Acute Appendicitis Associated with Hantaan Virus Infection

ABSTRACT.

Hantaviruses are Bunyaviridae viruses that cause hemorrhagic fever with renal syndrome. Appendicitis caused by Hantaan virus has not been reported previously. An 81-year-old man who underwent laparoscopic appendectomy for suspected appendicitis based on abdominal pain, fever, hypotension, and computed tomography findings. Based on a suspicion of hemorrhagic fever with renal syndrome, the patient’s plasma was simultaneously analyzed using an indirect immunofluorescent antibody assay and nested reverse transcription–polymerase chain reaction (RT-PCR). The appendix tissue was also analyzed using nested RT-PCR and immunohistochemical (IHC) staining to identify the presence of Hantaan virus. Nested RT-PCR detected the presence of Hantaan virus, and indirect immunofluorescent antibody assay results revealed the presence of elevated antibody levels. Furthermore, IHC staining of the appendix tissue confirmed Hantaan virus antigens in the peripheral nerve bundle. Based on these findings, we confirmed the nerve tropism of the Hantaan virus. Hantaan virus in plasma and appendix tissue samples was confirmed using PCR and phylogenetic tree analysis. Moreover, we detected hypertrophy of the submucosa and periappendiceal adipose tissue nerve bundle along with Hantaan virus antigens in peripheral nerve bundles using IHC staining. Hence, we report that Hantaan virus infection may be accompanied by appendicitis.

INTRODUCTION

Hemorrhagic fever with renal syndrome (HFRS) is a febrile disorder caused by the Hantaan and Seoul viruses in Korea and is characterized by fever, acute kidney injury, and hemorrhagic manifestations. Hantaviruses are single-stranded RNA viruses belonging to the family Bunyaviridae. Viral transmission to humans was acquired via inhalation of aerosolized excreta of infected rodents.^1–3 Other external manifestations, including acute impairment of visual function, acute myopia, central nervous system complications with seizures, myocarditis, and severe gastrointestinal hemorrhages, have been reported in various cases.⁴ In a previous study conducted in 94 patients with HFRS, abnormalities such as interstitial infiltrates and/or pleural effusions and atelectasis were found in many cases.⁵

The annual incidence of human HFRS in Korea has increased significantly. In particular, the Korea Centers for Disease Control and Prevention report shows that the number has risen sharply since early 2000. Clinical HFRS diagnosis was carried out based on patient exposure history, typical clinical manifestations, and serology, including ELISA, immunofluorescence antibody assay (IFA), and virus culture.⁶

Gastrointestinal manifestations such as nausea, vomiting, and abdominal pain are common, and infections accompanied by such symptoms are often misdiagnosed as surgical abdomen. Although cases of appendicitis caused by the Dobrava-Belgrade virus and Puumala viruses have been reported,⁷ to the best of our knowledge, there are no published reports of appendicitis caused by Hantaan virus infection. Here, we report a rare case of an elderly man with HFRS that manifested as appendicitis.

MATERIALS AND METHODS

Case description.

An 81-year-old male farmer, without any underlying disease except hypertension, fell on a ridge between rice paddies while driving a motorcycle owing to the aggravation of systemic weakness, poor oral intake, nausea, and dizziness that had developed 3 days prior. He visited a local hospital, where no trauma-related damage was observed. Once he returned home, his weakness worsened in the afternoon, necessitating admission to the same hospital. Appendicitis was suspected, and a computed tomography scan was obtained on day 2 of admission at the local hospital. Accordingly, he was held without food and treated with piperacillin/tazobactam. On the third day of admission to the local hospital, he developed fever, hypotension, and abdominal pain; therefore, he was transferred to Chosun University Hospital, South Korea.

On admission to Chosun University Hospital, the patient’s body temperature, blood pressure, and pulse rate were 36.6°C, 90/60 mm Hg, and 85 beats/min, respectively. The patient was alert and conscious, although he exhibited signs of acute illness. Physical examination revealed a soft and painful abdomen with maximum tenderness in the right lower quadrant (McBurney’s point). The patient also exhibited anuria. Results from laboratory tests, including clinical and biochemical parameters and blood gas measurements, are presented in Table 1.

