A Comprehensive Evaluation of a Loop-Mediated Isothermal Amplification Assay for the Diagnosis of Pulmonary Tuberculosis in Children Using Bronchoalveolar Lavage Fluid

Figures & data

Figure 1 Selection of the study population.

Abbreviations: BALF, bronchoalveolar lavage fluid; S-M, smear-microscopy; TB-LAMP, Loop-mediated Isothermal Amplification Assay; CCRS, the composite clinical reference standard.

Table 1 Clinical Characteristics of the Study Population (n=243)

Variable	No. (%)
Gender
- Male	108 (44.4)
- Female	135 (55.6)
Age (years old)
- ≤14	121 (49.8)
<5	8 (3.3)
5–9	27 (11.1)
10–14	86 (35.4)
- 15–18	122 (50.2)
Patient status
- In patients	235 (96.7)
- Outpatients	8 (3.3)
BCG vaccination	243 (100.0)
TB treatment history
-Previous TB treatment	8 (3.3)
-New TB treatment	235 (96.7)
Clinical diagnosis
- Pulmonary tuberculosis	133 (54.7)
- pneumonia	97 (39.9)
- NTM	3 (1.2)
- Lung cancer	1 (0.4)
- others	9 (3.7)

Abbreviations: BCG, Bacillus Calmette–Guérin; TB, tuberculosis; NTM, nontuberculous mycobacterium lung disease.

Table 2 Comparison of BALF and Expectorated Sputum for Detecting Mycobacterium tuberculosis in Pediatric Patients Using the Composite Clinical Reference Standard (CCRS) as a Reference

Number (%) of Childhood Pulmonary TB Patients (N = 34^a) Correctly Diagnosed Using
Diagnostic assay	BALF	Sputum
Smear-microscopy	10 (29.4)	3 (8.8)
Culture	13 (38.2)	5 (14.7)
TB-LAMP	24 (70.6)	15 (44.1)

Note: ^a34 out of the 65 patients recruited were diagnosed with pulmonary TB.

Abbreviations: BALF, bronchoalveolar lavage fluid; TB, tuberculosis.

Table 3 Performance of Smear-Microscopy, TB-LAMP and Their Combination for Detecting M. tuberculosis Using Culture as a Reference

Diagnostic Assay	Performance Parameters [Mean Percentage (N/N), 95% CI]
	Sensitivity	Specificity	PPV	NPV
Single tests
Smear-microscopy	40.0% (16/40) (25.3–56.6%)	96.1% (195/203) (92.1–98.2%)	66.7% (16/24) (44.7–83.6%)	89.0% (195/219) (83.9–92.7%)
TB-LAMP	77.5% (31/40) (61.1–88.6%)	81.8% (166/203) (75.6–86.7%)	45.6% (31/68) (33.6–58.1%)	94.9% (166/175) (90.2–97.5%)
Two tests in parallel^a	82.5% (33/40) (66.6–92.1%)	81.3% (165/203) (75.1–86.3%)	46.5% (33/71) (34.7–58.6%)	95.9% (165/172) (91.5–98.2%)

Notes: ^aResults of smear-microscopy and TB-LAMP were combined. Negative results of both assays were considered as a “negative” and any positive was taken as a “positive” for the combined assays.

Abbreviations: TB-LAMP, Loop-mediated Isothermal Amplification Assay; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value.

Figure 2 Receiver operating characteristic (ROC) curves for determining the sensitivity and specificity of smear-microscopy, TB-LAMP and the two tests in parallel using culture as a reference.

Abbreviation: TB-LAMP, Loop-mediated Isothermal Amplification Assay.

Table 4 Performance of TB-LAMP, Culture and Smear-Microscopy as Single Tests and Combined Tests for the Diagnosis of Pulmonary TB Using the Composite Clinical Reference Standard (CCRS) as a Reference Standard

Diagnostic Assay	Performance Parameters [Mean Percentage (N/N), 95% CI]
	Sensitivity	Specificity	PPV	NPV
Single tests
Smear-microscopy (SM)	16.5%(22/133) (10.9–24.2%)	98.2%(108/110) (92.9–99.7%)	91.7%(22/24) (71.5–98.5%)	49.3%(108/219) (42.5–56.1%)
Culture	30.1%(40/133) (22.6–38.7%)	100.0%(110/110) (95.8–100.0%)	100.0%(40/40) (89.1–100.0%)	54.2%(110/203) (47.1–61.1%)
TB-LAMP	51.1%(68/133) (42.0–60.5%)	99.1%(109/110) (94.3–100.0%)	98.6%(68/69) (90.2–100.0%)	62.6%(109/174) (57.2–72.2%)
Simultaneous combination^a
SM+TB-LAMP tests in parallel	53.4%(71/133) (44.6–62.0%)	99.1%(109/110) (94.3–100.0%)	98.6%(71/72) (91.5–99.9%)	63.7%(109/171) (56.0–70.8%)
Culture+TB-LAMP tests in parallel	57.9%(77/133) (49.0–66.3%)	99.1%(109/110) (94.3–100.0%)	98.7%(77/78) (92.1–99.9%)	66.1%(109/165) (58.2–73.1%)
SM+Culture+TB-LAMP tests in parallel	58.6%(78/133) (49.8–67.0%)	99.1%(109/110) (94.3–100.0%)	98.7%(78/79) (92.2–99.9%)	66.5%(109/164) (58.6–73.5%)
Sequential combination^b
SM (−) +TB-LAMP	43.2%(48/111) (34.0–53.0%)	99.1%(109/110) (94.3–100.0%)	98.0%(48/49) (87.8–99.9%)	63.4%(109/172) (55.7–70.5%)
Culture (−) +TB-LAMP	39.8%(37/93) (29.9–50.5%)	99.1%(109/110) (94.3–100.0%)	97.4%(37/38) (84.6–99.9%)	66.1%(109/165) (58.2–73.1%)

Notes: ^aSimultaneous combination: Results of SM and TB-LAMP were combined, Culture and TB-LAMP were combined, SM, Culture and TB-LAMP were combined. Both results being negative was considered as a “negative” and any positive result was taken as a “positive”; ^bsequential combination: TB-LAMP performed for pulmonary TB patients with negative (−) SM or Culture results.

Abbreviations: TB-LAMP, Loop-mediated Isothermal Amplification Assay; CI, Confidence interval; PPV, Positive predictive value; NPV, Negative predictive value.

Figure 3 Venn diagram showing results of different diagnostic tests on bronchoalveolar lavage fluid samples from children with suspected pulmonary tuberculosis (N = 243).

Abbreviation: TB-LAMP, Loop-mediated Isothermal Amplification Assay.

Figure 4 Receiver operating characteristic (ROC) curves for determining the sensitivity and specificity of smear-microscopy, TB-LAMP and the two tests in parallel using CCRS as a reference.

Abbreviations: TB-LAMP, Loop-mediated Isothermal Amplification Assay; CCRS, the composite clinical reference standard.

Figure 5 Receiver operating characteristic (ROC) curves for determining the sensitivity and specificity of culture, TB-LAMP and the two tests in parallel using CCRS as a reference.

Abbreviations: TB-LAMP, Loop-mediated Isothermal Amplification Assay; CCRS, the composite clinical reference standard.