Detection of probable hantavirus infections in clinically suspected dengue patients in a tertiary care hospital in Sri Lanka

Abstract

Background

Acute febrile illnesses, including dengue fever, are common causes of hospitalization in Sri Lanka. However, a significant proportion of clinically suspected dengue cases tested negative for dengue-specific markers, raising concerns about alternative infectious causes such as hantavirus. This study aimed to detect hantavirus infections among patients clinically suspected of dengue who tested negative for dengue NS1 and IgM antibodies and to analyze the epidemiology of hantavirus infections in febrile patients.

Methodology

A descriptive cross-sectional study was conducted at the Department of Virology, National Hospital Kandy, from January to August 2023. A total of 415 serum samples from clinically suspected dengue patients who tested negative for dengue NS1 and IgM antibodies were selected. Hantavirus detection was performed using real-time RT-PCR and IgM antibody testing. Additional tests for flavivirus, alphavirus, and leptospirosis were also conducted. Demographic, clinical, and laboratory data were collected from hantavirus-positive cases.

Results

Among the 415 samples, 21 (5.1%) tested positive for hantavirus IgM antibodies. None of the samples tested positive for hantavirus RNA. The demographic analysis revealed no significant difference in age or gender between hantavirus-positive and negative patients. Co-infection with leptospirosis was observed in one patient. In addition to fever, clinical features of hantavirus infection included dry cough (41.2%), vomiting (35.3%), and shortness of breath (23.5%). Laboratory findings showed elevated liver enzymes (AST: 61.5%, ALT: 53.8%), elevated creatinine (50%), and elevated CRP (72.7%).

Conclusion

The putative 5.1% hantavirus seropositivity rate among clinically suspected dengue patients suggests that hantavirus should be considered in differential diagnoses. Although no hantavirus RNA was detected, the presence of IgM antibodies and negativity to other potential infections indicates a potential recent infection or probable cases. The study highlights the challenge of diagnosing hantavirus due to its similar presentation to dengue fever and the need for improved diagnostic testing. Early diagnosis and tailored management can improve patient outcomes and guide public health responses. Further studies and expanded diagnostic capabilities are recommended to better understand hantavirus prevalence and to improve clinical care for affected patients.

Background

Acute febrile illnesses are a leading cause of hospitalization in Sri Lanka [1]. Among these, dengue fever is frequently suspected in patients presenting with fever and thrombocytopenia [2]. Routine diagnostic tests, such as NS1 antigen detection and IgM/IgG antibody assays, are commonly used to confirm the diagnosis. However, laboratory data indicate that a significant proportion of suspected dengue cases test negative, raising concerns about alternative infectious causes, including leptospirosis, chikungunya, typhus, and hantavirus infections, which share similar clinical features [3].

The Department of Virology at the National Hospital Kandy receives approximately 300 samples each month from clinically suspected dengue patients, yet around 70% of these test negative for dengue. Despite this high percentage, these cases are not routinely screened for other potential pathogens, including hantavirus. In the South Asian region, a considerable number of clinically suspected dengue patients were negative for laboratory testing for dengue [4–6].

Hantavirus is an emerging zoonotic infection transmitted by rodents and has become a growing public health concern in various parts of the world, including South Asia [7]. The virus causes two distinct clinical syndromes: Hemorrhagic Fever with Renal Syndrome (HFRS) and Hantavirus Pulmonary Syndrome (HPS) [8]. In addition to these classical forms, hantavirus infection can also present with mild or atypical symptoms [9–11]. Clinical manifestations such as fever, thrombocytopenia, severe headaches, nausea, and vascular permeability changes often resemble dengue fever, leading to frequent misdiagnosis [12]. Notably, early-stage HFRS often presents as an undifferentiated febrile illness, further complicating clinical differentiation [13]. Importantly, the aggressive fluid management recommended for dengue is not suitable for hantavirus infection, highlighting the critical need for accurate diagnosis and tailored clinical management.

Though early identification of hantavirus cases is essential for improving patient outcomes and guiding public health interventions, hantavirus infection remains underdiagnosed in Sri Lanka due to limited routine diagnostic availability [10, 14].

This study aimed to detect hantavirus infections among clinically suspected dengue patients who tested negative for dengue NS1 antigen and dengue-specific IgM antibodies. By analyzing the demographic, clinical, and laboratory characteristics of hantavirus-positive cases, this research sought to address a critical gap in the understanding of hantavirus epidemiology in Sri Lanka. Early detection and accurate diagnosis of hantavirus infection can improve clinical management, reduce mortality, and develop more effective preventive strategies. Additionally, this study’s findings are expected to raise awareness among healthcare professionals and support the expansion of diagnostic facilities for hantavirus infections in Sri Lanka.

