|Year : 2018 | Volume
| Issue : 1 | Page : 46-52
Comparative study of LoopampTM Mycobacterium tuberculosis Complex Kit for Rapid Detection of Mycobacterium tuberculosis complex in cameroon
Valerie Flore Donkeng Donfack1, Laure Ngando2, Eric Walter Yone Pefura3, Dieudonné Shubesi Che1, Ghislaine Ateba3, Jean Joel Rim Bigna4, Jean Louis Abena Foe5, Christopher Kuaban6, Sara Eyangoh1
1 Tuberculosis National Reference Laboratory, Centre Pasteur of Cameroon, International Pasteur Institutions Network, Yaoundé, Cameroon
2 Faculty of Medicine and Biomedical Sciences, University of Yaoundé, Yaoundé, Cameroon
3 Jamot Hospital, Tuberculosis Diagnostic and Treatment Center, Yaoundé, Cameroon
4 Epidemiology Unit, Centre Pasteur of Cameroon, Yaoundé, Cameroon
5 National Tuberculosis Program, Yaoundé, Cameroon
6 Faculty of Health Sciences, University of Bamenda, Bamenda, Cameroon
|Date of Web Publication||5-Mar-2018|
Dr. Valerie Flore Donkeng Donfack
Tuberculosis National Reference Laboratory, Centre Pasteur of Cameroon, International Pasteur Institutions Network, P.O. 1274 Yaoundé
Source of Support: None, Conflict of Interest: None
Background: The most practical test for identifying tuberculosis (TB) in developing countries remains smear microscopy. However, due to its low sensitivity, a new point-of-care diagnostic method has been developed. The purpose of this study was to assess the performance of TB-Loop-mediated isothermal amplification (TB-LAMP) test on sputum samples of suspected TB cases. Methods: Suspected pulmonary TB patients (527) from Jamot Hospital and without any history of anti-TB treatment were consecutively included in the study. Smear microscopy, TB-LAMP, GeneXpert® MTB/RIF, and liquid culture using BACTEC 960 Mycobacteria Growth Indicator Tube (MGIT) were performed on sputum samples collected from these patients. The sensitivity and specificity of TB-LAMP were compared with smear microscopy and GeneXpert® MTB/RIF. MGIT culture was the gold standard. Results: TB-LAMP and smear microscopy showed sensitivities of 82.6% (95% confidence interval [CI], 76.9–87.2) and 53.6% (95% CI, 46.8–60.3), respectively, and specificities of 96.0% (95% CI, 93.2–97.7) and 99.0% (95% CI, 97.1–99.7), respectively. The sensitivity and specificity of TB-LAMP were similar to GeneXpert®, (89.9%; 95% CI, 85.0–93.3 and 97.0%; 95% CI, 94.4–98.4). Conclusion: TB-LAMP is more sensitive than currently used microscopy. It presents a favorable diagnostic tool for TB in peripheral laboratories with limited equipment, such as those in developing countries.
Keywords: Tuberculosis, tuberculosis- Loop-mediated isothermal amplification, smear microscopy, GeneXpert® MTB/RIF, Mycobacteria Growth Indicator Tube culture
|How to cite this article:|
Donfack VF, Ngando L, Pefura EW, Che DS, Ateba G, Bigna JJ, Abena Foe JL, Kuaban C, Eyangoh S. Comparative study of LoopampTM Mycobacterium tuberculosis Complex Kit for Rapid Detection of Mycobacterium tuberculosis complex in cameroon. Biomed Biotechnol Res J 2018;2:46-52
|How to cite this URL:|
Donfack VF, Ngando L, Pefura EW, Che DS, Ateba G, Bigna JJ, Abena Foe JL, Kuaban C, Eyangoh S. Comparative study of LoopampTM Mycobacterium tuberculosis Complex Kit for Rapid Detection of Mycobacterium tuberculosis complex in cameroon. Biomed Biotechnol Res J [serial online] 2018 [cited 2020 Mar 29];2:46-52. Available from: http://www.bmbtrj.org/text.asp?2018/2/1/46/226580
| Introduction|| |
One of the main problems in global tuberculosis (TB) control is the lack of highly sensitive and specific point-of-care (POC) diagnostic tests. TB is a major global health problem, responsible for ill health among millions of people each year. In 2013, an estimated 9.0 million people developed TB and 1.5 million died from the disease, 360,000 of whom were HIV-positive.
