Low Fungal Load Research Articles

DIVERSITY OF FEEDSTUFF MYCOBIOME IS DETERMINED BY FEEDSTUFF COMPOSITION AND CULTURING CONDITIONS

Fungal contamination of feedstuffs is a serious threat impacting domestic animals via the production of mycotoxins. This study surveys the mycobiome diversity of some Egyptian feedstuffs in relation to the type of feed and some culturing conditions viz. type of nutrient medium and incubation temperature. Poultry, rabbit and cattle feeds from the Nile Delta, Egypt were assayed for nutritional components and fungal contamination. Feed inoculants were incubated on potato dextrose agar (PDA) and malt extract agar (MEA) at 25 °C and 30 °C to specify the appropriate culturing conditions for each feedstuff mycobiome. Poultry feed was relatively rich in protein, insoluble sugars and fat; cattle feed was rich in soluble sugars, fiber, moisture and ash; while rabbit feed had moderate composition. PDA rather than MEA favored maximal species richness, species diversity and germ load of feedstuffs. Despite its low fungal count, rabbit feed has high species diversity and species richness but cattle feed exhibited the opposite pattern. The most dominant fungal division was the Ascomycota, particularly Aspergillus spp. and Monascus ruber, followed by Zygomycota with a rarity of Basidiomycota. Some fungal species were confined to certain feedstuffs. The three fungal divisions exhibited different preferences for incubation temperature and nutrient medium. The low moisture content and fungal load refer to the hygienic nature of feedstuffs. Nevertheless, the prevalence of Aspergillus species points to a potential mycotoxin production. Each feedstuff has a unique fungal community, which is further screened by the culturing conditions. The species diversity and species richness of a feedstuff might contrast its fungal count. This study pays attention towards the consequences of seeping of mycotoxins, produced by the feed-born fungi, into the food chain from domestic animals to the humans.

Zoonotic sporotrichosis in humans and domestic cats: Profile of notifications and spatio-temporal distribution in Southeastern Brazil between 2017 and 2023

The research addresses zoonotic sporotrichosis in Brazil, particularly caused by Sporothrix brasiliensis, highlighting its epidemiological severity. Transmission occurs through contact with sick animals, especially felines, and diagnosis in humans is challenging due to the low fungal load in the lesions. The study analyzed data from Information System for Notifiable Diseases (SINAN) and Zoonosis Surveillance Unit (UVZ) from January 2017 to March 2023, carried out in Contagem, Minas Gerais. Geospatial tools and statistical analysis revealed a significant increase in cases, peaking in 2021 for felines and 2022 for humans. The geospatial analysis highlighted areas of higher incidence, suggesting a correlation between human and feline populations. The research contributes to the understanding of sporotrichosis in Contagem, emphasizing the importance of integrated approaches for surveillance and control strategies, aiming to mitigate impacts on the local community.

Feline sporotrichosis: Characterization of cutaneous and extracutaneous lesions using different diagnostic methods.

Sporotrichosis is a mycotic infection of the cutaneous and subcutaneous tissues caused by Sporothrix spp. that can also cause extracutaneous manifestations. This study aimed to characterize cutaneous and extracutaneous sporotrichosis lesions in cats. Over 1 year, 102 cats rescued by the Zoonoses Control Center of Belo Horizonte, Brazil, euthanized with clinical suspicion of feline sporotrichosis were evaluated. After euthanasia, the animals were evaluated by macroscopic, cytological, histopathological, and immunohistochemistry (IHC) examinations; fungal culture; and polymerase chain reaction (PCR). Sporothrix infection was identified by at least one diagnostic technique in all cats (n = 102) evaluated by postmortem examination, including 26/28 cases (93%) evaluated by IHC, 66/90 cases (73%) evaluated by cytology, 70/102 cases (68.6%) evaluated by histopathology, and 62/74 cases (84%) evaluated by fungal culture. Two cats had positive results only by fungal culture. Cytology and histopathology examinations were effective in diagnosing sporotrichosis, although IHC was needed to confirm the diagnosis in cats with low fungal loads. Sporothrix brasiliensis was confirmed by the sequencing of 3 samples. Skin lesions were characterized mainly by pyogranulomatous to granulomatous dermatitis (frequently with subcutaneous inflammation) with different intensities of Sporothrix spp. yeast. Extracutaneous findings associated with sporotrichosis included rhinitis or rhinosinusitis, lymphadenitis, pneumonia, meningitis, periorchitis, conjunctivitis, and glossitis. Extracutaneous infections were observed in 74/102 cases, and a possible association between the chronicity of the disease and the higher pathogenicity of this fungal species in cats requires further investigation.

