Evaluation of Automated Loop-Mediated Isothermal Amplification (LAMP) Malaria Test for the Parasite Detection in Vectors

Albert Eisenbarth, Hagen Frickmann

^a Bundeswehr Hospital Hamburg, Department of Microbiology and Hygiene
^b Bundeswehr Hospital Hamburg, Branch at Bernhard-Nocht-Institute for Tropical Medicine

Introduction

Malaria is a vector-borne infectious disease caused by members of the apicomplexan protozoan genus Plasmodium. It is a major global health concern with nearly 250 million infections and 600,000 deaths annually in tropical and subtropical countries, particularly in Sub-Saharan Africa (WHO 2023 [5]). Transmission is governed by the blood meal from an infected person of one of approximately one hundred competent female mosquito species of the genus Anopheles, where the parasite develops further to infective stages transmissible to a new human host while taking another blood meal. Therefore, the risk of infection for the population in an endemic area is dependent on

1. the diversity and abundance of competent malaria vectors,
2. the spatial and temporal biting dynamics of the vectors, and
3. the co-presence and frequency of persons infected with Plasmodium parasites.

To mitigate the risk for deployed military personnel during stabilizing missions in endemic regions for malaria and other vector-borne diseases, the Bundeswehr has established a vector monitoring program in and around the mission camps since 2007. The general procedure is that qualified and trained health personnel operate ventilation-based mosquito traps (generally a CDC model) distributed within the military camp perimeter. The catches are regularly emptied, and the insect samples are counted and sorted according to vector group. After the export to the Operations Control Laboratory (“Einsatzleitlabor”) located at the Bundeswehr Central Hospital in Koblenz, the vector species are identified by entomologically qualified staff using taxonomic keys to determine those responsible for the transmission of prevalent pathogens. For that subset, a molecular pathogen screening is performed to detect and classify selected genera of pathogens, including Plasmodiumspp. Although this method allows the depiction of vector occurrence and pathogen transmission dynamics with high precision for adequate risk assessment and mitigation, the delayed reporting time of the pathogen screening compromises efficient intervention times in response to a local infection event in proximity to the soldier camps, resulting in a highly elevated infection risk. A point-of-care system that immediately detects Plasmodium spp. in the vector would significantly accelerate the reaction time after parasite detection, contributing to health risk mitigation for soldiers during deployments.

Our research group has already confirmed the Alethia Malaria loop-mediated isothermal amplification (LAMP) system as a powerful reference tool for patient diagnostics [2]. This study aims to evaluate whether the same system is suitable for adapting parasite detection in vectors under typical field conditions.

Methods

The development was subdivided into four phases, as shown in Figure 1.

Fig. 1. Workflow for the adaption of the Alethia Malaria LAMP kit to be used on Anopheles vectors

Step 1: Adapting the protocol

As a first step, the protocol of the commercially available Alethia Malaria LAMP kit (Meridian Biosciences Inc., Cincinnati, United States) was adapted for malaria vectors and tested accordingly, including the homogenization and lysis of whole female mosquitoes with reaction-tube-sized pistils, and the supplementation of a human-derived mitochondrial gene fragment on a plasmid to mimic the presence of human internal control. The latter was necessary to circumvent invalid readouts due to the lack of human DNA in the specimens. All subsequent steps, like DNA extraction, LAMP reaction, and amplicon detection, were performed according to the manufacturer’s instructions. If the LAMP did not produce a valid diagnostic result, the test was repeated with residual homogenate.

Step 2: Proof-of-Priciple

Proof-of-principle of the assay was tested on homogenates of laboratory-bred Anopheles stephensi infected with the rodent malaria parasite Plasmodium berghei. The mosquitoes were kindly provided from the insectary of the Bernhard-Nocht-Institute for Tropical Medicine in Hamburg, a cooperation partner of the Bundeswehr hospital Hamburg in tropical medicine. As a reference standard, all LAMP results were verified by a Plasmodium-specific real-time PCR assay (RealStar Malaria PCR Kit 1.0, Altona Diagnostics GmbH, Hamburg, Germany) on a Rotor-Gene Q cycler (Qiagen AG, Hilden, Germany). Priorly, homogenate aliquots were subjected to DNA extraction (DNeasy Blood and Tissue Kit, Qiagen AG, Hilden, Germany) according to the manufacturer’s recommendation for insect samples.

Step 3: Performance test

After the proof-of-principle has been successful, the protocol was tested on pooled homogenates (10x) of caught Anopheles spp. with natural infections in hypo-, and hyperendemic regions in Colombia, South America, and Gabon, Central Africa. The vectors were available as part of entomological survey collections and shipped to our laboratory in Germany. The DNA of all individual homogenates was extracted and re-tested for those pools with a positive test result of LAMP, qPCR, or both. According to the manufacturer’s protocol, the results of the Plasmodium species of malaria-positive screening were differentiated with the RealStar Malaria Screen and Type 1.0 Kit (Altona Diagnostics, Hamburg, Germany).

Step 4: Field test

To assess performance under tropical field conditions, the LAMP test system was transferred to a basic entomological laboratory in a hyperendemic focus in Gabon during the rainy season and used to monitor freshly caughtAnophelesspp. in the adjacent area.

Results

Proof-of-principle LAMP test of experimentally infected Anopheles stephensi

In total, 43 femaleAn. stephensi from the insectarium, which were fed with P. berghei cultured blood were analysed. Of the 22 single-testedAn. stephensi with a positive qPCR result for the presence of Plasmodium DNA (51.2 % of total), 68.2 % (n = 15) were confirmed by LAMP, 9.1 % were invalid, and 22.7 % (n=5) were LAMP-negative (Table 1).

