Emerging evidence of sand fly fever virus transmission in Sri Lanka

Abstract

BACKGROUND

Sand fly fever virus (SFFV) infection is an acute self-limiting febrile illness caused by phleboviruses and transmitted by sand flies of the Phlebotomus species. The disease has been reported in the Mediterranean region, North Africa, the Middle East, and the Indian subcontinent, and is now considered to be expanding into new geographic areas. Clinically, SFFV infection closely resembles acute dengue fever.

AIM

To detect other possible infection including SSFV in patients with clinically suspected dengue fever with negative serology results for NS1 antigen.

METHODS

In this study, a total of 757 serum samples were collected from patients clinically suspected of dengue fever and admitted to three major hospitals in Sri Lanka between January and August 2023. Patients were from the Colombo and Kandy District, and demographic information was obtained from laboratory request forms, although detailed individual-level demographic data were limited due to anonymization in this laboratory-based study. All samples were initially screened for dengue NS1 antigen negativity and further tested for a panel of pathogens including SFFV immunoglobulin (Ig) M, hantavirus IgM, chikungunya IgM, and leptospira IgM antibodies, as well as molecular detection of hantavirus, flavivirus, and alphavirus RNA.

RESULTS

From tested samples, two samples were serologically positive for SFFV-specific IgM using a commercial immunoblot assay, while all other tests for dengue, chikungunya, leptospira, hantavirus, and related RNA viruses were negative. These two SFFV-positive cases were from Kandy District and included one child (6-8 years) and one adult (40-50 years), both presenting with uncomplicated dengue-like illness.

CONCLUSION

This study provides the first evidence of SFFV infection in Sri Lanka. Given the high endemicity of dengue in the country, SFFV infections may be clinically misdiagnosed as dengue and therefore remain under-recognized. Further studies involving febrile patients and sand fly vector surveillance are required to determine the true burden and epidemiological significance of SFFV in Sri Lanka.

Key Words: Sand fly fever virus; Sri Lanka; Dengue; Acute febrile illness; Sandfly

Core Tip: Sand fly fever virus (SFFV) infection is an acute self-limiting febrile illness caused by Phleboviruses and transmitted by sand flies of the Phlebotomus species. Clinically, SFFV infection closely resembles acute dengue fever and specific laboratory tests are required to diagnose SSFV. In Sri Lanka, a certain proportion of clinically suspected dengue patients were negative for dengue by laboratory investigation. We conducted this study to detect other possible pathogens in a group of patients admitted to hospitals with clinically dengue like illness. Seven hundred and fifty-seven patients’ samples were tested and two samples were positive for immunoglobulin M antibodies against SFFV, indicating recent or acute infection. The clinical and laboratory features of these patients were similar to those of uncomplicated dengue fever. Both patients were tested negative for flavivirus, alphaviruses, hantaviruses and leptospirosis. This study provides the first evidence of SFFV infection in Sri Lanka. As dengue fever is highly endemic in the country, SFFV infections may often be misdiagnosed as dengue and remain undetected. Further studies on febrile patients and sand fly vectors are needed to determine the disease burden and support effective control measures.

INTRODUCTION

Acute febrile illnesses are among the most common clinical presentations in tropical regions[1]. Sri Lanka is considered hyper-endemic for dengue fever, and most patients are clinically diagnosed. However, a significant proportion of patients suspected of having dengue test negative for dengue virus infection in laboratory investigations[2]. Other infections such as Zika, chikungunya, leptospirosis, hantavirus, rickettsioses, and other unidentified pathogens can clinically mimic acute dengue fever[1-5].

Sand fly fever virus (SFFV) is transmitted by sand flies of the Phlebotomus species. It belongs to the Phenuiviridae family, genus Phlebovirus, and includes several identified viruses such as sand fly fever Naples, Toscana, and Sicilian virus complexes[6-8]. Cases and outbreaks have been reported from the Mediterranean region, Europe, North Africa, the Middle East, and the Indian subcontinent, with recent evidence suggesting geographic expansion[8-10].

The Phlebotomus sand fly, which also transmits Leishmania, an emerging public health concern in Sri Lanka[8,11], acts as the vector for SFFV. Once infected, sand flies remain carriers for life, and transovarial transmission has been documented[7].

