Acute Encephalopathy in Children From Muzaffarpur, Bihar, India, and the Potential Role of Ambient Heat Stress-Induced Mitochondrial Dysfunction

Background: Periodic outbreaks of acute encephalopathy in children have been reported from Muzaffarpur, Bihar, India. No infectious cause has been identified for this. This study presents the clinical and metabolic profile of children hospitalized with acute encephalopathy and the potential role of ambient heat stress. Methods: This cross-sectional study included children (<15 years) with acute encephalopathy admitted from April 4, 2019, to July 4, 2019. The clinical and laboratory investigations included infections, metabolic abnormalities, and muscle tissue analysis. The children who had metabolic derangements but no infectious cause were labeled as acute metabolic encephalopathy. The descriptive analysis summarized the clinical, laboratory, and histopathology findings, and their association with the ambient heat parameters was explored. Results: Out of the 450 children hospitalized (median age, four years), 94 (20.9%) died. Children had early morning onset (89%), seizures (99%), fever (82%), hypoglycemia at admission (64%), raised aminotransferases (60%), and high blood urea (66%). Blood lactate (50%), lactate dehydrogenase (84%), pyruvate (100%), ammonia (32%), and creatinine phosphokinase (69%) were raised. Viral marker tests were negative. The patients had abnormal metabolic markers like decreased blood-free carnitine, elevated blood acylcarnitines, and elevated urinary lactate, oxalate, maleate, adipate, and fatty acid metabolites. Blood carnitine and acylcarnitine levels normalized in 75% of the patients treated with carnitine and coenzyme-Q. Muscle tissues showed megamitochondria on electron microscopy and reduced respiratory enzyme complex-I activity. A significant correlation between the number of admissions and ambient heat indices was observed. Conclusions: The findings suggest secondary mitochondrial dysfunction as a possible mechanism for acute encephalopathy in children from Muzaffarpur, Bihar, and ambient heat stress as a possible risk factor.

Heat stress exposure in animals and humans has been reported to cause metabolic derangements and mitochondrial dysfunctions, which can explain the majority of the biochemical derangements reported patients with the diagnosis of JE were excluded. The data for 450 cases were analyzed, out of which 94 (21%) died. In Muzaffarpur district, 70% of these cases were from three blocks: Meenapur, Mushahri, and Kanti. Almost all (n=447, 99%) cases occurred during June 2019, from June 9-21, 2019, with the peak during June 15-17, 2019 ( Figure 1). The expert team was present for two weeks at the hospital and facilitated better clinical record-keeping. Detailed clinical and investigation data for 150 patients could be retrieved from the case records.

Post-therapy only (n=30)
Pre-and post-therapy levels for the same child   Muscle histopathology (two cases) revealed fibre size variation and electron microscopy showed enlarged mitochondria, altered cristae pattern, presence of electron-dense material and clumped z-band ( Figures  3-6).

FIGURE 3: Muscle histopathology and electron microscopy findings from a patient with acute encephalopathy
Histochemical stains shows Type II (dark stained) fibre atrophy on ATPase 9.5 stain. There is no evidence of mitochondrial pathology or storage material (glycogen or lipid).   Grossly, low respiratory enzyme complex-I activity (0.07-0.08 µmols, 24-28% of control mean) was observed for the two patients ( Table 5).

Case management
Recurrent episodes of hypoglycemia were observed in 23% (34/150) of the patients when the dextrose infusion replacement was delayed or the infusion rate was tapered (four of them expired). Carnitine (50 mg per kg/day) and coenzyme-Q (10 mg per kg/day) were given to 37 patients. Repeat samples from 10 patients showed normalization of free carnitine (n=9) and acylcarnitines (n=7) levels after five to six days of therapy ( Figure 7). In these patients, the serum CPK, CPK-MB, LDH, ammonia, lactate, pyruvate levels normalized and urinary ketone disappeared. At discharge, 72% (108/150) of the patients had neurological deficits.

Sociodemographic parameters
A history of encephalopathy in siblings was noted in 5% (5/103) of patients. Parents gave a history of lychee intake within 24 hours before admission in 27% (28/103) of the cases. Most (91/106; 86%) of these patients belonged to poorer families and 45% (68/150) were underweight for age, although the detailed anthropometry could not be documented.

Relationship with ambient heat stress and meteorological parameters
The caseload of acute encephalopathy in children increased when the maximum ambient temperature crossed 40 0 C and sustained for two to three days, and declined with the drop in the temperature and onset of rain. Significant correlations between the acute encephalopathy caseload/admissions and weather parameters for the previous one, two, three, and seven days during the year 2019 were observed; positive with maximum (r=0.318 to 0.343) and minimum (r=0.371 to 0.395) ambient temperatures, and heat index (r=0.428 to 0.446) (p values <0.001). No significant correlation with rainfall/precipitation, humidity, wind speed, and sunshine hours was noted ( Table 6).

Reference period for meteorological parameters
The Pearson correlation coefficient (r) for parameters (95% CI) Maximum temperature  Regression analysis also documented a significant association between the acute encephalopathy caseload/admissions and the meteorological parameters for the previous one to seven days during 2019; positive association with the maximum (coefficient=0.774 to 0.896) and minimum (coefficient=1.251 to 2.033) temperatures, and heat index (coefficient=1.094 to 1.456) (p values <0.001) ( Table 7).

Reference period for meteorological parameters
The regression coefficient for the climate/meteorological parameters (95% CI)  * Correlation significant at the 0.05 level (2-tailed); ** Correlation significant at the 0.01 level (2-tailed).

