Epidemiological situation of human leptospirosis in Brazil and challenges in its diagnosis with a focus on molecular approaches

Leptospira interrogans is one of the causative agents of human leptospirosis, a zoonotic disease with worldwide distribution. Nowadays, this zoonosis is considered one of the biggest in terms of morbidity and mortality (even considering Dengue, the major arbovirosis affecting humans), having in Brazil 3,800 human cases per year. Currently, difficulties imposed by the absence of a rapid, sensitive diagnostic test that can be used as a routine test for the detection of leptospirosis lead to misdiagnosis and underreported cases. The gold standard diagnostic test for leptospirosis is the microscopic agglutination test (MAT), which presents difficulties


Leptospira and leptospirosis
Leptospirosis is a neglected zoonosis worldwide, caused by pathogenic spirochetes of the genus Leptospira, which is associated with the disease in humans or other mammals (LEVETT, 2015). The genus Leptospira includes 64 species, among pathogenic and saprophytic members (VINCENT et al., 2019). Pathogenic Leptospira spp. colonize the proximal renal tubules of the host, and then they are excreted to the environment through the urine, contaminating soil and water samples (KO et al., 2009). The host infection occurs indirectly by touching contaminated material or directly by contact with contaminated urine. Besides colonizing the kidneys, pathogenic leptospires can injure other organs, like the liver and lungs (ADLER; MOCTEZUMA, 2010).
Leptospirosis can occur from an asymptomatic form or mild flu symptoms to a severe clinical condition, known as Weil's disease (KO et al., 2009). This disease usually has a biphasic presentation, characterized by a septicemic acute stage, that persists for around one week, followed by an immune stage, which is defined by the production of antibodies and the end of the symptoms (convalescent phase). If not treated, leptospirosis can progress to its more severe form which, alongside the symptoms aforementioned, may develop kidney and liver failure, and pulmonary hemorrhage (HAAKE; LEVETT, 2015).
In the tropical regions, leptospirosis was estimated to cause 10 or more annual cases per 100,000 population (WHO, 2018). The World Health Organization's Leptospirosis Burden Epidemiology Group estimated that there are 1.03 million of leptospirosis cases per year worldwide, resulting in 58,900 deaths. Such numbers turn leptospirosis the leading zoonotic in terms of morbidity and mortality  resulting in approximately 2.9 million disability-adjusted life years (DALY) annually .
Leptospirosis has a high prevalence in the tropics, where its transmission is favored due to the prolonged survival of the pathogenic leptospires in warm and humid environments (HARTSKEERL et al., 2011). The disease is usually seasonal, presenting increased peak incidence during the rainy season (LEHMANN et al., 2014).
Aside from the rise of pluviometric precipitation values in some seasons of the year, the natural disasters, especially the ones with hydrologic focus, were already related in Brazil as public health emergencies. In the long term, the human population and other animals can be affected by transmissible diseases, like leptospirosis, in an intermediate time span (days and weeks) after disasters. Therefore it's necessary an approach to effectively reducing risk to health associated with disasters (REVISTA DO CENTRO BRASILEIRO DE ESTUDOS DE SAÚDE, 2014;FERENTZ et al., 2021).
