The Triphenyl Tetrazolium Chloride (Uroscreen) Test Alone and in Combination with the Gram Smear as a Screening Procedure for Significant Bacteriuria in Hospital Patients

Of 725 specimens of urine examined by the triphenyl tetrazolium chloride (TTC) [Uroscreen], pour plate and calibrated loop procedures, 30% yielded bacterial colony counts greater than 100,000/ml.; a 100% correlation was obtained among the three methods. Of 539 urine specimens containing more than 100,000 bacteria/ml., 517 (94.06%) gave a positive TTC test.Because of the high correlation between the TTC test and bacterial quantitative counts, the method of TTC in conjunction with smears was adopted as a routine procedure. Specimens which were TTC-negative and smear-negative were discarded. Of 1227 specimens from hospital in-patients and 349 outpatients, 369 urines showed significant bacteriuria (337 from hospital in-patients and 32 from outpatients). There was complete correlation between the TTC test and smear. Of 337 isolations, 27 (8.02%) gave a negative TTC test but a positive smear.     

Rapid screening for bacteriuria with a laser nephelometer

Detection of significant bacteriuria with a laser nephelometer was evaluated in this study and compared with the results obtained by the quantitative loop method. We screened 1002 urine specimens and 220 (21.95%) were found to be positive at greater than or equal to 10(5) colony-forming units (CFU)/mL of urine by the standard method. Of the 220 positive specimens, 210 (95.4%) were detected in 6 h or less and 177 (80.4%) were detected within 3 h. The false-positive rate was 2.3% at 3 h and 19.7% at 6 h. These findings suggest that a 6-h procedure is necessary to detect 95% or more of significant bacteriuria. Laser nephelometer is versatile and can be used for rapid screening of bacteriuria.     

Free DNA in urine: a new marker for bladder cancer? Preliminary data

The aim of the present preliminary study was to investigate the presence of free DNA (FDNA) in urine as a possible marker for the diagnosis of bladder cancer. Naturally voided morning urine specimens were collected from 57 patients with suspected bladder cancer before cystoscopy. A standard urine test was performed; the specimens were then processed in order to obtain a quantitative evaluation of the presence of free DNA in the urine. Twenty-two patients were excluded from the study because they had leukocyturia and/or bacteriuria. Free DNA concentrations higher than 250 ng/mL were found in all 16 patients showing bladder cancer at cystoscopy and in seven (36.8%) of the 19 patients with negative cystoscopy. Urinary FDNA seems to have an excellent sensitivity: we observed no false negative cases and 36.8% false positive cases. By contrast, only 6.25% of the bladder cancer patients had positive urine cytology. Our results seem promising, although further studies and larger numbers are needed to define urinary free DNA as a reliable marker of bladder cancer.     

Simple Disposable Method for Quantitative Cultures of Urine

A disposable kit was tested as a means of detecting significant bacteriuria by quantitative culture of urine. The total error in 3,563 specimens tested by five investigators was less than 1%. The method was very effective in differentiating significant bacteriuria, i.e., more than 100,000 bacteria per ml of urine from uninfected urine. In specimens from patients with urinary tract abnormalities who had mixed bacterial flora, the absolute numbers obtained with the dip-inoculum method had a 10% variation when compared to results obtained by calibrated loop or dilution pour plate methods. Therefore, the main utility of the kit is for screening and following patients after therapy. A significant delay in time between inoculation of the medium in the kit with the freshly voided urine and incubation of the kit to promote growth did not affect the reliability of the kit as a method of doing quantitative urine cultures to detect bacteriuria.     

Rapid Detection of Gram-Negative Bacteriuria by Use of the Limulus Endotoxin Assay

The Limulus in vitro endotoxin assay was evaluated as a possible method for the prompt detection of significant gram-negative bacteriuria in children. This assay is capable of detecting endotoxin associated with intact cell walls of viable gram-negative bacteria as well as free endotoxin. Quantitative results are obtained following a 1-h incubation of Limulus lysate and 10-fold dilutions of otherwise untreated urine. A standard curve of Limulus activity and viable cell counts of Escherichia coli and Klebsiella pneumoniae in urine demonstrated that a positive Limulus reaction at a dilution of 1:100 or 1:1,000 indicated a colony count of at least 100,000 bacteria/ml. A positive Limulus reaction only from undiluted urine or at a dilution of 1:10 indicated less than 100,000 cells/ml. These experimental observations were confirmed by comparing the Limulus test with quantitative plate counts on 209 urine specimens from a mixed pediatric population. These results indicate that the Limulus assay is a simple, accurate method for rapid presumptive detection of gram-negative bacteriuria in patients where an immediate diagnosis is needed. This test would also seem promising for screening large patient populations for bacteriuria or for monitoring the effectiveness of treatment of urinary tract infections.     

