Clin Chem Lab Med 2014; 52(10): 1507–1511
Alyexandra Arienzoa, Francesca Lositoa, Giorgia Bottini, Francesca Romana Priolisi, Alberto Mari, Paolo Visca and Giovanni Antonini*
A new device for the prompt diagnosis of urinary tract infections Abstract
Background: Urinary tract infections (UTIs) are among the most common infectious diseases. Results obtained from conventional microbiological analysis of urine and antibiotic susceptibility testing are available only after a few days, delaying precise diagnosis and appropriate therapy. Micro Biological Survey (MBS) srl (a spin-off of Roma Tre University, Rome, Italy) has developed and patented an automated colorimetric test for rapid bacterial counting. In a preliminary validation study it was demonstrated that the results obtained with the MBS method are equivalent to the results obtained with conventional culture-based microbiological analysis. Methods: In this study, sterile urine samples were artificially contaminated with bacterial species that are most frequently responsible for UTIs. The MBS method was used to evaluate the presence of bacteria and their sensitivity to some of the most commonly used antibiotics in UTIs. Results: The MBS method was able to detect in a few hours the presence or absence of bacteria at clinically significant concentrations ( > 105 CFU/mL), and to provide their susceptibility pattern to a limited panel of antibiotics. Discussion: The results obtained demonstrate that the MBS point-of-care testing (POCT) device could be developed into a valuable aid for the management of UTIs, possibly addressing more precise diagnosis and appropriate therapy.
Alyexandra Arienzo and Francesca Losito contributed equally to this work. *Corresponding author: Prof. Giovanni Antonini, MD, PhD, Department of Science, Roma Tre University, viale. G. Marconi 446, 00146 Rome, Italy, Phone: +39 3290570913, E-mail: [email protected]; Department of Science, Roma Tre University, Rome, Italy; and INBB Interuniversity Consortium of Structural and Systems Biology, Rome, Italy Alyexandra Arienzo, Francesca Losito and Paolo Visca: Department of Science, Roma Tre University, Rome, Italy Giorgia Bottini, Francesca Romana Priolisi and Alberto Mari: MBS srl, Rome, Italy a
Keywords: microbiological analysis; Micro Biological Survey (MBS); point-of-care testing (POCT); urinary tract infections (UTI). DOI 10.1515/cclm-2014-0294 Received March 17, 2014; accepted May 9, 2014; previously published online June 7, 2014
Introduction Urinary tract infection (UTI) represents one of the most common diseases encountered in medical practice today and occurs from the neonate to the geriatric age group [1]. UTIs are often empirically treated with antibiotics and, therefore, they represent a potential source for the emergence and/or selection of resistant bacteria [2, 3]. This empiric treatment is often guided by inadequate information about the rates of resistance, selection and emergence of resistance, even if there is a general consensus that there is a relationship between antibiotic misuse and resistance [4, 5]. In this context it could be useful to assess in a short time whether there is an infection and whether the infecting microorganisms are susceptible or resistant to a given antibiotic treatment. Point-of-care testing (POCT) is defined as medical testing at or near the site of patient care. This increases the likelihood that the patient, physician, and healthcare team will receive the results quicker, which allows for immediate clinical management decisions to be made. Microbiological POCTs are available for the detection of specific microorganisms in blood, urine or cerebral spinal fluid [6], and they represent the future of microbiological analysis, especially on patient admission [7]. To date there are no microbiological POCTs developed for the quantitative assessment of total viable bacteria in UTIs. The Micro Biological Survey (MBS) method is a new rapid method for selective counting of bacteria developed and patented by MBS srl (spin-off of Roma Tre University, Rome, Italy). It is based on a colorimetric survey performed in mono-use disposable reaction vials in which samples can be inoculated without any preliminary treatment. The operating principle of the MBS method is based
1508 Arienzo et al.: A new POCT for UTI on the capability to measure the catalytic activity of the redox enzymes in the main metabolic pathways of bacteria through a redox indicator that changes color according to the oxidative state of the medium. The time required for the color change is inversely related to the logarithm of bacterial concentration and this allows an unequivocal correlation between the observed enzymatic activity and the number of viable cells present in the samples. The analyses can be carried out where they are required, without the need for any other instrumentation than a thermostatic optical reader that can automatically detect the color change providing an estimate of the number of bacteria in the sample [8, 9]. In a previous study [10] a preliminary validation of the quantitative MBS method was carried out, in accordance with ISO Standards, in order to explore the possibility to use it as a microbiological POCT in UTIs. Precision, variance, uncertainty, linearity and accuracy were determined for all bacterial species tested with the MBS method, demonstrating that the MBS results were in good agreement with the ones obtained with the reference method of colony count on plate count agar for the bacterial species most commonly isolated in UTIs. In the present study, experiments were carried out using sterile urine samples artificially contaminated with different concentrations of bacteria belonging to the genera more frequently responsible for UTIs [11–13]. The main purpose was to test specific MBS reagents to detect the growth of bacteria in urine and to evaluate their sensitivity/resistance to a panel of antibiotics chosen among those more frequently used in the treatment of UTIs and prescribed from the Infectious Diseases Society of America [14]. The MBS vials for the detection of bacterial growth into urine, called urine bacterial count (UBC), are without antibiotics, while the same MBS vials for the evaluation of sensitivity/resistance are supplemented with the selected antibiotics. The antibiotics were added at the breakpoint concentrations according to standard therapy protocols in order to mimic the concentration range found in urine during therapy [15].
