Fluorescence In Situ Hybridization as Diagnostic Tool for Implant-associated Infections: A Pilot Study on Added Value.

Implant-associated infections are a devastating complication in surgery. Especially in infections with biofilm-forming microorganisms, the identification of the causing microorganism remains a challenge. However, the classification as biofilm is not possible with conventional polymerase chain reaction or culture-based diagnostics. The aim of this study was to evaluate the additional value of fluorescence in situ hybridization (FISH) and nucleic acid amplification technique (FISHseq) to discuss a diagnostic benefit of the culture-independent methods and to map spatial organization of pathogens and microbial biofilms in wounds. Methods: In total, 118 tissue samples from 60 patients with clinically suspected implant-associated infections (n = 32 joint replacements, n = 24 open reduction and internal fixation, n = 4 projectiles) were analyzed using classic microbiological culture and culture-independent FISH in combination with polymerase chain reaction and sequencing (FISHseq). Results: In 56 of 60 wounds, FISHseq achieved an added value. FISHseq confirmed the result of cultural microbiological examinations in 41 of the 60 wounds. In 12 wounds, one or more additional pathogens were detected by FISHseq. FISHseq could show that the bacteria initially detected by culture corresponded to a contamination in three wounds and could exclude that the identified commensal pathogens were a contamination in four other wounds. In five wounds, a nonplanktonic bacterial life form was detected. Conclusions: The study revealed that FISHseq gives additional diagnostic information, including therapy-relevant findings that were missed by culture. In addition, nonplanktonic bacterial life forms could also be detected with FISHseq, albeit less frequently than previously indicated.

Is There a Wound Recontamination by Eluates with High Bacterial Load in Negative-Pressure Wound Therapy with Instillation and Dwell Time?

BACKGROUND: This study investigated bacterial colonization of the foam eluate after negative-pressure wound therapy (NPWT) with instillation and dwell time (NPWTi-d) to obtain an indication of possible recontamination of the wound during NPWTi-d. To detect bacterial colonization and the extent of planktonic and nonplanktonic bioburden as comprehensively as possible, routine culture and molecular biology methods were used. METHODS: Before (time point 1) and after (median 3.0 days; time point 2) NPWT ( n = 15) and NPWTi-d with antiseptic installation ( n = 15), wound bed [22 acute, eight chronic wounds; median age, 51 years (range, 24 to 91); 26 men], foam, and eluate were examined by routine culture methods and fluorescence in situ hybridization (FISH), polymerase chain reaction, and FISH sequencing (FISHseq). RESULTS: At time point 2, 94.9% (37 of 39) of the pathogens identifiable in the eluate were also detected in the wound bed. Foam and eluate were always bacterially contaminated. NPWTi-d resulted in a significant reduction in the number of pathogen species compared with NPWT (NPWTi-d, time point 1 versus time point 2: P = 0.026; NPWT, time point 1 versus time point 2: not significant). Routine culture of wound bed samples at time point 2 identified only 28 of 52 (53.8%) of the pathogens, whereas examination of wound bed, foam, and eluate and additional FISHseq use detected 50 of 52 (96.2%) of the bacterial species. FISHseq identified biofilm in one and microcolonies in 10 wounds (time point 2). CONCLUSIONS: The bacterial load of the foam is flushed back into the wound during NPWTi-d. FISHseq should be used in addition to the routine culture method when pathogen identification and detection of nonplanktonic bacterial growth is particularly important for the patient's therapy. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Fluorescence in situ Hybridization (FISH) in the Microbiological Diagnostic of Deep Sternal Wound Infection (DSWI).