SFFV infection is an acute, self-limiting febrile illness characterized by high fever, headache, myalgia, arthralgia, and retro-orbital pain. Some patients may develop aseptic meningitis with mild neck stiffness. Most recover without complications[7,12,13]. Due to short viremia, diagnosis primarily relies on detecting specific immunoglobulin (Ig) M or IgG antibodies during the symptomatic phase[7]. No specific antiviral therapy exists, and management is supportive. No animal reservoirs have been identified to date, though serological evidence in animals has been reported[14].

This study presents the first serological evidence of SFFV infection in Sri Lanka, detected among patients clinically suspected patients having dengue fever.

MATERIALS AND METHODS

Study design

This was a retrospective, cross-sectional, observational study.

Ethical approval

This study protocol was approved by the Research and Ethics Committee of the National Hospital Kandy, Kandy (No. NHK/ERC/13/2023) and Ethic Review Committee, Medical Research Institute Colombo (No. ERC/14/2022). Informed consent was waivered due to retrospective nature of the study and data dissemination without any personal identification data.

Samples and study population

A retrospective analysis was carried out using blood samples that tested negative for the dengue NS1 antigen. These samples were collected from patients clinically diagnosed with dengue fever, based on the World Health Organization case definition, within the first five days of illness[15]. Samples originated from three major hospitals: (1) Lady Ridgeway Hospital for Children; (2) The National Institute of Infectious Diseases (Angoda) in Colombo District; and (3) The National Hospital Kandy in Kandy District. Samples were collected between January and August 2023. Patients were diagnosed by the treating physicians, and blood samples were referred to the respective virology or microbiology laboratories for dengue NS1 testing.

Routine dengue NS1 antigen testing was performed using a locally validated commercial rapid immunochromatographic test (STANDARD Q Dengue NS1 Ag ICT, Cat. No. 09DEN10D, SD Diagnostics, South Korea). The remaining serum samples were stored at -80 °C with continuous temperature monitoring.

Clinical and laboratory details were extracted from the laboratory investigation request forms.

Detection of SFFV IgM antibodies

Samples were tested for IgM antibodies against hantaviruses using a commercial immunoblot assay (recomLine HantaPlus IgM, Cat. No. 7673, Mikrogen, Germany). According to the manufacturer’s information, this assay also detects IgM antibodies against SFFV, as it contains complete nucleocapsid antigens from Toscana and Sicilian viruses.

Testing for other possible pathogens

All samples were further analyzed for hantavirus RNA using a locally validated in-house polymerase chain reaction[16], flavivirus RNA[17], alphavirus RNA[18], and for IgM antibodies to dengue (ELISA, Cat. No. 07DEN30, SD Diagnostics, South Korea), chikungunya (Chikungunya IgM Microlisa, J. Mitra & Co. Pvt. Ltd., India), and leptospira (Panbio Leptospira IgM ELISA, Abbott).

Statistical analysis

As this was an exploratory observational study intended to identify evidence of SFFV exposure among patients with dengue-like illness, no formal statistical analyses were performed. The study primarily reports descriptive laboratory findings and clinical characteristics of the identified positive cases.

RESULTS

Out of 757 tested samples, two samples were positive for SFFV IgM and negative for hantavirus IgM (Figure 1). Assay was repeated and confirmed the results. Both samples also tested negative for hantavirus, flavivirus, and alphavirus RNA, as well as for dengue, chikungunya, and leptospira IgM antibodies.

Figure 1 Photographs of immuno-blot assays conducted during this study. Top immuno-blot strip (LHAM 14) was positive for sand fly fever virus immunoglobulin M (IgM), middle one (LHAM 20) positive for hantavirus IgM and bottom one (LHAM 17) was negative for hantavirus and sand fly fever virus IgM; with the interpretation diagram given by the manufacture. Ig: Immunoglobulin.

The two SFFV IgM-positive samples were from Kandy District in central Sri Lanka (Figure 2), one from a child (6-8 years) and one from an adult (40-50 years). Both patients presented with typical dengue-like symptoms, including high fever, severe headache, and retro-orbital pain, and recovered uneventfully under standard dengue management protocol[19]. Mild leukopenia and thrombocytopenia were observed in both cases, but there were no signs of haemorrhage, shock, or organ failure (Table 1). Both samples also tested negative for hantavirus, flavivirus, and alphavirus RNA, as well as for dengue, chikungunya, and leptospira IgM antibodies (Table 2).