Impediments in diagnosis are considered as the prime obstacle to tackling the TB epidemic. Acid-fast bacteria (AFB) smear microscopy is commonly used as a primary diagnostic tool for the early detection of TB, especially in resource-limited countries with higher burden of TB., However, one weakness is its low sensitivity. AFB smear microscopy is even less sensitive when the suspected patient is HIV-positive or carrying a very small bacterial load. Culture methods are the gold standard for Mycobacterium TB complex (MTBC) diagnosis. However, culture is time-consuming, expensive due to biosafety requirements, and labor intensive due to the slow growth rate of MTBC. The implementation of advanced molecular-based methods like polymerase chain reaction can improve the efficiency and shorten the time of diagnosis. Accordingly, many molecular methods have been developed. In 2010, the WHO approved GeneXpert® MTB/RIF, an automated bench-top device which can simultaneously detect TB and rifampicin resistance in 2 h. It is relatively easy to use and can be used in primary healthcare settings.,, Despite its advantage, the sophisticated nature of the device and its maintenance are still challenging.
TB-Loop-mediated isothermal amplification (TB-LAMP) is a technology developed by Eiken Chemical Company in Japan. This technology has been recommended by the WHO in 2016, as a POC test for TB diagnosis applicable in resource-limited settings. This method is dependent on auto-cycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment which has high-strand displacement activity and is carried out isothermally at 67°C for 1 h. In the present study, the objective was to compare the performance of the commercial kit, Loopamp ™ MTBC Kit, on sputum samples of suspected TB cases, using auramine O smear microscopy, BACTEC 960 Mycobacteria Growth Indicator Tube (MGIT) culture, and GeneXpert® MTB/RIF.
| Methods|| |
The ethical clearance for this study was obtained from the Cameroon National Ethics Committee. All the participants signed the informed consent sheet before their inclusion for the study.
This cross-sectional study was carried out at Jamot Hospital between April and June 2014. This center has a purely microscopy laboratory. TB-LAMP was performed in a simple room without biosafety cabinets or other special equipment. Three demarcated benches served as working areas for sample processing, reaction mix preparation, amplification, and detection.
Patient inclusion and sample collection
Patients aged 15 years and above suspected to have pulmonary TB and without any previous anti-TB treatment was included in this study after they signed the informed consent. The information on age, sex, HIV serostatus, and the history of TB of the included patients was sought for and recorded. The included patients provided two consecutive early morning sputum samples as recommended in the Reference Center of Pneumopathy.
One collected sputum sample was used for routine auramine O smear microscopy and TB-LAMP at Jamot Hospital. The remaining sputum of the sample was temporarily stored at 4°C until shipment at 4°C–8°C to the National Reference Mycobacteriology Laboratory of Centre Pasteur of Cameroon (NRL-CPC) every evening to be tested with GeneXpert® MTB/RIF and MGIT culture. All remaining sputum samples were then stored at −20°C at NRL-CPC.
Auramine O smear microscopy
This semiquantitative method was performed as recommended by the WHO. The smear was flooded with auramine O stain, decolorized by 1% v/v acid-alcohol then counter-stained with methylene blue. After drying, the smears were examined under fluorescence microscope (20x). The result of smear was interpreted as positive if there was the presence of yellow fluorescent bacilli, and negative if there was the absence of fluorescent bacilli.
Tuberculosis-Loop-mediated isothermal amplification
TB-LAMP was carried out as described by the manufacturer (Eiken). A proprietary kit provided by Eiken Chemical made up of a DNA extraction component and a LAMP reaction tube. About 60 μl of untreated sputum from the same mug as for smear microscopy was transferred to a heating tube containing sodium hydroxide (NaOH) extraction solution. The mixture was properly mixed by repeated inversion and heated at 90°C for 5 min on the manufacturer's heating block to lyse and inactivate mycobacteria. After inactivation, the heating tube was cooled at room temperature for 2 min and the DNA purified using a procedure called PURE (Procedure for Ultra Rapid Extraction, Eiken Chemical) in what was a closed system. Approximately 30 ul of the extracted DNA was transferred to the LAMP reaction tube by firmly squeezing the body of the adsorbent tube. The reaction tube contained dried LAMP reaction mix and hence was given a proper mix by performing five repeated inversions. The reaction mix in the tube was made up of primers specific for MTBC, dNTPs, Bst DNA polymerase, and buffer. The LAMP reaction tube was then placed in the Eiken automated block for amplification at 67°C for 40 mins. The detection of positive results was performed using TB-LAMP that exploits a dye molecule, Calcein which fluoresces under Ultraviolet (UV) light. Under UV light, strong fluorescence was released from the positive reaction and no fluorescence was released from the negative reaction. Finally, the results were recorded and the reaction tubes discarded. A positive and negative control was included for each test batch of 14 samples or for each run.
