Epidemiology and management of tinea capitis in France: a 6-year nationwide retrospective survey.

Tinea capitis (TC) is still a frequent dermatophytosis in France, both autochthonous and imported. A nationwide retrospective survey was performed and a total of 4,395 TC cases were recorded within 36 French mycology laboratories during a 6-year period. TC is a disease that occurs in childhood with 85% of the cases occurring before 10 years old and 94% before the age of 15. Anthropophilic origin was predominant with 779 cases of Trichophyton tonsurans (32.6%), 738 cases of Trichophyton soudanense/T. violaceum (31%), and 445 cases of Microsporum audouinii (19.2%). Of note, T. tonsurans represents more than 80% of the cases in the French West Indies (Martinique and Guadeloupe). By contrast, zoophilic species were less prevalent with mainly M. canis (10.3%) confirming the shift from zoophilic to anthropophilic species observed in many centers during the last decades. During this survey, diagnosis methods were also collected. Most labs had a classical process for the diagnosis: microscopic direct examination associated to cultures on Sabouraud and Sabouraud-cycloheximide media (incubated between 25±5°C for 2 to 3 weeks) in all laboratories. Identification of the causal dermatophyte was performed by microscopic and macroscopic examination of the cultures in 100% of the labs, with various specific culture media available when fructification was insufficient (mainly malt or potato-dextrose agar, or Borelli medium). New techniques were also implemented with the introduction of MALDI-TOF mass spectrometry identification in more than two third of the labs, and molecular identification available if necessary in half of the labs.

A total of 4,395 tinea capitis cases were recorded within 36 French mycology laboratories during a 6-year period. An anthropophilic origin was predominant with 33%, 31% and 18.8% of cases due to Trichophyton tonsurans, T. soudanense/T. violaceum and Microsporum audouinii, respectively.

Emergence of Difficult-to-Treat Tinea Corporis Caused by Trichophyton mentagrophytes Complex Isolates, Paris, France.

We describe 7 cases of extensive tinea corporis since 2018 in a hospital in Paris, France, after failure to cure with terbinafine. Molecular analysis indicated Trichophyton mentagrophytes internal transcribed spacer type VIII (T. indotineae). This strain, which has mutations in the squalene epoxidase gene, is spreading on the Indian subcontinent.

Increased sensitivity of a new commercial reverse transcriptase-quantitative PCR for the detection of Pneumocystis jirovecii in respiratory specimens.

Optimal sensitivity to detect low Pneumocystis loads is of importance to take individual and collective measures to avoid evolution towards Pneumocystis pneumonia and outbreaks in immunocompromised patients. This study compares two qPCR procedures, a new automated RTqPCR using the GeneLEAD VIII extractor/thermocycler (GLVIII; ∼2.2 h workflow) and a previously validated in-house qPCR assays (IH; ∼5 h workflow) both targeting mtSSU and mtLSU for detecting P. jirovecii in 213 respiratory samples. GLVIII was found to be more sensitive than IH, detecting eight more specimens. Bland-Altman analysis between the two procedures showed a Cq bias of 1.17 ± 0.07 in favor of GLVIII. LAY SUMMARY: The fungus Pneumocystis needs to be detected early in respiratory samples to prevent pneumonia in immunocompromised hosts. We evaluated a new commercial RTqPCR on 213 respiratory samples to detect Pneumocystis and found it more sensitive and faster than our routine sensitive in-house qPCR assay.

Different repartition of the cryptic species of black aspergilli according to the anatomical sites in human infections, in a French University hospital.

