Features of cryptococcosis among 652 HIV-seronegative individuals in France: a cross-sectional observational study (2005-2020).

OBJECTIVES: We aimed to describe features and outcomes of cryptococcosis among HIV-seronegative individuals in a large surveillance network for cryptococcosis in France. METHODS: We included incident cases of cryptococcosis in HIV-seronegative individuals from 2005 to 2020. We compared patient characteristics, disease presentations, cryptococcal antigen results, and induction antifungal treatments according to underlying disease. We examined factors associated with 90-day mortality. Among patients with disseminated infections, we investigated whether receipt of flucytosine and polyene combination was associated with lower mortality. RESULTS: Among 652 individuals, 209 (32.1%) had malignancy, 130 (19.9%) were solid-organ transplant recipients, 204 (31.3%) had other immunocompromising conditions, and 109 (16.7%) had no reported underlying factor. The commonest presentations were disseminated infections (63.3%, 413/652) and isolated pulmonary infections (25.3%, 165/652). Solid-organ transplant patients were most likely to have disseminated infections and a positive serum cryptococcal antigen result. Patients with malignancy were older and less likely to receive a flucytosine-containing regimen for disseminated infections than others (58.7%, 78/133 vs. 73.2%, 194/265; p 0.029). The crude 90-day case-fatality ratio was 27.2% (95% CI, 23.5%-31.1%). Age ≥60 years (aOR: 2.75 [1.78-4.26]; p < 0.001), meningitis/fungaemia (aOR: 4.79 [1.80-12.7]; p 0.002), and malignancy (aOR: 2.4 [1.14-5.07]; p 0.02) were associated with higher 90-day mortality. Receipt of flucytosine and polyene combination was associated with lower 90-day mortality (aOR: 0.40 [0.23-0.71]; p 0.002) in multivariable analysis and inverse probability of treatment weighted analysis (aOR: 0.45 [0.25-0.80]; p 0.006). DISCUSSION: HIV-seronegative individuals with cryptococcosis comprise a wide range of underlying conditions with different presentations and outcomes, requiring a tailored approach to diagnosis and management.

Cryptococcus neoformans Infections Differ Among Human Immunodeficiency Virus (HIV)-Seropositive and HIV-Seronegative Individuals: Results From a Nationwide Surveillance Program in France.

Among 1107 cryptococcosis cases from the French surveillance network (2005-2020), the proportion of HIV-seronegative individuals has recently surpassed that of HIV-seropositive individuals. We observed marked differences in patient characteristics, disease presentations, cryptococcal antigen results, infecting species, and mortality according to HIV serostatus.

Invasive fungal diseases in patients with autoimmune diseases: a case series from the French RESSIF network.

OBJECTIVES: We aimed to describe patients with autoimmune diseases (AID) developing invasive fungal disease (IFD) and identify factors associated with short-term mortality. METHODS: We analysed cases of IFD associated with AID from the surveillance network of invasive fungal diseases (Réseau de surveillance des infections fongiques invasives, RESSIF) registry of the French national reference centre for invasive mycoses. We studied association of AID-specific treatments with 30-day mortality. We analysed total lymphocyte and CD4-T cell counts in patients with Pneumocystis jirovecii pneumonia (PCP). RESULTS: From 2012 to 2018, 549 individuals with IFD and AID were included, mainly with PCP (n=227, 41.3%), fungemia (n=167, 30.4%) and invasive aspergillosis (n=84, 15.5%). Rheumatoid arthritis (RA) and anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV) were the most frequent AID in PCP (n=55 and 25, respectively) and invasive aspergillosis (n=15 and 10, respectively), inflammatory bowel diseases (IBDs) were predominant in fungemia (n=36). At IFD diagnosis, 365 (66.5%) patients received glucocorticoids (GCs), 285 (51.9%) immunosuppressants, 42 (7.7%) tumor necrosis factor (TNF)-α blockers, 75 (13.7%) other biologics. Mortality at 30 days was 28.1% (143/508). Fungemia and high-dose GCs were independently associated with higher 30-day mortality. In PCP patients, lymphopenia <1500/mm3 was frequent (132/179, 73.7%) even if CD4+T cell count exceeded 200/mm3 in 56/78 patients (71.8%) (median 472.5/mm3, IQR 160-858). CONCLUSION: IFD associated with AID occurs primarily in RA, AAV and IBD, especially when treated with GCs and immunosuppressants. Mortality is high, especially for patients on high-dose GCs. Lymphopenia may help identify risk of PCP, but normal CD4+T cell count does not rule out the risk. Further studies are needed to assess the individual risk factors for IFD.

