Tickborne Relapsing Fever Caused by Borrelia persica, Uzbekistan and Tajikistan

International audienc

Copy Number Variation of Mitochondrial DNA Genes in Pneumocystis jirovecii According to the Fungal Load in BAL Specimens

Pneumocystis jirovecii is an unculturable fungus and the causative agent of Pneumocystis pneumonia, a life-threatening opportunistic infection. Although molecular diagnosis is often based on the detection of mtLSU rRNA mitochondrial gene, the number of copies of mitochondrial genes had not been investigated. We developed and optimized six real-time PCR assays in order to determine the copy number of four mitochondrial genes (mtSSU rRNA, mtLSU rRNA, NAD1, and CYTB) in comparison to nuclear genome (DHPS and HSP70) and tested 84 bronchoalveolar fluids of patients at different stages of the infection. Unexpectedly, we found that copy number of mitochondrial genes varied from gene to gene with mtSSU rRNA gene being more represented (37 copies) than NAD1 (23 copies), mtLSU rRNA (15 copies) and CYTB (6 copies) genes compared to nuclear genome. Hierarchical clustering analysis (HCA) allowed us to define five major clusters, significantly associated with fungal load (p = 0.029), in which copy number of mitochondrial genes was significantly different among them. More importantly, copy number of mtLSU rRNA, NAD1, and CYTB but not mtSSU rRNA differed according to P. jirovecii physiological state with a decreased number of copies when the fungal load is low. This suggests the existence of a mixture of various subspecies of mtDNA that can harbor different amplification rates. Overall, we revealed here an unexpected variability of P. jirovecii mtDNA copy number that fluctuates according to P. jirovecii's physiological state, except for mtSSU that is the most stable and the most present mitochondrial gene.This work was supported by research project PI14CIII/00045 from the Spanish Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III. CV is supported by research fellowships from the Fondo de Investigaciones Sanitarias of the Spanish Ministry of Economy and Competitiveness (FI12/00095).S

Continuous Decline of Toxoplasma gondii Seroprevalence in Hospital: A 1997–2014 Longitudinal Study in Paris, France

Background: The protozoan Toxoplasma gondii presents a risk for reactivation of latent cysts in immunocompromised patients. Anti-T. gondii antibodies are therefore usually screened before chemotherapy or transplantation to propose prophylactic measures against this parasite. We analyzed the results obtained in our hospital to study the epidemiological trend of T. gondii infection.Methods: We collected all the anti-Toxoplasma antibody titers from January 1, 1997 to December 31, 2013 using the Platelia IgG ELISA assay (Bio-Rad). The results were classified as positive when titers reached a concentration of ≥10 UI/ml. Only the first result obtained at entry for each patient was considered. T. gondii seroprevalence was estimated using a multivariate logistic regression model accounting for age, sex, and year in which the sample was collected.Results: A total of 21,480 patient samples were analyzed. The seroprevalence continuously decreased over time, from 64.5% in 1997 to 54.7% in 2013 (i.e., an average of 1.3% per year, p < 0.001). The decrease was 5.0% per year for patients <20 years. After 2013, the model predicts that the seroprevalence would continuously decrease. We also observed a higher proportion of seropositive men than women in the 15- to 45-year-old group (58.5% versus 52.0%, p < 10-3).Conclusion: The overall seroprevalence of toxoplasmosis at our hospital showed an accelerating downward trend over 17 years. The reason for this continuous decline is likely associated with the lower parasite presence within meat. Thus, although young immunocompromised patients are increasingly less at risk of reactivation in the near future, older immunocompromised patients will remain at high risk of reactivation. The reasons of the higher prevalence in men remain to be explored

Importance of operational factors in the reproducibility of Aspergillus galactomannan enzyme immune assay

