Comparison between the PaxView TB/NTM MPCR-ULFA Kit and current methods for Mycobacterium tuberculosis detection in
Malang, East Java, Indonesia
Agustin Iskandar
1, Putri Wulan Akbar
1,2, Andrea Aprilia
1, Young-Kil Park
3,
1 Faculty of Medicine, Brawijaya University, Malang, Indonesia
2 Faculty of Medicine, UIN Maulana Malik Ibrahim, Malang, Indonesia
3 Department of Research, PaxGenBio, Anyang, Republic of Korea
*Corresponding author. Email: [email protected]
ABSTRACT
The PaxView TB/NTM MPCR-ULFA Kit, which targets the IS6110 and mtp40 genes for Mycobacterium tuberculosis (MTB) detection, is a novel tool that substitutes gel electrophoresis for universal lateral flow assays. The sensitivity and specificity of this method were compared with those of established methodologies using Indonesian clinical isolates. In this study, 148 sputum specimens isolated from suspected tuberculosis (TB) carriers were examined to evaluate the performance of the PaxView TB/NTM MPCR-ULFA Kit compared to that of smear microscopy and the Xpert MTB/RIF assay. Out of 148 cases, the rate of TB-positive samples evaluated by different methods was 18.2% (27/148; 95% CI 11.9–24.4) for smear microscopy, 20.3% (30/148; 95% CI 13.8–26.8) for the Xpert MTB/RIF, and 34.5% (51/148; 95% CI 26.8–42.1) for the PaxView TB/NTM MPCR-ULFA Kit. Twenty sputum specimens from healthy subjects were also tested, all of which rendered negative results via the three diagnostic tools examined herein. Compared to the Xpert MTB/RIF, the PaxView TB/NTM MPCR-ULFA Kit was found to possess a 96.7%
sensitivity (29/30; 95% CI 90.3-100).Moreover, the PaxView TB/NTM MPCR-ULFA Kit detected 18.6% (22/118, 95% CI 11.6–
25.6) of Xpert MTB/RIF MTB negative samples and 20.7% (25/121, 95% CI 13.5–27.9) of smear microscopy negative samples as MTB positive. The PaxView TB/NTM MPCR-ULFA Kit could be a useful molecular diagnostic tool to identify MTB in clinical samples in resource-limited countries, as this procedure is more cost-effective and sensitive than the Xpert MTB/RIF, and more convenient than conventional PCR gel electrophoresis approaches.
Keyword
Mycobacterium tuberculosis, multi-plex PCR, universal flow lateral assay, Xpert MTB/RIF, smear microscopy
Introduction
To this day, tuberculosis (TB) continues to be a major chronic infectious disease worldwide. According to the 2019 Global TB Report, the WHO estimated that 1.5 million people died due to TB and 10.0 million people developed TB worldwide in 20181.
The current estimated tuberculosis incidence in Indonesia is 845,000, which ranks third globally, only surpassed by India and China. However, only 570,289 cases are reported, and rapid molecular diagnoses account for only 12% in 20181. Even though the coverage of molecular diagnosis is growing every year, a large gap remains between the notified cases and the estimated incidence. It has been estimated that 98,000 people died from tuberculosis in 2018 in Indonesia1. Most deaths from TB could be prevented with early diagnosis and appropriate treatment2,3, and therefore early TB diagnosis is essential to reduce its worldwide lethality.
There are many kinds of TB diagnostic tools, from the smear microscopy (SM) approach developed by Robert Koch in 1882 to modern sophisticated approaches including Xpert MTB/RIF (Cepheid, CA, USA). Each country implements these methods alone or in combination with other approaches, depending on nation-wide socioeconomic factors. However, an optimal (i.e., effective and cost-efficient) TB diagnostic tool is yet to be developed.
Among the various existing TB diagnostic tools, molecular diagnostic tools are the fastest methods for the detection of Mycobacterium tuberculosis (MTB) in clinical specimens, and also possess satisfactory sensitivity and specificity4. For example, the Xpert MTB/RIF, a current leading molecular diagnostic tool, has been extensively used not only for the detection of rifampicin-resistant MTB but also for primary MTB case detection5-8. However, due to the expensive equipment, cartridges, and installation costs required by the Xpert MTB/RIF approach, this testing solution is not suitable for implementation in developing countries unless international monetary aid is provided9,10.
