Over one-third of the worldwide population harbors a parasitic helminth; these parasitic diseases generate millions of deaths each year (1) and produce a series of morbidities that affect mostly vulnerable populations from low and medium income countries (LMIC), causing millions of disability-adjusted life years (DALYs) (2). Ancylostoma caninum is a zoonotic nematode, proven to cause local lesions (e.g., papular/pustular eruptions) rather than serpiginous tracks typical of cutaneous larva migrans. Other conditions occasionally attributed to A. caninum are myositis, unilateral subacute neuroretinitis, and eosinophilic enteritis (3). The filariform larvae stage penetrates the human epidermis but typically does not develop in the intestine, therefore, it becomes trapped in the skin and underlying muscles, causing irritation and itching (4–6). In some studies, few cases were reported where A. caninum was able to complete its migration to the human intestine (7), generating several eosinophilic gastroenteritis (8). Recent studies suggests that patent human infection is a possibility (9, 10).

Infection in dogs can occur through percutaneous penetration of third-stage larvae, orally or through lactogenic and trans-mammary route (11). Previously infected rodents and insects can act as paratenic and transport hosts, respectively. Canids ingesting such a host will develop patent infections due to reactivation of hypobiotic larvae in the prey (12). In puppies, symptoms can be extensive, including, anemia diarrhea, malnutrition and death. In older dogs, symptoms are mostly limited to anemia (13). Immature worms may still produce clinical disease (i.e., no eggs observed in feces). In Argentina, previous epidemiological studies showed a variable range of A. caninum prevalence in canine feces (14–26). A study performed in the central Buenos Aires province showed that 60.5% of dogs were parasitized with A. caninum (27). In contrast to the southern Patagonian region of the country, where the reported prevalence of A. caninum in canines was lower (0.41–6.2%) (14, 22).

The detection of parasitic structures in the feces is used for detection of infection, using different coproparasitological techniques to concentrate the sample and increase sensitivity (13). The most common coproparasitological techniques used for the detection of A. caninum eggs in canine feces is the standard fecal flotation technique with either saturated salt (28) or sucrose (29). The main disadvantages of these techniques, in epidemiological studies, is associated to false negative results due to low parasite burden, the biology of the parasite, and human resources that are not specifically trained or have little experience in the identification of parasitic structures under the microscope (30). Nonetheless, there are alternative coproparasitological methods that may be used to improve the sensitivity (31). Copro-antigen detection methods through Enzyme-Linked Immuno Sorbent Assay (ELISA) have shown a wide range of sensitivity and specificity values (32). Moreover, the detection of nucleic acids through the use of molecular techniques has generated improvements in sensitivity and specificity values (33–36). However, these techniques cannot generally be performed in endemic areas because they require sophisticated equipment and highly qualified personnel, complicating its implementation.

On the other hand, the isothermal amplification of nucleic acids has begun to be widely used since it can be carried out in laboratories without specialized equipment. Particularly, the LAMP (loop-mediated isothermal amplification) technique (37), uses 3 primer pairs that recognize a small DNA fragment, and generate looped structures that serve as a template to start a new polymerization cycle thus providing both higher specificity and sensitivity (38). The DNA polymerase I from Bacillus stearothermophilus (Bst) used in the technique causes DNA strand displacement and therefore does not require denaturing the double strand, thus the technique can be performed with any equipment that guarantees a constant temperature (37). The LAMP reaction characteristics (affordable, sensitive, specific, user-friendly, rapid, equipment-free) make it an attractive method for use in diagnosis and epidemiological surveillance (39).

Different protocols based on LAMP reactions, have been implemented for diagnosis of different parasites (30, 38, 40–49). Similarly, LAMP reactions have been shown to be useful for the detection of pathogens in food and surveillance of water quality (50–57). Nonetheless, this technique is not currently widely used in the diagnostic routine, probably due to the high cost of the visualization methods required and the cost of DNA extraction. Another complication is the contamination of the sample with unwanted amplification products which can be avoided by correcting the workflow.

In this work the development of an easy copro-LAMP reaction for A. caninum detection was performed. Furthermore, this reaction was evaluated using two different methods for DNA extraction in order to be able to implement it in laboratories without sophisticated equipment.

