Development of a Multiplex PCR Assay for Detection and Discrimination of Pathogenic and Saprophytic Leptospira in Water

Leptospirosis is a zoonosis prevalent in tropical countries and affects animals and humans alike. Leptospira interrogans, the causative organism for this waterborne infection, spreads through the urine of infected animals. There is a direct link between contaminated water and Leptospira outbreaks. This study reports a rapid assay to detect and differentiate pathogenic Leptospira from non-pathogenic in environmental water using multiplex PCR. The assay uses primers targeting the Lipl32 and Lipl21 gene. The multiplex PCR has been standardized using 11 pathogenic and one saprophytic serovar of Leptospira. The analytical sensitivity of the developed method was evaluated with different concentrations of template DNA. This method was used to screen water samples collected from 20 different sources from Chengalpattu town in Kancheepuram District, Tamil Nadu, India. Of the 20 water samples screened, 13 samples tested positive for pathogenic Leptospira, and seven samples tested negative. Four water samples were found to carry both pathogenic and saprophytic species. The developed multiplex PCR assay is highly useful for detecting and distinguishing pathogenic and saprophytic leptospires in water.


iNtROduCtiON
Leptospirosis is a zoonosis having a global presence with a higher prevalence in tropical regions. Heavy rainfall and flooding result in large epidemics causing deaths in developed and developing countries [1][2][3] . Humans are infected via exposure to the urine of infected animals or through contaminated soil or water 4 . Leptospira species display different affinities and adaptations in different hosts and exhibit clinical symptoms of varying degrees 5 . Rodents are reservoirs and carriers of Leptospira but rarely show symptoms of the disease.
Meanwhile, other animals like livestock, cattle, and pets exhibit severe symptoms, including multiple organ injury and abortion upon infection 6,7 . Similarly, in infected humans, the disease's clinical manifestations range from mild, flu-like symptoms to severe conditions like jaundice, renal impairment, and hemorrhage to life-threatening conditions 5 . A direct correlation between Leptospira epidemics and direct contact with contaminated environmental water has been reported. A damaged skin barrier like a cut or an open wound is the most common route of pathogen entry 2,8,9 .
Based on 16S rRNA phylogeny, the genus Leptospira is arranged into three large subgroups and includes at least 21 species. Pathogenic (Group I), Intermediate pathogenic (Group II), and saprophytes are the three subgroups. The infectious groups I and II include 14 species, and the non-infectious group III comprises 7 species referred to as saprophytes and is not known to cause infections 10 . Group I pathogens like L. kirschneri, L. noguchii, and L. interrogans have 250 distinct serotypes. They cause disease whose symptoms range from mild infections to severe disease and death. Intermediate pathogens mostly cause mild illnesses that are easily treatable and without fatal complications. Saprophytic Leptospira belongs to group III. They are freeliving environmental microorganisms and are morphologically similar to pathogenic Leptospira, making it hard to discriminate between them by microscopic observation alone 11. Currently, the microscopic agglutination test (MAT) is used to detect leptospirosis. Though this is deemed the gold standard, it lacks sensitivity and cannot be used for early diagnosis.
This study is designed to develop a multiplex PCR assay for rapid detection and differentiation of Leptospira strains in environmental water. In order to achieve this, a combination of primers that target a portion of the Lipl21 gene unique to saprophytic Leptospira and pathogen-specific Lipl32 gene were used. Lipl32 codes for a surface lipoprotein with a molecular weight of 32kDa and is present only in pathogenic Leptospira. Meanwhile, Lipl21 encodes another lipoprotein with a molecular weight of 21kDa. The study was performed using 12 Leptospira strains and was successfully used to detect and differentiate between the Leptospira strains in environmental water samples.

Leptospira strains
A total of 12 Leptospira strains obtained from Dr. Natarajasrinivasan, Department of Microbiology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India, were used in this study ( Table 1). The cultures were maintained in a liquid medium (Leptospira Medium Base M1009, Himedia) supplemented with 5-fluorouracil and Leptospira Enrichment (Himedia). All cultures were incubated at 30°C.

