Epidemiological Status of Leptospirosis in India

A corkscrew-shaped spirochaete named Leptospira interrogans causes the infectious disease called leptospirosis. Leptospirosis, a growing public health concern worldwide, from subclinical infections to potentially lethal pulmonary hemorrhage. The disease caused by the pathogenic Leptospira, poses a threat to both humans and animals, and its transmission occurs through contact with infected animals, contaminated water


INTRODUCTION
Leptospirosis is becoming a significant concern for public health, causing subclinical infections to lethal pulmonary hemorrhage. 1 The disease is widely distributed and occurs both in developed and developing countries in suitable habitat where the conditions are suitable for the growth of causative organisms. 2Incidences of the presence of pathogenic leptospires have been reported in a variety of domestic or pet animals, such as dogs, cattle, horses, rats, and pigs [3][4][5][6] and also in several wildlife animals including coyotes, sea lions, raccoons, opossums, bats, white-tailed deer and surprisingly in frogs and caimans. 7,8Rats, canines, buffaloes, and cattle are the hosts for leptospirosis. 9umans or animals are infected accidentally by direct or indirect contact with leptospira infected animals or contaminated water or urine. 10,11Infection is also possible by other methods, like handling clinically infected animal tissues, inhalation of droplets of urine, 12 and consuming contaminated food and water.Rodents, especially the common rat (Rattus norvegicus) is the most common host.Certain serovars are linked with particular animal hosts e.g., Icterohemorrhagiae with rats, canicola in dogs, and Hardjo in cattle. 13,14n humans, the early stages of leptospirosis are characterized by high fever, headaches, jaundice, and mucosal hemorrhages. 15For the survival of the leptospires, moisture is an essential key factor in the environment. 10ecent estimates show the annual incidence of leptospirosis worldwide is around one million cases and contributes to around 60,000 deaths. 19,20AT and ELISA are widely used tests, of which the microscopic agglutination test (MAT) is considered the most authentic.21 For the clinical detection of leptospirosis, molecular diagnostic methods are more popular as they are more sensitive and rapid than other methods, such as culture.22 Also, these techniques are less time-consuming and are more accurate than the conventional serological test, making the detection of organisms right in the samples in a short time.9 Among molecular biology techniques, real-time poly merase chain reaction (qPCR) is advantageous.It is more sensitive in diagnosing various infectious diseases compared to conventional PCR assays.

Etiology
The causative agent of leptospirosis is the spirochaete, which belongs to the order Leptospirales, family Leptospiraceae, and genus Leptospira containing 82 species (https://lpsn.dsmz.de/search?word=Leptospira).Based on their pathogenicity, the genus is divided into pathogenic and non-pathogenic strains.The differentiation of various serovars is due to the difference in agglutination reaction in MAT.Furthermore, 60 serovars for L. biflexa and above 300 serovars for L. interrogans have been reported Routray et al. 23 Leptospires are aerobic and motile, and they can sustain alkaline pH but cannot survive in the hot and dry environment.The bacteria are Gram-negative.In addition to Gram staining, silver impregnation staining and immunofluorescence are used, followed by visualization using a dark field microscope.Ellinghausen-McCullough-Johnson-Harris (EMJH) medium is the most popular medium for the culture of Leptospira.
As the pathogenic and non-pathogenic strains cannot be differentiated based on their morphology, a few tests, like testing in vivo pathogenicity and detection of outer membrane proteins, are done.[26][27][28]

Epidemiology
Leptospirosis outbreaks have been reported globally.Human infections are rampant in tropical regions with high rainfall, where the human population gets exposed to water contaminated with the urine of infected animals. 29eople who live in under developed nations with poor sanitation have a significant risk of contracting an infection through polluted water or soil.Leptospirosis is a serious issue in low-lying, densely inhabited parts of India that experience frequent flooding or water logging during the monsoon season.Farmers, sewage workers, butchers, and other occupational risk groups are most vulnerable to getting the illness.Swimming, sailing, and other water sports are among the leisure activities that are thought to increase the risk of contracting leptospirosis.
A recent meta-analysis in humans found that exposure to rain was a significant risk factor for leptospirosis and that the majority of cases were reported from the coastal zone and among farmers.97% of patients had fever, and 35% of them had conjunctival suffusion (red conjunctiva).In 34% and 35% of the patients, the liver and kidney, respectively, were affected.The combined mortality from multiple studies was estimated to be 11%. 30

Research gaps in epidemiology
In India, several research gaps exists that hampers understanding of epidemiology of leptospirosis in human and animals.Some of the key research gaps are; Addressing these research gaps in the epidemiology of diseases in India is essential for developing evidence-based strategies for disease prevention, control, and mitigation of leptospirosis in human and animals.

