoea cases and were examined to study the role ofCandida in the pathogenesis of diarrhoea in infants. The quantitative estimation of yeast population by simple gram stain smear revealed more than 70% of the cases had 3+ score. The isolates further screened for detection ofβ-lactamases. Among the isolatedCandida sp,β-lactamases was secreted byC. albicans, C. tropicalis, C. krusei andC. parapsilosis. Further, 46% of theCandida isolates were found to be produced 741–1110 mU/ml ofβ-lactamases, suggesting that these enzyme would inactivate penicillin group of drugs and cause failure in the therapy directed against other diarrhoegenic bacteria.

Bartonella quintana and Coxiella burnetii as Causes of Endocarditis, India

To the Editor: In industrialized countries, blood culture is negative for 2.5%–31% of infectious endocarditis cases (1). In developing countries such as South Africa (2), Algeria (3), and Pakistan (4), culture is negative for 48% to 56%. Negative cultures delay diagnosis and treatment, which profoundly affects clinical outcome. Negative blood cultures commonly result from previous administration of antimicrobial drugs, right-sided endocarditis, or fastidious or noncultivable pathogens (1). Our aim was to identify fastidious agents of blood culture–negative endocarditis by serology. Because of recent attention to zoonotic microorganisms as agents of this condition in developing countries (1), we investigated the prevalence of Coxiella burnetii, Bartonella spp., and Brucella melitensis among endocarditis patients in India. We cultured blood from 111 patients admitted to the Government General Hospital, Chennai, India, from August 2005 through December 2006, with a diagnosis of infectious endocarditis according to the Duke criteria (5). Informed consent was obtained from all patients. Three blood samples from each patient, collected at hourly intervals, were inoculated into brain–heart infusion broth supplemented with 0.04% sodium polyanethol sulfonate (HiMedia, Mumbai, India). Cultures were incubated at 37°C in a 5% CO2 atmosphere for 14 days and checked each day for turbidity. Subcultures were made on 5% sheep blood and MacConkey agar at 37°C at 24 hours, 48 hours, and when culture broth appeared turbid. Blood cultures were negative for 80 (72%) of the 111 patients. Serum from 63 patients was available for serologic testing. Of these patients, 30 were male and 33 were female; age range was 5–65 years and mean age was 25.5 years. Endocarditis involved the native valve for 60 (95.23%) and a prosthetic valve for 3 (4.76%). The most frequent predisposing factor was rheumatic heart disease, found in 38 (60.31%). Of the 60 with native valve endocarditis, the involved valve was mitral for most (36, 60.0%), followed by aortic (8, 13.33%), tricuspid (7, 11.66%), and pulmonary (1, 1.66%); 8 (13.33%) had both valvular and nonvalvular endocarditis. Of the 3 patients with prosthetic valve endocarditis, the involved valve was mitral for 2 and aortic for 1. Serum samples were screened for Bartonella spp. and C. burnetii by microimmunofluorescence (6,7). A diagnosis of endocarditis was based on an immunoglobulin (Ig) G titer >800 to phase I C. burnetii; this titer has a positive predictive value of 98% (6). A diagnosis of Bartonella infection was based on the combination of a positive microimmunofluorescence titer (IgG to B. quintana or B. henselae >200) and a Western blot profile consistent with endocarditis (8). Identification of the causative species was obtained by Western blot after cross-adsorption with either B. henselae or B. quintana antigens (8). Antibodies to B. melitensis were detected by agglutination by using the Rose Bengal and Brucella Wright tests (both from BioRad, Hercules, CA, USA). Of the 63 patients, 9 had a positive antibody response against a tested antigen (Table): 1 to phase I C. burnetii and 8 to Bartonella spp. (IgG >200). Of these, 7 had a 1-fold dilution higher titer to B. quintana than to B. henselae, including 1 with a low-level cross-reaction with C. burnetii, and 1 had identical titers to both. For all 8 patients, Western blot results were consistent with Bartonella endocarditis. For 7, cross-adsorption identified B. quintana as the causative species; for the other, the infecting Bartonella species remained undetermined because adsorption with B. quintana and B. henselae antigens removed all antibodies. Serologic results for B. melitensis were negative for all patients. Table Clinical findings and causative agent for 9 patients with blood culture–negative endocarditis, India, August 2005–December 2006* B. quintana is mostly associated with human body lice but has also been found in fleas (9). The predisposing factors for B. quintana endocarditis are homelessness, alcoholism, and exposure to body lice (10). For our patients, the common predisposing factors were poor hygiene and low socioeconomic status, which may expose them to ectoparasites including lice and fleas. In contrast with previous study findings, B. quintana infectious endocarditis developed on preexisting valvular lesions in all patients (10). This finding may reflect a different clinical evolution than in Europe, where studies have suggested that B. quintana infectious endocarditis followed chronic bacteremia in patients who did not have previous valvular defects (10). In summary, prevalence of negative blood culture among patients with infectious endocarditis was high (72%). The most commonly associated risk factor was rheumatic heart disease (Table). C. burnetii and Bartonella spp. were responsible for 8% of all infectious endocarditis cases and 14% of blood culture–negative cases. No case of infectious endocarditis caused by B. melitensis was identified. Our preliminary study suggests that zoonotic agents, especially Bartonella spp., are prevalent causative organisms of blood culture–negative endocarditis in India. We recommend serologic screening for antibodies to zoonotic microorganisms as diagnostic tools for this disease in India.

