Optimization of a Loop-Mediated Isothermal Amplification Assay as a Point-of-Care Tool for the Detection of Wuchereria bancrofti in Human Blood in Tana River Delta, Kenya

Abstract

Introduction: Accurate detection of filarial parasites in humans and vectors is essential for the implementation and evaluation of Global and National Programs to eliminate lymphatic filariasis. Immunological methods to detect infection are available; however, cross-reactivity issues have been reported in most of them. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity and can be used to detect the infections.

Methods: In this study, we evaluated loop-mediated isothermal amplification (LAMP) tests to amplify Wuchereria bancrofti DNA in patients' blood. The amplicons were tested by both pH-sensitive dyes for enhanced visual detection and agarose gel electrophoresis. A closed-tube LAMP assay was also evaluated. Cohen's Kappa statistics was used for statistical analysis of the assays. 125 patients consented for blood sampling which were used for clinical analysis of LAMP assays with the PCR method used as the "gold standard."

Results: The sensitivity of the evaluated Wuchereria bancrofti LAMP was 92.3%, with a specificity of 97.3% and kappa statistics value of 0.84, which is in a strong agreement.

Conclusion: In this study, LAMP assays coupled with fluorescence dye detection have been found to be suitable for diagnosis and monitoring of Wuchereria bancrofti infections in the Kenyan population.

Figure 1

Map of Tana River Delta in Tana River County, Kenya. Source: map generated by Jacob Mueti.

Figure 2

LAMP florescence dye detection (open tubes). Tube no. 1: W. bancrofti DNA-positive control; 2: negative control; 3, 4, 5, and 6: positive specimens; 7 and 8: negative specimens. Green color indicates that Wuchereria bancrofti DNA was present and was amplified (positive). Orange color means that there was no W. bancrofti DNA and hence no amplification that took place (negative).

Figure 3

LAMP amplification results on agarose gel. Lane 1: positive control of W. bancrofti DNA; lane 2: negative control; lanes 3, 4, 5, and 6: positive specimens; and lanes 7 and 8: negative results. Lane ML is the molecular weight marker of 100 bp.

Figure 4

(a) LAMP reaction mix before amplification. (b) LAMP mix reaction after amplification. (a) All reaction mix in tubes 1–5 was blue in color before incubation. On incubating the reaction mix at 63°C for 60 minutes, the results were as shown in (b) on the addition of 1 : 10 SYBR Green dye and viewed under a UV Light or under sunlight. Tubes 1 and 2 remained blue in color indicating the absence of Wuchereria bancrofti DNA (negative specimen), tube 3 turned green in color indicating positive W. bancrofti DNA amplification (positive specimen), tube 4 contained W. bancrofti DNA-positive control which turned green in color, and tube 5 was a negative control which remained blue in color.

Figure 5

LAMP sensitivity testing on gel electrophoresis. Lane NC represents negative control; lane PC represents positive control; lanes 10⁻¹–10⁻⁶ represented serial dilutions of DNA extracts for testing the assay sensitivity. 10⁻¹–10⁻⁶ showed amplification of W. bancrofti as bands appeared at different lengths indicating that our LAMP assay was sensitive and could detect to up to 1/1000000 (1/10⁶) DNA copies of the parasites in the diluent, while no band was observed in 1/10000000 (1/10⁷) copies in the diluent.

Figure 6

LAMP specificity testing on agarose gel electrophoresis. Lane ML is a molecular weight marker 100 bp; lane 1: W. bancrofti DNA-positive specimen; lanes 2–7: non-W. bancrofti DNA of other parasites which did not show any amplification; lane 2: Brugia Malayi; lane 3: Anopheles gambiae; lane 4: Schistosoma mansion; lane 5: Plasmodium falciparum; lane 6: Trichuris trachura; lane 7: Echinococcosis granulosus; lane 8: negative control; lane 9: W. bancrofti DNA-positive control.

Figure 7

PCR sensitivity testing on gel electrophoresis. Lane ML: molecular marker 100 bp; lane PC: positive control, W. bancrofti DNA; lanes 10⁻¹–10⁻⁷: serial dilutions of DNA extracts for testing the assay sensitivity; lane 10⁻⁸: no amplification; lane NC: represents negative control; 10⁻¹–10⁻⁷ showed amplification of W. bancrofti as bands appeared just below the 200 bp as the expected band size was 188 bp. This showed that PCR assay was sensitive and could detect to up to 1/1000000 (1/10⁷) copies of parasite DNA in the diluent, while no band was observed in 1/10000000 (1/10⁸) copies in the diluent.