In house-PCR is the cheapest molecular diagnostic method, but requires cumbersome gel electrophoresis procedures.
Therefore, PaxGenBio (Korea) developed the PaxView TB/NTM MPCR-ULFA Kit, which rapidly detects MTB
In this study, we utilized sputum specimens from suspected TB carriers, which were acquired from public TB diagnosis health centers, hospitals, or clinics from July to December of 2019 in the Malang region, Indonesia. The average age of the 148 TB suspects was 48.06 years (14<yrs<85), and the proportion of males and females among total TB suspects was 54% and 46% respectively. Additionally, 20 specimens were collected from healthy volunteers (laboratory staff and medical students) to serve as negative controls.
Ziehl-Neelsen Smear Microscopy
Ziehl-Neelsen direct AFB smear and grading was performed by the technicians from each institute according to the WHO/International Union Against Tuberculosis and Lung Disease method11.
DNA extraction from specimens
After SM, the remaining sputum specimen was treated with Xpert MTB/RIF buffer. Afterward, a portion of the pretreated specimen was tested with the Xpert MTB/RIF procedure. Another specimen sub-sample was analyzed with the PaxView DNA Extraction Kit (PaxGenBio, Korea). Briefly, 500 µl of the specimen pretreated with the Xpert MTB/RIF buffer was transferred into 1.5 ml screw capped tubes, to which 500 µl of washing buffer were added (provided by the PaxView DNA Extraction Kit). This mixture was then centrifuged at 13,000 rpm for 3 min, after which the supernatant was discarded. After washing the specimen once more, 100 µl of elution buffer were added into the tube, which was then transferred to a 95℃ heating block for 15 min. After centrifugation, 20 µl of supernatant were transferred into a new tube and used as a template.
Xpert MTB/RIF
Pretreated specimen was amplified with the GeneXpert kit following the manufacturer’s instructions.
PaxView TB/NTM MPCR-ULFA Kit PCR and interpretation of the results
The PaxView TB/NTM MPCR-ULFA Kit includes multiple primer pairs, including two for MTB-specific genes (IS6110 and mpt40), as well as for the mycobacteria rpoB gene. After multiplex PCR, the product identities were confirmed by universal lateral flow assay (ULFA), which is based on DNA-DNA hybridization with previously immobilized complementary DNA fragments on a nitrocellulose membrane (Figure 1). PCRs were performed with the following protocol: 50°C for 4 min; 95 °C for 10 min; 25 cycles of denaturation (95 °C for 15 sec), annealing and extension (71 °C for 60 sec); 20 cycles of denaturation (95 °C for 15 sec), annealing (60 °C for 30 sec), and extension (72 °C for 30 sec).
After PCR amplification, 5 µl of PCR solution were added to the ULFA device inlet, after which 50 µl of running buffer (provided by the manufacturer) were added immediately. After 5 minutes, 50 µl of washing buffer were added into the inlet and the results were then read within 15 minutes.
Bands 1 and/or 2 (with or without band 3) in the detection device indicate that the sample is MTB-positive, the presence of band 3 without band 1 and 2 means that the sample is NTM-positive, bands 4 and 5 without band 1 to 3 indicate that the sample is MTB/NTM-negative, and band 5 alone indicates that the test was invalid, indicating that the amplification process did not happen due to problems with the template or reagents (Figure 2)
Ethical statement
This study was approved by the Institutional Review Board of Brawijaya University, under the project title “The diagnosis of pulmonary TB using MPCR-ULFA targeting IS6110 and mtp40” (Approval number: 208/EC/KEPK- PPDS/07/2019).
Figure 1. Principles of the PaxView TB/NTM MPCR-ULFA Kit.
Figure 2. Interpretation of the PaxView TB/NTM MPCR-ULFA Kit results.
