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 Table of Contents  
Year : 2017  |  Volume : 1  |  Issue : 2  |  Page : 141-146

The diagnostic utility of anyplex plus™ MTB/NTM cycle threshold for detection of Mycobacterium tuberculosis complex among new and retreatment cases from a referral hospital in Limpopo Province, South Africa

1 Department of Pathology and Medical Sciences, University of Limpopo, Polokwane; TB Platform, n Medical Research Council, Pretoria, South Africa
2 Department of Pathology and Medical Sciences, University of Limpopo, Polokwane, South Africa
3 TB Platform, n Medical Research Council, Pretoria, South Africa

Date of Web Publication23-Nov-2017

Correspondence Address:
Lesibana Anthony Malinga
Private Bag X 385, Pretoria, 0001
South Africa
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_85_17

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Background: The objective of the study was to detect Mycobacterium tuberculosis (MTB) complex using a diagnostic Anyplex plus™ MTB/NTM (Seegene Incorporated, Korea) assay in new and retreatment cases of tuberculosis (TB) in rural South Africa. Methods: We prospectively collected 204 clinical isolates from patients with new and retreatment cases residing in rural to urban areas attending a tertiary hospital. The clinical isolates were decontaminated and DNA extracted according to manufactures instructions. GenoType MTBDRplus version 2 was performed on extracted DNA and positive MTB isolates were run on Anyplex plus™ MTB/NTM assay. Culture (gold standard) and smear microscopy were used as reference standards for correct diagnosis of TB. Results: A total of 101 (49.5%) clinical isolates had valid MTB results using Genotype MTBDRplus version 2. Anyplex plus™ MTB/NTM was able to detect 68 cases (67.3%) when compared to GenoType MTBDRplus version 2. Cycle threshold (CT) of Anyplex plus™ MTB/NTM assay was able to distinguish between true and false positives for both culture and smear microscopy. A CT cutoff value of 25.9 and 37.0 could be used to differentiate between the bacilliary load of new and retreatment cases, respectively, for true positives. Conclusion: The novel technique was able to detect TB bacilliary load from new and retreatment clinical specimens using CT values.

Keywords: Anyplex plus™ MTBMTB/NTM, culture, cycle threshold, Mycobacterium tuberculosis, smear microscopy, true positives

How to cite this article:
Mpanyane DM, Maguga-Phasha TN, Mashinya F, Malinga LA. The diagnostic utility of anyplex plus™ MTB/NTM cycle threshold for detection of Mycobacterium tuberculosis complex among new and retreatment cases from a referral hospital in Limpopo Province, South Africa. Biomed Biotechnol Res J 2017;1:141-6

How to cite this URL:
Mpanyane DM, Maguga-Phasha TN, Mashinya F, Malinga LA. The diagnostic utility of anyplex plus™ MTB/NTM cycle threshold for detection of Mycobacterium tuberculosis complex among new and retreatment cases from a referral hospital in Limpopo Province, South Africa. Biomed Biotechnol Res J [serial online] 2017 [cited 2022 Jan 24];1:141-6. Available from: https://www.bmbtrj.org/text.asp?2017/1/2/141/219116

  Introduction Top

The World Health Organization (WHO) has identified South Africa (SA) as one of the thirty high burden countries that contributes to more than 83% of the 10.4 million TB cases.[1] Despite the existing knowledge on treatment strategies, therapy, advanced diagnostic tools, and documented literature to overcome the burden of TB disease, it remains a health-threatening communicable disease.[2] Limpopo is one of the rural provinces of SA, with relatively poor infrastructure and public services including health. Tuberculosis (TB) retains its position as leading infectious disease causing death in Limpopo province.[2] Data captured from the electronic database in Limpopo province report a threefold increase in the number of TB cases between years 2000 and 2009.[3]

Adequate initial TB diagnosis is particularly important but remains a challenge in low-income areas.[4] In most rural settings, the diagnosis of TB is primarily performed by smear microscopy, which is affordable and easy to perform without high technological infrastructure.[5] The Xpert® MTB/RIF assay was then recommended as an initial diagnostic tool coupled with smear microscopy for TB diagnosis by the WHO is currently used in most of high burdened TB countries.[6] However, the low sensitivity and specificity of the results compared to culture method renders the use of smear microscopy inconsequential.[7] The slow turnaround time associated with culture method is limiting but still remains the gold standard. Lately, clinical diagnosis of TB is dependent on molecular assays despite the dearth of information on their sufficient standardization to enable reliability in results.[8],[9] The time consuming and high contamination is often associated with culture method. Better diagnostic technologies such Anyplex assays and GenoType MTBDRplus version 2 can detect Mycobacterium TB ( MTB) complex and its resistance to anti-TB drugs with high sensitivity and specificity are needed.[7] Novel assays are continuously developed for the detection of MTB complex and need to be evaluated in various geographic locations for their standardization.[8]

