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Keywords:

  • diagnosis;
  • loop-mediated isothermal amplification;
  • pleurisy;
  • real-time PCR;
  • tuberculosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Aims:  Tuberculous pleurisy is an important cause of pleural effusions in areas with a high incidence of tuberculosis. In this study, we developed an IS1081-based LAMP for the detection of Mycobacterium tuberculosis complex and investigated its usefulness in the diagnosis of tuberculous pleurisy.

Methods and Results:  Investigation of pleural effusion samples from patients with tuberculous pleurisy, majority of them smear-/culture-negative, and control individuals with non-TB diseases showed that the LAMP assay with incubation time of 60 min has much higher specificity and the LAMP assay with incubation time of 90 min has significantly higher sensitivity in the diagnosis of tuberculous pleurisy, as compared with fluorescent real-time PCR.

Conclusions:  The MTBC–LAMP is a useful assay for the diagnosis of tuberculous pleurisy, especially in pleural effusion smear-/culture-negative patients.

Significance and Impact of the Study:  Tuberculous pleural effusion usually contains low number of mycobacteria, which leads to low diagnostic sensitivity of acid-fast staining and mycobacterial culture methods. In this study, we developed a simple and sensitive LAMP assay for the diagnosis of tuberculous pleurisy. This assay should have broad application in resource-limited settings.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Tuberculosis (TB) is one of the most deadly infectious diseases. It is estimated that 9·27 million new cases of TB occurred in 2007, with 1·32 million deaths from TB in HIV-negative people and 456 000 deaths from TB in HIV-positive people (WHO 2009). Tuberculous pleurisy is an important cause of pleural effusions in the areas with a high incidence of TB (Udwadia and Sen 2010). The definitive diagnosis of tuberculous pleurisy is currently made by demonstrating the presence of tubercle bacilli or by histological examination of pleural tissue for granulomas (Gopi et al. 2007). Tuberculous pleural effusion usually contains very low number of mycobacteria, which leads to low diagnostic sensitivity of acid-fast staining and mycobacterial culture methods (Udwadia and Sen 2010). Collecting pleural tissue for histological examination is invasive and its use is restricted in resource-limited settings.

Several biological markers, such as adenosine deaminase (ADA) and IFN-γ, have been shown to be increased in tuberculous pleural effusion and are useful for the diagnosis of tuberculous pleurisy, although the specificity is suboptimal because these markers might increase in some other diseases (Dheda et al. 2009a,b; Porcel et al. 2010). Recently, Mycobacterium tuberculosis-specific IFN-γ release assay (IGRA) has been evaluated in the diagnosis of tuberculous pleurisy (Dheda et al. 2009a; Zhou et al. 2011). Although the reported results are variable, most studies indicate that pleural effusion mononuclear cell IGRA has poor accuracy in the diagnosis of tuberculous pleurisy because of high background IFN-γ level and inability to obtain enough pleural mononuclear cells in some patients (Dheda et al. 2009a; Zhou et al. 2011).

Specific nucleic acid amplification by real-time polymerase chain reaction (PCR) has relatively good sensitivity and specificity in the diagnosis of TB (Kim et al. 2011; Malbruny et al. 2011). However, real-time PCR requires expensive equipment, which limits its use in most developing countries where TB is most epidemic. Loop-mediated isothermal amplification (LAMP) is a gene amplification procedure in which the reaction can be processed at a constant temperature by one type of enzyme (Notomi et al. 2000), and its rapid and simple features make it a promising diagnostic method for point-of-care testing and for developing countries where resources are limited (Notomi et al. 2000; Tomita et al. 2008; Mori and Notomi 2009). In this study, we developed an IS1081-based LAMP for the detection of M. tuberculosis complex and investigated its usefulness in the diagnosis of tuberculous pleurisy.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Clinical specimens

