Mycobacterium tuberculosis and rifampin resistance, United Kingdom.

The United Kingdom Health Protection Agency Mycobacterium Reference Unit offers a national "Fastrack" molecular service for detecting Mycobacterium tuberculosis complex (MTBC) and rifampin resistance by using the INNO-LiPA Rif.TB assay. We analyzed the service in a routine, nontrial context of 1,997 primary clinical specimens, including 658 nonrespiratory specimens. The overall adjusted concordance, sensitivity, specificity, positive predictive value, and negative predictive value for detecting MTBC were 91.2%, 85.2%, 96.2%, 95.7%, and 86.7%, respectively (unadjusted, 86.7%, 85.2%, 88.2%, 86.9%, and 86.7%), when false-positive samples from patients (n = 83) with a known microbiologic diagnosis of MTBC or patients receiving current or recent antituberculous treatment were excluded. The parameters for detecting rifampin resistance were 99.1%, 95.0%, 99.6%, 92.7%, and 99.7%, respectively. The assay enabled earlier diagnosis of MTBC and rifampin resistance (15.2 days) compared with culture-based techniques (30.7 days).

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The increasing incidence of multidrug-resistant tuberculosis (MDRTB), defined as resistance to at least rifampin and isoniazid, is a notable global health problem (1). The rapid identification of patients with MDRTB enables early institution of appropriate treatment, which is associated with improved survival (2,3), and infection control procedures to minimize risk of transmission (4). The Centers for Disease Control and Prevention recommends that the culture/identification and susceptibility testing of Mycobacterium tuberculosis complex (MTBC) be completed within 21 and 30 days of specimen receipt, respectively (5). Molecular assays based on the genetics of drug resistance may considerably reduce these turnaround times.

In the United Kingdom, 82.5% of rifampin-resistant isolates are also resistant to isoniazid (6), making rifampin resistance a useful surrogate marker for MDRTB. Most rifampin-resistant MTBC strains have mutations in an 81-bp region of the rpoB gene that encodes the RNA polymerase [beta] subunit (7). This region is therefore an ideal target for molecular tests for rifampin resistance.

The United Kingdom Health Protection Agency Mycobacterium Reference Unit (MRU) offers a national molecular diagnostic service (Fastrack) for detection of MTBC and rifampin resistance (8) by using the INNO-LiPA Rif.TB assay (Innogenetics, Zwijndrecht, Belgium) and supplemented by DNA sequencing as needed. This assay is based on reverse hybridization between rpoB amplicons and membrane-bound capture probes (1 specific for MTBC, 5 overlapping wild-type probes spanning the rpoB target region, and 4 of the most common mutations). Genotypic resistance is indicated by absence of hybridization with wild-type probes or hybridization with resistance mutation probes (9).

A review of the line probe assay (LiPA) (10) found that most previous evaluations focused on mycobacterial isolates and culture-positive (mainly respiratory) specimens (9,11-14), but relatively little data exist on nonrespiratory and smear-negative specimens, which are often collected in routine clinical practice (8,15,16). The Fastrack service was initially targeted at smear-positive respiratory samples and mycobacterial isolates, but in response to widespread demand from other laboratories, was extended to all specimens, regardless of acid-fast bacilli (AFB) status. In January 2002, an in-house polymerase chain reaction (PCR) assay targeting the IS6110 insertion sequence (17) replaced LiPA for testing cerebrospinal fluid (CSF) samples. Therefore, CSF samples were not included in this study. This study evaluated LiPA in the context of a nontrial clinical service in one of the largest reported samples of 1,997 primary clinical specimens (including 658 nonrespiratory) and 290 clinical isolates tested from 1999 to 2002.

Materials and Methods

Clinical Specimens

From January 1999 through December 2002, the MRU received 2,287 consecutive non-CSF specimens from 2,110 patients (comprising 1,997 primary clinical specimens and 290 clinical isolates) from 152 centers in the United Kingdom and Ireland for Fastrack analysis. Specimens are submitted for analysis at the discretion of individual referring laboratories, usually when the diagnosis of MTBC is uncertain or when rifampin resistance is suspected. When multiple specimens were received from a single patient, each specimen was processed separately. Of the primary specimens, 1,339 respiratory specimens were sputum, bronchial washings, and bronchial and tracheal aspirates; 658 were nonrespiratory specimens. Samples were received only on weekdays, and routine processing and culture were initiated within 24 hours of receipt. Turnaround times for completion of analysis, culture, and identification of MTBC and drug-susceptibility testing were calculated from date of specimen receipt (5).

Routine Microscopy, Culture, Identification, and Susceptibility Testing

Samples were decontaminated by using the NaOH/N-acetyl-L-cysteine method in a 2-mL suspension, and AFB staining was performed with auramine-phenol and the Ziehl-Neelsen procedure (18,19). DNA was extracted from 1 mL of decontaminated specimen by using a previously described chloroform extraction technique (20), and the remaining 1 mL was added to 1 MB/BacT rapid culture vial (bioMerieux UK Ltd., Basingstoke, UK) and 1 Lowenstein-Jensen slope. Cultures were incubated for at least 8 weeks. Mycobacterial cultures were identified by microscopic and macroscopic appearances, biochemical tests, and DNA hybridization with Accuprobe (GenProbe, San Diego, CA, USA). Drug-susceptibility testing was carried out by the resistance ratio method (18).