There is a need to expand and improve MTB detection, especially in high prevalence areas with TB and human immunodeficiency virus (HIV) coinfection.[4] The most recently developed technique is the Anyplex plus™ NTM/MTB assay which has the ability to directly detect and discriminate MTB and non-TB Mycobacteria (NTM) in 2 h. Nucleic acid amplification tests have held a very promising future to improve TB management efforts. The WHO endorsed Xpert® MTB/RIF assay is an automated, real-time, cartridge-based PCR NAAT test. However, there is an emerging evidence of false positivity related to Xpert® MTB/RIF in retreatment cases in TB diagnosis.[4],[10] The cycle threshold (CT) that measures bacterial burden should be accurate, effective, and adaptable in molecular diagnostic tests to reduce false positivity.

The CT can be used as a determinant of bacillary load and is inversely proportional to the amount of target gene in a specimen. A single Xpert® MTB/RIF CT value is not adequate to capture the change in bacterial load that occurs with TB treatment.[11] There is evidence that indicated patients with false-positive Xpert® MTB/RIF results are likely to have TB history and low MTB bacillary load.[12] Similarly, to Xpert® MTB/RIF, the Anyplex plus™ MTB/NTM assay uses CT value in the detection of MTB. The aim of the study was to evaluate Anyplex plus™ MTB/NTM and assess the utility of CT values in discriminating between bacillary load of new and retreatment TB patients.

  Methods Top

Patient enrollment

Two hundred and four patients were enrolled from tertiary hospital, in a SA province. Three specimens were collected from each patient and two samples were sent to the national laboratory for routine diagnostic testing (located 180 km from hospital). The third sputum specimen was transported at ambient temperature to the research laboratory, during March 2013–August 2013.


New case-patient that has never been treated for TB or has been taking anti-TB treatment for less than a month.

Retreatment case-patient that received 1 month or more of anti-TB drugs in the past.

Specimen decontamination and GenoType MTBDRplus version 2

The clinical specimens were all subjected to quality and quantity check to be included into the study. A total of 169/204 (82.8%) cases were considered for the study and decontaminated with BBL MicroPrep kit based on N acetyl-L-cysteine-sodium hydroxide method (Becton Dickinson, BD Sparks, MD) and concentrated by maximum centrifugation for 15 min. DNA was extracted from the samples using GenoLyse (Hain LifeScience, Nehren, Germany) and performed according to manufacturer's instructions. Briefly, a volume of 500 μl of decontaminated sputum was centrifuged at 10,000 × g for 15 min and supernatant was discarded. The pellet was resuspended with 100 μl of alkaline-lysis buffer solution and incubated at 95°C for 5 min. A volume of 100 μl of neutralization buffer was added to the lysate, vortexed, and centrifuged at 13000 × g for 5 min. Therefore, 50 μl of supernatant was aliquoted to a sterile 1.5 microcentrifuge tube as template DNA and processed on GenoType MTBDRplus version according to manufacturer's instructions (Hain LifeScience, Nehren, Germany).

Smear microscopy and culture

At enrollment, the national laboratory staff performed smear microscopy and mycobacterial culture auramine and BACTEC MGIT 960, respectively (BD, Sparks, MD). The procedures were performed in a blinded manner and results released at the end of the study. Then, it was recorded as positive cultures for MTB when mycobacterial growth was observed within 6 weeks of incubation. Positive cultures were confirmed by Ziehl–Neelsen staining.

Anyplex plus™ MTB/NTM assay

The clinical isolates screened positive by GenoType MTBDRplus version 2 for MTB were further analyzed by Anyplex™ MTB/NTM (Seegene Incorporated, Korea). The Anyplex plus™ MTB/NTM master mix was a volume of 15 μl and was mixed with a DNA template of 5 μl to a final volume of 20 μl. The controls were included as well for this assay. The tubes were then loaded into the CFX 96 Real-Time PCR (Bio-Rad, Hercules, California, USA) instrument and subjected to a protocol provided by manufacturer and results were generated within 2 h and interpreted as directed in the protocol. The assay uses CT values to detect different amounts of DNA, i.e., higher CT values have low DNA whereas low CT have high DNA.

