Molecular and serological diagnostic of leptospirosis: a review (2014–2020)

Leptospirosis is a zoonosis caused by pathogenic bacteria of the genus Leptospira. This disease affects several animals and humans. Symptoms of leptospirosis can range from mild to severe, may in some cases lead to death. For the diagnosis of leptospirosis, the microscopic agglutination test is considered the “gold standard”; however, it has limitations and studies are being conducted to develop alternative methods of screening and diagnosis of leptospirosis. Serological and immunochemical tests using Leptospira recombinant antigens in combination to molecular tests may be an alternative. In this study, we reported the advances achieved from 2014 until 2020 in controlling leptospirosis based on serological tests using recombinant antigens and molecular diagnosis.

Pathogenic Leptospira spp. can infect humans and animals. Moreover, the infection can be direct, i.e., through the urine of infected animals, or indirectly, i.e., through water or soil contaminated with the urine of infected animals (Jorge et al., 2018;S. Faine, 1999), despite this, however, the transmission of human-human leptospirosis remains unclear (Victoriano et al., 2009). Climatic conditions strongly influence the transmission of leptospires, which require hot and humid conditions for survival, bacteria can persist for weeks to months after excretion in water or moist soil (Mwachui, Crump, Hartskeerl, Zinsstag, & Hattendorf, 2015;Sun, Liu, & Yan, 2020).
Leptospirosis is an emerging zoonotic disease leading to more than one million of serious cases and 60,000 deaths each year worldwide, mainly in tropical countries (Grippi et al., 2020;Guglielmini et al., 2019;Lam, Low, & Chee, 2020;Philip et al., 2020). In addition, leptospirosis is classified as a neglected disease of global distribution, which can affect a wide variety of domestic animals such as pets and those of economic interest, including dogs, cattle, and pigs, as well as wild animals, fish, and humans (Bharti et al., 2003;Mwachui et al., 2015;S. Faine, 1999;Vinetz, 2001). In humans, leptospirosis is considered a public health problem associated with a series of clinical signs affecting multiple organs, such as the liver, kidneys, lungs, and brain (Adler & de la Pena Moctezuma, 2010;Gouveia et al., 2008;S. Faine, 1999). In animals, leptospirosis is associated with poor reproduction, abortions, premature births, and stillbirths, which lead to economic damage to the agricultural sector (Levett, 2001;Padilha, Simao, Oliveira, & Hartwig, 2019;Petrakovsky, Bianchi, Fisun, Najera-Aguilar, & Pereira, 2014;S. Faine, 1999).
At present, the diagnosis of leptospirosis is based on both clinical examination and serological tests. Among all the tests used, the microscopic agglutination test (MAT) is considered the "gold standard" (Rajapakse et al., 2020). MAT test have as a principle to expose human or animal sera to a series of different strains of live leptospires and thus detects antibodies present in the sera samples (S. Faine, 1999). Although MAT is used as a reference method for the diagnosis of leptospirosis, it has several limitations, including low sensitivity in the acute phase of the disease (when antibodies are difficult to detect and there is a higher level of IgM) and incapable of differentiating IgM from IgG antibodies (highly present in other phases of the disease) (McBride, Athanazio, Reis, & Ko, 2005;Rajapakse, Rodrigo, Handunnetti, & Fernando, 2015). In addition, many serological variants produce highly diverse strains, thus making it extremely difficult to identify them using this serological approach, resulting in several cross-reactions (Adler & de la Pena Moctezuma, 2010). Also, MAT is an extremely laborious and expensive technique because of the need to maintain bacteria in cultivation (Caimi, Repetto, Varni, & Ruybal, 2017).
Thus, alternative methods for screening and diagnosis of leptospirosis are being developed. Serological and immunochemical tests using Leptospira recombinant antigens, as well as molecular tests, have been described as alternatives for the diagnosis of leptospirosis.

Study Criteria
This review included all studies published in PubMed (http://www.ncbi.nlm.nih.gov/pubmed/) from 2014 until 2020, which contained the following keywords: "Immunodiagnosis leptospirosis," "Serological diagnosis leptospirosis," "Detection Leptospira recombinant protein," "Diagnosis leptospirosis recombinant protein," "Immunodiagnosis leptospirosis recombinant protein," and "Molecular diagnosis leptospirosis," leading to 157, 103, 12, 32, 9, and 104 articles, respectively. The abstracts of these articles were analyzed, and all studies aimed at developing molecular and immunological diagnostic methods for leptospirosis were included in this review. Studies with other purposes were excluded. Thus, 67 articles were included in this review.

