S.N. Gaur

Isolation of multiple-triazole-resistant Aspergillus fumigatus strains carrying the TR/L98H mutations in the cyp51A gene in India

OBJECTIVES Azole resistance in Aspergillus fumigatus isolates impacts on the management of aspergillosis since azoles are primary agents used for prophylaxis and therapy. We report the emergence of resistance to triazoles in two A. fumigatus isolates from patients in Delhi, India. METHODS One hundred and three A. fumigatus isolates, collected from 85 patients suspected of bronchopulmonary aspergillosis during 2005-10, were investigated for susceptibility to itraconazole, voriconazole, posaconazole and isavuconazole. We undertook a mixed-format real-time PCR assay for the detection of mutations leading to triazole resistance in A. fumigatus. The resistant isolates were compared with 25 Dutch TR/L98H-positive isolates by microsatellite analysis. RESULTS Of the 103 A. fumigatus isolates tested, only 2 had high MIC values of itraconazole (>16 mg/L), voriconazole (2 mg/L), posaconazole (2 mg/L) and isavuconazole (8 mg/L). The resistant A. fumigatus isolates exhibited the TR/L98H genotype and showed identical patterns by microsatellite typing, but were different from 25 Dutch TR/L98H isolates. CONCLUSIONS We report for the first time from India the occurrence of TR/L98H mutations in the cyp51A gene (responsible for reduced azole susceptibility) in two A. fumigatus isolates from patients with chronic respiratory disease who had not previously been exposed to azoles. The presence of TR/L98H is consistent with a route of resistance development through exposure to azole compounds in the environment. Given the emergence of azole resistance in environmental strains, continued surveillance of resistance in clinical A. fumigatus strains is desirable for successful therapy of aspergillosis.

Clonal Expansion and Emergence of Environmental Multiple-Triazole-Resistant Aspergillus fumigatus Strains Carrying the TR34/L98H Mutations in the cyp51A Gene in India

Azole resistance is an emerging problem in Aspergillus which impacts the management of aspergillosis. Here in we report the emergence and clonal spread of resistance to triazoles in environmental Aspergillus fumigatus isolates in India. A total of 44 (7%) A. fumigatus isolates from 24 environmental samples were found to be triazole resistant. The isolation rate of resistant A. fumigatus was highest (33%) from soil of tea gardens followed by soil from flower pots of the hospital garden (20%), soil beneath cotton trees (20%), rice paddy fields (12.3%), air samples of hospital wards (7.6%) and from soil admixed with bird droppings (3.8%). These strains showed cross-resistance to voriconazole, posaconazole, itraconazole and to six triazole fungicides used extensively in agriculture. Our analyses identified that all triazole-resistant strains from India shared the same TR34/L98H mutation in the cyp51 gene. In contrast to the genetic uniformity of azole-resistant strains the azole-susceptible isolates from patients and environments in India were genetically very diverse. All nine loci were highly polymorphic in populations of azole-susceptible isolates from both clinical and environmental samples. Furthermore, all Indian environmental and clinical azole resistant isolates shared the same multilocus microsatellite genotype not found in any other analyzed samples, either from within India or from the Netherlands, France, Germany or China. Our population genetic analyses suggest that the Indian azole-resistant A. fumigatus genotype was likely an extremely adaptive recombinant progeny derived from a cross between an azole-resistant strain migrated from outside of India and a native azole-susceptible strain from within India, followed by mutation and then rapid dispersal through many parts of India. Our results are consistent with the hypothesis that exposure of A. fumigatus to azole fungicides in the environment causes cross-resistance to medical triazoles. The study emphasises the need of continued surveillance of resistance in environmental and clinical A. fumigatus strains.

