Rapid and Accurate Diagnosis of Dermatophyte Infections Using the DendrisCHIP® Technology.

Dermatophytosis is a superficial fungal infection with an ever-increasing number of patients. Culture-based mycology remains the most commonly used diagnosis, but it takes around four weeks to identify the causative agent. Therefore, routine clinical laboratories need rapid, high throughput, and accurate species-specific analytical methods for diagnosis and therapeutic management. Based on these requirements, we investigated the feasibility of DendrisCHIP® technology as an innovative molecular diagnostic method for the identification of a subset of 13 pathogens potentially responsible for dermatophytosis infections in clinical samples. This technology is based on DNA microarray, which potentially enables the detection and discrimination of several germs in a single sample. A major originality of DendrisCHIP® technology is the use of a decision algorithm for probability presence or absence of pathogens based on machine learning methods. In this study, the diagnosis of dermatophyte infection was carried out on more than 284 isolates by conventional microbial culture and DendrisCHIP®DP, which correspond to the DendrisCHIP® carrying oligoprobes of the targeted pathogens implicated in dermatophytosis. While convergence ranging from 75 to 86% depending on the sampling procedure was obtained with both methods, the DendrisCHIP®DP proved to identify more isolates with pathogens that escaped the culture method. These results were confirmed at 86% by a third method, which was either a specific RT-PCR or genome sequencing. In addition, diagnostic results with DendrisCHIP®DP can be obtained within a day. This faster and more accurate identification of fungal pathogens with DendrisCHIP®DP enables the clinician to quickly and successfully implement appropriate antifungal treatment to prevent the spread and elimination of dermatophyte infection. Taken together, these results demonstrate that this technology is a very promising method for routine diagnosis of dermatophytosis.

Evaluation of the Performance of the Novodiag® Stool Parasites Assay for the Detection of Intestinal Protozoa and Microsporidia.

OBJECTIVES: We aimed to assess the performance of the Novodiag® Stool Parasites (NSP) assay in the diagnosis of the most common intestinal protozoan and microsporidia infections. METHODS: A panel of 167 selected stool samples was retrospectively analysed with the NSP assay and compared to routine microscopy and qPCR methods for the detection of pathogenic protozoa and microsporidia. RESULTS: Whereas specificity was high for all protozoa and microsporidia, NSP sensitivity was strongly dependent on the comparative method used as reference. When compared to microscopic methods, NSP sensitivity was high (96.7 to 100%) for Blastocystis hominis, Entamoeba histolytica and Cyclospora cayetanensis but was lower for Giardia intestinalis (85.2%) and ≤50% for Cystoisospora belli and Dientamoeba fragilis. In comparison to conventional qPCR, the NSP assay demonstrated lower sensitivity characteristics dependent on parasite loads, reaching 60 to 70% for G. intestinalis, D. fragilis, Cryptosporidium spp. and E. histolytica. Sensitivity was 100% for Enterocytozoon bieneusi, but none of the five samples containing Encephalitozoon spp. were detected. CONCLUSIONS: The overall performance of the NSP assay in the diagnosis of gastrointestinal protozoa and microsporidia seems to be better than or equivalent to that observed with microscopic methods but inferior to that obtainable with classical targeted qPCR.

Fatal mucormycosis and aspergillosis coinfection associated with haemophagocytic lymphohistiocytosis: A case report and literature review.

Invasive mould infections are life-threatening and mainly occur in immunocompromised patients. Whereas aspergillosis is described during haemophagocytic lymphohistiocytosis (HLH), only a few cases of concomitant mucormycosis with HLH have been reported. Here, we present an uncommon coinfection of mucormycosis and aspergillosis associated with HLH probably due to a varicella zoster virus (VZV) viraemia which was unresponsive to triple antifungal therapy (liposomal amphotericin B combined with isavuconazole and caspofungin). A review of the cases of mucormycosis with HLH showed that this uncommon association was always lethal and underscored the relevance of screening for mould infections in patients with HLH.

