Abstract
The Russian Federation is a high-tuberculosis (TB)-burden country with high rates of Mycobacterium tuberculosis multidrug resistance (MDR) and extensive drug resistance (XDR), especially in HIV-coinfected patients. Rapid and reliable diagnosis for detection of resistance to second-line drugs is vital for adequate patient management. We evaluated the performance of the GenoType MTBDRsl (Hain Lifescience GmbH, Nehren, Germany) assay on smear-positive sputum specimens obtained from 90 HIV-infected MDR TB patients from Russia. Test interpretability was over 98%. Specificity was over 86% for all drugs, while sensitivity varied, being the highest (71.4%) for capreomycin and lowest (9.4%) for kanamycin, probably due to the presence of mutations in the eis gene. The sensitivity of detection of XDR TB was 13.6%, increasing to 42.9% if kanamycin (not commonly used in Western Europe) was excluded. The assay is a highly specific screening tool for XDR detection in direct specimens from HIV-coinfected TB patients but cannot be used to rule out XDR TB.
INTRODUCTION
Tuberculosis (TB) remains a serious health problem globally, killing 1.7 million people annually (1). Soaring HIV infection rates in many high-TB-burden areas, including countries of the former Soviet Union, pose a significant public health threat. HIV coinfection is known to be one of the most important factors promoting development of active TB; similarly, TB is recognized as the major contributor to morbidity and mortality in HIV-infected individuals (2, 3). Coinfection of HIV and multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB is largely lethal (4, 5).
The Russian Federation has arguably the highest rates of MDR and XDR TB globally. Increasing drug resistance poses enormous public health challenges due to problems in diagnosis and treatment, poor outcomes (especially in those coinfected with HIV), and high associated costs (1, 6). Although HIV rates in the Russian Federation have been relatively stable over the last 10 years (1), the total number of people living with HIV and AIDS is steadily increasing against a dramatic rise in MDR and XDR TB incidence due to insufficiencies of the national TB control program in the past (7, 8).
Smear microscopy is widely used for rapid diagnosis of TB. It does not, however, detect drug resistance and cannot be used as a tool to confirm or rule out MDR or XDR TB and cannot prevent delays in the initiation of appropriate treatment, leading to potential transmission of drug-resistant TB strains. Besides, smear microscopy is insensitive, often detecting acid-fast bacilli (AFB) in sputum samples of HIV-infected suspects in only 20% to 50% of cases due to a frequently lower bacillary load in coinfected patients (9). Recently reported associations between low CD4 counts and a decrease in AFB loads in sputum specimens obtained from HIV-infected individuals (10) demonstrate that the performance of any method based on direct detection of TB bacilli or its components in sputum samples could be severely compromised in those patients in greatest need of rapid diagnosis.
Although culture remains the gold standard for TB and MDR/XDR TB diagnosis, its complex requirements of laboratory infrastructure, equipment, and personnel, as well as biosafety considerations and relatively long turnaround times, limit its potential for rapid diagnosis, especially in resource-limited settings (11). Line probe assays (LPAs), endorsed by the World Health Organization, although still technically demanding, have been extensively evaluated for rapid detection of resistance to isoniazid (INH) and rifampin (RIF), demonstrating good performance on cultures and smear-positive samples (11). The GenoType MTBDRsl (Hain Lifescience GmbH, Nehren, Germany) assay is the only rapid commercial test for the detection of resistance to the main second-line drugs (SLD). The assay is potentially promising for use in suspected or confirmed MDR TB cases to diagnose further resistance and to rapidly identify XDR TB. It would be particularly useful for diagnosis of HIV-infected individuals, because undiagnosed XDR TB in HIV-coinfected patients is usually lethal (12). The assay has been evaluated in two low-incidence TB- and HIV settings, predominantly on cultures as well as patient specimens, and demonstrated good sensitivity and specificity (13, 14). Assay performance varied significantly depending on the genetic background of the strains and tended to be lower with Eastern European strains (15, 16).