BD BACTEC™ Mycosis IC/F culture vials for fungemia diagnosis and follow-up: a retrospective study from 2013 to 2020.

This retrospective study compared the BD BACTEC™ Mycosis IC/F with the BD BACTEC™ Plus Aerobic/F and BD BACTEC™ Lytic Anaerobic/F culture vials (i.e., standard vials) for fungemia diagnosis at Nîmes University Hospital, France. From 2013 to 2020, 57 blood samples were concomitantly collected in the 3 culture vial types. For 43.8% of these samples, all vials were positive for yeast. The mean time to positivity was shorter (32.0 hours vs 44.2 hours; -12.2 hours) and longer (89.4 hours vs 33.7 hours; +55.7 hours) with the BD BACTEC™ Mycosis IC/F culture vials than with the other culture vials in patients without and with antifungal treatment, respectively. Moreover 31.6% and 24.6% of samples were positive only with the standard vials and with the BD BACTEC™ Mycosis IC/F culture vials, respectively. The BD BACTEC™ Mycosis IC/F culture vials are useful for the initial fungemia diagnosis (before any treatment) because they provide faster results.

Gradient concentration strip-specific epidemiological cut-off values of antifungal drugs in various yeast species and five prevalent Aspergillus species complexes.

OBJECTIVES: To determine the epidemiological cut-off values (ECVs) of ten antifungal agents in a wide range of yeasts and Aspergillus spp. using gradient concentration strips. METHODS: The minimum inhibitory concentrations for amphotericin B, anidulafungin, caspofungin, micafungin, flucytosine, fluconazole, itraconazole, isavuconazole, posaconazole, and voriconazole, determined with gradient concentration strips at 35 French microbiology laboratories between 2002 and 2020, were retrospectively collected. Then, the ECVs were calculated using the iterative method and a cut-off value of 97.5%. RESULTS: Minimum inhibitory concentrations were available for 17 653 clinical isolates. In total, 48 ECVs (including 32 new ECVs) were determined: 29 ECVs for frequent yeast species (e.g. Candida albicans and itraconazole/flucytosine, and Candida glabrata species complex [SC] and flucytosine) and rare yeast species (e.g. Candida dubliniensis, Candida inconspicua, Saccharomyces cerevisiae, and Cryptococcus neoformans) and 19 ECVs for Aspergillusflavus SC, Aspergillusfumigatus SC, Aspergillusnidulans SC, Aspergillusniger SC, and Aspergillusterreus SC. CONCLUSIONS: These ECVs can be added to the already available gradient concentration strip-specific ECVs to facilitate minimum inhibitory concentration interpretation and streamline the identification of nonwild type isolates.

Diagnosis of Congenital Toxoplasmosis: Performance of Four IgG and IgM Automated Assays at Birth in a Tricentric Evaluation.

For postnatal diagnosis of congenital toxoplasmosis (CT), the gold standard for the detection of anti-Toxoplasma IgM in newborns relies on the immunosorbent agglutination assay (ISAGA), which is manufactured from whole Toxoplasma parasites that become difficult to maintain. For IgG, only the Platelia assay provides a validated assay for cord blood according to the manufacturer, allowing its use in this context. We compared the analytical performance of four commercialized automated assays, Platelia, Abbott, Vidas, and Liaison, for the detection of IgG and IgM in the cord blood or peripheral blood of newborns from women infected during pregnancy. The assays were performed on samples from 509 newborns, collected from the university hospitals of Montpellier, Nîmes, and Toulouse. For IgM, the four assays appeared to be sufficiently informative to be used for congenital toxoplasmosis diagnosis (area under the curve [AUC] > 0.8, receiver operating characteristic [ROC] analysis), with Platelia showing the best performance, similar to ISAGA with regard to accuracy (83%). For the Vidas (76%), Abbott (75%), and Liaison (74%) assays, the accuracy was significantly lower. Maternal treatment significantly decreased the sensitivity of all the assays. For IgG, the four evaluated assays showed a sensitivity of over 90%, with Abbott (95%) and Liaison (94%), exhibiting a significantly higher sensitivity than Platelia (90%). Furthermore, Abbott showed its superiority in the cases of maternal infection during the third trimester. In the context of the newborns of mothers infected by Toxoplasma gondii during pregnancy, to ensure efficient care, Platelia and Abbott seemed to be the most suitable reference tests for the detection of IgM for the former and IgG for the latter.

