Characterization of Ocular Fungal Infections in Egypt - The Egyptian ...

Egyptian Journal of Medical Microbiology, January 2006 Vol. 15, No 1came from agricultural areas with a history oftrauma with vegetable or animal matter. Fullclinical history was obtained as well as fullophthalmic examination and any previoustreatments were reported. Patients alreadyreceiving antifungal drugs were excludedfrom the study. Informed consent wasobtained from all subjects participating in thisstudy.Specimens: Three samples were taken fromeach subject: one sample for direct staining,the 2 nd sample for culture and the 3 rd samplewas collected in sterile phosphate bufferedsaline (PBS, Dulbecco's GIBCO, BRL,Paisely, Scotland) and preserved at -70 o C forPCR. Specimens included conjunctival swabsfrom cases of conjunctivitis (and also fromcontrols), scrappings from the base, marginand the leading edge of the infiltrate fromcases of corneal ulcers (by means of Kimuralikeplatinum spatula, using slit lampbimicroscopy) 14,15 and vitreous aspirate fromcases of endophthalmitis (using steriledispasable 27 gauge needle with a tuberculinsyringe) 16,17 .Methods:I. Identification of fungi by direct smearusing Spot Test Calcofluor White Reagent(Difco Lab., Detroit, Michigan, USA),according to manufacturer's instructions.Briefly, the spot test was done by transferringthe specimen on a clean glass slide and gentlymixing it with two drops of 10% KOH. Anequal volume of Calcofluor white reagent wasdispensed and mixed onto the slide thenmounted with a cover slip. The specimen wasfinally examined by a fluorescent microscope(Olympus BX40). Fungal elements display abrilliant apple-green fluorescence.II. Culture: Specimens were immediatelyinoculated onto Sabouraud's dextrose agar(SDA, Oxoid) 18 and Chromagar Candida(Chromagar Microbiology, France) 19 . Plateswere incubated aerobically at 37 o C. ChromagarCandida plates were incubated for 48 hourswhereas SDA plates were incubated for 3weeks at least. Plates were examined every 48hours for evidence of growth 14,17 . Suspectedcolonies on Chromagar Candida wereidentified according to manufacturer'sinstructions.III. Staining of the suspected fungal coloniesby Lactophenol Cotton Blue (BectonDickinson Microbiology Systems, Maryland,USA) and visualization by light microscopy(Olympus BX50).IV. Identification of the different yeastcolonies by API Candida (Biomerieux, Lyon,France) using a 3 McFarland turbidity standardyeast suspension. The strips were incubatedaerobically at 37 o C for 24-48 hours 20 .V. Detection of fungal DNA bysemi-nested PCR: A rapid semi-nested PCRapproach for the detection of C. albicans or A.fumigatus was applied for all specimens (fromcases and controls). A first round PCR wasachieved using two outer primerscorresponding to nucleotides 40 to 63(forward primer, UNI-F) and 637 to 654(reverse primer, UNI-R), derived from thelarge subunit ribosomal DNA (rDNA)complex of Saccharomyces cerevisiae V3region. Identification of C. albicans or A.fumigatus was then achieved by a 2 ndamplification reaction using species-specificinner forward primers (Ca-F or Af-F,respectively) in combination with the outerreverse primer (UNI-R) 21,22 . The followingselected oligonucleotide primers weresynthesized by a commercial vendor(Biosynthesis, Germany): Outer forwardprimer (UNI-F), 5'- GCA TAT CAA TAAGCG GAG GAA AAG -3', Outer reverseprimer (UNI-R), 5'- GGT CCG TGT TTCAAG ACG -3', C. albicans (inner) forwardprimer (Ca-F), 5'- TTG GAG CGG CAGCAG GAT AAT GG -3' and A. fumigatus(inner) forward primer (Af-F), 5'- GCA TTCGTG CCG GTG TAC TTC -3'.DNA extraction was done with aQIAamp DNA Mini Kit (Qiagen, UK). Outerprimers were included in a 50 µl first PCRreaction which contained 3 µl of extractedDNA, 50 mM KCl, 10 mM Tris-HCl, pH 8.3,1.0 mM MgCl 2 , 20 µM of each dATP, dCTP,dGTP and dTTP, 0.5 unit of Taq DNApolymerase (all Fermentas, Germany) and 2µM of each primer. The cycling parameters(using Perkin-Elmer 9600 thermal cycler)consisted of 30 cycles: 94 o C for 30 sec(except for 5 minutes for the first cycle),66 o C for 90 sec and 72 o C for 15 sec (exceptfor 5 min for the last cycle; final extension).Species-specific PCR (30 cycles) wasperformed in exactly the same way as the firstround PCR except that UNI-F was replacedwith either Ca-F or Af-F primer, in additionto UNI-R primer. Aliquots of 1 µl of the 1 st238