In the context of One Health, non-human animals (e.g. dogs) may be great sentinels in the detection of leptospirosis presence in the environment, for also having an important role in the transmission of the disease to humans (GHNEIM et al., 2007). As an example, there is a reported case of a dog which was rescued from the city of Brumadinho, Minas Gerais, after the rupture of the barricade of iron ore waste and exposed to the serovar Copenhageni, of the Icterohaemorrhagiae serogroup, showing a MAT titre ≥ 400. Thus, it reinforces the importance of the follow-through of cases and diagnosis of non-human leptospirosis for the control of new leptospirosis outbreaks (SILVESTRINI et al., 2020).
The diagnosis of leptospirosis requires compulsory notification since the year 2000 in Brazil (BRASIL, 2017). From that milestone until 2015, the country documented approximately 3,800 leptospirosis cases Leptospirosis in Brazil: current situation and main diagnostic challenges Agglutination Technique (MAT) or Martin and Pettit test (MARTIN;PETTIT, 1918). The MAT is based on the agglutination of the serological samples of the patient when confronted with antigens in suspensions of at least 18 alive leptospires from different serovars (serogroups) (WHO, 2003). However, MAT presents some limitations since (i) cultivation of leptospires is laborious and time-consuming due to the fastidious characteristic of the bacteria, which can result in frequent contaminations; (ii) high cost of available culture media and (iii) the interpretation of the result is subjective (50% of agglutination) (LIMMATHUROTSAKUL et al., 2012).
Other serological methods like latex agglutination, lateral flow, and Enzyme-Linked Immunosorbent Assay (ELISA) may circumvent those limitations. Rapid assays for specific antibody detection can be executed immediately after the collection of the biological sample, allowing for a quick diagnosis and early drug intervention, improving the treatment and prognosis of the patient. Additionally, ELISA is considered more sensitive when compared to MAT to the detection of Immunoglobulin M (IgM) during the acute phase of the disease in humans (RIBEIRO et al., 1995). On the other hand, the fact that the disease has a biphasic form (acute and convalescent phases) is a limitation to serological tests, since the antibodies in the acute phase are detected only around 4 to 5 days after the infection (PICARDEAU, 2013).
The application of molecular techniques for leptospirosis diagnosis is currently an important alternative (Table 2) (GUNASEGAR; NEELA, 2021). Tests based on the amplification of DNA segments like PCR (Polymerase Chain Reaction) are among the most efficient to confirm the diagnosis. However, this method requires technical knowledge to be executed, and it presents a high-cost in equipment and reagents, which can limit the implementation in resource-limited settings without specific competence in the field. (WAGGONER; PINSKY, 2016). Variations in the traditional PCR method like Nested PCR, PCR Multiplex, and Loop-Mediated Isothermal Amplification (LAMP) have been studied and can be applied to diagnosis with variable cost, sensibility and specificity.
Although some diagnostic techniques are commercially available for leptospirosis, new and improved methods are still required to increase the power of the diagnosis and diminish the underreport of the disease. Ideally, methods must be sensible and specific enough to detect the infection in different phases of the disease and to be affordable to be available for resource-limited settings.
The criteria used for the selection of the papers were: studies in Portuguese or English, that contemplates the leptospirosis and diagnosis relationship, which means that both subjects were addressed together in the study. For each paper selected, it was analyzed the title, abstract, keywords, and results. After this first analysis, the papers that corresponded to the proposed criteria were separated by year, the technique utilized, and main results. Such information was categorized and distributed together with the pros and cons of each diagnostic approach.

