Sensitivity and specificity of potassium hydroxide and calcofluor white stain to differentiate between fungal and Pythium filaments in corneal scrapings from patients of Pythium keratitis

Purpose: To assess the sensitivity of potassium hydroxide and calcofluor white (KOH+CFW) mount in the diagnosis of Pythium keratitis and concordance among microbiologists. Methods: Three microbiologists evaluated the microscopic images of KOH + CFW mounts of confirmed cases of Pythium and fungal keratitis seen between January 2019 and February 2021. The filaments were compared using specific differentiating features. The sensitivity and specificity of KOH + CFW in diagnosing Pythium infection were evaluated along with concordance among the microbiologists. Results: Sixty consecutive cases with confirmed growth of fungus or Pythium insidiosum (n = 29) were evaluated. The sensitivity of KOH + CFW in the correct identification of Pythium filaments ranged from 79.3% to 96.5% among three microbiologists. There was good interobserver (k = 0.76–0.90) and intraobserver (k = 0.70–0.97) agreements among three microbiologists. The differentiating findings (P < 0.0001) suggestive of Pythium filaments were the absence of septae in 23 (79.3%) and collapsed walls in 22 (75.9%) cases. Conclusion: KOH + CFW has good sensitivity and specificity in the diagnosis of Pythium keratitis with good interobserver and intraobserver concordance.

Effects of glyphosate on soil fungal communities: A field study / Efecto del glifosato en las comunidades fúngicas del suelo: estudio a campo

ABSTRACT The driving forces behind many soil processes are microorganisms and they are able to respond immediately to environmental changes. The soil microbial community impacts on many soil properties. More than one-third of the terrestrial ecosystems are semiarid. However, a limited number of studies have been conducted to characterize soil fungal communities in semiarid grasslands, in particular those of agricultural fields. The aim of this study was to explore changes in the diversity and structure of soil fungal communities in semiarid grasslands, after different doses of glyphosate were applied under field conditions. Changes in soil fungal communities were examined using different approaches including culturing, calcofluor white stain and denaturing gradient gel electrophoresis (DGGE). The different approaches complement each other, revealing different aspects of the effect of glyphosate on soil fungal communities. We demonstrated a negative effect of glyphosate on soil fungal biomass at high doses and an early and transitory stimulatory effect on soil fungal biomass. We also found a negative effect of glyphosate on the species richness of cultivable fungi and changes in the molecular structure of soil fungal communities after double doses or long-term glyphosate application. In summary, our findings demonstrate an overall negative effect of glyphosate on soil fungal communities.

RESUMEN Los microorganismos del suelo son los responsables de llevar a cabo la mayoría de los procesos biológicos que ocurren en el suelo, y son capaces de reaccionar ante el estrés ambiental. Más de un tercio de los ecosistemas terrestres son semiáridos. Sin embargo, son escasos los estudios realizados para caracterizar las comunidades fúngicas en suelos agrícolas en ecosistemas semiáridos. El objetivo del presente trabajo fue estudiar los cambios que se producen en la biomasa, la diversidad y la estructura de las comunidades fúngicas del suelo, luego de la aplicación de distintas dosis de glifosato en condiciones de campo. Se emplearon diferentes técnicas incluidas el cultivo, la tinción directa con blanco de calcoflúor y PCR acoplada a electroforesis en geles de gradiente desnaturalizante (DGGE). Las distintas metodologías empleadas se complementan entre sí al detectar cada una distintos aspectos del efecto del glifosato en las comunidades fúngicas del suelo. Se encontró que el glifosato produce un efecto negativo sobre la biomasa fúngica, también se encontró un efecto transitorio estimulante inmediatamente posterior a la aplicación del herbicida. Además, se vio un efecto negativo sobre la riqueza de hongos cultivables, así como también cambios en la estructura molecular de las comunidades luego de aplicaciones repetidas. En conclusión, se demostró un efecto negativo generalizado sobre las comunidades fúngicas del suelo.