PURPOSE: Postoperative mediastinitis after cardiac surgery is still a devastating complication. Insufficient microbiological specimens obtained by superficial swabbing may only detect bacteria on the surface, but pathogens that are localized in the deep tissue may be missed. The aim of this study was to analyze deep sternal wound infection (DSWI) samples by conventional microbiological procedures and fluorescence in situ hybridization (FISH) in order to discuss a diagnostic benefit of the culture-independent methods and to map spatial organization of pathogens and microbial biofilms in the wounds. METHODS: Samples from 12 patients were collected and analyzed using classic microbiological culture and FISH in combination with molecular nucleic acid amplification techniques (FISHseq). Frequency of and the time to occurrence of a DSWI was recorded, previous operative interventions, complications, as well as individual risk factors and the microbiologic results were documented. RESULTS: Tissue samples were taken from 12 patients suffering from DSWI. Classical microbiological culture resulted in the growth of microorganisms in the specimens of five patients (42%), including bacteria and in one case Candida. FISHseq gave additional diagnostic information in five cases (41%) and confirmed culture results in seven cases (59%). CONCLUSION: Microbial biofilms are not always present in DSWI wounds, but microorganisms are distributed in a "patchy" pattern in the tissue. Therefore, a deep excision of the wound has to be performed to control the infection. We recommend to analyze at least two wound samples from different locations by culture and in difficult to interpret cases, additional molecular biological analysis by FISHseq.

Identification of anti-microbial peptides and traces of microbial DNA in infrainfundibular compartments of human scalp terminal hair follicles.

BACKGROUND: The upper follicular compartment, a well-known reservoir of cutaneous microbiota, constitutes a space for intensive cross-barrier dialogue. The lower follicle comprises the bulb and bulge, structures with relative immune-privileged status, crucial for physiological cycling, and widely considered to be microbial-free. OBJECTIVES: Following our initial immunohistochemical screening for regulatory cytokines and defensin expression in anagen hair follicles, we aimed to confirm our results with a follow-up ELISA investigation. We postulated that exposure to microbial components may trigger expression, and thus opted to investigate microbial presence in this area. MATERIALS & METHODS: We performed immunohistochemical staining for selected cytokines and antimicrobial peptides, and Gram and Giemsa staining on tissue sections from healthy individuals. Based on ELISA analyses, we confirmed a marked presence of IL-17A- and HBD2 in infrainfundibular compartments from plucked anagen hair follicles of 12 individuals (six females, six males; frontal and occipital scalp sites). 16S rRNA sequencing on microbial DNA extracted from lower follicles, as well as fluorescence in situ hybridization (FISH) were applied to explore bacterial presence in the infrainfundibular compartments. RESULTS: 16S rRNA sequencing yielded reproducible data of bacterial presence in infrainfundibular compartments of plucked scalp follicles; Lawsonella clevelandensis, Staphylococcaceae and Propionibacteriaceae were the most abundant bacteria. Also, FISH revealed biofilm structures formed by Cutibacterium acnes (formerly Propionibacterium acnes) and Staphylococcus sp. below the infundibulum. CONCLUSION: As the skin microbiome largely influences the local immune system, the presence of bacteria in proximity to follicular immune-privileged areas may be of relevance to hair cycling in health and disease.

Fluorescence in situ hybridization for identification and visualization of microorganisms in infected heart valve tissue as addition to standard diagnostic tests improves diagnosis of endocarditis.

OBJECTIVES: In infective endocarditis (IE), identification of the causative organism and consecutive treatment are crucial for patient survival. Although the macroscopic aspect resembles infected tissue, standard diagnostic tests often fail to allow one to identify bacteria. Fluorescence in situ hybridization (FISH) is a molecular, culture-independent technique that allows one to identify and visualize microorganisms within tissue and to recognize their morphology, number and activity. We analysed the diagnostic benefit of FISH/polymerase chain reaction (PCR) by comparing its results to those of standard diagnostic tests. METHODS: From September 2015 to April 2018, 128 patients underwent first-time or redo valve surgery to treat IE. Patients were designated according to the modified Duke criteria as definite (n = 61), possible (n = 34) or rejected (n = 33) IE. Tissue specimens obtained intraoperatively were analysed using FISH/PCR in addition to undergoing standard diagnostic testing and PCR alone. RESULTS: We used blood cultures to detect microorganisms in 67/128 patients; valve cultures, in 34/128; PCR, in 67/128; histopathological diagnosis showed IE in 72/128 cases. We were able to detect microorganisms in 103/128 cases using FISH/PCR, with 55/61 in definite IE. Furthermore, we were able to identify 26 cases of bacterial biofilm using FISH/PCR, despite antibiotic treatment of 61 in the definite, 13 in the possible and 1 in the rejected group, including 8/33 patients in the rejected group with active bacteria. In all cases, the patient's therapy was altered. CONCLUSIONS: FISH/PCR was used to identify microorganisms in cases in which standard diagnostic tests failed to provide sufficient results for various reasons. Furthermore, FISH/PCR enabled us to identify bacterial biofilms and to differentiate between active versus degraded bacteria, thus indicating the impact of treatment. Therefore, we suggest FISH/PCR as an additional diagnostic tool in IE alongside standard diagnostic tests.

