RESUMO
Microsporidia are obligate intracellular parasites that infect eukaryotic cells and have emerged as major opportunistic human pathogens. Due to the difficulties in definitive laboratory diagnosis and insufficient knowledge, ocular microsporidiosis is infrequently reported in India. To improve diagnostic facilities, we have developed a novel duplex PCR (dPCR) for the simultaneous identification of both genera and species of isolates with microsporidian aetiology that cause keratitis. The material scraped from the corneas of 12 clinically diagnosed microsporidial keratitis patients was subjected to routine microbiological examinations and molecular diagnosis using a novel dPCR that targeted the small-subunit rRNA gene (SSU-rRNA) of microsporidia and Vittaforma corneae using genus- and species-specific primers. Of the 12 corneal scrapes, 6 showed positive results in smears, while dPCR provided positive amplification with both pan-microsporidial and V. corneae species-specific primers for 9 corneal scrapes. The results were validated by sequencing and blast analysis. The sensitivity of this novel dPCR method was higher than that of conventional microscopy in the diagnosis of corneal microsporidial infection. dPCR with specific primers is potentially more sensitive, specific and depends less on more complicated methods for exact identification of the aetiology of microsporidial keratitis.
Assuntos
Técnicas de Laboratório Clínico/métodos , Ceratite/diagnóstico , Microsporidiose/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Vittaforma/isolamento & purificação , Primers do DNA/genética , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genes de RNAr , Humanos , Índia/epidemiologia , Ceratite/microbiologia , Microsporidiose/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Dados de Sequência Molecular , Micologia/métodos , RNA Fúngico/genética , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Vittaforma/classificação , Vittaforma/genéticaRESUMO
PURPOSE: To evaluate resistances mediated by extended-spectrum ß-lactamases (ESBLs) and AmpC ß-lactamases among Gram-negative bacteria recovered from ocular infections. METHODS: As per the Clinical Laboratory Standards Institute M100-S-16 document, a total of 135 Gram-negative bacilli were recovered from ocular specimens and were subjected to phenotypic confirmation for ESBL production by the double-disc synergy test, cephalosporin and clavulanate combination disc test and E test, and, for AmpC ß-lactamase, the modified double-disc approximation method and AmpC disc test. RESULTS: In the double-disc synergy test, 21 (15.5%) isolates showed positive results against the cefpodoxime disc, 19 (14%) against cefpodoxime and cefotaxime, 15 (11%) against cefpodoxime, cefotaxime and ceftriaxone and 10 (7%) isolates were against cefpodoxime, cefotaxime, ceftriaxone and ceftazidime discs. In the cephalosporin/clavulanate combination disc test, 19 (14%) isolates showed positive results against cefotaxime with clavulanic acid and 10 (7%) isolates against ceftazidime with clavulanic acid. In the E test, 10 (7%) isolates displayed positive results against ceftazidime and 19 (14%) against cefotaxime. In the AmpC disc test for phenotypic confirmation, indentations were observed in 15 (11%) isolates with flattening also occurring in 10 (7%) isolates. CONCLUSION: The incidences of ESBL- and AmpC ß-lactamase-mediated resistances are found to be 7 and 18.5% among ocular isolates, respectively, and are more prevalent among the strains of Escherichia coli and Klebsiella pneumoniae.
