Direct Microscopic Methods Research Articles

Laboratory and clinical management capacity for invasive fungal infections: the Italian landscape.

We assessed the laboratory diagnosis and treatment of invasive fungal disease (IFD) in Italy to detect limitations and potential for improvement. The survey was available online at www.clinicalsurveys.net/uc/IFI management capacity/, and collected variables such as (a) institution profile, (b) perceptions of IFD in the respective institution, (c) microscopy, (d) culture and fungal identification, (e) serology, (f) antigen detection, (g) molecular tests, (h) susceptibility testing and (i) therapeutic drug monitoring (TDM). The laboratory capacity study received responses from 49 Italian centres, with an equitable geographical distribution of locations. The majority of respondents (n = 36, 73%) assessed the occurrence of IFD as moderate-high, with Aspergillus spp. being the pathogen of highest concern, followed by Candida spp. and Mucorales. Although 46 (94%) of the institutions had access to microscopy, less than half of them performed direct microscopy on clinical specimens always when IFD was suspected. Cultures were available in all assessed laboratories, while molecular testing and serology were available in 41 (83%), each. Antigen detection tests and antifungal drugs were also generally accessible (> 90%) among the participating institutions. Nevertheless, access to TDM was limited (n = 31, 63%), with a significant association established between therapeutic drug monitoring availability and higher gross domestic product per capita. Apart from TDM, Italy is adequately prepared for the diagnosis and treatment of IFD, with no significant disparities depending on gross domestic product. Future efforts may need to focus on enhancing the availability and application of direct microscopic methods, as well as TDM, to promote optimal treatment and better patient outcomes.

Detection of Gastrointestinal Parasites of Cats and Dogs in Basrah Province

The present study has been conducted for the detection of gastrointestinal parasites isolated from cats and dogs in Basrah province from November 2018 to January 2019. One hundred fecal samples were collected from cats and dogs. Samples were submitted to the parasite’s lab in college of veterinary medicine of Basrah university. for diagnoses by direct microscopic examination and concentration methods. Gastrointestinal parasites of cat identified in this study were: Toxascaris leonine 58.8%, Toxoplasma gondii 11.7, Isospora spp 11.7%, Entamoeba spp.17.6% , and for dog were Dipylidium caninum 21%, Toxocara canis 10.5%, Isospora canis 36.8%, Cryptosporidium spp. 5.2%, Giardia spp. 10.5%, Entamoeba spp. 5.2% , Ancylostoma caninum 10.5%,

Subatomic Distortion of Surface Monolayer Lattice Visualized by Moiré Pattern.

Mapping of the local lattice distortion is required to understand the details of the chemical and physical properties of thin films. However, the resolution by the direct microscopic methods was insufficient to detect the local distortion. Here, we have demonstrated that the local distortion of a monatomic film on a substrate is estimated with high resolution using the moiré pattern of the topographic scanning tunneling microscopy image. The analysis focused on the apparently low centers of the moiré pattern of the hexagonal iron nitride monolayer on the Cu(111) substrate. The local distortion was visualized by estimating the displacement of the experimentally detected center position from the ideal position that is extracted from the adjacent center positions. The map of the local distortion revealed that the subsurface impurities are preferentially located on the largely distorted areas. This approach is widely applicable to other thin films on the substrates.

Mycological profile of keratomycosis in a tertiary care centre from Pune Maharashtra

Introduction: Microbial keratitis remains a leading cause of corneal ulcer and blindness worldwide. Mycotic keratitis has emerged as a major ophthalmic problem and contributes to 6-53% of all corneal infections worldwide. Thus the present study was undertaken to assess the spectrum of fungal causes of keratitis in BJGMC & SGH, Pune. Aims and Objectives: 1. To establish the microbiological profile of keratitis. 2. To determine the mycological profile of keratitis. Materials and Methods: The present prospective study was conducted over a period of twelve months in a tertiary care hospital. The study was undertaken to evaluate the etiological profile of keratomycosis with a special reference to mycotic etiology. The corneal scrapings were collected aseptically and processed by direct microscopic methods and standard culture techniques. Results: Of 90 cases of keratitis screened, culture yielded growth in 50 cases (55.6%). These were included in the study. Out of these 50 cases of growth, fungi were isolated in 32% and bacteria in 64% cases. Mixed growth of bacteria and fungi was found in4% cases. Most common fungus found were Fusarium spp. (44.4%) followed by Aspergillus spp. (27.8%), Alternaria spp. (11.1%), Curvulariaspp (11.1%) and Acremonium spp (5.6%). Conclusion: Routine surveillance of fungal keratitis is necessary to know the existing and emerging pattern of pathogens and to prevent inappropriate use of antimicrobial therapy. Keywords: Keratitis, Fungus, Aspergellus, Keratomycosis.

