Short Communication: Coinfection of Japanese Encephalitis and Scrub Typhus in Acute Encephalitis Patients in North India

Abstract

Background:

Acute encephalitis syndrome (AES) is a significant public health issue in India, attributed to various etiologies. In eastern Uttar Pradesh, Japanese encephalitis (JE) was the leading cause of AES (10–14% of total AES) until scrub typhus (ST), caused by Orientia tsutsugamushi, was identified in cerebrospinal fluid and blood samples of AES patients contributing more than 60% of AES cases. This study investigates the prevalence of JE-ST coinfection and compares clinical outcomes among JE mono-infection, ST mono-infection, and JE-ST coinfection.

Materials and Methods:

AES cases admitted to BRD Medical College, Gorakhpur, Uttar Pradesh, India, from January 1, 2017, to December 31, 2017, were included. JE and ST diagnosis was confirmed by serological (IgM) and molecular (PCR) tests. Statistical analysis was done to correlate clinical outcomes and infection group.

Results:

Total 1180 cases were tested positive for JE and/or ST. The prevalence of JE-ST coinfection was 8.9% among AES cases. JE mono-infection showed a mortality rate of 34.5%, ST mono-infection 13.4%, and JE-ST coinfection 9.5%. JE-ST co-infected cases experienced less severe clinical outcomes compared to mono-infected cases.

Conclusion:

JE-ST coinfection in AES cases is relatively common, with better clinical outcomes and lower mortality rates compared to JE or ST mono-infections.

Acute encephalitis syndrome (AES) is a severe neurological condition and challenging to diagnose due to its association with various etiologies (Olsen et al., 2015). Japanese encephalitis (JE) virus is one of the leading causes of AES in India and Southeast Asia. The risk of JE infection was estimated to be one case/400,000 visits in travelers staying in rural settings of endemic regions (Lindquist, 2018). Recent studies from eastern Uttar Pradesh and Bihar showed that a high proportion of AES in this region is associated with scrub typhus (ST) caused by Orientia tsutsugamushi, indicating that scrub typhus is a cause of AES (Mittal et al, 2017; Jain et al., 2018). Travel-acquired ST infection is well reported in international travelers coming back from endemic areas (Nachega et al., 2007; Costa et al., 2021). A previous study from India has confirmed 33.3% copositivity for more than one pathogen among the AES cases from India (Jain et al., 2018). In this article, the AES cases were diagnosed with JE and ST to estimate the prevalence of coinfection. The monoinfected and coinfected cases were compared for demographic features, clinical characteristics, and laboratory findings.

Cases hospitalized between January 1, 2017 and December 31, 2017 at BRD Medical College, Gorakhpur, Uttar Pradesh, India were included (Fig. 1). The diagnosis is based on the AES case definition and laboratory diagnosis (Supplementary Data S1). Dengue is endemic in this region (Deval et al., 2021; Ranjan et al., 2014) Dengue NS-1 antigen-positive patients were excluded from this study; they will be discussed in another article. The cerebrospinal fluid (CSF) and/or serum were tested for anti-JE-virus IgM using National Institute of Virology (NIV) JE IgM Antibody Capture Enzyme Linked Immunosorbent Assay (MAC ELISA) kits (Ranjan et al., 2014), and anti-ST IgM was tested in serum samples using a commercial IgM ELISA Kit (InBios International, Inc, USA) as per Department of Health Research (DHR)-Indian Council of Medical Research (ICMR) guideline (Supplementary Data S1). In addition, we performed PCR in 480 blood specimens targeting the 56 kDa region of O. tsutsugamushi (Supplementary Data S1). In this article, JE, ST monoinfections and coinfection are represented as IgM and/or PCR positivity. Laboratory-confirmed AES cases of JE monoinfection (177 cases), ST monoinfection (898 cases), and cases positive for coinfection (105 cases) were included. Differential anti-JE virus IgM positivity in CSF and serum for both JE mono- and coinfections is given in Supplementary Data S1. Out of 480 specimens tested for ST by PCR, 397 specimens belong to ST-monoinfection category (397/898), and remaining 83 specimens were from the JE–ST coinfection category (83/105). In the ST-monoinfection category, PCR positivity was 23.43% (93/397). However, for coinfection category, this was 48.19% (40/83). Parameters were individually compared using chi-square or Fisher’s exact test when appropriate. Logistic regression was conducted to assess the association between the dependent and independent variables. The results are expressed as odds ratios with 95% confidence intervals. A p-value of <0.05 was considered statistically significant. This study was ethically approved by the institutional Human ethical committee of ICMR-National Institute of Virology, Pune, India (Approval no: NIV/IEC/2017/D-65/75). ICMR NIV unit (now ICMR RMRC Gorakhpur) and BRD MC are government-funded organizations that offer services free of cost to all patients admitted to BRD MC. All the data were anonymized. Clinicians took consent from the parent/guardian of the patient at the time of sampling.