Materials and methods Bacterial strains tested were: Escherichia coli (ATCC 25992), Salmonella enterica ser. Thyphimurium (ATCC 14028), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 12600). Artificially contaminated urine samples were prepared diluting bacteria coming from pure cultures of the above indicated strains in filter-sterilized urine from healthy donors.
The standard plate count dilution method for the enumeration of colony forming units (CFU) on selective media was used as the reference method for the assessment of the bacterial concentration in biological samples: 1 mL of the artificially contaminated urine samples was serially diluted in saline and then plated on a selective medium, according to the bacterial strain used for urine artificial contamination: Mac Conkey Agar [Merck, Vimodrone (MI), Italy] for E. coli, Brilliant Green Agar (Sigma Aldrich, St. Louis, MO, USA) for S. enterica ser. Thyphimurium, Bile Esculine Azide Agar (Sigma Aldrich) for E. faecalis, Pseudomonas Isolation Agar (Sigma Aldrich) for P. aeruginosa, Baird Parker Agar [Liofilchem, Roseto degli Abruzzi (Te), Italy] for S. aureus. The antibiotics added to the UBC vials were trimethoprimsulfamethoxazole 1+5 (TMP-SMZ) at a concentration of 4 mg/L; ciprofloxacin (CIP) at a concentration of 1 mg/L, and amoxicillinclavulanic acid 7+1 (BAN) at a concentration of 8 mg/L. Antibiotic susceptibility testing was performed by Muller-Hinton agar diffusion with commercial discs [16]. The colorimetric MBS method was used according to the previously described procedure [10]: 1 mL of artificially contaminated urine samples was analyzed with four different MBS vials (UBC, without antibiotics, UBC+BAN, UBC+CIP, and UBC+TMP-SMZ). The vials were then inserted into the MBS thermostatic optical reader at 37 °C which automatically detected the color change due to the growth of bacteria. The MBS POCT device used in the study consisted in the UBC MBS vials and the MBS optical reader (see Figure 1). The MBS vials’ shelf-life is up to 6 months at room temperature and the vials were always utilized within 3 months from production. The UBC MBS vials and the MBS optical reader are produced and commercialized by MBS srl (www.emmebiesse.net).
Results Urine samples artificially contaminated with different concentrations of pure cultures of the bacterial strains E. coli (ATCC 25992), S. enterica ser. Thyphimurium (ATCC 14028), E. faecalis (ATCC 29212), P. aeruginosa (ATCC 27853), S. aureus (ATCC 12600) were analyzed with both the UBC POCT device, made of vials and optical reader (see Figure 1), and with the traditional plate count method. The absence of interference due to different pH values, color and turbidity in the MBS analysis of different urine samples was firstly assessed. Table 1 reports the results obtained from the analysis of artificially contaminated urine samples using the UBC vials for the detection of bacterial growth and the CFU count on selective media. The analytical time for the detection of bacterial growth using the MBS optical reader is reported for each bacterial strain at different concentrations. The results show that in most cases the UBC POCT device is able to detect a concentration of bacteria ≥ 105 CFU/mL within 5 h. Table 1 also reports the results obtained from the analysis of artificially contaminated urine samples using
Arienzo et al.: A new POCT for UTI 1509
(ATCC 14028) and S. aureus (ATCC 12600) were sensitive to BAN CIP and TMP-SMZ; E. faecalis (ATCC 29212) was resistant to BAN, sensitive to CIP and has an intermediate sensitivity to TMP-SMZ; P. aeruginosa (ATCC 27853) was resistant to TMP-SMZ and BAN and sensitive to CIP.