Quick versus Quantitative: Evaluation of Two Commercial Real-Time PCR Assays for the Detection of Pneumocystis jirovecii from Bronchoalveolar Lavage Fluids.

Two commercial real-time PCR assays for the detection of Pneumocystis jirovecii were compared, the quantitative RealStar P. jirovecii assay and the qualitative DiaSorin P. jirovecii assay, the latter of which can be used without nucleic acid extraction. Archived bronchoalveolar lavage (BAL) specimens (n = 66), previously tested by molecular methods, were tested by both assays, and the results were compared to the respective original result. The RealStar P. jirovecii assay demonstrated good positive percent agreement (PPA) (90% [95% confidence interval (CI), 72 to 97%]; 27/30) and negative percent agreement (NPA) (100% [95% CI, 88 to 100%]; 36/36) with the reference method. The DiaSorin P. jirovecii assay concordantly detected P. jirovecii in 19 of 24 positive BAL samples (PPA = 73% [95% CI, 52 to 88%]). All negative BAL samples gave concordant results (NPA = 100% [95% CI, 87 to 100%]; 34/34). Discordant results occurred mostly in samples with low fungal loads. In conclusion, the RealStar assay demonstrated good concordance with reference results, and the DiaSorin P. jirovecii assay performed well for negative BAL and positive BAL samples with P. jirovecii concentrations of greater than 260 copies/mL. IMPORTANCE Pneumonia, caused by the opportunistic fungus Pneumocystis jirovecii, poses a significant risk for immunocompromised individuals. Laboratory testing for P. jirovecii is progressively shifting toward the use of molecular tests such as real-time PCR; however, this is often performed at reference laboratories. Many frontline laboratories are looking into improving their service and reducing turnaround times for obtaining P. jirovecii results by bringing molecular P. jirovecii testing in-house. We evaluated and compared two commercial real-time PCR assays with different workflows for the detection of P. jirovecii from bronchoalveolar lavage specimens. The RealStar P. jirovecii assay requires nucleic acid extraction and provides a quantification of fungal load for positive samples. The DiaSorin P. jirovecii assay offers a simple workflow without nucleic extraction from patient samples and qualitative results. Results from this study provide valuable information on performance and workflow considerations for laboratories that wish to implement P. jirovecii molecular testing.

Reducing environmentally mediated transmission to moderate impacts of an emerging wildlife disease

Abstract Emerging infectious diseases have caused population declines and biodiversity loss. The ability of pathogens to survive in the environment, independent of their host, can exacerbate disease impacts and increase the likelihood of species extinction. Control of pathogens with environmental stages remains a significant challenge for conservation and effective management strategies are urgently needed. We examined the effectiveness of managing environmental exposure to reduce the impacts of an emerging infectious disease of bats, white‐nose syndrome (WNS). We used a chemical disinfectant, chlorine dioxide (ClO2), to experimentally reduce Pseudogymnoascus destructans, the fungal pathogen causing WNS, in the environment. We combined laboratory experiments with 3 years of field trials at four abandoned mines to determine whether ClO2 could effectively remove P. destructans from the environment, reduce host infection and limit population impacts. ClO2 was effective at killing P. destructans in vitro across multiple concentrations. In field settings, higher concentrations of ClO2 treatment were needed to sufficiently reduce viable P. destructans conidia in the environment. The reduction in the environmental reservoir at treatment sites resulted in lower fungal loads on bats compared to untreated control populations. Survival following treatment was also higher in little brown bats (Myotis lucifugus), and trended higher for tricolored bats (Perimyotis subflavus). Synthesis and applications. Our results highlight that targeted management of sources for environmental transmission can be an effective control strategy for wildlife disease. We found that successfully reducing pathogen in the environment decreased disease severity and increased survival, but required higher treatment exposure than was effective in laboratory experiments, and the effects varied among species. More broadly, our findings have implications for other emerging wildlife diseases with free‐living pathogen stages by highlighting how the degree of environmental contamination can have cascading impacts on hosts, presenting an opportunity for intervention.

