Abstracts

INTRODUCTION

The urinary tract infection (UTI) is one of the most common infectious disease in humans, it occurs at high frequency in both inpatients and outpatients; it represents an important cause of morbidity and mortality⁽¹1 BROEREN, M. A. C. et al . Screening for urinary tract infection with the Sysmex UF-1000i urine flow cytometer. J Clin Microbiol, v. 49, n. 3, p. 1025-9, 2011.^,77 HÖRNER, R. et al . Prevalência de microrganismos em infecções do trato urinário de pacientes atendidos no Hospital Universitário de Santa Maria. RBAC, v. 38, n. 3, p. 147-50, 2006.^,88 JOLKKONEN, S. et al . Screening of urine samples by flow cytometry reduces the need for culture. J Clin Microbiol, v. 48, n. 9, p. 3117-121, 2010.⁾.

The clinical course of the disease is related to the virulence of microorganism involved, host resistance, effectiveness of used clinical and antimicrobial therapy⁽²2 CARVALHAL, G. F.; ROCHA, L. C. A.; MONTI, P. R. Urocultura e exame comum de urina: considerações sobre sua coleta e interpretação. Revista da AMRIGS , v. 50, n. 1, p. 59-62, 2006.^,1212 LUCCHETTI, G. et al . Infecções do trato urinário: análise da frequência e do perfil de sensibilidade dos agentes causadores de infecção do trato urinário em pacientes com cateterização vesical crônica. J Bras Patol Med Lab, v. 41, n. 6, p. 383-9, 2005.⁾. These factors make the appropriate diagnosis an important tool for infection resolution⁽⁶6 GRIEBLING, T. L. Urologic diseases in America project: trends in resource use for urinary tract infections in women. J Urol , v. 173, p. 1281-7, 2005.⁾.

The UTI diagnosis is performed based on the clinical condition of the patient and laboratory tests. The routine monitoring tests used for UTI diagnosis are: reactive strip, analysis urine test, smear microscopy, sediment analysis, and urine culture, which is considered the gold standard⁽⁵5 FOSTER, R. T. Uncomplicated urinary tract infections in women. Obstet Gynecol Clin North Am , v. 35, p. 235-48, 2008.⁾. The isolation and identification of bacteria, together with the antimicrobial susceptibility testing performance, are useful in the choice of drug therapy⁽¹1 BROEREN, M. A. C. et al . Screening for urinary tract infection with the Sysmex UF-1000i urine flow cytometer. J Clin Microbiol, v. 49, n. 3, p. 1025-9, 2011.⁾.

Samples of patients with strong suspicion of UTI that produced negative urine culture results may raise uncertainty about the reliability of the results released by the laboratory. Some studies suggest that the presence of inhibitory substances, such as antimicrobial residues in urine samples can interfere with the in vitrogrowth of the probable bacteria causing the infection, producing a false-negative result⁽¹⁰10 KUSSEN, G. M. B. Pesquisa de substâncias antimicrobianas em urinas destinas a cultura. In: ALBINI, C. A. A.; SOUZA, H. A. P H. M.; SILVEIRA, A. C.O. (Org.). Infecções urinárias: uma abordagem multidisciplinar. 1. ed. Curitiba: Editora CRV, 2012. p. 579-83.⁾.

OBJECTIVE

In order to verify the occurrence of false-negative urine cultures due to the presence of antimicrobial residues that interfere with bacterial growth in vitro, this study aimed to research, using two methods, the activity of such substances in urine samples of patients admitted to the Clinical Hospital of the Paraná Federal University (HC-UFPR).

MATERIAL AND METHODS

Automated technique for the research on antimicrobial residues

The automated research was performed using Alfred 60® equipment (ALIFAX - Padua, Italy) that offers the residual antimicrobial activity test (RAA). In this test, 500 µl of the urine sample were inoculated into a flask containing an enriched solution and a Staphylococcus epidermidissuspension, previously added. The flasks were incubated under constant shaking and controlled temperature at 37°C, for approximately 3 hours. Bacterial growth was monitored in real-time by turbidity compared to McFarland Scale. The same turbidity observed early in the reaction that does not change over time, indicates the absence of bacterial growth, resulting in positive RAA. The increase in suspension turbidity due to bacterial growth was reported as negative RAA test.