Egyptian Journal of Medical Microbiology, January 2006 Vol. 15, No 1PCR amplification products were used astargets in the species-specific PCR.Post-PCR gel electrophoresis wasperformed on 2% agarose gel (Hispan agar,Spain), containing 0.5 µg/ml ethidiumbromide (Amresco, USA) in 1% TBE buffer(GIBCO, BRL, Life Technologies, USA).The amplification products were visualizedusing Spectroline Bio-Vision UV/White LightTransilluminator and compared to acommercial molecular weight marker(ΦX174 DNA-HaeIII Digest, New EnglandBioLabs), which gives bands at 1353, 1078,872, 603, 310, 281, 271, 234, 194, 118, 72 bp(N.B., 281 and 271 bp bands appear as asingle band). The expected PCR productlengths were 156 bp and 192 bp for C.albicans and A. fumigatus, respectively.Negative (sterile distilled water) and positivecontrols [C. albicans standard strain (ATCC10232) and A. fumigatus standard strain(ATCC 10827)] were included in each PCRrun.Statistical Analysis: Data were analyzedusing Statistical Package for Social Sciences(SPSS) software, Version 11. P value < 0.05was considered significant.RESULTS:The present study included 50 cases (30males and 20 females) of clinically suspectedoculomycosis [39 cases (78%) ofkeratoconjunctivitis and 11 cases (22%) ofendophthalmitis] as the study group (cases),in addition to 20 healthy individuals (10males and 10 females) serving as the controlgroup.Table (1): Results of positive direct stain with Calcofluor white stain among cases and controlsFungusCases (N o = 50) Controls (N o = 20) P valueN o % N o %Yeasts 12 24 1 5 < 0.001Hyphae 20 40 1 5Total 32 64 2 101-a 1-b 1-cFigure (1): Calcofluor white stained specimens showing:1-a: Budding oval cells of C. albicans1-b: Germ tube of C. albicans1-c: Hyphal elements of septate filamentous fungi239

Egyptian Journal of Medical Microbiology, January 2006 Vol. 15, No 1Table (2): Comparison between the isolation rates of different fungal species on Sabouraud'sdextrose agar (SDA) and Chromagar Candida among cases and controlsSDAChromagar CandidaCases Controls Cases ControlsN o % N o % N o % N o %No growth 25 50 16 80 42 84 19 95Candida Spp.* 8 16 1 5 8 16 1 5Aspergillus spp.** 8 16 1 5 0 0 0 0Penicillium 4 8 1 5 0 0 0 0Alternaria 2 4 1 5 0 0 0 0Curvularia 2 4 0 0 0 0 0 0Rhodotorula rubra 1 2 0 0 0 0 0 0Total 50 100 20 100 50 100 20 100N.B.: *Isolated candida species included 4 C. albicans, 2 C. krusei, 1 C. parapsilosis and 1 C.tropicalis among cases, whereas only 1 C. parapsilosis was identified among controls(confirmed by API Candida).** Isolated Aspergillus species included 4 A. fumigatus, 2 A. niger and 2 A. flavus amongcases, whereas only 1 A. fumigatus was isolated from controls (confirmed bylactophenol cotton blue stain).2-a 2-b 2-c2-d 2-e 2-fFigure (2): Lactophenol cotton blue stain of:2-a: A. fumigatus (showing conidiophore having a flask-shaped swollen end called vesicle).2-b: A. flavus (showing conidiophore arising from a foot cell and terminating in a vesicle, whichproduces phialides in one or two series and unicellular conidia).2-c: A. niger (showing black spore heads).2-d: Penicillium (showing the brush appearance)2-e: Alternaria (showing multi-celled conidium).2-f: C. albicans (showing the characteristically asexual budding).240

