Abstract
We evaluated the performance of the molecular lab-on-chip-based VerePLEX Biosystem for detection of multidrug-resistant tuberculosis (MDR-TB), obtaining a diagnostic accuracy of more than 97.8% compared to sequencing and MTBDRplus assay for Mycobacterium tuberculosis complex and rifampin and isoniazid resistance detection on clinical isolates and smear-positive specimens. The speed, user-friendly interface, and versatility make it suitable for routine laboratory use.
Multidrug-resistant tuberculosis (MDR-TB) requires long and expensive treatment and often results in poor clinical outcome in both low- and high-income countries (1, 2). The World Health Organization (WHO) has endorsed specific molecular diagnostics to improve fast diagnosis of MDR-TB (3–5). However, the genotypic diversity and geographical distribution of Mycobacterium tuberculosis complex (MTBC), together with the inability to provide appropriate interpretation of silent mutations and the limited versatility are some of the restraints undermining the effectiveness of the current tools on a global scale (6–13).
In the present study, we evaluated a lab-on-chip (LoC) device, developed by STMicroelectronics (Geneva, Switzerland) and marketed by Veredus Laboratories (Republic of Singapore) as the VerePLEX Biosystem, for the diagnosis of MDR-TB and detection of common nontuberculous mycobacteria (NTM). The molecular assay was evaluated on both clinical isolates and direct specimens in low- and high-burden settings.
We tested 91 MTBC isolates (see Table S1 in the supplemental material) harboring different patterns of mutations in rpoB, katG, and inhA genes to evaluate the probes on the array listed in Table 1. Eighty respiratory specimens positive for acid-fast bacilli by smear microscopy and MTBC culture positive were decontaminated according to international guidelines and included in the study (Table S1) (14). An additional 116 MTBC culture-negative specimens were included in the analysis. DNA from isolates and specimens was extracted by thermal lysis and sonication as described elsewhere (15). Phenotypic drug susceptibility testing (DST) for rifampin (RIF) and isoniazid (INH) was performed according to international recommendations (16). Some of the specimens were tested in a representative high-burden setting in Uganda (Nsambya Hospital, Kampala, Uganda), by trained staff.
TABLE 1.
Probes spotted onto the array and targeted mycobacterial species and MDR-TB targets included in the assay
| Targeted mycobacterial species or MDR-TB target | Probe(s) |
|---|---|
| Targeted Mycobacterium species | |
| M. avium | MYC4a |
| M. intracellulare | MYC5a |
| M. simiae, M. kansasii, M. scrofulaceum | MYC6a |
| M. abscessus, M. chelonae | MYC8a |
| M. xenopi | MYC17a |
| M. haemophylum | MYC19a |
| M. fortuitum | MYC31a |
| M. tuberculosis complex | MYC15a-MYC16a |
| MDR-TB targets | |
| rpoB | |
| WT codons 510 to 513 | L511_w3a |
| L511P mutant | L511P_m3 |
| WT codons 515 to 518 | D516_w5 |
| D516V mutant | D516V_m1 |
| WT codons 523 to 526 | H526_w14 |
| H526D mutant | H526D_m2 |
| H526Y mutant | H526Y_m5 |
| WT codons 530 to 533 | S531L_w1 |
| S531L mutant | S531L_m2 |
| katG | |
| WT codons 313 to 317 | S315_w2 |
| S315T1 mutant | S315T1_m2 |
| S315T2 mutant | S315T2_m1 |
| inhA | |
| WT nucleotides −21 to −7 | inhA_w3 |
| T-8A mutant | InhA–8T>A_m2 |
| T-8C mutant | InhA–8T>C_m2 |
| C-15T mutant | InhA–15C>T_m3 |
DNA samples extracted from both isolates and specimens were tested in parallel, and results were compared with GenoType MTBDRplus (Hain Lifescience, Nehren, Germany) assay and Sanger sequencing performed as described elsewhere (17).
