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 ~  Abstract
 ~ Introduction
 ~ Methods
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 34  |  Issue : 3  |  Page : 322-327
 

Loop-mediated isothermal amplification assay for detection of Mycobacterium tuberculosis complex in infertile women


1 Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Obstetrics and Gynaecology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Cytology and Gynaecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Date of Submission12-Mar-2015
Date of Acceptance31-Mar-2016
Date of Web Publication12-Aug-2016

Correspondence Address:
S Sethi
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.188323

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 ~ Abstract 

Background: Female genital tuberculosis (FGTB) has a profound impact on the reproductive health of patients including infertility. Conventional diagnostic techniques have low sensitivity and specificity as well as long turnaround time. There is a need of developing newer, rapid and practically adaptable technique, especially in low-income countries. Objective: To standardize and evaluate loop-mediated isothermal amplification (LAMP) technique for diagnosis of FGTB. Methods: A total of 300 endometrial biopsy samples from infertile females were subjected to Ziehl-Neelsen (ZN) staining, Lowenstein-Jensen culture, automated culture (BACTEC mycobacterial growth indicator tube), histopathological examination (HPE), nucleic acid amplification by polymerase chain reaction (PCR) and LAMP technique. Composite gold standard (either smear/culture/HPE/PCR positive) was considered for calculation of outcome parameters. Results: The observed sensitivities of ZN smear, culture, HPE, PCR and LAMP were 2.94%, 10.29%, 8.82%, 95.59% and 66.18%, respectively. Overall concordance between PCR and LAMP was 63%, which shows a good agreement. Conclusion: This study is the first to evaluate LAMP in the diagnosis of FGTB and found it to be a rapid and convenient technique, especially in low resource endemic settings.


Keywords: Diagnosis, female genital tuberculosis, India, infertility, loop-mediated isothermal amplification


How to cite this article:
Sethi S, Dhaliwal L, Dey P, Kaur H, Yadav R, Sethi S. Loop-mediated isothermal amplification assay for detection of Mycobacterium tuberculosis complex in infertile women. Indian J Med Microbiol 2016;34:322-7

How to cite this URL:
Sethi S, Dhaliwal L, Dey P, Kaur H, Yadav R, Sethi S. Loop-mediated isothermal amplification assay for detection of Mycobacterium tuberculosis complex in infertile women. Indian J Med Microbiol [serial online] 2016 [cited 2019 Dec 9];34:322-7. Available from: http://www.ijmm.org/text.asp?2016/34/3/322/188323



 ~ Introduction Top


Female genital tuberculosis (FGTB) represents 15-20% of the extrapulmonary TB cases and generally affects patients of 20-40 years of age. [1] It has a profound impact on the reproductive health of these patients including infertility. The incidence of FGTB is high in India and accounts for 1-19% of cases of total infertile cases diagnosed each year. [2],[3] The diagnosis of FGTB is challenging due to its paucibacillary nature as well as the lack of any concrete laboratory investigation technique. Laparoscopy may miss the early changes of tubercular pathology whereas histopathological examination (HPE) can be confirmatory only when it demonstrates acid-fast bacilli (AFB) in the tissue which is very scarce in the case of the endometrial biopsy specimen. Certain tests such as Mantoux test, erythrocytic sedimentation rate (ESR) and serum adenosine deaminase levels have also shown poor sensitivity in this regard. Similarly, microbiological techniques including Ziehl-Neelsen (ZN) stain, Lowenstein-Jensen (LJ) culture and BACTEC Mycobacterial Growth Indicator Tube (MGIT) culture are associated with the disadvantages of low sensitivity and long turnaround time which prevents the early diagnosis of the disease when irreparable damage to the fallopian tubes and subsequent infertility can be avoided.

