Neurological Disease Associated with Chikungunya in Indonesia

ABSTRACT.

Chikungunya virus (CHIKV) is recognized but rarely considered as a cause of central nervous system infection in endemic areas. A total of 244 patients with acute meningoencephalitis in Indonesia were retrospectively tested to identify whether any CHIKV infection was associated with neurological manifestations, especially in provinces known for CHIKV endemicity. Cerebrospinal fluid (CSF) and blood specimens were tested using CHIKV-specific real-time reverse transcription polymerase chain reaction and IgM ELISA, alongside a panel of neurotropic viruses. We report four cases of suspected or confirmed CHIKV-associated neurological disease, including CHIKV RNA detection in CSF of one patient and in acute serum of another, and CHIKV IgM in CSF of three patients and in serum of a fourth. In conclusion, CHIKV should be considered as a cause of neurologic disease in endemic areas and especially during outbreaks, in addition to the more common arboviral diseases such as dengue and Japanese encephalitis viruses.

Chikungunya, a globally distributed arboviral infection, is usually a self-limiting disease associated with fever, rash, and debilitating joint pain. Although typically considered as a nonfatal disease, it is occasionally associated with life-threatening neurological complications, such as observed during the La Reunion Island outbreak.¹ To explore chikungunya virus (CHIKV)-associated neurological infection in Indonesia, we retrospectively tested 39 cerebrospinal fluid (CSF) and 161 serum specimens from 105 patients in Manado; 65 CSF and 39 plasma specimens from 67 patients in Bandung; 44 CSF, 39 plasma, and 4 serum specimens from 44 patients in Jakarta; and 11 CSF specimens from 11 patients in Jambi. The samples were obtained from central nervous system (CNS) infection studies where criteria for enrollment were acute onset of fever with altered mental state, accompanied by seizures, or focal neurological findings, or neck stiffness, as well as diagnostic specimens of neurological cases. All specimens were optimally maintained at the collection sites, shipped and stored at the Eijkman Institute at −80°C for retrospective testing with the exception of the diagnostic specimen from Jambi, which was kept at −20°C before shipping. This study was approved by the Medical Research Ethics Committee of R. D. Kandou General Hospital (066/EC-UPKT/III/2016), Ethics Committee of Faculty of Medicine Universitas Indonesia (1365/UN2.F1/ETIK/2018), Eijkman Institute for Molecular Biology Research Ethics Commission (78), and Oxford Tropical Research Ethics Committee (13-19).

To test for a current or recent CHIKV infection, both RT-PCR and ELISAs were performed on CSF and serum samples. In addition to CHIKV-specific real-time RT-PCR,² RT-PCR using genus-specific primers was performed to exclude dengue virus (DENV), Japanese encephalitis virus (JEV), and other neurotropic viruses such as herpesvirus, enterovirus, and paramyxovirus.³ The samples were also tested for IgM antibody against CHIKV, DENV, and JEV by in-house capture ELISAs.⁴ Whole CHIKV genome amplification of RT-PCR positive specimens was attempted using multiplex tiling PCR.⁵ Culture of the specimens was not conducted due to insufficient volumes.

Acute serum of case 1 and CSF of case 2 were positive by CHIKV RT-PCR. None of the four cases were positive by PCR for other neurotropic viruses, and neither DENV nor JEV IgM. One discharge serum from Manado and three CSF from Jambi, Bandung, and Jakarta were positive for CHIKV IgM. Cerebrospinal fluid of cases 2 and 4 were moderately positive for CHIKV IgM as was the plasma from case 4 (Table 1). Additionally, case 1 seroconverted for anti-CHIKV IgM in follow-up serum.

Table 1.