Clinical Manifestations
The symptoms of leptospirosis include fever, renal and hepatic insufficiency, pulmonary signs, and reproductive failure across human beings and domestic animals, primarily dogs, cattle, and pigs.Fever, jaundice, vomiting, diarrhea, intravascular disseminated coagulation, uremia followed by renal failure, hemorrhages, and death are possible symptoms of typical canine leptospirosis.Leptospirosis symptoms in cattle and pigs include reproductive failures, such as abortion, fetal mummification, weak new born, and agalactia. 31Humans may experience symptoms ranging from a mild, influenza-like illness to a severe infection with respiratory distress and renal and hepatic failure (Weil's disease).

Risk factors and transmission
The cases of leptospirosis are higher in the tropical regions than in the temperate parts because leptospires can survive longer in warm and humid conditions.Countries where there are chances of exposure of the human population to infected animals have greater chances of infection in humans.The host range of Leptospira spp is diverse because of its capability to infect and survive in different hosts.It occurs in a wide variety of wild, domestic, and marine animals.Rodents are a primary reservoir host, while other animals like cattle, pigs, and dogs may also act as carriers and sources of infection to other animals or humans. 32ontrolling rat infestation on farms and fencing to keep stray dogs out of the farm can be effective prevention strategies. 33In humans and animals, infections can occur by coming in contact with infected water and soil. 34,35ransmission of leptospirosis is either by direct contact with an infected animal or by indirect contact with soil or water contaminated with the urine of infected animals. 17Human-tohuman transmission is rarely reported.Infected urine is the source of infection in humans and animals coming in contact.In humans, the risk factors associated with leptospirosis are mainly occupational groups, i.e., farmers, abattoir workers, veterinarians, rice field workers, and animal handlers, or recreational activities like swimming and hunting.Hence, it is also known as paddy field worker disease, mud fever, or sewer worker disease.For animals, the important risk factors include shared grazing with common water resources, purchase or introduction of infected cattle, rodents in the farm, level of hygiene in milking and status of leptospiral vaccination, presence of other animals in the farms like dogs, sheep and goats, horse, pigs, and others (Figure 1).Leptospirosis can also be transmitted through the semen of infected animals. 36

METHODOLOGY Data source and study selection
The published data on leptospirosis was retrived from all animals (including dogs, cows, buffalo, pigs, and goats) reported from India using PubMed and Google search engines.Terms like "leptospira", 'leptospirosis" and "dogs" or "cows", or "buffaloes", or "pigs" or "goats" were searched.We included data from 2005 to 2023, along with a list of authors, year of publication, geographic location of Indian states, sample type, sample size, methods of detection, and prevalence (any kind, including culture/sero/molecular/others) of Leptospira spp.During the initial screening of titles and abstracts, criteria were determined to include based on their suitability.Inclusion criteria for detail analysis was defined for each species.Otherwise, review articles, metanalysis, and case reports were excluded from this analysis.The full-text documents were assessed, and data were retrieved from the table or the abstract.Publications in other languages were excluded from this study.Microsoft Excel using was used with "Data → Data Analysis" tool functions for the data analysis.
Apart from the analysis of retrieved data, some of the recent reports are mentioned below.
A study was conducted using animal samples from several districts in the south Gujarat region that were tested for leptospirosis.A total of 151 (or around 11%) of the 1406 animal samplesof goats, cows, buffalo, and bullocks tested positive.In cows, buffaloes, bullocks, and goats, the most prevalent serovars were Icterohemorrhagiae (22%), Patoc (58%), Hardjo (50%), and Autumonalis (50%), respectively. 37 recent study was conducted on a total of 300 cow samples from cases of mastitis, miscarriage, and recurrent breeding from both organized and unorganized farms in Jabalpur to check for leptospirosis.According to the findings, leptospirosis seroprevalence among suspected cases was 48.67%, and it was notably higher in the organized sector and with high seropositivity in older cattle. 38n another recent study conducted on canines in Gujarat, 45 of 410 sera were found to be positive, showing a seroprevalence of 10.98%, with serovarPyrogenes being the predominate serotype.According to MAT, ImmunoComb, and PCR, the total prevalence of leptospirosis was 16.59%. 39n the basis of our meta-analysis, the prevalence of leptospirosis was found to be highest in Andhra Pradesh (64.71%), followed by Kerala (60.5%),Chhatisgarh (54.7%), and Odisha (52.26%).The lowest prevalence of the disease was found in Assam (0.9%) and Haryana (1.4 %) (Figure 2).
The prevalence % of leptospirosis in humans is 9.47% as seen in Table .This disease is most prevalent in coastal areas.The reported incidence of human leptospirosis state-wise in India is described below.

