Detection of bacteriuria by bioluminescence: effect of pre-analysis centrifugation of specimens

N ichols , W.W., C urtis , G.D.W. & J ohnston , H.H. 1984. Detection of bacteriuria by bioluminescence: effect of pre-analysis centrifugation of specimens. Journal of Applied Bacteriology 56 , 247–257.
Three bioluminescence-based, rapid methods of detecting significant bacteriuria were applied in parallel to 514 urine specimens. The results were compared with those of a quantitative pour plate viable count method, defined as positive if ≥ 10⁵ c.f.u./ml of urine were observed. When adjusted to yield 21% falsely positive results the three rapid methods yielded 24%, 21% and 19% falsely negative results. If specimens with evidence of urethral or vaginal contamination were excluded (237 specimens remaining) the three methods yielded respectively 14%, 8% and 13% falsely negative results. A major source of disagreement between the bioluminescence-based methods and quantitative culture thus appeared to be contaminated urine specimens.     

Bacteriuria amongst Pregnant Women in the Buea Health District,Cameroon: Prevalence,Predictors, Antibiotic Susceptibility Patterns and Diagnosis

Background

Bacteriuria is associated with significant maternal and foetal risks. However, its prevalence is not known in our community.

Objectives

This study was carried out to determine the prevalence and predictors of bacteriuria in pregnant women of the Buea Health District (BHD) as well as the antibiotic sensitivity patterns of bacterial isolates. It also sought to determine the diagnostic performance of the nitrite and leucocyte esterase tests in detecting bacteriuria in these women.

Methods

An observational analytic cross-sectional study was carried out amongst pregnant women attending selected antenatal care centres in Buea. We recruited 102 consenting pregnant women for the study. Demographic and clinical data were collected using structured questionnaires. Clean catch midstream urine was collected from each participant in sterile leak proof containers. Samples were examined biochemically, microscopically and by culture. Significant bacteriuria was defined as the presence of ≥10⁸ bacteria/L of cultured urine. Identification and susceptibility of isolates was performed using API 20E and ATB UR EU (08) (BioMerieux, Marcy l''Etoile, France).

Results

Significant bacteriuria was found in the urine of 24 of the 102 women tested giving a bacteriuria prevalence of 23.5% in pregnant women of the BHD. Asymptomatic bacteriuria was detected in 8(7.8%) of the women. There was no statistically significant predictor of bacteriuria. Escherichia coli were the most isolated (33%) uropathogens and were 100% sensitive to cefixime, cefoxitin and cephalothin. The nitrite and leucocyte esterase tests for determining bacteriuria had sensitivities of 8%, 20.8% and specificities of 98.7% and 80.8% respectively.

Conclusion

Bacteriuria is frequent in pregnant women in the BHD suggesting the need for routine screening by urine culture. Empiric treatment with cefixime should be instituted until results of urine culture and sensitivity are available. Nitrite and leucocyte esterase tests were not sensitive enough to replace urine culture as screening tests.     

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%.     

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.     

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 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.     

Antibody-coated bacteria in the urine of preschool and school-aged girls with asymptomatic bacteriuria.

Urine samples from 3564 girls aged 2 to 13 years were screened for evidence of infection. Cultures were positive (bacteria count, more than 10(5)/ml) in 61 (1.7%) by the dipslide method and in 55 (1.5%) by standard culture techniques. In 13 (23.6%) of the 55, antibody-coated bacteria (ACB) were detected in the urine. The clinical, bacteriologic, radiologic and urinalysis findings in children with ACB were no different from those in children in whom the bacteria were not coated. Direct examination of uncentrifuged urine under high power revealed one or more bacteria per two high-power fields in 96% of infected urine samples and in only 7% of noninfected samples. Five or more leukocytes per high-power field in centrifuged urine were detected in 36.7% of infected urine samples but not in noninfected samples. The ACB test did not differentiate between asymptomatic bacteriuria with parenchymal scarring or vesicoureteral reflux or both and asymptomatic bacteriuria without these abnormalities.     

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.     

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.     

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.     

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.     

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.     

Evaluation of Miniature Test for Bacteriuria Using Dehydrated Media and Nitrite Pads

A new dip-inoculum method for detecting bacteriuria which utilizes dehydrated media pads and a nitrite pad attached to a small plastic strip was evaluated in hospitalized patients. Discrepant interpretations were made by independent observers in 9.3% of the specimens with > 10(5) colonies per ml. The media pads failed to support growth of yeast and gave variable results with Staphylococcus epidermidis and non-group D streptococci. False-negative culture results commonly occurred if the patients were receiving antibiotics. The nitrite test occasionally remained positive for brief periods after the elimination of bacteriuria by antibiotics. Conditions and drugs (especially phenazopyridine) which discolor urine interfered with reading both the culture and nitrite tests. Although not suitable for hospital use, or for monitoring therapy, the test strip is probably as reliable as the calibrated loop-streak plate culture for office screening.     

Is screening for bacteriuria in pregnancy worth while?

A total of 4470 pregnant women were screened for bacteriuria by the dipslide method and significant growth found in 226 (5.1%). In 198 cases the urine was re-examined, in 119 by using suprapubic aspiration or catheterisation (62 (52%) samples contained bacteria) and in 79 by using midstream urine samples (26 (33%) samples contained greater than 10(8) colony forming units/1), showing the maximum prevalence of confirmed bacteriuria to be 2.6%. Overt urinary tract infection developed later in four of 80 patients with proved bacteriuria who had been given antibiotics, in one of eight untreated patients with bacteriuria, in one of 110 patients with unconfirmed bacteriuria, and in one of 226 non-bacteriuric controls. A history of urinary tract infection was given by 18% of controls and 42% of women with confirmed bacteriuria. Screening for bacteriuria and treatment with antibiotics to prevent later overt infection is expensive. Whether it is worth while and cost effective depends largely on the prevalence of bacteriuria in the local population and the proportion who develop overt infection. The screening and treatment programme reported here appeared to prevent only six cases of overt infection.     

