Hagen Frickmann

Intracellular persisting Staphylococcus aureus is the major pathogen in recurrent tonsillitis.

Background The two major indications for tonsillectomy are recurrent tonsillitis (RT) and peritonsillar abscess (PTA). Unlike PTAs, which are primarily treated surgically, RT is often cured by tonsillectomy only after a series of failed drug therapy attempts. Although the bacteriological background of RT has been studied, the reason for the lack of success of conservative therapeutic approaches is not well understood. Methods In a prospective study, tonsil specimens from 130 RT patients and 124 PTA patients were examined for the presence of extra- and intracellular bacteria using antibiotic protection assays. Staphylococcus aureus isolates from RT patients were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing and MSCRAMM-gene-PCR. Their ability for biofilm formation was tested and their cell invasiveness was confirmed by a flow cytometric invasion assay (FACS), fluorescent in situ hybridization (FISH) and immunohistochemistry. Findings S. aureus was the predominant species (57.7%) in RT patients, whereas Streptococcus pyogenes was most prevalent (20.2%) in PTA patients. Three different assays (FACS, FISH, antibiotic protection assay) showed that nearly all RT-associated S. aureus strains were located inside tonsillar cells. Correspondingly, the results of the MSCRAMM-gene-PCRs confirmed that 87% of these S. aureus isolates were invasive strains and not mere colonizers. Based upon PFGE analyses of genomic DNA and on spa-gene typing the vast majority of the S. aureus isolates belonged to different clonal lineages. Conclusions Our results demonstrate that intracellular residing S. aureus is the most common cause of RT and indicate that S. aureus uses this location to survive the effects of antibiotics and the host immune response. A German translation of the Abstract is provided as supplementary material (Abstract S1).

Rapid Discrimination of Haemophilus influenzae, H. parainfluenzae, and H. haemolyticus by Fluorescence In Situ Hybridization (FISH) and Two Matrix-Assisted Laser-Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Platforms

Background Due to considerable differences in pathogenicity, Haemophilus influenzae, H. parainfluenzae and H. haemolyticus have to be reliably discriminated in routine diagnostics. Retrospective analyses suggest frequent misidentifications of commensal H. haemolyticus as H. influenzae. In a multi-center approach, we assessed the suitability of fluorescence in situ hybridization (FISH) and matrix-assisted laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) for the identification of H. influenzae, H. parainfluenzae and H. haemolyticus to species level. Methodology A strain collection of 84 Haemophilus spp. comprising 50 H. influenzae, 25 H. parainfluenzae, 7 H. haemolyticus, and 2 H. parahaemolyticus including 77 clinical isolates was analyzed by FISH with newly designed DNA probes, and two different MALDI-TOF-MS systems (Bruker, Shimadzu) with and without prior formic acid extraction. Principal Findings Among the 84 Haemophilus strains analyzed, FISH led to 71 correct results (85%), 13 uninterpretable results (15%), and no misidentifications. Shimadzu MALDI-TOF-MS resulted in 59 correct identifications (70%), 19 uninterpretable results (23%), and 6 misidentifications (7%), using colony material applied directly. Bruker MALDI-TOF-MS with prior formic acid extraction led to 74 correct results (88%), 4 uninterpretable results (5%) and 6 misidentifications (7%). The Bruker MALDI-TOF-MS misidentifications could be resolved by the addition of a suitable H. haemolyticus reference spectrum to the systems database. In conclusion, no analyzed diagnostic procedure was free of errors. Diagnostic results have to be interpreted carefully and alternative tests should be applied in case of ambiguous test results on isolates from seriously ill patients.

Nasal Screening for MRSA: Different Swabs – Different Results!

Objectives Swab-based nasal screening is commonly used to identify asymptomatic carriage of Staphylococcus aureus in patients. Bacterial detection depends on the uptake and release capacities of the swabs and on the swabbing technique itself. This study investigates the performance of different swab-types in nasal MRSA-screening by utilizing a unique artificial nose model to provide realistic and standardized screening conditions. Methods An anatomically correct artificial nose model was inoculated with a numerically defined mixture of MRSA and Staphylococcus epidermidis bacteria at quantities of 4×102 and 8×102 colony forming units (CFU), respectively. Five swab-types were tested following a strict protocol. Bacterial recovery was measured for direct plating and after elution into Amies medium by standard viable count techniques. Results Mean recovered bacteria quantities varied between 209 and 0 CFU for MRSA, and 365 and 0 CFU for S. epidermidis, resulting swab-type-dependent MRSA-screening-sensitivities ranged between 0 and 100%. Swabs with nylon flocked tips or cellular foam tips performed significantly better compared to conventional rayon swabs referring to the recovered bacterial yield (p<0.001). Best results were obtained by using a flocked swab in combination with Amies preservation medium. Within the range of the utilized bacterial concentrations, recovery ratios for the particular swab-types were independent of the bacterial species. Conclusions This study combines a realistic model of a human nose with standardized laboratory conditions to analyze swab-performance in MRSA-screening situations. Therefore, influences by inter-individual anatomical differences as well as diverse colonization densities in patients could be excluded. Recovery rates vary significantly between different swab-types. The choice of the swab has a great impact on the laboratory result. In fact, the swab-type contributes significantly to true positive or false negative detection of nasal MRSA carriage. These findings should be considered when screening a patient.