The VerePLEX Biosystem consists of a single disposable device comprising microfluidic PCR and microarray modules. The platform includes a temperature control system (TCS) and an optical reader (OR) which allows automatic analysis of the microarray, providing a user-friendly diagnostic report (see Fig. S2 in the supplemental material) (18). The protocols for MDR-TB assay are described in Text S3, and the primers are shown in Table S4. The assay allows detection of MTBC and other common NTM, together with the most frequent mutations affecting the rpoB, katG, and inhA genes, which are involved in phenotypic resistance to RIF and INH in MTBC.
Analysis of the diagnostic performance of the LoC assay on clinical isolates.
MTBC was detected in all 91 clinical isolates (Table 2). Concerning the rpoB and inhA targets, 100% concordance was observed between the MTBDRplus and LoC assay results. In one case, the LoC assay revealed both wild-type (WT) and mutated signals from probes targeting positions 523 to 526 in rpoB, which was not confirmed by MTBDRplus assay. A 95.74% concordance was observed between the MTBDRplus and LoC assay results for the katG target. In two cases, probes complementary to the WT sequence of codon 315 of katG were detected slightly over the on/off cutoff, but the MTBDRplus assay showed an absence of signal from the WT probe. In another two cases, a double pattern (mutated and WT) was detected by the LoC assay, but only the mutation was identified by the MTBDRplus assay.
TABLE 2.
Phenotypic DST, MTBDRplus, and VerePLEX Biosystem results for the 91 MTBC clinical isolates included in the study
| Phenotypic DST resulta for: |
MTBDRplus/sequencing resultb for the following gene: |
VerePLEX Biosystem resultb,c for the following gene: |
No. of isolatesd | |||||
|---|---|---|---|---|---|---|---|---|
| RIF | INH | rpoB | katG | inhA | rpoB | katG | inhA | |
| R | R | S531L | S315T1 | WT | S531L | S315T1 | WT | 15 |
| R | R | WT | WT | WT | WT | WT | WT | 1 |
| S | R | WT | WT | WT | WT | WT | WT | 5 |
| R | R | S531L | WT | C-15T | S531L | WT | C-15T | 16 |
| R | R | S531L | WT | WT | S531L | WT | WT | 7 |
| R | S | S531L | WT | WT | S531L | WT | WT | 2 |
| R | R | H526D | S315T1 | WT | H526D | WT* + S315T1 | WT | 1 |
| R | R | H526D | S315T1 | WT | WT + H526D | WT + S315T1 | WT | 1 |
| R | R | L511P | S315N | WT | L511P | WT* | WT | 1 |
| R | R | H526D | S315R | WT | H526D | Δ 313–317 WT | WT | 1 |
| R | R | H526Y | S315N | WT | H526Y | WT* | WT | 1 |
| R | S | D516V | WT | WT | D516V | WT | WT | 1 |
| R | R | S531L | S315T1 | T-8A | S531L | S315T1 | T-8A | 2 |
| R | R | L530M+S531P | S315T1 | T-8C | Δ 530–533 WT | S315T1 | T-8C | 1 |
| R | R | S531L | S315T2 | WT | S531L | S315T2 | WT | 2 |
| R | R | D516V | S315T1 | T-8A | D516V | S315T1 | T-8A | 3 |
| R | R | D516V | S315T1 | T-8C | D516V | S315T1 | T-8C | 1 |
| S | R | WT | WT | C-15T | WT | WT | C-15T | 11 |
| R | R | D516V | S315T1 | WT | D516V | S315T1 | WT | 5 |
| S | R | WT | S315T1 | WT | WT | S315T1 | WT | 5 |
| R | R | H526D | S315T1 | WT | H526D | S315T1 | WT | 1 |
| R | R | S531L | S315T1 | C-15T | S531L | S315T1 | C-15T | 3 |
| R | R | Q513P | S315T1 | WT | Δ 510–513 WT | S315T1 | WT | 1 |
| S | R | WT | S315N | WT | WT | Δ 313–317 WT | WT | 1 |
| R | R | H526Y | S315T1 | C-15T | H526Y | S315T1 | C-15T | 2 |
| S | S | WT | WT | WT | WT | WT | WT | 1 |
The phenotypic drug susceptibility testing (DST) results for rifampin (RIF) and isoniazid (INH) are given as follows: R, resistant; S, sensitive.
