Nested polymerase chain reaction for Mycobacterium tuberculosis DNA detection in aqueous and vitreous of patients with uveitis.
BACKGROUND
Tuberculosis may be a lethal disease. Its ocular manifestations are commonly associated with severe difficulties in diagnosis and therapy; furthermore, it may cause blindness. DNA amplification methods may allow early detection of small amounts of Mycobacterium tuberculosis DNA to afford the possibility of prompt diagnosis. We evaluated a nested polymerase chain reaction (nPCR) assay for detection of Mycobacterium tuberculosis DNA in aqueous and vitreous.


METHODS
In a case-control study, 22 cases of diagnosed TB uveitis (three HIV-infected patients) and 38 controls (18 HIV-infected patients) with other types of uveitis (syphilis, nine; cytomegalovirus, seven; toxoplasmosis, five; herpes simplex, one; autoimmune vasculitis, eight; Vogt-Koyanagi-Harada, four; pars planitis, one; serpinginous choroiditis, one; Wegener granulomatosis, one; and Fuchs iridocyclitis, one studied). Samples from aqueous or vitreous were cultured and analyzed by nPCR for presence of M. tuberculosis nucleic acids. We used two sets of primers corresponding to IS6110 region coding for 219 bp and 123 bp DNA sequences.


RESULTS
Results were confirmed by Southern blot. All samples were tested by PCR simultaneously for Herpes simplex I, Herpes zoster, cytomegalovirus (CMV) and Toxoplasma gondii. nPCR was positive in 17 cases (77.2%) and only in three controls (8.8%) p = 0.022. All cultures were negative. Southern blot confirmed all positive nPCR tests. According to our definition of cases, there were five false negative results: two in patients with pulmonary tuberculosis; two in patients with tuberculous lymphadenitis, and one with positive skin test and hematuria. There were three cases considered false positives for nPCR: one with autoimmune vasculitis, and two with toxoplasmic uveitis.


CONCLUSIONS
nPCR for TB in ocular fluids was positive in the majority of cases of ocular TB. This method is useful in early confirmation of ocular tuberculosis.
Ortega-Larrocea
Gabriela
G
Clínica de Enfermedades Oculares Inflamatorios, Asociación para Evitar la Ceguera en México Luis Sánchez Bulnes, Mexico City, Mexico.
Bobadilla-del-Valle
Miriam
M
Ponce-de-León
Alfredo
A
Sifuentes-Osornio
José
J
D000293:Adolescent; D000328:Adult; D000368:Aged; D015139:Blotting, Southern; D016022:Case-Control Studies; D004247:DNA / Q000032:analysis*; D017931:DNA Primers / Q000494:pharmacology; D005260:Female; D006679:HIV Seropositivity; D006801:Humans; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014376:Tuberculosis / Q000175:diagnosis; D014605:Uveitis / Q000378:metabolism* / Q000382:microbiology*
D017931:DNA Primers; D004247:DNA