Table 1.

Clinical and biochemical laboratory parameters of the patient with hemorrhagic fever with renal syndrome

Clinical and biochemical parameters	At diagnosis	Normal values
Hemoglobin	17.2 g/dL	11–17 g/dL
WBC	20,210/µL	4,000–10,800/µL
Neutrophils	84.5%	40–60%
Lymphocytes	7%	20–40%
Monocytes	2.3%	2–8%
Platelets	25,000/µL	150,000–400,000/µL
C-reactive protein	8.22 mg/dL	0–0.3 mg/dL
Alanine aminotransferase	44 U/L	< 40 U/L
Aspartate aminotransferase	108 U/L	< 40 U/L
Lactate dehydrogenase	1,215 U/L	200–450 U/L
Arterial blood gas analysis
pH	7.42	7.35–7.45
pCO2	32.9 mm Hg	35–45 mm of Hg
pO2	76.6 mm Hg	80–100 mm of Hg
HCO3−	19.8 mmol/L	24–28 mmol/L
O2 saturation	95.5%	95–100%

HCO₃⁻ = bicarbonate level; pCO₂ = partial pressure of CO₂; pO₂ = partial pressure of oxygen; WBC = white blood cells.

Based on the physical examination, laboratory diagnostic findings, and the presence of an enlarged appendix based on computed tomography scan (with contrast enhancement) and abdominal ultrasound, the patient was diagnosed with acute appendicitis (Figure 1) in the emergency room. The patient was transferred to the Department of General Surgery, where a laparoscopic appendectomy was performed. After surgery, the abdominal pain was alleviated; however, anuria and metabolic acidosis persisted, necessitating continuous renal replacement therapy. Hemorrhagic fever with renal syndrome was also suspected based on findings such as thrombocytopenia, elevated creatinine levels, fever, anuria, and hypotension. Moreover, the patient exhibited an altered mental state and stupor due to worsening metabolic acidosis from the third day post-surgery. Multiorgan failure rapidly progressed, and the patient died on day 4 of admission at Chosun University Hospital.

Figure 1.

Computed tomography and ultrasound images of the appendix. (A) Axial computed tomography image of the appendix with appendicular wall enhancement and thickening (arrow) without surrounding fat stranding or fluid. (B) Longitudinal and (C) transverse ultrasound images demonstrating a thickened, inflamed appendix that had increased in diameter. Periappendiceal hyperechogenicity was observed in keeping with the surrounding inflammation.

Diagnostic tests.

Serum Hantaan virus–specific immunoglobulin (Ig) G and IgM antibodies were detected using an indirect IFA.⁸ The Hantaan virus antigen slides were prepared by infecting the Vero E6 cell lines with 76–118 strains of the Hantaan virus, followed by inoculation of infected cells and fixation with 80% acetone on a Teflon-coated well slide. The prepared antigen slides were stored at −70°C until further use.

After 2-fold serial dilutions of 1:16 of the patient’s serum, 20 µL of diluted serum was incubated with the viral antigen on the slide wells. The test slides were incubated for 30 minutes at 37°C, washed with phosphate-buffered saline and distilled water, and air-dried. Slides were then incubated at 37°C for 30 minutes with fluorescein isothiocyanate–conjugated goat anti-human IgM and IgG (MP Biomedicals, Solon, OH) as secondary antibodies. After washing with phosphate-buffered saline and distilled water, specific fluorescence was observed under a fluorescence microscope (Olympus, ix73) at ×400 magnification. The final serum dilution factor at which the specific fluorescence observed was defined as the antibody titer.

Serum samples collected at the time of admission were submitted to Green Cross Corporation, a commercial laboratory in South Korea, for total Ig measurements. The laboratory used an indirect IFA to detect total Ig using anti-human IgM+G+A conjugate.