A study on the extent, aetiology and associated factors of dandruff in a group of medical students and the in vitro effects of antidandruff preparations

Introduction: Dandruff is a scalp disorder, characterized by itching and rapid skin flaking of the scalp of a person. Yeasts of the genus Malassezia are responsible for dandruff. The prevalence of dandruff in South Asia is 60.1%, whereas in the world it is 50%. The objectives of this study were to ascertain the prevalence, describe associated factors, isolate Malassezia sp. from dandruff samples of medical students and test the anti-fungal effects of commercially available shampoo and crude extracts of commonly used herbs against Malassezia. Methods: Ninety five students were chosen by systematic sampling. A self-administered questionnaire was used for data collection. Samples of skin flakes/swabs was collected from the scalp of participants. Specimens were examined using three direct microscopic methods. Fungal colonies isolated on Saborauds/Dixon Agar were identified using Gram stain and catalase test. Antifungal activity of six shampoo brands and crude extracts of eleven herbs were tested against isolated Malassezia sp. by agar well/disc diffusion method. Results: Prevalence of dandruff was 64.2% in the study group. The typical ‘spaghetti and meatball’ appearance was seen in the skin flakes taken from 44 of 61 (72.1%) students. Malassezia sp. was isolated only from 49 of the 61 students (80.3%). In-vitro, anti-malassezial effect (Inhibition zone (IZ)>10mm) was observed in 4 of the 7 tested ‘anti dandruff’ shampoos. Crude extracts of fresh leaves of Psidium guavava (guava), flowers of Punica grantum (pomegranate) and fruits of Solanum lycopersicum (tomato) and Citrus hystrix (Kaffir lime) showed IZs > 10mm. Conclusion: Prevalence of dandruff in the studied group of medical students was higher than in the Asian population. Four of the tested ‘anti dandruff’ shampoos and crude extracts of leaves of P. guavava (guava), fruits of S. lycopersicum (tomato), C. hystrix (kaffir lime), and flowers of P. grantum (pomegranate) showed an in-vitro inhibitory effect on the growth of Malassezia sp.

Automatic Segmentation and Classification of SEM Images of Bacteria Cells

In the field of microbiology, digital image analysis methods are receiving significant attention to automatically interpret images of bacterial cells. An automatic procedure to extract and classify images of lactic acid bacteria (LAB) is presented in this paper. Edge based watershed method with automatically generated markers were used to retain the image information at fine scales. The experiment was conducted on images containing one type of bacteria. The scanning electron microscopic (SEM) images of lactic acid bacteria (LAB) are used in this experiment. The image analysis and classification technique described in this paper is quick and simple to recognize organisms based on their morphological characteristics. The classification results indicate that routine methods for the detection, enumeration and identification of bacteria can be automated with use of direct microscopic methods

Isolation of acanthamoeba species from fresh water sources isn southern regions of Saudi Arabiass

Objective: The present study aimed to investigate the occurrence of Acanthamoeba species and other FLAs in freshwater sources in southern regions of the Kingdom of Saudi Arabia and to estimate the prevalence of their existence in these areas.Methods: Water samples from both fresh water course and hot springs (n = 15) were collected from five different zones in Aseer region, southern western parts of the kingdom. Isolation and identification of Acanthamoeba species and other FLAs were carried out from these water sources (fresh water course and hot spring). The methods included filtration, in vitro culture and incubation for up to three weeks for the selected samples according to standard methods.Results: Examination of water samples by direct microscopic methods showed no amoebic parasites among all of the 15 samples collected from five different sites. However, when the water samples were cultured on non-nutrient agar (NNA), four (26.7%) of the 15 samples showed growth of Acanthamoeba species. Principle component analysis (PCA) indicated that the water bodies/ sources that were positive for Acanthamoeba spp. shared a common natural characteristic that the water bodies were exposed to contamination.Conclusion: The study demonstrated that Acanthamoeba spp. were prevalent in some sites (3 out 5 sites were positives; 60%), low in two sites (1 out 3 samples) while absent in two sites (0 out of six samples). More comprehensive research is needed to find out the true prevalence of these parasites and factors affecting their existence.

Cicada Wing Surface Topography: An Investigation into the Bactericidal Properties of Nanostructural Features.