Statistical analysis

Patient and diagnostic data were entered into. Microsoft access and frequencies of variables were analyzed by Epi Info (version 8) (CDC, Atlanta, US). Diagnostic parameters of sensitivity, specificity, ROC were calculated by STATA (version 13). The ROC curve plays a central role in evaluating diagnostic ability of tests to discriminate the true state of subjects, finding the optimal cutoff values, and comparing two alternative diagnostic tasks when each task is performed on the same subject. The ROC curve is used to measure the performance of a test considering the size of area under the curve (AUC).


The study was approved by the Research and Ethics Committee (REC), MREC/HS/89/2013: PG, Department of Health and Social Development and National Laboratory for approval before sample collection. Patient's confidentiality was ensured through the use of laboratory numbers that were allocated by the researcher and no information by which samples could be traced back to patients was captured.

  Results Top

Clinical isolates and patient characteristics

Two hundred and four clinical isolates were collected, 169/204 (82.8%) of clinical isolates were eligible for inclusion in the study based on the quantity and quality scores of sputa. Of the 113 clinical isolates screened positive by GenoType MTBDRplus version 2 in the study, nine and three isolates, respectively, were excluded due to smear microscopy and culture missing results. The resulting 101 patients were analyzed. The detection rate of Anyplex plus™ MTB/NTM was 68/101 (67.3%) when compared to GenoType MTBDRplus version 2 [Figure 1].
Figure 1: Flow diagram of study specimens

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Patients had a median age of 38 (IQR 9–65) with more males (60%) than females (40%) and majority were retreatment cases, i.e. 84 (87.7%) compared to 17 (16.8%) that were new cases. Of the 101 clinical isolates included in the analysis, ninety (89.2%), seven (6.9%), and four (3.9%) were culture negative, positive, and contaminated, respectively. A total of ten (9.9%) patients had positive smear microscopy whereas 83 (82.2%) were smear negative and eight (7.9%) contaminated. Sixty-eight (67.3%) patients were Anyplex plus™ MTB/NTM positive and 33 (32.4%) had negative results [Table 1].
Table 1: Frequencies of Anyplex plus™ MTB/NTM, microscopy and culture processed isolates in new and retreatment cases

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Detection of MTB by Anyplex plus™ MTB/NTM, smear microscopy, and culture

A total of 101 clinical isolates were processed on Anyplex plus™ MTB/NTM, smear, and culture. The Anyplex™ MTB/NTM assay detected 14/17 (82.4%) and 54/84 (64.3%) of new cases and retreatment cases as MTB positives, respectively. There were only seven MGIT culture-positive specimens and Anyplex plus™MTB/NTM detected 7/7 (100%) as MTB positive in both new and retreatment cases. Culture identified 7/101 (6.9%) of those as MTB positive and 90/101 (21%) MTB negative and 4 (3.9%) as contaminated [Table 1]. A sensitivity and specificity of 100% and 34.3% was recorded.

Anyplex plus™ MTB/NTM false positivity in new and retreatment cases

We investigated the high number of false-positive isolates detected by Anyplex plus™ MTB/NTM. Since Anyplex plus™ MTB/NTM is a real-time assay with CT values, we correlated them with culture and smear microscopy. The mean CT (95% CI) for new and retreatment cases was 38.1 (35.27–40.84) and 39.9 (39.5–40.50), respectively. Using culture as the gold standard, we were able to detect 1 (8.33%) and 13 (78.6%) of new cases as true- and false-positive cases, respectively [Table 2]. For retreatment cases, 6 (11.3%) and 48 (88.7%) were true- and false-positive cases, respectively. No correlation was observed between CT values and time to positivity; however, most of the Anyplex plus™ MTB/NTM positive samples had an incubation time of ≥45 days [Figure 2]. Differences in CT in new and retreatment patients in culture and smear microscopy are shown in [Figure 3].
Table 2: Frequencies of true and false positives in culture and smear tests

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Figure 2: The Anyplex plus ct values against the time to positivity with the reference at 42 ct values

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Figure 3: Demonstrates the different case distribution according to smear and culture against the cut off values. (a) Smear retreatment cases (b) Smear new cases (c) Culture retreatment cases (d) Culture new cases

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Receiver operating curve analysis