Potential Role for Urine Polymerase Chain Reaction in the Diagnosis of Whipple's Disease.

BACKGROUND: Whipple's disease (WD) is a rare infection with Tropheryma whipplei that is fatal if untreated. Diagnosis is challenging and currently based on invasive sampling. In a case of WD diagnosed from a kidney biopsy, we observed morphologically-intact bacteria within the glomerular capsular space and tubular lumens. This raised the questions of whether renal filtration of bacteria is common in WD and whether polymerase chain reaction (PCR) testing of urine might serve as a diagnostic test for WD. METHODS: We prospectively investigated urine samples of 12 newly-diagnosed and 31 treated WD patients by PCR. As controls, we investigated samples from 110 healthy volunteers and patients with excluded WD or acute gastroenteritis. RESULTS: Out of 12 urine samples from independent, therapy-naive WD patients, 9 were positive for T. whipplei PCR. In 3 patients, fluorescence in situ hybridization visualized T. whipplei in urine. All control samples were negative, including those of 11 healthy carriers with T. whipplei-positive stool samples. In our study, the detection of T. whipplei in the urine of untreated patients correlated in all cases with WD. CONCLUSIONS: T. whipplei is detectable by PCR in the urine of the majority of therapy-naive WD patients. With a low prevalence but far-reaching consequences upon diagnosis, invasive sampling for WD is mandatory and must be based on a strong suspicion. Urine testing could prevent patients from being undiagnosed for years. Urine may serve as a novel, easy-to-obtain specimen for guiding the initial diagnosis of WD, in particular in patients with extra-intestinal WD.

Whipple's Disease: Diagnostic Value of rpoB Gene PCR from Peripheral Blood Mononuclear Cells.

INTRODUCTION: Chronic infection with Tropheryma whipplei, known as Whipple's disease (WD), classically affects the gastrointestinal tract, but any organ system may be affected, and isolated manifestations occur. Reliable diagnosis based on a combination of periodic acid-Schiff (PAS) staining, T. whipplei-specific immunohistochemistry (IHC), and polymerase chain reaction (PCR) from duodenal biopsies may be challenging in cases without classical gastrointestinal infection, so the need for additional diagnostic materials is urgent. OBJECTIVE: Our objective was to evaluate additional diagnostic possibilities for WD. METHODS: We analyzed samples from 20 patients with WD and 18 control subjects in a prospective observational pilot study. In addition to WD diagnosis by PAS staining, T. whipplei-specific IHC and PCR of duodenal or extra intestinal tissues, whole EDTA blood, peripheral blood mononuclear cells (PBMCs) and PBMC fractions enriched with or depleted of cluster of differentiation (CD)-14+ cells were examined using T. whipplei rpoB gene PCR. RESULTS: Tropheryma whipplei DNA was detected in 35 of 60 (58.3%) preparations from 16 of 20 patients with WD, most of whom lacked gastrointestinal signs and characteristic PAS-positive duodenal macrophages. CONCLUSION: This study provides evidence for the potential suitability of blood, particularly PBMCs, as material to assist in the diagnosis of WD via rpoB gene real-time PCR. Thus, PCR from blood preparations can be helpful for diagnostic decision making in atypical cases of WD.

Detection of Coxiella burnetii in heart valve sections by fluorescence in situ hybridization.