Comparison of a new system (Compactdry SCD) with conventional methods for quantitative urine cultures

AIMS: Compactdry SCD, a new quantitative, ready-to-use and self-diffusible dry medium sheet urine culture system, was compared with conventional methods to evaluate the results of quantitative urine cultures. METHODS & RESULTS: Compactdry SCD was tested on 25 urine specimens, and results compared with those of traditional culture methods. The results from Compactdry SCD analysis correlated well with those from the standard plate count (SPC) method. In fact, the correlation was stronger than that dipslide systems and SPC. Even low-count bacteriuria (< 103 cfu ml(-1) and mixed bacteriuria were detected by Compactdry SCD. CONCLUSIONS: The Compactdry SCD system provides results comparable to those obtained by SPC: simple interpretation, ease of use, long-term storage and good sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report suggesting that the Compactdry SCD system has many advantages over traditional quantitative urine culture methods and that it is both appropriate and practical for clinical use.     

Two rapid urine screens for detection of bacteriuria: An evaluation

Five hundred twenty-five random clean catch urine specimens, collected from 339 adult females, 137 adult males, and 49 pediatric patients, were screened for the presence of bacteriuria with the Uriscreen catalase test and with the Chemstrip 2 LN dipstick. Quantitative cultures were performed on all specimens. The sensitivity, specificity, positive predictive value, and negative predictive value for the catalase test, with 10⁵ CFU/ml as the threshold for significant bacteriuria, were 91.3%, 72.3%, 33.7%, and 98.0%, respectively. Values for the dipstick were 83.9%, 77.9%, 43.7%, and 96.0%. when 10⁴ CFU/ml was used as the threshold, the catalase test had a sensitivity of 89.2%, specificity of 70.4%, positive predictive value of 37.3%, and a negative predictive value of 97.0%. Values for the dipstick at that level were 82.3%, 77.5%, 48.6%, and 94.8%. While the catalase test was more sensitive than the dipstick, it was our opinion that high rates of false-negatives associated with these methods negated the convenience of these fast and simple urine screens.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

Comparison of 2 methods of qualitative bacteriological urinalysis

Two techniques of the quantitative bacteriological urinalysis were compared. Hundred seventy eight samples of the urine were analysed with routine technique and paper strip test "Mast Bacteriuritest". Hundred percent conformity of both techniques was obtained in case of insignificant bacteriuria. In case of significant bacteriuria the results differed: paper tests were negative in 10% of cases. Significant bacteriuria was diagnosed in the samples in which Gould's test was positive with routine technique in 22% and with paper test in 18% of the analysed samples. It seems that paper test is valuable quantitative technique of the urinalysis because of its simplicity and low cost. It should be used, however, for the detection of the significant bacteriuria.     

尿蛋白定性与定量检测结果的差异性和相关性分析

目的:探讨尿液干化学法及免疫透射比浊法检测尿白蛋白结果的差异性及相关性。方法:对514例住院患者随机尿标本进行尿液干化学法及免疫透射比浊法尿蛋白的检测。结果:尿液干化学法阳性率为82.1%,免疫透射比浊法阳性率为72.8%。两种方法检测结果均为阴性标本的符合率为98.9%,为(±)的标本二者符合率为69.7%,为(+)的标本二者符合率为75.6%,为(++)的标本二者符合率为67.2%,为(+++)标本中二者符合率为42.5%,为(++++)标本二者的符合率为37.5%。两种方法的检测结果有显著性差异(P0.05);UmAlb/Ucr、NAG、和NAG/Ucr与UmAlb具有显著相关(P0.05),且UmAlb/Ucr与UmAlb的相关性最高。两种方法所得等级结果比较,++~+++之间差异有统计学意义(P0.05),-~±、±~+、+~++、+++~++++之间差异均无统计学意义(P0.05)。结论:尿蛋白定性与定量检测结果存在显著性差异,而UmAlb/Ucr与UmAlb相关性较高。在泌尿系统疾病的诊断中,检测尿中UmAlb比尿常规更有意义。     

