A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis.

BACKGROUND: In-house real-time PCR (qPCR) is increasingly used to diagnose the so-called endemic mycoses as commercial assays are not widely available. OBJECTIVES: To compare the performance of different molecular diagnostic assays for detecting Histoplasma capsulatum and Coccidioides spp. in five European reference laboratories. METHODS: Two blinded external quality assessment (EQA) panels were sent to each laboratory that performed the analysis with their in-house assays. Both panels included a range of concentrations of H. capsulatum (n = 7) and Coccidioides spp. (n = 6), negative control and DNA from other fungi. Four laboratories used specific qPCRs, and one laboratory a broad-range fungal conventional PCR (cPCR) and a specific cPCR for H. capsulatum with subsequent sequencing. RESULTS: qPCR assays were the most sensitive for the detection of H. capsulatum DNA. The lowest amount of H. capsulatum DNA detected was 1-4 fg, 0.1 pg and 10 pg for qPCRs, specific cPCR and broad-range cPCR, respectively. False positive results occurred with high concentrations of Blastomyces dermatitidis DNA in two laboratories and with Emergomyces spp. in one laboratory. For the Coccidioides panel, the lowest amount of DNA detected was 1-16 fg by qPCRs and 10 pg with the broad-range cPCR. One laboratory reported a false positive result by qPCR with high load of Uncinocarpus DNA. CONCLUSION: All five laboratories were able to correctly detect H. capsulatum and Coccidioides spp. DNA and qPCRs had a better performance than specific cPCR and broad-range cPCR. EQAs may help standardise in-house molecular tests for the so-called endemic mycoses improving patient management.

Disseminated Emergomycosis in a Person with HIV Infection, Uganda.

We describe emergomycosis in a patient in Uganda with HIV infection. We tested a formalin-fixed, paraffin-embedded skin biopsy to identify Emergomyces pasteurianus or a closely related pathogen by sequencing broad-range fungal PCR amplicons. Results suggest that emergomycosis is more widespread and genetically diverse than previously documented. PCR on tissue blocks may help clarify emergomycosis epidemiology.

Molecular typing and in vitro resistance of Cryptococcus neoformans clinical isolates obtained in Germany between 2011 and 2017.

Cryptococcosis is a fungal infection of the central nervous system predominantly caused by Cryptococcus neoformans in immunocompromised patients. In several countries worldwide, up to 50% of isolates show in vitro resistance to clinically used antifungals including fluconazole. No prospective data on susceptibility to antifungal drugs are available for Germany. In this study, we characterised all C. neoformans isolates collected from individual patients' samples at the German reference laboratory for cryptococcosis 2011 and 2017 (n = 133) by multi-locus sequence typing and phenotypic drug susceptibility testing. We identified serotype A/genotype VNI isolates belonging to clonal complexes previously described from Europe, Africa, Asia and South America as the most prevalent agents of cryptococcosis in Germany. Overall, we observed minimal inhibitory concentrations (MICs) above the epidemiological cut-offs (ECVs) in 1.6% of isolates regarding fluconazole and 2.3% of isolates regarding 5-flucytosine. Here, two C. neoformans var. grubii isolates displayed decreased drug susceptibility to fluconazole, one of them additionally to 5-flucytosine. We also found 5-flucytosine MICs above the ECV for two C. neoformans var. neoformans isolates. We identified a novel mutation in the ERG11 gene which might be associated with the elevated fluconazole MIC in one of the isolates. The clinical importance of the detected in vitro resistance is documented by patient histories showing relapsed infection or primary fatal disease. Of note, sertraline demonstrated antifungal activity comparable to previous reports. Systematic collection of susceptibility data in combination with molecular typing of C. neoformans is important to comprehensively assess the spread of isolates and to understand their drug resistance patterns.

Progressive histoplasmosis with hemophagocytic lymphohistiocytosis and epithelioid cell granulomatosis: A case report and review of the literature.

Histoplasmosis in central Europe is a rare fungal disease with diverse clinical presentations. Apart from acute pulmonary histoplasmosis and involvement of the central nervous system, the most serious clinical presentation is progressive disseminated histoplasmosis which is generally associated with severe immunodeficiency and, in particular, advanced human immunodeficiency virus infection. Here, we report on an immunocompetent female residing in a non-endemic area, presenting with progressive disseminated histoplasmosis after a remote travel history to Thailand and Costa Rica. Diagnosis was delayed by several months due to misinterpretation of epithelioid cell granulomatosis of the intestine as Crohn's disease and of similar lung lesions as acute sarcoidosis. Prompted by clinical deterioration with signs and symptoms consistent with hemophagocytic lymphohistiocytosis, a bone marrow aspiration was performed that documented hemophagocytosis and intracellular organisms interpreted as Leishmania sp., but later identified by molecular methods as Histoplasma capsulatum. Treatment with liposomal amphotericin B followed by posaconazole led to prompt clinical improvement and ultimately cure.

