Diagnostic Significance of Fruiting Bodies in Pathology Specimens: A Series of 13 Patients.

Culture is the gold standard, while potassium hydroxide mount is simplest technique used for diagnosis of fungal pathogens. Histopathological examination is the only definitive means to identify certain uncultivable fungi. To analyse role of histopathological examination and potassium hydroxide (KOH) mount for diagnosing fungal infections by correlating them with culture. In this nine year retrospective study, all biopsy specimens submitted for microbiological examination were included. Histopathological examination of biopsies of cases with positive microbiological findings on either KOH mount or culture was carried out. Any discrepancy between histopathology interpretation and microbiology KOH or culture results, taking culture as the gold standard, were noted. Open Epi software was used for statistical analysis. Comparisons between groups were made by using the chi-square test. A p-value < 0.05 was considered statistically significant. Cohen's Kappa coefficient (κ) was calculated as a measure of agreement between different variables. Concurrent pathology specimen could be obtained in 70 samples positive for fungal elements in either KOH or culture. Thirty-two cases were positive for fungi in culture, of which 16 were correctly identified by histopathological examination. Histopathological examination was strongly associated with culture result. KOH mount was in good agreement with positive culture result for yeast. Eleven culture negative but KOH and histopathology positive cases included seven samples with hyphae suggestive of zygomycosis, and two cases of rhinosporidiosis. Allergic mucin was strongly associated with Aspergillus species. KOH mount and detection of allergic mucin on histopathological examination were found to be excellent complementary tools for diagnosing Aspergillus species. Necrosis was highly specific for fungal growth in culture and had good positive predictive value. We advocate using histopathology, culture and KOH examination in an integral manner to avoid potential lapses in patient management.

Concomitant infection with Aspergillus species and cervical squamous cell carcinoma in the female genital tract is a rare occurrence and attributed to the opportunistic nature of infection in the immunocompromised state due to the underlying malignancy. The contamination of smears with Aspergillus species should be excluded. The diagnosis of Aspergillus species infection along with squamous cell carcinoma was established on cervicovaginal pap smears in a 62-year-old female presented to gynecological clinic with complaints of stress urinary incontinence. Speculum examination revealed first-degree cervical descent. Smears showed features of squamous cell carcinoma along with fungal spores and fruiting body with hyphae of Aspergillus species. The presence of fruiting bodies and hyphae of Aspergillus species with coexisting squamous cell carcinoma is rare in routine pap smears. True infection needs to be distinguished from contamination by Aspergillus species. Early diagnosis can be established on routine cervicovaginal Pap smear examination.

ABSTRACTEndobronchial ultrasound (EBUS) guided fine needle aspiration is a modality for diagnosing lung nodules as well as fungal organisms. One of the most frequently isolated fungal organisms from immunocompromised patients or immunocompetent patients with severe COPD is Aspergillus spp., which is characterized by septate hyphae with progressive dichotomous branching. However, this finding is not entirely specific to Aspergillus spp. since it can be seen in non‐Aspergillus fungi. Nonetheless, the presence of fruiting bodies, background inflammation, and in correlation with the clinical and radiologic findings are supportive of Aspergillus infection. This case report describes a 73‐year‐old male smoker with severe COPD who presented with multiple lung nodules and hilar lymphadenopathy. An EBUS guided fine needle aspiration of the largest left lung cavitary nodule showed the presence of uniformly sized, septate hyphae with progressive dichotomous branching. There were randomly distributed fruiting bodies composed of a smooth stalk, a swollen vesicle at the terminal end of the stalk, and a single row of phialides occupying the upper two‐thirds of the vesicle. The background showed marked acute inflammation and singly dispersed conidia (spores). The GMS stain highlighted the presence of fruiting bodies, dichotomous branching septate hyphae, and singly dispersed conidia. The Fungal PCR result confirmed the presence of Aspergillus fumigatus. Hence, the presence of fruiting bodies is extremely helpful in the identification of the Aspergillus spp. over the other non‐Aspergillus fungi. While species identification is often performed in culture, the cytomorphology of the fruiting body could favor Aspergillus spp., which could facilitate prompt medical management.

