Aerobic Vaginitis Diagnosis Criteria Combining Gram Stain with Clinical Features: An Establishment and Prospective Validation Study

Wet-mount microscopy aerobic vaginitis (AV) diagnostic criteria need phase-contrast microscopy and keen microscopists, and the preservation of saline smears is less common in clinical practice. This research work developed new AV diagnostic criteria that combine Gram stain with clinical features. We enrolled 325 AV patients and 325 controls as a study population to develop new AV diagnostic criteria. Then, an independent group, which included 500 women, was used as a validation population. AV-related microscopic findings on Gram-stained and wet-mount smears from the same participants were compared. The accuracy of bacterial indicators from the two methods was verified by bacterial 16S rRNA V4 sequencing (n = 240). Logistic regression was used to analyse AV-related clinical features. The screened clinical features were combined with Gram-stain microscopic indicators to establish new AV diagnostic criteria. There were no significant differences in the leukocyte counts or the parabasal epitheliocytes (PBC) proportion between the Gram-stain and wet-mount methods (400×). Gram stain (1000×) satisfied the ability to identify bacteria as verified by 16S rRNA sequencing but failed to identify toxic leukocytes. The new criteria included: Lactobacillary grades (LBG) and background flora (Gram stain, 1000×), leukocytes count and PBC proportion (Gram stain, 400×), and clinical features (vaginal pH > 4.5, vagina hyperemia, and yellow discharge). These criteria satisfied the accuracy and reliability for AV diagnosis (Se = 86.79%, Sp = 95.97%, and Kendall’s W value = 0.899) in perspective validation. In summary, we proposed an alternative and valuable AV diagnostic criteria based on the Gram stain, which can make it possible to diagnose common vaginitis like AV, BV, VVC, and mixed infections on the same smear and can be available for artificial intelligence diagnosis in the future.

Lelliottia Rebaudianalis Sp. Nov. Isolated From Wild Stevia Rebaudiana Bertoni

A Gram-stain-negative, aerobic, chemoheterotrophic bacterium, characterized with rod shape and mobility, designated as LST-1T, was isolated from wild Stevia rebaudiana Bertoni and subjected to polyphasic taxonomic analysis. The LST-1T strain grew optimally at 37 °C and pH 6.0–7.0 in the presence of 0.5 % (w/v) NaCl. Phylogenetic sequence analysis based on 16S rDNA from LST-1 indicated that it is close to Lelliottia jeotgali (99.85%), Lelliottia nimipressuralis (98.82%), and Lelliottia amnigena (98.54%). Multi-locus sequence typing analysis of concatenated partial recA, atpD, and infB was performed to improve resolution, and clear distinctions between the closest related type strains were exhibited. Meanwhile, the results from average nucleotide identify analyses and DNA–DNA hybridization with four species (16S rDNA similarity > 98.65%) were less than 90% and 40% respectively, verifying the distinct characteristics from other species of Lelliottia, The cellular fatty acid profile of the strain consisted of C16:0, Summed Feature3, and Summed Feature8 (may be 16:1 w6c/16:1 w7c and 18:1 w6c) as major components. The major polar lipids included phosphatidylethanolamine, phosphatidylglycerol, aminophospholipid, three non-characteristic phospholipids, and a non-characteristic lipid. The genome of LST-1T is 4,611,055 bp, with a DNA G + C content of 55.02%. Combination of several phenotypic, chemotaxonomic, and genomic characteristics proved that the LST-1T strain does represent a novel genus, for which the name Lelliottia sp. LST-1 was proposed. The type strain is LST-1T (= CGMCC 1.19175T = JCM 34938T).

Differentiating necrotizing soft tissue infections from cellulitis by soft tissue infectious fluid analysis: a pilot study

Background We conducted this study to evaluate the characteristics of the infectious fluid in soft tissue infection and investigate the utility of the biochemical tests and Gram stain smear of the infectious fluid in distinguishing necrotizing soft tissue infection (NSTI) from cellulitis. Methods This retrospective cohort study was conducted in a tertiary care hospital in Taiwan. From April 2019 to October 2020, patients who were clinically suspected of NSTI with infectious fluid accumulation along the deep fascia and received successful ultrasound-guided aspiration were enrolled. Based on the final discharge diagnosis, the patients were divided into NSTI group, which was supported by the surgical pathology report, or cellulitis group. The t test method and Fisher’s exact test were used to compare the difference between two groups. The receiver–operator characteristic (ROC) curves and area under the ROC curve (AUC) were used to evaluate the discriminating ability. Results Total twenty-five patients were enrolled, with 13 patients in NSTI group and 12 patients in cellulitis group. The statistical analysis showed lactate in fluid (AUC = 0.937) and LDH in fluid (AUC = 0.929) had outstanding discrimination. The optimal cut-off value of fluid in lactate was 69.6 mg/dL with corresponding sensitivity of 100% and specificity of 76.9%. The optimal cut-off value of fluid in LDH was 566 U/L with corresponding sensitivity of 83.3% and a specificity of 92.3%. In addition, albumin in fluid (AUC = 0.821), TP in fluid (AUC = 0.878) and pH in fluid (AUC = 0.858) also had excellent diagnostic accuracy for NSTI. The Gram stain smear revealed 50% bacteria present in NSTI group and all the following infectious fluid culture showed bacteria growth. Conclusions The analysis of infectious fluid along the deep fascia might provide high diagnostic accuracy to differentiate NSTI from cellulitis.