Results
A total of 148 clinical specimens from hospitals and clinics in the Malang area were used to compare the performances of the PaxView TB/NTM MPCR-ULFA Kit, SM, and the Xpert MTB/RIF.Out of 148 cases, 18.2%
(27/148; 95% CI 11.9–24.4) were MTB positive according to SM, 20.3% (30/148; 95% CI 13.8–26.8) were positive according to the Xpert MTB/RIF, and 34.5% (51/148; 95% CI 26.8–42.1) were positive according to the PaxView TB/NTM MPCR-ULFA Kit.
Moreover, 20 specimens isolated from healthy individuals tested negative with all three methods analyzed herein.
Therefore, even though the number of negative samples was very limited, the specificity of the PaxView TB/NTM MPCR-ULFA Kit was flawless.
Compared to the Xpert MTB/RIF, the PaxView TB/NTM MPCR-ULFA Kit was found to possess a 96.7% sensitivity (29/30; 95% CI 90.3-100), as the PaxView TB/NTM MPCR-ULFA missed only one case out of the 30 samples that tested positive with the Xpert MTB/RIF (Table 1). This may have been caused by DNA loss during the DNA extraction step, as several manual washing steps are required for DNA purification.
Furthermore, the PaxView TB/NTM MPCR-ULFA Kit detected 18.6% (22/118, 95% CI 11.6–25.6) of Xpert MTB/RIF MTB negative samples and 20.7% (25/121, 95% CI 13.5–27.9) of SM negative samples as MTB positive.
Table 1. Comparison of MTB positive samples from suspected TB carriers depending on the diagnostic tool employed
PaxView MTB positive
PaxView
MTB negative Total
Z-N positive
Xpert MTB positive
26 1 27
Xpert MTB negative
0 0 0
Z-N Xpert 3 0 3
than the rpoB gene, which only has one copy.
According to Chaidir’s report, the distribution of MTB genotypes isolated in Indonesia consist of 46.9% of lineage 4, 33.7% of lineage 1, and 19.4% of lineage 212. This means that at least two-thirds of Indonesian MTB isolates have high IS6110 copy numbers, as lineages 2 and 4 are known to possess high IS6110 copy numbers13.
The ratio of positive cases determined by the PaxView TB/NTM MPCR-ULFA Kit to those of the Xpert MTB/RIF (34.5%/20.3%) was 1.7 (Table 1). This result has been confirmed in other previous reports.Reports of TB meningitis detection in Indonesia demonstrated that in-house PCR targeting IS6110 was superior to the Xpert MTB/RIF, with sensitivities of 43% for the Xpert MTB/RIF and 64% for in-house PCR (i.e., 1.52 ratio; 64%/43%)14. In Sharma’s report on the detection of pleural TB in India, the sensitivity of MPCR targeting for IS6110 and mpb64 was 2.7 times (89.6%/33.3%) superior to that of the Xpert MTB/RIF15.
In another group’s report on the detection of meningitis TB in India, the sensitivity of MPCR was 1.7 times (87.2%/50.5%) higher than that of the Xpert MTB/RIF16 and 1.2 times (100%/82.9%) higher for the detection of pulmonary TB in Nepal17.
In this study, the TB detection rate of the XpertMTB.RIF was only 1.12 times (20.3%/18.2%) higher than that of SM, which is only a slight difference compared the 2.16-fold (24.2%/11.2%) differences reported by Chaidir14, 1.75-fold (84%/48%) in a study in Thailand18, and 1.8-fold (65.5%/36.2%) in Ethiopia19. Generally, SM positive rates may vary depending on specimen quality, technician skill, and the number of samples treated in a day. In the latter case, having fewer samples may allow the technicians to observe the microscopy images more thoroughly.
