Prevalence of Leptospira spp. in Urine of Rats (Rattus spp.) in an Urban Village in the Philippines using LAMP and PCR Assays
Abstract
Rats are recognized as key reservoirs and potential transmission sources of leptospirosis. Despite this, limited data exist on the prevalence of Leptospira spp. among rats in urban villages of the Philippines. This study aimed to assess the presence of Leptospira spp. in rats from an urban village in Benguet Province. Urine samples from 50 rats were analyzed using Loop-Mediated Isothermal Amplification (LAMP) and Polymerase Chain Reaction (PCR) assays. Results showed a Leptospira spp. detection rate of 30% using LAMP and 18% using PCR. DNA sequencing confirmed the presence of Leptospira interrogans serovar Icterohemorrhagiae, suggesting that rats may playa significant role in leptospirosis transmission in the study area. Diagnostic performance analysis revealed that LAMP and PCR had substantial agreement, with a Kappa coefficient of 0.677. Compared to PCR, LAMP demonstrated a diagnostic sensitivity of 100% and a specificity of 85.37%. These findings underscore the importance of continuous monitoring of rodent populations in urban settings and highlight the potential utility of LAMPas a rapid screening tool for leptospiral infections.
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Introduction
Leptospirosis is a widespread bacterial zoonosis and a significant public health concern in Southeast Asia, including the Philippines. It is recognized both as a neglected zoonotic disease (NZD) and a neglected tropical disease (NTD), with increasing reports of outbreaks and severe cases globally. The disease primarily affects impoverished urban and rural populations, particularly in environments that support rodent-borne transmission.
Globally, leptospirosis accounts for over one million severe human cases annually, with the Philippines ranked among the countries with high incidence rates. Leptospirosis is endemic throughout the country and poses a serious health threat, especially in densely populated and flood-prone urban areas. Although most reported cases are concentrated in Regions VI, III, and the National Capital Region, the Cordillera Administrative Region (CAR) reports the highest case fatality rate (CFR) at 15%, despite a lower number of cases. This highlights the need for targeted surveillance and control strategies in this region. Rats are the primary reservoir hosts for pathogenic Leptospira spp., playing a critical role in the transmission cycle. Previous studies have detected multiple Leptospira serovars in both humans and rats in the Philippines, suggesting zoonotic transmission. However, data on the status of Leptospira infection in rats, particularly in urban villages of La Trinidad, Benguet, remain scarce.
This study aims to determine the prevalence of Leptospira spp. in rats in an urban village in Benguet Province using molecular detection techniques—Loop-Mediated Isothermal Amplification (LAMP) and Polymerase Chain Reaction (PCR). The study also evaluates the diagnostic agreement and performance of LAMP relative to PCR. By identifying the circulating Leptospira strains and estimating infection rates in rats, the study contributes valuable baseline data that may inform local public health interventions and surveillance strategies.
Conclusion
This study is the first to molecularly detect the presence of Leptospira spp. in rats within an urban barangay in La Trinidad, Benguet, Philippines, providing novel insights into the epidemiology of leptospirosis in this area. The use of LAMP and PCR assays revealed that 30% and 18% of the 50 captured rats tested positive for Leptospira DNA, respectively. LAMP demonstrated higher sensitivity, effectively identifying positive samples with low DNA concentrations that PCR failed to detect. Diagnostic evaluation of LAMP against PCR indicated a sensitivity of 100% and specificity of 85.37%, with substantial agreement between the two assays (κ = 0.677), highlighting LAMP'spotential as a practical and reliable diagnostic tool, particularly in low-resource settings and field conditions.
One of the most significant findings of this study was the identification of Leptospira interrogans serovar Icterohaemorrhagiae through DNA sequencing. This highly pathogenic strain is known to cause severe leptospirosis in both humans and animals. The identification of this serovar in rats suggests that it may be circulating within the local rodent population, particularly Rattus norvegicus, which are likely the primary reservoirs of the pathogen. Although sequencing was conducted on only one strongly positive sample, the result is consistent with previous reports of Icterohaemorrhagiae as a common cause of zoonotic transmission through rats.
The relatively high prevalence of Leptospira spp. detected in this study may be influenced by environmental and geographical factors in Betag, a low-lying area with relatively flat terrain, which may promote water stagnation and the persistence of leptospires, especially during the rainy season when the study was conducted. These conditions, combined with agricultural activities and backyard livestock farming, increase the risk of human and animal exposure to contaminated water and soil, underscoring the need for improved environmental management.