Point-of-Care Urine Gram Stain for Managing Febrile Urinary Tract Infection in Adults

Background Febrile urinary tract infections (fUTIs), which include pyelonephritis, prostatitis, and urosepsis, are the most common cause of sepsis. However, the treatment has become more complex because of the world-wide increase in antimicrobial resistance (AMR). The objective of this study was to clarify whether point-of-care Gram stain (PCGS) of urine contributed to fUTI diagnosis and treatment in adults. Methods This hospital-based observational study was undertaken between January 2013 and March 2015 in Okinawa, Japan. All enrolled patients were adults who had been admitted to the Division of Infectious Diseases with suspected fUTI. The usefulness of PCGS results were compared for urinalysis (U/A) and urine cultures (U/Cs). The targeted therapy type and its effectiveness based on PCGS were analyzed, and each was investigated in two groups: the uncomplicated pyelonephritis group and the complicated pyelonephritis/prostatitis group. Results Two hundred sixty-six patients were enrolled. The results of PCGS were closely correlated with those of U/A for pyuria and bacteriuria, and moderately correlated with the results of U/C for bacterial types. In the uncomplicated group, narrow-spectrum antimicrobials such as cefotiam were initially selected in 97.9% (47/48) of patients, and their effectiveness was 97.9% (47/48). In the complicated group, the effectiveness was 84.2% (186/221) (p=0.009) despite frequent AMRs (14.7%; 32/218) and low use of broad-spectrum antimicrobials such as carbapenems (7.7%; 17/221). Conclusion Urine PCGS led to a more precise fUTI diagnosis and prompted clinicians to select narrower-spectrum antibiotics with high effectiveness.

Arthrobacter cheniae and Arthrobacter frigidicola sp. nov., isolated from a glacier

Two Gram-stain-positive, aerobic, rod-shaped, pink and light pink colony-forming bacteria, designated as Hz2T and MDT2-14T, respectively, were isolated from glacier cryoconite samples. Comparisons based on 16S rRNA gene sequences showed that strains Hz2T and MDT2-14T take Arthrobacter bussei KR32T and Arthrobacter zhaoguopingii J391T as their closest neighbours, respectively. The average nucleotide identity values between the two novel strains and their closest relatives were 83.56 and 93.06 %, respectively. The two strains contain MK-9(H2) as their predominant menaquinone. The polar lipids of both strains were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. The major fatty acids of strain Hz2T were anteiso-C15 : 0, summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c) and iso-C15 : 0, while the major fatty acids of strain MDT2-14T were anteiso-C15 : 0 and anteiso-C17 : 0. Based on these data, we propose two novel species, Arthrobacter cheniae sp. nov. (Hz2T = CGMCC 1.9262T=NBRC 113086T) and Arthrobacter frigidicola sp. nov. (MDT2-14T=CGMCC 1.9882T=NBRC 113089T).

Development of Gram Stain Scoring System Based on Pro-Inflammatory Cytokines in the Sheep Model for Testing Toxicity of Vaginal Products