尿蛋白定性与定量检测结果的差异性和相关性分析

目的：探讨尿液干化学法及免疫透射比浊法检测尿白蛋白结果的差异性及相关性。方法：对514例住院患者随机尿标本进行尿液干化学法及免疫透射比浊法尿蛋白的检测。结果：尿液干化学法阳性率为82．1％,免疫透射比浊法阳性率为72．8％。两种方法检测结果均为阴性标本的符合率为98．9％,为（±）的标本二者符合率为69．7％,为㈩的标本二者符合率为75．6％,为（＋＋）的标本二者符合率为67．2％,为（卅）标本中二者符合率为42．5％,为（＋＋H）标本二者的符合率为37．5％。两种方法的检测结果有显著性差异（P〈O．05）;UmAlb／Ucr、NAG、和NAG／Ucr与UmAlb具有显著相关（P〈0．05）,且UmAlb／Ucr与UmAlb的相关性最高。两种方法所得等级结果比较,＋＋～卅之间差异有统计学意义（P〈0．05）,-～±、±～＋、＋～＋＋、＋＋＋～＋＋＋＋之间差异均无统计学意义（P〉0．05）。结论：尿蛋白定性与定量检测结果存在显著性差异,而UmAlb／Ucr与UmAlb相关性较高。在泌尿系统疾病的诊断中,检测尿中UmAlb比尿常规更有意义。     

Use of Pyromark Q96 ID pyrosequencing system in identifying bacterial pathogen directly with urine specimens for diagnosis of urinary tract infections

Detection and identification of bacterial etiology in urine is critical for accurate diagnosis and subsequent rational treatment of urinary tract infections (UTIs). Urine culture followed by a series of biochemical reactions is currently the standard method for detecting and distinguishing microorganisms associated with UTIs. The whole procedure commonly takes more than 24 h. Here we developed a new system combining 16S rRNA gene broad-range PCR with pyrosequencing technology that allows for bacteria detection and identification in urine in 5 h. To evaluate this system for rapid diagnosis of bacteriuria, 768 urine specimens were collected from patients with suspected UTIs and were tested side-by-side using standard urine culture-based identification method and the pyrosequencing method. The results from pyrosequencing correlated well with those from traditional culture-based identification method. The overall agreement between these two methods reached 98.0% (753/768). In addition, we tested the sensitivity of pyrosequencing method and determined that urine bacterial numbers as low as 10⁴ cfu/ml could be accurately detected and identified. In conclusion, compared with traditional biochemical method, the PCR-pyrosequencing system significantly improved the detection and identification of bacteriuria with shorter time, higher accuracy, and higher throughput, thus allowing earlier pathogen-adapted antibiotic therapy for patients.     

A Real-Time PCR-Based Semi-Quantitative Breakpoint to Aid in Molecular Identification of Urinary Tract Infections

This study presents a novel approach to aid in diagnosis of urinary tract infections (UTIs). A real-time PCR assay was used to screen for culture-positive urinary specimens and to identify the causative uropathogen. Semi-quantitative breakpoints were used to screen for significant bacteriuria (presence of ≥10⁵ CFU/ml of uropathogens) or low-level bacteriuria (containing between 10³ and 10⁴ CFU/ml of uropathogens). The 16S rDNA-based assay could identify the most prevalent uropathogens using probes for Escherichia coli, Pseudomonas species, Pseudomonas aeruginosa, Staphylococcus species, Staphylococcus aureus, Enterococcus species and Streptococcus species. 330 urinary specimens were analysed and results were compared with conventional urine culture. Using a PCR Ct value of 25 as semi-quantitative breakpoint for significant bacteriuria resulted in a sensitivity and specificity of 97% and 80%, respectively. In 78% of the samples with monomicrobial infections the assay contained probes to detect the bacteria present in the urine specimens and 99% of these uropathogens was correctly identified. Concluding, this proof-of-concept approach demonstrates that the assay can distinguish bacteriuria from no bacteriuria as well as detect the involved uropathogen within 4 hours after sampling, allowing adequate therapy decisions within the same day as well as drastically reduce consequent urine culturing.     