Allergic bronchopulmonary mycosis due to fungi other than Aspergillus: a global overview

Abstract Allergic bronchopulmonary mycosis (ABPM) is a hypersensitivity-mediated disease of worldwide distribution. We reviewed 143 reported global cases of ABPM due to fungi other than aspergilli. The commonest etiologic agent was Candida albicans, reported in 60% of the cases, followed by Bipolaris species (13%), Schizophyllum commune (11%), Curvularia species (8%), Pseudallescheria boydii species complex (3%) and rarely, Alternaria alternata, Fusarium vasinfectum, Penicillium species, Cladosporium cladosporioides, Stemphylium languinosum, Rhizopus oryzae, C. glabrata, Saccharomyces cerevisiae and Trichosporon beigelii. India accounted for about 47% of the globally reported cases of ABPM, attributed predominantly to C. albicans, followed by Japan (16%) where S. commune predominates, and the remaining one-third from the USA, Australia and Europe. Notably, bronchial asthma was present in only 32% of ABPM cases whereas its association with development of allergic bronchopulmonary aspergillosis (ABPA) is known to be much more frequent. The cases reviewed herein revealed a median IgE value threefold higher than that of ABPA, suggesting that the etiologic agents of ABPM incite a stronger immunological response than that by aspergilli in ABPA. ABPM is currently underdiagnosed, warranting comprehensive basic and clinical studies in order to elucidate its epidemiology and to devise a more effective therapy.

Identification of a serine protease as a major allergen (Per a 10) of Periplaneta americana

Background: Cockroach allergens are associated with the development of asthma, but none of these has been characterized for proteolytic activity. This study was undertaken to isolate and characterize a protease from Periplaneta americana and determine its allergenicity.

Schizophyllum commune as an emerging fungal pathogen: a review and report of two cases.

We report Schizophyllum commune as the aetiological agent of one case each of allergic broncho‐pulmonary mycosis (ABPM) and pulmonary fungal ball, and present a literature review. The fungus was characterised by clamp connections, hyphal spicules, and formation of basidiocarps with basidiospores. The phenotypic identification was confirmed by sequencing of the ITS region. To‐date, ABPM and pulmonary fungal ball to S. commune have been reported exclusively from Japan and North America respectively. Of the 71 globally reported cases due to S. commune, 45 (63%) were bronchopulmonary, 22 (31%) sinusitis and 4 extrapulmonary. Taken together, cases of bronchopulmonary disease and sinusitis numbered 67 (94%), indicating the respiratory tract as the primary target of disease. Concerning the country‐wise distribution, Japan topped the list with 33 cases (46%), followed by Iran – 7 cases (10%), U.S.A. – 6 cases (9%), and a lower prevalence of 1.4–6% for the remaining 12 countries. The preponderance of the disease in Japan may be attributed to its greater awareness vis‐à‐vis that in other countries rather than to any geographical/climatic factors. We believe that the burden of S. commune‐incited disease is currently underestimated, warranting comprehensive prospective studies to determine its prevalence.

Zygomycosis – a case report and overview of the disease in India

A case of zygomycosis caused by Rhizopus oryzae in a diabetic patient previously misdiagnosed as invasive pulmonary aspergillosis and an overview of the disease in India are presented. The case was diagnosed by direct microscopy, histopathologic examination and culture. Following surgical resection of pulmonary cavity under cover of amphotericin B administration, the patient recovered completely. Of 461 cases reported to‐date, approximately 70% had been diagnosed at the Postgraduate Institute of Medical Education and Research, Chandigarh, in north India. This may be attributed to better awareness, expertise and infrastructural facilities for mycological diagnosis than to any particular regional preponderance of the disease. Rhino‐orbito‐cerebral manifestations were the most common feature of zygomycosis (269 cases), followed by cutaneous disease (66 cases), which is in conformity with the pattern prevalent worldwide. The etiologic agents encountered were Rhizopus oryzae, Apophysomyces elegans, Saksenaea vasiformis, Cunninghamella bertholletiae, Absidia corymbifera, Basidiobolus ranarum and Conidiobolus coronatus. In contrast to cases from the developed world where transplant recipients and patients with haematological malignancies seem to be most vulnerable to zygomycosis, the most common risk factor in India was uncontrolled diabetes mellitus. Amphotericin B was the mainstay of various treatment modalities employed. The relevance of a strong clinical suspicion and early diagnosis of zygomycosis for favourable prognosis can hardly be over‐emphasised.