The AUC for new (0.5) and retreatment cases (0.6) was poor for detecting false-positive cases [Figure 4] and [Figure 5]. At a rule-in cutoff point of 25.95 CT value, Anyplex plus™ MTB/NTM had a sensitivity and specificity of 88.8% and 18.8%, respectively, in new cases. A cutoff point of >37 CT had a sensitivity and specificity of 100.0% and 47.7% in retreatment cases [Table 3].
Figure 4: Receiver operating characteristic curve of Anyplex plus™ MTB/NTM cyclethreshold values for prediction of false positive in new patients

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Figure 5: Receiver operating curve of Anyplex plus™ MTB/NTM cycle threshold values for prediction of false positives in retreatment patient

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Table 3: Accuracy of cycle threshold values for predicting Anyplex plus™ MTB/NTM assay true positive in new and retreatment patients

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  Discussion Top

The performance of the Anyplex plus™ MTB/NTM assay was evaluated on both new and retreatment TB cases. The assay identified 82.4% and 64.3% of new and retreatment, respectively, positive for MTB, suggesting that this assay could be a great tool for TB diagnostics. Compared to culture, Anyplex plus™ MTB/NTM was able to detect MTB with sensitivity of 100% and specificity of 34.4%, respectively, of all isolates that were culture positive. These findings are similar to those recorded by Perry et al. with a sensitivity of 100% in both smear positive and negative isolates. However, we had a lower detection rate of 67.3% for Anyplex plus™ MTB/NTM when compared to GenoType MTBDRplus version 2. The diagnostic sensitivity of this assay indicates that it could be a suitable and convenient tool for the detection of MTB.

We detected a high number of false positives in new and retreatment cases that resulted in low specificity. The performance of the assay is affected by false positivity due to homology between Mycobacterium species or dead bacilli.[13] The mean CT (95% CI) difference for new and retreatment cases of 38.1 (35.27–40.84) and 39.9 (39.5–40.50), respectively, was minimal. Sali et al. had Anyplex plus ™ MTB/NTM discrepant results with a CT >35 which suggest a low genome copy number.[14] Retreatment cases can consist of dead or nonviable which can lead to high false positives.

Discriminating between viable and nonviable bacteria is a priority for molecular diagnostics. This is important for new and retreatment cases to get proper diagnostics while awaiting culture and smear results or for treatment monitoring. However, approved assays such as Xpert® MTB/RIF lack that capabilities to use CT to differentiate between true positive or false positive.[11],[15]

In this study, a rule-in cut point of 25.95 CT was detected with high sensitivity and low specificity of 88.8% and 18.8%, respectively, in new cases. This may indicate that 17 in 20 culture positives will be positive by Anyplex plus™ MTB/NTM. A value of >37 CT with a sensitivity and specificity was 100.0% and 47.7%, respectively, in retreatment. This indicates that high number of true positives will be correctly identified than false-positive cases. Therefore, a cutoff value of 25.95 and 37 CT can be used by clinicians to treat new and retreatment patients, respectively. This can be done before culture results become available.

Our study was first to report the diagnostic utility of Anyplex plus™ MTB/NTM in new and retreatment TB cases. Using CT values to discriminate between true and false positives is an attractive prospect. Therefore, CT can be a useful marker to report routinely in real-time PCR molecular diagnostics. However, using reagents such as propidium monoazide in real-time PCR may be a useful approach for differentiating dead from live bacilli in sputum isolates and thus increasing specificity.[16]

The study had limitations:First, a central facility was used and there could have been referral bias involved in patient selection and respiratory samples were used to evaluate the assays performance. Second, treatment data from previous episode were not available for retreatment patients. The diagnostic data were only sourced from a referral laboratory leading to potential heterogeneity of results. Furthermore, a low sample size, low culture positives, and contamination reduced the statistical power of the study. Despite this limitation, the study provided insights into the utility of Anyplex plus™ MTB/NTM CT values in discriminating between true and false positives among new and retreatment cases, especially in high HIV/TB burden.

  Conclusion Top

The Anyplex plus™ MTB/NTM assay is indicative that it has high capabilities to detect MTB complex in pulmonary specimens, which considering its rapidity renders it an accurate diagnostic tool for TB in clinical specimens. The study suggests that Anyplex plus™ MTB/NTM assay is a good molecular test for routine use that can be applied in many several clinical laboratories. The study findings highlight the significance of molecular detection and rapid identification of MTB complex when compared to culture methods. However, the lower detection rate still needs further GenoType MTBDRplus version 2. The Anyplex plus™ MTB/NTM assay is sensitive and could still identify dead MTB bacilli, hence, a need for further evaluations.


