Detection of Mycobacterium tuberculosis transrenal DNA in urine samples among adults in Peru.
Author(s): Mesman AW, Calderon RI, Hauns L, Pollock NR, Mendoza M, Holmberg RC, Franke MF
Publication: Tuberculosis (Edinb), 2024, Vol. 148, Page 102549
PubMed ID: 39098064 PubMed Review Paper? No
Purpose of Paper
This paper investigated the sensitivity and specificity of a transrenal DNA (trDNA) assay for tuberculosis (TB) using two different cut-off thresholds. The authors also investigated potential associations between positivity of the TB assay and time of collection relative to unspecified patient treatment (pretreatment versus 4 or 7 days post-treatment), analysis of first morning versus a subsequent morning urine specimen, TB detection and grade in a sputum smear, patient age, and urinalysis parameters (specific density, pH and the presence of bacteria, protein, yeast, leukocytes or red blood cells).
Conclusion of Paper
The trDNA assay for TB had a specificity of 100%, regardless of which cut-offs were applied but an overall sensitivity of 38% (83 of 221 specimens) using the initial cutoff [crossing point (Cp) <35, threshold >10 units] and 58% (129 of 221 specimens) using a revised cutoff [Cp remained <35, threshold >5 units]. While the sensitivity was always higher when the revised threshold was used compared to the initial threshold, it was comparable among the treatment timepoints examined: before treatment (61% and 37%, respectively) and 4 days (58% and 40%, respectively) or 7 days (55% and 35%, respectively) post-treatment. The sensitivity was also higher in specimens from patients with a sputum smear that was TB-positive than one that was negative (38-47% versus 29% with an initial cut-off and 41-53% versus 11% with revised cut-off). When positivity was considered by patient (a patient was considered positive when any one of the three treatment timepoints was positive), the sensitivity was 73% with the initial cut-off and 88% with the revised cut-off. However, a TB-positive result in all three treatment timepoints only occurred in 11% of patients using the initial cut-off and 27% of patients using the revised cut-off.
Analysis by binomial generalized estimating equation (GEE) regression did not identify an association between the detection of TB in transrenal DNA and patient age (<22 years, 22-30 years, 31-40 years or >40 years); sputum smear grade; diagnosis with multi-drug resistant TB; use of first morning urine (versus subsequent morning collection); specific density of the urine (<1.01, 1.01-1.025, or >1.025), urine pH (acidic, neutral or alkaline; cut-offs not specified); or the presence of bacteria, protein, yeast, leukocytes or red blood cells in the specimen.
Studies
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Study Purpose
This paper investigated the sensitivity and specificity of a transrenal DNA (trDNA) assay for tuberculosis (TB) using two different cut-off thresholds. The authors also investigated potential associations between positivity of the TB assay and time of collection relative to unspecified patient treatment (pretreatment versus 4 or 7 days post-treatment), analysis of first morning versus a subsequent morning urine specimen, TB detection and grade in a sputum smear, patient age, and urinalysis parameters (specific density, pH and the presence of bacteria, protein, yeast, leukocytes or red blood cells). Urine (50 mL clean catch) was collected from 75 patients (18-76 years of age; 29 women and 46 men; two patients with HIV; two patients with diabetes) with a positive sputum culture for TB (19 with a TB negative sputum smear and 56 with a TB positive sputum smear; 10 with multi-drug resistant TB) between 6 and 11 AM (46 first morning urine specimens) before treatment and again on day 4 and 7 post-treatment (treatments not specified). An aliquot of each of the 221 specimens was used for urinalysis yielded the following results: 18 urine specimens were bacteria positive; 2 were protein positive; 6 were yeast positive; 53 were leukocyte positive; 49 were red blood cell positive; 208 were acidic, 8 were neutral and 4 were alkaline; and the specific density was <1.01 in 48 specimens, 1.01-1.025 in 168 specimens, and >1.025 in 4 specimens. EDTA was added to the remaining specimens to achieve a concentration of 10 mM, and the specimen was transported (cold-chain, temperature and duration not specified) and aliquoted before storage at -80°C. DNA was extracted from urine using an in-house anion exchange method that involved capturing DNA using Q-sepharose resin, washing in a low-salt solution (0.3 M LiCl, 10 mM NaOAc), eluting with a high salt solution (2 M LiCl, 10 mM NaOAc) and clean-up and concentration using QIAquick spin columns. Transrenal Tb DNA was quantified using semi-nested PCR to amplify a 39 bp region. The specificity of the assay was investigated using commercially acquired EDTA stabilized control urine specimens.