The PaxView TB/NTM MPCR-ULFA Kit incorporates multiplex PCR and universal lateral flow assay (ULFA) and uses the IS6110 and mtp40 genes for TB detection. Theoretically, the occurrence of multiple copies of IS6110 within the chromosomal DNA of MTB could increase test sensitivity when only a few of bacilli are present in a specimen compared that of single-copy genes such as rpoB gene or mpt40. However, given that strains lacking IS6110 have been identified in some countries (e.g., India), alternative PCR targets are necessary, and mpt40 can be a good candidate in such strains20. The mpt40 gene is exclusively present in MTB, i.e., not in M. bovis, M. bovis BCG, or NTM21. Therefore, the mpt40 gene is an excellent alternative PCR target to detect MTB bacilli in suspected TB- infected specimens. Unfortunately, some MTB strains do not possess an mpt40 gene in their chromosomal DNA22. In these strains, the IS6110 gene can serve as an alternative PCR target. Therefore, the use of multiple complementary PCR targets for IS6110 and mpt40 is critical to increase MTB detection sensitivity, as well as to accurately discriminate between MTB and NTM23. For this reason, the PaxView TB/NTM MPCR-ULFA kit uses these two genes as MTB detection targets.
We could not identify strains that contained exclusively mtp40 bands without the co-occurrence of IS6110 bands in this clinical trial, meaning that all tested strains had IS6110 copies in a given chromosome. However, the opposite happened in some strains. For example, in Figure 3, lane 4 and 6 showed only band 1 without band 2, whereas lane 3 exhibited band 1 and band 2. This highlights the advantage of having multiple copies of IS6110 in a given sample rather than only single-copy genes.
We found 2 invalid results (1.3%) out of 150 TB suspected samples, both of which were SM negative and Xpert MTB/RIF negative. This may be caused by incomplete removal of PCR inhibitors in the sputum samples during DNA extraction. Therefore, these two samples were excluded from the comparative analyses.
Figure 3. Results of PaxView TB/NTM MPCR-ULFA Kit.
Some cases of NTM were detected with the PaxView TB/NTM MPCR-ULFA Kit; however, NTM samples were excluded in comparative analyses, as SM and the Xpert MTB/RIF cannot detect NTM.The newly-developed PaxView TB/NTM MPCR ULFA Kit incorporates multiple polymerase chain reactions and simplifies the result-reading process by implementing ULFA instead of a cumbersome electrophoresis procedure. Another advantage of the PaxView MPCR-ULFA Kit is that it only requires a standard PCR device. In contrast, the implementation of XpertMTB/RIF requires specialized and expensive equipment (Table 2).
ULFA was designed as follows. One strand of the primer set was designed to link a universal probe that can hybridize to an immobilized oligomer at lines 1~ 4 on the membrane. This universal probe cannot be made into a double-strand during the PCR process due to interruption by a spacer. The other primer strand set was linked with biotin, which can bind with nano gold-streptavidin when the PCR product comes in contact with the sample pad. The PCR product then flows upward through the absorbent pad through the force of running buffer. During its flow, a universal probe binds with its specific complementary oligomers, which were previously immobilized on the membrane (Fig. 1). Usually, within 5 minutes of adding the PCR product to the pad, a gold color is revealed on the line. However, PaxGenBio recommends waiting 15 minutes for the final results. This procedure markedly shortens PCR product reading time and, compared to gel electrophoresis, does not require preparing a gel and reading it on a UV transilluminator.
Although in-house PCR or MPCR targeting IS6110 has satisfactory sensitivity and cost-effectiveness compared to the Xpert MTB/RIF targeting the rpoBgene, in-house PCR has the disadvantages of being time-consuming and requiring a cumbersome gel electrophoresis step. Therefore, the PaxView TB/NTM MPCR-ULFA Kit substitutes the gel electrophoresis step with ULFA for rapid PCR result detection.
The Xpert MTB/RIF has many advantages such as its simplicity and automatic procedures without requiring manual DNA extraction and reading PCR results. This considerably reduces the risk of sample contamination, as well as operation times. However, the reagent cost of the Xpert MTB/RIF amounts to approximately 10 USD per test in developing countries and up to 40 USD in other countries. Therefore, many developing countries implement this procedure only if financial support (e.g., Global Fund) is provided. The sensitivity of this assay is also lower than that of in-house MPCR using IS6110 as a target (Table 2). Therefore, a cost-effective, user-friendly, and highly sensitive and specific TB diagnostic tool continues to be critically necessary.
