Candida DNA Research Articles

Recent Challenges in Diagnosis and Treatment of Invasive Candidiasis in Neonates.

Invasive Candida infections represent a significant cause of morbidity and mortality in the neonatal intensive care unit (NICU), particularly among preterm and low birth weight neonates. The nonspecific clinical presentation of invasive candidiasis, resembling that of bacterial sepsis with multiorgan involvement, makes the diagnosis challenging. Given the atypical clinical presentation and the potential detrimental effects of delayed treatment, empirical treatment is often initiated in cases with high clinical suspicion. This underscores the need to develop alternative laboratory methods other than cultures, which are known to have low sensitivity and a prolonged detection time, to optimize therapeutic strategies. Serum biomarkers, including mannan antigen/anti-mannan antibody and 1,3-β-D-glucan (BDG), both components of the yeast cell wall, a nano-diagnostic method utilizing T2 magnetic resonance, and Candida DNA detection by PCR-based techniques have been investigated as adjuncts to body fluid cultures and have shown promising results in improving diagnostic efficacy and shortening detection time in neonatal populations. This review aims to provide an overview of the diagnostic tools and the current management strategies for invasive candidiasis in neonates. Timely and accurate diagnosis followed by targeted antifungal treatment can significantly improve the survival and outcome of neonates affected by Candida species.

Investigation of Candidemia Diagnosis by PCR Method in Febrile Neutropenic Patients with Hematological Malignancies

Objective: Candidiasis" is the predominant fungal infection that occurs invasively and opportunistically in individuals with compromised immune systems. Febrile neutropenia patients receiving cytotoxic chemotherapy due to malignancy, especially acute leukemia and stem cell transplantation are at greater risk among many others. In this study, it was aimed to search for Candida DNA to make an early diagnosis by using PCR method Materials & Method: This prospective study was conducted in Pediatric Hematology Transplantation Clinic of Akdeniz University. The study enrolled 78 patients with malignancies and candidiasis infection who were experiencing febrile neutropenia. The febrile neutropenic episodes were identified prospectively by monitoring the daily leukocyte counts and fever symptoms of the patients. Results: Candida was grown in the blood cultures of seven (9%) of the febrile neutropenic patients included in the study. All of the breeding Candidas were C.albicans. The underlying primary diagnosis of 6 (85.7%) of the febrile neutropenic patients with reproduction was hematological malignancy, while the underlying primary diagnosis of one (12.5%) was solid malignancy (Neuroblastoma). PCR detected Candida DNA in 9% of Febrile Neutropenic patients. C.albicans was grown in the blood cultures of all patients with Candida DNA. Conclusion: As a result, The conclusion drawn was that in cases where candidemia is suspected, particularly in febrile neutropenic patients with hematological malignancies who are immuno-compromised and at high risk of mortality, rapid and accurate diagnosis using Candida PCR with high sensitivity and specificity is necessary.

Detection of Candida DNA in peritoneal fluids by PCR assay optimizing the diagnosis and treatment for intra-abdominal candidiasis in high-risk ICU patients: A prospective cohort study.

Intra-abdominal candidiasis (IAC) is the predominant type of invasive candidiasis with high mortality in critically ill patients. This study aimed to investigate whether the polymerase chain reaction (PCR) assay for detecting Candida DNA in peritoneal fluids (PF) is useful in diagnosing and management of IAC in high-risk patients in intensive care unit (ICU). A prospective single-center cohort study of surgical patients at high risk for IAC was conducted in the ICU. PF was collected from the abdominal drainage tubes (within 24 h) or by percutaneous puncture. Direct PF smear microscopy, PF culture, blood culture, and serum (1-3)-β-D-glucan were performed in all patients. For Candida PCR assay, the ITS1/ITS4 primers that targeted the ITS1-5.8 s-ITS2 regions were used for PCR, and sequencing analysis was used to identify the pathogen at the species level. IAC was defined according to the 2013 European consensus criteria. Among 83 patients at high risk for IAC, the IAC criteria were present in 17 (20.5%). The sensitivity and specificity of the Candida PCR assay were 64.7 and 89.4%, respectively, and the area under the receiver operating characteristic curve was 0.77 (95% CI: 0.63-0.91). In this cohort, the positive predictive value and negative predictive value were 90.8% (95% CI: 80.3-96.2%) and 61.1% (95% CI: 36.1-81.7%), respectively. Diagnostic consistency was moderate (kappa 0.529, p < 0.001) according to the 2013 European consensus criteria. Detection of Candida DNA in PF using PCR can be considered an adjunct to existing routine diagnostic tools which may optimize the diagnosis and antifungal treatment of IAC in high-risk patients in the ICU.