FIG. 1.

Epidemiological curve of Japanese Encephalitis (JE), Scrub Typhus (ST) and their coinfection in Northern part of India.

The JE–ST coinfection was observed in 8.9% of AES cases. The demographic data and clinical manifestations of JE, ST monoinfection, and their coinfection are given in Table 1. Coinfection of JE and ST was seen in the 1.1–16 years of age group; however, JE monoinfection was significantly less common in the 1 to 5-year age group compared to ST monoinfection (OR [odds ratio] = 0.54, 95% CI: 0.38–0.77, p < 0.001.). While it is significantly more common in the 5 to 10-year age group compared to ST monoinfection (OR = 0.55, 95% CI: 0.40–0.77, p < 0.001). JE–ST coinfection cases were equally distributed across all the age groups. No significant difference in sex distribution between JE and ST monoinfection, JE and JE–ST coinfection, or ST and JE–ST coinfection was observed.

Table 1.

Comparison of Demographic, Clinical, and Biochemical Characteristics of Japanese Encephalitis (JE) Monoinfection, Scrub Typhus (ST) Monoinfection and Their Coinfection (JE–ST) Positive Cases

				JE–ST Monoinfection comparison		JE & coinfection comparison		ST & coinfection comparison
PARAMETERS^b	JE Monoinfection (n = 177)	ST Monoinfection (n = 898)	JE–ST coinfection (n = 105)	Odds ratio (Lower & Upper CI at 95%)	(p value)^c	Odds ratio (Lower & Upper CI at 95%)	(p value)	Odds ratio (Lower & Upper CI at 95%)	(p value)
Age group (years)
0–1	3 (1.7)	24 (2.7)	0 (0.0)	0.62 (0.18–0.2.0)	0.6	NA	0.2	NA	0.1
1.1–5.0	50 (28.2)	376 (42.1)	34 (32.4)	0.54 (0.38–0.77)	<0.001	0.82 (0.48–1.3)	0.5	1.5 (0.98–2.3)	0.06
5.1–10.0	80 (45.2)	282 (31.6)	43 (41.0)	0.55 (0.40–0.77)	<0.001	1.1 (0.72–1.9)	0.5	0.66 (0.44–1.0)	0.06
10.1–16.0	22 (12.4)	149 (16.7)	13 (12.4)	0.70 (0.43–1.1)	0.1	1.0 (0.48–2.0)	0.5	1.4 (0.77–2.6)	0.3
16.1–20	2 (1.1)	17 (1.9)	5 (4.8)	0.58 (0.13–2.5)	0.7	0.22 (0.04–1.1)	0.1	0.38 (0.14–1.0)	0.07
20.1–60	14 (7.1)	44 (22.2)	10 (9.5)	1.6 (0.88–3.0)	0.1	0.81 (0.34–1.9)	0.6	0.49 (0.24–1.0)	0.06
>60	6 (3.4)	1 (0.1)	0 (0.0)	0.93 (0.01–0.26)	<0.001	NA	0.08	NA	0.8
Sex (Female)	86 (48.6)	449 (50.3)	56 (53.3)	0.93 (0.67–1.2)	0.3	0.82 (0.50–1.3)	0.2	0.88 (0.59–1.3)	0.2
Onset of Fever (days) Mean (SD)	6.7 (5.8)	8.3 (4.1)	8.0 (3.8)	NA	<0.001	NA	<0.001	NA	<0.001
Altered Sensorium (days) Mean (SD)	2.1 (1.8)	1.8 (1.5)	1.8 (1.3)	NA	0.01	NA	0.01	NA	0.01
Seizure	91 (51.4)	452 (50.6)	49 (46.7)	1.03 (0.74–1.4)	0.8	1.2 (0.74–1.9)	0.4	1.7 (0.78–1.75)	0.2
Headache	36 (20.3)	163 (18.3)	24 (22.9)	0.87 (0.58–1.3)	0.2	0.86 (0.48–1.5)	0.3	0.75 (0.46–1.2)	0.1
Vomiting	99 (55.9)	495 (55.4)	59 (56.2)	1.0 (0.73–1.4)	0.9	0.98 (0.60–1.6)	0.4	0.96 (0.64–1.4)	0.4