Currently, only limited systematic data are available on the performance of molecular assays for the direct diagnosis of resistance to RIF and INH in specimens from patients in settings of high TB burden and high HIV prevalence (17, 18); the sensitivity and specificity of LPAs for the detection of resistance to SLD in specimens from HIV-infected TB patients are unknown. In our study, we aimed to assess the performance of the GenoType MTBDRsl assay for the rapid detection of TB and resistance to fluoroquinolones (FQ), aminoglycosides/cyclic peptides (AG/CP), and ethambutol (EMB) in sputum specimens in a cohort of HIV-positive Russian patients with MDR TB.
MATERIALS AND METHODS
The study was designed in accordance with the standards for the principles of the reporting of diagnostic accuracy study (STARD) for diagnostic accuracy studies (19).
Study material.
This study was conducted on microscopically smear-positive sputum specimens obtained from 90 consecutively recruited HIV-infected patients known to have MDR TB (see below). Fifteen specimens (16.7%) were graded AFB 1+, 17 (18.9%) were graded 2+, and the remaining 58 specimens (64.4%) were graded 3+.
Patients were recruited in Samara, Russia, between 2008 and 2010.
All patients were tested for HIV within the routine TB program initially by enzyme-linked immunosorbent assay (ELISA) and later by immunoblotting for confirmation of the diagnosis.
Bacteriological methods.
All of the included specimens had been identified as Mycobacterium tuberculosis using the GenoType MTBDRplus assay (HainLifescience GmbH, Germany).
Ziehl-Neelsen microscopy was performed on all specimens.
Cultures subsequently grown from the sputum specimens described above had first- and second-line drug susceptibility testing (DST) performed using solid and liquid culture media in Samara, Russia, as described below (see Tables 2 and 3). First-line drug (FLD) DST was performed either on Lowenstein-Jensen media (LJ) using the absolute concentration method or on Bactec MGIT 960. Drug concentrations (in milligrams per milliliter) used for FLD DST were as follows: for LJ media, streptomycin (STR), 10.0; INH, 1.0; RIF, 40.0; and EMB, 2.0; or for Bactec MGIT 960, STR, 1.0; INH, 0.1; RIF, 1.0; EMB, 5.0; and pyrazinamide (PZA), 100.0. Second-line drug (SLD) susceptibility testing was performed using Bactec MGIT 960 and the following drug concentrations: ofloxacin, 2.0; moxifloxacin, 0.25, amikacin (AMK), 1.0; capreomycin (CAP), 2.5; and kanamycin (KAN), 5.0 (20).
Table 2.
Drug resistance pattern for MTB strains grown from patients' smear-positive sputum samples used in the studya
| No. of strains | Resistance pattern |
||
|---|---|---|---|
| FQ | AG/CP | EMB | |
| 25 | S | S | S |
| 31 | S | S | R |
| 2 | S | R | S |
| 4 | S | R | R |
| 6 | R | R | S |
| 3 | R | R | R |
| 5 | R | S | S |
| 14 | R | S | R |
FQ, fluoroquinolones; AG/CP, aminoglycosides/cyclic peptides; EMB, ethambutol; S, sensitive; R, resistant.
Table 3.
Aminoglycoside/cyclic peptide resistance patternsa
| No. of strains | Resistance pattern |
||
|---|---|---|---|
| AMK | CAP | KAN | |
| Full cross-resistance (25/90; 27.7%) | |||
| 14 | S | S | S |
| 4 | S | S | NA |
| 5 | R | R | R |
| 2 | R | R | NA |
| No cross-resistance (65/90; 72.2%) | |||
| 57 | S | S | R |
| 2 | S | R | R |
| 5 | R | S | R |
| 1 | R | S | NA |
AMK, amikacin; CAP, capreomycin; KAN, kanamycin; S, sensitive; R, resistant; NA, not available.
The results of phenotypic DST and the GenoType MTBDRplus assay were consistent in terms of identifying resistance to both INH and RIF in all cases.