Egyptian Journal of Medical Microbiology, January 2006 Vol. 15, No 1Table (3): Results of culture in relation to the clinical diagnosis of casesCulture ResultsKeratoconjunctivitis EndophthalmitisN o % N o %No growth 22 56.4 3 27.2Aspergillus species 6 15.4 2 18.2A. fumigatus 2 5.1 2 18.2A. niger2 5.1 0 0A. flavus 2 5.1 0 0Candida species 4 10.3 4 36.4C. albicans 2 5.1 2 18.2C. krusei1 2.6 1 9.1C. tropicalis 1 2.6 0 0C. parapsilosis 0 0 1 9.1Penicillium 2 5.1 2 18.2Alternaria 2 5.1 0 0Curvularia 2 5.1 0 0Rhodotorula rubra 1 2.6 0 0Total 39 100 11 100N.B.: - Positive fungal culture was obtained in 17 cases (43.6%) of keratoconjunctivits and in 8 cases(72.7%) of endophthalmitis.Comparison between keratoconjunctivitis andendophthalmitis cases, regarding the commonpredisposing factors, revealed that out of the39 cases of keratoconjunctivitis, 22 cases(88%) had a definite history of trauma and 3cases (12%) were post-surgical. However, outof the 11 cases of endophthalmitis, 9 caseswere exogenous endophthalmitis [6 cases(54.5%); following cataract operations and 3cases (27.2%); following trauma] and 2 cases(18.2%) were endogenous endophthalmitis.Table (4): Comparison between direct smear and culture, regarding detection of mycosis amongcasesCultureTestPositiveNegativeTotalN o % N o % N o (%)Direct Positive 24 48 8 16 32 (64%)Smear a Negative 1 2 17 34 18 (36%)Total 25 50 25 50 50 (100%)aSensitivity = 96% Specificity = 68% Accuracy = 82%Positive Predictive Value (PPV) = 75% Negative Predictive Value (NPV) = 94.4%API Candida results revealed 4isolates of C. albicans, 2 isolates of C. krusei,1 isolate of C. tropicalis and 1 isolate of C.parapsilosis (a total of 8 Candida isolates)among cases, whereas among controls, APICandida could identify only one Candidaspecies (C. parapsilosis). There was acomplete agreement (100%) between theresults of Chromagar Candida and APICandida in the identification of the differentCandida species.Among the 50 cases of suspectedocular mycosis, PCR - performed directly onclinical specimens - revealed 6 positive (12%)C. albicans and 7 positive (14%) A.fumigatus. However, among controls, onlyone positive A. fumigatus (5%) was detectedby PCR. No statistically significant differencewas found - regarding the detection rates of A.fumigatus by PCR - between cases andcontrols (P value 0.28). Compared to culture,PCR proved to be 100% sensitive in thedetection of C. albicans and A. fumigatus.241

Egyptian Journal of Medical Microbiology, January 2006 Vol. 15, No 11 2 3 4 5 6 7 8Figure (3): Gel electrophoresis showing C. albicans and A. fumigatus - PCR productsLane 1: positive control for A. fumigatus (ATCC 10827), giving band at 192 bp. Lanes 2and 3: positive samples for A. fumigatus. Lane 4: PCR molecular weight marker(ΦX174 DNA-HaeIII Digest, New England BioLabs), which gives bands at 1353,1078, 872, 603, 310, 281, 271, 234, 194, 118, 72 bp (281 and 271 bp bandsappearing as a single band). Lane 5: positive control for C. albicans (ATCC10232), giving band at 156 bp. Lanes 6 and 7: positive samples for C. albicans.Lane 8: negative control (H2O).Table (5): Results of culture and PCR analysis for C. albicans of specimens from patients withpresumed oculomycosisSpecies-Specific PCRC. albicans CultureforPositiveNegativeTotalC. albicans a N o % N o % N o (%)Positive 4 8 2 4 6 (12%)Negative 0 0 44 88 44 (88%)Total 4 8 46 92 50 (100%)aSensitivity = 100% Specificity = 95.7% Accuracy = 97.9%Positive Predictive Value (PPV) = 66.7% Negative Predictive Value (NPV) = 100%for A. fumigatusTable (6): Results of culture and PCR analysis for A. fumigatus of specimens from patients withpresumed oculomycosisSpecies-Specific PCRA. fumigatus CulturePositiveNegativeTotalN o % N o % N o (%)Positive 4 8 3 6 7 (14%)Negative 0 0 43 86 43 (86%)Total 4 8 46 92 50 (100%)aSensitivity = 100% Specificity = 93.5% Accuracy = 96.8%Positive Predictive Value (PPV) = 57.1% Negative Predictive Value (NPV) = 100%DISCUSSIONOcular infections due to opportunisticfungal pathogens are steadily increasing 6,23 .Fungal keratitis is a major ophthalmologicproblem in the tropical regions of the worldand it is one of the frequent causes of cornealdamage in developing countries 24-26 .242