LiPA

LiPA was performed according to manufacturer's instructions. The first round of a nested PCR was performed with 10 [micro]L of DNA extract and outer primers (LiPA OP1, 5'-GAGAATTCGGTCGGCGAGCTGATCC-3' and LiPA OP2, 5'-CGAAGCTTGACCCGCGCGTACACC-3') for 30 cycles at 95[degrees]C for 60 s, 58[degrees]C for 30 s, and 72[degrees]C for 90 s. One microliter of first-round product was transferred to a 40-[micro]L PCR mixture containing inner primers (LiPA IP1, 5'-GGTCGGCATGTCGCGGATGG-3' and LiPA IP2, 5'-GCACGTCGCGGACCTCCAGC-3'), which were biotinylated at the 5' end, for the second round of amplification for 30 cycles at 95[degrees]C for 20 s, 65[degrees]C for 30 s, and 72[degrees]C for 30 s. Each PCR run included a duplicate and an inhibition control (100 genome copies of Mycobacterium bovis bacillus Calmette-Guerin [BCG]) for each sample, 5 extracted, water, negative controls, decontaminated, extracted, negative and positive controls (a known culture-positive clinical sample), and a positive control with a low amount of DNA (10 genome copies of BCG in 10 [micro]L). A 260-bp band on agarose gel electrophoresis confirmed successful amplification. The hybridization assay to determine genotypic rifampin resistance was then performed and analyzed as previously described (13). The MTBC result was then reported as positive (accompanied by a rifampin-susceptibility result), negative, equivocal, or inhibited. Results were considered equivocal if a sample tested PCR positive on 1 of 2 duplicates on 2 separate occasions. Extracted DNA was stored for retesting equivocal and inhibited results and for future resolution of discrepant susceptibility results.

Sequencing of rpoB PCR Product

Cultures of MTBC with discordant rifampin-susceptibility results by phenotypic and LiPA testing underwent automated sequencing of the rpoB PCR products with either the Long Read Tower System (Visible Genetics, Suwanee, GA, USA) or the CEQ 8000 Genetic Analysis System (Beckman Coulter, High Wycombe, UK). DNA was extracted from cultures and amplified in a PCR containing the outer primers OP1 and OP2 and sequenced with the inner primers IP1 and IP2.

Statistical Analysis

Data were entered into Microsoft Access (Microsoft Corp., Redmond, WA, USA) and analyzed with Microsoft Excel. Detection of MTBC and rifampin resistance by LiPA was compared with results by the accepted standards of culture and phenotypic susceptibility testing. Concordance, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. We excluded 85 (3.7%) samples from primary analysis because LiPA results could not be compared with culture results. These samples had equivocal PCR results (n = 27, 1.2%), were inhibitory to PCR (n = 22, 1.0%), could not be cultured (e.g., because of insufficient volume or histologic samples embedded in paraffin wax; n = 6, 0.3%), or were contaminated with bacteria or fungi (n = 30, 1.3%).

Results

Microscopy and Culture

Of the primary specimens tested by LiPA, the AFB smear microscopy was positive in 1,137 (56.9%), negative in 821 (41.1%), and not performed in 39 (2.0%). Specimen types are shown in Tables 1 and 2. Culture identification and drug susceptibility results are shown in Table 3. MTBC was cultured from 941 (47.1%) of 1,997 primary samples and 238 (82.1%) of 290 isolates. In 3 cases, both MTBC and nontuberculous mycobacteria were cultured. A total of 1,178 M. tuberculosis, 10 M. bovis (including 1 BCG), and 1 M. africanum cultures were identified in the 4-year study period. During this time, 6,500-7,000 cases of tuberculosis were reported in the United Kingdom per year, including 4,500-5,000 reported to be culture positive (6). The times taken to culture MTBC from primary specimens are shown in Table 4. There were 223 nontuberculous mycobacteria isolates: 80 M. avium complex, 38 M. kansasii, 26 M. xenopi, 23 M. malmoense, 20 M. chelonae, 10 M. fortuitum, 3 M. abscessus, 3 M. marinum, 2 M. gordonae, 2 M. simiae, 2 M. terrae, 1 M. szulgai, 1 M. vaccae, and 12 unidentified Mycobacterium species.

LiPA

Results of LiPA analysis for MTBC were positive in 1,153 (50.4%), negative in 1,085 (47.4%), equivocal in 27 (1.2%), and inhibited in 22 (1.0%) primary specimens. Of the 1,153 PCR-positive samples, 1,085 (94.1%) were reported as rifampin susceptible by LiPA, and 68 (5.9%) were reported as rifampin resistant. Of the 27 PCR-equivocal samples, 16 grew MTBC (6 AFB negative, 1 AFB unknown, 9 AFB positive), 3 grew M. avium complex, and 8 yielded no mycobacterial growth. Tables 1 and 2, respectively, show the results of LiPA in detecting MTBC from primary specimens and rifampin resistance from specimens that grew MTBC. Data on antituberculous treatment were incomplete, but when reported, 195 (9.8%) samples were from patients receiving treatment currently or within the last 3 months. A total of 309 (15.5%) had a history of antituberculous treatment (Tables 5 and 6).

Discrepant Results

There were 136 false-negative MTBC results, i.e., samples negative by LiPA that subsequently yielded MTBC on culture. There were 118 apparently false-positive MTBC results by LiPA, which were PCR positive but did not grow MTBC, although 88 were AFB positive. A total of 83 false-positive samples were considered to have correct molecular results because they were from patients with a microbiologic diagnosis of MTBC made at MRU from another sample (n = 61) or from patients who were receiving antituberculous treatment currently or had received it within the last 3 months (n = 22). These 83 samples were excluded from statistical analysis to give adjusted values for specificity and PPV (Table 1). Ten specimens were from 6 patients with discrepant results for rifampin susceptibility (Table 7).

Eight specimens had wild-type rpoB, and 2 had mutations not associated with rifampin resistance.