Figure 2  Map of the Sri Lanka, indicating Kandy District (in red color) where sand fly fever virus patients were located.

Table 1 Summary of demographic characteristics, clinical manifestations, and laboratory investigation results of patients seropositive for sand fly fever virus immunoglobulin M antibodies.

Parameter	Patient 1	Patient 2
Serial number	1445	1450
Age	40-50 years	6-8 years
Clinical features	Fever, headache, retro-orbital pain, joint pain, myalgia, vomiting	Fever, headache, retro-orbital pain, joint pain, vomiting
Total WBC count	4100 cells/μL	4700 cells/μL
Lowest platelet count	160000 cells/μL	142000 cells/μL
Packed cell volume	No increase	No increase
SGPT/SGOT	Mild elevation	Mild elevation

WBC: White blood cell; SGPT: Alanine transaminase; SGOT: Aspartate transaminase.

Table 2 Laboratory test results of the two sand fly fever virus immunoglobulin M-positive cases.

Test performed	Case 1 (child, 6-8 years)	Case 2 (adult, 40-50 years)
SFFV IgM antibody	Positive	Positive
Hantavirus IgM antibody	Negative	Negative
Dengue IgM antibody	Negative	Negative
Chikungunya IgM antibody	Negative	Negative
Leptospira IgM antibody	Negative	Negative
Hantavirus RNA	Negative	Negative
Flavivirus RNA	Negative	Negative
Alphavirus RNA	Negative	Negative

SFFV: Sand fly fever virus; IgM: Immunoglobulin M.

DISCUSSION

Acute febrile illnesses in Sri Lanka are most commonly attributed to dengue virus infection, which remains the leading cause of hospitalisation for undifferentiated fever in the country, with periodic epidemics contributing to a substantial disease burden. In addition to dengue, other viral pathogens such as chikungunya virus, leptospirosis-associated febrile illness, and sporadic cases of hantavirus, Zika virus infections have also been reported, although at significantly lower frequencies compared to dengue[20-23]. Previous hospital-based studies in Sri Lanka have demonstrated that a considerable proportion of patients presenting with dengue-like illness test negative for dengue virus, suggesting the presence of other undiagnosed viral or zoonotic infections contributing to the febrile illness spectrum. Similar observations have been reported in South Asia, where a substantial fraction of acute undifferentiated fever cases remain etiologically unresolved despite routine testing for common arboviruses[20]. In this context, the detection of SFFV IgM in patients clinically suspected of dengue fever highlights the possibility of an additional, previously unrecognised viral contributor to acute febrile illness in Sri Lanka, warranting further systematic surveillance and broader differential diagnostic approaches in future studies.

This study provides the first evidence of SFFV infection in Sri Lanka. The detection of SFFV-specific IgM indicates an acute or recent infection. Although IgM antibodies can persist for three to four months following infection, the absence of positive results for other common dengue-like illnesses supports the classification of these cases as probable acute SFFV infections. Serological cross-reactivity among Bunyaviruses, including hantaviruses, is possible[24]; however, neither of the positive samples showed reactivity with hantavirus antigens. Moreover, Rift Valley fever and Crimean-Congo hemorrhagic fever, two other Bunyaviridae-related infections, have not been reported in Sri Lanka or India, supporting the specificity of the results.

Although SFFV infection is self-limiting, it can cause significant morbidity and has the potential to cause outbreaks in areas where sand flies are abundant[25]. The study had several limitations. Convalescent-phase samples for IgG testing were not available. Due to the retrospective design and ethical constraints, detailed clinical and demographic data could not be obtained. SFFV RNA detection by polymerase chain reaction was not included because the viraemic phase is typically short. The exact SFFV strain could not be determined by the serological assay used, and neutralization testing would be required for strain identification[26].

CONCLUSION

In conclusion, this study demonstrates the first serological evidence of SFFV infection in Sri Lanka. Given the hyper-endemicity of dengue fever in the country, SFFV infections may often be misdiagnosed as dengue and therefore remain undetected. Further investigations on febrile patients and entomological studies on sand fly populations are essential to assess the disease burden and to support the implementation of effective control and surveillance strategies in Sri Lanka.

ACKNOWLEDGEMENTS

We acknowledge the Director, National Hospital Kandy grant permission to publish this data and Dr. Ishani De Silva to helping out trace the laboratory and clinical records.