Is the production of reactive oxygen and nitrogen species by macrophages associated with better infectious control in female mice with experimentally disseminated and pulmonary mucormycosis?

Different levels of resistance against Rhizopus oryzae infection have been observed between inbred (BALB/c) and outbred (Swiss) mice and are associated with the genetic background of each mouse strain. Considering that macrophages play an important role in host resistance to Rhizopus species, we used different infectious outcomes observed in experimental mucormycosis to identify the most efficient macrophage response pattern against R. oryzae in vitro and in vivo. For this, we compared BALB/c and Swiss macrophage activity before and after intravenous or intratracheal R. oryzae infections. The production of hydrogen peroxide (H2O2) and nitric oxide (NO) was determined in cultures of peritoneal (PMΦ) or alveolar macrophages (AMΦ) challenged with heat-killed spores of R. oryzae. The levels of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) were measured to confirm our findings. Naïve PMΦ from female BALB/c mice showed increased production of H2O2, TNF-α, and IL-10 in the presence of heat-killed spores of R. oryzae. Naïve PMΦ from female Swiss mice were less responsive. Naïve AMΦ from the two strains of female mice were less reactive to heat-killed spores of R. oryzae than PMΦ. After 30 days of R. oryzae intravenous infection, lower fungal load in spleen from BALB/c mice was accompanied by higher production of H2O2 by PMΦ compared with Swiss mice. In contrast, AMΦ from BALB/c mice showed higher production of NO, TNF-α, and IL-10 after 7 days of intratracheal infection. The collective findings reveal that, independent of the female mouse strain, PMΦ is more reactive against R. oryzae upon first contact than AMΦ. In addition, increased PMΦ production of H2O2 at the end of disseminated infection is accompanied by better fungal clearance in resistant (BALB/c) mice. Our findings further the understanding of the parasite-host relationship in mucormycosis.

Usefulness of ß-d-Glucan Assay for the First-Line Diagnosis of Pneumocystis Pneumonia and for Discriminating between Pneumocystis Colonization and Pneumocystis Pneumonia.

According to the immunodepression status, the diagnosis of Pneumocystis jirovecii pneumonia (PjP) may be difficult. Molecular methods appear very sensitive, but they lack specificity because Pj DNA can be detected in Pneumocystis-colonized patients. The aim of this study was to evaluate the value of a serum ß-d-Glucan (BDG) assay for the diagnosis of PjP in a large cohort of HIV-negative and HIV-positive patients, either as a first-line diagnostic test for PjP or as a tool to distinguish between colonization and PjP in cases of low fungal load. Data of Pj qPCR performed on bronchopulmonary specimens over a 3-year period were retrieved retrospectively. For each result, we searched for a BDG serum assay performed within ±5 days. Among the 69 episodes that occurred in HIV-positive patients and the 609 episodes that occurred in immunocompromised HIV-negative patients, we find an equivalent sensitivity of BDG assays compared with molecular methods to diagnose probable/proven PjP, in a first-line strategy. Furthermore, BDG assay can be used confidently to distinguish between infected and colonized patients using a 80 pg/mL cut-off. Finally, it is necessary to search for causes of false positivity to increase BDG assay performance. BDG assay represents a valuable adjunctive tool to distinguish between colonization and infection.

REPEATED SAMPLING OF WILD INDIVIDUALS REVEALS OPHIDIOMYCES OPHIDIICOLA INFECTION DYNAMICS IN A PENNSYLVANIA SNAKE ASSEMBLAGE.