Egyptian Journal of Medical Microbiology, January 2006 Vol. 15, No 1Preservation of vision requires early diagnosisand early institution of fungal therapy 6,27 .Non-specific fluorochromatic stainshave become popular for the detection offungi in ocular samples. Calcofluor whiteappears to be the most widely used of thesestains 28 . In the present study, 50 cases ofsuspected oculomycosis were investigated.Direct microscopic examination withCalcofluor white stain, mounted with 10%KOH revealed fungal elements in 32 cases(64%), whereas positive fungal cultures; ondifferent culture media; were obtained in 25cases (50%). Direct smear and fungal culturematched (were both positive or negative) in41 (82%) of the 50 specimens from cases.Similar results were reported by Nag et al. 29who stated that a wet mount with 10% KOHshowed fungal elements in 68% of cornealulcers, while culture was positive in only26.4% of the cases. Moreover, other studiesreported higher sensitivity of fungal detectionusing KOH and Calcofluor white stain thanculture 7,24 . Calcofluor white sensitivity of 80–90% in culture-proven mycotic keratitis hasbeen reported 28 . This agrees with our results,where Calcofluor white stain showed asensitivity of 96% in culture-proven cases ofoculomycosis. Calcofluor white fluorescentstaining proved to be a simple technique thatovercomes the difficulties and time involvedin staining 24,28,30 .Even with the advent of many newtechniques, culture remains the “goldstandard” and the cornerstone of the diagnosisof most ophthalmic mycoses, except forrhinosporidiosis, where direct microscopicexamination of samples yields more reliableresults 3,24,25,28,31 . Wherever possible, it is bestto use more than one culture medium, and toincubate these at 37°C and at 25-30°C for theoptimal recovery of ocular fungi 32 .Our culture results among cases(showing 50% positivity) are similar to thoseof El-Sawy et al. 33 and El-Mowafy et al. 34who detected fungal isolation from cultures in45% and 47.5% of cases of clinicallysuspected fungal keratitis, respectively.In the present study, positive fungalculture was obtained in 17 cases (43.6%) ofkeratoconjunctivitis. The most prevalentisolates were Aspergillus species thenCandida species, followed by Penicillium,Alternaria, Curvularia and lastlyRhodotorula rubra. This agrees with theresults of Tanure et al. 35 . On the other hand,different fungal species were isolated in 8cases (72.7%) of endophthalmitis; the mostcommon were Candida species followed byAspergillus fumigatus and lastly Penicillium.Internationaly, Aspergillus species isthe most common isolate (27%-64%) in casesof fungal keratitis worldwide, followed byfusarium (6%-32%) and penicillium (2%-29%) 1 . Similar studies also concluded thatfilamentous fungi are the principal causes ofmycotic keratitis in most parts of the world;either Aspergillus spp. or Fusarium spp. werethe most common isolates. Dematiaceousfungi, such as Curvularia spp. and Bipolarisspp., are the third most important cause ofkeratitis in a number of studies 3,24,25,36-39 . Astudy conducted in Saudi Arabia, concludedthat the higher prevalence of Aspergillusspecies; among cases of keratitis; could beexplained by the fact that its spores couldsurvive the hot dry weather 37 , as is the case inEgypt.Keratitis; due to yeasts and yeast-likefungi; is most frequently caused by C.albicans. Keratitis due to this organism tendsto occur more frequently in areas wheretraumatic keratitis is uncommon, but whereother predisposing factors causing epithelialor