Discussion

We assessed LiPA on the largest reported sample of 1,997 clinical specimens in a nontrial, routine context that would be meaningful to clinicians, especially those submitting samples other than AFB-positive respiratory specimens. The overall unadjusted concordance, sensitivity, specificity, PPV, and NPV were 86.7%, 85.2%, 88.2%, 86.9%, and 86.7%, respectively, for detecting MTBC in primary samples and 98.9%, 98.7%, 100%, 100%, and 93.3%, respectively, for isolates. Previous studies that tested mainly respiratory samples and isolates reported concordance rates with culture from 78.3% to 100% and were usually controlled studies (8-16).

When PCR was compared with culture for detecting MTBC, some false-positive results may, in fact, have been true-positive results. Of 118 samples classified as false positive, 83 were believed to be true positive on the basis of our planned protocol. These consisted of 61 samples from patients with a microbiologic diagnosis of MTBC at our laboratory in the last 18 or subsequent 3 months and an additional 22 samples from patients who were receiving antituberculous treatment or who had received it within the last 3 months. Patients who were receiving treatment currently or within the last 3 months were significantly less likely to have MTBC; of 195 samples from such patients, 70 (35.9%) had MTBC compared to 871 (49.3%) of 1,766 samples from patients with no reported treatment within the last 3 months ([chi square] = 12.7, p<0.001). Furthermore, a significantly higher proportion of rifampin-resistant MTBC was isolated from patients receiving treatment (12/70, 17.1%) compared with patients not reported to be receiving treatment (34/866, 3.9%, [chi square] = 24.2, p<0.001). In these 83 false-positive samples believed to represent true positive results, PCR detected nucleic acid from nonviable organisms (due to treatment) or viable organisms in insufficient numbers for successful culture. If these 83 samples are excluded from overall analysis, specificity improves for all primary specimens, AFB-positive specimens, and AFB-negative specimens from 88.2%, 70.8%, and 95.7%, respectively, to adjusted values of 96.2%, 91.8% and 97.7% (Table 1). PPV improves from 86.9%, 89.5% and 54.7%, respectively, to 95.7%, 97.5% and 70.0%. Other false-positive samples could probably be excluded; we only chose to exclude those with microbiologic diagnoses of MTBC at our laboratory because we had no data on microbiologic, histologic, or clinical diagnoses made by the other hospitals that submitted these samples. Furthermore, since relevant data were often not provided, many more patients likely were receiving antituberculous therapy that we were unaware of because treatment failure is a common reason for specimens being submitted for testing.

PCR-equivocal results were excluded from the primary analysis. However, a PCR-equivocal result may represent a lack of sensitivity. If PCR-equivocal results are considered PCR negative, the adjusted values for detecting MTBC in primary specimens were only marginally altered to 90.6%, 84.0%, 96.2%, 95.7%, and 85.7%, respectively, for concordance, sensitivity, specificity, PPV, and NPV.

A recent review of LiPA results reported that although little data on clinical specimens were available, sensitivity appeared lower than that of isolates (10). Our study confirmed this finding, with sensitivities of 85.2% for all clinical specimens and 98.7% for isolates. As with other PCR-based tests (21-23), sensitivities of LiPA for AFB-negative (29.4%) and nonrespiratory samples (61.1%) were low. Sensitivity was also reduced to 71.6% in patients receiving treatment at the time or within 3 months of the time the sample was obtained. Marttila et al. tested 75 clinical specimens with LiPA, including 66 from nonrespiratory sites, and reported a sensitivity of 58.8% compared with final clinical and pathologic diagnoses, whereas cultures showed a sensitivity of 35.3% (15). Several factors may explain the lower sensitivity of PCR-based methods in these samples. The mycobacterial load is lower, as demonstrated by the significantly shorter time taken to culture MTBC for AFB-positive samples than for AFB-negative samples (18.5 days vs. 29.5 days, z = 8.0, p<0.001), and respiratory samples than nonrespiratory samples (18.7 days vs. 25.0 days, z = 5.6, p<0.001). However, more respiratory samples were AFB positive (94.3% vs. 55.0%). Irregular clumping may take place within paucibacillary specimens, and small, suboptimal sample volumes often lead to sampling errors. Nonrespiratory specimens, especially pleural fluid, bone marrow, pus, and tissue biopsy specimens, may contain inhibitors of amplification (22,23). Inhibition rates in this study were 1.0% overall, with above-average rates in blood and feces (both 2/3, [66.7%]), pleural fluid (2/110 [1.8%]), bone marrow (1/23 [4.3%]), and pus/tissue (8/400 [2.0%]).

The nonrespiratory specimen types with the highest sensitivity rates were vertebral aspiratesbiopsy specimens (n = 30, sensitivity 83.3%), gastric aspirates (n = 18, sensitivity 80.0%), and lymph node aspirates/biopsy specimens (n = 144, sensitivity 72.5%). For pleural fluid, one of the most commonly submitted samples (n = 107), LiPA had one of the lowest sensitivity rates (21.7%) for detecting MTBC. The difficulties in detecting MTBC in pleural fluid are well recognized, with previous reported sensitivities of 20% (24) and 50% (23) with the Gen-Probe amplified M. tuberculosis direct test.

For detecting rifampin resistance in PCR-positive specimens yielding MTBC on culture, LiPA had concordance, sensitivity, specificity, PPV, and NPV values of 99.1%, 95.0%, 99.6%, 92.7%, and 99.7%, respectively. These results are consistent with previous studies that reported concordance rates of 90.2% to 100% (8-18). In this study, of the 69 rifampin-resistant MTBC strains cultured, 5 were PCR negative for MTBC. Of the remaining 64 that were PCR positive, 59 (93.7%) had detectable rpoB mutations and were reported as resistant. At least 90% of rifampin-resistant strains have mutations within the target rpoB region, although this proportion may vary in different populations (7).