[Differential diagnosis to malignant neoplasia: Nasopharyngeal mass in a cat with nasal cryptococcosis in Germany]. / Differenzialdiagnose zur malignen Neoplasie: Nasopharyngeale Masse bei einer Katze mit nasaler Kryptokokkose in Deutschland.

A 2-year-old Norwegian Forest cat was presented for evaluation of bilateral purulent nasal discharge and stertorous breathing. A computed tomography (CT) scan of the head revealed an intranasal mass of the left nasal cavity extending behind the tube openings and completely obstructing the nasopharynx. Rhinoscopy confirmed a pinkish, shiny mass. CT scan showed both compartments of the right middle ear filled with abnormal soft tissue attenuating material. There was no change in the bony outline of the middle ear. In the endoscopic examination, after endoscopically assisted tympanocentesis, this material in the accessible dorsolateral compartment proved to be classic polypous tissue in addition to highly viscous glue-like secretions. A secondary otitis media due to a drainage disorder was suspected.Using an endoscopic-interventional approach through the nostril, the nasopharyngeal mass was removed for histopathological examination, in order to restore the nasal airway, and to allow tube drainage. In contrast to cats with classical malignant nasal cavity masses, the cat showed several attachment points of the mass and multiple undulating elevations bilaterally in the nasopharyngeal mucosa.Cytological and histopathological examination identified the mass as a fungal granuloma in the context of a cryptococcus infection only rarely observed in Germany. Molecular genetic analysis confirmed an infection with Cryptococcus neoformans var. grubii.A single intranasal and nasopharyngeal endoscopic debridement resulted in a significant improvement of the clinical signs and a complete healing of the right middle ear (including the tympanic membrane) within 14 days, but not in a complete cure of the disease. The cat was therefore treated with oral itraconazole solution for several weeks.The case report shows that nasal cryptococcosis can also affect cats in Germany. Rhinoscopy reveals a nasopharyngeal mass with multiple attachment points, which is unusual for a neoplasia. In addition to the recommended removal of the mass, oral administration of systemic antimycotics is strongly advised.

Streptococcus gordonii septic arthritis: two cases and review of literature.

BACKGROUND: Despite advances in antimicrobial and surgical therapy, septic arthritis remains a rheumatologic emergency that can lead to rapid joint destruction and irreversible loss of function. In adults, Staphylococcus aureus is the most common microorganism isolated from native joints. Streptococcus gordonii is a prominent member of the viridans group of oral bacteria and is among the bacteria most frequently identified as being primary agent of subacute bacterial endocarditis. To the best of our knowledge, Streptococcus gordonii has not yet been described as agent of septic arthritis. CASE PRESENTATION: We describe here two cases of septic arthritis due to Streptococcus gordonii. It gives us an opportunity to review epidemiology, diagnosis criteria and management of septic arthritis. CONCLUSION: Although implication of S. gordonii as aetiologic agent of subacute endocarditis is well known, this organism is a rare cause of septic arthritis. In this case, the exclusion of associated endocarditis is warranted.

Osteomyelitis pubis caused by Kingella kingae in an adult patient: report of the first case.

BACKGROUND: Kingella kingae is the second most common pathogen causing paediatric arthritis and is described to be the causative bacteria in some paediatric osteomyelitis. Its microbiological detection is particularly difficult due to its slow growing. To our best knowledge this is the first case description of osteomyelitis pubis caused by this microorganism. CASE PRESENTATION: We report the unusual case of pubic osteomyelitis with soft tissue abcess caused by Kingella kingae in an adult patient of 66 years with a history of end-stage renal disease and breast carcinoma. Diagnosis was based on imaging and the microorganism was isolated from Computed Tomography-guided aspiration of synovial fluid. The infection resolved completely after twelve weeks of treatment with oral amoxicillin. CONCLUSION: This case description highlights the importance in osteoarticular infections of systematic inoculation of synovial liquid in BACTEC vials to optimise the detection of causative organisms, which can necessitate specific treatments.