stromal ulceration are important 40 , forexample, due to previous herpes simplexkeratitis, contact lens-induced cornealabrasions 14 or in patients with dry eyes 41 . C.albicans and related fungi have beeninfrequent isolates in most studies performedin tropical countries, possibly due to thepredominance of livelihoods, such asagriculture, which carry a higher risk for theoccurrence of trauma-related keratitis causedby filamentous fungi than for keratitis due toC. albicans 42 .In the present study, of the 39 cases ofkeratoconjunctivitis, 22 cases (88%) had adefinite history of trauma and 3 cases (12%)were post-surgical. Moreover, of the 11 casesof endophthalmitis, 6 cases (54.5%) wereclinically manifested following cataractoperations, 3 cases (27.2%) followed traumaand 2 cases (18.2%) occurred secondary tohematogenous dissemination in patientshaving debilitating diseases (endogenousendophthalmitis).Many studies reported that a definitehistory of ocular trauma (usually withvegetable matter) was the most common243

Egyptian Journal of Medical Microbiology, January 2006 Vol. 15, No 1predisposing factor for mycotic keratitis,(occurring in 44 to 55% of patients); lessfrequently reported risk factors includeprolonged use of topical corticosteroids oranti-bacterials, systemic diseases such asdiabetes mellitus, pre-existing ocular diseases,contact lens wear and previous ocularsurgery 2,24,39,42-44 . On the other hand, mycoticendophthalmitis has been reported to occurafter cataract surgery (61%), trauma (28%) oras metastatic endogenous endophthalmitis(11%) 45 . The yeast C. albicans is the mostcommon cause of endogenousendophthalmitis 46 . Moreover, Candidaspecies are particularly likely to causeexogenous endophthalmitis. In this setting,infection may be due to peri-operativecontamination of lens prostheses orcontamination of fluids used for irrigation ofthe eye. Infection may be also enhanced bythe pre- and post-operative use of topicalcorticosteroids and anti-bacterial agents 42 .However, Aspergillus endophthalmitis is thecommonest type of vision-threatening fungalendophthalmitis encountered in India 47 .Our results revealed that Sabouraud'sdextrose agar allowed for the recovery ofmost fungal pathogens within 2 weeks ofincubation. Generally, the duration ofincubation - for most yeasts - is one week,although the range of time for recovery ofdimorphic fungi may extend to 4-6weeks 25,48,49 .The present study also showed acomplete agreement (100%) between theresults of Chromagar Candida and APICandida in the identification of the differentCandida species, however, the yeast colonycolor development (on Chromagar Candida)or its proper identification (by API Candida)was better reported after 48 hours ofincubation, as previously noted 20,50 . Thisdelay in the identification by cultural and/orbiochemical procedures or the limited yield ofvitreous cultures (in cases of endophthalmitis)has led to a search for more rapidtechniques 51 . The speed and sensitivity ofPCR makes it an ideal choice for the basis ofa rapid identification system 51,52 . PCR hasbeen reported to be a more sensitive and rapiddiagnostic tool, compared to the conventionalmycologic methods in the diagnosis ofoculomycosis, where 55.8% of cases offungal endophthalmitis were positive byconventional methods versus 74.4% byPCR 47 . Similarly, Hidalgo et al. 51 andLohmann et al. 53 concluded that PCR had ahigher rate of positive fungal identificationthan by microscopy or diagnostic culture.This is similar to our results.Using species-specific primers in thepresent study, PCR could detect 6 positive C.albicans (12%) and 7 positive A. fumigatus(14%) among cases of suspectedoculomycosis; versus only 1 positive A.fumigatus (5%) among the control group. Nostatistically significant difference was found -regarding the detection rates of A. fumigatusby