Rapid preparation of Candida genomic DNA: combined use of enzymatic digestion and thermal disruption

Nucleic acid based molecular technologies are the most promising tools for the early diagnosis of Candida infection. A simple and effective DNA preparation method is of critical for standardizing and applying molecular diagnostics in clinic laboratories. The goal of this study was to develop a Candida DNA preparation method that was quick to do, easy to perform, and bio-safe. Snailase and lyticase were screened and combined in this work to enhance the lysis of Candida cells. The lysis solution composition and metal bath were optimized to boost amplification efficiency and biosafety. A duplex real-time PCR was established to evaluate the sensitivity and specificity of the preparation method. Using the supernatant from the rapid preparation method as templates, the duplex PCR sensitivities for five common Candida species were determined to be as low as 100 CFUs. When compared to conventional preparation methods, the samples prepared by our method showed higher PCR detection sensitivity. PCR identification and ITS sequencing were 100% consistent, which was better than biochemical identification. This study demonstrates a rapid method for Candida DNA preparation that has the potential to be used in clinical laboratories. Meanwhile, the practical application of the method for clinical samples needs to be proven in future investigations.

P452 Estimation of the detection limit of extracted Candida DNA from spiked EDTA blood sample

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMBackgroundCandidemia caused by genus Candida, are opportunistic pathogens with life-threatening infection in immunocompromised individuals with a mortality rate being 40%–54% in patients. Blood culture is not a reliable and rapid method for the diagnosis of candidemia as its sensitivity is as low as 50%. Molecular diagnosis is an alternative to conventional culture for early detection of candidemia. An appropriate DNA isolation technique is needed to obtain high purity DNA from blood specimens and improves the sensitivity of the polymerase chain reaction (PCR). In this study, two different methods were used to extract Candida DNA from spiked EDTA blood specimens, the In-house phenol-chloroform and potassium acetate method.ObjectivesDNA extraction from the spiked blood samplesQuantification of the Candida DNA extracted from the spiked sampleComparison of the extracted DNA by phenol-chloroform and potassium acetate methodMethodsA total of 5 ml of EDTA blood samples from healthy volunteers were spiked with 104-108 Candida albicans cells (ATCC 90028) to determine the detection limit of our extraction method. DNA was extracted from whole blood using phenol-chloroform and the potassium acetate methods which involves pretreatment with erythrocytes, leukocytes and fungal lysis buffer. DNA from C. albicans were amplified using ITS1 and ITS4 based primers. PCR products were visualized by agarose gel electrophoresis.ResultsThe spiked Candida cells taken were from 104-108 yeast cells. The total extracted DNA by phenol chloroform extraction method ranged from 18.29 to 51.64 ng/μl respectively. Whereas the total extracted DNA from potassium acetate extraction method ranged from 15.58 to 32.30 ng/μl respectively. The presence of a 535 base pair (bp) product was considered positive (Figs. 1 and 2). The lowest detection limit of PCR has been determined as 106-108 Candida cells for our spiked samples.ConclusionIn our study, till date, the extracted quantity of DNA was found to be higher by phenol-chloroform method as compared to the potassium acetate method. Greater numbers and processing are required to obtain conclusive data.

A pan-cancer mycobiome analysis reveals fungal involvement in gastrointestinal and lung tumors

Fungal microorganisms (mycobiota) comprise a small but immunoreactive component of the human microbiome, yet little is known about their role in human cancers. Pan-cancer analysis of multiple body sites revealed tumor-associated mycobiomes at up to 1 fungal cell per 104 tumor cells. In lung cancer, Blastomyces was associated with tumor tissues. In stomach cancers, high rates of Candida were linked to the expression of pro-inflammatory immune pathways, while in colon cancers Candida was predictive of metastatic disease and attenuated cellular adhesions. Across multiple GI sites, several Candida species were enriched in tumor samples and tumor-associated Candida DNA was predictive of decreased survival. The presence of Candida in human GI tumors was confirmed by external ITS sequencing of tumor samples and by culture-dependent analysis in an independent cohort. These data implicate the mycobiota in the pathogenesis of GI cancers and suggest that tumor-associated fungal DNA may serve as diagnostic or prognostic biomarkers.