Seven MDR TB strains (7.7%) were also XDR in blinded culture-based analysis.
DNA extraction and molecular methods.
Crude DNA extracts were isolated from the sputum samples by heating (95°C, 30 min) followed by sonication (20 min). All specimens were relabeled in a unique way by an independent staff member to avoid assay interpretation bias. Unblinding of the results was performed after completion of the experimental phase.
An experienced operator blinded with respect to the original phenotypical DST results ran the GenoType MTBDRsl (HainLifescience GmbH, Germany) assay for all 90 samples and read and interpreted results according to the manufacturer's instructions.
Assay results were considered uninterpretable when the band indicating the presence of TB (TUB band) was missing; also, assay results for individual groups of drugs (FQs, AG/CP, EMB) were considered unreadable if there were no positive hybridization/faint bands with respect to the locus control and wild-type or mutant probes preventing an operator from unambiguous reading and interpretation of test results.
Statistical analysis.
The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for all specimens to assess the performance of the GenoType MTBDRsl assay.
Statistical analysis was performed using Microsoft Excel (Microsoft) and PASW Statistics (release 18; SPSS Inc., Chicago, IL) software.
Ethics statement.
Ethics approval was obtained from the Bioethics Committee of Samara Oblast Ministry of Health and Social Development. All patients provided informed written consent.
RESULTS
Patient population.
Most of the patients were male, with a median age of 34.5 years. More than two-thirds of them lived in urban settings; 38.2% had a history of imprisonment. The majority of the patients admitted current or past recreational drug use (10.1% or 55.1%, respectively), most patients were unemployed, and 15.6% were homeless. Few patients (n = 13; 14.4%) had received antiretroviral therapy (Table 1).
Table 1.
Demographic characteristics of the patient populationa
| Characteristic | Value(s) |
|---|---|
| Male gender | 71/90 (78.9) |
| Settings | |
| Rural | 26/90 (28.9) |
| Urban | 64/90 (71.1) |
| Age: median | 34.5 yr |
| Imprisonment in the past | 34/89 (38.2) |
| Recreational drug use | |
| Current | 9/89 (10.1) |
| Previous | 49/89 (55.1) |
| Alcohol abuse | |
| Current | 7/90 (7.8) |
| Previous | 6/90 (6.7) |
| Homeless | 14/90 (15.5) |
| Unemployed | 72/90 (80.0) |
| Receiving HAART | 13/90 (14.4) |
| New case | 52/90 (57.8) |
| Retreatment case | 38/90 (42.2) |
| Contact with a TB patient | 10/90 (11.1) |
| Cough lasting more than 2 wk | 72/90 (80.0) |
| Fever | 77/90 (85.5) |
| Shortness of breath | 68/90 (75.5) |
| Chest pain | 24/90 (26.7) |
| Hemoptysis | 9/90 (10.0) |
| Sudden wt loss | 46/90 (51.1) |
| Presence of cavities in lungs | 63/90 (70.0) |
| Unilateral lesions | 39/90 (43.3) |
| Bilateral lesions | 51/90 (56.7) |
Values represent numbers of patients/total numbers of patients (percent) except where otherwise indicated. HAART, highly active antiretroviral therapy.
Almost two-thirds (57.8%) of the participants were newly diagnosed, and 11.1% reported previous contact with TB patients. A large proportion (70.0%) of the patients had advanced disease with cavities present; approximately half had both lungs affected.
Patient sputum: culture-determined drug resistance.
The GenoType MTBDRsl assay performance for drug resistance in patient sputum specimens was compared to the drug resistance of the cultures grown from the same patient sputum specimens represented in Tables 2 and 3.
Performance characteristics of the GenoType MTBDRsl assay for TB diagnosis and detection of drug resistance.