Ophidiomyces ophidiicola is an emerging fungal pathogen associated with infections in snakes across North America. Although documented in Pennsylvania, O. ophidiicola has not been found at Powdermill Nature Reserve (PNR) in southwestern Pennsylvania, where the snake assemblage has been studied since 2002 and several species have recently declined. We surveyed for O. ophidiicola and putative ophidiomycosis at PNR. We screened five species of free-ranging, wild snakes (n=34) for suspected ophidiomycosis by visually checking for dermatitis and swabbing for the presence of O. ophidiicola DNA. We found a moderate prevalence of snakes with skin lesions (n=15) but a low prevalence of snakes with O. ophidiicola DNA in traditional PCR assays (n=2). Both positive snakes belonged to the same species and only one presented with lesions. When quantitative PCR screens were performed on duplicate swabs, 19 snakes were positive for O. ophidiicola DNA, with positive individuals in two species. Mark-recapture methods revealed seasonal variability in disease dynamics for sampled snakes. One individual presented with less than five skin lesions and tested negative in May 2020, had more than five lesions with a high fungal DNA load in June 2020, and no lesions with a low fungal DNA load in July 2020. We also found that snakes sampled from under the same cover object at the same time either all tested positive or all negative, including one instance involving two species. Our results underscore the value of using multiple screening techniques for O. ophidiicola surveillance and repeated sampling of individuals to understand the dynamics of ophidiomycosis in wild populations as compared to single method and single timepoint approaches.

Development of a Droplet Digital Polymerase Chain Reaction for Sensitive Detection of Pneumocystis jirovecii in Respiratory Tract Specimens.

Background: Pneumocystis jirovecii is a human-specific opportunistic fungus that causes Pneumocystis pneumonia (PCP), a life-threatening opportunistic lung infection that affects immunocompromised patients. P. jirovecii colonization may be linked to the transmission of the infection. The detection of P. jirovecii in immunocompromised patients is thus especially important. The low fungal load and the presence of PCR inhibitors limit the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of P. jirovecii in specimens. Droplet digital PCR (ddPCR), however, presents a methodology that allows higher sensitivity and accuracy. Here, we developed a ddPCR method for detecting P. jirovecii DNA in respiratory specimens, and evaluated its sensitivity against qPCR.Materials and Methods: One bronchoalveolar fluid (BALF) sample each was collected from 82 patients with potential PCP to test the presence of P. jirovecii DNA using both ddPCR and qPCR, and samples with inconsistent results between the two methods were further tested by metagenomic next generation sequencing (mNGS). In addition, 37 sputum samples from 16 patients diagnosed with PCP, as well as continuous respiratory tract specimens from nine patients with PCP and treated with sulfonamides, were also collected for P. jirovecii DNA testing using both ddPCR and qPCR.Results: ddPCR and qPCR gave the same results for 95.12% (78/82) of the BALF samples. The remaining four specimens tested positive using ddPCR but negative using qPCR, and they were found to be positive by mNGS. Detection results of 78.37% (29/37) sputum samples were consistent between ddPCR and qPCR, while the other eight samples tested positive using ddPCR but negative using qPCR. The P. jirovecii load of patients with PCP decreased to undetectable levels after treatment according to qPCR, but P. jirovecii was still detectable using ddPCR.Conclusions: ddPCR was more sensitive than qPCR, especially at detecting low-pathogen-load P. jirovecii. Thus, ddPCR represents a useful, viable, and reliable alternative to qPCR in P. jirovecii testing in patients with immunodeficiency.

Rapid Genomic Diagnosis of Fungal Infections in the Age of Next-Generation Sequencing.

Next-generation sequencing (NGS) technologies have recently developed beyond the research realm and started to mature into clinical applications. Here, we review the current use of NGS for laboratory diagnosis of fungal infections. Since the first reported case in 2014, >300 cases of fungal infections diagnosed by NGS were described. Pneumocystis jirovecii is the predominant fungus reported, constituting ~25% of the fungi detected. In ~12.5% of the cases, more than one fungus was detected by NGS. For P. jirovecii infections diagnosed by NGS, all 91 patients suffered from pneumonia and only 1 was HIV-positive. This is very different from the general epidemiology of P. jirovecii infections, of which HIV infection is the most important risk factor. The epidemiology of Talaromyces marneffei infection diagnosed by NGS is also different from its general epidemiology, in that only 3/11 patients were HIV-positive. The major advantage of using NGS for laboratory diagnosis is that it can pick up all pathogens, particularly when initial microbiological investigations are unfruitful. When the cost of NGS is further reduced, expertise more widely available and other obstacles overcome, NGS would be a useful tool for laboratory diagnosis of fungal infections, particularly for difficult-to-grow fungi and cases with low fungal loads.

Role of IL-36γ/IL-36R Signaling in Corneal Innate Defense Against Candida albicans Keratitis.

