Differentiation of Enterobacteriaceae, Pseudomonas aeruginosa, and Bacteroides and Haemophilus species in gram-stained direct smears.

The accuracy of examination of the Gram-stained direct smear to classify presumptively Gram-negative rods into three morphotype groups, that is, (a) Enteric bacteria, (b) Pseudomonas, and (c) Bacteroides or Haemophilus, was evaluated. Randomly selected clinical strains (4-9) each of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Ps. aeruginosa, B. fragilis, and H. influenzae were used to produce peritonitis or subcutaneous abscesses in mice. A Gram-stained direct smear was prepared from exudate collected from each animal. The direct smears were examined to classify bacteria observed into one of the three morphotype groups. The percent accuracy was 82, 56, and 95, respectively, and 76 overall. The assumption was made that classification was based primarily on differences in length and width of the organisms. To test this hypothesis, we prepared scanning electron photomicrographs from each specimen of exudate and measured the lengths and widths of bacteria. Examination of the Gram-stained direct smear was more accurate for classification of enteric bacteria, H. influenzae, or B. fragilis. Electron microscopy was more accurate for classification of Ps. aeruginosa. The higher length-width radio should be helpful in recognizing Ps. aeruginosa in direct smears.     

自动化镜检结合人工智能分析系统对阴道分泌物革兰氏染色涂片形态学的准确性评价

目的 探究自动化镜检结合人工智能分析系统对阴道分泌物革兰氏染色涂片阴道微生态形态学评价的准确性.方法 收集2020年1—5月北京协和医院妇产科门诊患者的阴道分泌物标本.标本经涂片、革兰氏染色,分别进行人工镜检和自动化镜检.自动化镜检采用仕达思Comet-60au高倍镜检分析系统,并通过人工智能分析自动判定结果.对形态学...     

Use of lactophenol cotton blue mounts of corneal scrapings as an aid to the diagnosis of mycotic keratitis.

Lactophenol cotton blue (LPCB) mounts of corneal ulcer scrapings were assessed as a diagnostic tool in cases of mycotic keratitis over a period of 15 months. We investigated 568 cases of ulcerative keratitis by microbiological techniques consisting of direct microscopic examination of LPCB mounts and Gram-stained smears as well as culture of material scraped from the ulcer. Fungi were isolated in large numbers on multiple media from the corneal scrapings of 179 patients (culture-proven mycotic keratitis). Direct microscopic examination of LPCB mounts of corneal scrapings yielded positive results in 140 (78%) of 179 culture-positive patients and negative results in 371 (95%) of 389 culture-negative patients. There was a significant difference between the percentage of positive results obtained by LPCB mounts and by Gram-stained smears in the culture-positive cases. The LPCB mount was positive in greater than 80% of cases of keratitis due to Fusarium spp. and Aspergillus spp. The LPCB mount is strongly recommended as a simple, rapid, inexpensive, and sensitive diagnostic technique in cases of mycotic keratitis.     

革兰染色痰涂片镜检与痰培养的荟萃分析

目的:综合分析革兰染色痰涂片镜检的临床应用价值。方法:应用荟萃分析(meta-analysis)对国内6组(共3210例)痰涂片革兰染色镜检结果及痰培养结果随机对照分析,评价革兰染色痰涂片镜检的临床应用价值。结果:共有6篇纳入研究,包括3210例,其中3030例为检索相关文献,180例为现收集标本。分析表明,革兰染色痰涂片镜检结果具较重要的临床价值,与临床诊断结果密切相关。结论:应提倡进行革兰染色痰涂片镜检。     

The microbiology of bacteriuria in men: a 5-year study at a Veterans' Affairs hospital

Bacteriuria and urinary tract infection occur relatively frequently in older men, but data regarding the causative microorganisms are limited. We retrospectively identified all positive cultures of urine specimens (n = 4943) obtained over a 5-year period at our institution. We determined the frequency of causative microorganisms and grouped these by Gram type, setting of patient care, and method of urine specimen collection. We also assessed the performance characteristics of the Gram-stained smear of uncentrifuged urine. Among our patients, Gram-positive cocci (GPC) were isolated as often as Gram-negative rods (GNR). Escherichia coli was the single or predominant isolate in only 14% of cases, and Enterococcus was the single most commonly identified genus (22.5%). The Gram stain was accurate in predicting the culture results (positive likelihood ratio, 7.0 for GPC and 8.1 for GNR). We conclude that the microorganisms causing bacteriuria in older male veterans are substantially different from those found in women, and the Gram-stained smear provides useful information on the causative organisms.     