Kerala
Leptospirosis is common and endemic in most parts of Kerala.The Autumnalis, Australis and Icterohemorrhagiae, Canicola, Pomona, Shermani are the common serogroups.A study found that leptospirosis is also occupation-related, as it is common in oyster shell catchers (82%).There was an increase of 74 % in the monsoon and a sharp rise in cases in some areas where the disease was not found earlier, such as Kolenchery, a midland, and leptospirosis was rare before 1987. 23,40A postflood outbreak of human leptospirosis in Kerala claimed about 70 lives in 2018. 41

Andaman and Nicobar Islands
Andaman and Nicobar Islands have reported leptospirosis since the early part of the 20 th century and are endemic for leptospirosis.
There have been reports of outbreaks of Andaman hemorrhagic fever (AHF) since 1988.The disease surveillance system recorded 544 cases and 93 deaths in the Andamans between 2000 and 2004, with 2002 having the highest incidence. 42Five hundred twenty-four cases of AHF were reported from 1988-97. 23,43,44

Maharashtra
Leptospirosis in Maharashtra has been regularly reported Karande et al. 45 and Karande et al. 46 In 2005, because of the large outbreak during monsoon season and floods, 2355 cases and 167 deaths were reported.In Mumbai, during the course of ten days in July 2015, the Mumbai Corporation reported 15 leptospirosis fatalities after massive floods. 47

Gujarat
The disease is endemic in Gujarat, with the highest number of cases in Valsad, Navsari, and Surat since 1994. 48,49Situations such as heavy rainfall, high water table, and clay soil in south Gujarat favor endemicity for Leptospirosis. 42

Tamil Nadu
There have been reports of leptospirosis from Chennai since 1980s. 50,51To study leptospirosis, a laboratory at the Madras Medical College was established in 1994. 42In a study,a total of 2035 patient serum samples were tested for the presence of specific IgM antibodies using the Panbio Leptospira IgM ELISA kit to determine the seroprevalence of leptospirosis over a 10-year period from 2011 to 2021 in Chengalpattu district, Tamil Nadu, which revealed aprevalence rate of 9.14%. 52

Karnataka
In Karnataka, 152 cases and 11 fatalities were reported in 2004 while 224 cases and 19 fatalities were reported in 2005. 42In another study from the state, leptospirosis was shown to be substantially connected with environmental factors, proximity to an open sewer, occupational factors, the existence of skin cuts, direct contact with mud while working, and the presence of rats or rodent habitats, according to a populationbased case-control study in the Kodagu district. 53

Odisha
In Odisha, 142 patients with febrile sickness and hemorrhagic symptoms were assessed during October and November 1999, following the cyclone.Incidences of disease shoot-up during the rainy season.When tested, 28 (19.2%) were found positive for leptospirosis by MAT and 6 were positive by PCR/culture. 54

Other States
Data from other Indian states is reviewed as follows.The evaluation of acute febrile patients in Uttar Pradesh revealed that 7% had leptospirosis (25/ 346), and 17 of the 25 patients had jaundice. 55imilarly, in a study of 55 cases of leptospirosis in Hyderabad, 52% had renal failure, and 42% had jaundice.Apart from these, leptospirosis has also been reported in Rajasthan, Uttarakhand, Punjab, and Haryana.
Govt of India launched a pilot project on prevention and control of leptospirosis under the 11 th five-year plan, which in the next five-year plan launched a scheme called "Prevention and Control of Leptospirosis."In 2019, the National Center for Disease Control (NCDC) issued an advisory notice and preparatory checklist to control leptospirosis, especially during the outbreak in affected districts (https://ncdc.gov.in/showfile.php?lid=390).

Diagnosis of leptospirosis
Laboratory support is required for the diagnosis to find the serovar responsible for causing specific infection, a possible source of infection, location, and potential reservoir.The tests performed for leptospirosis are isolated by culture and PCR, while serological tests can be used for the detection of antibodies.For the confirmation of diagnosis, isolation of bacteria is the most definite way.However, blood culture takes much time and is not suitable for an early diagnosis, but it is considered valuable in patients who are at risk of losing life in the first week before developing antibodies.An advanced technique like PCR is promising, and it is sensitive and specific. 56,57he serological microscopic agglutination test (MAT) is the gold standard for the diagnosis.The main advantage of MAT is identifying serovar of epidemiological importance. 58Leptospires are cultured, and an agglutination reaction is carried out in laboratories to detect the antibodies.MAT is laborious and is not an economical method, and needs live bacterial antigen for the test.Because of these factors, MAT is conducted only in particularly designated labs. 59Various approaches to detect leptospirosis are presented in Figure 3.