Overall test readability (defined by the presence of a TUB band on the membrane) was excellent (98.9%), with all but one membrane readable. The unreadable membrane was obtained on an AFB 1+ sputum specimen. Sensitivity of detection of resistance to individual SLDs varied significantly, with the lowest value (9.4%) for KAN and the highest (71.4%) for CAP (Table 4). Specificity was excellent (>96.0%) for all drugs. The PPV ranged from 71.5% (CAP) to 100.0% (AMK); the NPV value was the lowest (17.4%) for KAN and the highest (97.4%) for CAP.
Table 4.
Performance of GenoType MTBDRsl assay compared to LJ and/or MGIT 960 phenotypical DST (N = 90)a
| Individual drug or drug group | No. concordant resistant (%) | No. concordant sensitive (%) | Total agreement (sensitive + resistant) (%) | Sensitivity (%) | 95% CI | Specificity (%) | 95% CI | PPV (%) | 95% CI | NPV (%) | 95% CI | No. of tests with uninterpretable results/total no. of results (%) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| FQ (n = 88) | 15/29 (51.7) | 59/73 (80.8) | 74/88 (84.1) | 15/27 (55.6) | 35.3–74.3 | 59/61 (96.7) | 88.7–99.6 | 15/17 (88.2) | 63.6–98.5 | 59/71 (83.1) | 72.3–91.0 | 2/90 (2.2) |
| AMK (n = 83) | 7/11 (63.6) | 72/76 (94.7) | 79/83 (95.2) | 7/11 (63.6) | 30.8–89.1 | 72/72 (100.0) | 95.0–100.0 | 7/7 (100.0) | 59.0–100.0 | 72/76 (94.7) | 87.1–98.5 | 7/90 (7.8) |
| CAP (n = 83) | 5/9 (55.6) | 74/78 (94.9) | 79/83 (95.2) | 5/7 (71.4) | 29.0–96.3 | 74/76 (97.4) | 90.8–99.7 | 5/7 (71.4) | 29.0–96.3 | 74/76 (97.4) | 90.8–99.7 | 7/90 (7.8) |
| KAN (n = 76) | 6/64 (9.4) | 12/70 (17.1) | 18/76 (23.7) | 6/64 (9.4) | 3.5–19.3 | 12/12 (100.0) | 73.5–100.0 | 6/6 (100.0) | 54.1–100.0 | 12/70 (17.1) | 9.2–28.0 | 7/90 (7.8) |
| AG/CP (n = 76) | 6/64 (9.4) | 12/70 (17.1) | 18/76 (23.7) | 6/64 (9.4) | 3.5–19.3 | 12/12 (100.0) | 73.5–100.0 | 6/6 (100.0) | 54.1–100.0 | 12/70 (17.1) | 9.2–28.0 | 7/90 (7.8) |
| EMB (n = 87) | 18/55 (32.7) | 32/69 (46.4) | 50/87 (57.5) | 18/50 (36.0) | 22.9–50.8 | 32/37 (86.5) | 71.2–95.5 | 18/23 (78.3) | 56.3–92.5 | 32/64 (50.0) | 37.2–62.8 | 3/90 (3.3) |
| XDR (n = 74) | 3/22 (13.6) | 52/71 (73.2) | 55/74 (74.3) | 3/22 (13.6) | 2.9–34.9 | 52/52 (100.0) | 93.2–100.0 | 3/3 (100.0) | 29.2–100.0 | 52/71 (73.2) | 61.4–83.1 | 7/90 (7.8) |
No. concordant resistant, number of concordant resistant results (MTBDRsl)/total number of resistant isolates by MGIT/LJ plus MTBDRsl; No. concordant sensitive, number of concordant sensitive results (MTBDRsl)/total number of sensitive isolates by MGIT/LJ plus MTBDRsl; FQ, fluoroquinolones; AMK, amikacin; CAP, capreomycin; KAN, kanamycin; AG/CP, aminoglycosides/cyclic peptides; EMB, ethambutol; XDR, extensively drug resistant; CI, confidential interval.