The results for the 91 MTBC isolates found by the MTBDRplus assay and sequencing or by the VerePLEX Biosystem are shown (wild type [WT] or mutant).
Symbols: *, probe signal was on at the cutoff; Δ, no WT signal.
The number of isolates apply to all the test results.
Other mutations identified by sequencing (L530M, S531P, and Q513 in rpoB and S315N and S315R in katG) were correctly detected on the chip by the absence of signal from respective WT probes.
Compared with DST, the sensitivity and specificity of the MTBDRplus assay for RIF were 98.53% and 100%, respectively, and the sensitivity and specificity for INH were 82.76% and 100%, respectively (Tables 3, 4, and 5).
TABLE 3.
Diagnostic performance of the phenotypic DST, MTBDRplus, VerePLEX Biosystem, and Xpert MTB-RIF for detecting rifampin resistance (rpoB) in clinical isolates and specimensa
| Parameter | Value (95% CI) for clinical isolates (n = 91) |
Value (95% CI) for clinical specimensb |
Method type and no. of indeterminate results/total (%) | ||
|---|---|---|---|---|---|
| MTBDRplus/seq | DST | MTBDRplus/seq/Xpert MTB-RIF (n = 71) | DST (n = 58) | ||
| Sensitivity (%) | 100.00 (94.58, 100.00) | 98.53 (92.13, 99.74) | 100.00 (77.19, 100.00) | 100.00 (75.75, 100.00) | Molecular 3/71 (4.23) |
| Specificity (%) | 100.00 (86.2, 100.00) | 100.00 (85.69, 100.00) | 100.00 (93.47, 100.00) | 100.00 (91.97, 100.00) | Phenotypic 2/58 (3.45) |
| PPV (%) | 100.00 (94.58, 100.00) | 100.00 (94.58, 100.00) | 100.00 (77.19, 100.00) | 100.00 (75.75, 100.00) | |
| NPV (%) | 100.00 (86.2, 100.00) | 95.83 (79.76, 99.26) | 100.00 (93.47, 100.00) | 100.00 (91.97, 100.00) | |
| Negative likelihood ratio | 0.00 (0.00, ?) | 0.01 (0.00, 0.10) | 0.00 (0.00, ?) | 0.00 (0.00, ?) | |
| Diagnostic accuracy (%) | 100.00 (95.95, 100.00) | 98.90 (94.03, 99.81) | 100.00 (95.95, 100.00) | 100.00 (93.58, 100.00) | |
The diagnostic performance of the MTBDRplus assay and sequencing (MTBDRplus/seq), phenotypic drug susceptibility testing (DST), and MTBDRplus assay, sequencing, and Xpert MTB-RIF assay (MTBDRplus/seq/Xpert MTB-RIF) for detecting rifampin resistance (rpoB) are shown. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), and diagnostic accuracy were calculated according to the Wilson score (www.OpenEpi.com). The positive and negative likelihood ratios were also calculated. The lower and upper limits of the 95% confidence interval (95% CI) are shown in parentheses. The effective number of samples considered for the analysis is reported for each target. The positive likelihood ratio cannot be computed, since specificity is always 100%.
There were a total of 80 M. tuberculosis-positive smear-positive clinical specimens and a total of 116 M. tuberculosis-negative clinical specimens.
TABLE 4.