Allergenicity assessment of transgenic mustard (Brassica juncea) expressing bacterial codA gene

Background:  Assessing the allergenicity and toxicity of genetically modified (GM) crops is essential before they become a regular part of our food supply. The present study aimed to assess the allergenicity of Brassica juncea (mustard) expressing choline oxidase (codA) gene from Arthrobacter globiformis that provides resistance against abiotic stresses.

Choline attenuates immune inflammation and suppresses oxidative stress in patients with asthma.

Asthma is a chronic immune inflammatory disease characterized by variable airflow obstruction and increased bronchial hyperreactivity (BHR). Therapeutic interventions reduce airway inflammation and relieve symptoms but associated with potential side effects that limit their usefulness. The present study was undertaken to assess the effect of choline on immune inflammation and BHR in asthma subjects. The patients of asthma (n=76) were recruited and treated with choline supplement (1500 mg twice) or standard pharmacotherapy for 6 months in two groups. The patients were evaluated by clinical, immunologic and biochemical parameters. The treatment with choline showed significant reduction in symptom/drug score and improvement in PC(20) FEV1 compared to baseline or standard pharmacotherapy (p<0.01). Choline therapy significantly reduced IL-4, IL-5 and TNF-alpha level as compared to baseline or standard pharmacotherapy after 6 months (p<0.01). Blood eosinophil count and total IgE levels were reduced in both the treatment groups. Cysteinyl leukotriene and leukotriene B4 were suppressed significantly by choline treatment (p<0.01). This was accompanied by decreased 8-isoprostanes, a biomarker for oxidative stress after choline treatment (p<0.01). Choline therapy modulates immune inflammation and suppresses oxidative stress in asthma patients. It can be used as an adjunct therapy for asthma patients.

Sensitization to blackgram in patients with bronchial asthma and rhinitis: clinical evaluation and characterization of allergens.

Background:  Legumes are important causative agents of type I hypersensitivity in south Asia and Europe but such studies are lacking in Indian population. The present study investigates blackgram sensitization in asthma and rhinitis patients and identifies immunoglobulin E (IgE)‐binding proteins.

Clinical Significance and Molecular Characterization of Nonsporulating Molds Isolated from the Respiratory Tracts of Bronchopulmonary Mycosis Patients with Special Reference to Basidiomycetes

ABSTRACT Nonsporulating molds (NSMs), especially basidiomycetes, have predominantly been reported as human pathogens responsible for allergic and invasive disease. Their conventional identification is problematic, as many isolates remain sterile in culture. Thus, inconclusive culture reports might adversely affect treatment decisions. The clinical significance of NSMs in pulmonary mycoses is poorly understood. We sequenced the internal transcribed spacer (ITS) region and D1/D2 domain of the larger subunit (LSU) of 52 NSMs isolated from respiratory specimens. The basidiomycetes were the predominant NSMs, of which Schizophyllum commune was the most common agent in allergic bronchopulmonary mycosis (ABPM), followed by Ceriporia lacerata in invasive fungal disease. Porostereum spadiceum, Phanaerochaete stereoides, Neosartorya fischeri, and Marasmiellus palmivorus were the other molds observed. Application of ITS and LSU region sequencing identified 92% of the isolates. The antifungal susceptibility data revealed that all basidiomycetes tested were susceptible to amphotericin B and resistant to caspofungin, fluconazole, and flucytosine. Except for 3 isolates of S. commune and a solitary isolate of M. palmivorus, all basidiomycetes had low MICs for itraconazole, posaconazole, and voriconazole. Basidiomycetes were isolated from patients with ABPM, invasive pulmonary mycosis/pneumonia, or fungal balls. In addition, the majority of the basidiomycetes were isolated from patients with chronic respiratory disorders who were sensitized to one of the basidiomycetous fungi and demonstrated precipitating antibodies against the incriminating fungi, indicating an indolent tissue reaction. Thus, isolation of basidiomycetes from the lower respiratory tract could be significant, and it is important to monitor these patients in order to prevent subsequent lung damage.