Detection of rifampin resistance by LiPA may be used as an early predictor of MDRTB before phenotypic susceptibilities are available, but this clearly depends on the prevalence of rifampin monoresistance in the study population. The diagnosis of rifampin monoresistance is also critical because this automatically invalidates the use of short-course chemotherapy (25). Of the 59 correctly identified rifampin-resistant MTBC isolates, 11 were rifampin monoresistant. The overall prevalence was 1.0% in this study, which was higher than the 0.3% reported in a national UK survey (6). This result reflects a common underlying reason for specimen referral for Fastrack analysis, i.e., failure of response to treatment.

For primary samples in which LiPA detected MTBC, diagnosis of tuberculosis was made an average of 15.2 days earlier than with automated liquid culture (14.8 days for AFB-positive specimens and 22.1 days for AFB-negative specimens). More days were saved with nonrespiratory samples (18.3 days) than with respiratory samples (14.7 days), although these samples had the lowest probability of detection. LiPA accurately determined rifampin susceptibility earlier than solid culture-based techniques by a mean of 30.7 days for all primary specimens. This compares favorably with a study that found that LiPA saved a median of 24 days compared with susceptibility testing with the BACTEC liquid culture system (Becton Dickinson, Sparks, MD, USA) and 54 days with solid media (11).

In summary, LiPA may be used with clinical samples for diagnosis of MTBC and rifampin resistance, saving, when positive results are obtained, an average of 15.2 days and 30.7 days, respectively, compared with conventional techniques. However, some limitations of LiPA are evident. As with other PCR-based assays, sensitivity is reduced in AFB-negative and nonrespiratory samples, such as paucibacillary forms of the disease, in which rapid diagnosis would be most helpful. Although the assay is a potential diagnostic route for patients receiving therapy, sensitivity is also reduced in these circumstances. The lower sensitivity rates for certain samples and the possibility of a PCR-equivocal or PCR-inhibited result also mean that conventional culture and sensitivity testing should still be used at the same time. Alternatives to LiPA may be useful, e.g., we used an IS6110-based PCR for diagnosis of tuberculous meningitis. Similarly, rifampin-resistance mutations can be detected by DNA sequencing (we now sequence all PCR products identified as MTBC with any form of rifampin probe mutations) or with noncommercial macroarrays (26,27). Thus, molecular results, as with any laboratory test, should be reviewed in the context of all clinical, microbiologic, and histologic results.

Acknowledgments

We thank the staff of the United Kingdom Health Protection Agency MRU for laboratory testing of samples.

Dr Sam is a medical microbiologist at the University of Malaya, Kuala Lumpur, Malaysia. His research interests include epidemiologic aspects of infectious diseases such as tuberculosis.

References

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(7.) Telenti AP, Imboden P, Marchesi F, Lowrie D, Cole S, Colston MJ, et al. Detection of rifampin-resistance mutations in Mycobacterium tuberculosis. Lancet. 1993;341:647-50.

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(10.) Morgan M, Kalantri S, Flores L, Pai M. A commercial line probe assay for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis. BMC Infect Dis. 2005;5:62.

(11.) Skenders G, Fry AM, Prokopovica I, Greekoseja S, Broka L, Metchock B, et al. Multidrug-resistant tuberculosis detection, Latvia. Emerg Infect Dis. 2005;11:1461-3.

(12.) Johansen IS, Lundgren B, Sosnovskaja A, Thomsen VO. Direct detection of multi-drug resistant Mycobacterium tuberculosis in clinical specimens in low- and high-incidence countries by line probe assay. J Clin Microbiol. 2003;41:4454-6.

(13.) Rossau R, Traore H, de Beenhouwer H, Mijs W, Jannes G, de Rijk P, et al. Evaluation of the INNO-LiPA Rif. TB assay, a reverse hybridization assay for the simultaneous detection of Mycobacterium tuberculosis complex and its resistance to rifampin. Antimicrob Agents Chemother. 1997;41:2093-8.

(14.) Traore H, Fissette K, Bastian I, Devleeschouwer M, Portaels F. Detection of rifampin resistance in Mycobacterium tuberculosis isolates from diverse countries by a commercial line probe assay as an initial indicator of multidrug resistance. Int J Tuberc Lung Dis. 2000;4:481-4.

(15.) Marttila HJ, Soini H, Vyshnevskaya E, Vyshnevskiy BI, Otten TF, Vasilyef AV, et al. Line probe assay in the rapid detection of rifampin-resistant Mycobacterium tuberculosis directly from clinical specimens. Scand J Infect Dis. 1999;31:269-73.

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(17.) Caws M, Wilson SM, Clough C, Drobniewski F. Role of IS6110-targeted PCR, culture, biochemical, clinical, and immunological criteria for diagnosis of tuberculous meningitis. J Clin Microbiol. 2000;38:3150-5.

(18.) Collins CH, Grange JM, Yates MD. Tuberculosis: bacteriology, organization and practice. 2nd ed. Oxford (UK): Butterworth-Heinemann; 1997.

(19.) Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. Atlanta (GA): Centers for Disease Control; 1985.

(20.) Watterson SA, Wilson SM, Yates MD, Drobniewski FA. Comparison of three molecular assays for rapid detection of rifampin resistance in Mycobaeterium tuberculosis. J Clin Microbiol. 1998;36:1969-73.