Methodology

This descriptive cross-sectional study was conducted at the Department of Virology, National Hospital Kandy. The study design was chosen for its simplicity and effectiveness in determining disease prevalence within a defined period.

The study population comprised clinically suspected dengue patients admitted to the National Hospital Kandy between January and August 2023. A total of 415 samples were selected through systematic sampling from patients who tested negative for dengue NSI and/or dengue-specific IgM by an immunochromatographic assay (STANDARD™ Q Dengue Duo, SD Diagnostics, South Korea). Patients included in the study were those presenting with fever and thrombocytopenia and clinically diagnosed with dengue fever by the treating physician according to the WHO case definition. Patients who tested positive for dengue NS1 antigen and/or dengue-specific IgM antibodies were excluded first and, then every. third sample with negative dengue serology was selected and samples with insufficient volume or clinical history was not included. Additionally, those with a confirmed diagnosis of another illness at the time of sample collection were also excluded.

After routine dengue NS1 and dengue-specific IgM testing, the remaining serum samples from suspected dengue cases were used for hantavirus detection through in-house real-time RT-PCR assay [15] and hantavirus-specific IgM antibody testing using a commercial immunoblot assay (Mikrogen recomLine HantaPlus IgM, Mikrogen, Germany). To rule out other viral and bacterial infections with similar clinical presentations, additional tests were conducted, including in-house RT-PCR assays for flavivirus RNA detection [16] and alphavirus RNA detection RT-PCR [17]. Furthermore, leptospirosis was excluded using real-time PCR following the World Organization for Animal Health (WOAH) guidelines and anti-leptospirosis IgM testing via a commercial ELISA kit (Panbio, Leptospira IgM ELISA, Abbott). For molecular assays, pathogen DNA/RNA was extracted and purified from blood samples by using commercial pathogen DNA/RNA extraction kit (Spin Star, ADT BioTec, Malaysia) according to manufacturer’s instruction.

Patient demographic information was retrieved from laboratory request forms, while clinical history and laboratory findings were extracted from hospital records for cases that tested positive for hantavirus by real-time RT-PCR or IgM detection. Data for each positive patient were recorded in an Excel sheet using laboratory reference numbers to maintain anonymity and confidentiality.

Ethical approval for the study was obtained from the Ethics Review Committees of the National Hospital Kandy (NHK/ERC/13/2023), Faculty of Allied Health Sciences, University of Peradeniya (AHS/ERC/2023/062) and Medical Research Institute (ERC/14/2022). The Director of the hospital granted permission to conduct the study. Since no personal identification or direct patient interaction was involved in the study, informed consent was waived by the Ethics Review Committees.

Results

From January 1st to August 15th, 2023, a total of 1,762 dengue detection samples were processed, of which 361 (20.5%) tested positive for dengue NS1 antigen and/or dengue-specific IgM antibodies. The remaining 1,401 samples (79.5%) were considered negative for dengue. Among them, 415 serum samples selected systematically for hantavirus testing, 21 individuals (5.1%) were found to be positive for anti-hantavirus IgM antibodies (Table 1). All IgM positive samples gave positive band for Hantaan (HaN) virus nucleocapsid antigen coated region and 19 of them gave bands on Dobrava antigen (DobN) and Seoul virus antigen (SeoN) coated region indicating infection could be due to hantavirus which produce IgM antibodies cross reacting with Hantaan, Dobrava and Seoul virus antigens. However, hantavirus RNA was not detected in any of the samples.

Table 1.

Hantavirus IgM seropositivity among clinically suspected dengue patients

Hanta IgM	Number	Percentage
Positive	21	5.1%
Negative	394	94.9%
Total	415	100

Demographic characteristics of patients with hantavirus IgM

Of the 415 samples tested, 58.8% (244) were males and 41.2% (171) were females. Among the 21 (5.1%) positive cases, 9 (42.9%) were males and 12 (57.1%) were females (Table 2). Pearson Chi-Square analysis did not reveal a significant relationship between gender and hantavirus positivity (p = 0.128).

Table 2.

Gender distribution of hantavirus IgM positive patients

Hanta IgM seropositivity	Number		Total number
	Male	Female
Positive	9 (42.9%)	12 (57.1%)	21 (100.0%)
Negative	235 (59.6%)	159 (40.4%)	394 (100.0%)
Total	244 (58.8%)	171 (41.2%)	415 (100.0%)

The age of patients ranged from 2 to 85 years (Fig. 1), with a mean age of 32.26. The mean age of hantavirus-positive cases was 34.71 years. There was no statistically significant difference in the mean age between positive and negative cases (p = 0.55).

