Culture-Independent Molecular Diagnosis in Routine

16 April 2018  
Karen an der Bench quadrIn his review, Lustig [1] compares, on a conceptual level, Molzym’s molecular based SepsiTest™-UMD with conventional culture diagnosis. Both test systems rely on the multiplication of targets – cell proliferation in culturing and DNA amplification of universally present microbial genes or parts thereof in SepsiTest™-UMD. While culture needs several days to become positive, molecular testing is completed within a few hours only. Also, fastidious strains and pathogens inhibited by applied antibiotics tend to be not able to grow in culture but are detectable by SepsiTest™-UMD.
   
 SepsiTest UMD routine
Fig. 1: Applications of SepsiTest™-UMD in routine molecular diagnosis.
   
Analysis of diverse specimens in clinical routine
SepsiTest™-UMD uses one protocol to process diverse samples for DNA extraction and PCR analysis. Tissue biopsies are shortly (10min) digested by a proteinase incubation before directed into DNA extraction. Human DNA is selectively depleted while enriched microbial DNA is purified by a bind-wash-elute procedure. Eluates are analysed by broad-range 16S and 18S rRNA gene PCR assays.  Amplicons from positive samples are sequence analysed for strain identification. This general approach allows for the unbiased diagnosis of a variety of infectious diseases without the need for cultivation (Fig. 1). In his application note, Lustig [1] summarises specimens that have been analysed by SepsiTest™-UMD as typical sampling material entering the routine laboratory. A basis of 12 reported studies using 1,179 clinical samples is used to evaluate the clinical performance of the test. Data analysis from studies including blood, cerebrospinal fluid and aspirates from orthopaedic samples indicates satisfying diagnostic sensitivity (median, 86%) and specificity (85.8%) against culture results. Importantly, the molecular test was positive with relevant pathogens in up to 26% of cases that were culture-negative. In infectious endocarditis, SepsiTest™-UMD found 1.9 to 4.9 times more relevant pathogens than culture.
   
The author comes to the conclusion that the test is a versatile tool for the culture-independent in vitro diagnosis of bacterial and fungal pathogens in daily routine.
   
Reference
[1] Lustig M (2017) SepsiTest™-UMD – Single protocol direct molecular diagnosis of pathogens in diverse samples. Trends Mol Diag 2/17, 1-2.
   
Application Note                List of Microbes