Animal Histoplasmosis in Europe: Review of the Literature and Molecular Typing of the Etiological Agents.

Histoplasmosis has been previously diagnosed in animals from Europe. The aim of this study is to review the literature on these reports, to analyze cases diagnosed at our laboratory (2000-2022) and to improve molecular typing of Histoplasma capsulatum directly from tissue to study the molecular epidemiology of Histoplasma capsulatum causing animal infections in Europe. Including 15 cases studied in our laboratory, we identified 39 cases of animal histoplasmosis between 1968 and 2022. They were diagnosed mostly in superficial tissue biopsies from cats and badgers from Central Europe. Using phylogenetic analyses of six partial genes, we were able to classify eight of the etiological agents as belonging to a highly supported lineage within the Eurasian clade. This study confirms the occurrence of autochthonous histoplasmosis in animals in Central Europe and proposes the addition of new loci to the MLST scheme to study the molecular epidemiology of histoplasmosis using either formalin-fixed paraffin-embedded tissue and fresh or cadaveric biopsies.

Splenic rupture and fungal endocarditis in a pediatric patient with invasive fusariosis after allogeneic hematopoietic stem cell transplantation for aplastic anemia: A case report.

Background: Invasive mold infections are a well-known and life-threatening condition after allogeneic hematopoietic stem cell transplantation (HSCT). While Aspergillus species are recognized as predominant pathogens, Fusarium species should also be considered due to their broad environmental distribution and the expected poor outcome of invasive fusariosis. Particularly, splenic rupture as a complication of disseminated disease has not been reported yet. Case presentation: Two weeks after allogeneic HSCT for severe aplastic anemia, a 16-year-old boy presented with painful, erythematous skin nodules affecting the entire integument. As disseminated mycosis was considered, treatment with liposomal amphotericin B and voriconazole (VCZ) was initiated. Invasive fusariosis was diagnosed after histological and previously unpublished polymerase chain reaction-based examination of skin biopsies. Microbiological tests revealed Fusarium solani species. Despite stable neutrophil engraftment and uninterrupted treatment with VCZ, he developed mold disease-associated splenic rupture with hypovolemic shock and fungal endocarditis. The latter induced a cardiac thrombus and subsequent embolic cerebral infarctions with unilateral hemiparesis. Following cardiac surgery, the patient did not regain consciousness because of diffuse cerebral ischemia, and he died on day +92 after HSCT. Conclusion: Invasive fusariosis in immunocompromised patients is a life-threatening condition. Despite antimycotic treatment adapted to antifungal susceptibility testing, the patient reported here developed uncommon manifestations such as splenic rupture and fungal endocarditis.

Pathological Findings in Eurasian Otters (Lutra lutra) Found Dead between 2015-2020 in Schleswig-Holstein, Germany.

In times of massive biodiversity loss and ongoing environmental crises, it is extremely important to ensure long-term conservation efforts of threatened species like Eurasian otters (Lutra lutra). To gain insights into the status of Northern Germany's otter population, 92 otters found dead in Schleswig-Holstein between 2015-2020 were collected and underwent detailed dissection with the aim to establish a monitoring program for this population. Examinations followed a protocol especially designed for otters, including various biological data assessments and extended sampling. The finding sites showed a clear concentration in the Continental region. Seasonal concentration differed among the years, yet peaks were seen from fall to winter. Overall, more males than females were found, although this differed among the years. The majority of otters that could be aged were between 1-3 years. Placental scars and pregnancy were recorded in only few females. Nutritional status was good in most cases. Infectious diseases found included Vagococcus lutrae, Toxoplasma gondii, and Emmonsia spp. A major cause of death was roadkill. Known sample bias in studies focusing on roadkill was considered in the finding interpretation. Continuation of the population health investigations is mandatory to analyze potential trends and to establish an actual monitoring program for Eurasian otters in Schleswig-Holstein.

Detection of Histoplasma DNA from Tissue Blocks by a Specific and a Broad-Range Real-Time PCR: Tools to Elucidate the Epidemiology of Histoplasmosis.

