Microbiological Profile of Culture-Positive Fungal Keratitis.

PURPOSE: To examine the microbiological profile of cases of culture-positive fungal keratitis presenting to a tertiary eye care center in eastern India. METHODS: Microbiology records of all culture-positive microbial keratitis patients presenting to L V Prasad Eye Institute, Bhubaneswar, between January 2020 and December 2021, were retrospectively reviewed. Collected data included smear results of culture-positive fungal or mixed infections, the species isolated, and the time taken for organisms to grow in each media. RESULTS: Fungal keratitis formed 36% of all culture-positive microbial keratitis, whereas mixed infections (fungi and other organisms) formed 8.5%. The most common fungal species isolated was Fusarium spp. (25.8%). The most common bacteria involved in mixed infection with fungi was Staphylococcus spp. (54.8%). The positivity of potassium hydroxide+calcofluor white stain in detecting fungal filaments was 89.0% and that of Gram stain was 76.1%. Culture-positive cases of fungal keratitis showed most frequent growth on potato-dextrose agar (77.6%). A similar pattern was observed in culture-positive mixed infections (Sabouraud dextrose agar [SDA]: 84%). Most frequent growth of bacteria in mixed infections was seen in thioglycolate broth (54.7%). The shortest time to achieve significant fungal growth was observed in blood agar (BA) and chocolate agar (CA) (2.2/2.3 days, and 1.8/2 days for fungal keratitis and mixed infections, respectively). Filamentous hyaline fungi took the shortest time to achieve significant growth (2.8 days), whereas yeast forms took the longest (5 days). CONCLUSION: This study highlights the importance of combined use of both solid and liquid culture media, especially potato dextrose agar (PDA)/SDA and CA, to arrive at a definitive diagnosis of fungal keratitis and possible bacterial co-infection, which forms a significant proportion of cases with fungal keratitis. In resource-poor laboratories, two culture media, either SDA or PDA, along with BA, may be plated to detect mixed infections. Examination of stained smears of corneal samples provides an inexpensive method of rapid diagnosis of fungal keratitis when culture media is not available.

Identification of population of bacteria from culture negative surgical site infection patients using molecular tool.

BACKGROUND: Managing surgical site infections, with negative culture report in routine diagnosis is a common dilemma in microbiology accounting more than 30% worldwide. The present study attempted to identify the presence of bacterial spp. if any in wound aspirates/swabs of culture negative surgical site infections of hospitalised patients using molecular tools. METHODS: Ninety-seven patients with post-operative SSI whose wound swabs/aspirate were negative in the conventional aerobic culture after 72 h of incubation were analysed by 16S rRNA gene specific broad range PCR. The amplified DNA fragments were sequenced by Sanger DNA sequencing method and homology of the sequence were matched using NCBI BLAST (NCBI, USA) RESULTS: Of the 97 patients, 16S rRNA based broad range PCR assay could identify the presence of bacterial pathogen in 53(54.63%) cases, of which 29 isolates were supposed to be of viable but non-culturable bacteria (VBNC), 07 were of obligatory anaerobes and 13 were of unculturable bacteria, 04 were with poly bacterial infections. CONCLUSIONS: Our study highlights the usefulness of PCR assay in detecting the presence of any VBNC, anaerobes and unculturable bacteria in SSI patients regardless of how well the bacteria may or may not grow in culture. Measures should be taken to use anaerobic culture system and PCR diagnosis along with conventional culture to detect the VBNC and unculturable bacteria where Gram stain is positive for better patient care.

Chlamydial eye infections: Current perspectives.

Chlamydia trachomatis, an obligate intraocular bacteria causing trachoma, adult and neonatal inclusion conjunctivitis, was the leading cause of blindness in the last century worldwide. Improvement in socioeconomic and living conditions, availability of antibiotics, and introduction of National Trachoma Control Programmes reduced the prevalence in developed countries, but it persisted in resource-poor settings of Africa and Asia, including India. In 2016, as per the WHO report, trachoma is restricted to 42 countries, causing blindness/visual impairment in ~1.9 million people. India is one of the five countries with nearly half of total active trachoma patients. Introduction of Global Elimination of Trachoma 2020 program by the WHO, using SAFE strategy (surgery for trachomatous trichiasis; Antibiotics for C. trachomatis; Facial cleanliness; and environmental improvement) greatly reduced the prevalence, but trachoma still persists in India. Global increase in the reproductive tract infection by C. trachomatis urogenital serotypes (D-K) has led to concurrent increase in C. trachomatis eye infections. Therefore, kerato eye infections due to chlamydial infections continue to be seen in hospitals. Over the years, there have been advances in laboratory diagnostics, in understanding the pathogenesis, tissue tropism, C. trachomatis genomics, and treatment modalities. Due attention and research is still needed for the study of C. trachomatis eye infections.

