Utility of point-of-care Gram stain by physicians for urinary tract infection in children ≤36 months.

Urinary tract infection (UTI) in children requires early diagnosis and treatment to prevent repeated UTI and renal scarring. This study aimed to evaluate the usefulness of the point-of-care Gram stain by physicians for suspected UTI in children at Okinawa Chubu Hospital as a rapid diagnostic test.A single-center, retrospective study was undertaken between January 2011 and December 2015. Patients aged 36 months or younger who were reviewed had suspected UTI in the emergency room or outpatient clinic. Urine culture, urinalysis, and point-of-care Gram stain were performed on a single specimen. Patients with structural or functional urological defects requiring routine catheterization were excluded. We compared the diagnostic performance among the rapid diagnostic tests (i.e., pyuria, point-of-care Gram stain, or both). Kappa statistics were used to evaluate the agreement between the results of point-of-care Gram stain and morphotypes of urine culture with the 95% CI (bias corrected bootstrap interval). We also analyzed which antibiotics were more susceptible to the bacteria of urine culture results, selected by the results of point-of-care Gram stain or empirical treatment based on the Japanese guidelines by McNemar test.Of 1594 patients reviewed in the study, 1546 were eligible according to our inclusion criteria. Using urine culture as the gold standard for UTI, the sensitivity and specificity of pyuria were 73.2% and 95.1%, whereas those of the point-of-care Gram stain were 81.4% and 98.2%, respectively. The concordance rate between the morphotypes of bacteria detected by point-of-care Gram stain and those of urine culture was 0.784 (kappa coefficient) (95% CI 0.736-0.831). Furthermore, the proportion of "susceptible" in the minimum inhibitory concentration of pathogen-targeted treatment based on the point-of-care Gram stain was higher than that of empirical therapy (exact McNemar significance probability: .0001).Our analysis suggests that the point-of-care Gram stain is a useful rapid diagnostic tool for suspected UTI in young children. Pathogen-targeted treatment based on the point-of-care Gram stain would lead to better antibiotic selection compared with empirical therapy.

Comparison of polymerase chain reaction and standard microbiological techniques in presumed bacterial corneal ulcers.

To assess utility of PCR in the diagnosis of bacterial corneal ulcer and to compare sensitivity and specificity of this technique with conventional laboratory methods. A prospective nonrandomized investigative study conducted on 122 eyes of presumed bacterial keratitis. Samples were collected for bacterial and fungal culture and Gram stain smear. A separate sample was taken for PCR with 26 gauge needle and was dipped directly into Eppendorff tube with lysis buffer in it. Diagnosis of culture proven bacterial keratitis was established in 53 (43.4 %) and most common bacterial isolate was staphylococcal sp. (83 %). Direct microscopic examination of Gram stained smear revealed presence of bacteria in 24 (23.9 %) specimens and PCR positivity was evident in 56 (45.5 %). In preantibiotic treated eyes culture was positive in 15 (30 %), Gram stain in 9 (18 %), and PCR in 18 (36 %). The same for untreated (fresh) eyes, positivity of culture as well as PCR was noted in 38 (52.7 %) and that of Gram stain was noted in 20 (27.7 %). Sensitivity of Gram stain and PCR was 45.28 and 88.68 % respectively; whereas specificity was 92.75 % for Gram stain and 86.96 % for PCR. The average time taken for PCR reaction was 4-8 h while culture reporting took at least 24-48 h. Our findings suggest that PCR is a good adjunct modality to the "Gold Standard" technique in the diagnosis of bacterial corneal ulcer.

A new rapid colourimetric method for testing Mycobacterium tuberculosis susceptibility to isoniazid and rifampicin: a crystal violet decolourisation assay

The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH) and rifampicin (RIF) resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA). Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution) was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple) was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.

A new rapid colourimetric method for testing Mycobacterium tuberculosis susceptibility to isoniazid and rifampicin: a crystal violet decolourisation assay.

The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH) and rifampicin (RIF) resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA). Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution) was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple) was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.

Re-evaluation of rejection criteria for endotracheal tube (ETT) specimens from adult patients.