Diagnosis of Congenital Toxoplasmosis: No Benefit of IgA Antibody Detection by Platelia ELISA in a Tricentric Evaluation.

The diagnostic accuracy of a commercial Toxoplasma gondii IgA antibody enzyme-linked immunosorbent assay (ELISA) was evaluated in the context of routine practice on 289 newborns with congenital toxoplasmosis (CT) and 220 healthy controls. The performance of this assay was compared to that of the current gold-standard test for anti-Toxoplasma IgM detection, an immunosorbent agglutination assay (ISAGA). IgM and IgA sensitivity and specificity were assessed in cord and postnatal samples. The sensitivity of IgA detection by ELISA on all serum and peripheral blood samples was 60.56% and 56.52%, respectively, which is low compared with the sensitivity of IgM detection by ISAGA (73.26% on serum samples, 82.35% on peripheral blood). Adding the T. gondii IgA antibody ELISA to the diagnostic panel did not significantly increase the overall performance of the serological diagnosis based on IgM detection.

Active Surveillance Program to Increase Awareness on Invasive Fungal Diseases: the French RESSIF Network (2012 to 2018).

The French National Reference Center for Invasive Mycoses and Antifungals leads an active and sustained nationwide surveillance program on probable and proven invasive fungal diseases (IFDs) to determine their epidemiology in France. Between 2012 and 2018, a total of 10,886 IFDs were recorded. The incidence increased slightly over time (2.16 to 2.36/10,000 hospitalization days, P = 0.0562) in relation with an increase of fungemia incidence (1.03 to 1.19/10,000, P = 0.0023), while that of other IFDs remained stable. The proportion of ≥65-year-old patients increased from 38.4% to 45.3% (P < 0.0001). Yeast fungemia (n = 5,444) was due mainly to Candida albicans (55.6%) with stable proportions of species over time. Echinocandins became the main drug prescribed (46.7% to 61.8%), but global mortality rate remained unchanged (36.3% at 1 month). Pneumocystis jirovecii pneumonia (n = 2,106) was diagnosed mostly in HIV-negative patients (80.7%) with a significantly higher mortality than in HIV-positive patients (21.9% versus 5.4% at 1 month, P < 0.0001). Invasive aspergillosis (n = 1,661) and mucormycosis (n = 314) were diagnosed mostly in hematology (>60% of the cases) with a global mortality rate of 42.5% and 59.3%, respectively, at 3 months and significant changes in diagnosis procedure over time. More concurrent infections were also diagnosed over time (from 5.4% to 9.4% for mold IFDs, P = 0.0115). In conclusion, we observed an aging of patients with IFD with a significant increase in incidence only for yeast fungemia, a trend toward more concurrent infections, which raises diagnostic and therapeutic issues. Overall, global survival associated with IFDs has not improved despite updated guidelines and new diagnostic tools. IMPORTANCE The epidemiology of invasive fungal diseases (IFDs) is hard to delineate given the difficulties in ascertaining the diagnosis that is often based on the confrontation of clinical and microbiological criteria. The present report underlines the interest of active surveillance involving mycologists and clinicians to describe the global incidence and that of the main IFDs. Globally, although the incidence of Pneumocystis pneumonia, invasive aspergillosis, and mucormycosis remained stable over the study period (2012 to 2018), that of yeast fungemia increased slightly. We also show here that IFDs seem to affect older people more frequently. The most worrisome observation is the lack of improvement in the global survival rate associated with IFDs despite the increasing use of more sensitive diagnostic tools, the availability of new antifungal drugs very active in clinical trials, and a still low/marginal rate of acquired in vitro resistance in France. Therefore, other tracks of improvement should be investigated actively.

COVID-19-Associated Pulmonary Aspergillosis, Fungemia, and Pneumocystosis in the Intensive Care Unit: a Retrospective Multicenter Observational Cohort during the First French Pandemic Wave.