Patients were identified through tracing specimens sent to Clinical TB Diagnostic Laboratory, Institute of Tuberculosis, or Department of Laboratory Medicine, 309 Hospital, Beijing, China, for laboratory diagnostic tests. In this study, pleural fluid specimens from 72 patients diagnosed as tuberculous pleurisy were investigated. The criterion for the diagnosis of tuberculous pleurisy was as following: (i) patients had clinical history of fever, pleuritic chest pain, cough and chest radiography for evidence of pleural effusion; (ii) culture of pleural fluid/pleural biopsy material or sputum yielded M. tuberculosis; or histology of pleural biopsy material showed granulomatous inflammation; (iii) there was obvious clinical improvement after anti-TB chemotherapy and there was no evidence of other causes of pleural disease in the case that mycobacteria were not identified in pleural fluid/pleural biopsy material or sputum. Among the 72 patients with pleural TB, one was pleural effusion culture-positive, 8 were sputum smear-/culture-positive. Pleural fluid specimens from patients with non-TB infection, cancer or other disease were used as controls. Sputum specimens from patients with pulmonary TB with positive acid-fast staining and from patients with non-TB pneumonia were also used in the study. All patients were HIV-negative. This study was evaluated and approved by the Ethics Committee of the 309 Hospital, Beijing, China.

Nucleic acids preparation

Mycobacterial reference strains M. tuberculosis (ATCC 27294), M. bovis (ATCC 19210), M. africanum (ATCC 25420), M. microti (ATCC 19422), M. avium (ATCC 25291), M. kansasii (ATCC 12478), M. intracellulare (ATCC 13950), M. ulcerans (ATCC 19423), M. abscessus (ATCC 23003), M. gordonae (ATCC14470), M. fortuitum (ATCC 6841), M. smegmatis (ATCC 19420), M. chelonae (ATCC 35752), M. gilvum (ATCC 43909), M. nonchromogenicum (ATCC 19530), M. phlei (ATCC 11758), M. terrae (ATCC 15755), M. scrofulaceum (ATCC 19981), M. xenopi (ATCC 19250), M. aurum (ATCC 23366), as well as Escherichia coli, Staphylococcus aureus and Acinetobacter baumannii (Table 2) were used in the study. The sputum specimens were treated with 4-volume 4% NaOH for 10 min and centrifuged at 12 400 g for 5 min to collect micro-organisms. Pleural fluid samples were centrifuged directly at 12 400 g for 5 min. The pellets were washed twice with sterile PBS and treated with 50 μl nucleic acid extraction buffer (Da An Gene Co. Ltd., Guangzhou, China) for 10 min at 100°C in heat block. After centrifugation at 12 400 g for 5 min, 1 μl of the extracted nucleic acids was used as templates for LAMP and fluorescent real-time PCR.

Table 2.   Specificity evaluation of the IS1081-based LAMP and fluorescent real-time PCR
Micro-organismsResults
LAMPReal-time PCR
Mycobacterium tuberculosis (ATCC 27294)++
M. bovis (ATCC 19210)++
M. africanum (ATCC 25420)++
M. microti (ATCC 19422)++
M. avium (ATCC 25291)
M. kansassi (ATCC 12478)
M. intracellulare (ATCC 13950)
M. ulcerans (ATCC 19423)
M. abscessus (ATCC 23003)
M. gordonae (ATCC14470)
M. fortuitum (ATCC 6841)
M. smegmatis (ATCC 19420)
M. chelonae (ATCC 35752)
M. gilvum (ATCC 43909)
M. nonchromogenicum (ATCC 19530)
M. phlei (ATCC 11758)
M. terrae (ATCC 15755)
M. scrofulaceum (ATCC 19981)
M. xenopi (ATCC 19250)
M. aurum (ATCC 23366)
Escherichia coli clinical isolate
Staphylococcus aureus clinical isolate
Acinetobacter baumannii clinical isolate

LAMP primer design

Using the online Primer Explorer V4 software (Eiken Chemical Co. Ltd., Tokyo, Japan) (http://primerexplorer.jp/elamp4.0.0/index.html), IS1081-specific LAMP primers were selected according to the general criteria described by Notomi et al. (Notomi et al. 2000). All the primers were purified by HPLC (Sangon Biotec, Shanghai, China) (Fig. 1 and Table 1).

image

Figure 1.  Location of LAMP primers used in the study. A right arrow indicates that a sense sequence is used for the primer. A left arrow indicates that a complementary sequence is used for the primer.