A Clustered Case Series of Mucorales Detection in Respiratory Samples from COVID-19 Patients in Intensive Care, France, August to September 2021.

While COVID-19-associated pulmonary aspergillosis is now well described in developed countries, COVID-19-associated mucormycosis (CAM) has seemed to remain quite rare in Europe. A retrospective study was performed between March 2020 to September 2021 among COVID-19 adult patients in the intensive care unit (ICU) at Toulouse Hospital (Southern France). PCR screening on respiratory samples, which target Aspergillus or Mucorales DNA, were performed, and the number of fungal detections was evaluated monthly during the study period. During the 19 months of the study, 44 (20.3%) COVID-19 ICU patients had a positive PCR for Aspergillus, an overall rate in keeping with the incidence of ICU COVID-19 patients. Ten patients (7.1%) had a positive Mucorales PCR over the same period. Surprisingly, 9/10 had a positive Mucor/Rhizopus PCR in August-September 2021, during the fourth Delta SARS-CoV-2 variant wave. Epidemic investigations have identified a probable environmental cause linked to construction works in the vicinity of the ICU (high levels of airborne spores due to the mistaken interruption of preventive humidification and summer temperature). Even if CAM are apparently rare in Europe, a cluster can also develop in industrialised countries when environmental conditions (especially during construction work) are associated with a high number of COVID-19 patients in the ICU.

New Insights into Blood Circulating Lymphocytes in Human Pneumocystis Pneumonia.

The host lymphocyte response is decisive in Pneumocystis pneumonia (PCP) pathophysiology but little is known of the specific roles of lymphocyte subpopulations in this fungal infection. Peripheral NK, NKT, B, TCD4+ and TCD8+ subpopulations were compared by immunophenotyping between 20 patients diagnosed with PCP (PCP(+)] and 20 uninfected immunosuppressed patients (PCP(-)). Among PCP(+) subjects, the lymphocyte populations were also compared between surviving and deceased patients. Low B cell count (<40 cells/µL) was more frequent in PCP(+) than in PCP(-) patients (p = 0.03), while there was no difference for the TCD4 count. Among the PCP(+) group, the 7 deceased patients had lower Th1 (p = 0.02) and Tc1 (p = 0.03) populations, higher Th2 response (p = 0.03), higher effector TCD8 (p < 0.01), lower central memory TCD8 (p = 0.04) and reduced NK cells (p = 0.02) compared with the 13 survivors. Th1/Th2 ratio < 17, CD8 Tc1 < 44%, effector TCD8 < 25%, central memory TCD8 < 4%, NK cells < 50 cells/µL and total lymphocytes < 0.75 G/L were associated with a higher risk of mortality (p = 0.003, p = 0.007, p = 0.0007, p = 0.004, p = 0.02 and p = 0.019, respectively). The traditional analysis of TCD4 and TCD8 populations may be insufficient in the context of PCP. It could be completed by using B cells to predict the risk of PCP, and by using lymphocyte subpopulations or total lymphocyte count, which are easy to obtain in all health care facilities, to evaluate PCP prognosis.

Misdiagnosis of imported falciparum malaria from African areas due to an increased prevalence of pfhrp2/pfhrp3 gene deletion: the Djibouti case.

Following the diagnosis of a falciparum malaria case imported from Djibouti and not detected by a pfHRP2-based rapid diagnostic test (RDT), we investigated the prevalence of the pfhrp2/pfhrp3-deleted parasites in Djibouti using 378 blood samples collected between January and May 2019, from Djiboutian patients with suspected malaria. Malaria diagnosis by quantitative PCR confirmed the presence of Plasmodium falciparum for 20.9% (79/378) samples while RDTs did not detect HRP2 antigen in 83.5% (66/79) of these samples. Quantitative PCRs targeting the pfhrp2/pfhrp3 genes confirmed the absence of both genes for 86.5% of P. falciparum strains. The very large number (86.5%) of falciparum parasites lacking the pfhrp2/pfhrp3 genes observed in this study, now justifies the use of non-HRP2 alternative RDTs in Djibouti. In this area and in most countries where HRP2-based RDTs constitute the main arsenal for falciparum malaria diagnosis, it is important to implement a systematic surveillance and to inform biologists and clinicians about the risk of malaria misdiagnosis. Further investigations are needed to better understand the mechanism of selection and diffusion of the pfhrp2/pfhrp3-deleted parasites.