Table 2. Pros and cons of various molecular MTB detection tools In-house PCR PaxView MPCR-
ULFA
Xpert MTB/RIF
Sensitivity compare to Xpert Higher Higher -
Reagent Cost (US$) 1 3 10~40
Conveniencecompared to Xpert 10% 50% 100%
Equipment General PCR General PCR Specialized
Equipment cost Low Low Very high
Ventilation system Very necessary Very necessary Necessary Skillful technician Very necessary Very necessary Necessary
Conclusion
In conclusion, the PaxView MPCR ULFA Kit has an excellent capacity for the detection of MTB in clinical specimens, and this kit could be implemented in laboratories with standard PCR equipment, bypassing the need to purchase expensive equipment. Moreover, the results of this kit can be obtained easily and quickly, without the need
Acknowledgements
We would like to thank to PaxGenBio, Republic of Korea, for supporting funding and equipments for this research.
We also thank to all of our colleagues of Soepraoen Army Hospital, Kanjuruhan General Hospital, Lawang General Hospital, Rampal-Celaket Public Health Center, and Dean of Medical Faculty of UniversitasBrawijaya for supporting cooperation between Faculty of Medicine UniversitasBrawijaya and PaxGenBio, Republic of Korea.
References
[1] World Health Organization (WHO) Global tuberculosis report 2019. Geneva: World Health Organization [2] Bhargava A, Bhargava M.Tuberculosis Deaths Are Predictable and Preventable: Comprehensive Assessment and
Clinical Care Is the Key. J Clin Tuberc OtherMycobact Dis Actions2020;19:100155.
[3] Min J, Kim JS, Kim HW, Shin AY, Koo HK, Lee SS, Kim YK, Shin KC, Chang JH, Chun G, Lee J, Park MS, Park JS. Clinical profiles of early and tuberculosis-related mortality in South Korea between 2015 and 2017: a cross-sectional study. BMC Infect Dis2019;19:735.
[4] Balcha TT, Sturegard E, Winqvist N, Skogmar S, Reepalu A, Jemal ZH, Tibesso G, Schon T, Bjorkman P.Intensified tuberculosis case-finding in HIV-positive adults managed at Ethiopian health centers: diagnostic yield of Xpert MTB/RIF compared with smear microscopy and liquid culture.PLoS One2014;9: e85478.
[5] Beyanga M, Kidenya BR, Gerwing-Adima L, Ochodo E, Mshana SE, Kasang C.Investigation of household contacts of pulmonary tuberculosis patients increases case detection in Mwanza City, Tanzania.BMC Infect Dis2018;18:110.
[6] Ikuabe PO, Ebuenyi ID.Prevalence of rifampicin resistance by automated Genexpert rifampicin assay in patients with pulmonary tuberculosis in Yenagoa, Nigeria.Pan Afr Med J 2018;29:204.
[7] Mesfin EA, Beyene D, Tesfaye A, Admasu A, Addise D, Amare M, Dagne B, Yaregal Z, Tesfaye E, Tessema B.
Drug-resistance patterns of Mycobacterium tuberculosis strains and associated risk factors among multi drug- resistant tuberculosis suspected patients from Ethiopia.PLoS One2018;13:e0197737.
[8] Sidharta SD, Yin JD, Yoong JS, Khan MS. High use of private providers for first healthcare seeking by drug- resistant tuberculosis patients: a cross-sectional study in Yangon, Myanmar. BMC Health Serv Res 2018;18:276.
[9] Abdurrahman ST, Emenyonu N, Obasanya OJ, Lawson L, Dacombe R, Muhammad M, Oladimeji O, Cuevas LE. The hidden costs of installing Xpert machines in a tuberculosis high-burden country: experiences from Nigeria. Pan Afr Med J.2014;18:277.
[10] Puri L, Oghor C, Denkinger CM, Pai M.Xpert MTB/RIF for tuberculosis testing: access and price in highly privatized health markets. www.thelancet.com/lancetgh 2016;e94~e95.