Black aspergilli of the section Nigri are rarely differentiated at the species level when originating from human specimens. We wondered whether some cryptic species could be more frequently observed in some clinical entities. We analyzed the 198 black isolates consecutively collected from the external ear canal (EEC; n = 66), respiratory specimens (n = 99), and environment (n = 33). DNA was extracted and species identification was performed upon the partial calmodulin gene. We identified by decreasing frequency: Aspergillus welwitschiae (35.3%), Aspergillus tubingensis (34.3%), Aspergillus niger (17.2%), Aspergillus luchuensis (4%), Aspergillus aff. welwitschiae (3%), Aspergillus neoniger (2%), Aspergillus piperis (1.5%), Aspergillus japonicus (1.0%), Aspergillus vadensis (0.5%), and two Aspergillus tubingensis clade (1%). The distribution of the three main cryptic species was different between EEC and respiratory samples (P < 0.001) but not different between respiratory and environment samples (P = 0.264). Aspergillus welwitschiae was more often associated with EEC (54.5%), whereas A. tubingensis and A. niger were predominant in respiratory samples (39.4 and 26.3%, respectively). Among the 99 respiratory isolates, only 10 were deemed responsible for probable invasive aspergillosis, of which six were mixed with other pathogenic moulds. This study shows the interest to pursue the identification of clinical isolates in the Aspergillus section Nigri to unravel some specific associations with clinical entities. The association of A. welwitschiae with otomycosis suggests a better fitness to infect/colonize the ear canal. Also, members of the Aspergillus section Nigri alone are rarely responsible for invasive aspergillosis. LAY SUMMARY: We analyzed 198 black aspergilli isolates collected from different samples type to determine their species identification. We observe a different distribution of species between ear canal and respiratory samples (P < 0.001), suggesting a better fitness of A. welwitschiae to infect the ear canal.

Prospective comparison of (1,3)-beta-D-glucan detection using colorimetric and turbidimetric assays for diagnosing invasive fungal disease.

Serum (1→3)-ß-D-glucan (BDG), an pan fungal antigen, is detected in some invasive fungal diseases (IFDs). We compared two commercial kits, the Fungitell assay (FA) (colorimetric) and the Wako assay (WA) (turbidimetric) over a 4-month period to prospectively test 171 patients who mainly had hematological conditions (62%) and experienced episodes (n = 175) of suspected invasive fungal infection. Twenty-three episodes due to BDG-producing fungi were diagnosed (pneumocystosis, n = 12; invasive aspergillosis, n = 5; candidemia, n = 3; invasive fusariosis, n = 2; hepato-splenic candidiasis, n = 1).Both assays provided similar areas under the curves (AUC = 0.9). Using the optimized positivity thresholds (≥120 pg/ml for FA and ≥ 4 pg/ml for WA), the sensitivity and specificity were 81.8% (CI95: 61.5-92.7), 94.8% (90.1-97.3) for FA and 81.8% (61.5-92.7), 95.4% (90.9-97.8) for WA. Negative predictive value was 97.3% (93.3-99.0) for both tests. If the manufacturer's positivity threshold (≥11 pg/ml) was applied, the WA sensitivity decreased to 50%. Among 71 patients with bacterial infections, 21.1% were FA-positive and 5.6% were WA-positive (p < 10-2).The WA performed similarly as compared to the FA with an optimized cutoff value. The WA is a single sample test that is clinically relevant when a prompt therapeutic decision is required. LAY SUMMARY: Serum (1→3)-ß-D-glucan testing is dominated by two kits including Fungitell colorimetric assay (FA) and the Wako turbidimetric assay (WA). We compared them prospectively and observed that they both perform similarly when selecting their optimal threshold (≥120 pg/ml for FA and ≥ 4 pg/ml for WA).

Imported leishmaniasis in travelers: a 7-year retrospective from a Parisian hospital in France.