For method validation, we added 10 µg/pl Gentamicin Solution to positive control flask, and 500 pl of sterile saline to negative control flask.

RESULTS

From the 188 studied samples, 44 (23.4%) showed positive urine culture, 121 (64.4%) showed negative urine culture, and 23 (12.2%) were considered contaminated because of the development of many bacterial species.

The negative cultures results associated with the presence of bacteriuria and RARA are inserted in Table 1.

From the 14 samples that showed presence of bacteria and positive RARA, different results were obtained from the techniques evaluated, and were divided in four profiles. Table 2 shows the profile of results and the percentage found, according to the microorganism used.

RARA was detected by all the techniques in 64.3% of samples (profile A + C). When analyzing the different microorganisms tested, we observed positivity for the three strains in only 35.7% of samples (profile A). S. epidermidis strain was more sensitive to antimicrobial activity detection, because in 92.8% of samples (profile A + C + D) the test was positive, followed by E. coli ATCC 25922 with 71.4% of positivity (profile A + B + C), and E. faecalis ATCC 29212 with 42.8% (profile A + B) of positivity.

DISCUSSION

Urine samples represent a large volume of specimens daily submitted to the microbiology laboratory for evaluation. Several factors such as use of antimicrobials, vaginal contamination and urethral colonization, may influence culture result and should be carefully evaluated during the exam to ensure its quality.

The positivity rate found in this study (23.4%) for urine culture was similar to that reported by other authors⁽³3 COSTA, L. C. et al. Infecções urinárias em pacientes ambulatoriais: prevalência e perfil de resistência aos antimicrobianos. RBAC , v. 42, n. 3, 2010.^{, 14}14 MULLER, E. V.; SANTOS, D. F.; CORRÊA, N. A. B. Prevalência de microrganismos em infecções do trato urinário de pacientes atendidos no laboratório de análises clínicas da Universidade Paranaense-Umuarama-PR. RBAC,v. 40, n. 1, 2008.⁾, ranging from 15.9% to 22.8%. Most samples showed negative culture result, which is consistent with many studies showing that up to 80% of samples submitted for culture has no bacterial growth, or show negligible growth⁽⁹9 KELLOGG, J. A. et al . Clinical relevance of culture versus screens for the detection of microbial pathogens in urine specimens. Am J Med, v. 83, n. 4, p. 739-45, 1987.^{, 15}15 OKADA, H. et al . Enumeration of bacterial cell numbers and detection of significant bacteriuria by use of a new flow cytometry-based device. J Clin Microbiol, v. 44, n. 10, p. 3596-9, 2006.⁾. This may be associated with excessive request filed by clinical material easily accessible, and due to UTI be one of the major infections acquired in hospitals environment⁽¹⁷17 VIEIRA, J. M. S. et al. Suscetibilidade antimicrobiana de bactérias isoladas de infecções do trato urinário de pacientes atendidos no Hospital Universitário Bettina Ferro de Souza, Belém PA. RBAC, v. 39, n. 2, 2007.⁾. However, the negative result must be carefully evaluated and correlated with other laboratory findings, such as the presence of bacteria and previous antimicrobial therapy⁽¹⁶16 SRIRANGARAJ, S.; KALI, A.; CHARLES, M. V. P. Antibiotic screening of urine culture as a tool for internal quality audit. Australas Med J, v. 7, n. 2, p. 73-7, 2014.^{, 18}18 WILSON, G.; BADARUDEEN, S.; GODWIN, A. Antibiotic screening of urine culture as a useful quality audit. J Infect Dev Ctries, v. 5, n. 4, p. 299-302, 2011.⁾.

The presence of bacteria and RARA positive in 14 samples with negative culture suggest a false negative result, representing 7.45% of studied samples (n = 188). In the study carried out by Botão et al, cited by Kussen⁽¹⁰10 KUSSEN, G. M. B. Pesquisa de substâncias antimicrobianas em urinas destinas a cultura. In: ALBINI, C. A. A.; SOUZA, H. A. P H. M.; SILVEIRA, A. C.O. (Org.). Infecções urinárias: uma abordagem multidisciplinar. 1. ed. Curitiba: Editora CRV, 2012. p. 579-83.⁾, the presence of antimicrobial residues was detected in 1% of urine samples, this value is lower than we found in this study. However, their sample included urine of both inpatients and outpatients, whereas in this study, only samples of hospitalized patients were included, in whom antimicrobial therapy is more widely used.