With regard to groups of SLDs, the sensitivity of the test for the FQs was substantially higher than for injectables, as the overall sensitivity to the latter group of drugs was significantly affected by a low sensitivity to KAN (55.6% and 9.4%, retrospectively). Specificity for all SLDs was excellent, ranging from 96.7% for FQs to 100% for AG/CP. PPV and NPV values were lower for injectables than for FQ. Total agreement between the molecular assay and phenotypic DST was the highest (84.1%) for FQs and lowest (23.5%) for the injectable drugs. Sensitivity, specificity, PPV, and NPV for EMB were 36.0%, 86.5%, 78.3%, and 50.0%, respectively. The overall sensitivity of detection of XDR was 13.6% (Table 4).
Performance of the test depending on sputum smear microscopy results.
Analysis of test performance stratified according to sputum smear positivity showed that the test readability for individual drugs and their drug groups ranged from 80.0% to 100.0%, with the lowest for specimens graded 1+ (Table 5). Within this group of specimens, lower readability rates were observed for the AG/CP group of drugs (n = 3; 20.0% of tests failed), with higher readability rates for FQ and EMB. Similar trends were observed in specimens graded 2+ and 3+ (Fig. 1).
Table 5.
Performance of GenoType MTBDRsl assay compared to LJ and/or MGIT 960 phenotypical DST depending on smear positivitya
| Individual drug or drug group | Smear positivity rate | No. concordant resistant (%) | No. concordant sensitive (%) | Total agreement (sensitive & resistant) (%) | Sensitivity (%) | 95% CI | Specificity (%) | 95% CI | PPV (%) | 95% CI | NPV (%) | 95% CI | No. of tests with uninterpretable results (%) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| FQ | 1+ | 0/2 (0.0) | 12/14 (85.7) | 12/14 (85.7) | 0/2 (0.0) | 0.0–84.2 | 12/12 (100.0) | 73.5–100.0 | 0/0 (NA) | NA | 12/14 (85.7) | 57.2–98.2 | 1/15 (6.7) |
| 2+ | 3/4 (75.0) | 13/14 (92.9) | 16/17 (94.1) | 0/2 (0.0) | 0.0–84.2 | 13/13 (100.0) | 75.3–100.0 | 3/3 (100.0) | NA | 13/14 (92.9) | 59.5–98.3 | 0/17 (0.0) | |
| 3+ | 12/23 (52.2) | 34/45 (75.6) | 46/57 (80.7) | 12/21 (57.1) | 34.0–78.2 | 34/36 (94.4) | 81.3–99.3 | 12/14 (85.7) | 57.2–98.2 | 34/43 (79.1) | 64.0–90.0 | 1/58 (1.7) | |
| AG/CP | 1+ | 0/9 (0.0) | 0/9 (0.0) | 0/9 (0.0) | 0/9 (0.0) | NA | 0/0 (NA) | NA | 0/0 (NA) | NA | 0/9 (0.0) | NA | 3/15 (20.0) |
| 2+ | 1/15 (6.7) | 2/16 (12.5) | 3/17 (17.6) | 1/15 (6.7) | 0.17–31.9 | 2/2 (100.0) | 15.8–100.0 | 1/1 (100.0) | 2.5–100.0 | 2/16 (12.5) | 1.55–38.3 | 1/17 (5.9) | |
| 3+ | 5/41 (12.2) | 10/46 (21.7) | 15/51 (29.4) | 5/41 (12.2) | 4.1–26.2 | 10/10 (100.0) | 69.2–100.0 | 5/5 (100.0) | 47.8–100.0 | 10/46 (21.7) | 10.9–36.4 | 3/58 (5.2) | |
| EMB | 1+ | 4/11 (36.4) | 3/10 (30.0) | 7/14 (50.0) | 4/8 (50.0) | 15.7–84.3 | 3/6 (50.0) | 11.8–88.2 | 4/7 (57.1) | 18.4–90.1 | 3/7 (42.9) | 9.9–81.6 | 1/15 (6.7) |
