Microbiology and Infectious Disease / INTEGRITY OF URINE SPECIMENS

Detection of Chlamydia trachomatis and/or Neisseria gonorrhoeae infection in urine using molecular amplification assays has permitted institutions with limited medical facilities to offer testing for these sexually transmitted diseases (STDs). The Nebraska Public Health Laboratory (NPHL) investigated the validity of urine samples submitted for C trachomatis and/or N gonorrhoeae amplification after receiving a substantial number of clear specimens. Approximately 75% of all urine specimens submitted for STD testing to the NPHL were from correctional facilities. The falsification of urine specimens submitted for microbiology studies is not evaluated routinely, and this problem was previously undocumented. By using the criteria for specific gravity of 1.001 or less and a creatinine concentration of less than 5 mg/dL (442 μmol/L), approximately 8% of all specimens submitted during the study interval were determined to be inconsistent with urine. The microbiology laboratory should be aware of the possibility for specimen manipulation to identify facilities submitting falsified specimens, to initiate appropriate intervention, and to minimize false-negative reporting. In the United States, sexually transmitted diseases (STDs) constitute an epidemic, with an estimated 15 million persons acquiring a new STD each year.1 Infections with Chlamydia trachomatis and/or Neisseria gonorrhoeae were the first and second most common STDs in the United States reported to the Centers for Disease Control and Prevention in 2000 and accounted for 80% of all notifiable diseases.2 The highest rates of chlamydial and gonococcal infection occur in the 20to 24-year age group, which accounts for approximately 75% of all cases. Small minorities of infected individuals maintain a high rate of new partner acquisition and, thus, enhance transmission of infection. In addition, a large number of asymptomatic or minimally symptomatic infected persons continue sexual activity, resulting in further transmission of disease. Infections acquired from asymptomatic persons underscore the importance of tracing sexual contacts and the detection of subclinical infection.3,4 The application of molecular amplification technologies has had a substantial impact on the detection rate of C trachomatis and N gonorrhoeae infections.5 Amplification assays such as ligase chain reaction and polymerase chain reaction have greater sensitivity than culture.6-8 First-generation amplification technologies used material collected on a swab from the cervix or the urethra. Such an approach necessitated appropriate examination equipment and trained medical personnel. The advent of high-sensitivity amplification procedures expanded the range of specimen types to include urine.9 Urine amplification testing not only offers a noninvasive means of specimen collection but also eliminates the need for a private examination room and medical personnel. Institutions Microbiology and Infectious Disease / ORIGINAL ARTICLE Am J Clin Pathol 2003;119:516-521 517 517 DOI: 10.1309/KQLD7JAE1B401CUN 517 © American Society for Clinical Pathology with a high prevalence of disease and limited medical facilities, such as state penitentiaries and youth correctional facilities, can provide routine STD testing with the expectation that expanded screening will reduce the number of subclinical infections and ultimately the number of new cases.10,11 While this approach has proved successful in small target communities, the ability to successfully implement urinebased screening for STDs on a larger and more sustained scale is unknown.10 The Nebraska Public Health Laboratory (NPHL), Omaha, provides infectious disease testing for a variety of state-sponsored medical clinics and institutions. Of the urine samples submitted for C trachomatis and/or N gonorrhoeae amplification, approximately 45% are from a youth correctional facility, with the state penitentiary accounting for an additional 30%. The validation of swab specimens submitted for the detection of chlamydial infection, using molecular techniques, has been a major focus of the public health community.12 Adulteration of urine used in screening for drugs of abuse is a well-known problem. However, to our knowledge, falsification of urine specimens submitted for C trachomatis or N gonorrhoeae amplification testing has not been described previously. Therefore, no procedures were in place for the detection of potentially manipulated specimens. The specific characterization of a specimen as urine is difficult, and no single test is able to validate or refute the source of a sample. The characteristic yellow color of urine is imparted by the presence of urobilinogen, a breakdown product of hemoglobin. Between 95% and 99% of random urine specimens submitted to the clinical laboratory for urinalysis testing are yellow.13 Interest in determining the validity of urine specimens developed when NPHL technologists noted a substantial number of colorless urine specimens submitted for C trachomatis and/or N gonorrhoeae amplification. To characterize the sample as compatible with urine or not, criteria derived from the studies of Cook et al14 were used. A specimen with a urine creatinine concentration of less than 5 mg/dL (442 μmol/L) and a specific gravity of 1.001 or less was judged to be incompatible with urine, and review of urine specimens using these criteria was initiated. Materials and Methods All testing was performed in 1 laboratory on urine samples submitted from public health clinics. Clients were instructed to urinate into a collection cup and deliver the sample to an attendant. All specimens were transported on ice to the laboratory and tested within 5 days of collection. The study consisted of 2 parts. The first determined the validity of the adopted visual screening criteria, and the second determined the prevalence of falsified urine samples from various submitting sites. All specimens were screened initially by appearance according to the criteria of Cook et al,14 which include color (nominally yellow, not colorless), clarity (normally transparent), and foaming properties (subjectively, those characteristic of a dilute protein solution rather than of detergent). Laboratory technologists performed visual screening based on these criteria, and all specimens were classified as visually consistent with or inconsistent with urine. Specific gravity measurements were performed on all flagged specimens. A concurrent urine creatinine concentration was obtained on visually suspect specimens with a specific gravity of 1.001 or less. To determine the validity of appearance alone as a screening tool, consecutive specimens, irrespective of visual classification, were submitted for paired measurements of specific gravity and creatinine concentration. The opacity of purulent specimens precluded a creatinine concentration measurement, and these samples were excluded from the validation study. Specific gravity was measured by using a light refractometer following the standard procedures recommended by the manufacturer (Reichert Cambridge, Buffalo, NY). Creatinine concentration measurements were performed with an enzymatic assay (creatinine amidohydrolase) on the Vitros 250 analyzer (Ortho Clinica Diagnostics, Rochester, NY) using the reagents and procedures developed by the manufacturer. The lower reportable limit of the assay, as determined by the manufacturer, was 0.05 mg/dL (4.4 μmol/L). Specimens with a specific gravity of 1.001 or less with a concurrent creatinine concentration of less than 5 mg/dL (442 μmol/L) were classified as inconsistent with urine. The reference range for human urine specific gravity is 1.003 to 1.030.15 Hyposthenuria, a potentially serious medical condition, is defined as a specific gravity of less than 1.007.16,17 Specimens with a specific gravity of less than 1.007 but greater than 1.001 were classified as hyposthenuric. Flagged samples with a subsequent specific gravity of 1.007 or more were classified as consistent with urine. A graphic representation of the classification scheme is provided in ❚Figure 1❚. Testing for C trachomatis and N gonorrhoeae was performed on all specimens, including those flagged for further evaluation. Testing using the BDProbeTec ET system (Becton Dickinson, Franklin Lakes, NJ) was performed according to the manufacturer’s recommended procedure. The BDProbeTec ET system has a reported sensitivity and specificity of 90.7% and 96.6%, respectively, in the detection of chlamydial infection, and a reported Birch et al / INTEGRITY OF URINE SPECIMENS 518 Am J Clin Pathol 2003;119:516-521 518 DOI: 10.1309/KQLD7JAE1B401CUN © American Society for Clinical Pathology sensitivity and specificity of 96.0% and 98.8%, respectively, in the detection of gonococcal infection. Coded specimen accession numbers were assigned during the initial screening, which permitted retrospective pairing of the sample with the submitting institution.