[Technical evaluation of nested PCR for the diagnosis of experimental sporotrichosis].
BACKGROUND
Sporotrichosis caused by the dimorphic fungus Sporothrix schenckii can presents in a variety of clinical forms. Routine diagnosis is made by mycology and serology studies. Few investigations have been focused on the evaluation of the molecular diagnosis.


OBJECTIVE
To determine the value of the nested PCR technique for the diagnosis of experimental sporotrichosis in organs of mice, and to compare the results with the established laboratory diagnostic procedures.


METHODS
BALB/c mice were inoculated with growing concentrations of the 2 morphological phases of the fungus. The infected animals were sacrificed one month later and specimens from liver, spleen, lung and testicle were obtained to perform wet mount, culture and molecular diagnosis by the nested PCR technique. Blood samples were obtained for determination of specific antibodies against S. schenckii by the double immunodiffusion procedure.


RESULTS
The pathogenicity observed with the different concentrations of the fungus inoculated and its isolation by culture, showed scarce differences in the study of specimens from organs infected with the 2 morphological phases of S. schenckii. Specimens from organs of mice inoculated with the mycelial phase when studied by wet mount and culture, showed a higher positivity (100 and 37.5%) than those from mice inoculated with the yeast phase (73 and 2%). However, diagnosis by the nested PCR molecular technique applied to the latter specimens showed a higher percentage of positivity (75%) and 43% of positive results coming from animals infected with the mycelial phase. Specific antibody detection was positive in 100% all groups of infected mice.


CONCLUSIONS
In the study of experimental sporotrichosis in mice, the culture, as well as the antibody detection, was an effective diagnostic procedure, while the nested PCR and microscopic studies had a lower diagnostic value.
Laboratorio de Micología, Instituto de Biomedicina, Caracas, Venezuela. mmendoz@movistar.net.ve
Mireya
M
Mendoza
Ana
A
Brito
Daniel Alejandro
DA
Schaper
Vanesa Alexandra
VA
Spooner
Primavera
P
Alvarado
Aura
A
Castro
Alexis
A
Fernández
D000818:Animals; D000908:Antibodies, Fungal / Q000097:blood; D001706:Biopsy; D004271:DNA, Fungal / Q000032:analysis*; D008099:Liver / Q000382:microbiology; D008168:Lung / Q000382:microbiology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D025282:Mycelium / Q000472:pathogenicity; D009172:Mycology / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D013154:Spleen / Q000382:microbiology; D013175:Sporothrix / Q000235:genetics / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification* / Q000472:pathogenicity; D013174:Sporotrichosis / Q000175:diagnosis*; D013737:Testis / Q000382:microbiology; D014774:Virulence
D000908:Antibodies, Fungal; D004271:DNA, Fungal