(21.) Sarmiento OL, Weigle KA, Alexander J, Weber DJ, Miller WC. Assessment by meta-analysis of PCR for diagnosis of smear-negative pulmonary tuberculosis. J Clin Microbiol. 2003;41:3233-40.

(22.) Honore-Bouakline S, Vincensini JP, Giacuzzo V, Lagrange PH, Herrman JL. Rapid diagnosis of extrapulmonary tuberculosis by PCR: impact of sample preparation and DNA extraction. J Clin Microbiol. 2003;41:2323-9.

(23.) Pfyffer GE, Kissling P, Jahn EM, Welscher HM, Salfinger M, Weber R. Diagnostic performance of amplified Mycobacterium tuberculosis direct test with cerebrospinal fluid, other nonrespiratory, and respiratory specimens. J Clin Microbiol. 1996;34:834-41.

(24.) Vlaspolder F, Singer P, Ruggeveen C. Diagnostic value of an amplification method (Gen-Probe) compared with that of culture for diagnosis of tuberculosis. J Clin Microbiol. 1995;33:2699-703.

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(26.) Nilolayevsky VT, Brown T, Balabanova Y, Ruddy M, Fedorin I, Drobniewski F. Detection of mutations associated with isoniazid and rifampin resistance in Mycobacterium tuberculosis isolates from Samara Region, Russian Federation. J Clin Microbiol. 2004; 42:4498-502.

(27.) Drobniewski F, Balabanova Y, Ruddy M, Weldon L, Jeltkova K, Brown T, et al. Rifampin- and multidrug-resistant tuberculosis in Russian civilians and prison inmates: dominance of the Beijing strain family. Emerg Infect Dis. 2002;8:1320-6.

Address for correspondence: Francis Drobniewski, Health Protection Agency National Mycobacterium Reference Unit, Centre for Infection, Institute of Cell and Molecular Science, Barts and the London School of Medicine, 2 Newark St, London E1 2AT, UK; email: [e-mail]

I-Ching Sam, * (1) Francis Drobniewski,* Philip More, * Melanie Kemp, * and Timothy Brown *

* Health Protection Agency, London, United Kingdom

(1) Current affiliation: University of Malaya, Kuala Lumpur, Malaysia

Table 1. Results of LiPA compared with culture in detecting
MTBC in primary clinical specimens *

                                   No. positive/no. tested (%)
Sample and AFB
smear result                          Concordance        Sensitivity

All primary LiPA                   1,667/1,922 (86.7)   782/918 (85.2)
  Positive                          960/1,099 (87.4)    747/798 (93.6)
  Negative                           679/792 (85.7)     35/119 (29.4)
  Not done                            28/31 (90.3)         0/1 (0)
All primary LiPA                   1,667/1,828 (91.2)   782/918 (85.2)
(adjusted values) ([dagger])
  Positive                          960/1,028 (93.4)    747/798 (93.6)
  Negative                           679/771 (88.1)     35/119 (29.4)
  Not done                            28/29 (96.6)         0/1 (0)
Respiratory                        1,168/1,298 (90.0)   672/738 (91.1)
  Positive                           827/915 (90.4)     657/696 (94.4)
  Negative                           328/369 (88.9)      15/42 (35.7)
  Not done                            13/14 (92.9)            --
Nonrespiratory                       499/624 (80.0)     110/180 (61.1)
  Positive                           133/184 (72.3)     90/102 (88.2)
  Negative                           351/423 (83.0)      20/77 (26.0)
  Not done                            15/17 (88.2)         0/1 (0)
Biopsy specimen$                     92/108 (85.2)       13/26 (50.0)
  Positive                            19/21 (90.5)      10/10 (100.0)
  Negative                            72/86 (83.7)       3/16 (18.8)
  Not done                            1/1 (100.0)             --
Gastric aspirate                      17/18 (94.4)        4/5 (80.0)
  Positive                            8/8 (100.0)        4/4 (100.0)
  Negative                            9/10 (90,0)         0/1 (0.0)
  Not done                                 --                 --
Lymph node                           103/142 (72.5)      50/68 (73.5)
  Positive                            54/79 (68.4)       42/48 (87.5)
  Negative                            48/62 (77.4)       8/20 (40.0)
  Not done                            1/1 (100.0)             --
Pleural fluid                        84/107 (78.5)       5/23 (21.7)
  Positive                            6/10 (60.0)         4/5 (80.0)
  Negative                            77/97 (79.4)        1/18 (5.6)
  Not done                            1/1 (100.0)             -
Psoas abscess                         8/15 (53.3)         3/6 (50.0)
  Positive                             2/3 (66.7)        2/2 (100.0)
  Negative                            6/12 (50.0)         1/4 (25.0)
  Not done                                 --                 --
Vertebral aspirate                    26/30 (86.7)       10/12 (83.3)
  Positive                            9/10 (90.0)        8/8 (100.0)
  Negative                            17/19 (89.5)        2/3 (66.7)
  Not done                             0/1 (0.0)          0/1 (0.0)
Other ([section])                    169/204 (82.8)      25/40 (62.5)
  Positive                            35/53 (66.0)       20/25 (80.0)
  Negative                           122/138 (88.4)      5/15 (33.3)
  Not done                            12/13 (92.3)            --

                                   No. positive/no. tested (%)