Evaluation of Droplet Digital PCR Assay for the Diagnosis of Candidemia in Blood Samples.

Numerous studies have shown that droplet digital PCR (ddPCR) is a promising tool for the diagnosis of pathogens, especially in samples with low concentrations of pathogenic DNA. An early diagnosis of candidemia is critical for the effective treatment of patients. In this study, we evaluated the sensitivity and specificity of ddPCR assay for Candida DNA detection both in vitro by mixing fungal cells with human blood and in vivo by analyzing blood samples from infected mice and patients with suspected candidemia. The results showed that ddPCR assay could detect a minimum of 4.5 DNA copies per reaction in blood samples. ddPCR showed higher sensitivity and specificity for Candida DNA detection than traditional culture and quantitative PCR (qPCR) methods and also exhibited significantly better positive and negative predictive values than the culture and qPCR methods that were commonly used in clinical practice. Hence, our study demonstrates that ddPCR assay is a promising method for the timely diagnosis of candidemia and could be useful for monitoring the treatment of candidemia.

Evaluation of 11 DNA Automated Extraction Protocols for the Detection of the 5 Mains Candida Species from Artificially Spiked Blood.

The molecular detection of Candida plays an important role in the diagnosis of candidaemia, a major cause of morbidity and mortality. The sensitivity of this diagnosis is partly related to the efficiency of yeast DNA extraction. In this monocentric study, we investigated the suitability of 11 recent automated procedures for the extraction of low and high amounts of Candida DNA from spiked blood. The efficacy of the DNA extraction procedures to detect Candida spp. in blood samples ranged from 31.4% to 80.6%. The NucliSENSTM easyMAGTM procedure was the most efficient, for each species and each inoculum. It significantly outperformed the other procedures at the lower Candida inocula mimicking the clinical setting. This study highlighted a heterogeneity in DNA extraction efficacy between the five main Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei). Up to five automated procedures were appropriate for C. krusei DNA extraction, whereas only one method yielded an appropriate detection of low amount of C. tropicalis. In the era of the syndromic approach to bloodstream infection diagnosis, this evaluation of 11 automated DNA extraction methods for the PCR diagnosis of candidaemia, puts the choice of an appropriate method in routine diagnosis within the reach of laboratories.

PCR-detectable Candida DNA exists a short period in the blood of systemic candidiasis murine model.

Invasive candidiasis is a major challenge to clinical medicine today. However, traditional fungal diagnostic techniques and empirical treatments have shown great limitations. Although efforts are necessarily needed in methodology standardization and multicenter validation, polymerase chain reaction (PCR) is a very promising assay in detecting fungal pathogens. Using a “heat-shock” DNA preparation method, a rapid and simple PCR protocol for quantification of the Candida albicans (C. albicans) ribosomal DNA was established. The PCR assay could detect Candida DNA as low as 10 CFU/mL in samples prepared by the heat-shock protocol, without any cross-reaction with DNA prepared from other Candida spp. and bacterial pathogens. For simulated blood samples, the PCR test sensitivity of whole blood samples was better than that of plasma and blood cells. In the systemic candidiasis murine model, detectable DNA was only observed within 24 h after C. albicans SC5314 injection, which is much shorter than that observed in the kidney.

Comparative molecular dynamics studies of heterozygous open reading frames of DNA polymerase eta (\u03b7) in pathogenic yeast Candida albicans

Genomic instability in Candida albicans is believed to play a crucial role in fungal pathogenesis. DNA polymerases contribute significantly to stability of any genome. Although Candida Genome database predicts presence of S. cerevisiae DNA polymerase orthologs; functional and structural characterizations of Candida DNA polymerases are still unexplored. DNA polymerase eta (Polη) is unique as it promotes efficient bypass of cyclobutane pyrimidine dimers. Interestingly, C. albicans is heterozygous in carrying two Polη genes and the nucleotide substitutions were found only in the ORFs. As allelic differences often result in functional differences of the encoded proteins, comparative analyses of structural models and molecular dynamic simulations were performed to characterize these orthologs of DNA Polη. Overall structures of both the ORFs remain conserved except subtle differences in the palm and PAD domains. The complementation analysis showed that both the ORFs equally suppressed UV sensitivity of yeast rad30 deletion strain. Our study has predicted two novel molecular interactions, a highly conserved molecular tetrad of salt bridges and a series of π–π interactions spanning from thumb to PAD. This study suggests these ORFs as the homologues of yeast Polη, and due to its heterogeneity in C. albicans they may play a significant role in pathogenicity.