Fig. 1.

Age distribution of study population

All 415 samples were subjected to RT-PCR testing for Alphavirus and Flavivirus. None of the samples were positive for Alphavirus RNA, while six samples (1.4%) tested positive for Flavivirus RNA (Table 3). Those six samples were further tested by dengue RT-PCR and were found positive for Dengue virus RNA, confirming the presence of active dengue infections in these cases. Further, a total of 415 samples were screened for Leptospira-specific IgM antibodies, with 16 samples (3.9%) testing positive. The PCR assay confirmed that two samples were positive for the presence of Leptospira DNA, indicating active infections. Interestingly, one patient tested positive for both hantavirus IgM and Leptospira-specific IgM, suggesting the possibility of co-infection.

Table 3.

Molecular and serological detection of other viral and bacterial pathogens in clinically suspected dengue patients

	Positive (n)	Negative (n)	Total(n)
Alphavirus RT-PCR	00	415	415
Flavi RT-PCR	06 (1.4%)	409	415
Dengue RT- PCR	06 (1.4%)	00	06
Leptospira specific IgM	16 (3.9%)	399	415
Leptospira DNA	02 (0.5%)	413	415

Clinical and laboratory characteristics of probable hantavirus infections

Among the 17 patients with available data, clinical and laboratory findings were analyzed.

Fever was reported in 100% of the patients, with headache present in 23.5% (4 patients). Vomiting and coughing were noted in 35.3% and 41.2% of patients, respectively. Abdominal pain was rare, reported in only 5.9% of patients. Arthralgia and myalgia were found in 35.3% of patients, while diarrhoea occurred in 17.6%. Back pain was present in 23.5%, and shortness of breath (SOB) was observed in 23.5%. Wheezing was noted in just one patient (5.9%). Anaemia was reported in one patient (5.9%).

Elevated WBC counts were observed in 23.5% of patients, while 29.4% had low total WBC counts. Among the elevated WBC, 53% were lymphocytes and 47% were neutrophils. Hematocrit (HCT) was decreased in 64.7% of patients, and 47.1% of the patients had a decreased platelet count. Elevated AST levels were found in 61.5% (8/13) of patients, while 53.8% (7/13) had elevated ALT levels. Elevated creatinine was seen in 50% of patients, and CRP levels were elevated in eight patients (72.7%). Chest X-ray changes indicative of pulmonary involvement were seen in 3 patients, along with crepitus and dry cough in over 40% of the cases. Additionally, two patients were diagnosed with renal failure (9.5%). These findings highlight the diverse clinical presentations of hantavirus infections, including respiratory and renal involvement, as well as laboratory abnormalities commonly associated with the disease (Tables 4 and 5).

Table 4.

Clinical features in hantavirus IgM positive patients

Clinical feature	Number of subjects (n = 17)	Percentage (%)
Abdominal pain	1/17	5.88%
Anemia	1/17	5.88%
Arthralgia/Myalgia	6/17	35.29%
Back pain	4/17	23.53%
Body ache	3/17	17.65%
Dry cough	7/17	41.18%
Crepts	8/17	47.06%
Diarrhea	3/17	17.65%
Headache	4/17	23.53%
Breathing difficulty	4/17	23.53%
Vomiting	6/17	35.29%

Table 5.

Laboratory parameters in hantavirus IgM-positive patients

Laboratory data	Frequency			Percentage (%)
	High	Normal	Low	High	Normal	Low
AST	8/13	5/13	-	61.5	38.5	-
ALT	7/13	6/13	-	53.8	46.2	-
Creatinine	5/10	5/10	-	50.0	50.0	-
CRP	8/11	3/11	-	72.7	27.3	-
WBC	4/17	8/17	5/17	23.5	47.1	29.4
Haematocrit	-	6/17	11/17	-	35.3	64.7
Platelet count	-	9/17	8/17	-	52.9	47.1

High, normal and low was decided by cut-off values given by manufacturer for the assay

During the follow-up period, two patients developed classical hemorrhagic fever with renal syndrome (HFRS). Of these, one patient experienced severe complications, requiring intensive care unit (ICU) admission and dialysis for renal support. Patient required intensive care unit (ICU) admission and dialysis unfortunately succumbed to death due to sepsis following secondary bacterial infection.