PurposeInterleukin (IL)-36 cytokines have been shown to play either beneficial or detrimental roles in the infection of mucosal tissues in a pathogen-dependent manner, but their involvement in fungal keratitis remains elusive. We herein investigated their expression and function in mediating corneal innate immunity against Candida albicans infection.MethodsGene expression in mouse corneas with or without C. albicans infection was determined by regular RT- and real-time (q)-PCR, Western blot analysis, ELISA or proteome profile assay. The severity of C. albicans keratitis was assessed using clinical scoring, bacterial counting, and myeloperoxidase (MPO) activity as an indicator of neutrophil infiltration. IL36R knockout mice and IL-33-specific siRNA were used to assess the involvement IL-33 signaling in C. albicans–infected corneas. B6 CD11c–DTR mice and clodronate liposomes were used to define the involvement of dendritic cells (DCs) and macrophages in IL-36R signaling and C. albicans keratitis, respectively.ResultsIL-36γ were up-regulated in C57BL6 mouse corneas in response to C. albicans infection. IL-36 receptor-deficient mice display increased severity of keratitis, with a higher fungal load, MPO, and IL-1β levels, and lower soluble sIL-1Ra and calprotectin levels. Exogenous IL-36γ prevented fungal keratitis pathogenesis with lower fungal load and MPO activity, higher expression of sIL-1Ra and calprotectin, and lower expression of IL-1β, at mRNA or protein levels. Protein array analysis revealed that the expression of IL-33 and REG3G were related to IL-36/IL36R signaling, and siRNA downregulation of IL-33 increased the severity of C. albicans keratitis. Depletion of dendritic cells or macrophages resulted in severe C. albicans keratitis and yet exhibited minimal effects on exogenous IL-36γ-induced protection against C. albicans infection in B6 mouse corneas.ConclusionsIL-36/IL36R signaling plays a protective role in fungal keratitis by promoting AMP expression and by suppressing fungal infection–induced expression of proinflammatory cytokines in a dendritic cell- and macrophage-independent manner.

β2 Integrin-Mediated Susceptibility to Paracoccidioides brasiliensis Experimental Infection in Mice.

The earliest interaction between macrophages and Paracoccidioides brasiliensis is particularly important in paracoccidioidomycosis (PCM) progression, and surface proteins play a central role in this process. The present study investigated the contribution of β2 integrin in P. brasiliensis-macrophage interaction and PCM progression. We infected β2-low expression (CD18low) and wild type (WT) mice with P. brasiliensis 18. Disease progression was evaluated for fungal burden, lung granulomatous lesions, nitrate levels, and serum antibody production. Besides, the in vitro capacity of macrophages to internalize and kill fungal yeasts was investigated. Our results revealed that CD18low mice infected with Pb18 survived during the time analyzed; their lungs showed fewer granulomas, a lower fungal load, lower levels of nitrate, and production of high levels of IgG1 in comparison to WT animals. Our results revealed that in vitro macrophages from CD18low mice slowly internalized yeast cells, showing a lower fungal burden compared to WT cells. The migration capacity of macrophages was compromised and showed a higher intensity in the lysosome signal when compared with WT mice. Our data suggest that β2 integrins play an important role in fungal survival inside macrophages, and once phagocytosed, the macrophage may serve as a protective environment for P. brasiliensis.

Diagnosis of Aspergillus Species in Paraffin-EmbeddedTissue with Low Fungal Loads.

Diagnosis of Aspergillus Species in Paraffin-EmbeddedTissue with Low Fungal Loads.

Low-temperature applied to rice seed storage: an efficient protection method against fungal contamination

Low-temperatures have long been thought to enhance seed health during storage. Here we tested the effect of low-temperature on the mycoflora associated with rice seeds. Seeds of the IRGA 423 and 424 cultivars were stored in refrigeration (8 and -50°C) or at room temperature (25 ± 10°C). Following storage (1, 45, and 90 days) was investigated the fungi associated with the seeds. We found that, for both cultivars, low-temperature stored seeds had a lower fungal load than the seeds at room temperature. After 90 days of storage, there is a decrease in its incidence rate average of more than 85% in the lower temperature (-50°C). All mycroflora was eliminated at 90 days except for Fusarium sp. The Trichoderma sp. was detected only in IRGA 423 seeds, whereas all other fungi were detected in the treatments in both cultivars. Only Bipolaris sp. was observed in seed-to-seedlings transmission analyses, in both cultivars, at 8°C, and was not detected at -50°C. Based on our results, we recommend low-temperature storage (at -50ºC) of rice seeds.