BACKGROUND: Leishmaniases are regularly seen in non-endemic areas due to the increase of international travels. They include cutaneous leishmaniases (CL) and mucocutaneous (MC) caused by different Leishmania species, and visceral leishmaniases (VL) which present with non-specific symptoms. METHODS: We reviewed all consecutive leishmaniasis cases seen between September 2012 and May 2020. The diagnostic strategy included microscopy after May-Grünwald-Giemsa staining, a diagnostic quantitative PCR (qPCR) assay, and species identification based on sequencing of the cytochrome b gene. RESULTS: Eighty-nine patients had a definitive leishmaniasis diagnosis. Nine patients had VL with Leishmania infantum. Eighty patients had CL. Twelve patients acquired CL after trips in Latin America (7 Leishmania guyanensis, 2 Leishmania braziliensis, 2 Leishmania mexicana, and 1 Leishmania panamensis). Species could be identified in 63 of the 68 CLs mainly after travel in North Africa (59%) with Leishmania major (65%), Leishmania tropica/killicki (24%), and L. infantum (11%), or in West Sub-Saharan Africa (32%), all due to L. major. The median day between appearance of the lesions and diagnosis was 90 [range 60-127]. CONCLUSIONS: Our diagnostic strategy allows both positive diagnoses and species identifications. Travelers in West Sub-Saharan Africa and North Africa should be better aware of the risk of contracting leishmananiasis.

Invasive Infections with Nannizziopsis obscura Species Complex in 9 Patients from West Africa, France, 2004-20201.

Nine new human invasive infections caused by the keratinophilic fungi Nannizziopsis obscura have been reported in France since 2004. The patients had variable clinical manifestations, had frequent dissemination, were mainly T-cell immunocompromised, and all originated from sub-Saharan West Africa. Before collection of the isolates, the etiologies of these infections were often misidentified, underscoring the extent of microscopic and cultural polymorphisms. All isolates but 1 had low MICs for the 8 antifungal drugs tested. When treated, patients received mainly azole therapy. Two of 7 patients with a known outcome died. We performed multilocus sequence analysis of N. obscura clinical strains and several strains of Nannizziopsis spp. isolated from reptiles. The human strains were clearly differentiated from the animal strains. N. obscura might be endemic to West Africa and responsible for undetected infections, which might become reactivated when immunosuppression occurs. N. obscura infection is probably underestimated because only sequencing enables proper identification.

Entamoeba histolytica DNA Detection in Serum from Patients with Suspected Amoebic Liver Abscess.

Amoebic liver abscess (ALA) is regularly seen in travelers or immigrants from tropical countries. The diagnosis relies on liver imaging that is not specific and on the detection of anti-Entamoeba histolytica antibodies, which cannot distinguish an acute from a former infection. We tested whether E. histolytica DNA detection in serum can improve the diagnosis of ALA. We retrospectively tested available serum samples taken from patients with ALA and non-ALA space-occupying lesions of the liver between 1 January 2010 and 30 November 2019. The quantitative PCR (qPCR) assay tested specifically amplifies a 99-bp fragment of the small-subunit rRNA gene of E. histolytica We analyzed 76 samples (19 ALA and 57 non-ALA samples) collected from 76 patients within 6 days before and after the antiamoebic treatment. Serum qPCR results were positive for 17 of 19 ALA patients and for none of the control patients (sensitivity and specificity were 89.5% and 100%, respectively). In parallel, the sensitivity and specificity of anti-E. histolytica antibody detection were 100% and 89.5%, respectively. The two false-negative qPCR results may be explained by ongoing metronidazole treatment or a possible persistent seropositivity that was not caused by the current liver abscess. Additionally, of 12 abscess pus aspirates (5 from ALA and 7 from non-ALA samples) tested, 5 were qPCR positive and 7 were qPCR negative, with concordant results in serum. This study demonstrates that cell-free circulating E. histolytica DNA can be detected in serum in ALA. This may assist in both positive diagnoses and treatment efficacy follow-up. The origin of this circulating DNA remains to be investigated.

The Fungal PCR Initiative's evaluation of in-house and commercial Pneumocystis jirovecii qPCR assays: Toward a standard for a diagnostics assay.

Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal PCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicenter and monocenter evaluation of PCP qPCR assays was performed. For the multicenter study, 16 reference laboratories from eight different countries, performing 20 assays analyzed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads, respectively). The monocenter study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, reverse transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation.

Time to and differential time to blood culture positivity for assessing catheter-related yeast fungaemia: A longitudinal, 7-year study in a single university hospital.