Using the Viral Gene-spin^TM Viral DNA/RNA Extraction Kit iNtRON, Seongnam, Korea), viral RNA was extracted from the patient’s plasma collected on admission and the appendix tissues obtained after surgery. cDNA synthesis from viral RNA was performed using the SuperScript VILO MasterMix (Invitrogen, Carlsbad, CA). Nested reverse transcription–polymerase chain reaction (nested RT-PCR) targeting the L-segment with a 380 bp amplicon and S-segment, using designed primers: HFRS-S-2F (5'-ARARRTCARBVCTHAGBTAYG-3') and HFRS-S-2R (5'-TGRTTVGAKATTTCCTTSAC-3') for the first PCR; HFRS-S2nd-1F (5'-GAYATTGAWGAACCWASWGGVC-3'), and HFRS-S2nd -1R (5'GAHGCCATKATKGTRTTYCKC-3') for nested PCR, with a 725 bp amplicon of the Hantavirus was performed along with Hantaan virus 76-118 cDNA as a confirmed positive control and sterile distilled water (molecular grade water) as negative controls. Polymerase chain reaction experiments were carried out using AmpliTaq Gold 360 Master Mix (Applied Biosystems, Foster City, CA) and a Veriti^TM 96-Well thermal cycler (Applied Biosystems).^9,10

Immunohistochemical staining.

The appendix tissues obtained after surgery were subjected to immunohistochemical staining. Automated immunohistochemical staining was performed using the polyclonal anti-N protein antibody of Hantaan virus 76-118 (kindly provided by BEI Resources; dilution, 1:200) on the Benchmark XT platform (BenchMark XT; Ventana Medical Systems, Tucson, AZ) with cell conditioning for 80 minutes, pre-peroxidase inhibition, and primary antibody incubation for 1 hour at 37°C. The OptiView DAB IHC Detection Kit and OptiView Amplification Kit (Ventana Medical Systems) were used to detecting the hantaviruses. Tissues were counterstained with hematoxylin II and bluing reagent for 12 and 4 minutes, respectively. Samples were dehydrated by sequential washing with 70% ethanol, 96% ethanol, and absolute ethanol and cleared in xylene, and then the slides were mounted.¹¹

RESULTS

Indirect immune fluorescence assay and immunohistochemistry studies.

Indirect IFA showed high IgM and IgG antibody titers in the patient serum collected on the first day (1:256 and 1:512, respectively) and on the third day (1:256 and 1:1,024, respectively) of admission. The same samples were sent to Green Cross Corporation, and indirect IFA results revealed that the total Ig titers were 1:40 and 1:80 on days 1 and 3 of admission, respectively.

Gross examination of the appendix tissues revealed hyperemia without purulent exudate. Microscopic examination revealed infiltration of inflammatory cells, particularly eosinophils and neutrophils, in the mucosa and muscularis propria (Figure 2A–C). Although these findings were similar to the histological findings of general appendicitis, a single vague granuloma in the mucosa was identified (Figure 2D). In addition, thickening of the vascular walls and hypertrophy of peripheral nerve bundles were observed in the submucosa of the appendix and periappendiceal adipose tissues; however, inflammatory cell infiltration was not observed (Figure 2E and F).

Figure 2.

Histopathologic findings and immunohistochemical staining of the appendix (B–F; hematoxylin and eosin staining). (A) Specimen of the inflamed turgid appendix. (B) Cross section of the appendix showing mild inflammatory cell infiltration in the mucosa and muscle. Scale bar: 1 mm. (C) Higher magnification showing acute inflammatory cell infiltration, mainly eosinophils and neutrophils. Scale bar: 500 µm. (D) Single vague granuloma (arrow) is identified in the mucosa. Scale bar: 100 µm. (E) Thickening of the vascular wall without vasculitis is noted in the submucosa and periappendiceal adipose tissue. Scale bar: 200 µm. (F) Hypertrophy of the peripheral nerve bundles is seen in the periappendiceal adipose tissue. Scale bar: 200 µm. (G) Positive immunostaining (arrows) for the hantavirus antigen identified in peripheral nerve bundles in the muscle layer. Inset: Higher magnification of the positive nerve bundle. Scale bar: 200 µm. (H) Hypertrophic nerve bundles (arrows) show a positive reaction to the hantavirus antigen. The thickened vascular wall (asterisks) shows negative immunostaining results. This figure appears in color at www.ajtmh.org.