International audienceBACKGROUND: The Platelia Aspergillus Ag assay (Bio-Rad) is designed for detecting Aspergillus galactomannan (GM) and is widely used for diagnosing invasive aspergillosis but is hampered by variable occurrences of unreproducible positive results. Frequency and origin of these unreproducible results have not been formally studied. METHODS: Different technicians simultaneously performed four tests on 550 consecutive sera from adult patients (Test#1-Test#2 for extraction#1 and Test#3-Test#4 for extraction#2). The samples were classified as confirmed negative [all tests with GM optical density index (GM-ODI) \textless0.5], confirmed positive (all tests with GM-ODI ≥0.5), extraction unreproducible positive (Test#1 and Test#2 ODIs ≥0.5, and Test#3 and Test#4 GM-ODIs \textless0.5, or conversely), and ELISA unreproducible positive (only one test with GM-ODI ≥0.5). The samples with positive and negative GM-ODIs within the assay coefficient of variation values were classified as non-conclusive. Four similar additional tests were performed after ≤72h storage at 4 °C and a new GM test after 8 months at -20 °C. RESULTS: Five-hundred-twenty sera (94.5%) were confirmed negative, 15 (2.7%) confirmed positive, 4 (0.7%) extraction unreproducible positive, 6 (1.1%) ELISA unreproducible positive, and 5 (0.9%) non-conclusive. Upon retesting, the unreproducible positive results turned negative except for one which turned non-conclusive. The confirmed positive and non-conclusive had similar GM-ODIs (p\textgreater0.4) upon retesting after storage ≤72h at 4 °C (n = 20) or eight months at -20 °C (n = 17). CONCLUSIONS: Operational unreproducible positives represent 33% of the GM-positive results and a second sample evaluation appears mandatory to avoid useless investigations or treatments. When operational artifacts are excluded, GM remains stable at standard storage conditions

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

International audienceOptimal 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 hrs workflow) and a previously validated in-house qPCR assays (IH; ∼5 hrs workflow) both targeting mtSSU and mtLSU for detecting P. jirovecii in 213 respiratory samples. GLVIII was found to be more sensitive than IH, detecting 8 more specimens. Bland-Altman analysis between the two procedures showed a Cq bias of 1.17 ± 0.07 in favor of GLVIII. 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

Bronchial aspirate obtained during bronchoscopy yields increased fungal load compared to bronchoalveolar lavage fluid in patients at risk of invasive aspergillosis and Pneumocystis pneumonia

Bronchoalveolar lavage fluid (BALF) is a standard respiratory sample for diagnosing invasive fungal diseases like Pneumocystis pneumonia (PCP) and invasive pulmonary aspergillosis (IPA). However, procedural variations exist across medical centers and wards. This study aimed to compare the diagnostic potential of BALF and bronchial aspirate (BA) obtained during bronchoscopy in 173 patients suspected of fungal infections. A prospective observational study was conducted from April 2020 to November 2021. BALF and BA were collected during bronchoscopy and subjected to direct examination, fungal culture, Aspergillus fumigatus qPCR (AfqPCR), and Pneumocystis jirovecii qPCR (PjqPCR). Galactomannan detection was performed on BALF. Patients were classified based on established European Organization for Research and Treatment of Cancer (EORTC) criteria. Out of 173 patients, 75 tested positive for at least one test in BA or BALF. For Aspergillus, proportion of positive AfqPCR (14.5% vs. 9.2%; P < 0.0001) and fungal loads (Cq of 31.3 vs. 32.8; P = 0.0018) were significantly higher in BA compared to BALF. For Pneumocystis, fungal loads by PjqPCR was also higher in BA compared to BALF (Cq of 34.2 vs. 35.7; P = 0.003). BA only detected A. fumigatus and P. jirovecii in 12 (42.9%) and 8 (19.5%) patients, respectively. BA obtained during a BAL procedure can be a suitable sample type for increased detection of P. jirovecii and A. fumigatus by qPCR. The use of BA in diagnostic algorithms requires further investigation in prospective studies