Performance of a rapid yeast test in detecting Candida spp. in the vagina

We compared the performance of a rapid vaginal yeast assay (Savvycheck) with that of microscopic examination of a Gram-stained smear and culture of vaginal discharge in detecting Candida spp. Two hundred thirty-one women with vaginal symptoms were studied prospectively. Vaginal specimens obtained from all participants were studied by the Savvycheck rapid yeast test, microscopic evaluation of Gram-stained vaginal smears, and yeast culture. Savvycheck rapid yeast test was positive in 79% of women with positive cultures and in 3.6% of women with negative cultures. The Savvycheck test detected yeasts in 93% of subjects with positive Gram stain and in 5.5% of subjects with negative Gram stain. The Savvycheck rapid yeast test showed 93% sensitivity, 95% specificity, and a 97% negative predictive value compared with the Gram stain. It revealed 79% sensitivity, 96% specificity, and an 87% negative predictive value compared with culture. The Savvycheck rapid yeast test can be used in the busy office instead of microscopy as a point-of-care tool for diagnosing vulvovaginal candidiasis. It can also reduce the need for yeast cultures in patients with vaginitis.     

Methanol fixation. An alternative to heat fixation of smears before staining

Methanol fixation of Gram-stained smears was compared to heat fixation. Smears were prepared in duplicate from direct clinical specimens, blood culture bottles, and bacterial colonies. Results from this study show that methanol fixation is superior in every instance to heat fixation. The morphology of bacteria and tissue cells was not distorted, little or no background debris was observed, and a better Gram reaction was noted.     

Diagnosis of necrotizing fasciitis in children.

Necrotizing fasciitis is a rare but progressive soft tissue infection. This condition is difficult to recognize in the early phase, when it is often confused with cellulitis. We report the cases of four children with necrotizing fasciitis. The initial presentation in these cases was cellulitis. Fever and soft tissue swelling occurred within 24 h and spreading erythema within 4 to 12 h. Radiologic studies of the lesions showed soft tissue thickening. Ultrasonography of the lesions demonstrated distorted, thickened fascia with fluid accumulation. Well-defined, loculated abscesses were demonstrated in two cases. Although typical dusky skin and purplish patches were not found in our cases, necrotizing fasciitis was strongly suspected on the basis of the clinical course and sonographic findings. Ultrasonography also was used as a guide for aspiration of pus. Gram-stained smears and bacterial cultures yielded the pathogens. The choice of antibiotic therapy was made on the results of smears and culture. All patients survived after immediate surgical debridement, intensive antibiotic therapy, and aggressive wound care. In conclusion, ultrasonography provides a rapid and valuable diagnostic modality for necrotizing fasciitis. The pus obtained through sonographically guided aspiration for bacterial culture can allow identification of the pathogenic organisms.     

Flow Cytometry-Based Analysis of Artemisinin-Resistant Plasmodium falciparum in the Ring-Stage Survival Assay

The ring-stage survival assay (RSA) is a powerful tool for phenotyping artemisinin-resistant Plasmodium falciparum but requires experienced microscopists to count viable parasites among 10,000 erythrocytes in Giemsa-stained thin blood smears. Here we describe a rapid flow cytometric assay that accurately counts viable parasites among 250,000 erythrocytes in suspension. This method performs as well as light microscopy and can be used to standardize the collection of RSA data between research groups in laboratory and field settings.     

The usefulness of changing focus during examination using Gram staining as initial diagnostic clue for infective tuberculosis

Gram staining is a useful technique for detecting bacteria but is highly questionable in detecting Mycobacterium tuberculosis. Its detection generally requires special staining, such as Ziehl–Neelsen staining. We experienced three cases in which tuberculosis was first suggested by Gram staining of sputum or pus, confirmed by Ziehl–Neelsen staining, and diagnosed by polymerase chain reaction or culture. To find colorless tubercle bacilli in clinical samples with various organisms, varying the focus to slightly longer and shorter during study of the slides is indispensable. We present criteria for detecting infective pulmonary tuberculosis in Gram staining. First, in the ordinary focus, weakly stained, thin, gram-positive bacilli are found; second, with a slightly longer focus distance, the thin, cord-like, conspicuous gram-positive bacilli can be observed; and third, with a shorter focus distance, the gram-positive bacilli have changed into the brightened, colorless, or ghost ones. Four laboratory technologists each evaluated 20 Gram-stained samples after being lectured on the criteria, with no prior information about the sample. They accurately evaluated the presence of the bacilli in Gram-stained preparations in more than 90% of samples containing 3+ bacilli on Ziehl–Neelsen staining. Gram staining is available as an easy and rapid initial clue to recognize highly infective tuberculosis.     