Discussion

Hantavirus infection is emerging as a global health concern, but the exact prevalence of the disease in Sri Lanka remains unclear. This study aimed to identify early-stage and mild/atypical forms of hantavirus infection among clinically suspected dengue patients who tested negative for dengue. Among the tested patients, 79.5% tested negative for dengue, highlighting the potential for misdiagnoses and delays in accurate diagnosis. These challenges are common in patients presenting with dengue-like symptoms, emphasizing the importance of prompt and accurate diagnoses. Early identification is crucial for effective patient management, epidemiological analysis, and the development of preventive strategies and future research.

Prevalence and demographic profile of probable hantavirus infections

The study found a 5.1% positivity rate for hantavirus among clinically suspected dengue patients, which is consistent with similar studies in the region [18, 19]. For instance, a study in Indonesia reported a 4.23% prevalence of hantavirus among dengue-like patients, while a Cambodia study showed a 4% prevalence [20, 21].

In-house real-time RT-PCR assay used for detection of hantavirus RNA was replication of method originally described by Mohamed et al., in 2013. It is SYBER green based real-time RT-PCR which has ability to detect L segment of the hantavirus genome which is the most conserved part of the viral genome in different type of hantavirus clade with analytical sensitive of less than 100 RNA copies/mL (15). Assay was validated locally by using synthetic genes of 12 different types of hantaviruses kindly provided by the authors described this assay originally. The commercial immunoblot assay (Mikrogen recomLine HantaPlus IgM, Mikrogen, Germany) has sensitivity of 98.3% and specificity of 94.1%, based on product broacher. There is no recorded cross reactivity with other virus or bacterial antibodies except antibodies against Sand fly fever virus (SSFV) and anti-malaria antibodies. Assay itself has test line for SSFV. And there was no evidence of other Bunyavirus infections in the country other than hantaviruses and malaria was eliminated since 2016. This test assay was used in previously published serological studies on hantaviruses in Europe [22, 23].

Interestingly, none of the study samples tested positive for hantavirus RNA, which could be due to factors such as low-level viremia or a short duration of viremia [24]. Hantavirus infection may present with a transient or low viral load, which may not be detectable during the sampling period [25]. In addition to that timing of sample collection and suboptimal sample handling before sending in to laboratory may have contributed for negative PCR results. Instead, Hantavirus IgM antibodies, which can remain detectable for 3–4 months after infection, may indicate recent past exposure to the virus [26]. Demonstration of four-fold rise of hantavirus IgG is confirmatory; however, it was not performed due to logistical reasons. Despite the presence of hantavirus IgM antibodies, these patients tested negative for other common febrile illnesses, including dengue, alphavirus infections, and leptospirosis, helping to narrow the potential causes of their symptoms. Therefore, these cases can be considered as probable or suspected hantavirus cases. All hantavirus IgM positive samples gave positive band on Hantaan virus nucleocapsid antigen coated region and majority has given bands on Hantaan, Dobrava and Seoul virus antigens coated region indicating infection could be due to hantavirus which antigenically related to Hantaan, Dobrava and Soul viruses. Serological evidence of Hantaan virus or Hantaan-like virus has detected in Sri Lanka previously and serological cross reactivity was noted [10, 14].

Additionally, six samples tested positive for flavivirus PCR, which confirmed these cases as dengue infections. Initially, these cases were negative for the NS1 antigen but were later confirmed as dengue through PCR. This discrepancy may be attributed to the low sensitivity of NS1 detection, particularly in secondary dengue cases, where the immune response might not produce detectable levels of the NS1 antigen [27]. This highlights the need for multiple diagnostic methods to accurately identify and differentiate between similar febrile illnesses.

Regarding demographic factors, gender was not significantly associated with hantavirus seropositivity. Among the 21 positive cases, 42.9% were male and 57.1% were female, which contrasts with most studies, where a higher proportion of males are affected [14]. A significant difference was found in age groups, with most positive cases occurring in individuals under 10 years and over 61 years. While hantavirus infections in most regions, including the Americas, Asia, and Europe, primarily affect adults, studies from Iran and Barbados have highlighted the occurrence of pediatric cases, suggesting varying age distributions of hantavirus infections across different regions [28].

Clinical and laboratory factors in probable hantavirus patients

Most hantavirus-positive patients (76.2%) did not require special care, and only 14.3% required intensive care. Two deaths were reported, corresponding to a mortality rate of 9.5%, which is slightly lower than that observed in similar studies. Fever was present in all positive cases, while other symptoms such as cough, myalgia, and arthralgia were reported in a subset of patients. The absence of severe symptoms in most cases suggests that the majority of infections were mild. Chest X-rays revealed pulmonary involvement in three patients, and renal failure was diagnosed in two patients, with symptoms consistent with both Hemorrhagic Fever with Renal Syndrome (HFRS) and Hantavirus Pulmonary Syndrome (HPS). These findings align with recent studies conducted in Sri Lanka, indicating that Hantavirus can present with a range of clinical manifestations [11]. Without specific efficient antiviral therapy, these patients were managed symptomatically with supportive care.