BACKGROUND: Time to positivity (TTP) and differential time to positivity (DTTP) between central and peripheral blood cultures are commonly used for bacteraemia to evaluate the likelihood of central venous catheter (CVC)-related bloodstream infection. Few studies have addressed these approaches to yeast fungaemia. OBJECTIVES: This study aimed to evaluate TTP and DTTP to assess CVC-related yeast fungaemia (CVC-RYF). PATIENTS/METHODS: We retrospectively analysed the results from 105 adult patients with incident fungaemia, with CVC removed and cultured, collected from 2010 to 2017. The bottles were incubated in a BioMérieux BacT/ALERT 3D and kept for at least 5 days. RESULTS: Of the 105 patients included, most were oncology patients (85.7%) and had of long-term CVC (79.6%); 32 (30.5%) had a culture-positive CVC (defined as CVC-RYF) with the same species as in blood culture, and 69.5% had culture-negative CVC (defined as non-CVC-RYF, NCVC-RYF). Candida albicans represented 46% of the episodes. The median TTP was statistically different between CVC-RYF and NCVC-RYF (16.8 hours interquartile range (IQR) [9.7-28.6] vs 29.4 hours [IQR 20.7-41.3]; P = .001). A TTP <10 hours had the best positive likelihood ratio (21.5) for CVC-RYF, although the sensitivity was only 28%. DTTP was available for 52 patients. A DTTP >5 hours had a sensitivity of 100% and a specificity of 71% for CVC-RYF. CONCLUSIONS: Since the median TTP was 17 hours and the most performing DTTP >5 hours, these delays are too long to take a decision in the same operational day. More rapid methods for detecting infected catheters should be tested to avoid unnecessary CVC withdrawal.

Outbreak-Causing Fungi: Pneumocystis jirovecii.

Pneumocystis jirovecii pneumonia (PCP) is an important cause of morbidity in immunocompromised patients, with a higher mortality in non-HIV than in HIV patients. P. jirovecii is one of the rare transmissible pathogenic fungi and the only one that depends fully on the host to survive and proliferate. Transmissibility among humans is one of the main specificities of P. jirovecii. Hence, the description of multiple outbreaks raises questions regarding preventive care management of the disease, especially in the non-HIV population. Indeed, chemoprophylaxis is well codified in HIV patients but there is a trend for modifications of the recommendations in the non-HIV population. In this review, we aim to discuss the mode of transmission of P. jirovecii, identify published outbreaks of PCP and describe molecular tools available to study these outbreaks. Finally, we discuss public health and infection control implications of PCP outbreaks in hospital setting for in- and outpatients.

Outcome and potentially modifiable risk factors for candidemia in critically ill burns patients: A matched cohort study.

BACKGROUND: Patients with extensive burns are at risk of developing candidemia. OBJECTIVES: To identify potentially modifiable risk factors and outcomes of candidemia in critically ill burns patients. PATIENTS AND METHODS: Retrospective matched cohort study including adult burns patients. Patients who developed candidemia were matched with burns patients with Candida spp colonisation and sepsis or septic shock without candidemia in a ratio of 1:3 (same severity scores and colonisation index). Univariate and multiple regression analyses were performed. RESULTS: Of 130 severely burned patients with Candida spp colonisation and at least one episode of sepsis or septic shock, 14 were diagnosed with candidemia. In the candidemia group, patients had a median (IQR) total burns surface area (TBSA) of 57 (38-68)%, SAPSII of 43 (36-58) and ABSI of 11 (8-13). Multiple regression analysis showed that only duration of prior antibiotic therapy was independently associated with candidemia. ICU mortality was higher in the candidemia group (71% vs 35% [P = 0.02]). The log-rank test for 28-day mortality comparing patients with candidemia treated with an empirical strategy vs a curative strategy did not reach significance (P = 0.056). CONCLUSIONS: Burns patients having received recent antibiotherapy have a higher risk of candidemia. Antifungal strategies did not influence outcome in this series.

Candiduria in kidney transplant recipients: Is antifungal therapy useful?

