TOP FOUR NEGLECTED RODENT-BORNE PATHOGENS IN INDONESIA

By: Upi Nurhayati, Aly Diana, Dewi Lokida, Adhella Menur

Revisiting the top four rodent-borne pathogens (Yersinia pestis, Leptospira spp., Rickettsia typhi, and Seoul Orthohantavirus) in Indonesia: neglected yet significant *Plus, a report from dr. Dewi Lokida, Sp.PK (K) supervision on the PESTO-RITA research in Pasuruan District, East Java

Each year, infectious diseases kill about 7 million people, devastating global health and creating significant economic losses. Zoonotic infections, which are naturally transmitted from vertebrate animals to humans, account for 70% of emerging infectious diseases. Rodents are well-known hosts and vectors for zoonotic infections, representing about 43% of all mammalian species worldwide. There are around 2,375 living species, spanning mice, rats, squirrels, hamsters, voles, beavers, chipmunks, capybara, and more. Rodents are being introduced from continent to continent along with human migration and trade. They are highly adapted to modified environments, making them one of the best-suited mammals for living in various habitats. Their capability to adapt within a relatively short period is due to their accelerated evolution, where the genomes evolve 4 to 6 times faster than primates. Interestingly, rats and mice are highly intelligent rodents. They are natural students who excel at learning and understanding concepts.

The combination of abundant food, global warming, and deforestation, which drove them to mi-grate from their natural habitats to human settlements, and a rapid reproduction rate has resulted in rodent’s explosion and increasing the risk of human exposure to the pathogens that rodents carry. Rodent-borne diseases can be spread via two different pathways: direct and indirect. Ro-dents can spread pathogens directly to humans, e.g., by biting them or because humans consume food products or water contaminated with rodent feces. Moreover, humans can encounter surface water contaminated with rodent urine or inhale germs from rodent excrements. Rodent-borne pathogens can also be spread indirectly to humans. They can serve as amplifying hosts of the pathogens and can bring them into contact with humans through ectoparasitic arthropod vectors (ticks, mites, and fleas). The rodent population in Indonesia is growing and becoming more brazen. So far, about 171 species of rodents have been identified in the country. The “commensal rodents” live near human and their surroundings, such as Rattus norvegicus (sewage rat), R. rattus diardii (roof rat), Mus musculus (house mouse), R. rattus brevicaudatus (ricefield rat), R. rattus roquei (wood rat), and R. exulans (polynesian rat). They can carry at least 85 unique zoonotic diseases from bacterial, viral, parasitic, and fungal infections. This article will briefly discuss the top four rodent-borne pathogens: Yersinia pestis, Leptospira spp., Rickettsia typhi, and Seoul Orthohantavirus, which are prevalent in Indonesia.

The first INA-RESPOND study, titled Acute Febrile Illness Requiring Hospitalization (AFIRE) and conducted from 2013-2016, revealed the critical problem of misdiagnosing rodent-borne diseases in Indonesia. The study recruited febrile subjects hospitalized at the country’s eight largest hospitals. Of the 156 rodent-borne disease cases identified (103 rickettsioses, 51 leptospirosis, and 2 Seoul ortho-hantavirus infections), 134 patients (85.9%) were misdiagnosed as other tropical diseases. Conse-quently, 48 subjects received treatment for Salmonella typhi, 33 for dengue virus, and 53 for other bacterial/ viral infections during their hospitalization.