The purpose of this study was to determine optimal criteria for microbiology laboratory screening of endotracheal tube (ETT) specimens submitted for bacterial culture from adult patients. ETT specimens from adult patients that were received by two microbiology laboratories were prospectively evaluated and subdivided into one of three study arms with the following criteria: <10 squamous epithelial cells (SECs) per low-power field with bacteria seen on Gram staining (arm 1), >10 SECs per low-power field with bacteria seen on Gram staining (arm 2) and <10 SECs per low-power field with no bacteria seen on Gram staining (arm 3). A fourth study arm (>10 SECs per low-power field with no bacteria seen on Gram staining) was planned but this arm was terminated due to the paucity of specimens meeting these criteria. Isolate evaluation was performed using standard microbiology protocols. A limited chart review was undertaken at one of the institutions, only reviewing patients from which a potential pathogen was recovered on culture. In total, 141 ETT specimens were evaluated. A potential respiratory pathogen was recovered from 54, 37 and 10 % of specimens in study arms 1, 2, and 3, respectively (P<0.0001, comparing between arm 1 and arm 3). For the 23 patients included in the chart review from whom a potential pathogen was recovered on culture, respiratory infection was considered to be present in 50 % (6/12) of patients in arm 1, 66.6 % (6/9) of patients in arm 2 and 100 % (2/2) of patients in arm 3. Therapy was rarely altered based on culture results. In this study, the ETT specimens submitted for bacterial culture were of limited benefit to clinicians. The data presented here support the use of an absence of bacteria on Gram staining as a rejection criterion for ETT specimens. The criterion of >10 SECs per low-power field should be further evaluated in a prospective study of patients with an unequivocal clinical diagnosis of pneumonia.

Mechanisms to assess Gram stain interpretation proficiency of technologists at satellite laboratories.

To address Gram stain interpretation proficiency in a satellite/centralized microbiology laboratory paradigm, two programs were devised. In quality assurance program 1, nonmicrobiology technologists at satellite laboratories were required to interpret standardized Gram-stained specimens of clinical material prepared by an experienced microbiologist at a central laboratory. In quality assurance program 2, clinical Gram stains prepared and read by the satellite laboratorians were reviewed by experienced microbiologists at the central laboratory. Satisfactory performance (94%) was achieved in quality assurance program 1. In contrast, quality assurance program 2 had a significantly lower overall performance (89%; P < 0.0001) due to poorer identification of host cells (93%) and bacteria (84%). A variety of intervention mechanisms, including continuous monitoring, resulted in overall performance improvement (P < or = 0.006). While a technologist challenge has educational merit, having a microbiologist review previously read slides is a better indicator of the technologist's Gram stain interpretation proficiency.

Errors in interpretation of Gram stains from positive blood cultures.

We reviewed major errors in Gram stain reports from positive blood cultures to identify patterns and potential clinical impact. During a 23-month period, blood cultures were misread for 57 (0.7%) of 8,253 patients. Of 5,885 read as gram-positive cocci, 6 (0.1%) had only gram-negative organisms by culture, 3 of which were Acinetobacter species Of 1,959 read as gram-negative bacilli, 25 (1.3%) had only gram-positive organisms by culture. Of these, 9 were Bacillus and 2 were Clostridium species. Nonrecognition of mixed Gram stains accounted for 28 errors that most often were associated with a reading of gram-positive cocci. In 4 cases, there were delays of 14 hours to 3 days in starting appropriate antibiotic treatment; 2 deaths occurred, although the erroneous Gram stain report probably was not contributory. Pathologists and laboratory personnel need to be aware of these types of misinterpretations and the potential effects on patient outcome.

Bacterial vaginosis in sexually experienced and non-sexually experienced young women entering the military.

