Validation ofa Loop-Mediated Isothermal Amplification (LAMP) Testfor Detection of Pulmonary TB in Kyeemyindaing and South Dagon Townships, Yangon, Myanmar

Saw Thein, Yin Myat Thwe, Kyaw Htet, Mikiko Araki, Kosuke Okada,Tetsuhiro Sugamoto, Norio Yamada & Zaw Myint

Special Issue May 2020

Early diagnosis of infectious TB cases is the key to accelerating a decline in incidence within the context of the End TB strategy. Loop-mediated isothermal amplification (LAMP) method is considered suitable in resource-limited settings. The study objective is to validate a LAMP test for diagnosing pulmonary TB in Myanmar. TB-LAMP study was conducted in two townships in Yangon in parallel with the routine diagnostic procedures starting with smear microscopy, followed by chest radiography (CXR) if those results were negative, and finallyXpertMTB/RIF assay (Xpert) was performed. The study compared the results of TB-LAMP with the results of TB diagnosis based on routine procedures. During Aug 2018- Jan 2019, 1,290 presumptive TB cases were enrolled in the study. TB-LAMP could detect 1.4 times more bacteriologically confirmed (BC) TB cases than smear microscopy did; among presumptive TB, 16.6% were smear-positive and 23.1% were LAMP-positive. Unexpectedly, 0.3% were LAMP-negative, but smear-positive. LAMP positive, smear negative among the presumptive TB were 6.7%. Xpert test was performed in 416 confirmed TB cases and a concordance rate between Xpert and LAMP was 91.3%. Out of 874 cases who were not registered as TB, 6.1% cases were LAMP-positive, meaning that 60.9% smear-negative cases were LAMP-positive. The proper initiation of LAMP test in high-burden countries like Myanmar will be anticipated to increase 1.4 times more BC TB case than smear microscopy.

Table 1. Comparison between TB-LAMP result and smear microscopy result

LAMP=Loop-mediated isothermal amplification

Of the 1,290 presumptive TB, 215(16.6%) were smear-positive and 1075(83.4%) were smear-negative while TB-LAMP was positive in 298(23.1%) presumptive TB and negative in 992(76.9%) presumptive TB. TB-LAMP showed 1.4 times higher positivity rate than smear microscopy. TB-LAMP tested positive in 87(8.0%) of smear-negative presumptive TB and in 211(98.1%) of the 215 smear-positive TB. The positivity rates by TB-LAMP were increased by smear grade; with 100% of TB cases with scanty smear, 96.8% of those with grade 1+ smear, 97% of those with grade 2+ smear, and 99% of those with grade 3+ smear (Table 1).

Table 2. Comparison between TB-LAMP assay and Xpert MTB/RIF assay

Concordance rate-91.3%

LAMP=Loop-mediated isothermal amplification

Out of the 416 diagnosed TB by routine procedures, 406 cases were compared between TB-LAMP and Xpert. TB-LAMP was positive in 230(90.2%) out of 255 Xpert-positive cases and 10 (6.6%) out of 151 Xpert-negative cases. There were 25 Xpert-positive/ LAMP-negative TB cases, and 10 Xpert-negative/ LAMP-positive TB cases. The concordance rate between TB-LAMP and Xpert was 91.3% (Table 2).

Table 3. Treatment category and the result of TB-LAMP

LAMP=Loop-mediated isothermal amplification

Out of 4 smear-positive LAMP-negative cases, 3 were Xpert positive. Out of 87 smear-negative LAMP-positive cases, only 30 cases can be tested by Xpert and 22 of them were Xpert positive. Out of the 416 TB cases in the study, 227 were BC TB, and 187 were CD TB. TB-LAMP was positive in 214(94.3%) of the 227 BC TB, and 31(16.4%) of 189 CD TB. Fifty-three (6.1%) out of 874 presumptive TB who were not registered as TB patients were LAMP positive (Table 3).

Tuberculosis (TB) is still a major global health problem as one-fourth of the global population are infected with M. tuberculosis and an estimated 1.6 million people including TB co-infected with HIV are killed by TB every year. Myanmar is ranked one of the 30 high-burden countries with an estimated incidence of 358 TB cases per 100,000 population and a mortality rate of 47 per 100,000 population in 2017.¹ The prevalence survey (2017-2018), Myanmar² showed that TB is more prevalent in urban areas than in rural areas in regions. Early diagnosis and proper treatment of TB is a key to prevent additional infection in the community.³ However, fluorescent smear microscopy is not sensitive in detecting bacteriologically positive TB, although it is used most commonly in resource-limited settings, resulting in limited case detection and the great number of false negatives.⁴

Liquid culture is more sensitive; however, its turnaround time is approximately up to 42 days and it requires specialized laboratory personnel.⁵ Although a large number of molecular methods including polymerase chain reaction (PCR) have been developed recently,⁶their sensitivities are greatly dependent on the efficiency in sample preparation, DNA extraction and the presence of PCR inhibitors, and the requirement of highly sophisticated laboratory settings lowers cost-effectiveness in resource-limited settings.⁷

A novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies the targeted DNA with high selectivity, efficiently and rapidly under iso-thermal condition was developed by Eiken Chemical Co. in Japan.⁸ LAMP has various attractive features that makeit suitablefor useinthe resource-limited settings.LAMP reaction does not require a denatured DNA template and relies on auto-cycling strand displacement DNA synthesis by a DNA polymerase.⁹

The huge amount of DNA generated in less than one hour and the fluorescent result could be interpreted with the naked eyes, without the need for gel electrophoresis.¹⁰The diagnostic accuracy of TB-LAMP was investigated in many studies. TB-LAMP can successfully identify and differentiate M. tuberculosis from another mycobacterium species with high specificity, simplicity, and superiority in the avoidance of aerosol contamination.¹¹

In addition, TB-LAMP has been highlighted as a candidate replacement for smear microscopy because of its simplicity, and higher sensitivity.¹² This study aimed at detecting the yield of bacteriologically confirmed (BC) TB compared to smear microscopy and accessing its effectiveness as an alternative test for smear microscopy at the township level.