Yersinia pestis, a member of the family Enterobacteriaceae, causes Plague disease with a case fatality rate (CFR) between 30–100% if left untreated. Plague is one of the most infamous and feared diseases, having caused three pandemics – the Justinian Plague in the 6th century, the Black Death in the 14th century, and another in China in the late 19th century – and resulted in more than 150 million deaths worldwide. People infected with Y. pestis often develop symptoms after an incubation period of one to seven days. Plague manifests itself in three main clinical syndromes: 1) bubonic Plague, 2) septicemic Plague, and 3) pneumonic Plague, which is transmissible from human-to-human. Bubonic Plague is the most common form and is characterized by painful swollen lymph nodes or ‘buboes’. Septicemic Plague presents as hypotension and shock without a bubo, and pneumonic Plague patients demonstrate high fever, cough, chest pain, and hemoptysis. Confirmation of Plague requires lab testing (staining, culture, molecular, and serology). The best practice is to identify Y. pestis from a sample of pus from a bubo, blood or sputum. There is a laboratory-validated antigen rapid dipstick test now widely used in Africa and South America, with the support of the WHO. Early antibiotics are the cornerstone of effective treatment, with aminoglycosides being considered first-line treatment. Alternative treatments include doxycycline, tetracycline, and levofloxacin. Vaccines to prevent Y. pestis infection exist, especially given concerns about the potential widespread dissemination of Plague via bioterrorism. However, the WHO only recommends vaccination for high-risk groups. Two types of Plague vaccines are currently used in various parts of the world. The live vaccine is derived from a Pgm2 attenuated strain, usually related to EV76, while the killed vaccine uses a for-malin-fixed virulent strain of Y. pestis. Natural transmission to humans remains a possibility in many regions of the world, where foci exist in infected rodent populations. Even today, there are an estimated 1000–3000 cases of the bubonic Plague each year worldwide, mainly in Africa, the Americas, and Asia. The three most endemic countries are the Democratic Republic of Congo, Madagascar, and Peru. In Indonesia, the main areas to monitor for Plague are villages located in the high valley that extends between the summits of two volcanoes. These fall into four foci which are in Selo and Cepogo in Boyolali District, Central Java; Cangkringan in Sleman District, Yogyakarta; Ciwidey in Bandung Regency, West Java; and Tutur Nongkojajar in Pasuran District, East Java.

Leptopira spp. are spirochete bacteria classified into L. interrogans (pathogenic) and L. biflexa (non-pathogenic). Human infections are caused by L. interrogans, of which there are over 300 known pathogenic serovars. It lives in the kidneys of natural hosts, predominantly in mammals, and is excreted with urine. Most infections caused by lepto-spires are either subclinical or of very mild severity and often overlap with other febrile illnesses such as dengue and typhoid. However, untreated cases are at an increased risk of progression to the severe manifestation of Weil’s disease (jaundice, renal impairment, and hemorrhages), or severe pulmonary hemorrhage syndrome (SPHS), which has a 10-70% CFR. Over 60,000 people die due to leptospirosis annually, and nearly one million are reported to be affected. Reliance on the Microscopic Agglutination Test (MAT), which detects serovar-specific antibodies and is the current gold standard for serology, is impractical due to technical requirements and expense. Since an accurate rapid diagnostic test (RDT) for leptospirosis is not available, molecular tests and ELISAs for IgM can be used to inform medical management in resource-limited settings. Doxycycline and azithromycin are the recommended treatments for leptospirosis, and empirical administration against suspected or probable leptospirosis cases may prevent the disease’s progression. Intravenous penicillin should be initiated for clinically severe forms of the disease, which may contribute to decreasing mortality. In most cases, ceftriaxone can be used as an alternative. Even after decades, we still do not have a universal vaccine for leptospirosis because the immune response is mainly dependent on serovar, does not cross-react, and is based on lipo-polysaccharide antigens. Several vaccine platforms have been widely used for many years to induce immunity in animals and humans, with limited success. In Indonesia, according to reports from 11 of 38 provinces, there were 1,408 cases of leptospirosis, with 139 fatal cases (CFR 9.87%) in 2022.

Rickettsia typhi is a gram-negative bacterium that causes murine or flea-borne typhus. It is one of the species from the Rickettsia genus. The genus is classified into four categories: the spotted fever group (SFG), the typhus group (which includes R. typhi), the ancestral group, and the transitional group. Murine typhus often goes unrecognized and is perceived as a clinically mild disease. However, untreated patients may have fever for 12–21 days and a 26% complication rate (including lung, central nervous system, and acute renal injury), indicating the pathogen’s substantial burden. There is a diverse range of presentations, from mild constitutional and gastrointestinal symptoms to severe sepsis-like physiology with multiorgan involvement. It is characterized by fever, headache, and macular rash, mainly on the trunk, along with a small black scab or eschar at the site of the insect bite, accompanied by local or general lymphadenopathy. The triad of fever, headache, and rash has historically been used as a clinical diagnostic tool; however, the triad is inconsistent and unreliable. Also, the rash appears an average of 5 days after the onset of symptoms, thus an unreliable early indicator. Ideally, laboratories should be equipped with valid diagnostic assays (PCR for molecular detection during acute illness and indirect fluorescent antibody (IFA) as the gold standard for serology). However, PCR and IFA have several disadvantages, including the need for an expensive thermal cycler or a fluorescence microscope. Therefore, ELISA can be an alternative when both are unavailable. The drug of choice for treating rickettsial infections is doxycycline; chloramphenicol and azithromycin are an option in the case of allergy and severe disease. Prevention relies on avoiding exposure to flea bites, mainly when residing or traveling to endemic areas, because no vaccine or prophylaxis is available. Murine typhus was first identified in Indonesia in 1951 from fever patients in Jakarta. The disease has been identified in Sumatera, Java, Bali, Sulawesi, Muna, Flores, Timor, and Papua. However, the epidemiology of human rickettsioses in Indonesia is still not well characterized.