The aim of this study was to evaluate diagnostic means, host factors, delay of occurrence, and outcome of patients with COVID-19 pneumonia and fungal coinfections in the intensive care unit (ICU). From 1 February to 31 May 2020, we anonymously recorded COVID-19-associated pulmonary aspergillosis (CAPA), fungemia (CA-fungemia), and pneumocystosis (CA-PCP) from 36 centers, including results on fungal biomarkers in respiratory specimens and serum. We collected data from 154 episodes of CAPA, 81 of CA-fungemia, 17 of CA-PCP, and 5 of other mold infections from 244 patients (male/female [M/F] ratio = 3.5; mean age, 64.7 ± 10.8 years). CA-PCP occurred first after ICU admission (median, 1 day; interquartile range [IQR], 0 to 3 days), followed by CAPA (9 days; IQR, 5 to 13 days), and then CA-fungemia (16 days; IQR, 12 to 23 days) (P < 10-4). For CAPA, the presence of several mycological criteria was associated with death (P < 10-4). Serum galactomannan was rarely positive (<20%). The mortality rates were 76.7% (23/30) in patients with host factors for invasive fungal disease, 45.2% (14/31) in those with a preexisting pulmonary condition, and 36.6% (34/93) in the remaining patients (P = 0.001). Antimold treatment did not alter prognosis (P = 0.370). Candida albicans was responsible for 59.3% of CA-fungemias, with a global mortality of 45.7%. For CA-PCP, 58.8% of the episodes occurred in patients with known host factors of PCP, and the mortality rate was 29.5%. CAPA may be in part hospital acquired and could benefit from antifungal prescription at the first positive biomarker result. CA-fungemia appeared linked to ICU stay without COVID-19 specificity, while CA-PCP may not really be a concern in the ICU. Improved diagnostic strategy for fungal markers in ICU patients with COVID-19 should support these hypotheses. IMPORTANCE To diagnose fungal coinfections in patients with COVID-19 in the intensive care unit, it is necessary to implement the correct treatment and to prevent them if possible. For COVID-19-associated pulmonary aspergillosis (CAPA), respiratory specimens remain the best approach since serum biomarkers are rarely positive. Timing of occurrence suggests that CAPA could be hospital acquired. The associated mortality varies from 36.6% to 76.7% when no host factors or host factors of invasive fungal diseases are present, respectively. Fungemias occurred after 2 weeks in ICUs and are associated with a mortality rate of 45.7%. Candida albicans is the first yeast species recovered, with no specificity linked to COVID-19. Pneumocystosis was mainly found in patients with known immunodepression. The diagnosis occurred at the entry in ICUs and not afterwards, suggesting that if Pneumocystis jirovecii plays a role, it is upstream of the hospitalization in the ICU.

Performance of the RealStar® Pneumocystis jirovecii PCR kit for the diagnosis of Pneumocystis pneumonia.

BACKGROUND: Pneumonia due to Pneumocystis jirovecii (PCP) is a frequent infection in HIV-positive and also in immunocompromised HIV-negative patients. PCR analysis of pulmonary samples has become an essential element in PCP laboratory diagnosis. Currently, many commercially PCR-based tests are available for P jirovecii detection and need to be evaluated. OBJECTIVES: We evaluated the performance of the RealStar® P jirovecii PCR kit for PCP diagnosis. METHODS: We performed the RealStar® P jirovecii PCR and an in-house PCR in 219 pulmonary samples. We then assessed the performance of the RealStar® P jirovecii PCR kit by classifying patients in proven, probable, possible PCP or no final diagnosis, on the basis of the clinical and radiological signs and direct examination of bronchoalveolar lavage samples. RESULTS: The results showed excellent concordance (96.8%) with another in-house PCR, previously used in the laboratory. The available clinical data allowed classifying 219 patients as having proven PCP (n = 6), probable PCP (n = 27), possible PCP (n = 29) and no final diagnosis of PCP (n = 157). The RealStar® P jirovecii PCR kit performed well with samples from patients with proven and probable PCP, as indicated by the detection of P jirovecii DNA in all these samples. The percentage of positive samples in the possible PCP category was 75.9%. In patients with no final diagnosis of PCP, P jirovecii DNA was detected in 13.4% of samples, indicating colonisation by this pathogen. CONCLUSIONS: The RealStar® P jirovecii PCR kit shows excellent performance for PCP diagnosis.

