The incidence of fungal infections has increased in recent years due to the onset of acquired immunodeficiency syndrome (AIDS) and improved medical techniques including the use of antibiotics, immunosuppressive therapy and organ transplantation.1 Infectious fungal keratitis and endophthalmitis cause extensive ocular morbidity worldwide.2 Early diagnosis and rapid intervention is a critical element for effective treatment of ocular infections. This has led to the development of culture-independent diagnostic tests such as polymerase chain reaction (PCR). The potential utility of PCR techniques for improving the diagnosis of fungal infections is well recognized3 and the use of PCR for this purpose is expanding. Assays targeting Candida, Aspergillus and Fusarium species have been tested in vitreous specimens and recently an assay using panfungal primers has been used more extensively.456
The ideal marker to detect a fungal infection should be present in all fungal genera (but should contain enough internal variation in its sequence to define a species) and should be a multicopy gene to maximize the sensitivity of detection. A PCR using universal fungal primers specific for 28SrRNA gene has been evaluated in our earlier study.6 The technique of PCR using primers amplifying conserved region of the 28SrRNA, 18SrRNA with further separation of genus and species targeting internal transcribed spacer (ITS) region, on the basis of exploiting small but phylogenetically important base pair difference of medically important fungi7 as well as real time PCR8 is being used for detection of fungal infections. The present study focused on the evaluation of PCR targeting ITS region for detection of fungal genome directly from specimens.
Materials and Methods
A total of 168 specimens consisting of 145 intraocular specimens and 23 corneal scrapings were processed for detection of fungal etiology for the period between 2003-2005. A total of 20 specimens consisting of 12 aqueous humor (AH) collected from patients undergoing uncomplicated cataract surgery and eight vitreous fluid (VF) from patients having vitreous hemorrhage, proliferative diabetic retinopathy, inflammation of the eye due to non-infectious etiology were used as controls. However, the break-up of endophthalmitis could be obtained only for 133 as against 145. This was because 12 specimens were received from other referral hospitals located in various parts of India. The exact clinical details could not be obtained for these 12 cases. The clinical details with regard to endophthalmitis and keratitis are provided in Table 1. Informed consent was obtained from the patients from whom the ocular specimens were collected.
Corneal scrapings were collected by the ophthalmologist after application of local anesthetic. The culture plates were inoculated in the form of 'C' curves by the ophthalmologist and the smears were processed for detection of fungal etiology in the microbiology laboratory. The AH samples (50 to 150 ml) were collected aseptically in a tuberculin syringe after application of topical anesthesia. The VF was aspirated by a syringe connected to the suction port of the vitreous cutter at the beginning of vitrectomy. A sterile disposable needle was fixed to the syringe, the air in it expelled carefully without causing aerosols; the needle was capped with a sterile rubber bung and sent to the lab immediately. About 50 to 150 ml of AH and 100 to 200 µl of VF was received in the laboratory for microbiological investigations. On receipt, the specimen was divided into two equal portions. One half was used for DNA extraction and the other half was subjected to conventional methods of direct smear and culture. The specimens (AH and VF, biopsy, corneal scrapings) were processed within 30 min after collection for direct microscopy and culture of bacteria and fungi. Direct microbiological investigations were carried out on cytospinned smears (Cytospin 2 Shandon, Southern Products Limited, Cheshire, England) of intraocular fluids and stained by KOH-Calcofluor white for detection of fungus and Gram''s stain for detection of bacteria. The culture media used for isolation of bacteria and fungi were blood agar and MacConkey agar (incubated aerobically at 37°C), Chocolate agar (10% carbon dioxide), Brucella blood agar (37° C at anerobic work station), Sabouraud's