Hookworms are nematodes that can affect human and animals (3, 64, 65). Ancylostoma diagnosis is not provided at the species level since the morphology of Ancylostoma egg species is often indistinguishable. The simplicity of copromicroscopy and its specificity makes it the gold standard method for the parasitic helminth egg detection. However, this copromicroscopy presents the disadvantage of low sensitivity in epidemiological studies, or in the identification of the hookworm species. These problems in sensitivity and specificity values can be resolved using molecular biology. Unfortunately, the high costs for its realization make it difficult to use in laboratories with poor resources or the lack of equipment specific for PCR. This is the reason why many PCR protocols specific for A. caninum detection that have been previously developed (33–35, 66, 67), are not currently available in some endemic areas.

Isothermal amplification techniques, such as LAMP, are able to specifically and sensitively amplify nucleic acids, without the need of expensive equipment. These characteristics make them an attractive alternative for molecular diagnosis in epidemiological studies (30, 68, 69). Different LAMP protocols for helminth DNA detection were developed. The LAMP assay for E. granulosus s. s. DNA detection developed by Ni et al. (70), was able to detect 10 pg of gDNA, and was more sensitive than both copro-ELISA and copro-PCR. While Bucher et al. (71) developed an easy and cost-effective protocol for E. multilocularis copro-detection, some LAMP reactions for the detection of nematode DNA were not able to overcome the sensitivity values of gold standard techniques (72) or PCR methods (73).

Herein, we developed for the first time, a simple LAMP reaction for A. caninum copro-detection. The Copro-LAMPAc was able to detect 100 fg of gDNA from A. caninum adult worms, which is more sensitive than previously reported values for other PCR assays (33–35, 66). This analytical sensitivity value is included in the femtograms range, similar to what has been obtained in other studies (38, 49, 74). In future studies, the A. caninum egg limit of detection could be determined.

The Copro-LAMPAc assay proved to be more sensitive than optical microscopy for the identification of A. caninum (Table 2). As previously described (38), commercial kits for DNA extraction from stool can be replaced by more accessible methods. This significant cost-reduction in sample processing increases the possibility of the Copro-LAMPAc assay implementation in practically any laboratory. It could be compared to techniques that increase the sensitivity of conventional coprological methods, such as the FLOTAC or mini-FLOTAC (31), although these methods cannot be used to distinguish hookworm species.

Due to the zoonotic potential of A. caninum, which can cause skin damage, eosinophilic enteritis or patent infection in humans, it is important to be able to have a tool to monitor the environmental contamination by hookworms (and other nematodes, such as Ascarids), since free-roaming domestic animals are a primary source of contamination (75, 76). Additionally, recent studies have demonstrated the presence of drug resistance to anthelminthics in dog hookworms (A. caninum) (77–79), where the authors suggest the use of the fecal egg count reduction test to evaluate the efficacy of drugs used (79). In epidemiological studies, the efficacy of this technique is lower. Therefore, we believe that other important uses for the copro-LAMPAc test could be for epidemiological studies, screenings, and when a species-diagnosis for hookworms is needed in the context of a veterinary clinic.

The Copro-LAMPAc assay developed herein is promising in the light of the sensitivity/specificity values herein obtained for A. caninum detection. The Copro-LAMPAc protocol may be implemented for epidemiological purposes in low complexity laboratories. Nevertheless, given the results obtained here are preliminary, future wider field studies should be performed to validate these findings. If the larger scale is confirmed, this method could also be implemented to determine environmental contamination. Adaptations to this protocol could be performed in order to detect A. caninum contamination in other matrixes (i.e., food, water, sand, etc.), in order to prevent infection in both animals and humans.

The Copro-LAMPAc technique provides a specific and sensitive method for the A. caninum detection, a zoonotic parasite that affects both humans and animals. Moreover, this technique uses an accessible method for DNA extraction, providing an easy and low cost tool for diagnosis. This study provides a new protocol to improve hookworm screening for epidemiological studies in basic laboratories from endemic areas.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The parasite material was obtained from the cadaver of animals previously donated by their respective owners, to the University Hospital from the School of Veterinary of the National University of La Plata.

MP and HA conceptualized and designed the experiments. HA performed the experiments and wrote the manuscript. MR, MC, PR, SR, MB, MT, and GS contributed reagents, materials, and/or analytical tools, and wrote the manuscript. MP and VP critically revised and approved the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by Fundación Mundo Sano, Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Ministerio de Salud Pública de la Provincia de San Juan and Universidad Católica de Cuyo.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.