Recently, the surface of the wings of the Psaltoda claripennis cicada species has been shown to possess bactericidal properties and it has been suggested that the nanostructure present on the wings was responsible for the bacterial death. We have studied the surface-based nanostructure and bactericidal activity of the wings of three different cicadas (Megapomponia intermedia, Ayuthia spectabile and Cryptotympana aguila) in order to correlate the relationship between the observed surface topographical features and their bactericidal properties. Atomic force microscopy and scanning electron microscopy performed in this study revealed that the tested wing species contained a highly uniform, nanopillar structure on the surface. The bactericidal properties of the cicada wings were investigated by assessing the viability of autofluorescent Pseudomonas fluorescens cells following static adhesion assays and targeted dead/live fluorescence staining through direct microscopic counting methods. These experiments revealed a 20-25% bacterial surface coverage on all tested wing species; however, significant bactericidal properties were observed in the M. intermedia and C. aguila species as revealed by the high dead:live cell ratio on their surfaces. The combined results suggest a strong correlation between the bactericidal properties of the wings and the scale of the nanotopography present on the different wing surfaces.

Clinico-demographical profile of keratomycosis in Delhi, North India

This study was undertaken to evaluate the clinico-demographical profile of keratomycosis. (January 2004 to January 2012). The corneal scrapings were processed by direct microscopic methods and standard culture techniques. Of 209 cases of keratitis studied, culture yielded growth in 80 cases (38.3%). Out of these 80 cases of growth, fungi were isolated in 77.5% and bacteria in 22.5%. The spectrum of keratomycosis was Aspergillus flavus (22.5%), Fusarium solani (16.1%), A. fumigatus (11.3%), Candida albicans (6.4%), etc., Routine surveillance of fungal keratitis is necessary to know the existing and emerging pattern of pathogens and to prevent use of un-warranted anti-microbial therapy.

Comparison of ELISA and direct microscopic examination for detecting Giardia intestinalis in children

Objective: Giardia intestinalis is the causes of giardiasis, which infects human fecally-orally. This leads to growth retardation in children, leading to malabsorption, which is widespread in Turkey, especially in the regions having low socio-economic status. In this study we aimed to compare results of Enzyme Linked Immunosorbent Assay (ELISA) and direct microscopic stool examination for detecting giardiasis. Methods: The feces samples of 761 children aged between 0 and 16 years who applied to Ondokuz Mayıs University Hospital with diarrhea were evaluated by using ELISA test and direct microscopic examination. Results: The study comprised 356 (46.7%) boys and 405 (53, 3%) girls. Both direct microscopy and ELISA gave positive results for giardiasis in 17 (2.2%) children. In 13 (17%) of the children ELISA was found positive but direct microscopy did not show Giardia intestinalis at stool samples. Both ELISA and direct microscopy of the remaining 731 (96.1%) of the patients gave negative results for giardiasis. Conclusion: Direct microscopic examination methods are often preferred because they are more cost-effective, and some other parasites can be found by examination of feces. Yet, in cases in which children have giardiasis symptoms but parasites cannot be determined by microscopic examination to search for G. intestinalis antigen by means of ELISA can be used as an alternative method.

Comparison of direct microscopic methods using potassium hydroxide, periodic acid Schiff, and calcofluor white with culture in the diagnosis of onychomycosis

Comparison of direct microscopic methods using potassium hydroxide, periodic acid Schiff, and calcofluor white with culture in the diagnosis of onychomycosis

Fungal biodeterioration of historical library materials stored in Compactus movable shelves

Mold deterioration of historical library materials in archives and libraries is a frequent and complex phenomenon that may have important economic and cultural consequences. Compactus shelving is one of the systems utilised for the conservation of library materials, because it allows for a more efficient use of space and protection against dust deposition. However, in the last ten years there have been many reports on single species mould infections within Compactus shelves in spite of conventional control of environmental temperature and humidity to recommended standards. Contamination was commonly characterized by white spots of mycelium, measuring 0.5–1.0 cm in diameter and observed on volume binding, especially those of leather, parchment or textile. Until now, the identification of the causal agent at species level has not been reported since attempts to grow it on media for subsequent identification were unsuccessful. Using a range of sampling techniques, including adhesive tape and nitrocellulose membrane, and a combination of conventional culturing methods, direct microscopic observations and molecular methods, we have for the first time identified to species level the fungus causing infections inside Compactus shelves.