Viral diagnostic assays performed in four CNS infection cases from Indonesia

			Molecular assays					ELISA assays
Case #	Type of sample	Study site	Flavivirus RT-PCR	CHIKV qRT-PCR	Enterovirus RT-PCR	Herpesvirus RT-PCR	Paramyxovirus RT-PCR	CHIKV IgM (CDC)*		DENV IgM (CDC)*		JEV-IgM (CDC)*
								P/N	Interpretation	P/N	Interpretation	P/N	Interpretation
Case 1	Acute serum	Manado	Negative	Positive	Negative	Negative	Negative	2.37	Equivocal	1.48	Negative	1.09	Negative
	Convalescent serum		Negative	Negative	ND	ND	ND	3.11	Positive	1.12	Negative	1.18	Negative
Case 2	CSF	Jambi	Negative	Positive	Negative	Negative	Negative	8.91	Positive	2.64	Equivocal	1.35	Negative
Case 3	Plasma	Bandung	Negative	Negative	Negative	Negative	Negative	4.04	Positive	1.83	Negative	1.45	Negative
	CSF		Negative	Negative	Negative	Negative	Negative	3.51	Positive	1.79	Negative	1.29	Negative
Case 4	Plasma	Jakarta	Negative	Negative	Negative	Negative	Negative	8.45	Positive	1.54	Negative	1.03	Negative
	CSF		Negative	Negative	Negative	Negative	Negative	7.57	Positive	1.18	Negative	1.04	Negative

CNS = central nervous system; CSF = cerebrospinal fluid; ND = not done; qRT-PCR = quantitative real-time reverse transcription PCR; RT-PCR = reverse transcription polymerase chain reaction.

*Interpretation for CDC MAC-ELISA: P/N < 2: negative, P/N ≥ 2 to < 3: equivocal, P/N ≥ 3: positive.⁴

All patients positive for CHIKV nucleic acid and IgM were hospitalized with suspected CNS infection. Case 1 was a 43-year-old man admitted with altered consciousness for 1 day, and left-sided weakness, slurred speech, continuous headache, and low-grade fever for 2 days (Table 2). The patient had a history of HIV infection, with presumptive toxoplasma encephalitis a year before, and pulmonary tuberculosis 2 years earlier. On admission, his Glasgow Coma Scale (GCS) was 13, temperature 37.8°C, with bilateral papilledema, left facial nerve palsy (central type), left hemiparesis, and without nuchal rigidity. Peripheral blood count showed mild anemia, normal white blood cells, and platelets. Brain imaging and lumbar puncture were not performed. Without CSF confirmation, he was presumptively diagnosed as having toxoplasma encephalitis and treated with pyrimethamine, clindamycin, cotrimoxazole, and initiation of antiretroviral drugs (tenofovir/lamivudine/efavirenz). After 3 days, his condition improved and he was discharged 5 days later with residual hemiparesis.

Table 2.

Clinical characteristics and laboratory investigations of CNS infection patients

Parameter	Case 1 43 y.o./M*	Case 2 19 y.o./F†	Case 3 32 y.o./F†	Case 4 21 y.o./M*
Clinical characteristics
Onset of fever (day)	3	2	7	1
Fever	+	+	+	+
Headache	+	+	−	−
Altered consciousness	+	+	+	+
Seizures	−	+	+	−
Nuchal rigidity	−	+	−	−
Joint pain	−	+	−	−
Skin rash	−	−	−	−
Diarrhea	−	−	+	+
Abdominal pain	−	−	−	+
Motor dysfunction	Facial nerve palsy, left hemiparesis	−	−	−
Glasgow Coma Scale	13	12	13	15
Blood pressure (mm/Hg)	120/80	110/70	140/90	100/62
Heart rate (beats/min)	80	107	102	125
Laboratory investigation
Blood test
Hemoglobin (mmoL/L)	6.21	6.27	6.7	11.8
Platelets(counts/μL)	181,000	310,000	516,000	46,000
White blood cells (cells/μL)	5,000	11,800	12,280	11,680
C-reactive protein (mg/L)	ND	ND	ND	35.4
Culture	ND	Negative	ND	ND
HIV test	Positive	Negative	Negative	Negative
CSF findings
White blood cells (cells/μL)	ND	100 (mononuclear cell)	61 (mononuclear cell)	11 (mononuclear cell)
Protein (mg/dL)	ND	30	120	ND
Glucose (mg/dL)	ND	100	24	44
CSF/blood glucose ratio	ND	0.82	0.22	0.55
Acid-fast bacilli stain	ND	Negative	ND	Negative
Gram stain	ND	Negative	ND	Negative
Real-time PCR (GeneXpert)	ND	ND	Negative	Negative
M. tuberculosis Culture	ND	ND	Positive	Negative
Chest X-ray	Normal	Normal	Pleural effusion	Normal
Brain CT scan	ND	Subdural hyperdensity in left frontotemporal lobe and mild diffuse cerebral edema	Multiple lacunar infarct	Normal
Comorbidities	HIV	Epilepsy	None	None
Presumptive clinical diagnosis	Toxoplasma encephalitis	TB meningitis	TB meningitis	Viral encephalitis
Treatment	Anti-toxoplasma and antiretroviral drugs	Anti-epilepsy, antibiotics, and anti-TB drugs	Anti-TB drugs	Acyclovir
Outcome at discharge	Residual hemiparesis	No neurological sequelae	Moderate neurological sequelae	No neurological sequelae