The presence of bacteria associated to negative result for RARA was detected in 4.1% of samples (n = 5), amounting 2.66% of samples (n = 188). This profile may occur due to an infection caused by anaerobic microorganisms that are not recovered from routine culture, or by low-growth bacteria that require specific diagnostic tests and appropriate isolation method⁽¹³13 McCARTER, Y. S. et al. Cumitech - laboratory diagnosis of urinary tract infections. American Society of Microbiology Press,2009.⁾. Other researchers also report the potencial presence of non-viable bacterial cells in urine⁽¹⁵15 OKADA, H. et al . Enumeration of bacterial cell numbers and detection of significant bacteriuria by use of a new flow cytometry-based device. J Clin Microbiol, v. 44, n. 10, p. 3596-9, 2006.⁾.

In the other samples evaluated, absence of bacteriuria and positive RARA occurred in 59.5% (n = 72), and absence of bacteriuria and negative RARA occurred in 24.8% (n = 30), which characterized evidenced negative results, because bacteria were not detected in the three different methods used in the study.

The evaluation of the samples with positive RARA, negative culture and bacteriuria (n = 14), the manual technique had a sensitivity of 71.4% (n = 9), and the automated technique had a sensitivity of 92.8% (n = 13). The low sensitivity of the manual technique can be explained by the small sample volume used for agar diffusion method⁽⁴4 DRISCOLL, A. J. et al . Disk diffusion bioassays for the detection of antibiotic activity in body fluids: applications for the pneumonia etiology research for child health project. Clin Infect Dis, v. 54, supl. 2, p. 159-64, 2012.⁾.

The use of E. faecalis strain did not increase the positivity of manual technique, demonstrating that this one, in subsequent studies, may be abolished or replaced. Some studies suggest the use of different microorganisms, such as Micrococcus luteus, Staphylococcus aureus, Bacillus stearothermophilus, among others, to improve the sensitivity of the method⁽⁴4 DRISCOLL, A. J. et al . Disk diffusion bioassays for the detection of antibiotic activity in body fluids: applications for the pneumonia etiology research for child health project. Clin Infect Dis, v. 54, supl. 2, p. 159-64, 2012.^{, 11}11 LIU, Y. C. et al. Detection of antimicrobial activity in urine for epidemiologic studies of antibiotic use. J Clin Epidemiol, v. 52, n. 6, p. 539-45, 1999.⁾.

Recent reports also suggest that the results of RARA may be interfered according to the antimicrobial class used, due to the presence of inert metabolites excreted in urine, or due to the use of antibiotics to treat infections in other anatomical locations⁽¹⁶16 SRIRANGARAJ, S.; KALI, A.; CHARLES, M. V. P. Antibiotic screening of urine culture as a tool for internal quality audit. Australas Med J, v. 7, n. 2, p. 73-7, 2014.^{, 18}18 WILSON, G.; BADARUDEEN, S.; GODWIN, A. Antibiotic screening of urine culture as a useful quality audit. J Infect Dev Ctries, v. 5, n. 4, p. 299-302, 2011.⁾. These studies reinforce the need for accurate information on antibiotic therapy at the time of exam request, since the use of antibiotics limits the sensitivity of the culture⁽¹⁸18 WILSON, G.; BADARUDEEN, S.; GODWIN, A. Antibiotic screening of urine culture as a useful quality audit. J Infect Dev Ctries, v. 5, n. 4, p. 299-302, 2011.⁾.

CONCLUSION

In this study, we demonstrated that in 7.45% of studied samples, the presence of antimicrobial residues compromised the recovery of uropathogens, producing a possible false negative result. The automated technique showed better performance when compared to the manual technique.

The research on antimicrobial residues activity can be considered a useful tool to rule out urinary infection, mostly in patients with bacteriuria which are under antimicrobial therapy, therefore, we suggest the implementation of the RARA as routine in microbiology laboratories.

AKNOWLEDGMENT

for the Diagnosis Support Unit of HC-UFPR for providing the structure for the accomplishment of the research, and to Alifax for materials and inputs supply.

REFERENCES

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