Rapid Perceptual Switching of a Reversible Biological Figure

Certain visual stimuli can give rise to contradictory perceptions. In this paper we examine the temporal dynamics of perceptual reversals experienced with biological motion, comparing these dynamics to those observed with other ambiguous structure from motion (SFM) stimuli. In our first experiment, naïve observers monitored perceptual alternations with an ambiguous rotating walker, a figure that randomly alternates between walking in clockwise (CW) and counter-clockwise (CCW) directions. While the number of reported reversals varied between observers, the observed dynamics (distribution of dominance durations, CW/CCW proportions) were comparable to those experienced with an ambiguous kinetic depth cylinder. In a second experiment, we compared reversal profiles with rotating and standard point-light walkers (i.e. non-rotating). Over multiple test repetitions, three out of four observers experienced consistently shorter mean percept durations with the rotating walker, suggesting that the added rotational component may speed up reversal rates with biomotion. For both stimuli, the drift in alternation rate across trial and across repetition was minimal. In our final experiment, we investigated whether reversals with the rotating walker and a non-biological object with similar global dimensions (rotating cuboid) occur at random phases of the rotation cycle. We found evidence that some observers experience peaks in the distribution of response locations that are relatively stable across sessions. Using control data, we discuss the role of eye movements in the development of these reversal patterns, and the related role of exogenous stimulus characteristics. In summary, we have demonstrated that the temporal dynamics of reversal with biological motion are similar to other forms of ambiguous SFM. We conclude that perceptual switching with biological motion is a robust bistable phenomenon.     

Nonspecific vaginitis and Gardnerella vaginalis: methodological and epidemiological notes on 328 cases

328 samples of vaginal discharges in adult fertile women, aged between 16 and 48, were examined in this trial. After excluding 95 women (39 were affected by vaginal Trichomoniasis, 55 whose discharge presented yeasts and 1 affected by gonococcal infection), the authors divided the remaining 233 cases into subjects affected or not affected by nonspecific vaginitis (NSV) only using a Gram-stained smear. In the 83 patients affected by NSV, the authors isolated Gardnerella vaginalis in the 90.4% of the cases. After statistically correlating the different parameters described in literature as associated to NSV (pH, KOH-test, Clue Cells, isolation of G. vaginalis, symptoms), the authors concluded that: a) Gram-stained smear may be used as a simple microscopical test for diagnosis, in most of the cases, NSV; b) in most of the cases of NSV are implicated more organisms (essentially G. vaginalis and anaerobic organisms); c) G. vaginalis can be considered as a marker of these infections.     

Reproducibility and observer variability of transrectal ultrasound measurements of prostatic volume

Transrectal ultrasound (TRUS) is an established investigation in benign and malignant prostatic disease though the level of reproducibility of TRUS volume measurements is not known. A group of observers in the UK and the USA who were participating in linked prospective studies of benign prostatic hyperplasia each carried out measurements during real-time scanning and on a series of static TRUS images. Results demonstrated good reproducibility for measurements of antero-posterior, transverse, and longitudinal dimensions of the prostate by an experienced UK urologist; only a minor degree of interobserver variation occurred in measurements made between experienced UK and USA observers. Using static images, USA observers obtained results that were in good agreement, whereas the UK observers, only two of whom used TRUS regularly, were in poor agreement with each other. This study demonstrates a high level of reproducibility for TRUS volume measurements performed by experienced observers.     

Diagnosis of pulmonary tuberculosis complicating HIV infection: superiority of sputum smear over bronchoalveolar lavage.

Tuberculosis should be prominently considered in the differential diagnosis when an HIV-infected patient has respiratory symptoms. Sputum smears and culture for acid-fast organisms should always be obtained. Fiberoptic bronchoscopy to exclude other concurrent opportunistic infections is appropriate because Pneumocystis carinii complicates as many as 25% of the cases of pulmonary TB, but acid-fast sputum smears should always be obtained. It should not be assumed that bronchoalveolar lavage is superior to expectorated sputum smear for the rapid diagnosis of pulmonary tuberculosis.     

Evaluation of the Gram stain of sputa from cystic fibrosis patients to predict the presence of Staphylococcus aureus

We evaluated the presence of Gram-positive cocci in cluster, seen in Gram-stained smears of cystic fibrosis (CF) sputa versus the microbiological culture for the prediction of the presence of S. aureus. Gram stain provided low accuracy (69.2%; odds ratio, 4.8; 95% confidence interval, 61.3–76.1) for predicting S. aureus in CF sputum.     

Demonstration and utility of clustered pseudohyphae on Gram-stained smears from Candida albicans-positive blood cultures

The presence of clustered and branched pseudohyphae was investigated on Gram-stained smears of 78 consecutive yeast-positive blood cultures. The accuracy of the method was 96.1%, with a positive predictive value of 96.6% for Candida albicans. These findings demonstrate that the presence of clustered and branched pseudohyphae on Gram stain may be used for the rapid and presumptive identification of C. albicans from yeast-positive blood culture bottles.     