Sample and AFB
smear result                          Specificity            PPV

All primary LiPA                    886/1,004 (88.2)    782/900 (86.9)
  Positive                           213/301 (70.8)     747/835 (89.5)
  Negative                           644/673 (95.7)      35/64 (54.7)
  Not done                            29/30 (96.7)         0/1 (0)
All primary LiPA                     886/921 (96.2)     782/817 (95.7)
(adjusted values) ([dagger])
  Positive                           213/232 (91.8)     747/766 (97.5)
  Negative                           644/659 (97.7)      35/50 (70.0)
  Not done                            29/30 (96.7)         0/1 (0)
Respiratory                          496/560 (88.6)     672/736 (91.3)
  Positive                           170/219 (77.6)     657/706 (93.1)
  Negative                           313/327 (95.7)      15/29 (51.7)
  Not done                            13/14 (92.9)          11.00
Nonrespiratory                       390/444 (87.8)     110/164 (67.1)
  Positive                            43/82 (52.4)      90/129 (69.8)
  Negative                           331/346 (95.7)      20/35 (57.1)
  Not done                           16/16 (100.0)            --
Biopsy specimen$                      79/82 (96.3)       13/16 (81.3)
  Positive                            9/11 (81.8)        10/12 (83.3)
  Negative                            69/70 (98.6)        3/4 (75.0)
  Not done                             1/4 (25.0)             --
Gastric aspirate                     13/13 (100.0)       4/4 (100.0)
  Positive                            4/4 (100.0)        4/4 (100.0)
  Negative                            9/9 (100.0)             --
  Not done                                 --                 --
Lymph node                            53/74 (71.6)       50/71 (70.4)
  Positive                            12/31 (38.7)       42/61 (68.9)
  Negative                            40/42 (95.2)       8/10 (80.0)
  Not done                            1/1 (100.0)             --
Pleural fluid                         79/84 (94.0)       5/10 (50.0)
  Positive                             2/5 (40.0)         4/7 (57.1)
  Negative                            76/78 (97.4)        1/3 (33.3)
  Not done                            1/1 (100.0)             -
Psoas abscess                          5/9 (55.6)         3/7 (42.9)
  Positive                             0/1 (0.0)          2/3 (66.7)
  Negative                             5/8 (62.5)         1/4 (25.0)
  Not done                                 --                 --
Vertebral aspirate                    16/18 (88.9)       10/12 (83.3)
  Positive                             1/2 (50.0)         8/9 (88.9)
  Negative                            15/16 (93.8)        2/3 (66.7)
  Not done                                 --                 --
Other ([section])                    145/164 (88.4)      25/44 (56.8)
  Positive                            15/28 (53.6)       20/33 (60.6)
  Negative                           117/123 (95.1)      5/11 (45.5)
  Not done                           13/13 (100.0)            --

                                     No. positive/
                                     no. tested (%)       Mean days
Sample and AFB
smear result                              NPV               saved

All primary LiPA                    886/1,022 (86.7)         15.2
  Positive                           213/264 (80.7)          14.8
  Negative                           644/728 (88.5)          22.1
  Not done                            29/30 (96.7)            --
All primary LiPA                    886/1,022 (86.7)         15.2
(adjusted values) ([dagger])
  Positive                           213/264 (80.7)          14.8
  Negative                           644/728 (88.5)          22.1
  Not done                            29/30 (96.7)            --
Respiratory                          496/562 (88.3)          14.7
  Positive                           170/209 (81.3)          14.5
  Negative                           313/340 (92.1)          20.5
  Not done                           13/13 (100.0)            --
Nonrespiratory                       390/960 (84.8)          18.3
  Positive                            43/55 (78.2)           17.2
  Negative                           331/388 (85.3)          23.4
  Not done                            16/17 (94.1)            --
Biopsy specimen$                      79/92 (85.9)           22.4
  Positive                            9/9 (100.0)            24.1
  Negative                            69/82 (84.1)           16.7
  Not done                            1/1 (100.0)             --
Gastric aspirate                      13/14 (92.9)           16.8
  Positive                            4/4 (100.0)            16.8
  Negative                            9/10 (90.0)             --
  Not done                                 --                 --
Lymph node                            53/71 (74.6)           18.7
  Positive                            12/18 (66.7)           16.6
  Negative                            40/52 (76.9)           29.6
  Not done                            1/1 (100.0)             --
Pleural fluid                         79/97 (81.4)           26.8
  Positive                             2/3 (66.7)            23.5
  Negative                            76/93 (81.7)           40.0
  Not done                            1/1 (100.0)             --
Psoas abscess                          5/8 (62.5)            19.0
  Positive                                 -                 17.0
  Negative                             5/8 (62.5)            23.0
  Not done                                 --                 --
Vertebral aspirate                    16/18 (88.9)           15.4
  Positive                            1/1 (100.0)            14.5
  Negative                            15/16 (93.8)           19.0
  Not done                             0/1 (0.0)              --
Other ([section])                    145/160 (90.6)          15.2
  Positive                            15/20 (75.0)           15.1
  Negative                           117/127 (92.1)          15.8
  Not done                           13/13 (100.0)            --

* MTBC excludes 75 specimens containing substances inhibitory
to the polymerase chain reaction (PCR), PCR-equivocal results, and
samples with no definitive culture results (i.e., contaminated or
not done). LiPA, line probe assay; MTBC, Mycobacterium
tuberculosis complex; AFB, acid-fast bacilli; PPV,
positive predictive value; NPV, negative predictive value.

([dagger]) Excludes samples from patients with a microbiologic
diagnosis of MTBC made at the Mycobacterium Reference Unit in
the last 18 or subsequent 3 months, and patients receiving
antituberculous treatment currently or within the last 3 months.

([double dagger]) Includes biopsy specimens from liver (n = 13),
kidney (n = 2), skin (n = 15), lung (n = 20), pleura (n = 13), and
miscellaneous sites (n = 45).

([section]) Includes ascites (n = 56), pericardial aspirates (n = 29),
aspirates from miscellaneous sites (n = 80), blood (n = 1),
bone marrow (n = 22), feces (n = 1), and urine (n = 15).