PURPOSE: Infective endocarditis is a severe and potentially fatal disease. Nearly a third of all cases remain culture-negative, making a targeted and effective antibiotic therapy of patients challenging. In the past years, fluorescence in situ hybridization (FISH) has proven its value for the diagnosis of infective endocarditis, particularly when it is caused by fastidious bacteria. To increase the number of infective endocarditis causing agents, which can be identified by FISH, we designed and optimized a FISH-probe for the specific detection of Coxiella burnetii in heart valve tissue. METHODOLOGY: Even with specific probes the detection and identification of bacteria can be complicated by the high autofluorescence due to calcification of the analysed tissue. To overcome this problem, we developed a protocol to detect C. burnetii by hybridizing, stripping and reprobing the identical section with different species-specific probes repeatedly.Results/Key findings. The newly designed specific FISH probe and the developed protocol exemplarily allowed us to unequivocally identify C. burnetii in tissue sections of a patient with infective endocarditis. CONCLUSION: This method provides an add-on to existing protocols for the unambiguous diagnosis of bacteria directly within tissues or other difficult tissue samples in cases with small sample size and limited sections.

Distribution and phylogeny of Brachyspira spp. in human intestinal spirochetosis revealed by FISH and 16S rRNA-gene analysis.

During six years as German National Consultant Laboratory for Spirochetes we investigated 149 intestinal biopsies from 91 patients, which were histopathologically diagnosed with human intestinal spirochetosis (HIS), using fluorescence in situ hybridization (FISH) combined with 16S rRNA gene PCR and sequencing. Aim of this study was to complement histopathological findings with FISH and PCR for definite diagnosis and species identification of the causative pathogens. HIS is characterized by colonization of the colonic mucosa of the human distal intestinal tract by Brachyspira spp. Microbiological diagnosis of HIS is not performed, because of the fastidious nature and slow growth of Brachyspira spp. in culture. In clinical practice, diagnosis of HIS relies solely on histopathology without differentiation of the spirochetes. We used a previously described FISH probe to detect and identify Brachyspira spp. in histological gut biopsies. FISH allowed rapid visualization and identification of Brachyspira spp. in 77 patients. In most cases, the bright FISH signal already allowed rapid localization of Brachyspira spp. at 400× magnification. By sequencing, 53 cases could be assigned to the B. aalborgi lineage including "B. ibaraki" and "B. hominis", and 23 cases to B. pilosicoli. One case showed mixed colonization. The cases reported here reaffirm all major HIS Brachyspira spp. clusters already described. However, the phylogenetic diversity seems to be even greater than previously reported. In 14 cases, we could not confirm HIS by either FISH or PCR, but found colonization of the epithelium by rods and cocci, indicating misdiagnosis by histopathology. FISH in combination with molecular identification by 16S rRNA gene sequencing has proved to be a valuable addition to histopathology. It provides definite diagnosis of HIS and allows insights into phylogeny and distribution of Brachyspira spp. HIS should be considered as a differential diagnosis in diarrhea of unknown origin, particularly in patients from risk groups (e.g. patients with colonic adenomas, inflammatory polyps, inflammatory bowel disease or HIV infection and in men who have sex with men).

Rapid molecular diagnosis of infective aortic valve endocarditis caused by Coxiella burnetii.

We describe a case of Q-fever endocarditis with severe destruction of the aortic valve with perivalvular abscess formation and cardiac failure. The patient needed urgent operative treatment and postoperative critical care. All specimens sent for microbiological examination were negative. Molecular analysis, including fluorescence in situ hybridization of aortic valve tissue combined with PCR and sequencing, led to the correct diagnosis and to appropriate anti-infective treatment. The patient subsequently recovered from complex cardiovascular surgery. This is the first report on Q-fever endocarditis that was rapidly diagnosed using these methods.

Validation of an rpoB gene PCR assay for detection of Tropheryma whipplei: 10 years' experience in a National Reference Laboratory.

The performance of a real-time PCR assay targeting the Tropheryma whipplei rpoB gene was evaluated using test strains and 1,236 clinical specimens in a national reference laboratory. The novel rpoB-PCR assay proved to be specific, revealed improved analytical sensitivity, and substantially accelerated detection of T. whipplei DNA in clinical specimens.