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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. This work was supported by research project PI21CIII/00007 from Spanish Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III Sí

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Introduction In-house real-time PCR (qPCR) is increasingly used for the diagnosis of endemic mycoses and diverse assays are in use in specialized laboratories. External quality control is currently lacking. Objective To compare the performance of different molecular detection protocols for the detection of Histoplasma capsulatum and Coccidioides spp. in a multicenter study involving five European laboratories. Methods Two test sample panels were sent to each laboratory which performed the analysis with their in-house assays. Recipients were blinded to sample content. The Histoplasma-panel included 14 samples representing a range of concentrations of Histoplasma DNA (n = 7), as well as a negative control and DNA from other fungi to test for specificity (Paracoccidioides lutzii n = 1; Blastomyces dermatitidis n = 1; Aspergillus fumigatus n = 1; Emergomyces spp. n = 2, and Candida albicans n = 1). The Coccidioides-panel included 10 samples representing a range of DNA concentrations of Coccidioides posadasii (n = 6), as well as a negative control and DNA from other fungi to test specificity (Uncinocarpus reesii n = 1; Trichophyton violaceum n = 1; and Candida albicans n = 1). Regarding techniques used, four laboratories used Histoplasma qPCRs, and one laboratory a conventional PCR and a broad-range PCR (brPCR) for fungal DNA. Four laboratories used different Coccidioides qPCRs and one laboratory a brPCR to detect Coccidioides DNA. Results Concerning the Histoplasma panel, qPCR assays were the most sensitives and agreement in the lowest detected amount of Histoplasma DNA was very suitable, ranging from 1 pg to 4 pg [ Conclusion Specific protocols based on qPCR showed better sensitivity than conventional and brPCR. These methods are useful for the rapid and sensitive detection Histoplasma and Coccidioides. Application of these tests on clinical samples may speed up diagnosis and potentially limit laboratory exposure to these fungi. Comparisons of in-house tests are essential to assess the performance and detect potential cross-reactivities and achieve a consensus.

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 → 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.

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.

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.

Bacterial pathogens are the most frequent cause of pneumonia after transplantation. Early after transplantation, recipients are at higher risk for nosocomial infections. The most commonly encountered pathogens during this period are gram-negative bacilli (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa …), but gram-positive coccus such as Staphylococcus aureus or Streptococcus pneumoniae and anaerobic bacteria can also be found. Empirical antibiotic therapy should be guided by previous colonisation of the recipient and bacterial resistance pattern in the hospital. Six months after transplantation, pneumonias are mostly due to community-acquired bacteria (S. pneumonia, H. influenza, Mycoplasma, Chlamydia and others). Opportunistic pathogens take advantage of the state of immunosuppression which is usually highest from one to six months after transplantation. During this period, but also occurring many years later in the setting of a chronically depressed immune system, bacterial pathogens with low intrinsic virulence can cause pneumonia. The diagnosis of pneumonia caused by opportunistic pathogens can be challenging. The delay in diagnosis preventing the early instauration of adequate treatment in kidney transplant recipients with a depressed immune system, frequently coupled with co-morbid conditions and a state of frailty, will affect prognosis and outcome, increasing morbidity and mortality. This review will focus on the most common opportunistic bacterial pathogens causing pneumonia in kidney transplant recipients: Legionella, Nocardia, Mycobacterium tuberculosis/nontuberculous, and Rhodococcus. Recognition of their specificities in the setting of immunosuppression will allow early diagnosis, crucial for initiation of effective therapy and successful outcome. Interactions with immunosuppressive therapy should be considered as well as reducing immunosuppression if necessary.

Fungal pneumonia is a dreaded complication encountered after kidney transplantation, complicated by increased mortality and often associated with graft failure. Diagnosis can be challenging because the clinical presentation is non-specific and diagnostic tools have limited sensitivity and specificity in kidney transplant recipients and must be interpreted in the context of the clinical setting. Management is difficult due to the increased risk of dissemination and severity, multiple comorbidities, drug interactions and reduced immunosuppression which should be applied as an important adjunct to therapy. This review will focus on the main causes of fungal pneumonia in kidney transplant recipients including Pneumocystis, Aspergillus, Cryptococcus, mucormycetes and Histoplasma. Epidemiology, clinical presentation, laboratory and radiographic features, specific characteristics will be discussed with an update on diagnostic procedures and treatment.

