Development of a loop-mediated isothermal amplification method for detection of Histoplasma capsulatum DNA in clinical samples.
Improved methods for the detection of Histoplasma capsulatum are needed in regions with limited resources in which the organism is endemic, where delayed diagnosis of progressive disseminated histoplasmosis (PDH) results in high mortality rates. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid inexpensive molecular diagnosis of this disease. Primers for LAMP were designed to amplify the Hcp100 locus of H. capsulatum. The sensitivity and limit of detection were evaluated using DNA extracted from 91 clinical isolates of known geographic subspecies, while the assay specificity was determined using DNA extracted from 50 other fungi and Mycobacterium tuberculosis. Urine specimens (n = 6) collected from HIV-positive individuals with culture- and antigen-proven histoplasmosis were evaluated using the LAMP assay. Specimens from healthy persons (n = 10) without evidence of histoplasmosis were used as assay controls. The Hcp100 LAMP assay was 100% sensitive and specific when tested with DNA extracted from culture isolates. The median limit of detection was ≤6 genomes (range, 1 to 300 genomes) for all except one geographic subspecies. The LAMP assay detected Hcp100 in 67% of antigen-positive urine specimens (4/6 specimens), and results were negative for Hcp100 in all healthy control urine specimens. We have shown that the Hcp100 LAMP assay is a rapid affordable assay that can be used to expedite culture confirmation of H. capsulatum in regions in which PDH is endemic. Further, our results indicate proof of the concept that the assay can be used to detect Histoplasma DNA in urine. Further evaluation of this assay using body fluid samples from a larger patient population is warranted.
Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Christina M
CM
Scheel
Yitian
Y
Zhou
Raquel C
RC
Theodoro
Bethany
B
Abrams
S Arunmozhi
SA
Balajee
Anastasia P
AP
Litvintseva
D003362:Cost-Benefit Analysis; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000032:analysis* / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D015658:HIV Infections / Q000150:complications; D006658:Histoplasma / Q000235:genetics / Q000302:isolation & purification*; D006660:Histoplasmosis / Q000175:diagnosis*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D014556:Urine / Q000382:microbiology
D017931:DNA Primers; D004271:DNA, Fungal; D005656:Fungal Proteins; C576552:Hcp100 protein, Histoplasma capsulatum