A Comparative Study of the Triphenyltetrazolium Chloride (Uroscreen) Test and Conventional Methods for the Detection of Bacteriuria

Acute urinary tract infection may be preceded by and active pyelonephritis may be associated with asymptomatic bacteriuria. Treatment of asymptomatic bacteriuria may prevent or arrest active, chronic pyelonephritis and its sequelae. Consequently, there is a need for a reliable and simple screening procedure to detect asymptomatic bacteriuria in large segments of the population.The reliability and practicability of tests advocated for the detection of bacteriuria, including the new chemical triphenyltetrazolium chloride (T.T.C.) (Uroscreen) test, were evaluated. Reliability was assessed by correlating results of these tests with bacterial counts of tested urines. Significant bacteriuria is defined as the presence of 100,000 or more organisms per ml. of urine.The T.T.C. (Uroscreen) test was positive in 92.5% of cases of bacteriuria; there were 7.5% false-negative and 2.8% false-positive results. Bacteria on Gram-stained smear were found in 95.5% of the cases of bacteriuria and in 14.6% of those with non-infected urine; pyuria (more than three leukocytes per high-power field), in 60% of those with bacteriuria and in 15.9% of those with presumably non-infected urine. Bacteria were conspicuous in the urinary sediment in 91.1% of cases of bacteriuria and in 3.7% of presumably non-infected urines.The T.T.C. (Uroscreen) test fulfilled the criteria for a reliable and simple screening procedure. It should be used concomitantly with other screening tests when the urine is examined routinely.     

Limitations of the direct immunofluorescence test for antibody-coated bacteria in determining the site of urinary tract infections in children.

The results of the direct immunofluorescence test for antibody-coated bacteria to determine the site of a urinary tract infection do not always correlate with the clinical data. When this test was performed on urine specimens from 282 children with significant bacteriuria, false-negative and false-positive results were observed in 20% (19/94) and 52% (19/188) of the specimens. Contamination of the specimen during collection and the presence of Fc receptors (receptors for the crystallizable fragment of the immunoglobulin molecule) on the surface of some strains of Staphylococcus aureus yielded false-positive results, and stools and vaginal secretions were shown to be potential sources of antibody-coated bacteria. It is suggested that for children this test be run on urine collected by bladder puncture. The use of conjugated anti-IgG antiserum containing only F(ab'')2 (the antigen-binding fragments of the IgG molecule) is also recommended to eliminate false-positive results due to the presence of Fc receptors on the bacterial surface.     

FUS-100尿沉渣分析仪在住院患者尿路感染筛检中的应用

目的 探讨FUS-100全自动尿沉渣分析仪(简称FUS-100)中的细菌和酵母定量计数在筛查住院患者尿路感染时的价值.方法 用定量细菌培养法和FUS-100检测505例疑似泌尿系统感染患者清洁中段尿标本.结果 505份尿标本培养阳性192份,阳性率为38.0％,其中36份标本有两种细菌生长,共分离出228株菌,革兰阳性球菌30株(13.2％),革兰阴性杆菌108株(47.4％),真菌90株(39.4％).以尿细菌计数≥23.72/μL、酵母计数≥0.65/μL为感染阳性标准,与中段尿培养相比较,FUS-100细菌定量计数的敏感性为85.5％,特异性为89.5％,阳性似然比为8.14,阴性似然比为0.16;酵母定量计数的敏感性为80.4％,特异性为97.2％,阳性似然比为28.71,阴性似然比为0.20.结论 FUS-100定量计数尿细菌和酵母可用于住院患者尿路感染的快速筛查.     

Application of Hyperbranched Rolling Circle Amplification for Direct Detection of Mycobacterium Tuberculosis in Clinical Sputum Specimens

Background

Global tuberculosis (TB) control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA), was optimized to detect Mycobacterium tuberculosis (Mtb) in clinical sputum specimens.

Methods

A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC) and eight strains of non-tuberculosis mycobacterium (NTM) were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb.

Results

The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136), which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136) and culture (80.9%,110/136). The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136). The overall specificity of HRCA was 98.6% (71/72) which was similar to that of quantitative real-time PCR (qRT-PCR) and smear/culture methods (100%, 72/72).

Conclusions

Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.     

Evaluation of microscopic examination for bacteriuria.

In order to find an easily available, simple, reliable and inexpensive method for demonstrating significant bacteriuria in routine urine examination, microscopic observation and bacteriological cultures have been made in parallel on total of 206 urine samples. Microscopic examinations of centrifuged deposit for both pus cells and bacteria were found to be more satisfactory in urine specimens with significant bacteriuria than the examinations for either of these elements alone. The criteria of more than five pus cells per high power field and organisms visible in methylene blue stain had sensitivity of 79% and a false positive rate of 13%.     

