More Pathogenicity or Just More Pathogens?—On the Interpretation Problem of Multiple Pathogen Detections with Diagnostic Multiplex Assays

Abstract

Modern molecular diagnostic approaches in the diagnostic microbiological laboratory like real-time quantitative polymerase chain reaction (qPCR) have led to a considerable increase of diagnostic sensitivity. They usually outperform the diagnostic sensitivity of culture-based approaches. Culture-based diagnostics were found to be insufficiently sensitive for the assessment of the composition of biofilms in chronic wounds and poorly standardized for screenings for enteric colonization with multi-drug resistant bacteria. However, the increased sensitivity of qPCR causes interpretative challenges regarding the attribution of etiological relevance to individual pathogen species in case of multiple detections of facultative pathogenic microorganisms in primarily non-sterile sample materials. This is particularly the case in high-endemicity settings, where continuous exposition to respective microorganisms leads to immunological adaptation and semi-resistance while considerable disease would result in case of exposition of a non-adapted population. While biofilms in chronic wounds show higher pathogenic potential in case of multi-species composition, detection of multiple pathogens in respiratory samples is much more difficult to interpret and asymptomatic enteric colonization with facultative pathogenic microorganisms is frequently observed in high endemicity settings. For respiratory samples and stool samples, cycle-threshold-value-based semi-quantitative interpretation of qPCR results has been suggested. Etiological relevance is assumed if cycle-threshold values are low, suggesting high pathogen loads. Although the procedure is challenged by lacking standardization and methodical issues, first evaluations have led to promising results. Future studies should aim at generally acceptable quantitative cut-off values to allow discrimination of asymptomatic colonization from clinically relevant infection.

Rapid molecular detection of macrolide resistance

“…A second, and more powerful strategy is to focus on levels of the genetic sequence observed. If mef(A) is helping a pathogen cause disease, it will be enriched to a higher copy number than it would be as a sporadic colonizer diluted into a healthy microbial community [65,66]. By providing quantitative data on relative levels of mef(A), our RPA assay is ideally suited to this approach, making the determination of an infection a matter of comparing the detected gene level with a threshold (after normalizing to total bacterial load).…”

“…By providing quantitative data on relative levels of mef(A), our RPA assay is ideally suited to this approach, making the determination of an infection a matter of comparing the detected gene level with a threshold (after normalizing to total bacterial load). Critically, future work must focus on empirically setting the threshold by testing many clinical samples, from both healthy and sick patients [65]. By providing a validated, easy-to-use rapid molecular assay, the present study represents a vital first step in this process.…”

Other Respiratory Viruses as a Cause of Community-Acquired Pneumonia

“…First, with increasingly sensitive diagnostic testing platforms, it can be difficult to distinguish viral carriage from true infection. 172 Rates of asymptomatic viral carriage range from 2 to 17%, depending on the patient population studied. 8,23,86,173 The specificity of PCR-based testing likely varies significantly, depending on both the age of the patient and the pathogen identified.…”

Novel diagnostics for point-of-care bacterial detection and identification

“…Further, the discrimination between an asymptomatic colonization and a clinically relevant infection relies upon the development of standardized quantitative cut off cyclethreshold (C t ) † values. 45 However, it is not clear that a relationship actually exists between the number of microorganisms present in an individual's specimen and the presence of healthy carriage or disease within the individual. One means of overcoming the problematic amplication of non-target DNA is to utilize multiplex-touchdown PCR (MT-PCR), which combines the features of a multiplex PCR (incorporating primers for a number of DNA targets) with touchdown PCR (which involves a cycling program, in which the annealing temperature is gradually reduced from a value above the estimated melting temperature (T m ) of the primers ‡ until it reaches the calculated annealing temperature [the touchdown temperature]), to increase the PCR specicity, sensitivity and yield.…”