Limitation of Screening of Different Variants of SARS-CoV-2 by RT-PCR.

Since January 2021, the diffusion of the most propagated SARS-CoV-2 variants in France (UK variant 20I/501Y.V1 (lineage B.1.1.7), 20H/H501Y.V2 (lineage B.1.351) and 20J/H501Y.V3 (lineage P.1)) were urgently screened, needing a surveillance with an RT-PCR screening assay. In this study, we evaluated one RT-PCR kit for this screening (ID SARS-CoV-2/UK/SA Variant Triplex®, ID Solutions, Grabels, France) on 2207 nasopharyngeal samples that were positive for SARS-CoV-2. Using ID Solutions kit, 4.1% (92/2207) of samples were suspected to belonged to B.1.351 or P.1 variants. Next-generation sequencing that was performed on 67.4% (62/92) of these samples confirmed the presence of a B.1.351 variant in only 75.8% of the samples (47/62). Thirteen samples belonged to the UK variant (B.1.1.7), and two to A.27 with N501Y mutation. The thirteen with the UK variant presented one mutation in the S-gene, near the ΔH69/ΔV70 deletion (S71F or A67S), which impacted the detection of ΔH69/ΔV70 deletion. Using another screening kit (PKampVariantDetect SARS-CoV-2 RT-PCR combination 1 and 3® PerkinElmer, Waltham, MA, USA) on the misidentified samples, we observed that the two mutations, S71F or A67S, did not impact the detection of the UK variant. In conclusion, this study highlights the limitations of the screening strategy based on the detection of few mutations/deletions as well as it not being able to follow the virus evolution.

Some undesirable traps which can mislead the pathologist.

In clinical laboratories, the diagnosis of parasite diseases can sometimes be challenging for non-expert microbiologists. Indeed, in spite of the advent of the molecular biology, macroscopic and microscopic examinations still remain essential. Nonetheless, it is usually not automated and requires great skills to complete the correct diagnosis. It is not infrequent that inert elements mislead to erroneous diagnoses. Through three different concrete examples, this article aims at underscoring the actual risk of parasite misidentification and at highlighting the systematic approach to be conducted in order to enable reliable diagnosis.

Kazachstania slooffiae: An unexpected journey to a human pleural sample.

We report a case of a 50-year-old shepherd hospitalized in intensive care unit for hiatal hernia complicated by an occlusive syndrome. In post-surgery, an acute respiratory distress occurs due to mediastinitis with large pleural effusion. At the laboratory, direct examination of the pleural sample revealed the presence of pseudohyphae. Kazachstania slooffiae was identified by Mass Spectrometry and confirmed by DNA sequencing. This uncommon yeast has never been previously described in human infections. Although its pathogenicity is not well known, K. slooffiae should be considered in the case of critically ill patients.

Rapid diagnostic tests failing to detect infections by Plasmodium falciparum encoding pfhrp2 and pfhrp3 genes in a non-endemic setting.

BACKGROUND: Rapid diagnostic tests (RDTs) detecting the histidine-rich protein 2 (PfHRP2) have a central position for the management of Plasmodium falciparum infections. Yet, variable detection of certain targeted motifs, low parasitaemia, but also deletion of pfhrp2 gene or its homologue pfhrp3, may result in false-negative RDT leading to misdiagnosis and delayed treatment. This study aimed at investigating the prevalence, and understanding the possible causes, of P. falciparum RDT-negative infections at Montpellier Academic Hospital, France. METHODS: The prevalence of falsely-negative RDT results reported before and after the introduction of a loop-mediated isothermal amplification (LAMP) assay, as part as the malaria screening strategy in January 2017, was analysed. Negative P. falciparum RDT infections were screened for pfhrp2 or pfhrp3 deletion; and exons 2 were sequenced to show a putative genetic diversity impairing PfHRP2 detection. RESULTS: The overall prevalence of P. falciparum negative RDTs from January 2006 to December 2018 was low (3/446). Whereas no cases were reported from 2006 to 2016 (0/373), period during which the malaria diagnostic screen was based on microscopy and RDT, prevalence increased up to 4.1% (3/73) between 2017 and 2018, when molecular detection was implemented for primary screening. Neither pfhrp2/3 deletion nor major variation in the frequency of repetitive epitopes could explain these false-negative RDT results. CONCLUSION: This paper demonstrates the presence of pfhrp2 and pfhrp3 genes in three P. falciparum RDT-negative infections and reviews the possible reasons for non-detection of HRP2/3 antigens in a non-endemic setting. It highlights the emergence of falsely negative rapid diagnostic tests in a non-endemic setting and draws attention on the risk of missing malaria cases with low parasitaemia infections using the RDT plus microscopy-based strategy currently recommended by French authorities. The relevance of a novel diagnostic scheme based upon a LAMP assay is discussed.