Detection of antibodies to HIV in homologous sets of plasma, urine and oral mucosal transudate samples using rapid assays in Tanzania

The aim of this study was to evaluate the performance of commercially available anti-HIV assays when testing plasma, urine and oral mucosal transudate (OMT) samples for the presence of antibodies to HIV. Homologous sets of plasma, urine and oral mucosal transudate specimens were collected from 288 hospitalized patients in northern Tanzania and tested for antibodies to HIV using a routine enzyme immunoassay (Recombinant 3rd Generation EIA, Abbott) and two rapid assays (Testpack HIV-1/HIV-2; Abbott and SUDS HIV-1, Murex). Incubation times and/or sample volumes when testing OMT or urine were increased as compared to those recommended for plasma. The corresponding plasma specimens from all repeatedly reactive samples and samples presenting discordant results were subjected to confirmational testing by an HIV-1/2 Western blot. A total of 15.3% (44/288) of the plasma samples were anti-HIV-1 positive by Western blot. The sensitivity using plasma was 100% by all assays, 69.7-97.7% using urine, and 92.7-100% using oral transudate specimens. The sensitivity of both rapid assays was excellent and higher than the EIA when testing OMT. Specificities ranged from 98.8-100% for plasma, 99-100% for urine and were 100% by all assays using oral samples. The results obtained using oral mucosal transudate specimens and rapid assays were at least comparable to those obtained with plasma, while the use of urine specimens produced suboptimal sensitivities with two of the three assays. The testing of alternative body fluids for antibodies to HIV is yet another strategy that may be applicable, particularly in developing countries.     

Diagnostic role of different immunologic methods for laboratory diagnostics of legionellosis

The aim of the study was a comparative analysis of diagnostic value of different laboratoty methods conducted on the basis of results of examination of patients during Legionnaires' disease outbreak in town Verkhnyaya Pyshma. Retrospective analysis of laboratory data from 74 patients with diagnosis of Legionnaires' disease was performed. Complex of laboratory methods was used (polymerase chain reaction (PCR), enzyme immunoassay (EIA), immunochromatography). In group of patients with Legionnaires' disease, the highest proportion of positive results (73%) was obtained by the EIA determining total specific antibodies in urine. Determination of antigen in urine by immunochromatographic express-test yielded 52% of positive results. PCR testing of blood specimens yielded positive results in 65% of samples but was low specific, due to that in 19% of patients from control group false-positive results were obtained. Testing of 3 autopsy samples showed that all specimens contained DNA of the causative agent. Performed analysis allowed to recommend complex use of immunochromatographic express-test of antigen detection and identification of total specific antibodies by EIA during mass people examination.     

Clinical Utility of the Cryptococcal Antigen Lateral Flow Assay in a Diagnostic Mycology Laboratory

Background

Cryptococcus neoformans causes life-threatening meningitis. A recently introduced lateral flow immunoassay (LFA) to detect cryptococcal antigen (CRAG) is reportedly more rapid and convenient than standard latex agglutination (LA), but has not yet been evaluated in a diagnostic laboratory setting.

Methods

One hundred and six serum, 42 cerebrospinal fluid (CSF), and 20 urine samples from 92 patients with known or suspected cryptococcosis were tested by LA and LFA, and titres were compared. Results were correlated with laboratory-confirmed cryptococcosis. Serial samples were tested in nine treated patients.

Results

Twenty-five of 92 patients had confirmed cryptococcosis; all sera (n = 56) from these patients were positive by LFA (sensitivity 100%, 95% confidence interval (CI) 93.6–100%) compared with 51/56 positive by LA (sensitivity 91.1%, 95% CI 80.7–96.1%). Fifty sera from 67 patients without cryptococcosis tested negative in both assays. While LA yielded more false negative results (5/56) this did not reach statistical significance (p = 0.063). Nine CSF samples from patients with cryptococcal meningitis yielded positive results using both assays while 17/18 urine samples from patients with cryptococcosis were positive by the LFA. The LFA detected CRAG in C. gattii infection (n = 4 patients). Agreement between titres obtained by both methods (n = 38 samples) was imperfect; correlation between log-transformed titres (r) was 0.84. Turn-around-time was 20 minutes for the LFA and 2 h for LA. The cost per qualitative sample was 18USD and 91 USD, respectively and per quantitative sample was 38USD and 144USD, respectively.

Conclusions

Qualitative agreement between the LFA and LA assays performed on serum and CSF was good but agreement between titres was imperfect. Ease of performance of the LFA and the capacity for testing urine suggest it has a role in the routine laboratory as a rapid diagnostic test or point-of-care test.