Diagnostic performance of the phenotypic DST, MTBDRplus, VerePLEX Biosystem, and Xpert MTB-RIF for detecting isoniazid resistance (katG and inhA) in clinical isolates and specimensa
| Parameter | Value (95% CI) for clinical isolates (n = 91) |
Value (95% CI) for clinical specimensb |
Method type and no. of indeterminate results/total (%) | ||||
|---|---|---|---|---|---|---|---|
| MTBDRplus/seq |
DST | MTBDRplus/seq/Xpert MTB-RIF |
DST (n = 57) | ||||
| katG | inhA | katG (n = 67) | inhA (n = 67) | ||||
| Sensitivity (%) | 95.74 (87.75, 98.83) | 100.00 (91.03, 100) | 82.76 (73.48, 89.26) | 93.75 (71.67, 98.89) | 90.91 (62.26, 98.38) | 73.33 (55.55, 85.82) | Molecular 3/67 (4.48) |
| Specificity (%) | 100.00 (91.97, 100.00) | 100.00 (93.12, 100.00) | 100.00 (51.01, 100.00) | 100.00 (92.59, 100.00) | 100.00 (93.24, 100.00) | 100.00 (86.68, 100.00) | Phenotypic 2/57 (3.5) |
| PPV (%) | 100.00 (92.13, 100.00) | 100.00 (91.03, 100.00) | 100.00 (94.93, 100.00) | 100.00 (79.61, 100.00) | 100.00 (72.25, 100.00) | 100.00 (85.13, 100.00) | |
| NPV (%) | 95.65 (85.47, 98.90) | 100.00 (93.12, 100.00) | 21.05 (8.51, 43.33) | 97.96 (89.31, 99.64) | 100.00 (90.23, 99.67) | 75.76 (58.98, 87.17) | |
| Negative likelihood ratio | 0.04 (0.02, 0.11) | 0.00 (0.00, ?) | 0.17 (0.15, 0.20) | 0.07 (0.009, 0.44) | 0.09 (0.01, 0.65) | 0.26 (0.21, 0.34) | |
| Diagnostic accuracy (%) | 97.8 (92.34, 99.4) | 100.00 (95.95, 100.00) | 83.52 (74.57, 89.75) | 98.44 (91.67, 99.72) | 98.44 (91.67, 99.72) | 85.45 (73.84, 92.44) | |
The diagnostic performance of the MTBDRplus/seq assays, phenotypic drug susceptibility testing (DST), and MTBDRplus/seq/Xpert MTB-RIF assays for detecting isoniazid resistance (katG and inhA) are shown. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), and diagnostic accuracy were calculated according to the Wilson score (www.OpenEpi.com). The positive and negative likelihood ratios were also calculated. The lower and upper limits of the 95% confidence interval (95% CI) are shown in parentheses. The effective number of samples considered for the analysis is reported for each target. The positive likelihood ratio cannot be computed, since specificity is always 100%.
There were a total of 80 M. tuberculosis-positive smear-positive clinical specimens and a total of 116 M. tuberculosis-negative clinical specimens.
TABLE 5.
Diagnostic performance of the phenotypic DST, MTBDRplus, and VerePLEX Biosystem for detecting M. tuberculosis in clinical isolates and specimensa
| Parameter | Value (95% CI) for clinical isolates (n = 91) |
Value (95% CI) for clinical specimens (n = 196)b by MTBDRplus/seq/Xpert MTB-RIF | No. of indeterminate results/total (%) | |
|---|---|---|---|---|
| MTBDRplus/seq | DST | |||
| Sensitivity (%) | 100.00 (95.95, 100.00) | 100.00 (95.95, 100.00) | 100.00 (95.31, 100.00) | 2/196 (1.02) |
| Specificity (%) | Undefined | Undefined | 100.00 (96.79, 100.00) | |
| PPV (%) | 100.00 (95.95, 100.00) | 100.00 (95.95, 100.00) | 100.00 (95.31, 100.00) | |
| NPV (%) | Undefined | Undefined | 100.00 (96.79, 100.00) | |
| Negative likelihood ratio | Undefined | Undefined | 0.00 | |
| Diagnostic accuracy (%) | Undefined | Undefined | 100.00 (98.06, 100.00) | |
The diagnostic performance of the MTBDRplus/seq assays, phenotypic drug susceptibility testing (DST), and MTBDRplus/seq/Xpert MTB-RIF assays for detecting M. tuberculosis are shown. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), and diagnostic accuracy were calculated according to the Wilson score (www.OpenEpi.com). The positive and negative likelihood ratios were also calculated. The lower and upper limits of the 95% confidence interval (95% CI) are shown in parentheses. The effective number of samples considered for the analysis is reported for each target. The positive likelihood ratio cannot be computed, since specificity is always 100%.