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.     

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.     

Suprapubic aspiration under ultrasound guidance in children with fever of undiagnosed cause.

OBJECTIVES--To assess the ease of use of suprapubic aspiration of urine under ultrasound guidance in babies with fever of uncertain cause and to assess the importance of bacterial counts and pyuria in relation to abnormalities of the urinary tract and the importance of pyuria in the absence of bacteriuria. DESIGN--Analysis of urine samples obtained by suprapubic aspiration in babies and children from July 1991 to June 1992. The clinical records of the children with bacteriuria and sterile pyuria were examined retrospectively. SETTING--Neonatal and paediatric wards of a district general hospital. SUBJECTS--508 babies and children who had fever of uncertain cause or were seriously ill. RESULTS--No difficulties arose in the collection of 545 specimens. Bacteria were isolated from the specimens of 44 children, 24 of whom had abnormalities of the urinary tract. The bacterial count was < 10(8)/l in 18 of the children with bacteriuria, 10 of whom had abnormalities. No white cells were seen in 22 of the 46 bacteriuric specimens; nine of the children with no pyuria had vesicoureteric reflux. 439 of the 499 non-bacteriuric specimens showed no white cells. 60 children had pyuria without bacteriuria. CONCLUSIONS--The use of ultrasound guidance simplifies suprapubic aspiration of urine in babies. Low bacterial counts may be associated with abnormalities of the urinary tract. Laboratory techniques capable of detecting such counts reliably should be used. Pyuria is absent in half of babies and very young children with bacteriuria. It rarely occurs without bacteriuria, and if it does an explanation should be sought.     

Bacterial profile and drug susceptibility pattern of urinary tract infection in pregnant women at University of Gondar Teaching Hospital, Northwest Ethiopia

ABSTRACT: BACKGROUND: Urinary tract infection (UTI) is a common health problem among pregnant women. Proper investigation and prompt treatment are needed to prevent serious life threatening condition and morbidity due to urinary tract infection that can occur in pregnant women. Recent report in Addis Ababa, Ethiopia indicated the prevalence of UTI in pregnant women was 11.6 % and Gram negative bacteria was the predominant isolates and showed multi drug resistance. This study aimed to assess bacterial profile that causes urinary tract infection and their antimicrobial susceptibility pattern among pregnant women visiting antenatal clinic at University of Gondar Teaching Hospital, Northwest Ethiopia. METHODS: A cross-sectional study was conducted at University of Gondar Teaching Hospital from March 22 to April 30, 2011. Mid stream urine samples were collected and inoculated into Cystine Lactose Electrolyte Deficient medium (CLED). Colony counts yielding bacterial growth of 105/ml of urine or more of pure isolates were regarded as significant bacteriuria for infection. Colony from CLED was sub cultured onto MacConkey agar and blood agar plates. Identification was done using cultural characteristics and a series of biochemical tests. A standard method of agar disc diffusion susceptibility testing method was used to determine susceptibility patterns of the isolates. RESULTS: The overall prevalence of UTI in pregnant women was 10.4 %. The predominant bacterial pathogens were Escherichia coli 47.5 % followed by coagulase-negative staphylococci 22.5 %, Staphylococcus aureus 10 %, and Klebsiella pneumoniae 10 %. Gram negative isolates were resulted low susceptibility to co-trimoxazole (51.9 %) and tetracycline (40.7 %) whereas Gram positive showed susceptibility to ceftriaxon (84.6 %) and amoxicillin-clavulanic acid (92.3 %). Multiple drug resistance (resistance to two or more drugs) was observed in 95 % of the isolates. CONCLUSION: Significant bacteriuria was observed in asymptomatic pregnant women. Periodic studies are recommended to check the outcome of asymptomatic bacteriuria and also monitor any changes in the susceptibility patterns of urinary tract pathogens in pregnant women.     

Asymptomatic Urinary Tract Infection in Gynaecological Outpatients

In this study mid-stream specimens of urine were collected from all new patients attending a gynaecological outpatient department and tested for significant bacteriuria. Those having an asymptomatic infection were folloWed up, treated, and investigated adiologically.Of 1,506 women screened for bacteriuria 82 (5·4%) were found to have a persistent infection. The predominant organism was Escherichia coli, present in 83% of infections. Treatment with sulphonamides produced a good cure rate, which was improved by ampicillin given to failures. Some patients, however, had infections that persisted or recurred despite several antibiotics. The radiological investigations showed that a high proportion of women with asymptomatic urinary infection had severe renal disease which was quite symptomless. This was more pronounced in those with persistent or recurrent infections.     

Isolation of L-Forms in a Clinical Microbiology Laboratory

Previous studies have demonstrated that L-forms of bacteria may play a role in persistent, chronic, or recurrent urinary-tract infections. A 2-year program was initiated to determine the feasibility of culturing for L-forms on a routine basis, and to determine the effectiveness of such a program. In relation to the total number of specimens, few L-forms were actually isolated. In comparison with the amount of equipment and technician time required, the return was negligible; only 0.5% of all urine specimens were positive for L-forms. An increase to only 1.2% was noted when culturing for L-forms was limited to patients with a diagnosis of bacteriuria or pyelonephritis. It is recommended that this technique be reserved for those patients with a long history of recurrent urinary-tract infections, after other attempts to cure the patient have met with failure.