Regarding laboratory results, thrombocytopenia was observed in 8 out of 17 positive cases, although it was not a common finding across all patients. Leukocytosis and elevated hematocrit were observed in a few patients; however, none of the cases showed a significant increase in hematocrit. These laboratory abnormalities, which are typically seen in the later stages of the disease, may have been missed because testing was conducted during the early or mild stages of infection [29]. Additionally, elevated ALT, AST, and CRP levels were noted in most positive cases, consistent with findings from a similar study conducted in Indonesia. These markers are indicative of liver involvement and systemic inflammation, reinforcing the need for comprehensive testing during the clinical evaluation of patients [30, 31].

Conclusion

This study determined that the positivity rate of probable hantavirus infection cases among dengue-suspected patients at the National Hospital Kandy was 5.1%. The majority of these patients experienced mild febrile illness, with many of the dengue-negative samples testing negative for other common pathogens, suggesting that the fevers in these patients were likely caused by illnesses other than the tested pathogens. There was no significant association between demographic, clinical, or laboratory factors and hantavirus positivity.

Limitations of the study

Based on IgM results alone are not sufficient to diagnose hantavirus infection with certainty as hantavirus IgM can persist 2–3 months following infection. None of the patients were positive for hanta virus RNA detection PCR. Demonstration of specific IgG was not available due to logistical reasons. Therefore, uncertainty of the confirmatory diagnosis is the key limitation in this study. The other limitation of this study was the incomplete documentation of laboratory-related factors. Some investigations were not ordered, and several patients ‘clinical records were misplaced, which may have affected the completeness of the data. Renal functions and liver functions tests routinely not performed in all dengue-like patients in the hospital especially in mild cases due to rational utilization of limited resources.

Implications and recommendations

Hantavirus infection should be considered as part of the differential diagnosis for dengue-like illnesses in Sri Lanka. The findings from this study provide valuable insights into the clinical spectrum of hantavirus infections, with the first demonstration of mild disease forms in the country, characterized by a lack of severe renal or pulmonary involvement. The study provides the foundation for future research and can help raise awareness within the medical community, potentially influencing preventive measures and improvements in hantavirus detection in laboratories.

Given the limited research on hantavirus in Sri Lanka, further studies are essential to determine its national prevalence and guide public health policies and preventive actions. Lack of adequate diagnostic facilities for detect hantavirus infection is one of the major issue to understand disease burden in the country. In addition to that, accessibility for certain confirmatory diagnostic assays like commercial hanta IgM/IgG Immuno fluorescence assays (IFA) and real-time RT-PCR kits complicate confirmatory diagnosis. The data generated from this study contribute to the growing body of knowledge about hantavirus infections in Sri Lanka and highlight the need for better diagnostics, continued surveillance and research to better understand the disease and its impact on public health.

Abbreviations

ALT

Alanine Aminotransferase

AST

Aspartate Aminotransferase

BHT

Bed Head Ticket

CRP

C-Reactive Protein

HFRS

Hemorrhagic Fever with Renal Syndrome

HPS

Hantavirus Pulmonary Syndrome

NHK

National Hospital, Kandy

RT-PCR

Reverse transcriptase polymerase chain reaction

Authors’ contributions

The study was conducted, and the data were analyzed by E.E., Y.S., M.G. and S.W. Clinical data collection and patient follow-up were carried out by L.R.; N.A. and A.W. optimized and validated the molecular assays. The study was conducted under the supervision of G.R. and R.M., with D.D. overseeing project administration. The original manuscript was drafted by T.P. and reviewed by R.M., and G.R. Revisions were done by R.M. and D.D. All authors have reviewed and approved the final manuscript.

Funding information

Medical Research Institute, Sri Lanka (RC/08/2023).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Ethical approval for the study was obtained from the Research and Ethics Review Committee National Hospital Kandy (NHK/ERC/13/2023), Faculty Ethics Review Committee Faculty of Allied Health Sciences, University of Peradeniya (AHS/ERC/2023/062) and Ethics Review Committee Medical Research Institute (ERC/14/2022). This study was conducted adherence to the Declaration of Helsinki on conduction of research using humans and/or human data, or human materials. Informed consent to participate was waived by the ethics review committees as this study did not use any data that leads in to any personal identifiers of the samples and patients.

The Director of the hospital granted permission to conduct the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.