GeneXpert® MTB/RIF assay
The GeneXpert® MTB/RIF assay was performed according to manufacturer's instruction and as previously described by others.,,, Briefly, untreated patient sample and sample reagent were added at a proportion of 1:2 in falcon tubes and left for 15 mins at RTP while agitating intermittently. Following inactivation, 2 ml of the specimen mixture was transferred to the cartridge (proprietary) containing all the reaction mixture and loaded to the GeneXpert ®. The results were interpreted as recommended by the manufacturer.
The culture was performed using the automated system BACTEC MGIT 960 Mycobacteria detection system as recommended by the manufacturer, using NALC-NaOH solution for decontamination. Growth was detected automatically by BACTEC MGIT 960 system. All MGIT positive tubes were tested for contamination using blood agar and confirmed for MTBC by Ziehl-Neelsen smear microscopy and the MPT64 antigen strip assay.
Data were entered using the Statistical Package for Social Science (SPSS) version 21.0 for Windows (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. IBM Corp., Armonk, NY, USA) and analyzed using Windows Program for Epidemiologists version 11.25. Categorical variables were expressed as numbers (N) with percentages (%). Continuous variables were expressed as means with standard deviation (SD). The agreement was measured using the Cohen Kappa statistic. Sensitivity, specificity  as well as positive and negative predictive values were calculated. All diagnosis elements were reported with their 95% confidence intervals (95% CI). The 95% CI for predictive values was calculated with the Fleiss-Levin-Paik method. The 95% CI for Kappa was calculated with the goodness-of-fit method.
| Results|| |
In this study, 527 consecutive suspected pulmonary TB patients without any previous anti-TB treatment were included over a period of 2 months. From these patients, nine did not bring their sputum samples and consequently were excluded from the study. Of the 518 specimen considered for the bacteriological study, nine showed contaminated cultures and were omitted from analysis. Finally, only 509 samples were considered for analysis. The mean age of the patients was 40.7 (SD 15.7) years, which represent the economically productive population age. Females were slightly less represented 245 (48.1%) than males 264 (51.9%). One hundred and fifty-three (30.05%) patients were HIV negative, 108 (21.21%) were HIV positive, and 248 (48.72%) were HIV unknown status. The performance of TB-LAMP for detection of MTBC was assessed using 509 sputum samples. In parallel, the performance of other identification methods (Auramine O smear microscopy, GeneXpert® MTB/RIF, and MGIT culture) was compared with TB-LAMP using the same set of samples. During the study, no contamination occurred on our negative control for TB-LAMP, meaning that the experimentation was carried out in the strict respect of good laboratory practice.
[Table 1] shows raw data of the study comparing the different methods used (Auramine O smear microscopy, TB-LAMP, and GeneXpert® MTB/RIF) to MGIT culture. A specimen was considered as MTBC negative if no growth was detected in MGIT. A specimen was considered as MTBC positive if growth was detected in MGIT and confirmed by Ziehl-Neelsen smear microscopy and MPT64 antigen strip assay. Of the 509 samples tested, 207 (40.7%) were culture positive and 302 (59.3%) were culture negative. One hundred and eighty-three (36.0%) samples were TB-LAMP positive and 326 (64.1%) were TB-LAMP negative. One hundred and fourteen (23.4%) samples were smeared positive and 395 (77.6%) were negative. One hundred and ninety-five (38.3%) samples were GeneXpert® MTB/RIF positive and 314 (61.7%) were GeneXpert® MTB/RIF negative.
|Table 1: Performance of TB-LAMP, Auramine O smear microscopy and GeneXpert® MTB/RIF compared to MGIT culture as gold standard|
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The performances of TB-LAMP, auramine O smear microscopy, and GeneXpert® MTB/RIF as compared to MGIT culture are also given in [Table 1]. The overall sensitivity of TB-LAMP, auramine O smear microscopy, and GeneXpert® MTB/RIF in the 207 culture positive specimens was 82.6%, (95% CI, 76.9–87.2); 53.6% (95% CI, 46.8–60.3); and 89.9% (95% CI, 85.0–93.3), respectively. Their overall specificity in the 302 culture negative specimens was 96.0% (95% CI, 93.2–97.7); 99.0% (95% CI, 97.1–99.7); and 97.0% (95% CI, 94.4–98.4), respectively.