Abdominal Pain	5 (2.8)	156 (17.5)	21 (20)	0.13 (0.05–0.33))	<0.001	0.11 (0.04–0.31)	<0.001	0.84 (0.50–1.4)	0.2
Rash	4 (2.3)	75 (8.4)	8 (7.6)	0.25 (.09–0.69)	<0.001	0.28 (0.08–0.95)	0.02	1.1 (0.52–2.3)	0.4
Hepatomegaly	15 (8.9)	189 (23.3)	20 (20.8)	0.32 (0.18–0.56)	<0.001	0.37 (0.18–0.76)	0.003	1.1 (0.68–1.9)	0.2
GCS (n = 884) ^a
3 to 7	19 (12.8)	49 (6.6)	6 (7.14)	0.48 (0.27–0.84)	0.007	0.52 (0.2–1.3)	0.09	0.92 (0.38–2.2)	0.4
8 to 11	116 (78.4)	505 (68.6)	64 (76.2)	0.60 (0.39–0.91)	0.008	0.88 (0.46–1.6)	0.3	0.68 (0.40–1.1)	0.07
12 to 15	13 (8.8)	182 (24.7)	14 (16.7)	0.29 (0.16–0.53)	<0.001	0.48 (0.21–1.08)	0.04	0.60 (0.33–1.1)	0.05
≤ 8 GCS	89 (60.4) [148]	319 (43.3) [736]	36 (42.8) [84]	0.50 (0.35–0.72)	<0.001	0.49 (0.28–0.85)	0.01	1.2 (0.64–1.6)	0.9
Abnormal DTR	107 (65.6)	435 (54.6)	48 (51.1)	0.62 (0.44–0.89)	0.004	0.54 (0.32–0.91)	0.01	1.1 (0.75–1.7)	0.2
Abnormal Tone	118 (80.8)	415 (55.3)	53 (63.9)	0.29 (0.18–0.45)	<0.001	0.41 (0.22–0.77)	0.002	0.69 (0.43–1.1)	0.06
Plantar extensor	117 (66.1)	497 (55.7)	55 (52.4)	0.64 (0.45–0.90)	0.01	0.56 (0.34–0.92)	0.01	1.1 (0.76–1.7)	0.2
Neck rigidity	13 (8.2)	78 (10.9)	8 (8.6)	0.74 (0.40–1.3)	0.1	0.95 (0.37–2.3)	0.4	0.78 (0.36–1.6)	0.2
Kernig’s Sign	12 (7.6)	66 (9.1)	7 (7.5)	0.82 (0.44–1.5)	0.2	1.0 (0.38–2.6)	0.5	0.81 (0.36–1.8)	0.3
Anemia	120 (77.9)	688 (91.6)	83 (91.2)	0.32 (0.20–0.50)	<0.001	0.30 (.14–0.77)	0.008	1.05 (0.48–2.2)	0.8
Leucocytosis	94 (61.1)	487 (64.6)	66 (73.3)	0.85 (0.59–1.2)	0.4	0.56 (0.32–1.0)	0.06	0.66 (0.40–1.1)	0.1
Leukopenia	3 (2.0)	12 (1.6)	0	1.2 (0.34–4.4)	0.7	NA	0.1	NA	0.1
Lymphocytosis	15 (8.9)	300 (40)	29 (31.9)	0.16 (.09–0.2)	<0.001	0.23 (0.11–0.46)	<0.001	1.4 (0.89–2.2)	0.1
Lymphocytopenia	89 (58.5)	103 (13.7)	17 (18.7)	0.11 (0.07–0.16)	<0.001	0.16 (0.08–0.30)	<0.001	1.4 (0.81–2.5)	0.2
Elevated SGOT	104 (71.7)	637 (88.8)	88 (86.3)	0.31 (0.20–0.48)	<0.001	0.40 (0.19–0.81)	0.004	1.2 (0.65–2.4)	0.4
Elevated SGPT	47 (32.4)	559 (78.7)	65 (73.9)	0.13 (0.09–0.19	<0.001	0.16 (0.09–0.30)	<0.001	1.2 (0.75–2.0)	0.4
SGOT/SGPT >1.5	83 (79)	356 (54.4)	45 (58.4)	0.31 (0.19–0.51)	<0.001	0.37 (0.19–0.71)	0.003	0.84 (0.52–1.3)	0.5
Thrombocytopenia	30 (19.6)	391 (52.2)	46 (50.6)	0.22 (0.14–0.34)	<0.001	0.23 (0.13–0.42)	<0.001	1.06 (0.69–1.6)	0.8
Increased CSF cell	122 (83.0)	630 (91.7)	80 (96.4)	0.44 (0.26–0.73)	0.003	0.18 (0.05–0.62)	0.002	0.41 (0.12–1.3)	0.1
Abnormal CSF Glucose	66 (43.1)	275 (39.7)	35 (41.6)	1.1 (0.80–1.6)	0.4	1.0 (0.61–1.8)	0.8	0.92 (0.58–1.4)	0.7
Abnormal CSF Protein	131 (85.6)	660 (95.1)	83 (98.8)	0.30 (0.17–0.54)	<0.001	0.07 (0.01–0.54)	<0.001	0.23 (0.03–1.7)	0.1
Mortality	61 (34.5)	120 (13.4)	10 (9.5)	0.29 (0.20–0.42)	<0.001	0.20 (.09–0.41)	<0.001	0.67 (0.34–1.3)	0.4