Background: Development of safe, effective products to prevent the sexual transmission of HIV remains a priority. Prior to clinical testing, the products must undergo strict safety evaluations to avoid mucosal drug toxicity, inflammation, and vaginal microbiome (VMB) shifts. Based on the Food and Drug Administration (FDA) guidance, we designed a study to measure the inflammatory markers and VMB changes after intravaginal treatment with products that have been associated with toxicity, with the objective to develop a Gram stain slide scoring system, similar to Nugent scoring, correlated with the proinflammatory cytokines in sheep.Methods: Non-pregnant Dorset ewes (n = 34) were randomized to receive 5 ml intravaginal 4% nonoxynol-9 (N9) contraceptive gel, positive control (0.2% benzalkonium chloride), placebo control [hydroxethyl cellulose (HEC)], or no application daily for 10 days, with 11-day post-treatment follow-up. The vaginal swabs were collected for the cytokines, VMB, and Gram-stained slides. An enzyme-linked immunosorbent assay (ELISA) analysis of cytokines interleukin (IL)-1β, IL-8, CXCL10, and tumor necrosis factor-α (TNF-α) was used to determine inflammatory state of the sample. Vaginal microbiome community types (CT) were utilized to create five equivalent slide subsets for iterative development of a Gram-stained slide scoring system with comparisons with inflammatory state based on the cytokine levels.Results: Digital images of the Gram-stained slides were scored based on Gram staining and morphology of bacteria, presence of sheep epithelial cells, and immune cells. The scoring system was modified in an iterative fashion with weighting based on cytokine categorization of inflamed samples, with three of four cytokine values above the mean indicating that the sample was inflamed. The parameters in the final version of the scoring system included mature epithelial cells, Gram-negative rods, and Gram-positive diplococci indicating normal and immune cells indicating inflammation. The area under the receiver operator characteristic curve (ROC AUC) was 0.725 (ROC AUCs range between 0.5 and 1.0) with a greater area indicating higher diagnostic ability of a test with a binary outcome: inflamed or normal.Conclusion: The scoring system, derived from the advanced VMB and cytokine analyses, provides a validated, practical method for quantification of Gram-stained slides that can be performed in most laboratories, increasing the potential for standardization. The training plan can assist laboratories to determine the safety of intravaginal products in their sheep studies or the methodological approach can be applied to other animal models where such data are also needed.

What are the best diagnostic tests for diagnosing bacterial arthritis of a native joint?

Aims This study aimed to answer two questions: what are the best diagnostic methods for diagnosing bacterial arthritis of a native joint?; and what are the most commonly used definitions for bacterial arthritis of a native joint? Methods We performed a search of PubMed, Embase, and Cochrane libraries for relevant studies published between January 1980 and April 2020. Of 3,209 identified studies, we included 27 after full screening. Sensitivity, specificity, area under the curve, and Youden index of diagnostic tests were extracted from included studies. We grouped test characteristics per diagnostic modality. We extracted the definitions used to establish a definitive diagnosis of bacterial arthritis of a native joint per study. Results Overall, 28 unique diagnostic tests for diagnosing bacterial arthritis of a native joint were identified. The following five tests were deemed most useful: serum ESR (sensitivity: 34% to 100%, specificity: 23% to 93%), serum CRP (sensitivity: 58% to 100%, specificity: 0% to 96%), serum procalcitonin (sensitivity: 0% to 100%, specificity: 68% to 100%), the proportion of synovial polymorphonuclear cells (sensitivity: 42% to 100%, specificity: 54% to 94%), and the gram stain of synovial fluid (sensitivity: 27% to 81%, specificity: 99% to 100%). Conclusion Diagnostic methods with relatively high sensitivities, such as serum CRP, ESR, and synovial polymorphonuclear cells, are useful for screening. Diagnostic methods with a relatively high specificity, such as serum procalcitonin and synovial fluid gram stain, are useful for establishing a diagnosis of bacterial arthritis. This review helps to interpret the value of various diagnostic tests for diagnosing bacterial arthritis of a native joint in clinical practice. Cite this article: Bone Joint J 2021;103-B(12):1745–1753.

Gram stain and addition of amphotericin B to improve the microbial safety of human donor corneas

To determine the effectiveness of two methods to improve the microbial safety of human corneas preserved in organ culture. We compared the number of positive preservation solutions of corneas in organ culture in which the initial short-term hypothermic corneal maintenance solution was supplemented with amphotericin B 2.5 µg/mL and the historical data of microbial test results (2015–2019). In addition, we appraised the efficacy of Gram stain to detect bacterial or fungal contamination in the organ culture solutions of corneas from at-risk donors compared to the culture tests of corneas from not-at-risk donors. Statistical analysis was performed using STATA and statistical significance set at p < 0.05. The number of positive culture tests after preservation was 15 (0.5%) in 2020 compared to a mean of 37 (1.2%) in the period 2015–2019 (p < 0.01), with 10 (1.0%) positive samples in the cohort of 998 corneas from at-risk donors and 5 (0.2%) in the 2046 corneas from not-at-risk donors (p < 0.01). All corneas from at-risk donors tested positive at Gram stain and the results were available 1–3 days before those of the conventional culture tests. Amphotericin B supplementation in the short-term maintenance solution markedly reduced the number of positive microbial tests after organ culture and the early detection of contaminants, including slow-growing microorganisms, by Gram stain before the standard culture results. This meant fewer corneas being discarded and a greater likelihood of preventing post-graft infections.