Non-Invasive Methods to Diagnose Fungal Infections in Pediatric Patients with Hematologic Disorders.

BackgroundInvasive fungal infection (IFIs) is a major infectious complication in immunocompromised patients. Early diagnosis and initiation of antifungal therapy is important to achieve the best outcome.ObjectivesThe current study aimed to investigate the incidence of IFIs and evaluate the diagnostic performance of non-invasive laboratory tests: serologic (β-D-glucan, galactomannan) and molecular (nested polymerase chain reaction) tests to diagnose fungal infections in hematologic pediatric patients.Patients and MethodsIn a cross-sectional study from October 2014 to January 2015, 321 blood samples of 62 pediatric patients with hematologic disorders and at high risk for fungal infections were analyzed. Non-invasive tests including the Platelia Aspergillus enzyme immunoassay (EIA) to detect galactomannan antigen, Glucatell for β–D–glucan and nested PCR to detect Candida and Aspergillus species-specific DNA were used in a weekly screening strategy.ResultsTwenty six patients (42%) were considered as proven and probable IFIs, including 3 (5%) proven and 23 (37%) probable cases. Eighteen patients (29%) were considered as possible cases. The sensitivity, specificity, positive and negative predictive values for galactomannan test in 26 patients with proven and probable fungal infections were 94.4%, 100%, 100% and 94.7%; for β-D-glucan test 92.3%, 77.7%, 85%, 87.5% and for nested-PCR were 84.6%, 88.8%, 91.7% and 80%, respectively.ConclusionsThe rate of IFIs in pediatric patients with hematologic disorders is high, and sample collection from the sterile sites cannot be performed in immunocompromised patients. Detection of circulating fungal cell wall components and DNA in the blood using non-invasive methods can offer diagnostic help in patients with suspected IFIs. Their results should be interpreted in combination with clinical, radiological and microbiological findings.

Prevalence of Candida Infection at the Intensive Care Unit with Nested Polymerase Chain Reaction (PCR) Using Primer Mixes Specific to Candida DNA Topoisomerase II Genes

Background: The occurrence of invasive Candida infections has increased during the past two decades as a result of increasing in the number of immunocompromised patients. Objectives: In this study (cross sectional design), during six months, the prevalence patterns of Candida species isolated from sterile body sites of patients admitted to the general hospital of Milad Intensive Care Units (ICUs) in Tehran (Iran), were determined. Methods: Candidal isolates were obtained from 50 patients admitted to Milad ICUs from April to September 2013. Identification of the isolates was performed using morphological and Polymerase Chain Reaction assay. For identification of Candida at the species level, degenerated and specific primers based on the genomic sequences of DNA topoisomerase II of Candida species were used and their specificities tested by PCR-based identifications. Results: A total of 67 Candida isolates were obtained from 50 patients. Out of 67 Candida isolates, 47.8% were C. glabrata, 28.3% were C. albicans, 7.5% were C. tropicalis, 7.5% were C. guilliermondii, 3% were C. krusei and 4.4% were C. dubliniensis. The main patient group affected by candidal infections was aged 50 to 70 years. Overall, 11.7% of patients had cancer while other diseases such as diabetes were less reported. The mean time of stay at the ICU before identification was 25.3 days (ranging from 2 to 120 days). Conclusions: Increase in the prevalence of non-C. albicans species in the recent years has become a problematic event amongst clinicians caring for ICU patients. Candida glabrata is the most common species isolated from ICU patients in comparison with other species in this study. These findings emphasized on the significance of organizing treatable prevention programs.

Contribution of Candida biomarkers and DNA detection for the diagnosis of invasive candidiasis in ICU patients with severe abdominal conditions.