Discussion and conclusions
Figure 1 MBS POCT consisting of vials and an optical reader. The color change of the mono-use disposable reaction vial (provided with sterile MBS original reagents) occurs in the presence of bacteria. The starting color blue (left) changes to yellow (right) in the presence of bacteria. In the absence of bacteria the color remains blue. The time required for color change is inversely related to bacterial concentration, almost independently on the bacterial strains present into the vial. The optical reader can allocate up to eight different vials and can automatically detect the color change and can calculate the concentration of bacteria present in each vial on the basis of the time required for their color change. The PC connected with the optical reader provides a PDF file with the analytical results.
the UBC vials for the evaluation of sensitivity/resistance of bacteria to the antibiotics under study: UBC+amoxicillinclavulanic acid (BAN), UBC+ciprofloxacin (CIP), and UBC+trimethoprim-sulfamethoxazole (TMP-SMZ). The antibiotics were added at breakpoint concentrations [15]. “S” means that the optical reader did not detect bacterial growth and hence the bacteria were found to be sensitive to the antibiotic under study while “R” means that the optical reader detected bacterial growth and hence that bacteria were found to be resistant to the antibiotic under study. “I” means that the optical reader detected bacterial growth only at the highest bacterial concentration and thus that bacteria displayed an intermediate sensitivity to the antibiotic under study. The results obtained with this new method are in agreement with those obtained with antibiotic susceptibility tests performed using Muller Hinton agar diffusion with commercial disks (data not shown). The results indicate that E. coli (ATCC 25922), S. enterica ser. Thyphimurium
The aim of the present study was to evaluate a new method for microbiological analysis (UBC device) for a prompt diagnosis of acute UTIs, giving at the same time an estimate of bacterial susceptibility to a small panel of selected antibiotics. Experiments have been carried out in vitro, using sterile urine samples artificially contaminated with bacteria among those most frequently isolated in UTIs, in the presence or absence of selected antibiotics. The antibiotics were chosen among those most frequently used for the treatment of UTIs. The MBS analysis was carried out in comparison with the traditional method of antibiotic susceptibility testing (Muller Hinton agar diffusion test). The data here reported indicate that the MBS POCT device consisting of the UBC vials and MBS optical reader is able to detect the presence of bacteria in urine. According to the quantitative criterion for the microbiological identification of significant bacteriuria, that is generally considered to be ≥ 105 CFU/mL [17], it was demonstrated that when bacteria are present at clinically significant concentrations, the analytical time required for growth detection with the MBS method is in most cases
Alyexandra Arienzoa, Francesca Lositoa, Giorgia Bottini, Francesca Romana Priolisi, Alberto Mari, Paolo Visca and Giovanni Antonini*
A new device for the prompt diagnosis of urinary tract infections Abstract
Background: Urinary tract infections (UTIs) are among the most common infectious diseases. Results obtained from conventional microbiological analysis of urine and antibiotic susceptibility testing are available only after a few days, delaying precise diagnosis and appropriate therapy. Micro Biological Survey (MBS) srl (a spin-off of Roma Tre University, Rome, Italy) has developed and patented an automated colorimetric test for rapid bacterial counting. In a preliminary validation study it was demonstrated that the results obtained with the MBS method are equivalent to the results obtained with conventional culture-based microbiological analysis. Methods: In this study, sterile urine samples were artificially contaminated with bacterial species that are most frequently responsible for UTIs. The MBS method was used to evaluate the presence of bacteria and their sensitivity to some of the most commonly used antibiotics in UTIs. Results: The MBS method was able to detect in a few hours the presence or absence of bacteria at clinically significant concentrations ( > 105 CFU/mL), and to provide their susceptibility pattern to a limited panel of antibiotics. Discussion: The results obtained demonstrate that the MBS point-of-care testing (POCT) device could be developed into a valuable aid for the management of UTIs, possibly addressing more precise diagnosis and appropriate therapy.
