Methicillin-resistant Staphylococcus aureus (MRSA) poses an infection risk for international military deployments. In the presented mini-review, the history of MRSA in the medical service and modern warfare is highlighted. To allow rapid diagnosis, various molecular diagnostic point-of-care solutions are available. Most evaluation studies, however, are focused on screening swabs rather than clinical materials and evaluation data from harsh environments are widely lacking. Accordingly, studies with complex sample materials under difficult environmental conditions, e.g., in the desert or in the tropics, are desirable to close this gap of knowledge regarding the diagnostic reliability of such modern molecular point-of-care devices.
Authors:Ralf Matthias Hagen, Hagen Hinz, and Hagen Frickmann
ESBL (extended-spectrum-β-lactamase)-positive Enterobacteriaceae, which colonized European soldiers in tropical Western African Mali, were subjected to a molecular assessment of their resistance determinants. By doing so, a better insight into the locally endemic pattern of ESBL-associated β-lactamase genes was aspired.
From a previous study on diarrhea in European soldiers on deployment in tropical Mali, 15 ESBL-positive Escherichia coli with demonstrated high clonal diversity and one positive Klebsiella pneumoniae were assessed. Polymerase chain reactions (PCRs) for blaTEM and blaSHV β-lactamase genes with subsequent sequencing for the discrimination of ESBL- and non-ESBL variants were performed, followed by four group-specific PCRs for blaCTX-M genes.
Non-ESBL-associated blaTEM-1 was identified in six out of 15 (40%) E. coli strains, while 100% of the assessed strains were positive for group I blaCTX-M.
Considering the known clonal diversity of the assessed strains, the striking restriction to one group of blaCTX-M genes accounting for the ESBL phenotypes of the isolates suggests little genetic exchange in the local setting. Under such circumstances of restricted numbers of locally endemic target genes, PCR-based screening approaches for ESBL colonization might be promising.
Rickettsiae tend to have a rapid decrease of viability outside living cells. Therefore, the transport of samples containing viable rickettsiae for culturing in cell culture for diagnostic purposes is challenging.
The viability of rickettsiae in different transport media (commercially available transport medium COPAN “UTM-RT transport medium for viruses, chlamydia, mycoplasma, and ureaplasma,” minimal essential medium (MEM) with and without 10% foetal calf serum) at various time points at 4 °C and at ambient temperature (22 °C) was compared. Rickettsia honei was used as model organism.
After 2 weeks of storage at room temperature, no viable rickettsiae were detectable any more while storage at 4 °C kept rickettsiae viable for up to 4 weeks. The commercially available COPAN medium showed similarly good or slightly better stabilizing effects on rickettsiae compared with MEM + 10% foetal calf serum, pure MEM demonstrated the poorest results.
It is important to transport and store media with potentially rickettsiae-containing samples at 4 °C to prevent inactivation. MEM + 10% foetal calf serum can be used if no commercial medium is available with similarly good results.
A reliable and complete inactivation is an indispensable premise for any concentration of rickettsiae or for the development of diagnostic strategies based on their antigens. This study deals with the testing of methods to inactivate rickettsiae.
Rickettsia honei was used as a model organism. The inactivating potency of formalin, Qiagen® antiviral lysozyme (AVL) buffer, heating to 56 °C, and β-propiolactone was analyzed in cell culture.
The inactivation limits for rickettsiae were 0.1% formalin about 10 min, Qiagen AVL buffer about 5 min, 56 °C about 5 min, 0.125% β-propiolactone about 1 h, and 0.0125% β-propiolactone overnight. The interpretation was limited by cytotoxic effects of the inactivation procedures and by the culturally achievable rickettsial density in the cell culture supernatants that were used for the inactivation experiments.
Reliable modes of inactivation were identified, allowing for the secure handling of rickettsial antigens for diagnostic purposes.
As therapy-refractory giardiasis is an emerging health issue, this review aimed at summarizing mechanisms of reduced antimicrobial susceptibility in Giardia duodenalis and strategies to overcome this problem.
A narrative review on antimicrobial resistance in G. duodenalis was based upon a selective literature research.
Failed therapeutic success has been observed for all standard therapies of giardiasis comprising nitroimidazoles like metronidazole or tinidazole as first line substances but also benznidazoles like albendazole and mebendazole, the nitrofuran furazolidone, the thiazolide nitazoxanide, and the aminoglycoside paromomycin. Multicausality of the resistance phenotypes has been described, with differentiated gene expression due to epigenetic and post-translational modifications playing a considerable bigger role than mutational base exchanges in the parasite DNA. Standardized resistance testing algorithms are not available and clinical evidence for salvage therapies is scarce in spite of research efforts targeting new giardicidal drugs.
In case of therapeutic failure of first line nitroimidazoles, salvage strategies including various options for combination therapy exist in spite of limited evidence and lacking routine diagnostic-compatible assays for antimicrobial susceptibility testing in G. duodenalis. Sufficiently powered clinical and diagnostic studies are needed to overcome both the lacking evidence regarding salvage therapy and the diagnostic neglect of antimicrobial resistance.