Available patient record.

Parameters were taken at the time of hospitalization.

chi-squared test is performed for individual variable to observe significance among monoinfections and coinfection group and findings at p < 0.05 were considered as significant.

Data are number (percentage) or Mean (SD); OR, Odds ratio, GCS, Glasgow Coma Scale; DTR, deep tendon reflex; CSF, cerebrospinal fluid; TLC, total leukocyte count; SGOT, glutamic-oxalacetic transaminase; and SGPT, glutamic-pyruvic transaminase.

Values in bold indicate statistical significance (p < 0.05).

Onset of fever (Mean±SD) was significantly shorter in JE monoinfection compared to ST monoinfection and JE–ST coinfection (p < 0.001 for both comparisons). The duration of altered sensorium (Mean±SD) was significantly longer in JE monoinfection compared to ST monoinfection and JE–ST coinfection (p = 0.01 for both comparisons). No significant differences in seizure occurrence between the comparison groups were observed. Abdominal pain was found to be significantly less common in the JE monoinfection group compared to ST monoinfection and JE–ST coinfection (JE vs. ST: OR = 0.13, 95% CI: 0.05–0.33, p < 0.001; JE vs. coinfection: OR = 0.11, 95% CI: 0.04–0.31, p < 0.001). Hepatomegaly was significantly more common in the ST monoinfection group compared to the JE monoinfection (OR = 0.32, 95% CI: 0.18–0.56, p < 0.001) and JE–ST coinfection (OR = 0.37, 95% CI: 0.18–0.76, p = 0.003). JE monoinfection showed significantly lower Glasgow Coma Scale (GCS) (≤8) compared with ST monoinfection (OR = 0.50, 95% CI: 0.35–0.72, p < 0.001) and no significant difference with JE–ST coinfection (OR = 1.2, 95% CI: 0.64–1.6, p = 0.9) was observed. Abnormal deep tendon reflex was significantly more common in the ST monoinfection group compared to the JE monoinfection (OR = 0.62, 95% CI: 0.44–0.89, p = 0.004). While abnormal tone was significantly more common in the JE monoinfection group compared with ST monoinfection (OR = 0.29, 95% CI: 0.18–0.45, p < 0.001). However, ST monoinfection and coinfection positive cases presented with clinical symptoms such as abdominal pain, rashes, and hepatomegaly. Further comparison of biochemical parameters among JE, ST monoinfections, and coinfection patients showed that anemia and lymphocytosis were significantly more common in the ST monoinfection group compared to the JE monoinfection group, and the ST monoinfection group had significantly elevated glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase compared with JE monoinfection group. while, thrombocytopenia was significantly more common in ST-monoinfection group and JE–ST coinfection group compared with JE monoinfection group. CSF analysis revealed abnormally increased CSF cell count in the ST monoinfection group compared with JE monoinfection (OR = 0.44, 95% CI: 0.26–0.73, p = 0.003). Abnormal CSF protein was significantly more common in ST monoinfection compared with JE monoinfection (OR = 0.30, 95% CI: 0.17–0.54, p < 0.001) A significant reduction in lymphocyte counts in peripheral blood has been observed in JE monoinfection cases compared with ST monoinfection and JE–ST coinfection cases.