Seoul Orthohantavirus (SEOV) is a (-) single-stranded RNA-enveloped virus and one of the species from the family Hantaviridae of order Bun-yaviridae. SEOV belongs to the “Old World” hantaviruses group along with Hantaan (HTNV), Dobrava -Belgrade (DOBV), and Puumala (PUUV), generally affect blood vessels and kidneys and can manifest as hemorrhagic fever with renal syndrome (HFRS). The other group, the “New World” hantaviruses, is generally associated with lungs and can manifest as hantavirus pulmonary syndrome (HPS). HFRS has become a significant epidemic, primarily in Asia and Europe. SEOV manifests as moderate HFRS and is associated with a mortality rate of ±1%. The disease has a 1 – 5-week incubation period, starting with a fever and influenza-like symptoms. Classically, HFRS occurs in five distinct phases: febrile (3-5 days), hypotensive (few hours to few days), oliguric (3-7 days), polyuric (1-2 weeks), and convalescent (3-6 months). The oliguric stage accounts for approximately one-half of all hantavirus-related deaths. Interestingly, in SEOV infection, gastrointestinal symptoms and elevated liver enzymes are prominent, but kidney dysfunction is mild, imitating the hepatitis virus. Dual infection with Leptospira spp. may occur since they need a similar rodentia as a reservoir. Evidence of viral antigen in tissue by immunohistochemistry (IHC), the presence of viral RNA in blood or tissue, or positive serological test (ELISA or IFA) result, with compatible history of HFRS, is considered diagnostic for the disease. The treatment of HFRS is mainly supportive and dependent on the clinical manifestations. Currently, no antivirals, vaccines, or immuno-therapeutics are approved by the U.S. FDA. Several inactivated vaccines have been generated from hantavirus in cell cultures or the rodent brain, and a few of these have been licensed for use in humans in Korea and China. The existence of Ortho-hantavirus in rodents in Indonesia was confirmed in 29 provinces based on MoH special research on vectors and reservoirs (Rikhus Vektora) in 2015-2018. About 16 Orthohantaviruses infections (by serology and/or molecular testing) in human manifested as HFRS have been reported since 1995. Three patients were confirmed as SEOV infection by sequencing, with one of the cases co-infected with Leptospira spp.

The top four rodent-borne pathogens are often overlooked due to their similar clinical manifestations with other tropical diseases, even though they can potentially cause considerable epidemics in human populations. The underreporting of cases is due to a lack of integrated surveillance, clinicians’ unawareness, unreliable RDTs, and limited access to standard diagnostics. Underdiagnoses could engender inappropriate management, treatment delays, prolonged hospitalization, and increased morbidity and mortality. Further comprehensive research is urgently needed in every aspect: epidemiological, diagnosis, treatment, and vaccine. One Health approach includes human, animal and environmental health must be considered to understand better the epidemiology, clinical burden, and transmission of these neglected yet significant diseases.

Supervision on PESTO-RITA research in Pasuru-an Regency, East Java

In 2023, The Eijkman Institute for Molecular Biology and The National Research and Innovation Agency (BRIN) granted support from the WHO to conduct research, namely “Development of Detection and Genomic Characteristics and Molecular Epidemiology of Plague, Leptospirosis, Rickettsioses, and Hantaviruses in East Java and Central Java” or PESTO-RITA research. The head of INA-RESPOND Reference Laboratory, dr. Dewi Lokida, Sp.PK(K) is appointed as the Supervisor. The PESTO-RITA research aims to identify the top four rodent-borne pathogens in humans, ectoparasites, and rodents in hot-spot areas in East and Central Java, Indonesia. Dr. Dewi is responsible for assisting in protocol development, specimen handling from collection to testing (molecular and IFA), and results analysis.