Optimization of MALDI-ToF mass spectrometry for yeast identification: a multicenter study.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) is routinely used in mycology laboratories to rapidly identify pathogenic yeasts. Various methods have been proposed to perform routine MS-based identification of clinically relevant species. In this study, we focused on Bruker technology and assessed the identification performance of three protocols: two pretreatment methods (rapid formic acid extraction directly performed on targets and full extraction using formic acid/acetonitrile in tubes) and a direct deposit protocol that omits the extraction step. We also examined identification performance using three target types (ground-steel, polished-steel, and biotargets) and two databases (Bruker and online MSI [biological-mass-spectrometry-identification application]) in a multicenter manner. Ten European centers participated in the study, in which a total of 1511 yeast isolates were analyzed. The 10 centers prospectively performed the three protocols on approximately 150 yeast isolates each, and the corresponding spectra were then assessed against two reference spectra databases (MSI and Bruker), with appropriate thresholds. Three centers evaluated the impact of the targets. Scores were compared between the various combinations, and identification accuracy was assessed. The protocol omitting the extraction step was inappropriate for yeast identification, while the full extraction method yielded far better results. Rapid formic acid extraction yielded variable results depending on the target, database and threshold. Selecting the optimal extraction method in combination with the appropriate target, database and threshold may enable simple and accurate identification of clinically relevant yeast samples. Concerning the widely used polished-steel targets, the full extraction method still ensured better scores and better identification rates.

Real-time PCR for diagnosis of imported schistosomiasis.

BACKGROUND: The diagnosis of schistosomiasis currently relies on microscopic detection of schistosome eggs in stool or urine samples and serological assays. The poor sensitivity of standard microscopic procedures performed in routine laboratories, makes molecular detection methods of increasing interest. The aim of the study was to evaluate two in-house real-time Schistosoma PCRs, targeting respectively S. mansoni [Sm] and S. haematobium [Sh] in excreta, biopsies and sera as potential tools to diagnose active infections and to monitor treatment efficacy. METHODS: Schistosoma PCRs were performed on 412 samples (124 urine, 86 stools, 8 biopsies, 194 sera) from patients with suspected schistosomiasis, before anti-parasitic treatment. Results were compared to microscopic examination and serological assays (enzyme-linked immunosorbent assay (ELISA), indirect haemagglutination (HA) and Western Blot (WB) assay). RESULTS: Compared to microscopy, PCRs significantly increased the sensitivity of diagnosis, from 4% to 10.5% and from 33.7% to 48.8%, for Sh in urine and Sm in stools, respectively. The overall sensitivity of PCR on serum samples was 72.7% and reached 94.1% in patients with positive excreta (microscopy). The specificity of serum PCR was 98.9%. After treatment, serum PCR positivity rates slowly declined from 93.8% at day 30 to 8.3% at day 360, whereas antibody detection remained positive after 1 year. CONCLUSION: Schistosoma PCRs clearly outperform standard microscopy on stools and urine and could be part of reference methods combined with WB-based serology, which remains a gold standard for initial diagnosis. When serological assays are positive and microscopy is negative, serum PCRs provide species information to guide further clinical exploration. Biomarkers such as DNA and antibodies are of limited relevance for early treatment monitoring but serum PCR could be useful when performed at least 1 year after treatment to help confirm a cured infection.

Performance of seven commercial automated assays for the detection of low levels of anti-Toxoplasma IgG in French immunocompromised patients.