PCR - between cases and controls (P value0.28), which might be explained by the smallsample size included in the study. However,using culture, 4 C. albicans (8%) and 4 A.fumigatus (8%) isolates were recovered fromcases and only one A. fumigatus isolate wasrecovered from controls. Among the 50 casesin the present study, PCR and fungal culturefor C. albicans and A. fumigatus matched in48 (96%) and 47 (94%) of the specimens,respectively. In 2 (4%) and 3 (6%) casespecimens, PCR detected C. albicans and A.fumigatus, respectively, where no organismwas found in culture. All of these patientsappeared clinically to have ocular fungalinfections, and fungi were present onCalcofluor white smear for all.PCR would probably be most valuablein providing a positive result in a shorterperiod than that required for culture 6,12,53 andin identification of a fungal isolate whichdoes not sporulate 54 . Moreover, PCR offersthe ability to analyze specimens far fromwhere they are collected 11,13 . Eventually, PCRmight solidly complement the current “goldstandard” diagnostic techniques for guidingmanagement or supporting research studies ofoculomycosis 13 . However, concern persistsregarding the specificity of this technique andthe problems that may arise from theproduction of false-positive results. PCR doesnot distinguish viable from non-viableorganisms; it may therefore be difficult toassess the relevance of a positive PCR resultin a healing corneal ulcer, where culture isnegative 55 , or in locations such as theconjunctival sac, where fungi may be foundas transient commensals 13 . Although PCR isextremely sensitive and specific, it cannot beused to monitor the patient’s response totreatment. Moreover, A few culture mediawill suffice to detect and grow the common244

Egyptian Journal of Medical Microbiology, January 2006 Vol. 15, No 1ocular pathogens, but PCR must bemultiplexed for each microorganism that issuspected 56 . The difficulty of DNA extraction(some filamentous fungi have a sturdy cellwall; which is resistant to standard DNAextraction procedures) and the presence ofPCR inhibitors in human specimens are someof the difficulties encountered with fungaldetection in ocular samples 57 . Finally, PCRcan detect only fungi for which the DNAsequence is known and primers areavailable 58 .To conclude, the increased awarenessof ocular fungal infections, the betterrecognition of their clinical features and theimproved laboratory diagnostic techniques,will all lead to an increase in the frequency ofcorrect diagnosis. A rapid and accuratediagnosis of oculomycosis will improve thechances of a complete recovery, especially inthe tropics, where patients may delaypresenting to an ophthalmologist. Calcofluorwhite fluorescent staining proved to be asimple and reliable technique for initiation ofantifungal therapy. Culture methods remainimportant diagnostic tools that add to theproper identification and characterization ofoculomycosis, however, new culture medianeed to be developed. PCR is a potentiallyvaluable technique for diagnosing ocularfungal pathogens, particularly, if panfungalprimers are used and optimized. AlthoughPCR is expensive and requires laboratoryexpertise, yet the loss of an eye, due to delayin laboratory diagnosis, is devastating.References:1. Alexandrakis G, Kanellopoulous J,Donald S, et al. (2002): Fungalkeratitis. In: Kuhn F and Pieramici D(eds.): Ocular trauma: Principles andPractice, P. 293-301, Thieme MedicalPublishers, New York.2. Srinivasan R, Kanungo R and GoyalJL (1991): Spectrum of oculomycosis inSouth India. Acta Ophthalmologica, 66:774-779.3. Garg P, Gopinath G, Choudhary Kand Rao GN (2000): Keratomycosis:clinical and microbiological experiencewith dematiaceous fungi. Ophthalmol.,107: 574-580.4. Bloom PA, Laidlaw DA, Easty DL, etal. (1992): Treatment failure in a case offungal