Discussion
The 2019 outbreak investigation revealed that almost all children had early morning onset seizures and altered sensorium with hypoglycemia at admission. The multiorgan involvement (brain, liver, kidney, and muscles), hypoglycemia, ketonemia, and metabolic derangements involving fatty acids, tricarboxylic acid, ammonia, and ketone metabolism pathways indicated possible mitochondria dysfunction. The muscle histopathology and enzyme complex assay also supported mitochondrial pathology. The clinical and metabolic improvements with carnitine/coenzyme-Q therapy support this. Significant correlations between the number of admissions and the ambient temperatures (maximum and minimum) and the heat index were observed.
Higher associations with litchi consumption (OR=9.6), orchard visit (OR=6.0), and missing evening meals (OR=2.2) were noted. With documentation of hypoglycin A and/or MCPG in 44-64%, Lychee consumption was proposed as the factor for encephalopathy [5].
The reports for 2011, 2013, 2014, and 2019 indicated non-infective and metabolic aetiology for encephalopathy during the May-June months. Multiorgan (brain, liver, kidney, muscle and heart) involvement with abnormal CPK-MB, LDH, lactate, urinary organic aciduria and plasma acylcarnitine profiles were documented in these children. During the 2019 outbreak, ketonemia/ketonuria was documented in several cases, but with MCPG, hypoketonemia is expected [6]. The history of lychee intake was noted in 27% of cases in 2019. Many parents denied lychee intake by their children, as the local government campaigned against children consuming lychee after 2017. Urinary lychee metabolites detection may not correlate with fruit intake amount. In absence of the lethal dose (LD50) and effective dose (ED50) of lychee for humans, considering the doses for rats, about 300 lychees are to be consumed, which is difficult for young children [5,7,16,17]. Minimal involvement of children from upper socioeconomic strata does not support the lychee consumption hypothesis, as lychee is consumed by all.
The electron microscopy findings and reduced respiratory chain enzyme complexes improvements in metabolic parameters after carnitine/coenzyme-Q therapy are pointers toward potential secondary mitochondrial dysfunction. Seizures in children with carnitine/acylcarnitine deficiency have been reported [18].
During May-June, the Muzaffarpur climate becomes extremely hot (highest: 40-46 0 C, lowest 28-30 0 C) and humid (50-60%). More cases were observed when the highest temperature was above 40 0 C for some days [3]. A recent report by our group reported positive correlations between acute encephalopathy caseload (n=1318, during 2012-2020) with maximum (r=0.275) and minimum (r=0.306) temperatures and heat index (r=0.325) and negative correlation with humidity (r=−0.222) and rainfall (r=−0.183) during past 24 hours (all p < 0.05). The correlation was also consistent for these climate parameters for the past 48 and 72 hours before admission [19]. The significant positive correlation between acute encephalopathy caseload and ambient climate parameters (maximum and minimum temperatures and heat index) suggests a possible relationship, if not causative, a contributory factor for mitochondrial pathology.
Heat-related morbidity and mortality have been observed from the third day of high temperatures [8].
Mitochondrial dysfunction with reduced adenosine triphosphate (ATP) production, raised serum CPK, LDH, and transaminases have been documented with heat stress [9][10][11]. In animals, exposure above 39 0 C has shown nuclear chromatin condensation and mitochondrial alterations (reduced number of cristae membranes, membrane disruptions, and swelling) in the myocardium [12]. Cerebral oedema with changes in axons, nerves, glial cells, and vascular endothelium was observed in rats with exposure to ≥38 0 C [13,14].
JE has been the leading infectious cause of acute encephalitis in children in India followed by scrub typhus and dengue, while the aetiology remains elusive in half of the cases. There is inter-state variation in the proportion of infectious aetiologies contributing to acute encephalitis in children. The aetiological profile is also changing with the introduction of the JE vaccination in selected areas. There is also clinical ambiguity in labelling the cases, in most scenarios the cases are clubbed as AES, which includes both encephalitis and encephalopathy, which makes it difficult to differentiate between these entities, whether a different neurological disorder or any combination. Despite these variations, the acute encephalopathy outbreak in children from Muzaffarpur and nearby areas remains a challenge, with limited success from infectious aetiological investigations.
These indicate the ongoing debate and confusion regarding the aetiology across India and different neighbouring countries. The efforts for viral and other infectious aetiological isolation have been partially successful. Thus, there is a need to think beyond the infectious causes for finding the aetiology.
The study has several limitations. This study collected data during the response to the acute encephalopathy outbreak and not as a systematically designed study. The detailed clinical and nutritional assessment and investigations could not be undertaken for all patients and only a small number of patients could be subjected to detailed biochemical, metabolic, and mitochondria histopathology evaluation. It didn't include any control participant or community investigation to identify risk factors.

Conclusions
The findings suggest the possible role of secondary mitochondrial dysfunction for acute encephalopathy in children from Muzaffarpur during the summer. Although the exact cause of mitochondrial dysfunction could not be established, heat stress with underlying malnutrition and micronutrient deficiencies may be possible explanations. Carefully designed studies using a robust methodology, markers of metabolic encephalopathy and mitochondrial derangements linked to the environmental heat stress indices, and individual-level factors (sociodemography, nutrition, and other contributors) are needed to confirm the association or causal linkages.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Institute Ethics Committee, Sri Krishna Medical College, Muzaffarpur, Bihar, India issued approval 17/20 (IEC). Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.