Efficacy of fluconazole and itraconazole in the treatment of oral candidiasis in HIV patients

A total of 46 strains of Candida were collected from HIV infected patients, of which 25 strains were isolated from patients with oral candidiasis, and 21 strains were from mouthwash samples of asymptomatic carriers. The most common species isolated was Candida albicans (73.9%), followed by Candida tropicalis (21.7%). In vitro susceptibility of the strains to fluconazole and itraconazole was tested using minimum inhibitory concentration (MIC) studies by agar dilution technique. Out of the 18 strains of C. albicans isolated from mouthwash samples, four were resistant to fluconazole whereas only two were resistant to itraconazole. Out of 16 strains of C. albicans isolated from oral lesions, one was resistant to fluconazole where as all were sensitive to itraconazole. Among the other species of Candida tested, C. tropicalis gave higher MIC values to both drugs than other species such as Candida guillermondii and Candida krusei. In vitro MIC values correlated well with in vivo responses in patients. Hence, itraconazole may be used as an alternative in the treatment of candidiasis, which does not respond to fluconazole therapy.

Environmental isolation of Cryptococcus neoformans and Cryptococcus gattii from living trees in Guindy National Park, Chennai, South India

We report for the first time the environmental isolation of Cryptococcus neoformans from decaying wood and bark debris of living trees in Guindy National Park, Chennai, South India. Of the 40 trees screened, four isolates of Cryptococcus species were recovered of which two were Cryptococcus gattii, one was C. neoformans and one was untypable. The isolation of C. neoformans from Eucalyptus globulus and C. gattii from Cassia marginata in this study constitutes the first record of the natural occurrence of C. neoformans varieties in these tree species anywhere in the world. The isolation of C. gattii from Syzygium cumini represents the first isolation from South India.

Mustard seed agar, a new medium for differentiation of Cryptococcus neoformans.

Cryptococcosis due to Cryptococcus neoformans has emerged as the fourth most common lethal infection among AIDS patients (2). The formation of brown pigment due to phenol oxidase activity on medium containing seed extracts of Guizotia abyssinica (Staib’s niger seed agar) or Helianthus annus (Pal’s sunflower seed agar) or on medium containing chemicals such as caffeic acid or L-3,4-dihydroxyphenylalanine (L-DOPA) has been widely used as a criterion for the identification of this fungus (1, 3). In a search for a simple alternative seed-based medium for identification of C. neoformans, mustard seed extracts were found to induce the brown color effect (BCE) in C. neoformans. Brassica juncea or brown mustard is a cash crop widely cultivated, especially in India, for oil extraction and culinary purposes. Preparation of mustard seed agar involved procurement of dried mustard seeds from the local market followed by grinding in a domestic blender (Preethi Chefpro, Chennai, India). First, an aqueous extract of ground seeds (including kernels and shells) was prepared by adding the ground seeds to distilled water at a ratio of 1:20 (wt/vol), followed by boiling for 30 min. Next, the seed extract was cooled and filtered through gauze. The volume was then readjusted to 1 liter, and 20 g of agar (HiMedia, India) was added before the mixture was autoclaved at 121°C for 15 min. The medium was allowed to cool to between 45 and 55°C and dispensed into sterile 90-mmdiameter petri dishes (25 ml agar per plate). Five isolates of C. neoformans (consisting of three isolates of C. neoformans var. gatti and two isolates of C. neoformans var. neoformans) and five isolates of Candida albicans were inoculated on the mustard seed agar and incubated at 30°C on the laboratory bench. In addition, two strains comprising C. neoformans var. grubii (H-99) and C. neoformans var. neoformans (B-3501), kindly provided by J. Kwon-Chung (National Institutes of Health, Bethesda, Md.), were used as reference strains. At 48 h postinoculation, all seven isolates of C. neoformans could be easily identified by the BCE on mustard agar and differentiated from the white colonies of C. albicans. Mustard agar plates were held for up to 7 days to check for any variations in the colony color. The BCE was observed to intensify progressively during the entire period of incubation. Strains were also seeded on Pal’s agar and were observed to produce BCE. Repeated testing of different batches of mustard seed agar confirmed the ability of the new medium to support growth and pigment production of C. neoformans. Studies using mustard seed agar prepared according to Staib’s recommended formula with 0.1% glucose–0.1% creatinine– 0.1% KH2PO4 were also attempted. Growth of C. neoformans was observed within 48 h, but pigment production appeared to be delayed (72 h). Hence, mustard seed agar without supplements appeared to be more suitable for differentiation of C. neoformans. Thus, to the best of our knowledge, this is the first report on the prospective usage of a mustard seed-based agar in the identification of C. neoformans. The BCE of C. neoformans on media such as Staib’s or Pal’s agar is attributed to the presence of diphenolic substrates such as L-DOPA. However, mustard does not contain such compounds. Hence, the BCE on mustard agar may not be the same as that one observes on standard L-DOPA-containing media. This phenomenon has to be analyzed further for the elucidation of the exact mechanism of BCE on mustard agar. Furthermore, evaluation of this medium with more strains, including melanin-deficient mutants, would confirm its utility in diagnostic laboratories.

ABO blood groups in relation to the infection rate of dermatophytosis

Summary. The role of ABO blood groups in the carriage rate of dermatophytosis was studied. Blood grouping was done for 108 culture‐proven dermatophytosis patients. Forty‐nine patients belonged to blood group A, 54 to blood group O, three to blood group B and two to blood group AB. The incidence of dermatophytosis was found to be high in patients of blood group O and A. However, chronicity of the disease was more frequent in those in blood group A. The control group consisted of 100 healthy subjects. Sixteen out of 29 control subjects belonging to blood group A had a history of skin infections. None of the O blood group control subjects had a history of skin infections. Our study suggests that A blood group subjects may be prone to chronic dermatophytosis.