One of the PESTO-RITA research sites is Pasuruan District, East Java. There are Plague hot-spot areas under five Puskesmas (primary health centers/ PHCs) in Pasuruan District, i.e., Nongkojajar, Tutur, Tosari, Puspo, and Pasrepan, which are now have been updated as “Low List Extreme” by the WHO. Plague was brought into East Java by infected rats and fleas in cargoes of rice imported from Ran-goon (Myanmar) into the eastern seaport of Sura-baya in November 1910. From 1910 to 1920, a total of 240,375 Plague deaths were reported in Java. In 1987, there was a Plague outbreak in Tutur Nongkojajar subdistrict, Pasuruan District, in which 248 cases with 21 deaths were reported. In the same district, one case occurred in 1997, and Plague bacilli were isolated from rodents in one of the villages (Sulorowo village). The last human Plague case from Pasuruan District was reported in 2007, which caused one death of 82 suspects. The government collaborates with the people to strengthen rodent surveillance (rodent live-trapping and fleas’ identification) and human sur-veillance (active case finding). Fortunately, until now, there have been no reports of human Plague cases anymore. However, the surveillance should be consistently performed, as the absence of new Plague cases could also be a sign of a ‘silent peri-od,’ which may last as long as ten years or even longer, after which sudden explosions of rodents or human Plague may occur.

On 26th-28th June 2023, dr. Dewi visited Pasuruan District, East Java. She discussed research preparation with the Pasuruan Health Officials and field team from the PHCs and Hospitals. She had a chance to lecture about the PESTO-RITA briefly and presented INA-RESPOND AFIRE study results emphasizing rodent-borne pathogens detection. She also visited P2P BBTKLPP Surabaya Laboratory in Nongkojajar to supervise research testing preparation. The activity will continue until next years.

References

• Barazzone, et al, 2022. Revisiting the development of vaccines against pathogenic Leptospira: innovative approaches, present challenges, and future perspectives. Frontiers in immunology.
• BBLKPP Surabaya, 2023. Collaboration between BBKLPP Surabaya and BRIN on PESTO-RITA diseases molecular research. [Internet]. Available from: https://www.btklsby.go.id/
• Dillard RL, Juergens AL, 2023. Plague. In: StatPearls [Internet] . Available from: https://www.ncbi.nlm.nih.gov/books/NBK549855.
• Doppler JF, Newton PN, 2020. A systematic review of the untreated mortality of murine typhus. PLoS Neglected Tropical Diseases.
• Gasem MH, et al, 2020. An observational prospective cohort study of the epidemiology of hospitalized patients with acute febrile illness in Indonesia. PLoS Neglected Tropical Diseases.
• Gasem MH, et al, 2020. Leptospirosis in Indonesia: diagnostic challenges associated with atypical clinical manifestations and limited laboratory capacity. BMC infectious diseases.
• Griffiths J, et al, 2022. Survey of rodent-borne pathogens in Singapore reveals the circulation of Leptospira spp., Seoul hantavirus, and Rickettsia typhi. Scientific Reports.
• Jonsson CB, et al, 2010. A global perspective on hantavirus ecology, epidemiology, and disease. Clinical microbiology reviews.
• Liat, et al, 1986. Status of commensal rodent-borne diseases research in Indonesia. Buletin Penelitian Kesehatan.
• Lokida D, et al, 2020. Underdiagnoses of Rickettsia in patients hospitalized with acute fever in Indonesia: Observational study results. BMC infectious diseases.
• Lukman N, et al, 2019. A review of Hantavirus research in Indonesia: Prevalence in humans and rodents, and the discovery of Serang Virus. Viruses.
• Meerburg BG, et al, 2009. Rodent-borne diseases and their risks for public health. Critical reviews in microbiology.
• Pasuruan District Health Office, 2021. Even though 10 years with no Plague cases, Pasuruan District Health Office urges people to be aware of Plague. [Internet]. Available from: https://www.pasuruankab.go.id/
• Rabiee MH, et al, 2018. Rodent-borne diseases and their public health importance in Iran. PLoS neglected tropical diseases.
• Thybert D, 2019. Rodents are awesome: Extreme evolution. [Internet]. Available from: https://www.earlham.ac.uk/articles/rodents-are-awesome-extreme-evolution
• Sebbane F, Lemaître N, 2021. Antibiotic therapy of plague: A review. Biomolecules.
• Snowden J, Ladd M, King KC, 2023. Rickettsial Infection. In: StatPearls [Internet]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK431127/.
• WHO, 2006. WHO International meeting on preventing and control plague: the old calamity still has a future. Wkly Epidemiol Rec.
• WHO SEARO, 2002. Plague surveillance and outbreak response report of an informal intercountry consultation, Bangalore, India.
• Widianto E, 2020. Dealing with the COVID-19 Pan-demic, Learning from the Plague in Java. [Internet]. Available from: https://www.mongabay.co.id/