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Table 1.   Primer sets designed for LAMP and fluorescent real-time PCR
MethodsPrimer designationPrimer sequencesIS1081-Target positionAmplicon size (bp)
LAMPIS1081-F35′-TCTCATCTTATCGACGCCGA-3′135–154203
IS1081-B35′-CGGGTGTCGAAATCACGGT-3′337–319
IS1081-FIP (F1c + F2 primers)5′-GGCGATGAACGTCGAGAGCA227–208
GCAGCTTCTGGCTGACCAA-3′155–173
IS1081-BIP (B1c + B2 primers)5′-TTGATGGGGGCTGAAGCCGA230–249
TTGCGCTGATTGGACCG-3′306–290
Fluorescent real-time PCRIS1081-1F5′-CTGCGCGGGCTGCTCTC-3′197–213116
IS1081-1R5′-TAGCCGTTGCGCTGATTGG-3′313–295
IS1081 Taqman probe5′-CGCTCATCGCTGCGTTCGCGGT-3′292–271

LAMP reactions

The LAMP reactions were performed using Loopamp DNA amplification kit with addition of loopamp fluorescent detection reagent (both from Eiken Chemical, Tochigi, Japan). The reaction mixture (25 μl total volume) contained 1 μl of template, 5 μl of primer mixture (1·6 μmol l−1 for IS1081-FIP and IS1081-BIP primers, 0·2 μmol l−1 for IS1081-F3 and IS1081-B3 primers), 12·5 μl of 2 × reaction buffer, 1 μl of enzyme (Bst DNA polymerase) and 1 μl of fluorescent detection reagent. The reaction tubes were incubated at 65°C in a real-time turbidity meter (LA-320; Teramecs, Kyoto, Japan) for 60 or 90 min and terminated by heat inactivation of the enzyme at 90°C for 5 min. After the amplification reaction, results were read under visible light or UV light using handheld UV-lamp (wavelength: 365 nm). To confirm the amplification of target DNA, 0·5 μl of the reaction solution was added to 1·5% agarose gel and run at 140 V for 40 min in 1 × TAE buffer.

Real-time PCR

Fluorescent real-time PCR primers and Taqman probe were designed based on IS1081 sequence (Table 1). The probe was labelled with 6-carboxyfluorescein (FAM) and black hole quencher 1 (BHQ1). A M. tuberculosis fluorescent polymerase chain reaction diagnostic kit was also purchased from Da An Gene Co. Ltd. (Guangzhou, China). Real-time PCR was performed on an iQ5 PCR instrument (Bio-Rad, Hercules, CA, USA).

Preparation of nucleic acids for sensitivity determination of LAMP and real-time PCR

The concentration of extracted nucleic acids from M. tuberculosis strain H37Rv was calculated after measuring absorption at 260 nm using a DU600 UV spectrophotometer (Beckman Coulter, Brea, CA, USA) and was adjusted with sterile Milli-Q water to 1 ng μl−1. This template was used to make 10-fold serial dilutions in 10 mmol l−1 Tris–HCl (pH 8·8), ranging in concentration from 1 ng μl−1 to 10 fg μl−1. One microlitre of each dilution was used as template for LAMP reaction and fluorescent real-time PCR. To determine real analytic sensitivity of the LAMP reaction, the M. tuberculosis strain H37Rv was mixed with negative pleural fluid sample, and LAMP reaction and real-time PCR were performed with 10-fold serial dilutions of DNA templates.

Statistical analysis

The sensitivity, specificity, positive predict value (PPV) and negative predict value (NPV) were calculated by using spss program (ver. 13.0) (Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Development of LAMP for the detection of Mycobacterium tuberculosis complex

By using online LAMP primer designing software, four primers that recognize six distinct regions in M. tuberculosis IS1081 sequence were selected (Fig. 1 and Table 1). BLASTn searching suggested that these primers targeted M. tuberculosis complex only. Amplification of M. tuberculosis genomic DNA with the set of primers in the LAMP reaction at 65°C for 60 or 90 min was demonstrated by the appearance of colour change in the tubes under visual light and fluorescence under UV light, which were not observed in negative control without M. tuberculosis genomic DNA template (Fig. 2a). Agarose gel electrophoresis also confirmed the presence of amplified DNA in the tubes with M. tuberculosis genomic DNA and absence in the negative control tube (Fig. 2b). Furthermore, the pattern of amplified DNA by LAMP on agarose gel electrophoresis was similar to that reported previously (Iwamoto et al. 2003).

image

Figure 2.  Analysis of detection limit of IS1081-based LAMP and fluorescent real-time PCR. Serial 10-fold diluted genomic DNA extracted from M. tuberculosis H37Rv reference strain was used for testing. (a) Visual inspection of colour change under daylight and fluorescence under UV light; (b) Electrophoretic analysis of LAMP products; (c) The amplification curves of the IS1081-based fluorescent real-time PCR. NC is negative control and PC is positive control.