CNS = central nervous system; CSF = cerebrospinal fluid; CT = computed tomography; ND = not done; y.o. = year old.

^*M = Male.

^†F = Female.

Case 2 was a 19-year-old woman, known to have epilepsy, who presented with multiple seizures the night before admission and headache, fever, and joint pain 2 days previous (Table 2). On admission, the patient was conscious, but deteriorated to a GCS of 12 four hours later. She also had fever (38.0°C) and neck rigidity. No other focal neurological deficits were found. She was treated with a provisional diagnosis of status epilepticus and CNS infection. Peripheral blood testing showed mild anemia and leukocytosis, with normal platelets. CSF investigation showed pleocytosis (100 cells/µL) with a predominance of mononuclear cells (97%), and mildly elevated glucose (100 mg/dL), CSF/blood glucose ratio of 0.82, and normal protein (30 mg/dL). Cerebrospinal fluid Gram’s and acid-fast bacillus (AFB) stains yielded negative results. Computed tomography (CT) scan showed appearance of subdural hyperdensity in left frontotemporal lobe and mild diffuse cerebral edema (Figure 1). The patient was treated with first-line tuberculosis drugs based on Thwaite’s score and other clinical considerations, in addition to ceftriaxone and phenytoin. She was discharged 6 days after admission without any neurological sequelae.

Figure 1.

Computed tomography scan of case 2. Axial non contrast-enhanced computed tomography (CT) scan showed diffuse cerebral edema and subdural hyperdensity in the left frontotemporal region (arrow).

Case 3 was a 32-year-old woman who was admitted with focal bilateral seizures for 1 day, altered consciousness for 1 day, and fever and diarrhea for 1 week (Table 2). On admission, she felt unwell, vital signs were normal, and laboratory analysis showed a mild anemia with normal blood leukocytes and platelets. There was a right pleural effusion on the admission chest x-ray, and brain CT scan showed multiple lacunar infarct. Cerebrospinal fluid analysis showed pleocytosis (61 cells/µL), glucose level of 24 mg/dL, CSF/blood glucose ratio of 0.22, and protein level of 120 mg/dL. CSF was negative for Mycobacterium tuberculosis by GeneXpert real-time PCR (Cepheid, USA), but positive by culture on day 21. Antituberculosis drugs were administered and she was discharged from the hospital with maximum GCS and a Glasgow Outcome Scale (GOS) of 4 (moderate disability).

Case 4 was a 21-year-old man admitted with altered consciousness 6 hours before admission (Table 2), following high fever, abdominal pain, and diarrhea, one day before admission. Physical examination revealed a maximum GCS with slow mentation and occasional agitation, and high fever (40°C). Peripheral blood count showed thrombocytopenia (84,000, dropping to 46,000/uL after 5 days), with elevated C-reactive protein (CRP) (35.4 mg/L), procalcitonin (11.04 ng/mL), and serum creatine kinase (4,740 U/L). Computed tomography scan was normal and CSF investigation showed mild pleocytosis (11 cells/µL, mononuclear cells) with normal protein, CSF glucose of 44 mg/dL, and CSF/blood glucose ratio of 0.55. Patient was presumptively diagnosed with viral encephalitis and was treated with acyclovir. His condition improved after 5 days of treatment and he was discharged without any sequelae.