dextrose agar incubated at 25° C in a cooling incubator and liquid medium brain heart infusion broth (BHIB), Thioglycollate broth incubated at 37° C. Culture plates and specially the liquid medium BHIB were incubated for a period of one month to isolate fungus. The fungal isolates were identified using standard mycological methods.91011 The DNA extraction from intraocular fluids and corneal scrapings was carried out using Biogene kit (Biogene, USA), for tissue and purulent specimens, Qiamp DNA minikit (Qiagen, Cat. No. 51304, Germany) was used. The DNA extraction was carried out on fresh specimens according to manufacturer''s instructions. Sensitivity of PCR was determined using serial tenfold dilutions of standard strain of Candida albicans ATCC 24433. Ten mg of C. albicans, A. fumigatus and F. lichenicola DNA was used as the template. Specificity of primers was verified by using DNA extracts of C. albicans (ATCC 90028), C. tropicalis (ATCC 750), C. parapsilosis (ATCC 90018), C. krusei (ATCC 6258) and laboratory isolates of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Fusarium, Curvularia and Alternaria species, Staphyloccus aureus (ATCC 12228), Pseudomonas aeruginosa (ATCC 27853), Mycobacterium tuberculosis H37Rv, Mycobacterium fortuitum (ATCC 1529), Mycobacterium chelonae (ATCC 1524) and laboratory isolates of Nocardia asteroides, Actinomyces species, herpes simplex virus (HSV) and human leukocyte DNA. For a 50 µl reaction, 8 µl of dNTPs (200µmoles), 5 µl of 1 X PCR buffer (1.5 mM MgCl2, 50 mM KCl, 10 mM Tris Cl,0.001% gelatin), 6 µl of 25 mM MgCl2 (1 in 10 diluted to get 1.5 mM), 10 picomoles of forward primer, ITS1 - 5' tcc gta ggt gaa cct gcg g 3'and reverse primer ITS4 - 5' tcc tcc gct tat tat gc 3'targeting ITS region and 10µl of template DNA was used.12 Amplification was carried out in Perkin Elmer thermal cycler (Model 2700). The first round of amplification yielded 520 - 611 bp product according to different fungal species for ITS region. Thermal cycling consisted of initial denaturation at 95o for 5 min followed by 35 cycles of denaturation at 95o C for 30 seconds, annealing at 55o C for 60 seconds and extension at 72o C for 60 seconds followed by final extension at 72o C for 6 min. Semi-nested amplification was carried out using the same PCR conditions as that of the first round with forward primer ITS4 and reverse primer ITS86 - 5' gtg aat cat cga atc ttt gaa c 3'. Five µl of amplified product was transferred from the first round to the second round and amplification was carried out in initial denaturation at 95o C for 5 min followed by 35 cycles of denaturation at 95o C for 30 seconds, annealing at 55o C for 30 seconds and extension at 72o C for 30 seconds followed by final extension at 72o C for 5 min. The amplified products were detected using 2% agarose incorporated with 0.5 µg/ml of ethidium bromide. The electrophoresis was carried out at 100 volts and documented using Gel documentation system (Vilber Lourmat).
The sensitivity of semi-nested PCR after two rounds of amplification is shown in [Fig. 1]. The ITS primers had a sensitivity of 1 femtogram of C. albicans DNA (Single C. albicans cell) and a sensitivity to detect 10fg of Aspergillus fumigatus and Fusarium lichenicola DNA. The specificity of ITS PCR is shown in [Fig. 2]. The primers were specific amplifying all the fungal DNA. No amplification was obtained with bacterial, viral and human DNA. The results of conventional microbiological investigations done on clinical specimens is given in Table 2. The conventional microbiological investigations revealed fungal etiology in 34 (20.23%) by smear and in 42 (25%) by culture. There was only a single failure in smear positive case (corneal scraping 1). Fungi were isolated in 33 specimens in which the direct smear revealed the presence of fungal elements. Culture was additionally positive in nine cases. The fungal isolates were A. flavus 14, A. fumigatus 5, A. niger 5, Fusarium species 4 C. parapsilosis 6, C. tropicalis 3, C. albicans 2, T. beigellei 2, A. terreus 1. The results of PCR using ITS primers applied on 168 clinical specimens for detection of fungal genome are given in Table 3. Fungal genome was not detected in all the 20 control specimens. The results of PCR applied on clinical specimens for fungal genome detection are shown in Fig. 3.