keratitis caused byPseudallescheria boydii. Br. J.Ophthalmol., 76 (6): 367-368.5. Thomas PA (2003): Fungal infection ofthe cornea. Eye, 17: 852-862.6. Kumar M and Shukla PK (2005): Useof PCR targeting of internal transcribedspacer regions and single-strandedconformation polymorphism analysis ofsequence variation in different regions ofrRNA genes in fungi for rapid diagnosisof mycotic keratitis. J. Clin. Microbiol.,43(2): 662-668.7. Sharma S, Kunimoto DY, Gopinthan Uet al. (2002): Evaluation of cornealscraping smear examination methods inthe diagnosis of bacterial and fungalkeratitis: a survey of eight years oflaboratory experience. Cornea, 21(7):643-647.8. Chandler FW (1991): Histologicdiagnosis of mycotic diseases, p. 235-242. In: Gatti F, de Vroey C and Persi A(eds.): Human mycoses in tropicalcountries. Health Cooperation Papers N o13. Organizzazione per la CooperazioneSanitaria Internazionale, Bologna, Italy.9. Mendonza L, Ajello L and McGinnisMR (1996): Infections caused by theoomycetous pathogen Pythiuminsidiosum. J. Mycol. Med., 6: 151-164.10. Ahmad, S, Khan Z, Mustafa AS et al.(2002): Semi-nested PCR for diagnosisof candidemia: comparison with culture,antigen detection, and biochemicalmethods for species identification. J.Clin. Microbiol., 40: 2483-2489.11. Wu Z, Tsumura Y, Blomquist G, et al.(2003): 18S rRNA gene variation amongcommon airborne fungi anddevelopment of specific oligonucleotideprobes for the detection of fungal isolate.Appl. Environ. Microbiol., 69: 5389-5397.12. Ferrer C, Munoz G, Alio JL et al.(2002): Polymerase chain reactiondiagnosis in fungal keratitis caused byAlternaria alternata. Am. J. Ophthalmol.,133: 398-399.13. Gaudio, PA, Gopinathan U, SangwanV, et al. (2002): Polymerase chainreaction based detection of fungi ininfected corneas. Br. J. Ophthalmol.,86(7): 755-760.14. Forster RK (1994): Fungal keratitis andconjunctivitis: Clinical aspects. In:245

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Egyptian Journal of Medical Microbiology, January 2006 Vol. 15, No 1توصيف عدوى فطريات العين في مصر٢١١١إيمان أحمد الصعيدي ، زينب عبد الخالق إبراهيم ، شروق خميس الحدقي ، هشام عادل حسنقسم الميكروبيولوجيا الطبية٢١والمناعة وقسم الرمد – كلية الطب -جامعة القاهرةيعتبر التهاب العين الفطرى من الأمراض المتزايدة والتى تمثل تحديا ً كبيرا ً وذلك لتأخر تشخيصهاوبالتالى تأخر علاجها مما قد يؤدى إلى مضاعفات كثيرة بالعين.‏ ويعد التهاب القرنية أكثر هذه الأمراضشيوعا"،‏ ولكن يمكن أن تحدث مثل هذه الإصابة في أى جزء آخر من العين.‏ وتمثل الإصابات الفطرية للقرنيةحواليلها.‏%٥٠من كل حالات العدوى الميكروبية للقرنية والتي تم إثباتها فعليا"‏ بالزرع.‏تحدث الإصابات الفطرية بواسطة عدد كبير من أنواع وسلالات الفطريات،‏ تبعا"‏ للمنطقة الجغرافيةوهذه الأعداد في تزايد مستمر.‏ ويمكن أن يساعد التشخيص السريع والدقيق لهذه الفطريات في بدء العلاج الفعالكان الهدف من هذه الدراسة هو تقييم استعمال التفاعل المتسلسل لخميرة البلمرة في تشخيص عدوىفطريات العين،‏ بالمقارنة بالطرق التشخيصية التقليدية الآخرى،‏ وذلك من خلال عدد ٥٠ حالة بها احتمالالتهاب فطرىبالعين تم تشخيصها إكلينيكيا"‏ وأيضا"‏ عدد٢٠ شخصا"‏وجودكمجموعة ضابطة.‏ تم أخذ مسحات منالملتحمة أو عينات من القرح القرنية أوالسائل الزجاجى بالعين من الحالات،‏ بينما تم أخذ مسحات من الملتحمةمن المجموعة الضابطة.‏تشخيص عددعن نتائجحالات إيجابيةوقد أظهرت نتائج البحث أنه تم تشخيص عدد٣٢ حالة (%٦٤)٢٥ حالة (%٥٠)عن طريق صبغة الكلكوفلور و كذلكعن طريق الزرع في المرضى.‏ أما بالنسبة للمجموعة الضابطة،‏ فقد تم الكشفإيجابية بنفس هذه الصبغة وكذلك بالزرع في عدد٢ و٤ أشخاص فقط،‏ على التوالي.‏تم تشخيص نسبة أكبر من الحالات عن طريق التفاعل المتسلسل لخميرة البلمرة،‏ حيث وجد عدد٦(%١٢)للفطور البيض وكذلك عدد٧ حالاتإيجابيةأما بالنسبة للمجموعة الضابطة،‏ فقد تم تشخيص حالة واحدة فقط فيها(%١٤)(%٥)للرشاشيات الدخناء في المرضى.‏للرشاشية الدخناء.‏خلص البحث إلى أن صبغة الكلكوفلور وكذلك التفاعل المتسلسل لخميرة البلمرة من الطرق الواعدة فيتشخيص حالات عدوى فطريات العين،‏ ولكن يبقى للزرع أهمية تشخيصية كبرى.‏248