Authors:Hagen Frickmann, Rebecca Hinz, and Ralf Hagen
Here, we assessed the extraction efficiency of a deployable bench-top nucleic acid extractor EZ1 in comparison to the column-based approach with complex sample matrices.A total of 48 EDTA blood samples and 81 stool samples were extracted by EZ1 automated extraction and the column-based QIAamp DNA Mini Kit. Blood sample extractions were assessed by two real-time malaria PCRs, while stool samples were analyzed by six multiplex real-time PCR assays targeting bacterial, viral, and parasitic stool pathogens. Inhibition control PCR testing was performed as well.In total, 147 concordant and 13 discordant pathogen-specific PCR results were obtained. The latter comprised 11 positive results after column-based extraction only and two positive results after EZ1 extraction only. EZ1 extraction showed a higher frequency of inhibition. This phenomenon was, however, inconsistent for the different PCR schemes. In case of concordant PCR results, relevant differences of cycle threshold numbers for the compared extraction schemes were not observed.Switches from well-established column-based extraction to extraction with the automated EZ1 system do not lead to a relevantly reduced yield of target DNA when complex sample matrices are used. If sample inhibition is observed, column-based extraction from another sample aliquot may be considered.
Little is known on the abundance of the pathogens Bacillus anthracis and Burkholderia pseudomallei in environmental samples in Cameroon. Therefore, 100 respective samples were assessed in a proof-of-principle assessment.
DNA residuals from nucleic acid extractions of 100 environmental samples, which were collected between 2011 and 2013 in the Mapé Basin of Cameroon, were screened for B. anthracis and B. pseudomallei by real-time PCR. The samples comprised soil samples with water contact (n = 88), soil samples without water contact (n = 6), plant material with water contact (n = 3), water (n = 2), and soil from a hospital dressing room (n = 1).
B. anthracis and B. pseudomallei were detected in none of the samples assessed.
The results indicate that at least a quantitatively overwhelming, ubiquitous occurrence of B. anthracis and B. pseudomallei in the environment in Cameroon is highly unlikely. However, the number and choice of the assessed samples limit the interpretability of the results.
Authors:Konstantin Tanida, Andreas Hahn, and Hagen Frickmann
The aim of the study was a comparative evaluation of in-house real-time PCR and commercial real-time PCR (Fast Track Diagnostics (FTD), ampliCube/Mikrogen) targeting enteropathogenic bacteria from stool in preparation of Regulation (EU) 2017/746 on in vitro diagnostic medical devices.
Both 241 stool samples from patients and 100 samples from German laboratory control schemes (“Ringversuche”) were used to comparatively assess in-house real-time PCR, the FTD bacterial gastroenteritis kit, and the ampliCube gastrointestinal bacterial panels 1&2 either with the in-house PCRs as gold standard and as a test comparison without gold standard applying latent class analysis. Sensitivity, specificity, intra- and inter-assay variation and Cohen’s kappa were assessed.
In comparison with the gold standard, sensitivity was 75–100% for strongly positive samples, 20–100% for weakly positive samples, and specificity ranged from 96 to 100%. Latent class analysis suggested that sensitivity ranges from 81.2 to 100% and specificity from 58.5 to 100%. Cohen’s kappa varied between moderate and nearly perfect agreement, intra- and inter-assay variation was 1–3 to 1–4 Ct values.
Acceptable agreement and performance characteristics suggested replaceability of the in-house PCR assays by the commercial approaches.
Authors:Hagen Frickmann, N. Chantratita, Y. Gauthier, H. Neubauer, and R. Hagen
Discrimination of Burkholderia (B.) pseudomallei and B. mallei from environmental B. thailandensis is challenging. We describe a discrimination method based on sequence comparison of the ribosomal protein S21 (rpsU) gene.The rpsU gene was sequenced in ten B. pseudomallei, six B. mallei, one B. thailandensis reference strains, six isolates of B. pseudomallei, and 37 of B. thailandensis. Further rpsU sequences of six B. pseudomallei, three B. mallei, and one B. thailandensis were identified via NCBI GenBank. Three to four variable base-positions were identified within a 120-base-pair fragment, allowing discrimination of the B. pseudomallei/mallei-cluster from B. thailandensis, whose sequences clustered identically. All B. mallei and three B. pseudomallei sequences were identical, while 17/22 B. pseudomallei strains differed in one nucleotide (78A>C). Sequences of the rpsU fragment of ‘out-stander’ reference strains of B. cepacia, B. gladioli, B. plantarii, and B. vietnamensis clustered differently.Sequence comparison of the described rpsU gene fragment can be used as a supplementary diagnostic procedure for the discrimination of B. mallei/pseudomallei from B. thailandensis as well as from other species of the genus Burkholderia, keeping in mind that it does not allow for a differentiation between B. mallei and B. pseudomallei.
Authors:Sonja Obersteller, Heinrich Neubauer, Ralf Matthias Hagen, and Hagen Frickmann
The extraction and further processing of nucleic acids (NA) from formalin-fixed paraffin-embedded (FFPE) tissues for microbiological diagnostic polymerase chain reaction (PCR) approaches is challenging. Here, we assessed the effects of five different commercially available nucleic acid extraction kits on the results of real-time PCR.
FFPE samples from organs of Burkholderia pseudomallei-infected Swiss mice were subjected to processing with five different extraction kits from QIAGEN (FFPE DNA Tissue Kit, EZ1 DNA Tissue Kit, DNA Mini Kit, DNA Blood Mini Kit, and FlexiGene DNA Kit) in combination with three different real-time PCRs targeting B. pseudomallei-specific sequences of varying length after 16 years of storage.
The EZ1 DNA Tissue Kit and the DNA Mini Kit scored best regarding the numbers of successful PCR reactions. In case of positive PCR, differences regarding the cycle-threshold (Ct) values were marginal.
The impact of the applied extraction kits on the reliability of PCR from FFPE material seems to be low. Interfering factors like the quality of the dewaxing procedure or the sample age appear more important than the selection of specialized FFPE kits.