[Table 2] shows the comparison of TB-LAMP results with auramine O smear microscopy and MGIT culture. The sensitivity of TB-LAMP in the 114 smear positive specimens was 60.1%, (95% CI, 52.9–66.9) and its specificity in the 395 smear negative specimens was 98.8% (95% CI, 96.9–99.5). More ever, TB-LAMP displayed the sensitivity of 85.1% (95% CI, 75.3–91.5) in 99 smear and culture positive samples, and the specificity of 88.9% (95% CI, 85.0–91.8) in 299 smear and culture negative samples. For smear negative and culture positive samples, TB-LAMP presented higher sensitivity of 98.4% (95% CI, 91.5–99.7).
|Table 2: Comparison of TB--LAMP, Auramine O smear microscopy and MGIT Culture|
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In [Table 3], the comparison of TB-LAMP results with GeneXpert® MTB/RIF and culture is shown. TB-LAMP displayed the sensitivity of 95.6% (95% CI, 91.6–97.8) in 195 GeneXpert MTB/RIF positive specimens and the specificity of 93.9% (95% CI, 90.7–96.0) in 404 GeneXpert® MTB/RIF negative specimens. TB-LAMP showed higher sensitivity of 96.1% (95% CI, 92.1–98.1) in culture and GeneXpert® MTB/RIF positive specimens and higher specificity of 94.7% (95% CI, 91.6–96.7) in 301 culture and GeneXpert® MTB/RIF negative specimens. However, its sensitivity and specificity were very low (16.7%) in culture negative and GeneXpert® MTB/RIF positive specimens as well as culture positive and GeneXpert® MTB/RIF negative specimens. In addition, TB-LAMP also demonstrated higher sensitivity of 93.9% (90% CI, 88.0–97.0) in 110 culture, GeneXpert® MTB/RIF, and smear-positive specimens, and higher specificity of 99.3% (90% CI, 97.5–99.8) in 291 culture, GeneXpert® MTB/RIF, and smear-negative specimens.
|Table 3: Comparison of TB--LAMP, Gene Xpert MTB/RIF, Auramine O smear and Culture|
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The performances of TB-LAMP, auramine O smear microscopy, and GeneXpert® MTB/RIF on HIV positive and culture positive patients are shown in [Table 4]. Of the 47 HIV-positive and culture positive patients, GeneXpert® MTB/RIF detected more positive cases (42) follow by TB-LAMP (36) and auramine O smear microscopy (22).
|Table 4: Performance of TB--LAMP, Auramine O Smear microscopy and GeneXpert® MTB/RIF on HIV+ and Culture+ patients|
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| Discussion|| |
We conducted a cross-sectional study to assess the performance of TB-LAMP and compared with auramine O smear microscopy and GeneXpert® MTB/RIF in the diagnosis of pulmonary TB using MGIT culture as gold standard. The GeneXpert® MTB/RIF method had the highest sensitivity (89,9%), followed by TB-LAMP (82,6%), and smear microscopy (53,6%). The smear microscopy test exhibited the highest specificity (99%), followed by GeneXpert® MTB/RIF (97%), and TB-LAMP (96%). The main finding of this study is that the sensitivity of TB-LAMP was similar to that of GeneXpert® MTB/RIF , but higher than that of auramine O smear microscopy. All three tests demonstrated similar specificity. A concordance of 0.88 was obtained between the two molecular test (TB-LAMP and Gene Xpert MTB/RIF) using the κ statistic.