In fungi, little is known about connections between volatile organic compound (VOC) formation and developmental stages that are amongst others triggered by fruiting-related genes (FRGs). We analysed the volatilomes of Schizophyllum commune during different developmental stages in a variety of FRG-deletion strains and wild-type strains. The deletion strains Δtea1Δtea1, Δwc-2Δwc-2 and Δhom2Δhom2 were unable to develop fruiting bodies, and Δfst4Δfst4 formed only rudimentary fruiting body structures. Early developmental stages of these strains were dominated by esters, including methyl 2-methylbutanoate, ethyl 2-methylbutanoate, isobutyl 2-methylpropionate, and 2-methylbutyl acetate, of which the last three were not found in the headspace (HS) of the wild-type samples. Compared to the wild type, in the HS of hom2con samples, that are able to form fruiting bodies, methyl 2-methylbutanoate was the most abundant substance at early stages (68–81% of the total peak area). In contrast to fruiting body forming strains, Δtea1Δtea1, Δwc-2Δwc-2, Δhom2Δhom2 and Δfst4Δfst4 showed less sesquiterpenes in the HS. However, the sesquiterpenes found in the HS of FRG-deletion strains, namely, (E)-nerolidol, δ-cadinene, L-calamenene, α-bisabolol and β-bisabolene, were not present in hom2con or wild-type strains that mainly formed fruiting bodies and barely mycelium. Several sesquiterpenes, including α-guaiene, chamigrene and γ-gurjunene, were only found in presence of fruiting bodies. Our results show remarkable connections between FRGs, fruiting body development and VOC production in S. commune, especially counting for sesquiterpenes. Future studies are needed to reveal whether FRGs directly regulates VOC formation or indirectly by changing the phenotype.

Host-microbial interactions in patients with chronic rhinosinusitis

To investigate the effect of topically applied proparacaine on bacterial and fungal culture results and to compare cytologic and culture results in patients with ulcerative keratitis. Corneal samples were collected from 33 dogs, 19 horses, and 12 cats with spontaneously arising ulcerative keratitis. Samples for bacterial (dogs, cats, horses) and fungal (horses) cultures were collected prior to and following application of 0.5% proparacaine or saline. All patients then received a topical anesthetic, and samples were collected for cytology. Frequency of cultivatable bacteria before (Swab 1) and after (Swab 2) application of proparacaine or saline was compared using Fisher's exact test. Homogeneity of culture and cytology results was assessed using McNemar's test. No difference was detected in number of animals from which bacteria were isolated from Swab 1 or Swab 2 for proparacaine (21/37 and 17/37, respectively) or saline (10/27 and 12/27, respectively). Small numbers prevented analysis of fungal culture results in horses between Swab 1 and Swab 2 for proparacaine (2/12 and 1/12, respectively) or saline (both, 1/8). Bacteria were isolated from 10 of 20 horses and detected cytologically in 3 of these; fungi were isolated from 3 of 20 horses and detected cytologically in 2 of these. Bacteria were detected more frequently using culture (31/64) than cytology (19/64). Proparacaine did not significantly alter bacterial or fungal culture results in cats, dogs, or horses; however, clinical significance warrants investigation. Culture and cytology provided complementary data; both should be performed to maximize organism detection in patients with ulcerative keratitis.

Low positivity rate and high percentage of nondermatophyte molds in an analysis of 35,257 fungal nail culture results from a United States national commercial laboratory, 2019-2022

BackgroundInvasive mold infections are challenging to diagnose and in part relies on fungal cultures. A large proportion of mold isolates are recovered on routine bacterial cultures in our medical center, thus we sought to define the utility of bacterial versus fungal cultures for isolation of mold from clinical specimens. MethodsRoutine bacterial and fungal culture results from wound, tissue, body fluid, and respiratory specimens from Jan 2019-Dec 2020 from Keck Medical Center of USC (Los Angeles, CA) were retrospectively reviewed. Cases were excluded if specimens were collected specifically for dermatophyte recovery or for blood culture. Cultures in which mold, including dimorphic fungi, were isolated were included in the evaluation. ResultsMold was isolated from 612 specimens from 408 patients, with recovery from 329 bacterial and 450 fungal cultures. Among the 329 bacterial cultures, fungal cultures were not requested in 119 (36.2%) while the remaining 210 had concurrent fungal cultures which recovered mold in 167 cases (79.5%). Of 450 fungal cultures recovering mold, a corresponding bacterial culture was performed in 445, isolating mold in 181 (38.8%) of these cases. Two or more molds were found in 28 fungal cultures and in 5 bacterial cultures. Of positive specimens with both fungal and bacterial cultures performed (n=488), mold was isolated in fungal cultures in 446 (91.4%) and in bacterial cultures in 209 (42.9%) (Table).Yield of molds in 488 specimens with concomitant bacterial and fungal culturesConclusionAlthough a significant number of molds are recovered in routine bacterial cultures, over half would be missed without concomitant fungal cultures. Conversely, recovery of clinically relevant mold species was optimal when both bacterial and fungal cultures were requested on a specimen. This may be related to increased specimen sampling and incubation conditions allowing for broader organism recovery. DisclosuresAll Authors: No reported disclosures