Case fatality rate (or ratio) in JE monoinfection (34.5%) was significantly higher than that in ST monoinfection (13.4%) (OR = 0.29, 95% CI: 0.20–0.42, p < 0.001) and coinfection positive (9.5%) (OR = 0.67, 95% CI: 0.34–1.3, p = 0.4) AES cases. JE monoinfection has a significantly longer time to discharge compared with ST monoinfection (p < 0.001) and JE–ST coinfection (p = 0.004).

It is known that the JE and ST seasonality coincides with the monsoon season in this region (Fig. 1). Mittal et al. also reported the same trend in their study (Mittal et al., 2017). It has been reported previously that JE incidence is highest in under-five children (Ranjan et al., 2014). Whereas this study observed a higher proportion of JE in those above 5 years of age (Table 1). This may be due to the introduction of the JE vaccine in the routine immunization program in JE endemic regions of India.

Severe neurological manifestations were associated with JE monoinfection patients, which is in agreement with earlier reports of prominent predictors of JE infections from this region (Ooi et al., 2008). Whereas abnormal liver function and milder neurological manifestations (GCS >8) were associated with ST monoinfection and coinfection cases.

Lymphocytopenia was observed predominantly in JE monoinfection. A 100% decrease in lymphocyte count was also observed in JE cases from China (Su et al., 2021). This condition may facilitate ST bacteria to infect the person who was already infected with the JE virus, as this area is known to be coendemic for both the etiologies (Mittal et al., 2017). Apparent lower mortality among coinfection patients may be due to asymptomatic JEV infection. JE could have worse mortality than ST because ST is treatable, and JE has no specific therapy available, only supportive care.

There are many limitations to this article. First, we were unable to collect convalescent samples to confirm JE positivity by measuring a four-fold rise in antibody titers in the 63 JE-IgM serum-positive samples. Meningitis presents with peculiar signs, and the treatment protocols are different for meningitis of bacterial and other etiologies, which is not a subject matter of the present article. Dengue NS1-positive patients were not included in the present analysis; hence, the cross-positivity issues were not alluded too.

In summary, this investigation shows that the coinfection cases have a significantly different clinical presentation from that of JE monoinfection cases. Coinfection patients are likely exhibiting only ST pathophysiology, or this might be due to asymptomatic/silent JE infection after ST infection; at present, no pathophysiologically plausible explanation is available to understand this phenomenon. Hence, it is necessary to increase awareness among clinicians in the nonendemic area about the epidemiology of these infections. To avoid severe consequences, travelers must follow Advisory Committee on Immunization Practices (ACIP) recommendations on JE vaccination. Considering ST as a major cause of AES, a specific guideline for travelers visiting the AES endemic regions should be recommended that may include Azithromycin antibiotics for empirical treatment. JE and ST should be considered in all travelers coming back from endemic regions and presenting with acute encephalitis illness.

Footnotes

Acknowledgments

The authors extend our sincere thanks to all Scientists, Technical, and administrative Staff of ICMR-Regional Medical Research Center, Gorakhpur. The contribution of medical and nonmedical staff of AES Cell, Department of Paediatrics and Department of Medicine of Baba Raghav Das Medical College, Gorakhpur is also gratefully acknowledged.

Authors’ Contributions

V.P.B., H.D., B.R.M., and N.S. had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis and H.D., B.R.M., and N.S. share first authorship. V.P.B. and H.D. contributed equally as corresponding author in this article. V.P.B., H.D., A.R.D., B.R.M., and N.S.: Concept and design. B.R.M., A.R.D., N.S., N.K., S.P.B., K.Z., R.S., and A.K.P.: Acquisition, analysis, or interpretation of data. B.R.M., N.S., and H.D.: Drafting of the article. R.S., A.K.P., M.M., and R.K.: Critical review of the article for important intellectual content B.R.M., A.R.D., and N.S.: Statistical analysis. V.P.B. and H.D.: Obtained funding. H.D., M.M., and R.K.: Administrative, technical, or material support. V.P.B., R.K., and M.M.: Supervision. All authors have approved the final version of this article.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This work is supported by Indian council of Medical Research (Grant Number: vir/RCI/6/2016-ECD-2580).

Supplementary Material

Supplementary Data S1

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