| 2+ | 3/9 (33.3) | 7/13 (53.8) | 10/16 (62.5) | 3/9 (33.3) | 7.5–70.1 | 7/7 (100.0) | 59.0–100.0 | 3/3 (100.0) | 29.2–100.0 | 7/13 (53.8) | 25.1–80.8 | 1/17 (5.9) | |
| 3+ | 11/35 (31.4) | 22/46 (47.8) | 33/57 (57.9) | 11/33 (33.3) | 18.0–51.8 | 22/24 (91.7) | 73.0–99.0 | 11/13 (84.6) | 54.6–98.1 | 22/44 (50.0) | 34.6–65.4 | 1/58 (1.7) | |
| XDR | 1+ | 0/2 (0.0) | 7/9 (77.8) | 7/9 (77.8) | 0/2 (0.0) | 0.0–84.2 | 7/7 (100.0) | 59.0–100.0 | 0/0 (NA) | NA | 7/9 (77.8) | 40.0–97.2 | 3/15 (20.0) |
| 2+ | 0/3 (0.0) | 13/16 (81.3) | 13/16 (81.2) | 0/3 (0.0) | 0.0–70.8 | 13/13 (100.0) | 75.3–100.0 | 0/0 (NA) | NA | 13/16 (81.2) | 54.4–96.0 | 1/17 (5.9) | |
| 3+ | 3/17 (17.6) | 33/47 (70.2) | 36/50 (72.0) | 3/17 (17.6) | 3.8–43.4 | 33/33 (100.0) | 89.4–100.0 | 3/3 (100.0) | 29.2–100.0 | 33/47 (70.2) | 55.1–82.7 | 3/58 (5.2) |
No. concordant resistant, number of concordant resistant results (MTBDRsl)/total number of resistant isolates by MGIT/LJ plus MTBDRsl; No. concordant sensitive, number of concordant sensitive results (MTBDRsl)/total number of sensitive isolates by MGIT/LJ plus MTBDRsl; FQ, fluoroquinolones; AMK, amikacin; CAP, capreomycin; KAN, kanamycin; AG/CP, aminoglycosides/cyclic peptides; EMB, ethambutol; XDR, extensively drug resistant; NA, not available; CI, confidential interval.
Fig 1.
Readability of the MTBDRsl assay corresponding to sputum smear-positivity status (n = 90). TUB, band indicating the presence of TB; FQ, band indicating the presence of the FQ locus control; AG/CP, band indicating the presence of the AG/CP locus control; EMB, band indicating the presence of the EMB locus control.
Specificity to detect resistance to individual drugs was not affected by the number of TB bacilli in the test specimen and was excellent for all drugs (91.8% to 100.0%) except EMB (50.0% specificity in specimens graded 1+). In contrast, sensitivity and other performance characteristics, including PPV and NPV, varied significantly depending on the AFB count, being generally higher in specimens graded 2+ and 3+, especially for injectable drugs (Table 5). In specimens with lower AFB counts (graded 1+), sensitivity was poor or suboptimal (0% to 50.0%) for all drugs except CAP (100.0% sensitivity). Sensitivity for the detection of resistance to EMB was the lowest, ranging between 33.3% and 50.0% in all groups.
DISCUSSION
This was the first study to assess the diagnostic accuracy of a novel rapid test detecting resistance to SLDs when applied directly on sputum specimens obtained from patients with MDR TB who were coinfected with HIV. Recruited patients came from Russia, a country with a rapidly escalating problem of TB drug resistance and growing numbers of people living with HIV and AIDS.