Variable Correlation between Bronchoalveolar Lavage Fluid Fungal Load and Serum-(1,3)-β-d-Glucan in Patients with Pneumocystosis—A Multicenter ECMM Excellence Center Study

Pneumocystis jirovecii pneumonia is a difficult invasive infection to diagnose. Apart from microscopy of respiratory specimens, two diagnostic tests are increasingly used including real-time quantitative PCR (qPCR) of respiratory specimens, mainly in bronchoalveolar lavage fluids (BAL), and serum β-1,3-d-glucan (BDG). It is still unclear how these two biomarkers can be used and interpreted in various patient populations. Here we analyzed retrospectively and multicentrically the correlation between BAL qPCR and serum BDG in various patient population, including mainly non-HIV patients. It appeared that a good correlation can be obtained in HIV patients and solid organ transplant recipients but no correlation can be observed in patients with hematologic malignancies, solid cancer, and systemic diseases. This observation reinforces recent data suggesting that BDG is not the best marker of PCP in non-HIV patients, with potential false positives due to other IFI or bacterial infections and false-negatives due to low fungal load and low BDG release.

Serum (1,3)-Beta-d-Glucan has suboptimal performance for the diagnosis of Pneumocystis jirovecii pneumonia in cancer patients and correlates poorly with respiratory burden as measured by quantitative PCR

ObjectiveNon-HIV immunocompromised patients with Pneumocystis jirovecii pneumonia (PCP) have lower fungal load than those with AIDS, potentially affecting the accuracy of diagnostic biomarkers. Therefore, we investigated the performance of serum (1,3)-Beta-d-Glucan (BDG) in conjunction with quantitative Pneumocystis jirovecii PCR (qPCR) in non-HIV cancer patients. MethodsWe reviewed records of non-HIV cancer patients and classified them as definite, probable, or possible PCP cases, according to clinicoradiological features, microscopy findings, and qPCR results in bronchoscopy specimens. We evaluated the diagnostic performance of serum BDG and its correlation with qPCR results. ResultsWe identified 101 PCP patients (73 definite/probable, 28 possible) and 74 controls. Correlation of BDG and qPCR was low among all 101 qPCR-positive patients (Spearman's = 0.38) and in definite/probable PCP cases (Spearman's = 0.18). Considering all qPCR-positive patients, BDG showed consistently low sensitivity at different cutoffs. Among definite/probable cases, the diagnostic accuracy of BDG remained poor, yet slightly improved with high qPCR thresholds (AUC = 0.86 at ≥2000 DNA copies/mL). BDG had a low PPV but excellent NPV across different qPCR and BDG cutoffs. ConclusionsBDG and qPCR levels correlate poorly in non-HIV cancer patients with PCP. BDG diagnostic performance is suboptimal but a negative test may be useful to rule out PCP in this population.

Quantification of Pneumocystis jirovecii: Cross-Platform Comparison of One qPCR Assay with Leading Platforms and Six Master Mixes

Diagnosis of Pneumocystis jirovecii pneumonia relies on nucleic acid quantification in respiratory samples. Lack of standardization among molecular assays results in significant differences among assays/centers. To further promote standardization, we compared four thermocyclers and six master mixes for the detection of P. jirovecii. Whole nucleic acid (WNA) was extracted from broncho-alveolar lavages. Positive and negative sample extracts were pooled to get enough homogeneous materials. Three master mixes were tested to detect DNA by qPCR (D1, D2, and D3), and three to detect WNA by reverse transcriptase qPCR (W1, W2, and W3) manufactured by Roche, Eurogentec, Applied Biosystem, Invitrogen and Thermofischer Scientific. Experiments were performed on four thermocyclers (Roche LightCycler 480, Qiagen Rotor-Gene Q, Applied Biosystem ABI7500, and QuantStudio). Comparison of quantitative cycle (Cq) values between the methods targeting WNA versus DNA showed lower Cq values for WNA, independently of thermocycler and master mix. For high and low fungal loads, ∆Cq values between DNA and WNA amplification were 6.97 (±2.95) and 5.81 (±3.30), respectively (p < 0.0001). Regarding DNA detection, lower Cqs were obtained with D1 compared to D2 and D3, with median ∆Cq values of 2.6 (p = 0.015) and 2.9 (p = 0.039) respectively. Regarding WNA detection, no mix was superior to the others. PCR efficiency was not significantly different according to the qPCR platform (p = 0.14). This study confirmed the superiority of WNA over DNA detection. A calibration method (e.g., an international standard) for accurate comparative assessment of fungal load seems necessary.