Immunohistochemical staining revealed that the hypertrophic nerve bundles in the muscle layer were positive for Hantaan virus (Figure 2G). However, the thickened vascular walls did not show positive staining (Figure 2H).

Nested RT-PCR assay for the specimens collected from the patient.

The nested RT-PCR targeting the L-segment and S-segment of the hantavirus from the plasma and appendix tissues was positive, and the PCR-positive products were purified and sequenced. DNA sequencing analysis of the PCR-positive samples confirmed the presence of the Hantaan virus in both the plasma and appendix.

Phylogenetic analysis of the sequences.

Partial L-segment and S-segment sequences obtained from patient samples along with GenBank reference sequences were used to construct a phylogenetic tree using ClustalX (Ver 2.0; www.clustal.org/) and Tree Explorer (DNASTAR, Madison, WI). The sequences of the L and S segments were aligned using Clustal X, and neighbor-joining trees were generated based on 1,000 bootstrap replicates according to the Clustal X software program (http://www.clustal.org/clustal2/). The Tree Explorer was used to identify the strain pedigree. The results of the phylogenetic tree analysis based on the nucleotide sequences of the partial L-segment (360 bp) and partial S-segment (650 bp) are shown in Figure 3A and B, respectively.¹²

Figure 3.

(A) Phylogenetic tree based on the partial L-segment (360 bp) and (B) partial S-segment sequences (650 bp) from the plasma and appendix of an 81-year-old man and from GenBank as reference sequences., The numbers at the nodes represent bootstrap confidence levels for 1,000 replicates. AND = Andes virus; DOB = Dobrava–Belgrade virus; HTN = Hantaan virus; PUU = Puumala virus; SEO = Seoul virus; SN = Sin Nombre virus; SOO = Soochong virus; TUL = Tula virus.

The partial L-segment and S-segment sequences (accession nos. MW349026, MW349027, and MW349028) obtained from patient samples formed a cluster with Hantaan virus on the phylogenetic trees. For the L-segment phylogenetic tree, we did not add the sequences of the Hantaan virus Maaji strain due to the unavailability of sequences at the NCBI site.

Similarity analysis carried out using the NCBI BlastN network service with partial L-segment sequences from the plasma sample sequence (accession no. MW349026) showed 83.8% homology with the Hantaan virus isolate Galkino/AA57/2002 (accession no. AB620033); 83.3% homology with the Hantaan virus strain Z10 (accession no. AF189155); and 82.7%, 80.5%, 80.5%, and 78.6% homology with the Hantaan virus strains TJJ16, 76-118, LR1, and A9 (accession nos. KU215675, NC_005222, AF288292, and AF293665), respectively. Furthermore, there was 80.8% and 79.9% homology with the Soochong virus strains SOO-1 and SOO-2 (accession nos. DQ056292 and AY675354), respectively.

Similarity analysis of the partial L-segment sequences (accession no. MW349027) from the appendix tissues was 83.6% homologous with the Hantaan virus isolate Galkino/AA57/2002 (accession no. AB620033) and 83.1% homologous with the Hantaan virus strain Z10 (accession no. AF189155). In addition, there was 82.5%, 80.3%, 80.3%, 80.3%, and 78.3% homology with the Hantaan virus strains TJJ16, 76-118, LR1, and A9 (accession nos. KU215675, NC_005222, AF288292, and AF293665), respectively, and 80.6% and 79.7% homology with the Soochong virus strains SOO-1 and SOO-2 (accession nos. DQ056292 and AY675354), respectively.

Based on the DNA similarity analysis of the partial S-segment from the plasma sample (accession no. MW349028), there was 87.8% homology with the S segment nucleocapsid protein gene of the Hantaan virus strain Maaji-1 (accession no. AF321094.1) reported in Korea.