Recently, nucleic acid amplification techniques (NAAT) such as polymerase chain reaction (PCR) targeting various genes such as IS6110, mpt64 and ESAT-6 have been reported to be more sensitive, specific and less time consuming. [1],[3],[4] However, these methods are accompanied by limitations of the requirement of expensive equipment and difficulty to perform in remote and peripheral settings. [5] A novel NAAT, loop-mediated isothermal amplification (LAMP) has been found to be a useful alternative to overcome the above-mentioned problems. It is a simple technique in which template DNA is amplified under isothermal conditions by enzyme Bst DNA polymerase which has high strand displacement activity. This technique uses four to six sets of primers that recognize six to eight regions of target DNA. It has a wide range of applicability in the detection of a variety of microorganisms. LAMP targeting gyrB, 16S rRNA (rrs), rim and IS6110 has been reported in diagnosis of pulmonary TB. [6],[7],[8],[9],[10],[11],[12],[13],[14],[15] However, very few studies regarding its applicability in the diagnosis of extrapulmonary TB are available in literature. [16],[17],[18] To the best of our knowledge, LAMP has not been standardised for the diagnosis of FGTB from endometrial biopsy samples. Therefore, this study was planned to evaluate and standardise LAMP for diagnosis of FGTB and to compare the technique with conventional and rapid diagnostic methods.


 ~ Methods Top


Study population and sample collection

A total of 300 women aged between 20 and 40 years with complaints of infertility presenting to the infertility clinic were included in the study. World Health Organization defines infertility as failure to conceive despite over 12 months of regular and unprotected intercourse. Other known causes of infertility such as anovulation, hypothyroidism, hyperprolactinaemia, infection and previous surgery on fallopian tube, behaviour factors and male causes were ruled out in the study group. Written informed consent was obtained from each patient and the study was approved by the Institute Ethics Committee. Endometrial biopsy was taken aseptically in late premenstrual phase or on day 1 of the menstrual cycle under paracervical block or sedation in the minor operation theatre of gynaecology outpatient department. The adequate sample was collected in a sterile container in normal saline for processing in Department of Medical Microbiology and in formalin for HPE in Department of Histopathology.

Histopathological examination

Any features of tuberculoma/granulomas and endometritis with inflammatory cell infiltration were considered suggestive of TB.

Sample processing in microbiology laboratory

The samples were digested-decontaminated and concentrated by adding N-acetyl L-cysteine-sodium hydroxide. The sediment was resuspended in 1-3 ml of phosphate buffer which was further used for making ZN smear and inoculating LJ and BACTEC MGIT 960 culture using the standard methods. LJ culture was observed every day for the 1 st week and then weekly up to 8 weeks for the presence of any growth suggestive of Mycobacterium tuberculosis BACTEC MGIT cultures were looked for any positive signals upto 6 weeks. A volume of 1 ml of sample was used for DNA extraction for PCR and LAMP.

DNA extraction

Cetyl trimethyl ammonium bromide and chloroform-isoamyl alcohol method of DNA extraction described by van Embden et al. [19] with minor modifications was followed to extract DNA from endometrial biopsy samples.

Polymerase chain reaction

DNA amplification of 123 bp IS6110 (insertion sequence) of M. tuberculosis complex was carried out by method described by Thangappah et al. [3] The master mix (25 μl) was prepared by adding PCR buffer ×10, dNTP mix (10 mM) (Thermo Fisher Scientific, Waltham, Massachusetts, United States), primers in equimolar concentrations (10 pM/μl) (Sigma-Aldrich, St. Louis, Missouri, United States), Taq polymerase (2U/μl) (Bangalore Genei Private Limited, Bengaluru, Karnataka, India), DNA template and PCR grade water. DNA amplification was carried out as follows: initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 68°C for 45 s and extension at 72°C for 45 s and then a final extension at 72°C for 10 min. The positive and negative control consisted of M. tuberculosis H37Rv strain and PCR grade water, respectively. The amplicons were run on 1.5% agarose gel stained with ethidium bromide and examined under ultraviolet (UV) light to look for bands of 123 bp using MW marker of 100 bp ladder.

Loop-mediated isothermal amplification assay

The LAMP reaction was designed using six primers (inner, outer and loop primers) targeting M. tuberculosis IS6110 gene as previously described by Aryan et al. [12] The reaction was carried out in a volume of 25 μl consisting of thermopol buffer (×10), dNTP mix (Thermo Fisher Scientific, Waltham, Massachusetts, United States) and each of FIP, BIP (20 pmol), each of FLP, BLP (20 pmol), each of FOP, BOP (5 pmol) (Sigma-Aldrich, St. Louis, Missouri, United States), Betaine (0.8 M), Bst polymerase large fragment (8 U) (New England Biolabs, Ipswich, Massachusetts, United States), DNA template and PCR grade water.

