Utility of a lateral flow assay for culture confirmation of Mycobacterium tuberculosis complex

Abstract

Background

Therapy for the clinical management of patients with Mycobacterium tuberculosis complex (MTBC) and non tuberculous mycobacteria (NTM) is different. Prompt detection and discrimination is necessary for administration of suitable therapy.

Methods

The aim of the study was to evaluate the performance of a Immunochromatographic Test (ICT) in rapid differentiation of MTBC from NTM grown in Lowenstein-Jensen medium and MGIT broth in comparison to molecular methods.

Results

Of the 106 isolates in this study, 96 and 95 were identified as MTBC by 16S rRNA PCR and MPT64 respectively. The sensitivity and specificity of MPT64 was found to be 99% and 100% respectively.

Conclusion

The Lateral Flow Assay Test is a useful and specific tool in rapid differentiation of M. tuberculosis complex from culture. Therefore proper identification avoids unnecessary ATT to patients infected with NTM.

Introduction

Non tuberculous mycobacterial (NTM) species have shown an increasing association with clinical infections.^{1, 2} These NTM species are resistant to first-line anti-TB drugs and when erroneously identified as Mycobacterium tuberculosis, give rise to a mistaken diagnosis of multidrug-resistant TB (MDR-TB) when treated with ATT. Therefore, it is important to differentiate M. tuberculosis complex from NTM as soon as possible in both pulmonary as well as extrapulmonary clinical specimens.

The identification of M. tuberculosis using biochemical methods is a complex, labor-intensive, and time-consuming process.³ Nucleic acid amplification (NAA) methods, such as real time PCR, AccuProbe Mycobacterium tuberculosis complex culture identification test (Gen Probe Inc., CA), the GenoType Mycobacterium CM test (Hain Lifescience, GmbH, Germany) are both rapid and specific but are technically challenging, and they require the use of sophisticated instruments. Antigen detection tests have not been widely used, although they are rapid, simple, and more affordable.

Amongst M. tuberculosis antigens, the most often studied have been LAM and MPT64.^{4, 5} Lateral flow assays, also called immunochromatographic assays, have been developed for the discrimination between M. tuberculosis complex and non tuberculous mycobacteria. These include the SD Bioline Ag MPT64 Rapid assay (Standard Diagnostics, Kyonggi-do, Korea), Capilia TB (TAUNS, Numazu, Japan), and the MGIT TBc Identification Test (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD). These sandwich-type assays use a monoclonal antibody to detect the MPB64 protein (Rv1980c; also termed as MPT64), which is specifically secreted during growth by the M. tuberculosis complex.^{4, 5} The MPB64 is a 24 kDa protein, highly specific for the M. tuberculosis complex and differentiates between MTBC and NTM. Rapid immunochromatographic tests (ICT) that detect the M. tuberculosis complex (MTBC) MPT64 protein are cheaper and simpler to use and thus may have an important role in resource poor settings and also early diagnosis.

Material and Methods

The study was carried out in a tertiary care teaching centre between January 2012 and May 2013. MGIT TBc identification test (TBc ID; Becton Dickinson, USA) was compared with Real time PCR targeted at 16S rRNA for identification of MTB complex after growth on liquid as well as solid media.

Assuming a sensitivity of 99% for the new test with 85% for the reference test and a power of 90% within confidence interval of 95% the estimated sample size required was 77. One hundred and thirty smear positive sputum specimens were processed by modified Petroff's method and then cultured on LJ medium and MGIT broth (Middlebrook 7H9). Of these, 106 specimens showed growth of AFB in both solid and liquid culture media and were included in the study. Two LJ cultures showed contamination and were not included in the study. All the 106 isolates were subjected to the ICT Lateral Flow Assay and real time PCR targeting 16S rRNA.

The TBcID assay (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) consists of a nitrocellulose membrane on a test device with immobilized anti-MPB64 mouse monoclonal antibodies conjugated with gold colloidal for the detection of the MPB64 protein. When samples are added to the test device, MPT64 antigen binds to anti-MPT64 antibodies conjugated to visualizing particles on the test strip. The antigen-conjugate complex migrates across the test strip to the reaction area and is captured by a second specific MPT64 antibody applied to the membrane. If the MPT64 antigen is present in the sample, a color reaction is produced by the labelled colloidal gold particles and is visualized as a pink to red line. The Lateral flow assay was done using 100 μl volume of liquid culture dropped directly onto the sample well. Colonies confirmed to be AFB positive from the LJ Medium were suspended in 100 μl of extraction buffer supplied with the kit before being placed in the sample well. The result was read after 20 min and appearance of a band in both control and test region was interpreted as a valid test positive for M. tuberculosis and the absence of a band in the test region was interpreted as indicative of NTM. (Fig. 1). All handling of cultures was done in a Class II Biosafety cabinet with standard safety precautions.

Fig. 1.