Isolation and genotyping of Acanthamoeba spp. from Acanthamoeba meningitis/ meningoencephalitis (AME) patients in India.

BACKGROUND: Acanthamoeba spp. are free-living ubiquitous protozoans capable of causing Acanthamoeba meningitis/meningoencephalitis (AME) of the central nervous system in humans. Acanthamoeba spp. are divided into 20 different genotypes (T1-T20) on the basis of variation in nucleotide sequences of the 18S rRNA gene. The objective of this study was to identify the genotypes of Acanthamoeba spp. in patients of Acanthamoeba meningitis/meningoencephalitis (AME) using 18S rRNA gene-based PCR assay. The present study provides information regarding the involvement of the most prevalent and predominant genotype of Acanthamoeba spp. in Acanthamoeba meningitis/meningoencephalitis infections in India. METHODS: Cerebrospinal fluid (CSF) was collected from 149 clinically suspected Acanthamoeba meningitis/meningoencephalitis (AME) patients reporting to the outpatient department/causality services of the Neurosciences Centre, AIIMS, New Delhi, India during the past five years. Samples were inoculated onto 2 % non-nutrient agar plates overlaid with E. coli and incubated at 30 °C for 14 days. Among 149 suspected patients, ten were found culture-positive for Acanthamoeba spp. out of which six isolates were established in axenic culture for molecular analysis. DNA was isolated and a PCR assay was performed for amplification of the Diagnostic fragment 3 (DF3) (~280 bp) region of the 18S rRNA gene from axenic culture of six Acanthamoeba spp. isolates. Rns genotyping was performed on the basis of the variation in nucleotide sequences of DF3 region of the 18S rRNA gene. RESULTS: In the phylogenetic analysis, all of the six Acanthamoeba spp. isolates were found to belong to genotype T4. The sequence homology search for these six isolates in the NCBI databank showed homology with the available strains of Acanthamoeba spp. The newly generated sequences are available in the GenBank database under accession numbers KT004416-KT004421. CONCLUSIONS: In the present study, genotype T4 was found as the most prevalent and predominant genotype in Acanthamoeba meningitis/ meningoencephalitis infections. Hence further studies are needed to develop optimal therapeutic strategy against Acanthamoeba spp. of genotype T4 to combat against the infections.

Genotyping of Acanthamoeba spp. and characterization of the prevalent T4 type along with T10 and unassigned genotypes from amoebic keratitis patients in India.

Free-living amoebae of the genus Acanthamoeba are the causative agents of severe sight-threatening infection of the cornea. This study was designed to characterize the genotype of 20 Acanthamoeba spp. isolates obtained from corneal scrapings of 183 suspected Acanthamoeba keratitis patients reporting to the Outpatient Department/Casualty Services of Dr R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India during the period 2011-2015. Corneal scrapings were inoculated onto 2 % non-nutrient agar plates overlaid with Escherichia coli and incubated at 30 °C for 15 days. Amongst 183 suspected Acanthamoeba keratitis patients, 29 were found culture-positive for Acanthamoeba spp. out of which 20 samples were established in axenic culture for molecular analysis. DNA was isolated and PCR assay was performed for the amplification of the diagnostic fragment 3 (DF3) (∼280 bp) region of the 18S rRNA gene from axenic culture of 20 Acanthamoeba spp. isolates. Rns genotyping was performed on the basis of variation in nucleotide sequences of the DF3 region of the 18S rRNA gene. In the phylogenetic analysis, 16 of the 20 isolates were found to be of prevalent genotype T4, two were of genotype T10 and the remaining two isolates were of unassigned genotypes. Hence, it was concluded that genotype T4 was found as the most predominant genotype involved in Acanthamoeba keratitis infections. Genotype T10, which had not been reported from India, was detected for the first time in two patients. Two isolates were found to be unique, which shared < 95 % homology with all the known genotypes (T1-T20) of Acanthamoeba spp.