DEVELOPMENT AND EVALUATION OF A LOOP-MEDIATED ISOTHERMAL

AMPLIFICATION ASSAY FOR RAPID DETECTION OF LEISHMANIA INFANTUM

Introduction Leishmaniasis constitute a group of parasitic diseases caused by protozoan parasites of the genus Leishmania. We have developed a

species-specific L. infantum LAMP assay for the diagnosis of clinical canine leishmaniasis using the cysteine protease B (cpb) multi-

copy gene as target. This technique was compared to serological, PCR and parasitological diagnostics.

Materials and Methods

- The LAMP primer sets were designed using Primer Explorer Ver.4.

- The L. infantum specific LAMP reaction was standardized for optimal temperature and time.

- Seventy-five DNAs extracted from Blood of suspicious Leishmania infected dogs from different zones in and around Tunis.

- The dogs were examined for occurrences of visible clinical signs of the disease.

- The samples were tested by : Microscopy (Gold standard), Serology (IFAT), PCR and LAMP.

- Statistical Analysis.

Results

1. Sensitivity and Specificity of the L. infantum specific LAMP

A set of oligonucleotide primers targeting L. infantum cpb genes

sequences were designed for LAMP reaction. Serially diluted

samples were assessed for the presence of the L. infantum strain

(MHOM/TN/80/IPT1) promastigote DNA used as reference. LAMP

was able to detect up to 50 fg of DNA against up to 100 pg

detected by conventional cpb PCR (Chaouch et al., 2013).

To evaluate the specificity of the LAMP reaction, DNA samples from

other Leishmania and Trypanosoma species (L. major, L. tropica,

L. turanica, L. gerbilii, L. tarentolea and T.cruzi) were examined. No

amplification product was detected even when using up to 50 ng

of DNA showing that the LAMP cpb was specific for L. infantum

detection (data not shown).

Conclusion

MELEK CHAOUCH1, MOEZ MHADHBI2, GERARD J SCHOONE3, SASSI LIMAM2, EMILY R ADAMS3, MOHAMED-AZIZ

DARGHOUTH2, IKRAM GUIZANI1, SOUHA BEN ABDERRAZAK1

Acknowledgments : This study received financial support from the International Atomic Energy Agency (IAEA Contract

n°15137 - Contract n°15111) and from Ministère de l’Enseignement et de la Recherche Scientifique, Tunisia

LR00SP04).

1 INSTITUT PASTEUR DE TUNIS, TUNIS – TUNISIA

2 ECOLE NATIONALE DE MÉDECINE VÉTÉRINAIRE, SIDI-THABET– TUNISIA

3 ROYAL TROPICAL INSTITUTE, AMSTERDAM - NETHERLANDS

2. Hydroxy Naphthol Blue Staining

Fig. 3 : Flow-diagram of the results recorded by the diagnostics techniques applied to clinical samples.

A simpler colorimetric assay using a metal ion indicator, namely,

hydroxy naphthol blue (HNB) was used for the detection of the LAMP

reaction. This HNB staining is superior to the existing colorimetric

assays for LAMP (such as SYBR Green, agarose gel….). It also

presents reduced contamination risks and is helpful in high-

throughput DNA and RNA detection.

Fig 2: Visual examination of LAMP products by HB. A positive reaction is indicated by a color change

from violet to sky blue

3. Microscopy, IFAT, PCR and LAMP reactions

Parasitological examination of the 75 dogs’ samples by

microscopy shows that 35 animals were infected with Leishmania.

The remaining 40 dogs were considered as microscopically

negative. All these animals were tested by IFAT and the detection

of Leishmania DNA was run on the 75 dogs’ biological samples

using the PCR and LAMP assays described previously. The results

obtained from the different diagnostic techniques are presented in

the Flow diagram

Fig 1: Sensitivity of cpb LAMP. Total DNA of L. infantum reference strain (MHOM/TN/80/IPT1) was serially

diluted from 5 ng down to 5 fg (Lanes 1 to 7) - negative control (Lane 8).

4. Statistical analysis

To our knowledge this is the first study that evaluates the L.

infantum specific LAMP alongside other diagnostics tools for CanL.

The relative stability of the reagents indicates that LAMP may be a

good alternative to a conventional PCR, especially under field

conditions. Being able to detect and differentiate Leishmania

species with a technique that combines the reliability of molecular

techniques together with the low cost and technicality, opens

horizons for production of high precision LAMP-based devices.

IFAT PCR cpb LAMP cpb

Sensitivity (%) 88.57 37.14 54.29

Specificity (%) 52.5 82.5 80

Positive predictive value (%) 62 65 70.37

Negative predictive value (%) 84 60 66.67

X ² 14.17 3.86 9.52

The IFAT gave the best sensitivity (88.57%). However, this test

produce a high number of false-positive (22 animals), resulting in a

much lower specificity (52.5%).The PCR assay targeting the cpb

gene, gave the lower sensitivity (37.14%) and specificity around

82.5%.The practical evaluation of the LAMP gave more suitable

sensitivity and specificity.

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