The optimum temperature and duration for LAMP assay were found out to be 64°C for 60 min. M. tuberculosis H37Rv and PCR grade water were used as positive and negative control respectively. The amplicons were run on 1.5% agarose gel stained with ethidium bromide. It was examined under UV light to look for multiple DNA bands appearing as ladder-like pattern.

Statistical analysis

The results were analysed using Chi-square test and P value was evaluated at the <0.05 level of significance. Sensitivity, specificity, positive and negative predictive value were calculated using a composite gold standard, i.e., either of acid-fast smear/culture/histopathological examination or PCR positive sample. To see the agreement between PCR and LAMP, kappa test of agreement was applied using SPSS software (Statistical Package for Social Sciences, SPSS Inc., Chicago, IL, USA, version 13.0 for Windows).


 ~ Results Top


Clinical profile of study subjects

About 75.67% of the 300 females, belonged to age group of 21-30 years. A total of 214 (71.33%) patients presented with primary infertility whereas 86 (28.67%) had secondary infertility. The common symptoms included menstrual disturbances (21.66%), pelvic pain (9%), fever (3.3%) and weight loss (2.3%). Among menstrual complaints, oligomenorrhoea was the chief presentation in 77% of patients. ESR was raised (>20 mm/h) in 27% patients while Mantoux test was positive in 16% patients. 36 (12%) patients had history of TB (pulmonary, gastrointestinal or lymph node) and intake of ATT whereas 11 (3.7%) patients had a history of ectopic pregnancy. Contact history of TB was present in 12 (4%) patients.

Diagnostic test results

As for the diagnosis of FGTB, there is no absolute gold standard test available. Therefore, a composite gold standard was considered for calculations. In this study, total cases (n = 300) were divided into two groups, in which samples which were showing positive results by composite gold standard (ZN smear or LJ culture or BACTEC culture or HPE or PCR) were included under Group - I (n = 68) and samples which were showing negative results by composite gold standard were included under Group - II (n = 232). Of total 300 endometrial biopsy samples, 2 (0.7%) were positive by ZN smear, 4 (1.3%) were positive by LJ culture, whereas BACTEC could detect M. tuberculosis in 7 cases (2.3%), PCR using IS6110 showed positive results in 65 cases (21.7%), whereas LAMP using IS6110 primers was positive in 62 cases (20.7%). Six (2%) samples were positive by HPE. Sensitivity, specificity, positive and negative predictive value of all diagnostic tests are presented in [Table 1]. All smear positive and LJ positive samples were also BACTEC culture positive. All samples which were ZN smear and LJ culture positive were also PCR and LAMP positive. Five of 7 BACTEC positive samples were both PCR and LAMP positive whereas rest 2 were negative. Of the six HPE positive samples, 5 were culture, PCR and LAMP positive while 1 was negative by these tests. The correlation of LAMP with other diagnostic techniques is given in [Table 2]. The results of both PCR and LAMP are shown in [Figure 1] and [Figure 2].
Figure 1: Agarose gel showing results of IS6110 polymerase chain reaction. Lane 1-100 bp DNA ladder; Lane 2 o template control; Lane 3 ositive control: 123 bp product; Lane 4, 5 ositive result of clinical samples; Lane 6, 7 egative result of clinical sample

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Figure 2: Agarose gel showing results of IS6110 loop-mediated isothermal amplification. Lane 1-100 bp DNA ladder; Lane 2 o template control; Lane 3 ositive control showing loop-mediated isothermal amplification products; Lane 4-7 ositive clinical sample showing ladder like pattern; Lane 8, 9 egative results of clinical samples

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Table 1: Sensitivity, specificity, positive predictive value and negative predictive value of various diagnostic tests applied in the present study for diagnosis of female genital tuberculosis


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Table 2: Correlation of loop - mediated isothermal amplification with other diagnostic techniques


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The concordance between PCR and LAMP is considered good as shown in [Table 3] (Kappa 0.63).
Table 3: Concordance between IS6110 and loop-mediated isothermal amplification


