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.
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.
Authors:Ralf Matthias Hagen, Rebecca Hinz, Egbert Tannich, and Hagen Frickmann
We compared the performance of an in-house and a commercial malaria polymerase chain reaction (PCR) assay using freeze–thawed hemolytic blood samples.
A total of 116 freeze–thawed ethylenediamine tetraacetic acid (EDTA) blood samples of patients with suspicion of malaria were analyzed by an in-house as well as by a commercially available real-time PCR.
Concordant malaria negative PCR results were reported for 39 samples and malaria-positive PCR results for 67 samples. The inhouse assay further detected one case of Plasmodium falciparum infection, which was negative in the commercial assay as well as five cases of P. falciparum malaria and three cases of Plasmodium vivax malaria, which showed sample inhibition in the commercial assay. The commercial malaria assay was positive in spite of a negative in-house PCR result in one case. In all concordant results, cycle threshold values of P. falciparum-positive samples were lower in the commercial PCR than in the in-house assay.
Although Ct values of the commercial PCR kit suggest higher sensitivity in case of concordant results, it is prone to inhibition if it is applied to hemolytic freeze–thawed blood samples. The number of misidentifications was, however, identical for both real-time PCR assays.
Authors:Rebecca Hinz, Andreas Zautner, Ralf Hagen, and Hagen Frickmann
Haemophilus influenzae is a key pathogen of upper respiratory tract infections. Its reliable discrimination from nonpathogenic Haemophilus spp. is necessary because merely colonizing bacteria are frequent at primarily unsterile sites. Due to close phylogenetic relationship, it is not easy to discriminate H. influenzae from the colonizer Haemophilus haemolyticus. The frequency of H. haemolyticus isolations depends on factors like sampling site, patient condition, and geographic region.Biochemical discrimination has been shown to be nonreliable. Multiplex PCR including marker genes like sodC, fucK, and hpd or sequencing of the 16S rRNA gene, the P6 gene, or multilocus-sequence-typing is more promising. For the diagnostic routine, such techniques are too expensive and laborious. If available, matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry is a routine-compatible option and should be used in the first line. However, the used database should contain well-defined reference spectra, and the spectral difference between H. influenzae and H. haemolyticus is small. Fluorescence in-situ hybridization is an option for less well-equipped laboratories, but the available protocol will not lead to conclusive results in all instances. It can be used as a second line approach. Occasional ambiguous results have to be resolved by alternative molecular methods like 16S rRNA gene sequencing.
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.
Authors:Maria Franziska Ostermann, Heinrich Neubauer, Hagen Frickmann, and Ralf Matthias Hagen
This study assessed the variation of phenotypic features of clinical isolates of Burkholderia spp. from common rpsU gene sequence clusters.
A total of 41 clinical Burkholderia spp. isolates from German mucoviscidosis patients was subjected to rpsU gene sequencing. Biochemical assessment included the API systems 20 NE and 50 CHE as well as the Micronaut NF system. Fatty acid patterns were assessed using gas chromatography—mass spectrometry (GC—MS). Broth microdilution was used to identify minimum inhibitory concentrations.
Five rpsU gene sequence clusters comprised more than one clinical isolate. Altogether, assignments to three species and seven clusters comprising more than one Burkholderia species were performed. Inhomogeneity of biochemical reactions within the clusters ranged from 0/28 to 45/50 reactions. The standard deviation for fatty acid distributions ranged from 0% to 11.5%. Minimum inhibitory concentrations within the clusters showed a wide variation but only minor differences between the clusters.
Broad variations within identified rpsU gene sequence clusters regarding biochemical reactions, fatty acid patterns, and resistance patterns of clinical Burkholderia spp. isolates make the application of rpsU gene sequence analysis as a stand-alone procedure for discriminations within the Burkholderia cepacia complex unreliable.
Authors:Carola Edler, Henri Derschum, Mirko Köhler, Heinrich Neubauer, Hagen Frickmann, and Ralf Matthias Hagen
Reliable identification of pathogenic Burkholderia spp. like Burkholderia mallei and Burkholderia pseudomallei in clinical samples is desirable. Three different selective media were assessed for reliability and selectivity with various Burkholderia spp. and nontarget organisms.
Mast Burkholderia cepacia agar, Ashdown + gentamicin agar, and B. pseudomallei selective agar were compared. A panel of 116 reference strains and well-characterized clinical isolates, comprising 30 B. pseudomallei, 20 B. mallei, 18 other Burkholderia spp., and 48 nontarget organisms, was used for this assessment.
While all B. pseudomallei strains grew on all three tested selective agars, the other Burkholderia spp. showed a diverse growth pattern. Nontarget organisms, i.e., nonfermentative rod-shaped bacteria, other species, and yeasts, grew on all selective agars. Colony morphology did not allow unambiguous discrimination.