A French single-centre retrospective study between 2010 and 2014 was undertaken to assess candiduria's incidence in kidney transplant recipients (KTR), and the use and impact of antifungal treatment on outcome. Candiduria was defined as a urine culture with ≥103  cfu/mL of Candida species. Candiduria clearance, severe complications and death rates were estimated by Kaplan-Meier methods and the effect of treatment by Cox models. 52/1223 (4.3%) KTR had ≥1 episode of candiduria, 42 (81%) were females, 18 (35%) had diabetes, with an incidence of 2.3/100 person-year of follow-up. Candiduria was asymptomatic in 51 (98%) patients. Candida glabrata was the most frequent pathogen identified. Overall fungal clearance rate was 89%. Antifungal therapy was initiated in only 14 episodes (12%), according to guidelines. Three patients (6%) developed severe complications in the first 2 weeks after transplantation, and 8 (15%) died. Antifungal treatment had no impact on candiduria clearance (HR, 0.6; 95% CI, 0.3-1.1; P = .10), on recurrence rate (HR, 0.5; 95% CI, 0.1-2.3; P = .41) and on the risk of severe complications or death (HR, 1.1; 95% CI, 0.3-4.8; P = .89). Candiduria is rare and usually asymptomatic among KTR. Candiduria management in the immediate post-transplant period deserves careful attention.

Continuous increase of Trichophyton tonsurans as a cause of tinea capitis in the urban area of Paris, France: a 5-year-long study.

Tinea capitis (TC) is a highly contagious fungal infection of the scalp due to dermatophytes in children. To obtain information on the epidemiology of TC in the urban area of Paris, we analysed the microbiological results of 3090 patients seen with suspected TC from October 2010 to September 2015 at Saint Louis hospital, Paris, France. A peak of TC was observed in 3-6 year-old children, followed by a progressive decrease until 16 years of age. Of the 1311 positive cultures, 95% (1246) yielded one of the three anthropophilic species [Trichophyton tonsurans (33.5%), Trichophyton soudanense (38.3%), or Microsporum audouinii (28.2%)]. When considering one TC case per family, we observed a significant increase of T. tonsurans (P = .018) during these 5 years. The increase was more pronounced (P = .0047) in patients of West-African descent (n = 666), and was at the expense of M. audouinii and T. soudanense. On the other hand, the Caribbean patients (n = 85) remained predominantly (72.9%) infected by T. tonsurans. Our results show a better virulence of T. tonsurans over other species as already reported. Since T. tonsurans has not been reported in Africa, the infection of patients of West-African descent probably took place in the Paris area by exchanges with Caribbean patients. This increase of TC due to T. tonsurans was observed in the context of griseofulvin being the only licensed paediatric treatment for TC in France, which should deserve reappraisal because terbinafine may be more efficacious.

Detection of Circulating Mucorales DNA in Critically Ill Burn Patients: Preliminary Report of a Screening Strategy for Early Diagnosis and Treatment.

BACKGROUND: Invasive wound mucormycosis (IWM) is associated with an extremely poor outcome among critically ill burn patients. We describe the detection of circulating Mucorales DNA (cmDNA) for the early diagnosis of IWM in those patients and report the potential value of detecting cmDNA for treatment guidance. METHODS: Severely ill burn patients admitted to our tertiary referral center between October 2013 and February 2016 were included. Retrospective plasma samples were tested for the presence of cmDNA by quantitative real-time polymerase chain reaction (qPCR). Patients were then prospectively screened twice a week, and liposomal amphotericin-B therapy initiated based on a positive qPCR. The primary endpoint was the time between cmDNA detection and standard diagnosis. Secondary endpoints were the time from cmDNA detection and treatment initiation and mortality. RESULTS: Seventy-seven patients (418 samples) were included. The average age was 46 (28-60) years, abbreviated burn severity index was 8 (7-10), and simplified acute physiology score was 33 (23-46). The total body surface area was 33% (22%-52%). cmDNA was detected 11 (4.5-15) days before standard diagnosis. The in-hospital mortality was 62% for patients with IWM and 24% for those without (P = .03). The mortality due to IWM was 80% during period A and 33% during period B (P = .46). CONCLUSIONS: This study suggests that the detection of cmDNA allows earlier diagnosis of IWM in severely ill burn patients and earlier initiation of treatment. Further studies are needed to confirm the impact of earlier treatment initiation on patient outcome.