Besides the more prevalent deep fungal infections, a variety of infections are diagnosed rarely in most areas. This chapter discusses the more common infections including the endemic mycoses caused by dimorphic pathogens, implantation mycoses, and infections defined by appearance of fungal elements in tissue sections (Phaeohyphomycosis and hyalohyphomycosis) and rarely recovered yeasts.

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.

Establishing the etiology of invasive fungal infections is important to guide therapeutic options and for epidemiologic purposes. Formalin-fixed, paraffin-embedded (FFPE) tissue specimens from patients with proven invasive fungal infections are valuable to determine the etiology of systemic fungal infections. We compared different polymerase chain reaction (PCR) amplification strategies from FFPE tissue blocks to identify agents of invasive fungal infections. We found that specific PCR assays show superior sensitivity in the identification of DNA of Mucorales and Aspergillus and mixed infections caused by both as compared to broad-range PCR assays. Shorter amplicon lengths and less detection of contaminating fungal DNA are potential factors involved. However, detection of fungal DNA by highly sensitive specific PCR assays in the absence of demonstration of fungal elements in tissue suggests that PCR results should be interpreted in the context of the histopathology and clinical findings.

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.

BACKGROUND: Antiretroviral therapy reduces mortality and morbidity in HIVinfected individuals, most markedly when initiated early, before advanced immunodeficiency has developed. Although the international guidelines recommend starting antiretroviral therapy ART with a high CD4 cell count level, in the practice, this is particularly challenging to achieve, especially in late presentation of HIV diagnosis. The aim of this study was to determine the frequency and the demographic features associated with late presentation for HIV diagnosis in our Centre. METHODS: All newly diagnosed patients with HIV between January 2007 and December 2011 in our AIDS Reference Centre, were included. Late presenter patient was defined as patient with CD4 count 350/mm(3) at the time of diagnosis. Demographic age, sex, ethnicity, migration and clinical characteristics transmission mode, CD4 cell count, viral load were collected. We also collected data on outcome median day of hospitalization, mortality, virological response to ART and lost to followup LTFU. LTFU was defined as patient without any medical contact and viral load measurements during two consecutive years in our centre. RESULTS: From 2007 to 2011, 154 429 out of 359 patients newly diagnosed with HIV were late presenters. According to univariate analysis, age 50, female gender, migrant from subSaharan Africa and heterosexual contact were associated with late presentation for HIV diagnosis. In the multivariate analysis, age 50, heterosexual contact and migrant status particularly women were the only independent risk factors for late presentation. Late presenters tend to have a worse outcome than nonlate presenters. CONCLUSION: A considerable proportion of patients continue to be diagnosed with advanced HIV disease, despite the fact that risk factors for late presentation have been clearly identified. Despite high testing rate for HIV in Belgium, highrisk population like migrant, heterosexual contact, remain under tested. In order to be able to detect and treat all patients with high CD4 cell count as recommended by all international guidelines, we recommend developing testing policies specifically focused on these categories at high risk for late presentation.

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.

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.

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.

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 byBartonella henselaewhich illustrates this diagnostic difficulty.

OBJECTIVE AND IMPORTANCE: Bartonella henselae infections are among the most common causes of fever and lymphadenopathies, but can lead to severe complications in immunocompromised hosts; early recognition of these infections is of paramount importance in immunocompromised patients. CLINICAL PRESENTATION: Here we report the case of a renal transplant recipient who presented with fever, lymphadenopathies, and a splenic abscess secondary to Bartonella henselae infection, successfully treated with doxycycline. DISCUSSION AND CONCLUSIONS: We discuss the various clinical presentations of Bartonella henselae infections in immunocompromised patients and the available diagnostic tools for this potentially severe complication.

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.

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.