Table 2. Results of LiPA in detecting rifampin resistance
in specimens from which MTBC was correctly identified
and cultured *

Sample and AFB
smear result          Cordance       Sensitivity

All primary LiPA   775/782 (99.1)    38/40 (95.0)
  Positive         743/747 (99.5)    35/36 (97.2)
  Negative          32/35 (91.4)      3/4 (75.0)
  Not done               --               --
Respiratory        669/672 (99.6)    32/33 (97.0)
  Positive         654/657 (99.5)    31/32 (96.9)
  Negative         15/15 (100.0)     1/1 (100.0)
  Not done               --               --
Nonrespiratory     106/110 (96.4)     6/7 (85.7)
  Positive          89/90 (98.9)     4/4 (100.0)
  Negative          17/20 (85.0)      2/3 (66.7)
  Not done               --               --
Clinical isolate   229/235 (97.4)    21/23 (91.3)

Sample and AFB
smear result        Specificity          PPV

All primary LiPA   737/740 (99.6)    38/41 (92.7)
  Positive         708/710 (99.7)    35/37 (94.6)
  Negative          29/30 (96.7)      3/4 (75.0)
  Not done               --               --
Respiratory        637/639 (99.7)    32/34 (94.1)
  Positive         623/625 (99.7)    31/33 (93.9)
  Negative         14/14 (100.0)     1/1 (100.0)
  Not done               --               --
Nonrespiratory     100/101 (99.0)     6/7 (85.7)
  Positive         85/85 (100.0)     4/4 (100.0)
  Negative          15/16 (93.8)      2/3 (66.7)
  Not done               --               --
Clinical isolate   208/211 (98.6)    21/24 (87.5)

Sample and AFB                        Mean days
smear result            NPV             saved

All primary LiPA   738/740 (99.7)        30.7
  Positive         709/710 (99.9)        30.4
  Negative          29/30 (96.7)         37.2
  Not done               --               --
Respiratory        637/638 (99.8)        30.4
  Positive         623/624 (99.8)        30.2
  Negative         14/14 (100.0)         39.1
  Not done               --               --
Nonrespiratory     101/102 (99.0)        32.5
  Positive         86/86 (100.0)         32.0
  Negative          15/16 (93.8)         35.5
  Not done               --               --
Clinical isolate   208/210 (99.0)        16.3

* LiPA, line probe assay; MTBC, Mycobacterium tuberculosis complex;
AFB, acid-fast bacilli; PPV, positive predictive value;
NPV, negative predictive value.

Table 3. Final culture identification results for all specimens *

                           Primary
                          specimens,   Isolates,          Total,
Result                     no. (%)      no. (%)           no. (%)

MTBC
  Rifampin sensitive
    ([dagger])            892 (94.9)   214 (90.9)      1,106 (93.9)
  Rifampin resistant
    only ([dagger])        9 (1.0)      3 (1.3)          12 (1.0)
  MDR-TB ([dagger])        37 (3.9)     20 (8.4)         57 (4.8)
  Susceptibilities not
    determined
    ([dagger])             2 (0.2)      1 (0.4)           3 (0.3)
  Total MTBC              940 (47.1)   238 (82.1)      1,178 (51.5)
NTM                       181 (9.1)    42 (14.5)         223 (9.8)
Contaminated               22 (1.1)     8 (2.8)     30[subsection](1.3)
Culture not done           5 (0.3)      1 (0.3)     6[subsection](0.3)
No mycobacterial spp.     849 (42.5)    1 (0.3)         850 (37.2)
Total                       1,997         290              2,287

MTBC, Mycobacterium tuberculosis complex; MDR-TB,
multidrug-resistant tuberculosis; NTM, nontuberculous mycobactena.

([dagger]) Percentages of total MTBC cultures.

([double dagger]) These 36 (1.6%) cases without definitive culture
results were excluded from analyses of assay performance.

Table 4. Mean time in days to culture MTBC from all primary
specimens (including those from patients receiving treatment),
stratified according to smear microscopy result *

                                       PCR result
AFB stain
result      Positive (n)   Negative (n)   Equivocal (n)   Total (n)

Positive     18.0 (747)     23.3 (51)       22.0 (9)      18.4 (807)
Negative     25.4 (35)      31.3 (84)       30.0 (6)      29.6 (125)
Not done       0 (0)         22.0 (1)       21.0 (1)       21.5 (2)
Total        18.4 (782)     28.2 (136)      24.9 (16)     19.9 (934)

* MTBC, Mycobacterium tuberculosis complex; PCR, polymerase
chain reaction;
AFB, acid-fast bacilli; n, no. of samples.

Table 5. Results of LiPA in detecting MTBC in clinical specimens in
which MTBC was correctly identified and cultured, stratified by
history of antituberculous treatment'

                                   No. positive/no. tested (%)
Treatment history/
AFB smear result                Concordance       Sensitivity

Current or within 3 mo         86/182 (47.3)      48/67 (71.6)
  Positive                     61/132 (46.2)      46/55 (83.6)
  Negative/not done            25/50 (50.0)        2/12(16.7)
Adjusted values ((dagger])     86/139 (61.9)      48/67 (71.6)
  Positive                     61/99 (61.6)       46/55 (83.6)
  Negative/not done            25/40 (62.5)        2/12(16.7)
>3 mo ago                      85/106 (80.2)      42/53 (79.2)
  Positive                     54/65 (83.1)       40/45 (88.9)
  Negative/not done            31/41 (75.6)        2/8(25.0)
Adjusted values ((dagger])     85/102 (83.3)      42/53 (79.2)
  Positive                     54/63 (85.7)       40/45 (88.9)
  Negative/not done            31/39 (79.5)        2/8(25.0)
No stated treatment          1,497/1,634 (91.6)  692/798 (86.7)
  Positive                    845/902 (93.7)     661/698 (94.7)
  Negative/not done           652/732 (89.1)     31/100 (31.0)
Adjusted values ((dagger])   1,497/1,634 (91.6)  692/798 (86.7)
  Positive                    845/892 (94.7)     661/698 (94.7)
  Negative/not done           652/728 (89.6)     31/100 (31.0)