Alyexandra Arienzo and Francesca Losito contributed equally to this work. *Corresponding author: Prof. Giovanni Antonini, MD, PhD, Department of Science, Roma Tre University, viale. G. Marconi 446, 00146 Rome, Italy, Phone: +39 3290570913, E-mail: [email protected]; Department of Science, Roma Tre University, Rome, Italy; and INBB Interuniversity Consortium of Structural and Systems Biology, Rome, Italy Alyexandra Arienzo, Francesca Losito and Paolo Visca: Department of Science, Roma Tre University, Rome, Italy Giorgia Bottini, Francesca Romana Priolisi and Alberto Mari: MBS srl, Rome, Italy a
Keywords: microbiological analysis; Micro Biological Survey (MBS); point-of-care testing (POCT); urinary tract infections (UTI). DOI 10.1515/cclm-2014-0294 Received March 17, 2014; accepted May 9, 2014; previously published online June 7, 2014
Introduction Urinary tract infection (UTI) represents one of the most common diseases encountered in medical practice today and occurs from the neonate to the geriatric age group [1]. UTIs are often empirically treated with antibiotics and, therefore, they represent a potential source for the emergence and/or selection of resistant bacteria [2, 3]. This empiric treatment is often guided by inadequate information about the rates of resistance, selection and emergence of resistance, even if there is a general consensus that there is a relationship between antibiotic misuse and resistance [4, 5]. In this context it could be useful to assess in a short time whether there is an infection and whether the infecting microorganisms are susceptible or resistant to a given antibiotic treatment. Point-of-care testing (POCT) is defined as medical testing at or near the site of patient care. This increases the likelihood that the patient, physician, and healthcare team will receive the results quicker, which allows for immediate clinical management decisions to be made. Microbiological POCTs are available for the detection of specific microorganisms in blood, urine or cerebral spinal fluid [6], and they represent the future of microbiological analysis, especially on patient admission [7]. To date there are no microbiological POCTs developed for the quantitative assessment of total viable bacteria in UTIs. The Micro Biological Survey (MBS) method is a new rapid method for selective counting of bacteria developed and patented by MBS srl (spin-off of Roma Tre University, Rome, Italy). It is based on a colorimetric survey performed in mono-use disposable reaction vials in which samples can be inoculated without any preliminary treatment. The operating principle of the MBS method is based
1508 Arienzo et al.: A new POCT for UTI on the capability to measure the catalytic activity of the redox enzymes in the main metabolic pathways of bacteria through a redox indicator that changes color according to the oxidative state of the medium. The time required for the color change is inversely related to the logarithm of bacterial concentration and this allows an unequivocal correlation between the observed enzymatic activity and the number of viable cells present in the samples. The analyses can be carried out where they are required, without the need for any other instrumentation than a thermostatic optical reader that can automatically detect the color change providing an estimate of the number of bacteria in the sample [8, 9]. In a previous study [10] a preliminary validation of the quantitative MBS method was carried out, in accordance with ISO Standards, in order to explore the possibility to use it as a microbiological POCT in UTIs. Precision, variance, uncertainty, linearity and accuracy were determined for all bacterial species tested with the MBS method, demonstrating that the MBS results were in good agreement with the ones obtained with the reference method of colony count on plate count agar for the bacterial species most commonly isolated in UTIs. In the present study, experiments were carried out using sterile urine samples artificially contaminated with different concentrations of bacteria belonging to the genera more frequently responsible for UTIs [11–13]. The main purpose was to test specific MBS reagents to detect the growth of bacteria in urine and to evaluate their sensitivity/resistance to a panel of antibiotics chosen among those more frequently used in the treatment of UTIs and prescribed from the Infectious Diseases Society of America [14]. The MBS vials for the detection of bacterial growth into urine, called urine bacterial count (UBC), are without antibiotics, while the same MBS vials for the evaluation of sensitivity/resistance are supplemented with the selected antibiotics. The antibiotics were added at the breakpoint concentrations according to standard therapy protocols in order to mimic the concentration range found in urine during therapy [15].
Materials and methods Bacterial strains tested were: Escherichia coli (ATCC 25992), Salmonella enterica ser. Thyphimurium (ATCC 14028), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 12600). Artificially contaminated urine samples were prepared diluting bacteria coming from pure cultures of the above indicated strains in filter-sterilized urine from healthy donors.
The standard plate count dilution method for the enumeration of colony forming units (CFU) on selective media was used as the reference method for the assessment of the bacterial concentration in biological samples: 1 mL of the artificially contaminated urine samples was serially diluted in saline and then plated on a selective medium, according to the bacterial strain used for urine artificial contamination: Mac Conkey Agar [Merck, Vimodrone (MI), Italy] for E. coli, Brilliant Green Agar (Sigma Aldrich, St. Louis, MO, USA) for S. enterica ser. Thyphimurium, Bile Esculine Azide Agar (Sigma Aldrich) for E. faecalis, Pseudomonas Isolation Agar (Sigma Aldrich) for P. aeruginosa, Baird Parker Agar [Liofilchem, Roseto degli Abruzzi (Te), Italy] for S. aureus. The antibiotics added to the UBC vials were trimethoprimsulfamethoxazole 1+5 (TMP-SMZ) at a concentration of 4 mg/L; ciprofloxacin (CIP) at a concentration of 1 mg/L, and amoxicillinclavulanic acid 7+1 (BAN) at a concentration of 8 mg/L. Antibiotic susceptibility testing was performed by Muller-Hinton agar diffusion with commercial discs [16]. The colorimetric MBS method was used according to the previously described procedure [10]: 1 mL of artificially contaminated urine samples was analyzed with four different MBS vials (UBC, without antibiotics, UBC+BAN, UBC+CIP, and UBC+TMP-SMZ). The vials were then inserted into the MBS thermostatic optical reader at 37 °C which automatically detected the color change due to the growth of bacteria. The MBS POCT device used in the study consisted in the UBC MBS vials and the MBS optical reader (see Figure 1). The MBS vials’ shelf-life is up to 6 months at room temperature and the vials were always utilized within 3 months from production. The UBC MBS vials and the MBS optical reader are produced and commercialized by MBS srl (www.emmebiesse.net).