The SA Medical Research Council, TB platform, National Health Laboratory Service (Braamfontein), and Modimolle MDR-TB Hospital Staff (Dr. Lesufi and staff), for support and corporation during conduction of this study.

Financial support and sponsorship

This work was supported by the National Research Foundation (reference number: SFH14080184872) and TB grant (reference: C153 RG2-TBM/XDR) by Department of Science and Technology through the University of Limpopo. The study was approved by REC, MREC/HS/89/2013: PG.

Conflicts of interest

There are no conflicts of interest.

  References Top

World Health Organization. Global Tuberculosis Report. Geneva: World Health Organization; 2016.  Back to cited text no. 1
Mabunda JT, Khoza LB, Van den Borne HB, Lebese RT. Needs assessment for adapting TB directly observed treatment intervention programme in Limpopo Province, South Africa: A community-based participatory research approach. Afr J Prim Health Care Fam Med 2016;8:e1-7.  Back to cited text no. 2
Mabunda TE, Ramalivhana NJ, Dambisya YM. Mortality associated with tuberculosis/HIV co-infection among patients on TB treatment in the Limpopo Province, South Africa. Afr Health Sci 2014;14:849-54.  Back to cited text no. 3
Pai M, Schito M. Tuberculosis diagnostics in 2015: Landscape, priorities, needs, and prospects. J Infect Dis 2015;211 Suppl 2:S21-8.  Back to cited text no. 4
Steingart KR, Schiller I, Horne DJ, Pai M, Boehme CC, Dendukuri N, et al. Xpert® MTB/RIF assay for pulmonary tuberculosis and rifampicin resistance in adults. Cochrane Database Syst Rev 2014;1:1-166.  Back to cited text no. 5
World Health Organization. Global Tuberculosis Report. Geneva: World Health Organization; 2015.  Back to cited text no. 6
Noor KM, Shephard L, Bastian I. Molecular diagnostics for tuberculosis. Pathology 2015;47:250-6.  Back to cited text no. 7
Perry MD, White PL, Ruddy M. Potential for use of the Seegene Anyplex MTB/NTM real-time detection assay in a regional reference laboratory. J Clin Microbiol 2014;52:1708-10.  Back to cited text no. 8
Matabane MM, Ismail F, Strydom KA, Onwuegbuna O, Omar SV, Ismail N, et al. Performance evaluation of three commercial molecular assays for the detection of Mycobacterium tuberculosis from clinical specimens in a high TB-HIV-burden setting. BMC Infect Dis 2015;15:508.  Back to cited text no. 9
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Theron G, Venter R, Calligaro G, Smith L, Limberis J, Meldau R, et al. Xpert MTB/RIF results in patients with previous tuberculosis: Can we distinguish true from false positive results? Clin Infect Dis 2016;62:995-1001.  Back to cited text no. 11
Shenai S, Ronacher K, Malherbe S, Stanley K, Kriel M, Winter J, et al. Bacterial loads measured by the Xpert MTB/RIF assay as markers of culture conversion and bacteriological cure in pulmonary TB. PLoS One 2016;11:e0160062.  Back to cited text no. 12
Sanjuan-Jimenez R, Toro-Peinado I, Bermudez P, Colmenero JD, Morata P. Comparative study of a real-time PCR assay targeting senX3-regX3 versus other molecular strategies commonly used in the diagnosis of tuberculosis. PLoS One 2015;10:e0143025.  Back to cited text no. 13
Sali M, De Maio F, Caccuri F, Campilongo F, Sanguinetti M, Fiorentini S, et al. Multicenter evaluation of anyplex plus MTB/NTM MDR-TB assay for rapid detection of Mycobacterium tuberculosis complex and multidrug-resistant isolates in pulmonary and extrapulmonary specimens. J Clin Microbiol 2016;54:59-63.  Back to cited text no. 14
Ssengooba W, Respeito D, Mambuque E, Blanco S, Bulo H, Mandomando I, et al. Do Xpert MTB/RIF cycle threshold values provide information about patient delays for tuberculosis diagnosis? PLoS One 2016;11:e0162833.  Back to cited text no. 15
Kim SY, Ko G. Using propidium monoazide to distinguish between viable and nonviable bacteria, MS2 and murine norovirus. Lett Appl Microbiol 2012;55:182-8.  Back to cited text no. 16


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3]


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