                                   No. positive/no. tested (%)

Treatment history/
AFB smear result                Sensitivity       Specificity

Current or within 3 mo         48/67 (71.6)      38/115 (33.0)
  Positive                     46/55 (83.6)       15/77 (19.5)
  Negative/not done             2/12(16.7)        23/38 (60.5)
Adjusted values ((dagger])     48/67 (71.6)       38/72 (52.8)
  Positive                     46/55 (83.6)       15/44 (34.1)
  Negative/not done             2/12(16.7)        23/28 (82.1)
>3 mo ago                      42/53 (79.2)       43/53 (81.1)
  Positive                     40/45 (88.9)       14/20 (70.0)
  Negative/not done              2/8(25.0)        29/33 (87.9)
Adjusted values ((dagger])     42/53 (79.2)       43/49 (87.8)
  Positive                     40/45 (88.9)       14/18 (90.9)
  Negative/not done              2/8(25.0)        29/31 (93.5)
No stated treatment           692/798 (86.7)     805/836 (96.3)
  Positive                    661/698 (94.7)     184/204 (90.2)
  Negative/not done            31/100 (31.0)     621/632 (98.3)
Adjusted values ((dagger])    692/798 (86.7)     805/836 (96.3)
  Positive                    661/698 (94.7)     184/194 (94.8)
  Negative/not done            31/100 (31.0)     621/628 (98.9)

                                   No. positive/no. tested (%)
Treatment history/
AFB smear result                    PPV               NPV

Current or within 3 mo         48/125 (38.4)      38/57 (66.7)
  Positive                     46/108 (42.6)      15/24 (62.5)
  Negative/not done             2/17(11.8)        23/33 (69.7)
Adjusted values ((dagger])     48/82 (58.5)       38/57 (66.7)
  Positive                     46/75 (61.3)       15/24 (62.5)
  Negative/not done              2/7(28.6)        23/33 (69.7)
>3 mo ago                      42/52 (80.8)       43/54 (79.6)
  Positive                     40/46 (87.0)       14/19 (73.7)
  Negative/not done              2/6(33.3)        29/35 (82.9)
Adjusted values ((dagger])     42/48 (87.5)       43/54 (79.6)
  Positive                     40/44 (90.9)       14/19 (73.7)
  Negative/not done              2/4(50.0)        29/35 (82.5)
No stated treatment           692/723 (95.7)     805/911 (88.4)
  Positive                    661/681 (97.1)     184/221 (83.3)
  Negative/not done            21/42 (73.8)      621/690 (90.0)
Adjusted values ((dagger])    692/723 (95.7)     805/911 (88.4)
  Positive                    661/671 (98.5)     184/221 (83.3)
  Negative/not done            31/38 (81.6)      621/690 (90.0)

* LiPA, line probe assay; MTBC, Mycobacterium tuberculosis complex;
AFB, acid-fast bacilli; PPV, positive predictive value;
NPV, negative predictive value.

([dagger]) Excludes samples from patients with a microbiologic
diagnosis of MTBC made at the Mycobacterium Reference Unit in
the last 18 or subsequent 3 months.

Table 6. Results of LiPA in detecting rifampin resistance in
clinical specimens in which MTBC was correctly identified and
cultured, stratified by history of antituberculous treatment *

                No. positive/no. tested (%)
Treatment
history          Concordance      Sensitivity     Specificity

Current or
  within 3 mo    46/48 (95.8)     8/10 (80.0)     38/38 (100)
>3 mo ago        41/42 (97.6)      7/7 (100)      34/35 (97.1)
None stated     689/691 (99.7)    23/23 (100)    666/668 (99.7)

                No. positive/no. tested (%)
Treatment
history              PPV              NPV

Current or
  within 3 mo     8/8 (100)      38/40 (95.0)
>3 mo ago         7/8 (87.5)      34/34 (100)
None stated      23/25 (92.0)    666/666 (100)

* LiPA, line probe assay; MTBC, Mycobacterium tuberculosis complex;
AFB, acid-fast bacilli; PPV, positive predictive value;
NPV, negative predictive value.

Table 7. Sequence analysis of 10 discrepant
rifampin-susceptibility results *

         Rifampin susceptibility

Sample   LiPA result       Phenotypic
no.                        result

1-3      Sensitive         Resistant
4        Sensitive         Resistant
5-7      Resistant (AS4)   Sensitive

8        Resistant (AS1)   Sensitive
9        Resistant (R5)    Sensitive
10       Resistant (AS2)   Sensitive

Sample               Conclusion after
no.                      sequencing

1-3          From the same patient; wild-type
                      rpoB test region

4             Mycobacterium bovis; wild-type
                      rpoB test region

5-7          From the same patient; synonymous
                  substitution (R528R) not
            associated with rifampin resistance

8             2 genotypes present: wild-type
              (predominant) and mutant (L511P)

9                S531L mutation; wild-type
                  rpoB on retesting, thus
                  likely laboratory error

10             D516A mutation; no high-level
                 resistance when seen alone

* LiPA, line probe assay.