Multi-modal registration for correlative microscopy using image analogies

Correlative microscopy is a methodology combining the functionality of light microscopy with the high resolution of electron microscopy and other microscopy technologies for the same biological specimen. In this paper, we propose an image registration method for correlative microscopy, which is challenging due to the distinct appearance of biological structures when imaged with different modalities. Our method is based on image analogies and allows to transform images of a given modality into the appearance-space of another modality. Hence, the registration between two different types of microscopy images can be transformed to a mono-modality image registration. We use a sparse representation model to obtain image analogies. The method makes use of corresponding image training patches of two different imaging modalities to learn a dictionary capturing appearance relations. We test our approach on backscattered electron (BSE) scanning electron microscopy (SEM)/confocal and transmission electron microscopy (TEM)/confocal images. We perform rigid, affine, and deformable registration via B-splines and show improvements over direct registration using both mutual information and sum of squared differences similarity measures to account for differences in image appearance.     

Aortic aneurysm volume calculation: effect of operator experience

Background We have successfully applied sequential volumetric analysis of abdominal aortic aneurysms to exclude endoleak in patients who have an aortic endostent. This study compared the effect of variable operator experience on volumetric calculation accuracy.Methods Four operators with different experience levels calculated abdominal aneurysm volumes in 10 patients at two different times (≥ 1 week apart). The four reviewers were ranked as having a high level of experience (one full-time laboratory worker specializing in three dimensions with 3 years of experience), a moderate level of experience (one part-time laboratory worker specializing in three dimensions/computed tomographic technician with 1 year of part-time experience), and a low level of experience (two individuals taught volumetric measurements for the purposes of this study: a fellow in abdominal imaging and a computed tomographic technician). All volumes were calculated with a GE Advantage 4.0 workstation (General Electric, Waukesha, WI, USA).Results Mean aneurysm volume and volume difference between two measurements were calculated for four operators. The average (standard deviation) percent volume differences were 1.2% (0.2%) for the experienced reader, 3.2% (0.3%) for the moderately experienced reader, and 6.0% (1.0%) and 5.8% (1.1%) for the two readers with light experience. Differences between averages were statistically significant (p < 0.005).Conclusion We have defined a percent margin of error for aortic aneurysm volume measurement and have shown a direct correlate to level of experience. Diagnosis of endoleak based on aneurysm volume enlargement on serial scans needs to account for the level of operator experience.     

Classification of two species of Gram-positive bacteria through hyperspectral microscopy coupled with machine learning

Gram stain is one of the most common techniques used to visualize bacteria under microscopy and classify bacteria into two large groups (Gram-positive and Gram-negative). However, such an inaccurate classification is unfavorable for bacterial research. For instance, soil-rhizosphere bacteria, Bacillus megaterium (B. megaterium) and Bacillus cereus (B. cereus) have different effects on plants, nonetheless, they are both Gram-positive and difficult to be differentiated. Here, we present a method to precisely classify Gram-positive bacteria via hyperspectral microscopy. The pH-value differences in the intracellular environment of various types of bacteria can lead to different ionization of the auxochrome of crystal violet (CV) molecules during the Gram stain process. Consequently, there is a subtle difference in the absorption peak of Gram-stained bacteria. Harnessing hyperspectral microscopy can capture this subtle difference and enable precise classification. Besides the spectral features, the spatial features were also used to improve the quality of bacterial identification. The results show that the classification accuracy of two species of Gram-positive bacteria, B. megaterium and B. cereus, is up to 98.06%. We believe this method can be used for other Gram-positive bacteria and Gram-negative bacteria, realizing a more elaborate classification for Gram-stained bacteria.     

False-positive blood culture results in patients with hematologic malignancies

Blood cultures are the most valuable tool when bacteremia is clinically suspected. Technical advances have led to the development of automated blood culture systems to detect bacterial infections. Usually positive signals in automated blood culture systems result from the proliferation of microorganisms. Cases are classified as false-positive when the automated blood culture system produces a positive signal but no microorganisms are detected on Gram-stained smears and no microorganism growth is observed in blood subcultures. False-positive blood culture results are very rare in patients with hematologic malignancies. Recently, we encountered four patients who had false-positive blood culture results. Two of the patients were diagnosed with acute leukemia, involving hyperleukocytosis and an excess of blasts. The other two patients were diagnosed with acute leukemia and diffuse large B cell lymphoma with leukocytopenia. Although hypercapnia or acidosis, apart from hyperleukocytosis, might also cause false-positive results, our cases clearly did not have these conditions. We should be aware of the possibility that false-positive blood culture results can occur in patients with leukocytopenia, as well as hyperleukocytosis. To understand the mechanisms responsible for the observed false-positive results, additional studies are needed after the accumulation of similar cases.