title: Application of semi-nested polymerase chain reaction targeting internal transcribed spacer region for rapid detection of panfungal genome directly from ocular specimens.
abstract: BACKGROUND
  The incidence of fungal endophthalmitis has dramatically increased in recent years and rapid detection of fungi using nucleic acid-based amplification techniques is helpful in management.
  
  
  OBJECTIVE
  To evaluate semi-nested polymerase chain reaction (PCR) targeting internal transcribed spacer (ITS) region for detection of panfungal genome in ocular specimens.
  
  
  METHODS
  Z test for two proportion.
  
  
  METHODS
  Standardization of PCR targeting ITS primers was carried out by determining analytical sensitivity and specificity. The sensitivity and specificity of PCR was determined by serial tenfold dilutions of C. albicans (ATCC 24433) DNA and DNA extracts of laboratory isolates of Aspergillus fumigatus, Fusarium lichenicola (4), other fungal and closely related bacterial strains and also human DNA. Semi-nested PCR was applied onto a total of 168 ocular specimens with clinically suspected fungal etiology during 2003-2005.
  
  
  CONCLUSIONS
  PCR was specific and sensitive to detect 1fg of fungal DNA with ITS primers. PCR detected fungal genome in 90 (53.57%) in comparison with the conventional technique, positive in 34 (20.23%) by smear examination and in 42 (25%) by culture. The increase in clinical sensitivity by 28.57% using PCR was found to be statistically significant { P < 0.001 using Z test for two proportion}. The accuracy of the test was found to be 70.85%. PCR proved to be a rapid diagnostic technique for detection of panfungal genome directly from clinical specimens.
authors.affiliation: Larsen and Toubro Microbiology Research Centre, Vision Research Foundation, No.18, College Road, Sankara Nethralaya, Chennai - 600 006, India.
authors.forename: R
authors.initials: R
authors.lastname: Bagyalakshmi
authors.forename: K L
authors.initials: KL
authors.lastname: Therese
authors.forename: H N
authors.initials: HN
authors.lastname: Madhavan
mesh_terms: D001082:Aqueous Humor / Q000382:microbiology; D003315:Cornea / Q000382:microbiology; D004271:DNA, Fungal / Q000235:genetics*; D003937:Diagnosis, Differential; D009877:Endophthalmitis / Q000175:diagnosis / Q000382:microbiology*; D015821:Eye Infections, Fungal / Q000175:diagnosis / Q000382:microbiology*; D005658:Fungi / Q000235:genetics* / Q000302:isolation & purification; D016681:Genome, Fungal / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014822:Vitreous Body / Q000382:microbiology
chemical_list: D004271:DNA, Fungal