There were a total of 80 M. tuberculosis-positive smear-positive clinical specimens and a total of 116 M. tuberculosis-negative clinical specimens.
Analysis of the diagnostic performance of the LoC assay on clinical specimens.
DST results for RIF and INH were available for 58 and 57 samples, respectively. The chips presenting incomplete results were repeated once and then included in the analysis (Table 6).
TABLE 6.
Phenotypic DST, MTBDRplus, Xpert MTB-RIF, and VerePLEX Biosystem M. tuberculosis results for the 80 smear-positive MTBC culture-positive clinical specimens included in the study
| Phenotypic DST resulta for: |
MTBDRplus/sequencing resultb for the following gene: |
Xpert MTB-RIF resultc for: |
VerePLEX MTB resultb,d for the following gene: |
No. of clinical specimense | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| RIF | INH | rpoB | katG | inhA | MTB | RIF | rpoB | katG | inhA | |
| S | R | WT | WT | C-15T | WT | WT | C-15T | 9 | ||
| R | R | S531L | S315T1 | WT | S531L | S315T1 | WT | 2 | ||
| R | R | S531L | WT + S315T1 | WT | S531L | WT + S315T1 | WT | 1 | ||
| S | R | WT | WT | WT | WT | WT | WT | 6 | ||
| R | R | D516V | S315T1 | WT | D516V | S315T1 | WT | 2 | ||
| R | R | S531L | WT | WT | S531L | WT | WT | 2 | ||
| S | R | WT | S315T1 | WT | WT | S315T1 | WT | 4 | ||
| R | R | S531L | S315T1/T2 | WT | S531L | S315T1/T2 | WT | 1 | ||
| R | R | Q513P | S315T1 | WT | Δ 510–513 WT | S315T1 | WT | 1 | ||
| S | R | WT | S315N | WT | WT | Δ 313–317 WT | WT | 1 | ||
| R | S | S531L | WT | WT | S531L | WT | WT | 1 | ||
| R | R | S531L | WT | C-15T | S531L | Δ 313–317 WT | WT | 1 | ||
| S | S | WT | WT | WT | WT | WT | WT | 15 | ||
| R | R | Δ 518–525 WT, Δ 530–533 WT | S315T1 | WT | Δ 523–526 WT, S531L | S315T1 | WT | 1 | ||
| D516V | S315T1 | T-8C | D516V | S315T1 | T-8C | 1 | ||||
| WT | WT | WT | WT | WT | WT | 15 | ||||
| WT | S315T1 | WT | WT | S315T1 | WT | 1 | ||||
| S | S | WT | WT | WT | 9 | |||||
| pos | WT | WT | WT | WT | 4 | |||||
| WT | WT | WT | PCNV | PCNV | PCNV | 1 | ||||
| S | S | ND | S315T1 | WT | MTBND | MTBND | MTBND | 1 | ||
| S | S | ND | WT | WT | ND | ND | ND | 1 | ||
The phenotypic drug susceptibility testing results for rifampin and isoniazid are given as follows: R, resistant; S, sensitive.
The results for the 80 smear-positive, MTBC culture-positive isolates found by the MTBDRplus assay and sequencing or by the VerePLEX Biosystem are shown (wild type [WT] or mutant). Δ, no WT signal; ND, not detected.
MTB, M. tuberculosis; pos, positive.
PCNV, PCR controls not valid; MTBND, M. tuberculosis not detected; ND, not detected.
The number of smear-positive, MTBC culture-positive clinical specimens applies to all the tests.