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To evaluate specificity of the newly developed LAMP, we tested 20 mycobacterial reference strains, as well as Escherichia coli, Staphylococcus aureus and Acinetobacter baumannii strains (Table 2). Amplification was observed in the tubes containing genomic DNAs from M. tuberculosis, M. bovis, M. africanum and M. microti at incubation time of 60 and 90 min (Table 2). No amplification was found with all other strains tested after 60 and 90 min of incubation. Same results were also observed by fluorescent real-time PCR with primers targeting the same insertion element sequence (Table 2). The results indicate that the IS1081-based LAMP assay specifically detects only M. tuberculosis complex.

Sensitivity of LAMP for the detection of Mycobacterium tuberculosis

To evaluate sensitivity of the MTBC–LAMP assay, we tested serial 10-fold diluted genomic DNA extracted from culture of M. tuberculosis H37Rv reference strain. Positive LAMP reactions were detected by visual inspection and electrophoretic analysis. The MTBC–LAMP assay was able to detect up to 1 pg of purified M. tuberculosis DNA with visual inspection (Fig. 2a) and 100 fg with electrophoretic analysis (Fig. 2b), and the detection limit of fluorescent real-time PCR using IS1081-specific primers described in Table 1 was 100 fg (Fig. 2c). When negative pleural fluid sample was added to M. tuberculosis strain, the real analytic sensitivity was reduced by 10-fold to 1 pg for both MTBC–LAMP assay and fluorescent real-time PCR. These results indicate that the MTBC–LAMP assay and fluorescent real-time PCR has similar sensitivity in the detection of M. tuberculosis.

To further determine the application and sensitivity of the MTBC–LAMP assay in detecting clinical samples, sputum specimens with positive acid-fast staining from 18 patients with pulmonary TB were tested. DNA was extracted from the sputum specimens and tested by the MTBC–LAMP. To control possible cross-contaminations, negative control was included for each clinical sample reaction. The MTBC–LAMP with 60-min incubation (MTBC–LAMP60) was positive in 13 of 18 specimens (72·2%), while MTBC–LAMP with 90-min incubation (MTBC–LAMP90) was positive for all sputum specimens tested. Two sputum specimens from patients with non-TB pneumonia were obtained as well, and there was no amplification by either MTBC-LAMP60 or MTBC–LAMP90. These results indicate that LAMP90 has good sensitivity in detecting clinical sputum specimens from patients with TB.

Comparison of LAMP and real-time PCR for the diagnosis of tuberculous pleurisy

To evaluate the usefulness of the LAMP assay in the diagnosis of tuberculous pleurisy, pleural fluid specimens from 96 patients were obtained, 72 from patients with tuberculous pleurisy, 10 from patients with non-TB pleural infection, 13 from patients with cancer and one from patient with undetermined aetiology. Among the pleural fluid specimens tested, MTBC–LAMP60 was positive in 18/72 (25%) specimens from patients with tuberculous pleurisy, majority of them smear-/culture-negative, while no positive reaction was observed in specimens from patients with non-TB infection, cancer or other disease (Table 3). The calculated sensitivity, specificity, PPV and NPV were 25%, 100%, 100% and 30·8% respectively (Table 3).

Table 3.   Analysis of 96 pleural fluid specimens by MTBC–LAMP and fluorescent real-time PCR
TestsResultsClinical diagnosisSensitivity (%)Specificity (%)PPV (%)NPV (%)
Patients with tuberculous pleurisy (n = 72)Patients with non-TB diseases (n = 24)
  1. PPV, positive predict value; NPV, negative predict value; MTBC–LAMP60, LAMP with incubation time of 60 min; MTBC–LAMP90, LAMP with incubation time of 90 min.

MTBC-LAMP60Positive18025·010010030·8
Negative5424
MTBC-LAMP90Positive35448·683·389·735·1
Negative3720
Real-time PCRPositive20327·887·587·028·8
Negative5221

The positive rate of MTBC–LAMP90 for pleural fluid specimens from patients with tuberculous pleurisy was 48·6%, which is significantly higher than MTBC–LAMP60 (Table 3). However, 4 patients with non-TB diseases were also positive, and the specificity decreased to 83·3% (Table 3).