[11] Van Rie A, Fitzgerald D, Kabuya G, Van Deun A, Tabala M, Jarret N, F Behets, E Bahati. Sputum smear microscopy: evaluation of impact of training, microscope distribution, and use of external quality assessment guidelines for resource-poor settings. J Clin Microbiol 2008;46:897–901.
[12] Chaidir L, Sengstake S, de Beer J, Oktavian A, Krismawati H, Muhapril E, Kusumadewi I, Annisa J, Anthony R, van Soolingen D, et al. Predominance of modern Mycobacterium tuberculosis strains and active transmission of Beijing sublineage in Jayapura, Indonesia Papua. Infect Genet Evol. 2016;39:187-193.
[13] Gonzalo-Asensio J, Pérez I, Aguiló N, Uranga S, Picó A, Lampreave C, Cebollada A, Otal I, Samper S, Martín C.New insights into the transposition mechanisms of IS6110 and its dynamic distribution between Mycobacterium tuberculosis Complex lineages.PLoS Genet. 2018 Apr 12;14(4):e1007282.
[14] Chaidir L, Annisa J, Dian S, Parwati I, Alisjahbana A, Purnama F, van der Zanden A, Ganiem AR, van Crevel R.
Microbiological diagnosis of adult tuberculous meningitis in a ten-year cohort in Indonesia. Diagn Microbiol Infect Dis 2018;91:42-46.
[15] Sharma S, Dahiya B, Sreenivas V, Singh N, Raj A, Sheoran A, Yadav A, Gupta KB, Mehta PK.Comparative Evaluation of GeneXpert MTB/RIF and Multiplex PCR Targeting mpb64 and IS6110 for the Diagnosis of Pleural TB.Future Microbiol 2018;13:407-413.
[16] Sharma K, Sharma M, Chaudhary L, Modi M, Goyal M, Sharma N, Sharma A, Jain A, Dhibar DP, Jain K, Khandelwal N, Ray P, Lal V, Salfinger M. Comparative evaluation of Xpert MTB/RIF assay with multiplex polymerase chain reaction for the diagnosis of tuberculous meningitis. Tuberculosis 2018;113:38-42.
[17] Sah AK, Joshi B, Khadka DK, Gupta BP, Adhikari A, Singh SK, Rai G, Vaidya GS, Rajbhandari R, Pant B, Rai SK.Comparative study of GeneXpert MTB/RIF assay and multiplex PCR assay for direct detection of Mycobacterium tuberculosis in suspected pulmonary tuberculosis patients.Curr Microbiol 2017;74:1026-1032.
[18] Reechaipichitkul W, Suleesathira T, Chaimanee P. Comparison of GeneXpert MTB/RIF assay with conventional AFB smear for diagnosis of pulmonary tuberculosis in northeastern Thailand.Southeast Asian J Trop Med Public Health2017;48:313-321.
[19] Geleta DA, Megerssa YC, Gudeta AN, Akalu GT, Debele MT, Tulu KD.Xpert MTB/RIF assay for diagnosis of pulmonary tuberculosis in sputum specimens in remote health care facility. BMC Microbiol Actions 2015;15:220.
[20] Kathirvel M, Kommoju V, Brammacharry U, Ravibalan T, Ravishankar N, Radhakrishnan B, Muthaiah M.
Clinical evaluation of mtp40 polymerase chain reaction for the diagnosis of extra pulmonary tuberculosis. World J Microbiol Biotechnol2013:s11274-013-1566-z.
[21] Parra CA, Londono LP, Portillo PD, Patarroyo ME. Isolation, characterization, and molecular cloning of a specific Mycobacterium tuberculosis antigen gene: identification of a species-specific sequence. Infect Immun1991;59:3411-3417.
[22] Weil A, Plikaytis BB, Butler WR, Woodley CL, Shinnick TM. The mtp40 gene is not present in all strains of Mycobacterium tuberculosis. J Clin Microbiol1996;34:2309-2311.
[23] Sinha P, Gupta A, Prakash P, Anupurba S, Tripathi R, Srivastava GN. Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments. BMC Infect Dis 2016;16:123.