BackgroundTo assess the performance of Candida albicans germ tube antibody (CAGTA), (1 → 3)-ß-D-glucan (BDG), mannan antigen (mannan-Ag), anti-mannan antibodies (mannan-Ab), and Candida DNA for diagnosing invasive candidiasis (IC) in ICU patients with severe abdominal conditions (SAC).MethodsA prospective study of 233 non-neutropenic patients with SAC on ICU admission and expected stay ≥ 7 days. CAGTA (cutoff positivity ≥ 1/160), BDG (≥80, 100 and 200 pg/mL), mannan-Ag (≥60 pg/mL), mannan-Ab (≥10 UA/mL) were measured twice a week, and Candida DNA only in patients treated with systemic antifungals. IC diagnosis required positivities of two biomarkers in a single sample or positivities of any biomarker in two consecutive samples. Patients were classified as neither colonized nor infected (n = 48), Candida spp. colonization (n = 154) (low-grade, n = 130; high-grade, n = 24), and IC (n = 31) (intra-abdominal candidiasis, n = 20; candidemia, n = 11).ResultsThe combination of CAGTA and BDG positivities in a single sample or at least one of the two biomarkers positive in two consecutive samples showed 90.3 % (95 % CI 74.2–98.0) sensitivity, 42.1 % (95 % CI 35.2–98.8) specificity, and 96.6 % (95 % CI 90.5–98.8) negative predictive value. BDG positivities in two consecutive samples had 76.7 % (95 % CI 57.7–90.1) sensitivity and 57.2 % (95 % CI 49.9–64.3) specificity. Mannan-Ag, mannan-Ab, and Candida DNA individually or combined showed a low discriminating capacity.ConclusionsPositive Candida albicans germ tube antibody and (1 → 3)-ß-D-glucan in a single blood sample or (1 → 3)-ß-D-glucan positivity in two consecutive blood samples allowed discriminating invasive candidiasis from Candida spp. colonization in critically ill patients with severe abdominal conditions. These findings may be helpful to tailor empirical antifungal therapy in this patient population.

Lethal and sub-lethal effects of UV-B radiation exposure on the collembolan Folsomia candida (Willem) in the laboratory

Abstract This study assessed the effects of UV-B irradiation on the physiology, life history, DNA, and behavior of the blind non-pigmented soil-dwelling collembolan Folsomia candida (Willem). In three sets of controlled laboratory studies, adult F. candida were reared in darkness and exposed to light with no UV-B (filtered), low UV-B or high UV-B (weighted irradiance energy of 3.12 mW/m 2 and 22.1 mW/m 2 , respectively). Study I investigated UV-B impacts on mortality, egg viability, and egg development time of F. candida under continual exposure. Study II assessed the movement patterns, mortality, egg production and fecundity of adult F. candida in response to UV-B exposure within choice environments. Study III determined the degree of UV-B induced DNA damage (thymine dimer formation) on F. candida and subsequent DNA repair. Continuous irradiation resulted in increased mortality, under high and low UV-B conditions compared to controls. Consistent with other studies on the impact of light on collembola, we found that F. candida avoided light when given the option. Though preferring darkness, animals were more likely to venture into lighted regions with no UV-B (filtered) and experienced higher mortality rates than in regions with high or low UV-B. Eggs were laid preferentially in the dark, with fewer total eggs observed in the habitat with the highest UV-B. We further demonstrated that UV-B induced the formation of thymine dimers in a dose dependent manner. DNA repair was not evident in animals that had returned to the dark after a brief intense UV-B irradiation. These findings indicate that UV-B exposure and exposure to longer wavelengths of light has both lethal and sub-lethal effects on F. candida that can adversely affect its survival.

Clinical evaluation of the Magicplex Sepsis Real-time Test (Seegene) to detect Candida DNA in pediatric patients

Clinical evaluation of the Magicplex Sepsis Real-time Test (Seegene) to detect Candida DNA in pediatric patients

Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood

Species of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5-10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.

Assessment of a real-time PCR method to detect human non-cryptococcal fungal meningitis.