An overview of a diagnostic and epidemiologic reappraisal of cutaneous leishmaniasis in Iran

Cutaneous leishmaniasis (CL) is a widespread tropical infection which has a high incidence rate in Iran. Leishmania tropica, the causative agent of anthroponotic cutaneous leishmaniasis (ACL), and Leishmania major, which causes zoonotic cutaneous leishmaniasis (ZCL), are endemic in various parts of Iran with a high incidence rate. The aim of this study was to evaluate the reappraisal of the diagnosis and epidemiology of CL in Iran, by different clinical, parasitological and molecular assays among patients suspected of CL referred to the Department of Parasitology, at the Pasteur Institute of Iran during 2006–2009. Two hundred samples from patients with ulcerative skin lesions were collected, clinical analyses were applied, data questionnaire was completed and samples were examined for CL by using both direct microscopic and culture methods. Moreover, PCR assay was applied for detection of Leishmania species in CL isolates resulting from parasitological assay. Clinical observation revealed that the majority (58%) of lesions was single; double lesions were observed in 22% of patients, and only 20% of CL had multiple lesions. Out of 200 patients, Leishman body was observed in 77 samples (38.5%) by direct smear and 40% by cultivation assay. Most patients (21.3%) had a travel history to the Isfahan province, one of the most important endemic areas of CL located in center of Iran. PCR assay by kDNA indicated 32 and 18 out of 50 isolates respectively had similar patterns with standard L. major and L. tropica. In conclusion, clinical manifestations and an appropriate diagnostic assay with a parallel molecular characterization of CL may lead to a screening evaluation of disease, prognosis, treatment and control strategies.

Soil microbial biomass: The eco-physiological approach

Abstract In the 1980s ecosystem research projects were implemented world-wide since there was a pressing need to quantify the impacts of anthropogenic pollutants. Soil ecosystem analyses concentrated first on the quantification of the element and energy transfer between pools. Since mineralization of organic substrates and the release of nutrients and elements are due to the heterotrophic activity of the microbial decomposer compartment, this subsystem of terrestrial ecosystems gained importance. Direct microscopic observation methods were inadequate for the quantification of environmental impacts on the microflora. We adopted the maintenance requirement concept for the quantification of environmental impacts or stress effects on the soil microbial community. The paper gives a brief inside to the concept of maintenance from autecological studies and describes the underlying points which lead to our experimental approach of its application at the synecological level (i.e., microbial biomass as a single ecological entity) – a process which rested on long-term continuous research.

Correlation of Trichomonas vaginalis to bacterial vaginosis: a laboratory-based study

This study aimed to define the occurrence of different organisms causing vulvovaginitis; to evaluate different laboratory methods used for diagnosis of Trichomonas vaginalis (T. vaginalis); and to evaluate the direct score system and clue cell method compared with culture for diagnosis of bacterial and T. vaginalis vaginosis. Clinical and laboratory evaluations were performed for 110 patients. Laboratory methods used for bacteriological diagnosis were direct Gram staining for clue cells and scoring by Nugent score system and bacterial culture. T. vaginalis was identified by wet mount microscopic examination, culture, direct Gram, Giemsa staining and acridine orange (AO). The Nugent score method revealed that the sensitivity and specificity for diagnosis of vaginal discharge by direct rapid microscopic methods were 30% and 80% and for clue cells sensitivity and specificity were 37% and 75% respectively for diagnosis of bacterial vaginosis compared to culture. For diagnosis of T. vaginalis, the Nugent score method revealed that the sensitivity and specificity were 60% and 90% respectively, and for clue cells 75% and 80% respectively. For microcopic methods used for T. vaginalis only, the Gram stain and Giemsa stain sensitivities were poor (15.2% and 48.5%, respectively). Wet mount showed reasonable sensitivity of 75.8%. Acridine orange sensitivity was 93.9% and specificity was 97.5%, Prevalent pathogens associated with vaginitis were (Gardnerella vaginalis) G. vaginalis, T. vaginalis and Mycoplasma hominis (M. hominis). Wet mount microscopic examination, acridine orange, and high Nugent score were found as rapid and sensitive methods for diagnosis of T. vaginalis.

Behaviors of physiologically active bacteria in water environment and chlorine disinfection

Direct microscopic methods using several fluorescent staining were applied to estimate the proportion of physiologically active bacteria in the water environment and evaluate the efficacy of disinfection with chlorine. 4',6-diamidino-2-phenylindole (DAPI) was used to determine total bacterial numbers, and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was chosen for direct detection of respiring bacteria. BacLight kit was used to assess bacterial membrane integrity. Bacteria with growth potential were enumerated using the DVC method and microcolony technique. The total bacterial number in river was 8 x 10(6)-3 x 10(10) cells/mL, and colony forming units on R2A medium were 1 x 10(4)-4 x 10(5) cfu/mL. In the case of wastewater treatment plant, 1-10% of total bacterial cells could form colonies. Physiologically active bacteria in river and wastewater treatment plant determined by fluorescent staining were much higher than those obtained by plate counting. The effect of chlorine on the physiological viability of Escherichia coli was also investigated. Microscopic viable bacteria were even more chlorine resistant than culturable bacteria. The inactivation rate coefficients of direct viable bacteria were one-second to third those of culturable bacteria.