The findings of this study offer significant contributions to our understanding of the role of rats as reservoirs of Leptospira spp. in urbanizing agricultural settings and suggest a potentially underestimated public health threat in La Trinidad. However, there are limitations in this study, such as the small sample size and the lack of analysis of rat species, age, and sex, which have been shown to influence the carriage of Leptospira (Ivanova et al., 2012; Levett, 2001). Future studies with larger sample sizes, incorporating these variables, and including human and other animal populations, would provide a more comprehensive understanding of the transmission dynamics of leptospirosis in the region.
Based on the results of this study, several potential applications emerge. First, LAMP’shigh sensitivity and practical applicability in low-resource settings suggest it could be widely used for early diagnosis and surveillance of leptospirosis in rural and urban settings. This diagnostic approach could prove invaluable for rapidly detecting and controlling outbreaks, especially in areas where PCR testing is not available. Additionally, the identification of Leptospira interrogans serovar Icterohaemorrhagiae as circulating in rats underscores the need for ongoing molecular surveillance of the pathogen in rodent populations, which could help monitor the spread of this zoonotic disease. The use of LAMP in regular monitoring programs, alongside molecular surveillance of different Leptospira serovars, would help public health authorities assess and manage the risk of leptospirosis more effectively, ultimately contributing to better prevention and control strategies in endemic regions. In conclusion, the results of this study highlight the need for continued vigilance and action to mitigate the risk of leptospirosis in La Trinidad, Benguet, and similar urban agricultural settings. The use of LAMPas a diagnostic tool, along with improved rodent control, environmental management, and public health education, will be crucial in reducing the burden of leptospirosis in both human and animal populations. Future research with larger sample sizes and broader surveillance will be essential for further elucidating the transmission dynamics of Leptospira spp. and for developing effective control strategies. RECOMMENDATIONS Based on the findings of this study, several recommendations are made to mitigate the risk of leptospirosis transmission in La Trinidad, Benguet, and similar areas. First, effective rodent control measures should be implemented in both urban and agricultural areas, including targeted rodent trapping, improved environmental sanitation, and better waste management, to reduce the rat population, which serves as a significant reservoir for Leptospira. Additionally, improving flood control infrastructure and drainage systems is crucial, as the low-lying terrain and flooding in the study area may contribute to the persistence of leptospires in the environment. This can help minimize standing water, a key habitat for the survival of Leptospira, and reduce the risk of environmental contamination.
Community awareness and education are also critical. Local public health campaigns should focus on educating people, particularly those working in agriculture and areas with high rat populations, about the risks of leptospirosis and preventive measures, such as using protective gear, properly disposing of waste, and avoiding contact with potentially contaminated water sources. Regular surveillance of rodent populations is also recommended to monitor the spread of leptospirosis and identify areas of higher risk. Rapid diagnostic methods such as LAMP can be used for ongoing monitoring to guide public health responses more effectively.
Given the potential for leptospirosis transmission to livestock and domestic animals, veterinary monitoring and health programs should be strengthened. Livestock owners should be educated about the risks of leptospirosis and encouraged to adopt preventive measures to protect both animals and humans. Furthermore, continued molecular surveillance is essential to explore the full range of Leptospira serovars circulating in the area, providing valuable information for targeted diagnostic and control strategies. Sequencing additional positive samples will deepen our understanding of the strains in circulation and help inform future public health interventions.
In addition, future research should consider including variables such as the species, age, and sex of rats, as these factors have been shown to influence the likelihood of Leptospira carriage (Ivanova et al., 2012; Levett, 2001). Incorporating these variables would provide more detailed insights into the epidemiology of leptospirosis and improve understanding of how the disease is transmitted in urban and agricultural environments. Furthermore, studies with larger sample sizes, including both humans and other animals, would be beneficial to determine the full transmission cycle of leptospirosis and assess the status of this zoonosis. Lastly, a One Health approach that integrates human, animal, and environmental health sectors should be adopted. This approach would facilitate collaboration between local health authorities, agricultural departments, environmental agencies, and the community, ensuring a coordinated response to leptospirosis and enhancing overall public health management. By addressing these recommendations, local authorities can better manage and reduce the risk of leptospirosis in both human and animal populations, ultimately improving public health in La Trinidad and surrounding areas.