OBJECTIVE: To estimate the prevalence of bacterial vaginosis by Nugent Gram stain criteria in a nonclinic national sample of young women entering recruit training; to examine clinical associations with bacterial vaginosis; and to evaluate the performance of a pH test card and Papanicolaou smear against Gram stain as screening tools for bacterial vaginosis. METHODS: A cross-sectional study of 1938 women was conducted. Self-collected vaginal swabs were applied to a colorimetric pH test card and a glass slide for Gram stain evaluation according to the Nugent criteria. Papanicolaou smears and samples for sexually transmitted diseases screening were collected during routine entry pelvic examinations. RESULTS: Bacterial vaginosis prevalence was 27%, with 28% in sexually experienced and 18% in non-sexually experienced women (P = .001). Bacterial vaginosis prevalence was 11% in Asian/Pacific Islanders, which was lower than in other nonwhite ethnic groups (P = .004). Clinically, bacterial vaginosis was directly related to multiple sexual partners (P = .026), self-report of vaginal discharge (P = .001), self-report of vaginal odor (P < .001), and concurrent Chlamydia trachomatis infection (P = .002), and inversely related to hormonal contraceptive use (P = .013). Vaginal discharge did not achieve statistical significance in multivariate analysis. Compared with the Nugent criteria, the sensitivities and specificities for bacterial vaginosis diagnosis were as follows: colorimetric pH test: 72% and 67%; Papanicolaou smear: 72% and 79%, respectively. CONCLUSION: Among these diverse young women, bacterial vaginosis occurs commonly in both sexually experienced and inexperienced young women and differs by race and ethnicity. The pH colorimetric test and Papanicolaou smear performed moderately well as screening tools for bacterial vaginosis. The inverse relationship of bacterial vaginosis with hormonal contraceptive use and its direct relationship with C. trachomatis need further study.

Trypanocidal constituents in plants 2.xanthones from the stem bark of Garcinia subelliptica.

The constituents of the stem bark of Garcinia subelliptica (Guttiferae) were investigated based on its trypanocidal activity against epimastigotes of Trypanosoma cruzi, the etiologic agent for Chagas' disease. As the active components, nine xanthones were isolated including two new ones, 4-hydroxybrasilixanthone B and 1,3,5,6-tetrahydroxy-4,7,8-tri(3-methyl-2-butenyl)xanthone. Their structures were determined by spectroscopic analysis. Trypanocidal activity against trypomastigotes, an infectious form of T. cruzi, was also estimated as well as cytotoxic activity. Fukugetin, the major component of the bark, showed no activity.

Use of urinary gram stain for detection of urinary tract infection in childhood.

In this study, urinary culture, urinary Gram stain, and four tests within the urinalysis, leukocyte esterase, nitrite, microscopyfor bacteria, and microscopyforpyuria, were examined in 100 children with symptoms suggesting urinary tract infection. Our purpose was to determine the validity of the urinary Gram stain compared with a combination of pyuria plus Gram stain and overall urinalysis (positiveness of nitrite, leukocyte esterase, microscopy for bacteria, or microscopy for white blood cell). Of 100 children, aged two days to 15 years, 70 (70 percent) had a positive urinary culture: 40 girls (57 percent) and 30 boys (43 percent). Escherichia coli was the most common isolated agent. The sensitivity and specificity of the urinary Gram stain were 80 percent and 83 percent, and that of the combination of pyuria plus Gram stain 42 percent and 90 percent, and that of the overall urinalysis 74 percent and 3.5 percent respectively. Our findings revealed that neither method of urine screen should substitute for a urine culture in the symptomatic patients in childhood.

Improving Gram-stained reproducible result by further adding clue cells in diagnosing bacterial vaginosis.

The reproducibility of interpretation in diagnosing bacterial vaginosis may be enhanced by adding pus cells and clue cells into two different criteria, developed by Spiegel et al. and Nugent et al. The purpose of study was designed to find out which parameter was more reproducible. 100 patients were collected with the diagnosis of bacterial vaginosis as an experimental group, while the other 100 patients who were with routine Papanicolaou smears in gynecologic clinic the collected as a control group. Two slides, including the original and reproducible ones, were obtained from vaginal smears for each patient. Three technicians read the slides randomly by using two different criteria, plus pus cells and clue cells. This showed the agreement for clue cells is the best method regardless of experimental group or control group (Kappa values between 0.708 and 1.000). The intra-observer agreement for the diagnosis of bacterial vaginosis by the method of Nugent et al. is superior to the method of Spiegel et al. Our data show the comparison of Amsel criteria versus Nugent criteria for the diagnosis of bacterial vaginosis with sensitivity of 88.9%, specificity of 55.4%, negative positive value of 62.1%, and positive predictive value of 85.8%. Moreover, our data also demonstrate the comparison of Amsel criteria versus the diagnosis either based on Nugent criteria or the presence of clue cells with sensitivity of 95.7%, specificity of 56.7%, negative positive value of 81.2%, and positive predictive value of 87.1%. The results demonstrate further adding score of the clue cells can enhance the reproducible diagnosis of bacterial vaginosis, which is superior to the methods of Nugent et al. and Spiegel et al.