Valid results were obtained in 99.00%, 95.80%, and 95.50% of the cases for MTBC, rpoB, katG, and inhA targets, respectively. MTBC was detected with 100% sensitivity and specificity on the LoC, as well as resistance to RIF (Tables 3, 4, and 5). One discrepant result was detected for the katG and inhA genes, leading to a sensitivity of 93.75% and 90.91%, respectively, compared to the MTBDRplus assay. Overall, the sensitivity and specificity of katG and inhA targets were 73.33% and 100%, respectively, compared to DST. Three specimens gave invalid values by the LoC assay. One sample gave an invalid result for PCR controls, possibly due to inhibitors affecting the reaction in the microfluidic environment. The remaining two specimens also yielded invalid results with the MTBDRplus assay. All 116 MTBC culture-negative specimens were classified correctly.
In the current study, we developed and evaluated a LoC-based assay for the diagnosis of MDR-TB. LoC devices represent promising tools to fill the diagnostic gap in low-income countries: they integrate many of the laboratory components on a small chip, thus reducing infrastructure and technical requirements but preserving analytical capabilities. In addition, the operating speed, ease of modification (addition/removal of probes), and ability to perform multiplex tests and to scale down costs represent other relevant features of LoCs (19, 20).
Our results showed high specificity and sensitivity of the semiautomated VerePLEX Biosystem for the MDR-TB targets, thus suggesting an usefulness of the platform for fast and simple diagnosis of MDR cases in centralized laboratories. The sensitivity and specificity of the NTM probes on the same platform were evaluated by Lazzeri et al. (21). The assay allowed us to identify correctly MTBC in 100% of the smear-positive samples tested independently of the smear microscopy score, with a small number of indeterminate results due most likely to the low quality of DNA extracted. Resistance to RIF and INH was detected by the chip with high sensitivity and specificity in agreement with the minimal requirements established by the WHO for molecular tools, comparable to the sensitivity and specificity of the MTBDRplus assay (12). The limit of detection of the assay was observed in the range of 101 genome copies/reaction, as reported in Table S5 in the supplemental material.
A separate array layout for spoligotyping of MTBC was also developed in the TM-REST Project (data not shown). The possibility of integrating the probes for spoligotyping, MDR- and extensively DR-TB in one medium-density microarray layout by using separate multiplex-PCR would enhance the benefits of the microarray assays and would enable the reduction of time to results compared to other available tests (22–24).
The ease of customization of the array design makes the LoC a versatile tool for easy integration of relevant targets for local genetic variants, new genes and/or mutations, and novel key drugs included in new therapeutic regimens. In addition, the LoC can be adapted for other diagnostic or research needs, thus providing a multipurpose platform suitable for other relevant diseases (e.g., influenza, malaria, tropical diseases) (25, 26).
Supplementary Material
ACKNOWLEDGMENTS
This study was supported by FP7 EU grant TM-REST (HEALTH-F3-2008-202145) and European and Developing Countries Clinical Trials Partnership as part of the TB CHILD project (IP.2009.32040.007).
The members of the TM-REST Consortium follow: Patrizia Di Pietro, Floriana San Biagio, Enrico Alessi, and Tony G. Barbuzzi (Analog, MEMS & Sensor Group, HealthCare Business Development Unit, STMicroelectronics, Catania, Italy); Silva Tafaj (University Hospital Shefqet Ndroqi, Tirana, Albania); Elizabetha Bachiyska (National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria); Irina Kontsevaya (Samara TB Service, Samara, Russian Federation); Yanina Balabanova (Clinical TB and HIV Group, Blizard Institute, Queen Mary University of London, United Kingdom, and Department of Infectious Diseases and Immunity, Imperial College London, United Kingdom); and Elisa Lazzeri (Laboratory of Molecular Microbiology and Biotechnology, Department of Medical Biotechnologies, University of Siena, Siena, Italy). The members of the TB-CHILD Consortium follow: Joseph Sserunkuma, Francesco Aloi, and Martin Nsubuga (Laboratory Department, St. Raphael of St. Francis Nsambya Hospital, AISPO, Kampala, Republic of Uganda) and Mohamed Sasamalo (Ifakara Health Institute, Bagamoyo, United Republic of Tanzania).
We thank Tanja Ubben and Tanja Struwe Sonnenschein for excellent technical assistance and Enrico Tortoli for valuable support.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.01824-15.
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