Summary of Findings:
The trDNA assay for TB had a specificity of 100% as TB was not detected in any of the commercially acquired control urine specimens, regardless of the cut-off applied. The trDNA assay with the initial cutoff [crossing point (Cp) <35 and threshold >10 units] had a sensitivity of 38% (83 of 221 specimens), with similar sensitivity before (37%; 28 of 75 specimens) and 4 days (40%; 29 of 72 specimens) or 7 days (35%; 26 of 74 specimens) post-treatment. The sensitivity of the initial cut-off was highest among urine specimens collected pre-treatment from patients with a TB-positive sputum smear that was classified as grade 3+ (50%; 6 of 12 specimens) and lowest in urine specimens collected on day 4 post-treatment from patients with a TB-negative sputum smear (24%; 4 of 17 specimens). When positivity was considered by patient (a patient was considered positive when any one of the three treatment timepoints was positive), the sensitivity was 73% (55 of 75 patients), but only 11% (8 of 75 patients) had a positive assay at all three timepoints.
When the positivity threshold was reduced to an increase of >5 instead of >10 (Cp still <35), the sensitivity of the trDNA assay increased to 58% (129 of 221 specimens), with similar sensitivity still observed among urine specimens collected before (61%; 46 of 75 specimens) and 4 days (58%; 42 of 72 specimens) or 7 days (55%; 41 of 74 specimens) post-treatment. Importantly, the change in positivity cut-off threshold to >5, increased the assay sensitivity at each timepoints both in patients with a TB-negative sputum smear (41-53%) and those with a TB-positive sputum smear positive (50-68%). When positivity was considered by patient (any one of the three specimens positive using revised cut-off) with the revised cut-off, the sensitivity was 88% (66 of 75 patients), with a slightly higher sensitivity amongst patients with a TB-positive smear than a TB-negative sputum smear (92% versus 79%), but only 27% (20 of 75 patients; 16% of smear negative patients and 47% of smear positive patients) had a assay result that was positive at all three timepoints. Analysis using a binomial generalized estimating equation (GEE) regression did not identify an association between detection of TB in trDNA and patient age (<22 years, 22-30 years, 31-40 years or >40 years); sputum smear grade; diagnosis with multi-drug resistant TB; use of first morning urine (versus subsequent morning collection); specific density of the urine (<1.01, 1.01-1.025, or >1.025), urine pH (acidic, neutral or alkaline cut-offs not specified); or the presence of bacteria, protein, yeast, leukocytes or red blood cells in the specimen.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Other diagnoses
Platform:
Analyte Technology Platform DNA Real-time qPCR Electrolyte/Metal Clinical chemistry/auto analyzer Cell count/volume Clinical chemistry/auto analyzer Small molecule Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient age <22 years
22-30 years
31-40 years
>40 years
Preaquisition Diagnosis/ patient condition Tuberculosis
Multi-drug resistant tuberculosis
Preaquisition Prognostic factor Sputum smear negative
Sputum smear positive 1+
Sputum smear positive 2+
Sputum smear positive 3+
Biospecimen Acquisition Time of biospecimen collection First morning urine
Subsequent morning urine
Pretreatment
4 days post-treatment
7 days post-treatment
Biospecimen Aliquots and Components pH High pH
Low pH
Neutral
Biospecimen Aliquots and Components Biospecimen components Effects of presence of bacteria, protein, yeast, leukocytes or red blood cells investigated
Real-time qPCR Specific Data handling Threshold of Cp <35 and a threshold >10
Threshold of Cp <35 and a threshold >5