Burden of respiratory viruses in patients with acute respiratory failure.

Respiratory viruses (RVs) are ubiquitous pathogens that represent a major cause of community-acquired pneumonia and chronic pulmonary diseases exacerbations. However, their contribution to acute respiratory failure events requiring intensive care unit admission in the era of rapid multiplex molecular assay deserves further evaluation. This study investigated the burden of viral infections in non immunocompromised patients admitted to the intensive care unit for acute respiratory failure using a multiplex molecular assay. Patients were investigated for RVs using immunofluoresence testing and a commercial multiplex molecular assay, and for bacteria using conventional culture. Half the patients (34/70, 49%) had a documented RVs infection. No other pathogen was found in 24 (71%) patients. Viral infection was detected more frequently in patients with obstructive respiratory diseases (64% vs. 29%; P = 0.0075). Multiplex molecular assay should be considered as an usefull diagnostic tool in patients admitted to the intensive care unit with acute respiratory failure, especially those with acute exacerbations of chronic obstructive pulmonary disease and asthma.

Evaluation of the inoculum effect of new antibiotics against carbapenem-resistant enterobacterales.

OBJECTIVES: New antibiotics have been developed to treat multidrug-resistant Enterobacterales. We evaluated the impact of the inoculum size on minimal inhibitory concentrations (MICs) of recently commercialized antibiotics. METHODS: We focused on 40 clinical carbapenemase-producing Enterobacterales and evaluated the impact of the inoculum size on the MICs to cefiderocol and to new ß-lactams/ß-lactamase inhibitors (ceftolozane-tazobactam, ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam) at usual and high inocula (105 and 107 CFU/mL, respectively). RESULTS: At usual inoculum, 15% were resistant to cefiderocol (n = 6), 30% to meropenem-vaborbactam (n = 12), 42.5% to ceftazidime-avibactam (n = 17), 55% to imipenem-relebactam (n = 22), and 90% to ceftolozane-tazobactam (n = 36). At higher inoculum, a switch from susceptible to resistant category was observed for 88% (n = 30/34; CI, 71.6-96.2), 75% (n = 3/4; CI, 21.9-98.7), 72% (n = 13/18; CI, 46.4-89.3), 50% (n = 14/28; CI, 31.1-68.9), and 8.7% (n = 2/23; CI, 1.5-29.5) isolates regarding cefiderocol, ceftolozane-tazobactam, imipenem-relebactam, meropenem-vaborbactam, and ceftazidime-avibactam, respectively. DISCUSSION: Cefiderocol and meropenem-vaborbactam were the most efficient against carbapenemase-producing Enterobacterales at usual inoculum. When increasing inoculum to 107 CFU/mL, all of the molecules were impacted, particularly cefiderocol and imipenem-relebactam, while others, such as ceftazidime-avibactam, remain mildly affected. Our in vitro results deserved to be confirmed in vivo.

Cryptococcus neoformans meningitis in kidney transplant recipients: A diagnostic and therapeutic challenge.

