Bacterial and fungal infections in the blood are dangerous, particularly to the very young, the very old and those who are immunocompromised. For effective treatment, it’s important to know exactly which infectious agent is behind the sepsis as soon as possible, and researchers in Poland are looking into real-time PCR (qPCR) as a potential solution for simultaneous detection of both bacteria and fungi.
The standard route to identification of bacteria and fungi in blood samples is by carrying out blood cultures using specialised media. This is low cost, but the results can take a long time and the sensitivity is low, both of which could delay antibiotic or antifungal treatment at a critical time. The results can also be affected by early broad-spectrum therapy while the physicians are waiting for specific results
Molecular methods such as the polymerase chain reaction (PCR) have potential in faster and more accurate diagnosis and aren’t affected by any antibiotic or antifungal treatments. However, lysis of bacterial and fungal cells to release the DNA can be challenging and amplification of microbial DNA can be inhibited by haem. To meet these challenges, the team developed a form of nested multiplex real-time PCR and assessed it on sterile blood samples inoculated with four species of microorganisms (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus) and on blood collected from 102 patients with clinical symptoms of sepsis. The results were published in BMC Microbiology.
The researchers created four external primer sequences corresponding to Gram-positive bacteria, Gram-negative bacteria, yeast fungi, or filamentous fungi. The PCR technique was able to detect the four species of microorganisms in the sterile blood samples at levels of 101 CFU/ml, and came up with positive results in 70% of patient blood samples, compared with 19% for the microbiological culture method and 18% for non-nested multiplex PCR. Negative controls tested using the nested PCR technique showed as negative each time, reducing the risk of false positives. The nested PCR technique also designated microorganisms correctly as Gram-positive bacteria, Gram-negative bacteria, yeast fungi, or filamentous fungi.
According to the researchers, the method improved sensitivity and allowed detection across the whole panel of bacterial and fungal microorganisms, with differentiation of Gram-negative bacteria, Gram-positive bacteria, yeast fungi, and filamentous fungi. They added that the results would be “very useful to the physician when selecting the appropriate treatment before he receives the final identification from microbiological laboratory.”