Enhanced recovery of fungal isolates from blood by using the Isolator system has been reported previously. We examined bacterial and fungal blood cultures during a 14-month period to determine if this enhanced recovery required a separate fungal culture and to determine the differential utility between a fungal blood culture and a routine bacterial culture. During this period, 84 of 5,196 (1.6%) fungal blood cultures and 170 of 25,702 (0.6%) bacterial blood cultures were positive for yeast or filamentous fungi. Thirty-seven positive fungal cultures, simultaneously collected, had correspondingly positive bacterial cultures. An additional 15 positive fungal cultures yielded isolates that had either been previously recovered from a bacterial culture or were recovered from a bacterial culture collected within 48 h. Of the 32 unpaired fungal cultures remaining, 5 were Candida albicans whose unique isolation was believed to be the result of specimen sampling variance rather than any enhanced recovery characteristics of fungal culture methods. Examination of patient data relating to the 27 remaining isolates (24 patients episodes) showed that only five fungal blood cultures (0.096% of all collected) had any impact on patient therapy decisions, and one of these was judged to be the cause of unnecessary therapy. Our data suggest that separate fungal cultures of blood are not cost-effective for those laboratories using the Isolator for routine blood cultures and furthermore may not be cost-effective for laboratories using automated broth systems that are comparable to the Isolator in recovery of fungi.

Clinical Utility of Routine Use of Fungal Blood Cultures

Objective To explore bronchoalveolar lavage fluid (BALF) positive rate in the etiological diagnosis of community-acquired pneumonia (CAP), healthcare associated pneumonia (HCAP) and immunocompromised hosts (ICH) in the real world of China, and to investigate influencing factors of the positive rate. Methods A retrospective study of 13 hospitals in 2014 hospitalized with pulmonary infection, all patients with community acquired pneumonia (CAP), HCAP or immunocompromised host pulmonary infection, and BALF examination was adopted within 1 weeks after admitted.The general data were collected including age, gender, underlying diseases, symptoms, the use of antibiotics, the severity of the disease, imaging manifestations, hospital mortality, length of stay in ICU, with the results of sputum culture were collected, and bronchoalveolar lavage (BAL) inspection time, BALF bacterial culture, fungal culture, nucleic acids of respiratory viruses, atypical pathogens (mycoplasma, chlamydia), Legionella and Pneumocystis jiroveci. Results 6 056 cases of pneumonia were collected, including 4 869 cases of CAP, 848 cases of HCAP, 338 cases of ICH pneumonia, 616 cases (10.2%) underwent BALF examination 1 week within admitting, 50 cases had severe pneumonia, 95% had used antibacterial drugs before BALF examination; 95% BALF were performed during antimicrobial treatment of the patient.The mean duration from the onset of the symptoms to the performance of BAL was 14(10, 24) d and the mean duration of the hospitalization before BAL was 4(2, 6) d. Untreated patients exhibited positive quantitative bacterial cultures (30.4%), as compared to patients with prior antimicrobial therapy having positive cultures (14.8%, P=0.041). The bacterial and fungal culture positive rates in severe patients were higher than the non-severe patients with CAP, HCAP (P<0.05). The positive rate of BALF bacterial and fungal culture was 32.3% in immunocompromised host (ICH), it was higher than that of HCAP patients (17.1%) and CAP patients (13.8%) (P<0.05).31 cases (37.8%) had adjust antibiotics according to the results of culture and drug sensitivity in 82 cases of pneumonia which pathogen was bacteria; All 17 cases targeted anti-infection drugs in the pneumonia which pathogen etiology were fungal or viral. Conclusions Immunocompromised host and severe pneumonia patients have positive rate of BALF pathogen.Identification of pathogens can guide the treatment of anti-infective drugs. Key words: Pneumonia; Bronchoalveolar lavage fluid; Etiology

BackgroundBlood culture techniques have improved to the point where they are considered sensitive enough for detection of Candida. Expert guidelines clarifying the utility of use of dedicated fungal isolator cultures are lacking, and we wondered what utility, if any, they add for the diagnosis of candidemia.MethodsAll patients with cultures between March 2016-February 2020 positive for Candida were examined via manual chart review, noting time to positivity and time of initiation of antifungal therapy.ResultsWe focused on cases of candidemia where a fungal culture was ordered and turned positive (59 out of the total 181 cases of candidemia). We eliminated an additional 10 cases where fungal cultures were sent while already on antifungal therapy or in patients already known to be fungemic, given our interest in de novo diagnoses. Another case was removed due to lack of clinical details, as the patient was discharged prior to culture results and managed at a different medical facility.There were 14 cases with discordant growth (fungal culture positive, bacterial culture negative). One patient passed away prior to culture results, but in the remaining 13 cases, the fungal culture changed clinical management, in most cases by prompting initiation of antifungal therapy.The remaining 36 cases involved with concordant growth between bacterial and fungal cultures. In 11 of those cases, the fungal culture isolated yeast 12 or more hours faster than its paired bacterial culture (average 40.7 +/- 26.6 hours). In 7 of these cases, the fungal culture changed management – in the remaining cases, the patient was already on empiric therapy.Among all cultures sent in patients not receiving antifungals that isolated Candida, the overall time to positivity for fungal cultures was 37.2 +/- 13 hours, while bacterial cultures took 54 +/- 26.4 hours.Fungal Culture ResultsConclusionFungal cultures changed management in 20/59 cases of candidemia (34%) either by making the diagnosis faster than a bacterial culture or making it outright. Given the morbidity and mortality associated with candidemia, rapid diagnosis is critically important. More specific guidelines optimizing how to best utilize fungal cultures to help standardize practice among clinicians will be critical going forward.DisclosuresOmai Garner, PhD, D(ABMM), Beckman Coulter (Scientific Research Study Investigator)

