Broad-range bacterial and fungal DNA amplification on vitreous humor from suspected endophthalmitis patients.
BACKGROUND
Prompt and accurate diagnosis of infectious endophthalmitis is crucial for rapid and effective treatment. By identifying whether the causative pathogen is bacterial or fungal, a rational approach for the use of antibacterials or corticosteroids, respectively, can be followed.


OBJECTIVE
To assess the clinical utility of broad-range bacterial and fungal DNA amplification in the detection of endophthalmitis (postoperative, posttraumatic, and endogenous).


METHODS
In a prospective study, vitreous humor samples from 70 patients with the clinical diagnosis of presumed endophthalmitis, and from 30 patients undergoing surgery for non-infectious causes, were subjected to routine microbiologic and molecular investigation. DNA extracted from a 50 microL sample was amplified by primers targeting the conserved 16S and 18S ribosomal RNA gene sequences of bacteria and fungi, respectively. Reagents for bacterial DNA amplification were decontaminated of endogenous DNA using 8-methoxypsoralen and long wave UV treatment.


CONCLUSIONS
A total of 35 specimens were positive for bacteria or fungi by culture. Of these, Gram-positive organisms were isolated in 19 specimens, Gram-negative organisms in 13 specimens and fungi in 3 specimens. Pseudomonas species, coagulase-negative Staphylococcus, and Streptococcus species were the main etiological agents isolated. Bacterial DNA amplification resulted in 49 positive specimens, compared with 32 positive specimens by culture; and fungal DNA amplification resulted in 11 positive specimens, compared with 3 positive specimens by culture. All control specimens were negative for both culture and DNA amplification.


CONCLUSIONS
DNA extracted using a single-extraction protocol from 50 microL of vitreous humor and amplified with broad-range bacterial and fungal primers will enable the rapid differentiation (within 14 hours) between bacterial and fungal endophthalmitis and allow tailoring of therapy to individual patients.
Varghese
Betsy
B
Department of Microbiology and Research, P.D. Hinduja National Hospital and Medical Research Center, Mumbai, India.
Rodrigues
Camilla
C
Deshmukh
Minal
M
Natarajan
S
S
Kamdar
Paritosh
P
Mehta
Ajita
A
D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D009877:Endophthalmitis / Q000175:diagnosis* / Q000382:microbiology; D005658:Fungi / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D014822:Vitreous Body / Q000382:microbiology*
D012336:RNA, Ribosomal, 16S; D012337:RNA, Ribosomal, 18S