Fungal endophthalmitis is often difficult to diagnose and missed, unless proper microbiological studies are performed. Laboratory diagnosis is of great importance and the confirmation of the organism is the key to appropriate therapy. The laboratory diagnosis of fungal infection is highly dependent on traditional methods of microscopy and culture. Culture is considered as the 'gold standard' but its true sensitivity is not known and it is also time-consuming. The eye hospital in which the study was carried out is one of the major referral hospitals and the majority of the patients referred might have undergone several regimes of therapy including antifungal therapy resulting in negative culture results. In addition, the indolent nature and localized infection and fastidious nature of the fungi could have attributed to the lower yield by culture. Since PCR is a sensitive technique and requires the mere presence of DNA, more number of samples were positive as against culture in which the presence of viable organism is essential for isolation. These are the valid findings supporting the high positivity by PCR.
The major drawback of fungal culture is the prolonged period of time, minimum of two to four days and up to three weeks, required for isolation of slow-growing fungus on culture medium. In this study it varied from one to 15 days with an average of three days whereas the results of PCR were available within 5h from the time of collection of specimen. The negative results may be due to less sample volume, prior therapy with antifungal agents and fastidious nature of the organism. Molecular microbiological methods have greater sensitivity and specificity than conventional methods. Multicopy genes, 28SrRNA and ITS region were used as targets to increase the clinical sensitivity and universal panfungal primers were used to broaden the range of detectable fungi. We have proven in our earlier study6 that clinical sensitivity by using PCR has been increased. Our results of PCR on intraocular specimens to detect fungal genome indicate not only high analytical specificity but also increased sensitivity compared to the conventional methods, which was statistically significant correlating with the study conducted by Ferrer et al.12 The clinical sensitivity of PCR was increased by 30% as against conventional methods and it was found to be statistically significant (P < 0.001) using Z (normal approximation) test for two proportion. The DNA sequencing by Microseq kit was carried out on 11 fungal PCR positive AC tap specimens and five vitreous aspirate specimens. The results of PCR were validated by DNA sequencing of 11 fungal PCR positive AC tap specimens and five vitreous aspirate specimens. The 11 fungi from AH were identified as Fusarium falciforme (3), Candida albicans (2) Aspergillus flavus (2), Fusarium lichenicola (1) Candida tropicalis (1) Aspergillus fumigatus (1) and Trichosporon species (1). The five fungi in VF were Candida albicans (2), Aspergillus flavus (1), Aspergillus fumigatus (1) and Fusarium falciforme (1). In patients where the PCR result was positive and culture results negative, the identification of the fungi was obtained by the sequencing of the DNA followed by treatment with the appropriate antifungal therapy. Twenty-five per cent (the remaining were dropouts) of these patients improved with this therapy confirming the PCR result.
The reason for targeting ITS region in the study is that apart from imparting greater threshold for detection as it is a multicopy gene, it also exhibits sequence variation unique for species determination of fungi. Though the other molecular targets like 28SrRNA and 18SrRNA genes are being used for panfungal genome detection, ITS offers an added advantage for determining the species. By application of PCR rapid diagnosis was available within six hours of specimen collection. Furthermore, the results of PCR correlated well with specimens which were culture positive for fungus. Other proposed molecular techniques for fungal identification such as the use of RFLP (Zarzoso et al.13 Williams et al.14 Morace et al.15), hybridization with specific probe (Sandhu et al.16 Lindsley et al.17) and specific nested PCR (Hidalgo et al.18) could be useful in confirming specific fungal infection.
The development of semi-nested PCR for detection of panfungal genome proves to be a useful rapid diagnostic test for detection of fungal infection. However, ITS PCR needs to be applied to a large number of clinical specimens to determine its clinical specificity. To conclude, to the best of our knowledge this is the first study in India conducted applying semi-nested PCR targeting ITS region for detection of fungal genome which proved useful as a rapid diagnostic tool.
Source of Support:
Conflict of Interest:
We thank Alcon Research Laboratories for their technical assistance in carrying out the DNA sequencing work.
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