Although not traditionally considered to be a neurotropic virus, CHIKV-associated CNS infection could have a broad spectrum of neurological presentations including meningitis, encephalitis, meningoencephalitis, encephalomyelitis, or Guillain–Barré syndrome.⁶ Chikungunya virus–associated CNS infection has been reported to range widely across studies between 0.14% and 47% depending upon the study parameters.^7–10 Encephalitis (as in cases 1 and 4) is reportedly the most common neurological presentation.^8,9 Young children and elderly people are more at risk for CHIKV-associated CNS infection,¹ so the adult and young adult cases in our study are unusual. Underlying comorbidities as in case 1 have been suggested to play a role in neurological complications of CHIKV infection.⁶ However, the mechanism of neurotropism of CHIKV remains unclear.⁶

The only recent reports of CHIKV-associated meningoencephalitis in the Southeast Asian region are from Thailand and Cambodia,^8,11 and these are usually linked with outbreaks associated with a variant of the East Central South African genotype (ECSA). Chikungunya virus is endemic in a number of provinces in Indonesia¹² and is mostly with the Asian lineage, whereas the ECSA genotype is rarely detected.^13,14 It is not known whether there are virulence differences for CNS infection among the different genotypes. Occasional outbreaks have been reported in Jambi Province and Northern Sulawesi, where a seroprevalence rate of 8% has been found.^12,15 Chikungunya virus was also found to be a common cause of acute febrile illness in Jakarta and Bandung.¹⁶

Presenting symptoms in the reported cases included fever and altered mental status (all), polyarthralgia, motor dysfunction, and facial nerve palsy (each in one case). The lack of classical features (arthralgia and rash) concurs with findings from a study in India.¹⁷ Third nerve palsy has been associated with neuro-chikungunya,⁶ but facial nerve palsy has been rarely reported.¹⁸ The peripheral blood picture was unremarkable as in most CHIKV infections¹⁹ except in case 4 who showed marked thrombocytopenia. Cerebrospinal fluid pleocytosis, which is not a consistent feature for CHIKV encephalitis,⁹ was observed in all three cases with available CSF data. Chikungunya virus is rarely detected directly in the CSF and there is no knowledge of how long it remains detectable in the CSF.²⁰ In our study, only a single case was positive for CHIKV RNA in CSF, which could be due to transient viral circulation in CSF; however, with the exception of a few reports on CHIKV detection in the early days of infection, there is limited information on viral persistence in CSF in CHIKV encephalitis. Although diagnosis of CHIKV infection in cases 3 and 4 were only by antibody detection, the use of a high-performance, sensitive IgM capture ELISA enhanced its credibility.⁴ Additionally, no other common causes of neurological infection were detected with the exception of case 3 where tuberculous meningitis is likely to be the predominant cause of the CNS disease with chikungunya as a concomitant infection, given that there was quite strong evidence of intrathecal production of CHIKV IgM in both admission and discharge sera using the CDC reference assay. Complete neurological recovery was observed for cases 2 and 4 as is generally reported,^6,9 while case 1 had facial paresis and hemiparesis on discharge, which may have been exacerbated by the patient’s immunocompromised status. Sporadic reporting of neurological sequelae due to CHIKV has been noted since the 2006 La Reunion Island outbreak.

There are some notable limitations in our study. In our retrospective analysis, some relevant clinical data related to CHIKV were not available, and except for case 3, follow-up clinical evaluations to determine long-term sequelae were not recorded. Sequence data were not available to confirm the RT-PCR findings. Cerebrospinal fluid was taken only once and the convalescent blood specimens were not available to study the dynamics of CHIKV CNS infection. Magnetic resonance imaging (MRI), although a valuable tool in the diagnosis of CNS infection and monitoring response to treatment, is not routinely conducted in Indonesia due to lack of resources. Additionally, the confirmatory neutralization assay for CHIKV was not conducted due to limited sample. However, the CDC IgM assay has a high specificity for the region as it was reported to cross-react only with o’nyong-nyong (African alphavirus) and Mayaro (tropical America alphavirus) viruses.²¹

To our knowledge, this is the first report of CHIKV-associated CNS infection in Indonesia. Chikungunya virus still remains an under-recognized cause of neurological complications, especially since it could mimic other CNS infections with nonspecific manifestations and might be treated empirically with antimicrobials as with these cases. In conclusion, CHIKV should be considered as a cause of neurologic disease in endemic areas and especially during outbreaks in addition to the more common arboviral causes of neurologic diseases such as DENV and JEV.