MPT64 ICT – Identification of Mtb complex A: Negative for Mtb B: Positive for Mtb complex.

Real time PCR for Identification of MTBC was done by using a LightMix Kit (Tibmolbiol, Germany) employing a primers set designed to amplify 16S rRNA gene specific to mycobacterial species and M. tuberculosis alongwith hybridization probes (Lachnik et al, 2002). DNA was extracted by the spin column method (Qiamp DNA Mini Kit, Qiagen, Germany). Following amplification, a melt curve analysis was carried out which differentiated MTBC and NTM based on the melting peaks generated (Fig. 2).

Fig. 2.

16S rRNA Real Time PCR: Panel A shows lack of Melting peak for NTM (Blue), Panel B shows peak for Mtb.

Results

Of the 106 isolates, 95 were identified as M. tuberculosis complex by MPT64 ICT while 96 were identified as M. tuberculosis complex by 16S rRNA PCR. Eleven isolates did not give a band in the MPT64 ICT while 10 isolates were identified as NTM by 16S rRNA PCR. Taking Real Time 16S rRNA PCR as the reference test, sensitivity, specificity, PPV and NPV of the ICT in both solid and liquid media was 99%, 100%, 100% and 91% respectively (Table 1).

Table 1.

Comparison of sensitivity, specificity, PPV and NPV of ICT in present study with other studies.

Author	Sensitivity (%)	Specificity (%)	PPV	NPV	Reference method used
Ismail et al Int J Tuberc Lung Dis 2009 (South Africa)	97	100	100	92	Accuprobe molecular probe assay
Brent et al JCM 2011 (Kenya)	97.6	100	100	98.4	Accuprobe molecular probe assay
Maurya et al Ind J Med Res 2011 (India)	99.1	100	–	–	IS 6110 PCR and biochemical reactions
Prasad & Mukhopadhyay JCDR 2014 (India)	100	100	–	–	Accuprobe molecular probe assay
Present study	99	100	100	91	Real time PCR melt curve analysis

Discussion

M. tuberculosis poses a diagnostic and therapeutic problem due to the long turnaround time of the diagnostic tools available for its identification and discrimination with NTM. Rapid identification of mycobacteria is important and a simple, sensitive, and specific identification method is required for proper treatment. Traditional biochemical tests take a long time in differentiation of MTBC from NTM. Newer techniques like chemiluminescent DNA probes, nucleic acid amplification, high-performance liquid chromatography and sequencing of 16S rRNA genes are more sophisticated methods but are not cost-effective and require expensive equipment.⁶

All members of the MTBC have identical 16S rRNA gene sequences. Therefore amplification of mycobacterial 16S rRNA gene can discriminate MTBC from NTM. Real Time PCR assay using 16S rRNA is a rapid and accurate method for detection of MTBC.⁷ In the present study, 16S rRNA Real Time PCR was taken as the reference method and the sensitivity, specificity, PPV and NPV of MPT64 ICT was found to be 99%, 100%, 100% and 91% respectively. These results were comparable to results of studies by Brent et al⁹ and Ismail et al,¹⁰ Maurya et al⁶ and Prasad et al⁸ (Table 1) who have used molecular methods as the reference method for statistical analysis. Studies have been published employing MPT64 ICT direct on clinical samples but have demonstrated low sensitivity.¹¹ Low sensitivity for detection of MTBC isolates by MPT64 ICT may be due to deletion or mutation of the MPB64 gene or to low MPT64 concentrations in early cultures or mixed cultures.⁹

ICT test can be applied directly to cultured organisms, and does not require any special equipment. It can discriminate between MTBC and NTM. The low cost, simplicity, rapidity, high sensitivity and high specificity for the MPT64 antigen detection make the ICT a useful diagnostic tool for differentiation between MTBC and NTM in laboratories having culture facilities but lacking a molecular diagnostic setup. The cutting down of the time for further identification would enable timely administration of appropriate therapy for M. tuberculosis or Non tuberculous Mycobacteria.

Conflicts of interest

All authors have none to declare.

Corrigendum

1. The correct designation and address of second author in the article ‘Gross and microscopic study of insertion of levator palpebrae superioris and its anatomical correlation in superior palpebral crease formation and its clinical relevance’ by Lt Col Subhendu Pandit and Lt Col Manish Singh Ahuja (Retd) published in Med J Armed Forces India 2015;71:330–336 is Col MS Ahuja, Commanding Officer, Military Hospital Dhrangdhara, Gujarat, India.



2. The correct title of the article, ‘Objective criteria for diagnosing high altitude pulmonary edema in acclimatized patients at altitudes between 2700m and 3500m’ by Col Anuj Chawla and Gp Capt KK Tripathi published in Med J Armed Forces India 2015; 71:345–351 is ‘Objective criteria for diagnosing high altitude pulmonary edema in low landers at altitudes between 2700m and 3500m‘.