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 ~ Discussion Top


Genital TB generally affects women of reproductive age group, most of which are asymptomatic. The majority of the patients in this study were in the age group of 21-30 years. Epidemiological profile of infertility patients in previous studies has also shown maximum cases of infertility in the age group of 25-35 years. [20],[21],[22] The most common feature of the presentation was menstrual irregularities similar to previous studies. [3] The primary infertility of 71.33% of patients reported in our study lies in the similar range (72-73%) of previous studies. [20],[21]

The gold standard method for diagnosis of TB is demonstration or culture isolation of the causative agent. Due to paucibacillary nature, AFB staining has very low sensitivity (10 4 organism/ml). Similarly, the sensitivity of culture is also low (10 2 organism/ml) in addition to its long turnaround time of 6-8 weeks. [21],[23] Laparoscopy has been taken as gold standard in certain studies, but these procedures are associated with surgical risks, may result in the spread of infection and overlook the initial changes of disease in uterus. It is also not feasible to perform these procedures on every patient in practice. [20] Therefore, it is very difficult to take a single test as the gold standard in the diagnosis of FGTB. [3] To overcome this problem, many previous studies have taken combined gold standard. [21],[24],[25] Similarly, in this study, a composite gold standard was considered which included any case positive by smear or culture or HPE or PCR which accounted for 68 patients.

The AFB smear showed a low sensitivity of 2.94% in our study which is similar to previously reported a range of 0.1-2% in FGTB due to sparse and uneven distribution of bacilli in sample tissue. [21],[26],[27] LJ culture and BACTEC MGIT culture were also associated with lower sensitivity of 10.29% which is also explained by sparsely distributed AFB in the tissue sampled. Culture takes a long time of 6-7 weeks for positive results which cannot be afforded in infertile cases as fallopian tubes may get permanently damaged. Previous studies have also shown lower sensitivity in both LJ (1-7%) and BACTEC culture (3-13%). [20],[23],[24],[26],[27] HPE of the tissue is only suggestive and not confirmatory unless AFB are demonstrated in the lesion. Many nontubercular lesions including actinomycosis and sarcoidosis may mimic TB. In this study, HPE showed a better sensitivity of 8.82% as compared to previous studies reporting sensitivity range of 0.6-5%. [20],[21],[26],[28] The variation of HPE sensitivity may be attributed to the irregular shedding of granulomas during menstrual cycles.

PCR has shown to be reasonably good alternative in the diagnosis of FGTB in previous studies using different primers. It demonstrated a good sensitivity of 95.59% in this study which is quite higher than already available literature (13-59%). [3],[24],[27],[29] The PCR positivity is hindered by the presence of blood and human genome. PCR was unable to pick up two BACTEC positive samples and one HPE positive sample which may be due to inappropriate specimen or presence of inhibitors. The major drawback of PCR is its requirement for thermal cycler which is expensive and difficult to perform in peripheral settings.

LAMP technique has been developed to overcome the drawbacks of PCR. This technique amplifies DNA under isothermal conditions like a simple water bath. It has shown very good sensitivity of 85-100% and specificity of >95% in sputum samples from pulmonary TB cases. [7],[8],[10],[13] It has also been standardised for TB meningitis, ocular TB and TB lymphadenitis. [16],[17],[18] Balne et al. have reported the use of LAMP targeting mpb64 primers in the diagnosis of intraocular TB with a sensitivity and specificity of 85.7% and 100%. In another study by Nagdev et al., LAMP targeting IS6110 primers was used to diagnose TB meningitis with a sensitivity of 88.23%, and specificity of 80%. Titipungul et al. carried out the technique using 16S rRNA in Formalin-fixed, paraffin-embedded samples of TB lymphadenitis cases and showed a sensitivity of 30.4% and specificity of 100%. This study therefore is the first to standardise LAMP targeting IS6110 for diagnosis of FGTB although with a lower sensitivity of 66.18%. It has shown a higher sensitivity as compared to conventional techniques. The results of LAMP and PCR have shown good concordance, and thus, LAMP can act as an alternative to expensive PCR techniques in resource-limited endemic settings.


 ~ Conclusion Top


This study is the first in literature for evaluating and standardizing LAMP for FGTB diagnosis. LAMP is a rapid and sensitive economical method which can be used in low-income countries as an alternative to PCR and obviously better than conventional methods.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

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  [Table 1], [Table 2], [Table 3]



 

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