Microsporidial keratitis in India: 16S rRNA gene-based PCR assay for diagnosis and species identification of microsporidia in clinical samples

被引:45
作者
Joseph, Joveeta
Sharma, Savitri [1 ]
Murthy, Somasheila I.
Krishna, Pravin V.
Garg, Prashant
Nutheti, Rishita
Kenneth, John
Balasubramanian, Dorairajan
机构
[1] LV Prasad Eye Inst, Jhaveri Microbiol Ctr, Hyderabad 500034, Andhra Pradesh, India
[2] LV Prasad Eye Inst, Cornea Serv, Hyderabad 500034, Andhra Pradesh, India
[3] LV Prasad Eye Inst, Int Ctr Adv Rural Eye Care, Hyderabad 500034, Andhra Pradesh, India
[4] LV Prasad Eye Inst, Res Biochem, Hyderabad 500034, Andhra Pradesh, India
关键词
POLYMERASE-CHAIN-REACTION; STOOL; EXTRACTION; DNA; SPECIMENS; ANTIBODY; PATIENT; AIDS;
D O I
10.1167/iovs.06-0376
中图分类号
R77 [眼科学];
学科分类号
100212 ;
摘要
PURPOSE. To evaluate 16S rRNA-based polymerase chain reactions for the detection and species identification of the microsporidia that cause keratitis. METHODS. Of the 5892 cases of microbial keratitis seen between September 2002 and December 2005, 31 (0.5%) microscopically diagnosed cases of microsporidial keratitis were included in the test group; 103 patients with nonmicrosporidial keratitis constituted the control group. A 16S rRNA-based pan-microsporidian PCR was chosen for the detection of microsporidian DNA. Species level identification was made using species-specific primer sets of Encephalitozoon spp (E. cuniculi, E. hellem, and E. intestinalis). Sequencing and BLAST analysis of amplicons obtained with pan-microsporidian primers were performed for validation. RESULTS. The corneal scrapings from 26 of 31 cases in the test group and 2 of 103 cases in the control group showed a 250- to 280-bp amplicon in PCR by pan-microsporidian primers (sensitivity of 83% and specificity of 98%). The amplicons of 13 of 26 test group samples were identified by species-specific PCR: E. cuniculi, n = 7 (549 bp); E. hellem; n = 3 (549 bp); E. intestinalis; n = 1 (520 bp). The two cases in the control group were identified to be E. cuniculi. The remaining 15 cases (test group) were confirmed to be Vittaforma corneae by sequencing and BLAST analysis. All species were confirmed by sequencing and database homology comparison. CONCLUSIONS. This study is the first to validate PCR-based assays for detection of microsporidial DNA in corneal scrapings. Pan microsporidian PCR can be a useful adjunct with smear examination in the diagnosis of microsporidial keratitis.
引用
收藏
页码:4468 / 4473
页数:6
相关论文
共 20 条
[1]   DETECTION OF MICROSPORIDIA BY INDIRECT IMMUNOFLUORESCENCE ANTIBODY-TEST USING POLYCLONAL AND MONOCLONAL-ANTIBODIES [J].
ALDRAS, AM ;
ORENSTEIN, JM ;
KOTLER, DP ;
SHADDUCK, JA ;
DIDIER, ES .
JOURNAL OF CLINICAL MICROBIOLOGY, 1994, 32 (03) :608-612
[2]  
ASUBEL FM, 1990, CURRENT PROTOCOLS MO, V2
[3]   Detection of BK virus in urine by polymerase chain reaction: a comparison of DNA extraction methods [J].
Behzadbehbahani, A ;
Klapper, PE ;
Vallely, PJ ;
Cleator, GM .
JOURNAL OF VIROLOGICAL METHODS, 1997, 67 (02) :161-166
[4]   DNAzol(R): A reagent for the rapid isolation of genomic DNA [J].
Chomczynski, P ;
Mackey, K ;
Drews, R ;
Wilfinger, W .
BIOTECHNIQUES, 1997, 22 (03) :550-553
[5]   Detection of Septata intestinalis (Microsporidia) Cali et al. 1993 using polymerase chain reaction primers targeting the small subunit ribosomal RNA coding region [J].
DaSilva, AJ ;
Slemenda, SB ;
Visvesvara, GS ;
Schwartz, DA ;
Wilcox, CM ;
Wallace, S ;
Pieniazek, NJ .
MOLECULAR DIAGNOSIS, 1997, 2 (01) :47-52
[6]   POLYMERASE CHAIN-REACTION AND CULTURE CONFIRMATION OF DISSEMINATED ENCEPHALITOZOON-CUNICULI IN A PATIENT WITH AIDS - SUCCESSFUL THERAPY WITH ALBENDAZOLE [J].
DEGROOTE, MA ;
VISVESVARA, G ;
WILSON, ML ;
PIENIAZEK, NJ ;
SLEMENDA, SB ;
DASILVA, AJ ;
LEITCH, GJ ;
BRYAN, RT ;
REVES, R .
JOURNAL OF INFECTIOUS DISEASES, 1995, 171 (05) :1375-1378
[7]   Diagnosis of herpes simplex virus-1 keratitis using Giemsa stain, immunofluorescence assay, and polymerase chain reaction assay on corneal scrapings [J].
Farhatullah, S ;
Kaza, S ;
Athmanathan, S ;
Garg, P ;
Reddy, SB ;
Sharma, S .
BRITISH JOURNAL OF OPHTHALMOLOGY, 2004, 88 (01) :142-144
[8]   IDENTIFICATION OF MICROSPORIDIA IN STOOL SPECIMENS BY USING PCR AND RESTRICTION ENDONUCLEASES [J].
FEDORKO, DP ;
NELSON, NA ;
CARTWRIGHT, CP .
JOURNAL OF CLINICAL MICROBIOLOGY, 1995, 33 (07) :1739-1741
[9]   Molecular techniques for detection, species differentiation, and phylogenetic analysis of microsporidia [J].
Franzen, C ;
Müller, A .
CLINICAL MICROBIOLOGY REVIEWS, 1999, 12 (02) :243-+
[10]   Laboratory identification of the microsporidia [J].
Garcia, LS .
JOURNAL OF CLINICAL MICROBIOLOGY, 2002, 40 (06) :1892-1901