Diagnosis of yeast infection is typically accomplished by fungal smear and culture, histopathologic examination, and/or serologic studies. Newer assays based on mass spectrometry may be useful for yeast identification when histologic examination is inconclusive, fungal cultures are not ordered, or cultures fail to yield a causative agent. The purpose of this study was to evaluate the ability of the PLEX-ID broad fungal assay to accurately detect and identify yeasts in formalin-fixed paraffin-embedded (FFPE) tissues. Tissue samples from 78 FFPE specimens with both histopathology and corresponding culture results for a variety of yeasts were tested using the PLEX-ID broad fungal assay. A 40-μm FFPE tissue section from each specimen was digested with proteinase K followed by nucleic acid extraction and PCR using broad-range fungal primers. Yeast DNA in amplified products was identified using electrospray ionization mass spectrometry. Discordant results were resolved by D2 rRNA gene sequencing. PLEX-ID analysis detected yeast DNA in 78.2% (61/78) of the cases, of which 91.8% (56/61) were concordant with culture results. Of the 5 discordant positive results, 4 PLEX-ID results were considered to result from environmental contaminants, while 1 clinically important discrepancy was observed (Blastomyces dermatitidis by culture and Cryptococcus neoformans by PLEX-ID). Sequencing of the discordant sample was unsuccessful. The majority of histopathology results (89.7% [70/78]) correlated with culture results. The PLEX-ID broad fungal assay identifies fungi directly from FFPE tissues and can be a useful adjunct to traditional culture and histopathology tests.

Mature tropical urban trees are susceptible to root and trunk rot caused by pathogenic fungi. A metagenomic survey of such fungi was carried out on 210 soil and tissue samples collected from 134 trees of 14 common species in Singapore. Furthermore, 121 fruiting bodies were collected and barcoded. Out of the 22,067 OTUs (operational taxonomic units) identified, 10,646 OTUs had annotation information, and most were either ascomycetes (63.4%) or basidiomycetes (22.5%). Based on their detection in the diseased tissues and surrounding soils and/or the presence of fruiting bodies, fourteen basidiomycetes (nine Polyporales, four Hymenochaetales, one Boletales) and three ascomycetes (three species of Scytalidium) were strongly associated with the diseased trees. Fulvifomes siamensis affected the largest number of tree species surveyed. The association of three fungi was further supported by in vitro wood decay studies. Genetic heterogeneity was common in the diseased tissues and fruiting bodies (Ganoderma species especially). This survey identified the common pathogenic fungi of tropical urban trees and laid the foundation for early diagnosis and targeted mitigation efforts. It also illustrated the complexity of fungal ecology and pathogenicity.

Slow decline was observed in five mature and unmanaged hazelnut orchards in central Italy. Trees were trained into multi-branch bushes, and declining trees showed sparse crowns with chlorotic leaves and dead branches. Severely damaged trees died. Dead and declining branches were affected by a yellowish wood decay, especially at the base. Resupinate fruiting bodies, typical of Fomitiporia mediterranea or F. punctata, were observed on the dead branches. Vegetative Fomitiporialike isolates were also obtained from the decayed wood of declining/living branches. Thirty four fungal isolates, either derived from the fruiting bodies or the vegetative isolates, were identified as F. mediterranea by ITS sequencing, specific primer-based PCRs and RFLP. A reliable PCR-based detection method from decayed wood was developed. Based on the presence of fruiting bodies, the incidence of the fungus was 18% and 14% in orchards 1 and 4, respectively. However, based on F. mediterranea detection by PCR from decayed wood samples (26 trees), the incidence was 100% in orchard 1 and 99% in orchard 4. F. mediterranea was also found on wild hazelnut growing in forests located in the same area as the hazelnut orchards. Furthermore, the hymenomycetes Coniophora puteana, a known agent of wood decay, was found in the decayed wood of one declining tree. This is the first time F. mediterranea and C. puteana have been reported on hazelnut.