DISCUSSION

HFRS is caused by hantavirus. The infection is characterized by fever, acute kidney injury, and hemorrhagic manifestations and can cause a variety of clinical symptoms throughout the body. Gastrointestinal manifestations are very common, with abdominal pain and tenderness observed in over two-thirds of patients, and many patients had been admitted to surgical wards due to severe abdominal pain.^13–15 Specifically, pain limited to the right lower quadrant can be misdiagnosed as acute appendicitis.

Hantaan and Dobrava viruses are typically associated with a severe form of HFRS. Nephropathia epidemica, a mild form of HFRS, is mainly caused by Puumala virus. Appendicitis associated with HFRS caused by Dobrava and Puumala viruses has been reported. Jakab et al.¹⁶ reported acute renal failure following an exploratory laparotomy for appendicitis in a patient. Hantavirus infection was suspected, and Dobrava virus infection was confirmed by serum ELISA and RT-PCR, where the final diagnosis was HFRS.

In another report, a patient developed thrombocytopenia and acute renal failure after laparoscopic appendectomy for appendicitis. Serological testing for hantaviruses revealed positive results for Puumala virus IgG and IgM. In addition, IHC staining for appendix tissues revealed Puumala virus antigen in the vascular endothelial cells, and the Puumala virus-specific real-time RT-PCR showed negativity for Puumala virus RNA.¹⁷ In another study in which a patient with nephropathia epidemica underwent gastroscopy and colonoscopy during the acute phase, Latus et al.¹⁸ found Puumala virus RNA positivity on RT-PCR of biopsy tissues. In addition, IHC staining detected the presence of Puumala virus nucleocapsid antigen in the endothelial cells of capillaries and in the larger vessels of the lamina.

In the present case, vascular wall thickening in the submucosa and periappendiceal adipose tissue was observed, although there was no vasculitis. Results from immunohistochemical staining revealed that hantavirus did not infect the endothelial cells. Hantavirus antigens were detected by immunostaining, along with hypertrophy of peripheral nerve bundles located in the muscle layer of the appendix tissue.

Although some patients with HFRS have severe abdominal pain, their underlying pathophysiology remains unclear. Further studies are needed to determine whether the invasion of nerve bundles by hantavirus, as observed in the present case, is related to gastrointestinal manifestations such as abdominal pain.

Neurological symptoms are frequently observed in patients with HFRS. In a study in which 811 cases of Puumala virus infection were reviewed, neurological symptoms included headaches, blurred vision, and vomiting. Potential life-threatening neurological complications associated with HFRS include meningism, cerebral hemorrhage, epileptiform seizures, and urinary bladder paralysis.¹⁹ A case of isolated abducens nerve palsy in a patient with HFRS has also been reported.²⁰

In the present case, positive immunostaining using the Hantaan virus antibody was observed in the peripheral nerve bundles in the muscle layer. Moreover, hypertrophic nerve bundles in the periappendiceal adipose tissue were also observed. Thus, Hantaan virus nerve tropism was identified in the patient, which has not been previously reported in the literature. Further studies may determine whether this nerve tropism is one of the characteristics of Hantaan virus or exhibited by other hantaviruses. Furthermore, the association between neurological manifestations and nerve invasion by Hantaan virus should be investigated. The single granuloma found in the mucosa of our patient is a rare finding in general appendicitis, and the possibility of coinfection with mycobacteria, fungi, or parasites cannot be ruled out.

CONCLUSION

To the best of our knowledge, no studies have demonstrated clinical manifestations of Hantaan virus infections similar to those of appendicitis, as documented by immunohistochemical staining and PCR. We confirmed the presence of the same Hantaan virus in the plasma and in the appendix tissue using PCR. We report hypertrophy of the nerve bundle in the muscle layer and periappendiceal adipose tissue along with Hantaan virus Ag in the peripheral nerve bundles using IHC staining. Hence, our findings call for research on the nerve tropism of the Hantaan virus.