Diagnosis of leptospirosis 4.1 Polymerase chain reaction
Methods based on DNA amplification, such as polymerase chain reaction (PCR), are crucial tools for the detection of Leptospira spp. during the acute phase of the disease wherein antibodies are below the detection limit of the most serological tests (Ahmed, van der Linden, & Hartskeerl, 2014;Denipitiya et al., 2016). The PCR is a fast, sensitive, and specific technique; however, it remains expensive (Courdurie et al., 2017;Waggoner & Pinsky, 2016).
The biological samples used in most of these studies were blood or its fractions (serum or plasma) (Ahmed et al., 2014;Denipitiya et al., 2016;Gokmen et al., 2016;Nhan et al., 2014;Waggoner et al., 2015) and urine (Esteves et al., 2018;Podgorsek et al., 2020;Suwancharoen et al., 2016).  Techniques such as real-time PCR are promising, faster, highly sensitive, and require less product handling than conventional PCR (Denipitiya et al., 2016). In general, real-time PCR is performed on the basis of the 16S gene, which can be amplified only in the presence of pathogenic Leptospira DNA (Waggoner et al., 2015). However, it has been suggested that which proved to be faster, more sensitive, specific, and cheaper, with 90% sensitivity and 97% specificity.
As an alternative to the high cost of implementing real-time PCR in the routine, the RPA technique (Ahmed et al., 2014) proved to be accurate, with sensitivity and specificity of 94.7% and 97.7%, respectively. The 16SrRNA-PCR-RFLP (Gokmen et al., 2016) and

Serological and immunochemical tests
Serological tests are based on the detection of antibodies that are produced because of the stimulation of the host's humoral immune response generated by Leptospira spp. infection (S. Faine, 1999).  Wynwood et al., 2016). Nonetheless, other tests aim for a more specific response, with the detection of IgG in the convalescent phase (Alizadeh et al., 2014;Anita et al., 2016;Brownlow et al., 2014;Nagalingam et al., 2015;Padilha et al., 2019;Shiokawa et al., 2016;Wynwood et al., 2016;Ye et al., 2014).
The LipL32 protein is the lipoprotein expressed in higher quantity in the outer membrane of leptospires, thus making it extremely immunogenic (Chang et al., 2016;Haake et al., 2000). In addition, LipL32 is conserved among pathogenic species and can be used as a distinctive marker for leptospirosis (Haake & Matsunaga, 2002;Shiokawa et al., 2016). Another protein that has been widely used in the development of serological tests is LipL21, a crucial immunodominant protein expressed during infection (Nally, Whitelegge, Bassilian, Blanco, & Lovett, 2007). Thus, these proteins are considered crucial targets in the serological diagnosis of leptospirosis (Table 3). As shown in Table 3, most serological tests being developed are on the basis of enzyme-linked immunosorbent assay (ELISA) (Alizadeh et al., 2014;Courdurie et al., 2017;Deneke et al., 2014;Kitashoji et al., 2015;Padilha et al., 2019;Shiokawa et al., 2016;Ye et al., 2014) or in the latex agglutination test (LAT) (Brownlow et al., 2014;Deneke et al., 2014;Nagalingam et al., 2015). In addition, Wynwood and colleagues (2016) validated a microsphere immunoassay, which can simultaneously test many samples against a large number of serovars, as well as determine individual IgG and IgM titers.
Overall, these factors would be extremely beneficial in laboratory diagnostics and in epidemiological studies of leptospirosis.
Vanithamani and colleagues (2015) developed a lateral flow type assay based on LPS immunochromatography for the detection of IgM antibodies, which was sensitive and specific (>90%). Furthermore, this test is simple and fast and allows diagnosis of serogroup in endemic regions.
Raja and colleagues (2019) used gold and silver nanoparticles associated with different proteins (some well-known and others are little studied) to develop a dot blot that was fast, reliable, and sensitive for the early detection of leptospirosis.
Moreover, the lipL32 gene was described in an optimized DNA-based bioassay for the detection of Leptospira interrogans, which used gold nanoparticle-embedded carboxylated multiwalled carbon nanotubes electrode (Nagraik et al., 2019).
Nanotechnology has enhanced our ability to diagnose, treat, and prevent infectious diseases. However, there are concerns to be addressed and studied with respect to its limitations, as their toxicity, their effect on the environment and effective cost of this technology.

Final Considerations
Controlling leptospirosis is still a challenge and, therefore, several studies have been evaluated with the aim to develop more sensitive and applicable diagnostic tests. Serological methods based on specific recombinant antigens are widely studied. However, molecular methods have been widely used because they can be effective as the acute phase of the disease anticipates treatment and prevents evolution and its complications. Considering the importance of leptospirosis both in public health and the agricultural sector encourages the scientific community to identify methods that aim to improve and facilitate Research, Society andDevelopment, v. 11, n. 2, e19511225471, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i2.25471 the control of the disease. Therefore, several tests have been described to improve diagnostic accuracy, and several studies have shown promising results, bringing new perspectives for the diagnosis of leptospirosis.