While the assessed selective media reliably allowed the growth of a wide range of B. pseudomallei strains, growth of other Burkholderia spp. is only partially ensured. Growth of various nontarget organisms has to be considered. Therefore, the assessed media can only be used in combination with other confirmative tests in the diagnostic procedure for the screening for melioidosis or glanders.
Authors:Matthias Halfter, Ulrich Müseler, Ralf Matthias Hagen, and Hagen Frickmann
The study was performed to assess the infection risk of German police officers on predominantly tropical deployments, mostly United Nations missions, with gastrointestinal pathogens.
Police officers were offered PCR-based screening for gastrointestinal pathogens before and after deployment. The screening panel comprised enteroinvasive bacteria (Salmonella spp., Shigella spp./enteroinvasive Escherichia coli, Campylobacter jejuni, and Yersinia spp.), enteropathogenic protozoa (Entamoeba histolytica, Giardia duodenalis, Cryptosporidium spp., and Cyclospora cayetanensis), as well as enteric helminths (Ancyclostoma spp., Ascaris lumbricoides, Enterobius vermicularis, Hymenolepis nana, Necator americanus, African Schistosoma spp., Strongyloides stercoralis, Taenia saginata, Taenia solium, and Trichuris trichiura).
G. duodenalis (n = 3), C. jejuni (n = 2), Salmonella spp. (n = 1), Shigella spp./enteroinvasive E. coli (n = 3), and S. stercoralis (n = 3) were detect in 12 out of 133 (9.0%) police officers. The majority had shown gastrointestinal symptoms on deployment and all were asymptomatic at the time of medical assessment. The major infection sites were Sub-Saharan Africa followed by Northern Africa and the Middle East.
Deployment of police officers to tropical deployment sites on United Nations missions is associated with a considerable acquisition risk of gastrointestinal pathogens in a quantitatively relevant minority. Post-deployment screening is advisable to facilitate therapeutic and hygiene-related consequences.
Authors:Heike Granzer, Ralf Matthias Hagen, Philipp Warnke, Wolfgang Bock, Tobias Baumann, Norbert Georg Schwarz, Andreas Podbielski, Hagen Frickmann, and Thomas Koeller
This study addressed carbapenem-resistant Acinetobacter baumannii complex (ABC) isolates from patients that were injured during the military conflict in the Eastern Ukraine and treated at German Armed Forces Hospitals in 2014 and 2015. Clonal diversity of the strains and potential ways of transmission were analyzed.
Patients with one or several isolation events of carbapenem-resistant ABC were included. Isolates were characterized by VITEK II-based identification and resistance testing, molecular screening for frequent carbapenemase genes, and DiversiLab rep-PCRbased typing. Available clinical information of the patients was assessed.
From 21 young male Ukrainian patients with battle injuries, 32 carbapenem- and fluoroquinolone-resistant ABC strains were isolated. Four major clonal clusters were detected. From four patients (19%), ABC isolates from more than one clonal cluster were isolated. The composition of the clusters suggested transmission events prior to the admission to the German hospitals.
The infection and colonization pressure in the conflict regions of the Eastern Ukraine with ABC of low clonal diversity is considerable. Respective infection risks have to be considered in case of battle-related injuries in these regions. The low number of local clones makes any molecular exclusion of transmission events difficult.
Authors:Hans Kollenda, Ralf Matthias Hagen, Miriam Hanke, Sandra Rojak, Rebecca Hinz, Lars Wassill, Sven Poppert, Egbert Tannich, and Hagen Frickmann
Background: The objective of this study was to assess an in-house loop-mediated isothermal amplification (LAMP) platform for malaria parasite detection and identification on species level.
Methods: LAMP primers specific for the human Plasmodium spp., namely, P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, as well as genus-specific primers, were tested against a composite gold standard comprising microscopy from thick and thin blood films, commercial genus-specific Meridian illumigene Malaria LAMP, in-house real-time polymerase chain reaction (PCR), and commercial fast-track diagnostics (FTD) Malaria differentiation PCR.
Results: Of the 523 blood samples analyzed, the composite gold standard indicated 243 Plasmodium-species-DNA-containing samples (46.5%). Sensitivity and specificity of the analyzed genus- and species-specific LAMP primers were 71.0%–100.0% and 90.8%–100.0%, respectively. The influence of parasitemia was best documented for P. falciparum-specific LAMP with sensitivity values of 35.5% (22/62) for microscopically negative samples containing P. falciparum DNA, 50% (19/38) for parasitemia ≤50/μL, 84% (21/25) for parasitemia ≤500/μL, and 100% (92/92) for parasitemia >500/μL.
Conclusions: In our hands, performance characteristics of species-specific in-house LAMP for malaria lack reliability required for diagnostic laboratories. The use of the easy-to-apply technique for surveillance purposes may be considered.