Development of a Multilocus Sequence Typing (MLST) scheme for Leptospira spp.
Genotyping is a process to analyze single strains of bacteria at the genomic level (based on the DNA sequences). It is important to reliably differentiate among related bacterial isolates of the same species, which is essential for epidemiological surveillance, detection of the sources of outbreaks and to study bacterial population and transmission dynamics ( VAN BELKUM et al., 2007;WOLSKA;SZWEDA, 2012). Therefore, genotyping methods are critical to the understanding of the dynamic of Leptospira in the environment and in the different hosts. Among the main techniques that have been employed to genotype Leptospira strains, Amplified fragment length polymorphism (AFLP) (VIJAYACHARI et al., 2004), Multilocus Variable Number of Tandem Repeat Analysis (MLVA) (MAJED et al., 2005), and Pulsed-Field Gel Electrophoresis (PFGE) (GALLOWAY; LEVETT, 2010) are the most studied. Currently, AFLP and PFGE present limited discriminatory power and low reproducibility for certain groups of strains, while the MLVA presents the disadvantage of being accurate only for strains of L. A. P. P. Estrella et al.  Immunoglobulin G (IgG), including the pros and cons.

Method
Target Pros Cons Sources

IgM
IgM antibodies can be detected in the first week of the disease.
During the elapse of the disease, the levels of IgG become higher, evidencing the convalescent phase of leptospirosis.
Variable Specificity and sensibility.
An epidemiologic study of the strains present in the region may be needed.
MLST allows to gather more accurate clinical data that permits better studies about this genus virulence, and epidemiologic surveillance, that allows to detect outbreaks and the dynamic of transmission among hosts.
MLST is a genotyping method based on the sequencing of single-nucleotide polymorphisms (SNPs) of housekeeping genes or essential genes, with each PCR fragment named as a distinct allele. From the upload of the set of alleles of the selected genes, the sequence typing (ST) is determined. Therefore, isolates with the same allelic profile (or the same ST) are described as being part of the same clone. The first MLST scheme was described at the end of the 1990s for Neisseria meningitidis and it is largely employed nowadays as a typing method for many bacterial species (MAIDEN et al., 1998;. The MLST method usually presents higher resolution and higher replicability when compared to other traditional typing methods. Additionally, to find the corresponding ST, MLST requires the submission of the sequencing of the housekeeping genes in online databases, making the results available through a collaborative network. On the other hand, variability of housekeeping genes among bacteria strains still presents itself as a bottleneck for the development of MLST schemes for certain species (MAIDEN et al., 2013).
Currently, there are 3 MLST schemes available for Leptospira spp. that can be accessed through the following website link <https://pubmlst.org/organisms/ leptospira-spp/> (JOLLEY et al., 2018). During the selection of a gene for a MLST scheme, it's important that the gene presents a slow evolution among the same species (AHMED et al., 2006). Therefore, the main genes used in the available MLST schemes for Leptospira spp. are usually genes encoding outer membrane proteins, 16S rRNA and housekeeping genes (Table 3). Boonsilp et al. (2013) reported that even with the small number of samples of Leptospira submitted to the MLST scheme it was possible to assign the samples to distinct clades with 100% of precision, suggesting the potential for global epidemiological survey, including the main pathogenic species in the Leptospira genus. This same study also demonstrated a potential for this approach in defining the species' phylogeography through time and linking the species to their maintenance hosts (BOONSILP et al., 2013).

MLST Scheme Characterization and species assignment Authors
all clades of Leptospira, since the most recent scheme was described in 2014.
The analysis of the genes encoding the 16s rRNA is largely employed for phylogenetic and typing studies, since those sequences are less susceptible to horizontal gene transfer and variations along evolution (ACINAS et al., 2004). The precision of the phylogenetic analysis based on 16S rRNA usually decreases among specific species or among the serovar of Leptospira (TAN et al., 2013), requiring other gene markers to better solve those taxa. Furthermore, the analysis of 16S rRNA can be challenging when working with certain taxonomic groups, since many bacteria have multiple copies of those sequences in the genome (ACINAS et al., 2004).
Despite the efforts, the scheme presenting the best discriminatory power among the species of the genus was the one described from Boonsilp et al. (2013), which can be used for seven species, whoever Leptospira is a genus with 64 species in four sub-clades, therefore new schemes with a better discriminatory power are needed to avoid false negatives.
For this reason, recent studies propose the analysis of complete genome sequences called core genome multilocus sequence typing (cgMLST) as an efficient, accurate and reproducible method for genotyping of Leptospira isolates. In contrast to MLST, the cgMLST analyses hundreds of loci for the comparison of genes of the assembled genome, allowing the identification of species, clades, clonal groups, and sequence types, turning this method one of the most straightforward ways to explore complex genomic data in an epidemiological context (GUGLIELMINI et al., 2019; GRILLOVÁ; PICARDEAU, 2020). Guglielmini et al. (2019) contributed to the collection of scientific data to the development of a cgMLST scheme from comparative analysis using Leptospira strains for many sources and geographic locations, by identifying 764 core genes for the genus, being 545 of those considered suitable for cgMLST genotyping (GUGLIELMINI et al., 2019).

Closing remarks
The unspecific clinical symptoms of leptospirosis and the diversity among the species of the Leptospira genus led to limitations in the clinical and laboratory diagnosis, often causing the zoonosis to be underreported. Due to these challenges, many studies have been developed to identify novel diagnostic targets capable of inclusion and classification at serovar level. The cost per test and facility structure are of extreme importance when developing a leptospirosis diagnostic method, since outbreaks of this disease are more frequent in developing countries, which commonly present limited resources. It is important to notice that rapid methods need to consider rural areas and cities located away from the certificated laboratories, allowing local diagnosis. Studies to improve the sensibility and specificity rates of the diagnostic tests are also required.
The availability of genotypic analysis though online databases, like PubMLST, opens the door for sharing data among groups of various locations, supporting epidemiological data on local, global, and long-term scales. Currently, the cgMLST may represent a promising scheme for the genotyping of Leptospira isolates and offers an opportunity to better understand those Spirochaetes, since cgMLST can be performed to study transmission among hosts and detection and surveillance of outbreaks.