Expanding the diagnostic use of PCR in leptospirosis: improved method for DNA extraction from blood cultures

Abstract

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).

Methodology/principal findings: 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/significance: 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.

Figure 1. Flowchart for experiment 1–3.

Figure shows flow over three independent experiments performed in the evaluation of five methods for DNA extraction from blood cultures media. BD-AE: BACTEC™ aerobic Plus, BD-AN: BACTEC™ anaerobic Plus, BD-Ped: BACTEC™ Paed Plus, BacT-SA: BacT/ALERT® SA, BacT-SN: BacT/ALERT® SN,BacT-PF: BacT/ALERT® PF, BacT-FA: BacT/ALERT® FA, BacT-FN: BacT/ALERT® FN. M1: Qiagen, DNeasy® Blood & Tissue Kit; M2: Molzym, MolYsis Plus Kit; M3: Protocol described by Fredricks and Relman; M4: Improved method for anaerobic and paediatric blood culture media; M5: Improved method adapted for all blood culture media.

Figure 2. Leptospira sp. DNA recovery and inhibition of PCR.

Estimated total copy-number of Leptospira interrogans DNA recovered from a spiked sample of BACTEC™ anaerobic Plus containing approximately 50.000 Leptospira/5 µl by five DNA extraction methods. The mean copy-numbers are given above each column. Error bars indicate the 95% confidence interval. M1: Qiagen, DNeasy® Blood & Tissue Kit; M2: Molzym, MolYsis Plus Kit; M3: Protocol described by Fredricks and Relman; M4: Improved protocol for anaerobic and paediatric blood culture media; M5: Improved protocol adapted for all blood culture media.

Figure 3. DNA recovery from different blood culture media.

Estimated total copy-number of Leptospira interrogans DNA recovered from spiked samples of six different blood culture media containing approximately 50.000 Leptospira/5µl. All DNA extractions were performed with M5, the improved protocol adapted for all blood culture media. The mean copy-numbers are given above each column. Error bars indicate the 95% confidence interval. BD-AE: BACTEC™ aerobic Plus, BD-AN: BACTEC™ anaerobic Plus, BD-Ped: BACTEC™ Paed Plus, BacT-SA: BacT/ALERT® SA, BacT-SN: BacT/ALERT® SN and BacT-PF: BacT/ALERT® PF.