Molecular diagnosis of Pneumocystis pneumonia in immunocompromised patients.

Pneumocystis pneumonia (PCP) is a frequent opportunistic infection associated with a high mortality rate. PCP is of increasing importance in non-HIV immunocompromised patients, who present with severe respiratory distress with low fungal loads. Molecular detection of Pneumocystis in broncho-alveolar lavage (BAL) has become an important diagnostic tool, but quantitative PCR (qPCR) needs standardization. Despite a high negative predictive value, the positive predictive value of qPCR is moderate, as it also detects colonized patients. Attempts are made to set a cut-off value of qPCR to discriminate between PCP and colonization, or to use noninvasive samples or combined strategies to increase specificity. It is easy to set a qPCR cut-off for HIV-infected patients. In non-HIV IC patients, a gain in specificity could be obtained by combining strategies, that is, qPCR on BAL and a noninvasive sample, or qPCR and serum beta-1,3-D-glucan dosage.

Genotyping Pneumocystis jirovecii: Impacting Our Understanding of Interhuman Transmission

Pneumocystis jirovecii is an atypical fungus transmitted via interhuman airborn contact. This fungus is exclusively associated with humans and almost each individual has encountered it at least once before reaching the age of two. Pneumocystis jirovecii can be responsible for spontaneously resolutive symptoms in infants and for severe pneumocystis pneumonia (PCP) in immunocompromised patients. The increase of the population of immunocompromised patients would probably lead to rising of PCP infections in the next decades. With the advance of molecular technologies and Polymerase Chain Reaction (PCR) assays, the detection of low fungal load has been improved and several physiopathological questions has raised. For instance, is PCP due to reactivation of a colonizing strain or acquisition of a new one? Are the strains in PCP and non-PCP patients similar? Are outbreaks due to a specific strain? Most of these questions can be addressed using genotyping, which must be directly performed on clinical specimens because of the absence of reliable culture methods. In this review we will resume the main typing methods developed for P. jirovecii characterization, with their pros and cons, in insisting on the typing of the low fungal loads, a subject rarely underlined. Secondarily, we will present the main results obtained and the importance to include these patients with low fungal load in the analyses because of their possible role as reservoirs and their impact on P. jirovecii transmission. Eventually, we will open the debate on systematic treatment of the patients with low fungal load not only to prevent full-blown PCP, but also to block transmission chain.

10.21926/obm.genet.1804049

Pneumocystis jirovecii pneumonia (PcP) remains a major cause of respiratory illness among immunocompromised patients. PcP is difficult to diagnose, in particular in non-HIV-infected patients due to the lack of specific clinical data associated. Since P. jirovecii could not be cultivated for many years, microscopic visualization of cystic or trophic forms in respiratory specimens based on cytochemical or immunofluorescence staining are the standard procedures to identify this fungus. Polymerase chain reaction (PCR)-based methodologies have been developed to overcome the low sensitivity of microscopy in respiratory specimens especially those with low fungal load, and in non-HIV-infected patients. Real-time quantitative PCR is the only format suitable for a quantitative diagnosis and these results have been used to differentiate PcP (high fungal load) from carriage/colonization (low fungal load), however, it is inconclusive with limited results in intermediate fungal loads. New strategies based on measurement of blood biomarkers could be a way to perform PcP diagnosis non-invasively. Several studies explored the usefulness of candidate serum biomarkers, such as (1-3)-β-D-Glucan, Krebs von den Lungen-6 antigen, lactate dehydrogenase or S-adenosylmethionine, with the former presenting the most promising results. More recently, approaches based on the detection of specific anti-P. jirovecii antibodies in patients’ sera are showing promising results that could enable a faster and inexpensive screening/diagnosis of this opportunistic infectious disease, helping better-tailored therapeutic interventions, the disease control and retrenchment to healthcare systems.