BACKGROUND: Immunocompromised patients are at high risk for the development of severe toxoplasmosis from tissue cyst reactivation, the most frequently, or from recently acquired acute infections. Knowledge of serologic status is therefore crucial. Screening for toxoplasmosis is sometimes performed while patients are already immunocompromised and have a low or even undetectable IgG titer by routine automated enzyme immunoassays. The aim of this study was to assess the sensitivity and specificity of seven reagents for the detection of low levels of IgG. Sera from 354 patients were collected and analysed. RESULTS: Elecsys® offered the best analytic performances, superior to those of Architect® and Platelia®, which were superior to those of Access II® and TGS TA®. Vidas II® and Liaison II® reagents exhibited poor analytical performances in this cohort. For Elecsys®, Platelia® and Architect®, new thresholds for the grey zone and positive zone have been defined to improve the sensitivity of these reagents while maintaining excellent specificity. CONCLUSIONS: Commercialized assays for toxoplasmosis screening are not suitable for IgG low-level detection in patients without adapting the supplier thresholds to avoid false negative results and risk generalized toxoplasmosis.

TITLE: Performance de sept tests automatisés du commerce pour la détection de faibles taux d'IgG anti-Toxoplasma chez des patients immunodéprimés français. ABSTRACT: Contexte : Les patients immunodéprimés courent un risque élevé de développer une toxoplasmose grave résultant de la réactivation de kystes tissulaires, le plus souvent, ou d'infections aiguës récemment contractées. La connaissance du statut sérologique est donc cruciale. Le dépistage de la toxoplasmose est parfois effectué chez des patients déjà immunodéprimés et dont le titre en IgG est faible, voire indétectable, par les immunoessais enzymatiques automatisés de routine. Le but de cette étude était d'évaluer la sensibilité et la spécificité de sept réactifs pour la détection de faibles taux d'IgG. Les sérums de 354 patients ont été recueillis et analysés. Résultats : Elecsys® offre les meilleures performances analytiques, supérieures à celles d'Architect® et de Platelia®, supérieures à celles d'Access II® et de TGS TA®. Les réactifs Vidas II® et Liaison II® ont présenté des performances analytiques médiocres dans cette cohorte. Pour Elecsys®, Platelia® et Architect®, de nouveaux seuils pour la zone grise et la zone positive ont été définis pour améliorer la sensibilité de ces réactifs tout en maintenant une excellente spécificité. Conclusions : Les tests commercialisés pour le dépistage de la toxoplasmose ne conviennent pas à la détection de faibles taux d'IgG chez les patients sans adaptation des seuils des fournisseurs pour éviter les résultats faux négatifs et le risque de toxoplasmose généralisée.

Time before anti-Toxoplasma IgG seroconversion detection by 7 commercial assays in French pregnant women.

We assessed the ability to early detect a toxoplasmic seroconversion between 1 immunoblot (LDBIO II®) and 6 automated assays (TGS TA®, Architect®, Vidas II®, Liaison II®, Platelia®, and Elecsys®), comparing the time before anti-Toxoplasma gondii IgG detection during infection in pregnant women. From 2007 to 2015, 620 sera of 269 women were included. The median durations before positive IgG detection with Vidas II®, Liaison II®, Platelia®, and Elecsys® were significantly longer than Architect® with differential times from 11 to 28days (P<0.001). This time was significantly shortened by the use of LDBIO®, resulting in a saving of 13days (P<0.001). The detection of a positive rate of IgG with TGS TA® was as early as Architect® (P=0.105). The ability to early detect a toxoplasmic seroconversion is not equivalent between the assays and has to be considered when selecting the reagents to reduce the time to therapeutic intervention.

Prevalence of Plasmodium falciparum parasites resistant to sulfadoxine/pyrimethamine in pregnant women in Yaoundé, Cameroon: emergence of highly resistant pfdhfr/pfdhps alleles.