Accelerated identification of Staphylococcus aureus from blood cultures by a modified fluorescence in situ hybridization procedure.

This study evaluated fluorescence in situ hybridization (FISH) for rapid identification of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) directly from blood cultures. Initially, 360 blood cultures containing Gram-positive cocci were investigated by a previously described microwave-FISH procedure: 44/49 (89.8 %) S. aureus and 298/299 (99.7 %) CoNS were correctly identified. Because FISH proved useful and reliable but handling was found to be inconvenient, the method was modified by employing a recently developed slide chamber. This reduced the time required from 60 to 30 min. The simplified execution allowed integration of the method into the workflow of a routine laboratory without difficulty. The modified method proved to be highly reliable, identifying 37/37 (100 %) S. aureus and 169/172 (98.2 %) CoNS directly from blood cultures.

Rapid identification of Burkholderia pseudomallei and Burkholderia mallei by fluorescence in situ hybridization (FISH) from culture and paraffin-embedded tissue samples.

We evaluated newly developed probes for rapid identification of Burkholderia (B.) pseudomallei and B. mallei and differentiation from B. thailandensis by fluorescence in situ hybridization (FISH). FISH correctly identified 100% of the tested B. pseudomallei (11), B. mallei (11), and B. thailandensis (1) strains, excluded 100% of all tested negative controls (61), and allowed demonstration of B. pseudomallei infection in a paraffin-embedded spleen tissue sample of an experimentally infected mouse.

Fluorescence in situ hybridization (FISH) in the microbiological diagnostic routine laboratory: a review

Abstract Early identification of microbial pathogens is essential for rational and conservative antibiotic use especially in the case of known regional resistance patterns. Here, we describe fluorescence in situ hybridization (FISH) as one of the rapid methods for easy identification of microbial pathogens, and its advantages and disadvantages for the diagnosis of pathogens in human infections in the laboratory diagnostic routine. Binding of short fluorescence-labeled DNA or nucleic acid-mimicking PNA probes to ribosomes of infectious agents with consecutive analysis by fluorescence microscopy allows identification of bacterial and eukaryotic pathogens at genus or species level. FISH analysis leads to immediate differentiation of infectious agents without delay due to the need for microbial culture. As a microscopic technique, FISH has the unique potential to provide information about spatial resolution, morphology and identification of key pathogens in mixed species samples. On-going automation and commercialization of the FISH procedure has led to significant shortening of the time-to-result and increased test reliability. FISH is a useful tool for the rapid initial identification of microbial pathogens, even from primary materials. Among the rapidly developing alternative techniques, FISH serves as a bridging technology between microscopy, microbial culture, biochemical identification and molecular diagnostic procedures.

PCR for enteric pathogens in high-prevalence settings. What does a positive signal tell us?

Abstract Background: Molecular methods, in particular PCR, are increasingly used for the diagnosis of enteric pathogens in stool samples. In high-endemicity settings, however, asymptomatic carriage or residual DNA from previous infections will hamper the interpretation of positive test results. We assessed the quantitative dimension of this problem in schoolchildren in the rural highlands of Madagascar. Methods: Stool samples were collected from 410 apparently healthy Madagascan schoolchildren and analysed by multiplex real-time PCR for enteroinvasive bacteria (Salmonella spp., Shigella spp./enteroinvasive Escherichia coli (EIEC), Campylobacter jejuni, Yersinia spp.), enteric protozoa (Entamoebea histolytica, Giardia duodenalis, Cryptosporidium spp., Cyclospora spp.), and helminths (Ascaris lumbricoides, Ancylostoma spp., Necator americanus, Strongyloides stercoralis). Symptoms of gastrointestinal disease were assessed. Results: Among the 410 samples, we detected Giardia duodenalis in 195, Campylobacter jejuni in 91, Ascaris lumbricoides in 72, Cyclospora cayetanenesis in 68, Shigella spp./EIEC in 56, and Strongyloides stercoralis and Cryptosporum spp. in 1 case each. Salmonella spp., Yersinia spp. and hookworms were not observed. Relative risk assessment suggested few and incoherent associations with pathogen detections, indicating asymptomatic carriage or DNA residuals. Only 26.1% of the schoolchildren were tested negative for all analysed pathogens. Conclusions: The very high risk of detecting traces of asymptomatic carriage or residual DNA from previous infections limits the value of highly sensitive PCR for the causal attribution of detected enteric pathogens from stool samples to an infectious gastrointestinal disease in the high-endemicity setting. Evaluated standards for the interpretation of such results are needed both for the diagnostic routine and for epidemiological assessments.