Leptospirosis research at IIT Guwahati
Molecular characterization for a comprehensive understanding of the CRISPR/ Cas system of Leptospira is being carried out at the Indian Institute of Technology, Guwahati.The long-term goal of the lab is to utilize the outer membrane proteins (OMPs) of the spirochete to develop a rapid diagnostic assay and vaccine to control the loss incurred by the disease.The overall goal is to elucidate the role of Cas proteins (Cas 6/7/8) in CRISPR adaptive immunity of the spirochetes and develop a CRISPR-Cas, an emerging microbial adaptive immune defense system of microbes (including spirochetes) against the foreign genetic elements that were being transferred horizontally from viruses or plasmid.Recent studies (from the lab of Manish Kumar, the 3 rd author) include transcriptional analysis of CRISPR 1-B array of L. interrogans, 60 assembly of Cas7 subunit of Leptospira, 61 and Cas2 protein of subtype I-C. 62

Leptospirosis research at the vet versity
The Guru Angad Dev Veterinary and Animal Sciences University started working on leptospirosis in 2018, collaborating with IIT Guwahati, a joint collaborative project funded by the Department of Biotechnology-North East Region (DBT-NER).
In 2018, DBT launched a DBT-Canine Research Center and Network, and univeristy is a collaborating institution of this mega project.The overall goal of this mega project was focused on the development of canine diagnostics and vaccines, under which a lab on leptospirosis has been established at the College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University.Under this project, a dedicated Bio-safety lab for diagnosing leptospirosis has been set up for serological diagnosis.Detection of anti-leptospiral antibodies using the Microscopic agglutination test (MAT) and molecular detection by PCR is carried out, in which about 10 percent seropositivity was found in animals using microscopic agglutination test. 63he samples from various parts of the state are coming for the diagnosis of leptospirosis.
Only a few referral labs maintain the leptospiral serovars because there is no longterm preservation method, and it has to be continuously sub-cultured every week.There is a high risk of contamination and loss of culture.We maintain reference cultures obtained from ICAR-NIVEDI and seven reference serovars that are commonly prevalent in domestic species in India, i.e., Australis, Autumnalis, Canicola, Hardjo, Icterohaemorrhagiae, Pomona, and Pyrogenes are being maintained.These reference cultures are used as antigen panels in the MAT and positive control in the molecular detection of leptospires in samples by PCR.

CONCLUSION AND FUTURE PROSPECTIVE
Due to the wide distribution of the disease in domestic and wild animals, prevention and control of leptospirosis are difficult.Control of leptospirosis includes identification of infected and carrier animals, immunization through vaccination, suitable drainage systems, and disinfection of the contaminated source of water.In the future, the adoption of prevention strategies, immunization of susceptible populations using current or nextgeneration vaccines, and timely reporting of outbreaks could mitigate disease incidences.

Figure 1 .
Figure 1.A flow diagram of transmission of Leptospira and organisms in humans and animals showing the zoonotic importance of the disease.Bacteria are shed into the water and soil from the mammalian reservoir host from where humans and animals (cattle, dogs, pigs, horses, and others) pick infection, and further shed organisms in the urine.Pathogen-containing urine further contaminates water and soil.Leptospirosis is considered an occupational health hazard because the farmers, personnel working in the slaughterhouse, veterinarians and other workers coming in close proximity to infected animals are prone to get the disease.

Figure 2 .
Figure 2. State wise prevalence of leptospirosis in India evidenced by the data published from 2005-2023.Data analysis showed that coastal states of India like Andhra Pradesh (64.7%),Kerala (60.6%),Orissa (52.3%),Tamil Nadu (45%), Andaman Nicobar (42.15%) have a high prevalence of leptospirosis.High to the low prevalence of the disease is indicated by the red to green spectrum of color.White color indicates insufficient or no data available for the state.

Figure 3 .
Figure 3. Various approaches to detect leptospires in the blood and urine of humans and animals.PCR is a rapid test for the detection of the nucleic acid of leptospires in blood and urine.In contrast, MAT is the standard gold method for the detection of antibodies in the serum.Isolation and identification of Leptospira species are done by the culture of samples (blood, urine, CSF).