Expanding the diagnostic use of PCR in leptospirosis: improved method for DNA extraction from blood cultures.
BACKGROUND
Leptospirosis is a neglected zoonosis of ubiquitous distribution. Symptoms are often non-specific and may range from flu-like symptoms to multi-organ failure. Diagnosis can only be made by specific diagnostic tests like serology and PCR. In non-endemic countries, leptospirosis is often not suspected before antibiotic treatment has been initiated and consequently, relevant samples for diagnostic PCR are difficult to obtain. Blood cultures are obtained from most hospitalized patients before antibiotic therapy and incubated for at least five days, thus providing an important source of blood for PCR diagnosis. However, blood cultures contain inhibitors of PCR that are not readily removed by most DNA-extraction methods, primarily sodium polyanetholesulfonate (SPS).


RESULTS
In this study, two improved DNA extraction methods for use with blood cultures are presented and found to be superior in recovering DNA of Leptospira interrogans when compared with three previously described methods. The improved methods were easy and robust in use with all tested brands of blood culture media. Applied to 96 blood cultures obtained from 36 patients suspected of leptospirosis, all seven patients with positive convalescence serology were found positive by PCR if at least one anaerobic and one aerobic blood culture, sampled before antibiotic therapy were tested.


CONCLUSIONS
This study suggests that a specific and early diagnosis can be obtained in most cases of severe leptospirosis for up to five days after initiation of antimicrobial therapy, if PCR is applied to blood cultures already sampled as a routine procedure in most septic patients.
Villumsen
Steen
S
Department of Microbiological Surveillance and Research, Statens Serum Institut, Copenhagen, Denmark.
Pedersen
Rebecca
R
Krogfelt
Karen Angeliki
KA
Jensen
Jørgen Skov
JS
D005591:Chemical Fractionation / Q000379:methods*; D046508:Culture Techniques; D004269:DNA, Bacterial / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007919:Leptospira / Q000187:drug effects / Q000254:growth & development; D007922:Leptospirosis / Q000097:blood* / Q000175:diagnosis*; D011074:Polyanetholesulfonate / Q000494:pharmacology; D016133:Polymerase Chain Reaction
D004269:DNA, Bacterial; D011074:Polyanetholesulfonate