In our study, the overall interpretability of the test (defined as the ability of the operator to read and unambiguously interpret the test results using the manufacturer's criteria) was good, ranging from 80.0% to 100.0%, being higher in samples with a greater bacterial load. Reduced diagnostic accuracy of molecular tests in samples with lower AFB counts is consistent with the results of previously reported studies (18). The excellent specificity of the GenoType MTBDRsl assay demonstrated in the studies that used both cultures and direct specimens (13–16, 21, 22) was confirmed in our work; the exception was EMB, which may reflect the imperfect reference standard currently available for this drug. The sensitivity of the assay was more problematic that the specificity, detecting approximately half of culture-confirmed FQ resistance and only about one-tenth of resistance to injectable agents due to the very low ability to detect resistance to KAN. Consecutively, the assay is able to detect less than a quarter of XDR cases. When KAN is excluded from the analysis, the sensitivity of the test for detection of XDR TB increases to 42.9% while specificity remains 100.0%. This is important in countries outside Eastern Europe, where clinical and programmatic use of and resistance to KAN are not significant.
Controversies regarding sensitivity rates of the assay reported in previous studies, especially for AGs and CPs and for KAN in particular (15, 16, 21–23), could be attributed to the diverse genetic background of strains and different distribution of mutations conferring drug resistance in different geographic settings. In recent studies conducted on clinical strains isolated in Estonia, Vietnam, and South Africa, the sensitivity of the assay for the detection of resistance to KAN was very low (reaching 43% at its maximum) (16, 22, 23), probably due to the high prevalence of Beijing strains harboring mutations in eis genes not recognized by the assay and responsible for conferring resistance to KAN only, as was shown in Vietnam (23). Absence of cross-resistance to AMK and CAP in strains harboring mutations in the eis promoter region has been recently demonstrated in a genomic study of resistance in Samara, Russia (24), and in another study conducted in Russia (25). In a previous large study conducted in Samara, a significant proportion of Beijing KAN-resistant and AMK-sensitive strains was found to contain mutations in the eis gene but not in the rrs gene (24). The isolates obtained from the study population had a very high proportion (71.0%) of strains sensitive to AMK but resistant to KAN as determined by phenotypical DST. It has also been shown that the Beijing family of strains dominates TB epidemiology not just in Samara and Russia overall but also in other former Soviet Union countries and is significantly associated with drug resistance (26–28).
However, as probes for the eis gene are not included in the current version of the GenoType MTBDRsl assay, this compromises its ability to detect XDR overall. As similar epidemiologies of Mycobacterium tuberculosis strains can be assumed across other Eastern European settings, the test could be improved for use by adding probes for eis.
Suboptimal performance characteristics (sensitivity, NPV, and agreement rates) demonstrated in our study for detection of resistance to EMB are consistent with previous studies performed on clinical specimens and highlight the necessity of further studies to identify the molecular basis of resistance to EMB and/or the imperfect reference standard for this drug.
Although the performance of the assay was affected by smear-positivity status, the assay is a useful screening tool for rapid XDR detection in direct specimens in HIV-positive patients due to its high specificity. Based on this and previous work, we suggest that, in settings with high rates of drug resistance, rapid molecular assays should be used directly on specimens to detect MDR (or RIF resistance) and that, once resistance is detected, the MTBDRsl assay could be applied to smear-positive samples. Once a culture is ready, FLD and SLD testing can be set up in parallel with and potentially accompanied with the MTBDRsl assay applied to any cultured isolate if it was not performed earlier.
One of the study limitations was a relatively small sample size that might have affected diagnostic parameters in samples with different smear-positivity grades. Nevertheless, HIV-positive patients tend to produce sputum samples with a low bacillary load.
The current assay may be more useful in countries where KAN resistance is rare and resistance to injectable drugs is mainly conferred by the rrs gene mutations and where the strain populations are not dominated by the Eastern European Beijing clade (24). However, one should bear in mind that, due to much lower rates of XDR TB in these settings, the PPV would be different, possibly resulting in a much higher number of falsely diagnosed XDR cases. Settings with high rates of drug resistance and especially HIV would benefit from the assay, which could be improved by the addition of eis mutation gene probes and improved sensitivity for smear-negative samples.
ACKNOWLEDGMENT
The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement FP7-223681.
Footnotes
Published ahead of print 14 November 2012
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