Changes in the distribution of colonising and infecting Candida spp. isolates, antifungal drug consumption and susceptibility in a French intensive care unit: A 10-year study.

Monitoring fungal ecology and resistance to antifungal agents within intensive care units (ICU) is essential for the management of invasive fungal infections. Therefore, a retrospective descriptive study was carried in the ICU of Nimes University Hospital, France, from 2007 to 2016. As the majority of invasive fungal infections in ICU are caused by Candida species, the study objectives were to describe Candida species distribution, to assess candidaemia incidence and to monitor the antifungal drug susceptibility of Candida isolates and the consumption of antifungal agents. Among the recorded invasive Candida infections (n=244), 43% were intra-abdominal and 22% bloodstream infections. Candida albicans was the most frequent species (55.8%), followed by Candida glabrata (14.1%), Candida tropicalis (10%), Candida parapsilosis (8%) and Candida krusei (5.3%). Candidaemia incidence was 4.49 per 1000 admissions. The mean consumption of antifungal agents was of 170.5 defined daily doses (DDD) for 1000 hospital days (HD) per year. Changes in antifungal drug consumption were observed, with an increased use of echinocandins (from 17.96 DDD/1000 HD in 2007 to 48.76 DDD/1000 HD in 2016), and the total treatment cost tripled during the study period. No significant change in fungal ecology or in the emergence of resistant species was observed; indeed, only 1.1% of isolates presented an unusual resistance to antifungal agents.

Rapid emergence of FKS mutations in Candida glabrata isolates in a peritoneal candidiasis.

We report a rapid acquisition of echinocandin resistance after 12 days of micafungin treatment, without prior exposure, in a patient with peritoneal candidiasis due to C. glabrata. Isolates recovered before and after treatment were compared by multilocus sequence typing. Results of antifungal susceptibility testing and FKS mutations were reported. The interest of repeating antifungal susceptibility testing for echinocandin molecules during the treatment is discussed and a strategy to research FKS mutations proposed.

Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia.

Pneumonia due to Pneumocystis jirovecii (PCP) is a frequent infection among HIV-positive or other immunocompromised patients. In the past several years, PCR on pulmonary samples has become an essential element for the laboratory diagnosis of PCP. Nevertheless, very few comparative studies of available PCR assays have been published. In this work, we evaluated the concordance between four real-time PCR assays, including three commercial kits, AmpliSens, MycAssay, and Bio-Evolution PCR, and an in-house PCR (J. Fillaux et al. 2008, J Microbiol Methods 75:258-261, doi:http://dx.doi.org/10.1016/j.mimet.2008.06.009), on 148 pulmonary samples. The results showed concordance rates ranging from 81.6% to 96.6% (kappa, 0.64 to 0.93). Concordance was excellent between three assays: the in-house assay, AmpliSens, and the MycAssay PCR (kappa, >0.8). The performances of these PCR assays were also evaluated according to the classification of the probability of PCP (proven, probable, possible, or no final diagnosis of PCP) based on clinical and radiological signs as well as on the direct examination of bronchoalveolar lavage samples. In the proven PCP category, Pneumocystis jirovecii DNA was detected with all four assays. In the probable PCP category, the in-house PCR, AmpliSens, and the MycAssay PCR were positive for all samples, while the Bio-Evolution PCR failed to detect Pneumocystis jirovecii DNA in two samples. In the possible PCP category, the percentage of positive samples according to PCR varied from 54.5% to 86.4%. Detection of colonized patients is discussed. Finally, among the four evaluated PCR assays, one was not suitable for colonization detection but showed good performance in the proven and probable PCP groups. For the three other assays, performances were excellent and allowed detection of a very low fungal burden.