In brief, 60µl of sputum from a specimen container was transferred to the heating tube containing the extraction solution, mixed thoroughly and was placed in the heating block of 90°C for 5 minutes to lyse and inactivate mycobacteria, then cooled down for 2 minutes. The heating tube was attached to the adsorbent tube and mixed by shaking until all the powder had been completely mixed with the solution.

An injection cap was placed onto the adsorbent tube, the nozzle was then inserted into a reaction tube and drops of solution were transferred to the reaction tube. For the amplification step, the reaction tube was loaded into the heating block and the reaction was started and then, it will be stopped automatically after40 minutes. The final result could be interpreted under ultraviolet light visually. If one or both smear (on-spot and early morning specimens) tests showed positive, the case was defined as a smear-positive pulmonary TB (BC TB).

If both sputum specimens were negative, chest radiography (CXR) was carried out to clinically diagnose TB by township medical officers. After diagnosing active TB with smear microscopy or CXR, Xpert MTB/RIF (Xpert) was tested to detect rifampicin resistance. If M. tuberculosis detected by Xpert from a clinically-diagnosed TB (CD TB) with negative smear and CXR suggestive of active TB, the case was changed from CD TB to BC TB.

Statistical analysis

Data analysis was done by using SPSS software version 20.2. The performance between TB-LAMP and Xpert was compared using a concordance rate (a proportion of cases with matching classification) of results.

Ethical consideration

The study proposal was approved by the Ethical Review Committee, Department of Medical Research and the approved ERC number was 2018-101.

In recent years, TB-LAMP, a novel method of nucleic acid amplification has been developed and its effectiveness has been demonstrated in several studies. One study in Northern India evaluated that the sensitivity and specificity of TB-LAMP assay in culture-positive samples were 100% and 99.2%, respectively while that of Xpert were 82.6% and 94.9% promising the diagnostic value of TB-LAMP superior to smear method.¹³ Feasibility and operational performance of tuberculosis detection by TB-LAMP was conducted in the Uganda, Peru, and India to those patients with symptoms suggestive of TB and revealed the sensitivities of 97.2% and 62.0% for smear-positive and smear-negative TB patients whereas the specificity of ranging from 94.5% to 98.0%.¹⁴ The WHO has recommended the TB-LAMP assay as a replacement test or follow-on test for sputum smear microscopy pulmonary TB diagnosis.¹⁵

This study was the pilot study which was conducted for the first time in Myanmar, and could obviously show that the performance of TB-LAMP was superior to smear microscopy, yielding 87 smear-negative/ LAMP-positive cases of which 30 cases were tested for Xpert and 22 (73%) were Xpert-positive. Of the 406 TB who were examined for both TB-LAMP and Xpert, the concordance rate was 91.3% which was similar to the findings in Malawi and Northern India (92.1% and 93.2%).^13,16

The TB-LAMP was positive in 31 (16.4%) of 189 CD TB which would have been changed to BC TB. Fifty-three cases with positive LAMP were not registered as TB patients, pointing out the fact that early case detection of TB was not accomplished and additional 6.1% BC TB were under-diagnosed through the current diagnostic procedures. These 53 LAMP-positive cases were still followed-up and accessed their clinical status.

TB-LAMP has some unique features as TB diagnostics; it does not have a computerized system or any mechanically operated parts, and it just controls the temperature of machine to performPCR amplification.
It can conduct 16 tests including a positive and a negative control at one time within one hour. Therefore, for example, it can be used for active case finding which needs many sputum examinations for bacteriological diagnosis with higher sensitivity than smear microscopy in the field where Xpert is challenging in being implemented.

There are some limitations in this study; it could not be able to evaluate the sensitivity and specificity because there was no reference test such as culture examination. Another limitation is that because of the insufficient human resources in South Dagon, the same laboratory technician performed both fluorescent microscopy and Xpert. Therefore, anonymity was not ensured because the same technician performed both smear microscopy and Xpert due to lack of additional staff. Thirdly, it is technical competency of the laboratory staff although 3-day training was conducted for TB-LAMP before its introduction in the two townships.

In conclusion, the proper initiation of LAMP test to high-burden countries like Myanmar will be anticipated to increase 1.4 times more BC TB case than smear microscopy. Some 6.1% of presumptive TB cases who were missed by clinical diagnosis with CXR were identified as BC TB cases by TB-LAMP. However, further researchis required to examine the sensitivity and specificity of TB-LAMP by comparing culture examination as the reference test.

The authors acknowledge Dr. Aung Thu (National Professional Officer) and Dr. Ikushi Onozaki (Medical Officer) of WHO, Myanmar for their technical supports of the study design and protocol development. The study was funded by Japan Anti-TB Association (JATA) and the performing smear microscopy, CXR, and Xpert was supported by National TB Program, Myanmar.

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