Lack of sensitive diagnostic tests impairs the understanding of the epidemiology of histoplasmosis, a disease whose burden is estimated to be largely underrated. Broad-range PCRs have been applied to identify fungal agents from pathology blocks, but sensitivity is variable. In this study, we compared the results of a specific Histoplasma qPCR (H. qPCR) with the results of a broad-range qPCR (28S qPCR) on formalin-fixed, paraffin-embedded (FFPE) tissue specimens from patients with proven fungal infections (n = 67), histologically suggestive of histoplasmosis (n = 36) and other mycoses (n = 31). The clinical sensitivity for histoplasmosis of the H. qPCR and the 28S qPCR was 94% and 48.5%, respectively. Samples suggestive for other fungal infections were negative with the H. qPCR. The 28S qPCR did not amplify DNA of Histoplasma in FFPE in these samples, but could amplify DNA of Emergomyces (n = 1) and Paracoccidioides (n = 2) in three samples suggestive for histoplasmosis but negative in the H. qPCR. In conclusion, amplification of Histoplasma DNA from FFPE samples is more sensitive with the H. qPCR than with the 28S qPCR. However, the 28S qPCR identified DNA of other fungi in H. qPCR-negative samples presenting like histoplasmosis, suggesting that the combination of both assays may improve the diagnosis.

Disseminated coccidioidomycosis: Monitoring of serologic markers for treatment response.

We describe a patient with a disseminated coccidioidomycosis. Biomarkers in serum during itraconazole therapy showed a rapid clearing of Coccidioides DNA as detected by PCR. Coccidioides antibody detection by lateral flow assay became negative after one year and decreased from 1:64 to 1:8 in the complement fixation test after two years. The (1 â†’ 3)-ß-D-glucan levels normalised after two years without increase after cessation of antifungal therapy. Biomarkers in serum may guide treatment decisions in disseminated coccidioidomycosis.

Cutaneous Mycobacterium chelonae infection distal to the arteriovenous fistula.

A few single cases of Mycobacterium chelonae skin infection have been reported in haemodialysis patients. We report three additional cases that share peculiar clinical characteristics, pointing to diagnostic clues. All three cases presented as erythematous nodules developing distally to a proximal arteriovenous fistula (AVF). This presentation was identical to that of two published cases. A survey of all Belgian haemodialysis units during the period 2007-11 yields an estimated incidence of ∼0.9/10 000 patient-years. Although the source of M. chelonae remains unclear, this specific clinical presentation should be added to the listing of potential complications of an AVF and should be recognized, as it is fully treatable if diagnosed by culture and tissue biopsy.

Necrotizing ANCA-Positive Glomerulonephritis Secondary to Culture-Negative Endocarditis.

Infective endocarditis (IE) and small-vessel vasculitis may have similar clinical features, including glomerulonephritis. Furthermore the association between IE and ANCA positivity is well documented, making differential diagnosis between IE- and ANCA-associated vasculitis particularly difficult, especially in case of culture-negative IE. We report on one patient with glomerulonephritis secondary to culture-negative IE caused by Bartonella henselae which illustrates this diagnostic difficulty.

Osteonecrosis of the femoral head in patients with type 1 human immunodeficiency virus infection: clinical analysis and review.

Osteonecrosis of the femoral head (ONFH) typically affects relatively young, active patients and frequently follows an unrelenting course resulting in considerable loss of function. In human immunodeficiency virus-infected patients, ONFH is a growing problem. Etiology, pathogenesis, and treatment of ONFH in these patients remain controversial. We analyzed retrospectively patients with ONFH in a series of 815 patients followed in our AIDS reference center. Six patients out of the 815 were affected by ONFH (0.74%). The sex ratio was 1. Two of the six patients (33.3%) had no evidence of risk factor, whereas four patients (66.6%) had risk factors. One patient had three cumulated risk factors which were corticosteroids, chemotherapy, and radiotherapy. For this patient, the onset time for ONFH was shorter (36 months). It is difficult to attribute the effect to any single class of antiretroviral agents because combination therapy is standard of care, and a change in therapies is common. All classes of antiretroviral drugs have been used: protease inhibitors (mean use duration of 15.2 months before the ONFH onset), non-nucleoside reverse transcriptase inhibitors (12 months), and nucleoside reverse transcriptase inhibitors (40.5 months). ONFH was bilateral in four cases (66.6%) and unilateral in two cases (33.3%). One patient had other osteonecrosis location (both shoulders). ONFH was classified on plain radiography stage IV in five patients and stage III in one patient. All patients received initial medical treatment. It consisted of painkillers and non-weight bearing of the hip. All were finally operated on by total hip arthroplasty (THA). The average interval between ONFH diagnosis and the first THA was 10.3 months for the six patients. A controlateral THA was performed for three patients after a mean interval of 23.3 months after ONFH diagnosis. Of the nine implanted prostheses, four were cemented, four were cementless, and one was resurfacing prosthesis.