In this study, the sensitivity of TB-LAMP was similar to that reported from some previous studies.,,,,, However, the studies conducted by Bojang et al., and N'guessan et al. displayed higher sensitivity of TB-LAMP (99% and 92%, respectively) compared to our finding (82.6%)., Regarding specificity of TB-LAMP, previous studies shown similar specificity to our finding.,,,,,,, Concerning GeneXpert® MTB/RIF assay, the sensitivity (89.9%) and specificity (97.0%) were similar to the one obtained in a meta-analysis, which showed the pooled sensitivity and specificity of 89% and 98.0%, respectively.
TB-LAMP and GeneXpert® MTB/RIF also demonstrated higher sensitivity compared to smear microscopy. These two molecular techniques improve the diagnostic of TB compared to microscopy. In the smear-negative specimens, TB-LAMP was able to detect 73 positive samples that were missed by smear microscopy. This can perhaps be ascribed to the fact that the limit of detection of TB-LAMP and GeneXpert® MTB/RIF is lower than microscopy. Direct sputum smear microscopy is naturally insensitive when the number of acid-fast bacilli (AFB) in the sputum is <105 organisms/ml. This impacts particularly on the diagnosis of TB in children and the diagnosis of HIV-associated TB, which are often paucibacillary. The lower sensitivity observed for smear microscopy may be also related to reader bias. In addition, some discordant results between the TB-LAMP, GeneXpert® MTB/RIF, and auramine O smear microscopy or culture can happen in the presence of nontuberculous mycobacteria. TB-LAMP and GeneXpert® MTB/RIF detect the presence or absence of the genetic material of MTBC. While smear microscopy detects the morphology of mycobacteria and culture methods make a distinction based on the physiology of the viable organism.
Twelve samples were positive with TB-LAMP but negative with culture. In fact, the NaOH use to pretreat samples during the culture procedure could stress the MTBC growth or may kill up to 60% of tubercle bacilli in clinical specimens and may give a false negative result of culture, especially in cases of paucibacillary disease as seen in early disease, or in many HIV-positive patients.
In the smear negative and culture positive specimens, TB-LAMP was able to detect 62 positive samples that were missed by smear microscopy. In addition, TB-LAMP presented higher sensitivity (98.8%) in smear-negative samples. This means that TB-LAMP is useful as a confirmatory test for all patients suspected of pulmonary TB with negative microscopy. Furthermore, TB-LAMP could really replace microscopy for pulmonary TB diagnostic in remote areas to increase the number of TB patients diagnosed as recommended by the WHO.
TB-LAMP also showed higher sensitivity (95.6%) and specificity (93.9%) in GeneXpert® MTB/RIF positive and negative specimens, respectively. Therefore, despite the fact that TB-LAMP is unable to detect drug-resistant patterns, this latter might replace GeneXpert® MTB/RIF in low-income peripheral area where multidrug-resistant TB is not prevalent.
In this study, not all the included patients knew their HIV status, 248 (48.72%) were HIV unknown status. Accordingly, we could not present the sensitivity and specificity of the TB-LAMP in HIV ± cases. However, TB-LAMP improves the diagnostic of TB in HIV + patients compare to microscopy.
TB-LAMP is a manual assay that requires <1 h to perform and the result can be read with the naked eye under UV light. The technicians found it easier to use without molecular training and more rapid, and the results are easier to evaluate. In 2016, the WHO recommends TB-LAMP as a replacement for microscopy for the diagnosis of pulmonary TB in adults with signs and symptoms of TB. It can also be considered as a follow-on test to microscopy in adults with signs and symptoms of pulmonary TB, especially when further testing of sputum smear-negative specimens is necessary.
| Conclusion|| |
In this study, TB-LAMP was performed and compared to auramine O smear microscopy and GeneXpert® MTB/RIF for TB diagnostic using MGIT culture as gold standard. TB-LAMP demonstrated to have a great potential to be used for clinical diagnosis of TB in peripheral laboratories with limited equipment, such as those in developing countries. Hence, TB-LAMP is a promising diagnostic tool for TB in smear-negative sample. In addition, it would help the National TB program in the early diagnosis and treatment of patients and in a long run probably contributes to eliminating TB as part of the STOP TB vision.
We are grateful to the Foundation for Innovative New Diagnostic (FIND) for its financial support and Eiken Chemical Company for the material support. We also thank all TB-NRL staff for their technical contribution to this work. Moreover, finally, the patients and staff of Jamot Hospital.
Financial support and sponsorship
FIND for the financial support and Eiken Chemical Company for the material support.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]