The signs and symptoms associated with fungal meningitis are similar to those seen with more common bacterial infections. In this study, we investigate whether Aspergillus or Candida DNA can be detected in cerebrospinal fluid (CSF) samples from patients suspected of fungal meningitis using real-time PCR assay. From April 2007 to November 2009, we evaluated CSF samples and sera from patients with risk factors for cerebral fungal meningitis in Nemazi Hospital, Shiraz University of Medical Sciences, Iran, by real-time PCR assay and routine mycological studies (direct microscopy examination and culture). Two CSF and two serum samples from each patient were examined. CSF and serum samples from 38 patients (total: 152) suspected of fungal meningitis were examined. India ink staining and KOH smear were negative for all patients. C. albicans was isolated from two CSF samples. There were ten patients with positive real-time PCR results in their CSF samples: three patients had C. albicans, one with C. glabrata, four with Aspergillus species and two with both C. albicans and Aspergillus species DNA. Four patients had positive serum results for Aspergillus or Candida infections. Considering the findings, it seems that molecular examination can help in the diagnosis of fungal meningitis in patients with clinical and radiological presentations. Further studies should be conducted in other regions and settings to confirm these findings.

Value of (1-3)-β-D-glucan, Candida mannan and Candida DNA detection in the diagnosis of candidaemia

This study determined the value of (1,3)-β-d-glucan (BDG), Candida mannan (MN) and Candida species-specific DNA as surrogates for diagnosis of candidaemia. Thirty-nine patients yielding Candida species in blood cultures were investigated for presence of BDG, MN and Candida species-specific DNA in serum samples. The Candida spp. bloodstream isolates included C. albicans (n = 16), C. tropicalis (n = 10), C. parapsilosis (n = 7), C. glabrata (n = 3) and C. dubliniensis (n = 3). Positivity of the three markers was as follows: Candida DNA for corresponding Candida species, 100%; BDG, 87%; MN, 59%. Despite varying sensitivities of these bio-Markers, they provided a useful adjunct to the diagnosis of candidaemia.

Development of a novel quantitative real-time assay using duplex mutation primers for rapid detection of Candida species

We developed a novel quantitative real-time PCR for quantitating Candida DNA based on the duplex mutation primer principle, in which a signal is generated by melting a duplex mutation primer during renaturation. The duplex mutation primers are much more specific than double-stranded DNA dyes such as SYBR-Green I and, unlike other probes, do not require the double-labeled synthesis of fluorophore and a quencher on the same molecule. A total of 176 clinical blood specimens were obtained from patients hospitalized in our hospital with clinically proven or suspected systemic Candida infection. The presence of DNA from pathogens in the Candida species was detected using real-time PCR targeting of an internal transcribed spacer region of a fungal gene. The assay exhibited a low limit of detection (10 CFU/ml of blood), an excellent reproducibility and specificity. Twenty-eight positive samples exhibited a wide range of Candida species loads, extending from 13 to 90,528 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97.4%, respectively, compared with the results of blood culture. Our data suggest that this assay may be appropriate for use in clinical laboratories as a simple, low-cost and rapid screening test for the most frequently encountered Candida species.

Detection of Candida and Aspergillus species DNA using broad-range real-time PCR for fungal endophthalmitis

The goal of this work is to establish a broad-range real-time polymerase chain reaction (PCR) diagnostic system for ocular fungal infection and to measure Candida and Aspergillus DNA in the ocular fluids obtained from unknown uveitis/endophthalmitis patients. After obtaining informed consent, intraocular fluids (aqueous humor and vitreous fluid samples) were collected from 54 patients with idiopathic uveitis or endophthalmitis. Samples were assayed for Candida or Aspergillus DNA using broad-range (18S rRNA sequences) quantitative real-time PCR. Candida or Aspergillus DNA was detected in seven out of 54 patient ocular samples (13%). These PCR-positive samples showed significantly high copy numbers of Candida or Aspergillus DNA. On the other hand, fungal DNA was not detected in any of the other 46 samples collected from these idiopathic uveitis or endophthalmitis patients. In the one PCR-negative case, PCR did not detect any fungal genome in the sample, even though this patient was clinically suspected of having Candida endophthalmitis. Real-time PCR results were negative for fungal DNA in the bacterial endophthalmitis patients and in various uveitis patients. In addition, fungal DNA was also not detected in patients without ocular inflammation (controls). Analysis of ocular samples by this broad-range real-time PCR method can be utilized for rapid diagnosis of patients suffering from unknown intraocular disorders such as idiopathic uveitis/endophthalmitis.