Comparison of Gram stain and Pap smear procedures in the diagnosis of bacterial vaginosis.

OBJECTIVE: The purpose of this study was to examine the characteristics of Gram stain versus Pap smear in diagnosis of bacterial vaginosis (BV). METHODS: One-thousand and sixty women were enrolled in this study. All cases with symptoms of BV were determined by Amsel's criteria, which were accepted as the gold standard for diagnosis of BV. Pap smear and Gram stain evaluations were compared according to Amsel's criteria, without viewing the clinical results of the patients. Gram stain and Pap smear results were determined as negative or positive according to Amsel's criteria. Sensitivity, specifity and positive predictive values were calculated. RESULTS: After accepting the cases that were diagnosed as BV according to Amsel's criteria as reference cases, the sensitivity of the Gram stain method was calculated as 97% and the sensitivity of the Pap smear method as 93%. Similar specificity rates were obtained with both methods in diagnosis of BV related to the clinical results. There were no statistically significant differences in diagnosis of BV between these two groups. CONCLUSION: If Amsel's criteria are accepted as the gold standard for diagnosis of BV, Gram stain and Pap smear methods will give similar results in diagnosis.

Gram stain versus culture in the diagnosis of vulvovaginal candidiasis.

We evaluated Gram stain as a rapid diagnostic method for vulvovaginal candidiasis (VVC). Vaginal swabs were taken from 100 pregnant women and subjected to Gram stain and culture; 39% were diagnosed as having VVC (29% had symptomatic VVC and 10% had asymptomatic VVC). Candida albicans was isolated from 94.9% of all cultures while other Candida species were isolated from 5.1% of cases. Multigravidae were significantly more affected than primigravidae. Gram stain is a valuable method in rapid accurate diagnosis of symptomatic VVC and even superior to culture as it demonstrates the invasive forms of the yeast. However, its low sensitivity could restrict its use in routine practice. A combination of culture and Gram stain is the ideal approach for the diagnosis of VVC.

Quantitative gram stain interpretation criteria used by microbiology laboratories in Alberta, Canada.

Microbiology laboratories in Alberta, Canada, were surveyed to determine the quantitative interpretive criteria used to routinely read and report Gram stains. There was a wide variability in the quantitative reporting criteria cited for both cells and bacteria, with only 11 of 32 (34.4%) laboratories surveyed using the criteria recommended by the external proficiency-testing program. Lack of standardized criteria not only poses a problem in the grading of proficiency testing results but may also impact the quality of patient care.

Evaluating rapid diagnostic tests of intra-amniotic infection: Gram stain, amniotic fluid glucose level, and amniotic fluid to serum glucose level ratio.

OBJECTIVE: The aim of the study was to compare the diagnostic utility of the Gram stain, the amniotic fluid glucose level, and the ratio of amniotic fluid glucose level to serum glucose level in detecting intra-amniotic infection. STUDY DESIGN: We conducted a prospective study of 127 patients with preterm labor and 26 patients with preterm premature rupture of the membranes (153 total). All patients underwent amniocentesis to diagnose intra-amniotic infection. The diagnostic criterion for intra-amniotic infection was a positive amniotic fluid culture result. RESULTS: The Gram stain is 80% sensitive and 91% specific when a positive is considered the presence of white blood cells or bacteria. Amniotic fluid glucose level and the ratio of amniotic fluid glucose level to serum glucose level are significantly lower when amniotic fluid culture results are positive, but as diagnostic tests they are inferior to the Gram stain. Logistic regression models that combine predictors yield superior accuracy with respect to individual tests. The most accurate combination was amniotic fluid glucose level and Gram stain with white blood cells or bacteria. Although the number of patients with preterm premature rupture of the membranes was small in this study (n = 26), analysis of our data suggests that the diagnostic performance levels of these tests were similar when used in patients with preterm labor and intact membranes and in patients with premature rupture of the membranes. CONCLUSIONS: The amniotic fluid glucose level and the ratio of amniotic fluid to serum glucose level have equivalent diagnostic utility and are inferior to the Gram stain. The combination of Gram stain with amniotic fluid glucose level is superior to any individual test.