Comparison of four DNA extraction methods from cerebrospinal fluid for the detection of Toxoplasma gondii by polymerase chain reaction in AIDS patients.
BACKGROUND
Toxoplasmosis is a serious and often life-threatening disease in immunodeficient patients. Polymerase chain reaction (PCR) assays allow a rapid diagnosis of Toxoplasma infection by direct detection of the parasite's DNA. To perform a sensitive, specific, and reliable PCR-based diagnostic test, the availability of pure DNA lacking PCR inhibitors as well as a rapid and easy-to-perform DNA extraction protocol are essential. The aim of the present study was to compare four DNA extraction methods for the detection of T. gondii on cerebrospinal fluid (CSF) using the PCR technology.


METHODS
Four DNA extraction methods (boiling, lysis + centrifugation, the miniMAG commercial system, and phenol-chloroform) were compared with respect to the time of completion, the manual labor involved, and PCR analytical sensitivity for the detection of T. gondii in CSF. The optimal DNA extraction method for the detection of the parasite was evaluated in CSF from 43 AIDS patients using the nest-PCR B1 assay.


RESULTS
According to the time required for completion, labor, and PCR analytical sensitivity, the lysis + centrifugation protocol proved to be a simple, efficient, and economical in-house procedure to recover the T. gondii DNA present in the CSF. The diagnostic sensitivity of nest-PCR, according to Centers for Disease Control and Prevention (CDC) criteria, was 86.3% and the diagnostic specificity was 100%.


CONCLUSIONS
We report a simple, rapid, reproducible, and economical in-house method for T. gondii DNA extraction from CSF. This method is recommended for diagnostic PCR of Toxoplasmic encephalitis (TE) in places with economical shortage.
Alfonso
Yenisey
Y
Department of Parasitology, Institute of Tropical Medicine Pedro Kouri, Ciudad de La Habana, Cuba.
Fraga
Jorge
J
Cox
Raymundo
R
Bandera
Francisco
F
Pomier
Olga
O
Fonseca
Carlos
C
Ginorio
Dora
D
Torres
Griselda
G
Capo
Virginia
V
D017088:AIDS-Related Opportunistic Infections / Q000134:cerebrospinal fluid / Q000469:parasitology*; D000818:Animals; D016054:DNA, Protozoan / Q000134:cerebrospinal fluid* / Q000302:isolation & purification; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D014122:Toxoplasma / Q000235:genetics* / Q000302:isolation & purification; D016781:Toxoplasmosis, Cerebral / Q000134:cerebrospinal fluid / Q000469:parasitology*
D016054:DNA, Protozoan