Results Urine samples artificially contaminated with different concentrations of pure cultures of the bacterial strains E. coli (ATCC 25992), S. enterica ser. Thyphimurium (ATCC 14028), E. faecalis (ATCC 29212), P. aeruginosa (ATCC 27853), S. aureus (ATCC 12600) were analyzed with both the UBC POCT device, made of vials and optical reader (see Figure 1), and with the traditional plate count method. The absence of interference due to different pH values, color and turbidity in the MBS analysis of different urine samples was firstly assessed. Table 1 reports the results obtained from the analysis of artificially contaminated urine samples using the UBC vials for the detection of bacterial growth and the CFU count on selective media. The analytical time for the detection of bacterial growth using the MBS optical reader is reported for each bacterial strain at different concentrations. The results show that in most cases the UBC POCT device is able to detect a concentration of bacteria ≥ 105 CFU/mL within 5 h. Table 1 also reports the results obtained from the analysis of artificially contaminated urine samples using
Arienzo et al.: A new POCT for UTI 1509
(ATCC 14028) and S. aureus (ATCC 12600) were sensitive to BAN CIP and TMP-SMZ; E. faecalis (ATCC 29212) was resistant to BAN, sensitive to CIP and has an intermediate sensitivity to TMP-SMZ; P. aeruginosa (ATCC 27853) was resistant to TMP-SMZ and BAN and sensitive to CIP.
Discussion and conclusions
Figure 1 MBS POCT consisting of vials and an optical reader. The color change of the mono-use disposable reaction vial (provided with sterile MBS original reagents) occurs in the presence of bacteria. The starting color blue (left) changes to yellow (right) in the presence of bacteria. In the absence of bacteria the color remains blue. The time required for color change is inversely related to bacterial concentration, almost independently on the bacterial strains present into the vial. The optical reader can allocate up to eight different vials and can automatically detect the color change and can calculate the concentration of bacteria present in each vial on the basis of the time required for their color change. The PC connected with the optical reader provides a PDF file with the analytical results.
the UBC vials for the evaluation of sensitivity/resistance of bacteria to the antibiotics under study: UBC+amoxicillinclavulanic acid (BAN), UBC+ciprofloxacin (CIP), and UBC+trimethoprim-sulfamethoxazole (TMP-SMZ). The antibiotics were added at breakpoint concentrations [15]. “S” means that the optical reader did not detect bacterial growth and hence the bacteria were found to be sensitive to the antibiotic under study while “R” means that the optical reader detected bacterial growth and hence that bacteria were found to be resistant to the antibiotic under study. “I” means that the optical reader detected bacterial growth only at the highest bacterial concentration and thus that bacteria displayed an intermediate sensitivity to the antibiotic under study. The results obtained with this new method are in agreement with those obtained with antibiotic susceptibility tests performed using Muller Hinton agar diffusion with commercial disks (data not shown). The results indicate that E. coli (ATCC 25922), S. enterica ser. Thyphimurium
The aim of the present study was to evaluate a new method for microbiological analysis (UBC device) for a prompt diagnosis of acute UTIs, giving at the same time an estimate of bacterial susceptibility to a small panel of selected antibiotics. Experiments have been carried out in vitro, using sterile urine samples artificially contaminated with bacteria among those most frequently isolated in UTIs, in the presence or absence of selected antibiotics. The antibiotics were chosen among those most frequently used for the treatment of UTIs. The MBS analysis was carried out in comparison with the traditional method of antibiotic susceptibility testing (Muller Hinton agar diffusion test). The data here reported indicate that the MBS POCT device consisting of the UBC vials and MBS optical reader is able to detect the presence of bacteria in urine. According to the quantitative criterion for the microbiological identification of significant bacteriuria, that is generally considered to be ≥ 105 CFU/mL [17], it was demonstrated that when bacteria are present at clinically significant concentrations, the analytical time required for growth detection with the MBS method is in most cases