Characterization of nanostructured GaSb: comparison between large-area optical and local direct microscopic techniques

Low energy ion-beam sputtering of GaSb results in self-organized nanostructures with the potential of structuring large surface areas. Characterization of such nanostructures by optical methods is studied and compared to direct (local) microscopic methods. The samples consist of densely packed GaSb cones on bulk GaSb, approximately 30, 50, and 300 nm in height, prepared by sputtering at normal incidence. The optical properties are studied by spectroscopic ellipsometry, in the range 0.6-6.5 eV, and with Mueller matrix ellipsometry in the visible range, 1.46-2.88 eV. The optical measurements are compared to direct topography measurements obtained by scanning electron microscopy, high resolution transmission electron microscopy, and atomic force microscopy. Good agreement is achieved between the two classes of methods when the experimental optical response of the short cones (<55 nm) is inverted with respect to topological surface information, via a graded anisotropic effective medium model. The main topological parameter measured was the average cone height. Optical methods are shown to represent a valuable characterization tool of nanostructured surfaces, in particular when a large coverage area is desirable. Because of the fast and nondestructive properties of optical techniques, they may readily be adapted to in situ configurations.

Structural approach to the study of deformation mechanism of amorphous polymers

A new microscopic procedure for the visualization of structural rearrangements in amorphous polymers during their deformation to high strains is described. This approach involves the deposition of thin (several nanometers) metallic coatings onto the surface of the deformed polymer. Subsequent deformation entails the formation of a relief in the deposited coating that can be studied by direct microscopic methods. The above phenomenon of relief formation provides information concerning the deformation mechanism of the polymer support. Experimental data obtained with the use of this procedure are reported, and this evidence allows analysis of the specific features of structural rearrangements during deformation of the amorphous polymer at temperatures above and below its glass transition temperature under the conditions of plane compression and stretching, uniaxial tensile drawing and shrinkage, rolling, and environmental crazing. This direct structural approach originally justified in the works by Academician V.A. Kargin appears to be highly efficient for the study of amorphous polymer systems.

Sodium chloride-enhanced adherence of Edwardsiella tarda to HEp-2 cells

The adherence of Edwardsiella tarda strains to HEp-2 cells was studied by the viable cell count and direct microscopic count methods. Three selected E. tarda strains having different hemagglutination activities against guinea pig erythrocytes were cultured in a peptone-yeast extract broth supplemented with 3% NaCl (3%-NaCl culture) or without NaCl (0%-NaCl culture). Strain FK1051 showed significantly higher adherence in the 3%-NaCl culture than in the 0%-NaCl culture. A similar adherence pattern was observed in strain KG8401, though the adherence of the 0%-NaCl culture was higher than that of FK1051. In contrast, both cultures of strain SU166 were low in the adherence. These adherence patterns to the HEp-2 cells correlated well with the previously reported hemagglutinating activities of the strains. N-acetylneuraminic acid markedly inhibited the adherence as well as the hemagglutination. This NaCl-enhanced adherence may be involved in pathogenesis of E. tarda infection particularly in marine fish.

Predominant cultivable subgingival microbiota of healthy and HIV-infected ethnic Chinese.

Although clinically distinctive types of periodontal disease are known to be associated with HIV infection, the pathogenesis remains unclear. In the present study, the subgingival microflora of 21 HIV-infected and 11 HIV-free ethnic Chinese were studied using the direct microscopic and anaerobic culture methods. Motile curved rods were found to be three times higher in the HIV-infected group under direct microscopy. Otherwise, there were no significant differences between the diseased and healthy groups when analyzed either in relation to the morphotype distribution or Gram stain morphology or oxygen tolerance. The most common bacteria isolated were Capnocytophaga species followed by Prevotella loescheii, Streptococcus sanguinis, Lactobacillus spp. and Fusobacterium spp. Although there were 20 bacterial species that were strictly limited to the HIV-infected group, and 5 limited to the healthy group, none of the species was a predominant isolate in either group (p>0.05). These findings agree with previous studies which report that subgingival bacteria of HIV-infected individuals (with or without periodontal disease) are similar to those found in healthy HIV-free individuals (with or without periodontal disease). Further work examining the subgingival microflora from patients with specific and/or severe forms of HIV-associated periodontal disease is required to shed light on the pathogenesis of this complex clinical entity.