OBJECTIVES: To determine, 6 years after the adoption of intermittent preventive treatment of pregnant women with sulfadoxine/pyrimethamine (IPTp-SP) in Cameroon, (i) the polymorphism and prevalence of Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) gene mutations associated with sulfadoxine/pyrimethamine resistance and (ii) the consequences of sulfadoxine/pyrimethamine use in the selection of pfdhfr/pfdhps alleles. METHODS: pfdhfr and pfdhps genes from P. falciparum isolates collected in Yaoundé (Cameroon) from pregnant women with symptomatic malaria before taking IPTp-SP [SP- group (control) (n = 51)] or afterwards [SP+ group (n = 49)] were sequenced. RESULTS: The pfdhfr N51I, C59R, S108N triple mutant had a prevalence close to 100% (96/100) and no mutations at codons 50 and 164 were detected in either of the groups. The most frequent pfdhps mutation was A437G with a prevalence of 76.5% (39/51) in the SP- group, which was significantly higher in pregnant women who took sulfadoxine/pyrimethamine [95.9% (47/49)] (P = 0.012). Our study confirmed the presence of the pfdhps K540E mutation in Cameroon, but it remained rare. The prevalence of pfdhps A581G and A613S mutations had increased [5.9% (3/51) and 11.8% (6/51) in the control group, respectively] since the last studies in 2005. Surprisingly, the new pfdhps I431V mutation was detected, at a prevalence of 9.8% (5/51), and was found to be associated with other pfdhfr/pfdhps alleles to form an octuple N51I, C59R, S108N/I431V, S436A, A437G, A581G, A613S mutant. CONCLUSIONS: Significant changes were found in pfdhps polymorphism. In particular, we observed several parasites carrying eight mutations in pfdhfr/pfdhps genes, which are very susceptible to having a high level of resistance to sulfadoxine/pyrimethamine.

A complementary tool for management of disseminated Histoplasma capsulatum var. capsulatum infections in AIDS patients.

In South America, disseminated histoplasmosis due to Histoplasma capsulatum var. capsulatum (H. capsulatum), is a severe and frequent opportunistic infection in AIDS patients. In areas outside the USA where specific-Histoplasma antigen detection is not available, the diagnosis is difficult. With the galactomannan antigen (GM) detection, a test commonly used for invasive aspergillosis diagnosis, there is a cross-reactivity with H. capsulatum that can be helpful for the diagnosis of histoplasmosis. The aim of this study was to evaluate the GM detection for the diagnosis of disseminated histoplasmosis in AIDS patients. The performance of the GM detection was evaluated with serum collected in French Guiana where H. capsulatum is highly endemic. Sera from AIDS patients with disseminated histoplasmosis occurring from 2002 to 2009 and from control HIV-positive patients without histoplasmosis were tested with the GM detection and Histoplasma-specific antibody detection (IEP). In 39 AIDS patients with proven disseminated histoplasmosis, the sensitivity of the Histoplasma IEP was only 35.9% and was linked to the TCD4+ lymphocyte level. For the GM detection, the sensitivity (Se) was 76.9% and specificity (Sp) was 100% with the recommended threshold for aspergillosis diagnosis (0.5). The test was more efficient with a threshold of 0.4 (Se: 0.82 [95% CI: 0.66-0.92], Sp: 1.00 [95% CI: 0.86-1.00], LR+: >10, LR-: 0.18). This study confirms that the GM detection can be a surrogate marker for the diagnosis of disseminated histoplasmosis in AIDS patients in endemic areas where Histoplasma EIA is not available.

An extraction method of positive blood cultures for direct identification of Candida species by Vitek MS matrix-assisted laser desorption ionization time of flight mass spectrometry.

Candida spp. are an important cause of nosocomial bloodstream infections. Currently, complete identification of yeasts with conventional methods takes several days. We report here the first evaluation of an extraction method associated with the Vitek MS matrix-assisted laser desorption ionization time of flight mass spectrometry for direct identification of Candida species from positive blood cultures. We evaluated this protocol with blood cultures that were inoculated with reference and routine isolates (eight reference strains, 30 patients isolates and six mixed cultures containing two strains of different Candida species), or from patients with candidemia (28 isolates). This method performed extremely well (97% correct identification) with blood cultures of single Candida spp. and significantly reduced the time of diagnosis. Nevertheless, subculture remains indispensable to test fungal resistance and to detect mixed infections.