Diagnosis of neuroschistosomiasis by antibody specificity index and semi-quantitative real-time PCR from cerebrospinal fluid and serum.

We describe the case of a 16-year-old German male expatriate from Ghana who presented with obstipation, dysuria, dysaesthesia of the gluteal region and the lower limbs, bilateral plantar hypaesthesia and paraesthesia without pareses. A serum-cerebrospinal fluid (CSF) Schistosoma spp. specific antibody specificity index of 3.1 was considered highly suggestive of intrathecal synthesis of anti-Schistosoma spp. specific antibodies, although standardization of this procedure has not previously been described. Diagnosis was confirmed by detection of Schistosoma DNA in CSF by semi-quantitative real-time PCR at 100-fold concentration compared with serum. Accordingly the two diagnostic procedures, which have not previously been applied for routine diagnosis, appear to be useful for the diagnosis of neuroschistosomiasis. Clinical symptoms resolved following anthelmintic and anti-inflammatory therapy.

Increased detection of invasive enteropathogenic bacteria in pre-incubated blood culture materials by real-time PCR in comparison with automated incubation in Sub-Saharan Africa

Abstract Background: Invasive enteropathogenic bacteria can cause systemic infections. Data from studies with PCR detection suggest, at least for Salmonella enterica, that blood culture may lead to underestimation in the tropics. Corresponding data are lacking for other invasive enteropathogenic bacteria. We compared classical blood culture and molecular methods for the diagnosis of blood infections. Methods: A real-time multiplex PCR for Salmonella spp., Shigella spp./entero- invasive Escherichia coli (EIEC), Yersinia spp., and Campylobacter jejuni was applied to 2321 retained blood culture samples from Ghanaian patients, after enrichment by automated culture. Results: PCR detected Salmonella DNA in 56 out of 58 pre-incubated Ghanaian blood cultures with growth of S. enterica. In 2 samples molecular diagnosis was only possible after 1:10 dilution. Twenty-two samples negative by blood culture and 1 positive with Micrococcus spp. were PCR-positive for Salmonella spp. In addition, 3 Shigella spp./EIEC, 2 Yersinia spp., and 1 C. jejuni were detected by PCR but not by culture growth. Conclusions: Real-time PCR was more sensitive in identifying invasive enteropathogenic bacteria than automated blood culture, which is hampered by a lack of evidence-based standardization of pre-analytic conditions in the tropics. Primary agar culture and Gram-staining prior to automated blood culture is advisable in cases where transportation times are long.

Food and drinking water hygiene and intestinal protozoa in deployed German soldiers

This report analyzes the occurrence of Cryptosporidium spp., E. histolytica, and G. intestinalis in stool of returnees from military deployments and the impact of hygiene precautions. Between 2007 and 2010, stool samples of 830 returnees that were obtained 8-12 weeks after military deployments in Afghanistan, Uzbekistan, the Balkans, Democratic Republic of the Congo/Gabonese Republic, and Sudan and 292 control samples from non-deployed soldiers were analyzed by PCR for Cryptosporidium spp., E. histolytica, G. intestinalis, and the commensal indicator of fecal contamination E. dispar. Data on hygiene precautions were available. The soldiers were questioned regarding gastrointestinal and general symptoms. Among 1122 stool samples, 18 were positive for G. intestinalis, 10 for E. dispar, and no-one for Cryptosporidium spp. and E. histolytica. An increased risk of acquiring chronic parasitic infections in comparison with non-deployed controls was demonstrated only for G. intestinalis in Sudan, where standardized food and drinking water hygiene precautions could not be implemented. Standard food and drinking water hygiene precautions in the context of screened military field camps proved to be highly reliable in preventing food-borne and water-borne chronic infections and colonization by intestinal protozoa, leading to detection proportions similar to those in non-deployed controls.