The pleural fluid specimens were also tested simultaneously with the fluorescent real-time PCR. Among the pleural fluid specimens tested from patients with pleural TB, 20 were positive (27·8%) (Table 3). Three specimens from non-TB patient group also showed positive in the real-time PCR amplification (Table 3).

Taken together, the results demonstrated that MTBC–LAMP60 has much higher specificity with slightly lower sensitivity, while MTBC–LAMP90 has significantly higher sensitivity with slightly lower specificity in the diagnosis of pleural TB as compared with fluorescent real-time PCR.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The first report on mycobacteria-specific LAMP used primers targeting the gyrB gene of certain mycobacterial strains, and amplification was visualized by adding SYBR green to the tubes (Iwamoto et al. 2003). This LAMP assay had positive reactions in 16 of 18 sputum specimens in which M. tuberculosis complex strains were isolated (Iwamoto et al. 2003). Since then, several studies have established LAMP for the diagnosis of TB (Enosawa et al. 2003; Boehme et al. 2007; Pandey et al. 2008; Zhu et al. 2009; Geojith et al. 2011; Nagdev et al. 2011; Neonakis et al. 2011). Boehme et al. performed a relatively large evaluation on the gyrB gene-based LAMP for the diagnosis of pulmonary TB (Boehme et al. 2007). The sensitivity in smear-positive sputum specimens was 97·7% and in smear-negative/culture-positive specimens 48·8%, and the specificity was 99% (Boehme et al. 2007). Pandey et al. developed a LAMP that targeting 16S rRNA gene of M. tuberculosis and it had a detection limit of 100 fg for purified M. tuberculosis DNA by measuring turbidity at 590 nm (Pandey et al. 2008). The sensitivity of the LAMP for culture-positive sputum specimens was 100% and the specificity was 94·2% (Pandey et al. 2008). Nagdev et al. compared LAMP and nested PCR, both targeting M. tuberculosis IS6110 genomic sequence, for the diagnosis of tuberculous meningitis and found a sensitivity and specificity of 88·23% and 80% by LAMP, 52·9% and 90% for nested PCR, respectively (Nagdev et al. 2011). To our knowledge, there is no report on evaluating LAMP for the diagnosis of tuberculous pleurisy so far.

The IS1801-based MTBC–LAMP developed in this study had similar sensitivity in detecting purified mycobacterial DNA as reported by others (Iwamoto et al. 2003; Pandey et al. 2008). One of the most attractive characteristics of LAMP is the visual judgement of nucleic acid amplification; however, detecting a small amount of the white precipitate by the naked eye is not always easy. To facilitate easy recognition by the naked eye, we added fluorescent detection reagent to the reaction solution and there is slightly improvement in the detection limit. To achieve best specificity, 60 min of incubation in the LAMP assay is recommended.

The reason to choose IS1081 as amplification target in the study is that this insertion element is carried by all M. tuberculosis complex strains investigated, with stable 5–6 copies (Collins and Stephens 1991; van Soolingen et al. 1992, 1993; Dziadek et al. 2001). IS6110 has high copy numbers in most M. tuberculosis strains and is used as target by many nucleic acid amplification assays; however, strains with low copies or lacking IS6110 completely have been identified (van Soolingen et al. 1993; Flores et al. 2010). Recently, an IS6110-related element in a strain of M. smegmatis was identified and it has 67% amino acid identity and 79% similarity (Coros et al. 2008). This IS1801-based MTBC–LAMP assay could be used with sputum samples for the diagnosis of pulmonary TB as well.

In conclusion, an IS1801-based LAMP assay for the detection of M. tuberculosis complex was developed in this study. This MTBC–LAMP provides a useful tool for the diagnosis of tuberculous pleurisy, especially in patients with acid-fast staining-negative and mycobacterial culture-negative pleural effusions. Because LAMP reaction and detection need only conventional equipments, such as water bath/heat block and UV light, this technique should have broad applications in resource-limited settings.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The study was supported by a grant for infectious diseases from Ministry of Health, China, to X.C. (2008ZX10003-012) and a grant from Beijing Natural Science Foundation, China (7092100).

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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