LAMP performance test of naturally infected Anopheles spp. from endemic regions

All samples from Columbia (n = 24) were tested negative for LAMP and qPCR. From a hyperendemic focus in Gabon, Central Africa, 1771 wild-caught female Anopheles spp. were collected in the vicinity of human settlements. Whereas the species identification and preparation for LAMP is ongoing, 789 individuals divided into 79 pools have been analyzed. Six individuals (7.6 %) were tested positive by LAMP, of which only 5.1 % (n = 4) were confirmed by qPCR. Single testing of the pools confirmed malaria infection in 5 out of 6 LAMP-positives (83.3 %, Table 1).

Table. 1. Test performances of the Alethia Malaria LAMP compared to the reference standard Plasmodium qPCR.

Numbers and percentages (in brackets) of female Anopheles spp. used for Plasmodium spp. screening with the newly adopted Alethia Malaria LAMP assay compared to the qPCR reference assay. +ve: positive test result; -ve: negative test result; inv: invalid test result; n.d.: assay not done

LAMP robustness test in a tropical field setting

Only 18Anophelesspp. samples were collected during the field trial in Gabon. LAMP analyses revealed a valid readout rate of 94.5 % (n = 17). One individual (5.6 %) tested positive for malaria parasites. No qPCR reference has been conducted.

Discussion

Table 1 summarizes the test performances of the Alethia Malaria LAMP in comparison to the reference standard Plasmodium qPCR. For the lab-reared An. stephensi vectors, the sensitivity was significantly reduced to 68 % in the LAMP assay compared to the reference. No false negatives were detected, but two samples were invalid in LAMP even after repeating the test. The result of the qPCR reference was, in both cases, Plasmodium-positive. A plausible explanation for the lower sensitivity and robustness is that the LAMP assay has not been adapted to detect animal-borne Plasmodium spp., such as P. berghei. However, animal-borne malaria is usually irrelevant in the clinical context, except for primate-borne Plasmodiumspp. in Asia, which has not been included in this study [4].

For the human-pathogenicPlasmodiumspp., all samples detected by the reference qPCR were also detected by LAMP. Species differentiation of these pool samples revealed the presence of 1 or 2 out of 10 vectors infected with the predominant human-pathogenic species in the region Plasmodium falciparum – in one case a double infection ofP. falciparum with P. ovale. The latter is known to occur in Gabon but is generally prone to be underreported due to low blood parasitaemia in patients [3]. Two malaria-positive LAMP pool samples were negative in the qPCR reference (Table 1). LAMP re-testing of the single mosquito homogenates was positive in one pool sample (3 out of 10 individuals) and negative in the other. That finding indicates

1. a higher sensitivity of LAMP over qPCR and
2. a potentially slightly reduced specificity.

Given that the mosquitoes used for the performance test stem from entomological surveys, we cannot know the infection status completely. Infection experiments with human-pathogenic Plasmodiumspp. in competent Anophelesspp. in controlled environments would be appropriate to overcome such caveats.

The aptitude of the LAMP assay under field laboratory and tropical climate conditions was tested in a hyperendemic focus in Gabon. Unfortunately, the number of vectors collected during the observation period was low (n = 18, Table 1). Nonetheless, the assay showed impressive performance with only one invalid test dropout (5.6 % of total) and the detection of one female Anopheles moucheti harboring Plasmodium sp. Although the low sample size does not permit extrapolation, the frequency aligns with the overall estimated infection rate of 2.2 % for malaria vectors in Gabon [1].

Conclusion

The newly adapted Malaria LAMP for vectors shows good to excellent performance regarding sensitivity, specificity and robustness under tropical field conditions. Further studies shall evaluate the integration capacity into the current vector monitoring as well as how much prevention and control for deployed soldiers in malaria-endemic settings shall benefit.

References

1. Boussougou-Sambe ST, Woldearegai TG, Doumba-Ndalembouly AG, et al: Assessment of malaria transmission intensity and insecticide resistance mechanisms in three rural areas of the Moyen Ogooué Province of Gabon. Parasit Vectors 2022; 20; 15(1): 217. mehr lesen
2. Frickmann H, Wegner C, Ruben S, et al: Evaluation of the multiplex real-time PCR assays RealStar malaria S&T PCR kit 1.0 and FTD malaria differentiation for the differentiation of Plasmodium species in clinical samples. Travel Med Infect Dis 2019; 3 1: 101442. mehr lesen
3. Lalremruata A, Jeyaraj S, Engleitner T, et al: Species and genotype diversity of Plasmodium in malaria patients from Gabon analysed by next generation sequencing. Malar J. 2017; 16(1): 398. mehr lesen
4. Mewara A, Sreenivasan P, Khurana S: Primate malaria of human importance. Trop Parasitol. 2023; 13(2): 73-83. mehr lesen
5. World Malaria Report 2023. Geneva: World Health Organization; 2023.  mehr lesen

For the authors

Oberregierungsrat Dr. rer. nat. Albert Eisenbarth
Bundeswehr Hospital Hamburg
Department of Microbiology and Hospital Hygiene
Branch at Bernhard-Nocht-Institute for Tropical Medicine
Bernhard-Nocht-Str. 74, D-20359 Hamburg
E-Mail: alberteisenbarth@bundeswehr.org