Primers used
Oligonucleotide primers used in this study ( Table 2) were synthesized by Priority Life Science, Coimbatore, Tamil Nadu, India. These primers were used to amplify Lipl21 (582 bp) and Lipl32 (719 bp) regions from L.meyeri serovar Semarangaa and L.interrogans serovar Icterohaemorrhagiae respectively. The 582 bp region amplified by Lipl21 FP and Lipl21 RP is specific to saprophytic Leptospira 21 .

DNA Extraction
All genomic DNA used in the study was isolated using DNeasy Blood and Tissue kit by Qiagen (Germany). 100 ng of genomic DNA from the different pathogens was used for subsequent PCR reactions.

Sample collection
Twenty different water samples were collected from in and around Chengalpattu town in Kancheepuram District, Tamil Nadu, India. The collected water samples were stored in a sterile glass container at -20°C. The water samples were concentrated for 45 minutes using a vacuum concentrator (Eppendorf, India) and used as templates for multiplex PCR.

Results
All 12 cultures used in this study were screened for the presence of their respective marker gene (i.e., Lipl32 in the case of pathogenic species and a stretch of DNA sequences in Lipl21 that is found explicitly in non-pathogens). A multiplex PCR technique was developed using recombinant plasmids containing genes Lipl21 and Lipl32. The amplification products of 582 bp (Lipl21) and 719 bp (Lipl32) were observed in a single PCR reaction. The products were confirmed by comparing the uniplex PCR products of Lipl21 L. interrogans serovar Hebdomadis -+ 3.
All the pathogenic cultures used in this study amplified the Lipl32 region except the saprophytic Leptospira serovar Semaranga, which tested positive for Lipl21. None of the cultures showed amplification products of both genes (Fig. 3). These outcomes indicate the specificity of the developed method.
In this study, the water samples were concentrated and used as a template for a multiplex PCR. Thirteen water samples tested positive for pathogenic Leptospira, and seven samples showed negative amplification for pathogenic and saprophytic Leptospira. Four water samples were found to carry both pathogenic and saprophytic Leptospira. Few amplification products of negative and positive reactions are shown in Fig. 4.

disCussiON
The misdiagnosis of leptospirosis can be attributed to the similarity between the clinical symptoms of leptospirosis and other febrile diseases like dengue and malaria. This, in turn, has led to the underestimation of the prevalence of the disease 22 . Long incubation periods required for the growth of Leptospira make culturing difficult for diagnosis, and observation using dark-field microscopy is unreliable. The serological test MAT, which is considered the benchmark technique for diagnosing this disease, has several drawbacks as it is arduous, slow, and requires maintaining an elaborate collection of reference strains 23,24 .
The extensive use of molecular techniques has upgraded the speed, sensitivity, and specificity of leptospirosis diagnosis. Many PCR-based methods have been reported for Leptospira detection, but each technique comes with limitations 16 . The sensitivity and specificity of the PCR significantly depend on choosing reliable target genes and primer optimization. The marker gene Lipl32 used in this study is specific to pathogenic Leptospira species. The Lipl21 gene is present in both pathogenic and non-pathogenic Leptospira, but sequence variations exist between the Lipl21 sequence of these two Leptospira groups. We exploited this sequence variation and developed a multiplex PCR targeting Lipl32 and Lipl21 genes to screen for pathogenic and saprophytic Leptospira species. When performed using genomic DNA from pathogenic and non-pathogenic cultures, the assay showed an analytical specificity of 100%. Water samples from multiple sources were tested using this multiplex PCR method to determine the assay's effectiveness in the field. It was observed that the multiplex PCR assay successfully detected & differentiated between the two groups, even in water samples that contained both pathogenic and non-pathogenic Leptospira. These results indicate the usefulness of this method for screening environmental samples. This will help take necessary measures to prevent exposure to Leptospira-contaminated water.