The First Acanthamoeba keratitis Case of Non-Contact Lens Wearer with HIV Infection in Thailand

Acanthamoeba keratitis (AK) is a rare sight-threatening corneal infection, often reporting from contact lens wearers. An asymptomatic human immunodeficiency virus (HIV)-infected Thai male without history of contact lens use complained foreign body sensation at his left eye during motorbike riding. He had neither specific keratitis symptoms nor common drugs responding, which contributed to delayed diagnosis. By corneal re-scraping, Acanthamoeba-like cysts were detected by calcofluor white staining and agar culture. The etiological agent obtained from the culture was molecularly confirmed by Acanthamoeba spp.-specific PCR, followed by DNA sequencing. The results from BLAST and phylogenetic analysis based on the DNA sequences, revealed that the pathogen was Acanthamoeba T4, the major genotype most frequently reported from clinical isolates. The infection was successfully treated with polyhexamethylene biguanide resulting in corneal scar. This appears the first reported AK case from a non-contact lens wearer with HIV infection in Thailand. Although AK is sporadic in developing countries, a role of free-living Acanthamoeba as an opportunistic pathogen should not be neglected. The report would increase awareness of AK, especially in the case presenting unspecific keratitis symptoms without clinical response to empirical antimicrobial therapy.

Growth and Adhesion to Cells of Candida albicans Labeled by Calcofluor White and Fluorescent Concanavalin A / 中山大学学报(医学科学版)

@#【Abstract】 【Objective】To assess effect of calcofluor white and fluorescent dye labeled concanavalin A in Candida albicans growth and adhesion of cells.【Methods】Yeast cells of 20 strains of Candida albicans were labeled by calcofluor white and Alexa fluor 488 conjugated concanavalin A respectively. The growth of labeled Candida albicans were tested by counts of colony forming unit. Then yeast cells of Candida albicans were co-cultured with macrophages and enterocytes for half an hour. The adhesion of labeled Candida albicans to macrophages and enterocytes were observed by fluorescence microscopy.【Results】No difference was observed on the number of colony forming units between calcofluor white-labeled groupand control group(P=0.942). Also,no difference was observed on the number of colony forming units between Alexa fluor 488 conjugated concanavalin A-labeled group and control group. However,either the number of calcofluor white-labeled Candida albicans or Alexa fluor 488 conjugated concanavalin A-labeled Candida albicans that bound to macrophages was less than that in control group(P=0.000,respectively). Either the number of calcofluor white-labeled Candida albicans or Alexa fluor 488 conjugated concanavalin A-labeled Candida albicans that bound to enterocytes was less than that in control group(P = 0.000,respectively). Hyphae were observed in control group but not in calcofluor white group after yeast cells of Candida albicans were co-cultured with cells for half an hour.【Conclusions】Growth of Candida albicans was not changed,while its adhesion to cells was reduced after its labeling by calcofluor white and Alexa fluor 488 conjugated concanavalin A.The germination of Candida albicans was affected when it had been labeled by calcofluor white.

Fluorescent staining with hypersensitivity and enhancement for early diagnosis of superficial fungal infection / 临床检验杂志

Objective To evaluate the application of novel fluorescent staining method with hypersensitivity and enhancement for microscopic examination of superficial fungal.Methods A total of 200 cases with clinically suspected superficial fungal infection were screened by the hypersensitive,enhanced fluorescent staining,calcofluor white staining and microscopic examination with 1.78 mol/L potassium hydroxide (KOH) based smear assay,respectively.The positive percentages of the different methods were calculated and compared.Results In the developed fluorescent staining examination,clean background was shown in the view field.The hyphae and spores displayed in bright blue,thus fungal morphology was so distinct that they were easy to be distinguished.Compared with calcofluor white staining and KOH based smear,the fluorescent staining showed high positive rate with significant difference respectively (x2 =17.984,P ＜ 0.05;x2 =32.063,P ＜ 0.05).Conclusion The novel fluorescent staining should be a rapid,accurate method for microscopic examination of fungi,which is worthy to be widely used in clinical laboratories for early diagnosis of fungal infection.

Acanthamoeba Keratitis: Case Report

We experienced chronic relapsing central corneal ulcer and chronic conjunctivitis with mucoid discharge. Both patients were transfered to our hospital because of no improvement with long term treatment of broad spectrum antibiotics topically and systemically Specimens from corneal scraping and conjunctival mucoid discharge were examined with calcofluor white staining method. We could find a narrow rim of light green colored cystic wall and orange red colored cytoplasm in dark field fluorescent microscopy in both cases, and those were diagnosed as Acnthamoebic cyst by pathologist. We couldn't find any report about ocular Aanthamoeba infections in Korea. To our knowledge, this is the first case report about ocular Acanthamoebic infections in Korea.