Cryptococcosis is the third most common invasive fungal infection in solid organ transplant recipients. We describe three cases of neuro-meningeal cryptococcosis occurring among kidney transplant (KT) patients, and discuss the diagnostic and therapeutic challenges in this context. Median time from KT to infection was 6 months [range: 3-9]. The most common clinical manifestations at diagnosis were fever (2/3), headache (2/3), and confusion (2/3); none had extra-neurological involvement. CrAg was positive in all cases at diagnosis both in serum and cerebrospinal fluid (CSF). For two patients, analysis of previous samples showed that CrAg was detected in plasma up to 4 weeks before diagnosis. All patients received induction treatment with liposomal amphotericin-B (L-AmB) and flucytosine for a median duration of 10 days [range: 7-14], followed by fluconazole maintenance therapy. Acute kidney injury secondary to L-AmB therapy was observed in only one case, but all patients had a tacrolimus overdose following initiation of maintenance therapy due to drug-drug interactions between fluconazole and tacrolimus. Among KTR, early detection of Cryptococcus meningitis using serum CrAg is possible. Close monitoring of renal function during treatment is essential due to the nephrotoxicity of L-AmB, but also drug-drug interactions between fluconazole and calcineurin inhibitors.

Evaluation of a New Histoplasma spp. Quantitative RT-PCR Assay.

Laboratory diagnosis of histoplasmosis is based on various methods, including microscopy, culture, antigen, and DNA detection of Histoplasma capsulatum var. capsulatum or Histoplasma capsulatum var. duboisii. To improve sensitivity of existing real-time quantitative PCR (qPCR) assays, we developed a new RT-qPCR assay that allows amplification of whole nucleic acids of Histoplasma spp. validated on suspected cases. The limit of detection was 20 copies, and the specificity against 114 fungal isolates/species was restricted to Histoplasma spp. Whole nucleic acids of 1319 prospectively collected consecutive samples from 907 patients suspected of having histoplasmosis were tested routinely between May 2015 and May 2019 in parallel with standard diagnostic procedures performed in parallel. Forty-four had proven histoplasmosis attributable to H. capsulatum var. capsulatum (n = 40) or H. capsulatum var. duboisii (n = 4) infections. The results of RT-qPCR were positive in 43 of 44 patients (97.7% sensitivity) in at least one specimen. Nine of 863 cases (99% specificity) were RT-qPCR positive and therefore classified as possible cases. RT-qPCR was positive in 13 of 30 (43.3%) blood samples tested in proven cases. A positive RT-qPCR result in blood was significantly associated with H. capsulatum var. capsulatum progressively disseminated histoplasmosis with a positive RT-qPCR result in 92.3% of the immunocompromised patients with disseminated disease. This new Histoplasma RT-qPCR assay enabling amplification of H. capsulatum var. capsulatum and H. capsulatum var. duboisii is highly sensitive and allows the diagnosis of histoplasmosis advantageously from blood and bronchoalveolar lavage fluid.

Comparison of MultiLocus Sequence Typing (MLST) and Microsatellite Length Polymorphism (MLP) for Pneumocystis jirovecii genotyping.

Pneumocystis jirovecii is an atypical fungus responsible for severe respiratory infections, often reported as local outbreaks in immunocompromised patients. Epidemiology of this infection, and transmission risk emphasises the need for developing genotyping techniques. Currently, two methods have emerged: Multilocus Sequence typing (MLST) and microsatellite length polymorphism (MLP). Here we compare an MLST strategy, including 2 nuclear loci and 2 mitochondrial loci, with an MLP strategy including 6 nuclear markers using 37 clinical PCR-positive respiratory samples from two French hospitals. Pneumocystis jirovecii MLST and MLP provided 30 and 35 different genotypes respectively. A higher number of mixed infections was detected using MLP (48.6% vs. 13.5% respectively; p = 0.002). Only one MLP marker (STR279) was statistically associated with the geographical origin of samples. Haplotype network inferred using the available genotypes yielded expanded network for MLP, characterized by more mutational steps as compared to MLST, suggesting that the MLP approach is more resolutive to separate genotypes. The correlation between genetic distances calculated based on MLST and MLP was modest with a R 2 value = 0.32 (p < 0.001). Finally, both genotyping methods fulfilled important criteria: (i) a discriminatory power from 97.5% to 99.5% and (ii) being quick and convenient genotyping tools. While MLP appeared highly resolutive regarding genotypes mixture within samples, using one genotyping method rather than the other may also depend on the context (i.e., MLST for investigation of suspected clonal outbreaks versus MLP for population structure study) as well as local facilities.