Association of acute toxic encephalopathy with litchi consumption in an outbreak in Muzaffarpur, India, 2014: a case-control study

Background Outbreaks of unexplained illness frequently remain under-investigated. In India, outbreaks of an acute neurological illness with high mortality among children occur annually in Muzaffarpur, the country’s largest litchi cultivation region. In 2014, we aimed to investigate the cause and risk factors for this illness. Methods In this hospital-based surveillance and nested age-matched case-control study, we did laboratory investigations to assess potential infectious and non-infectious causes of this acute neurological illness. Cases were children aged 15 years or younger who were admitted to two hospitals in Muzaffarpur with new-onset seizures or altered sensorium. Age-matched controls were residents of Muzaffarpur who were admitted to the same two hospitals for a non-neurologic illness within seven days of the date of admission of the case. Clinical specimens (blood, cerebrospinal fluid, and urine) and environmental specimens (litchis) were tested for evidence of infectious pathogens, pesticides, toxic metals, and other non-infectious causes, including presence of hypoglycin A or methylenecyclopropylglycine (MCPG), naturally-occurring fruit-based toxins that cause hypoglycaemia and metabolic derangement. Matched and unmatched (controlling for age) bivariate analyses were done and risk factors for illness were expressed as matched odds ratios and odds ratios (unmatched analyses). Findings Between May 26, and July 17, 2014, 390 patients meeting the case definition were admitted to the two referral hospitals in Muzaffarpur, of whom 122 (31%) died. On admission, 204 (62%) of 327 had blood glucose concentration of 70 mg/dL or less. 104 cases were compared with 104 age-matched hospital controls. Litchi consumption (matched odds ratio [mOR] 9·6 [95% CI 3·6 – 24]) and absence of an evening meal (2·2 [1·2–4·3]) in the 24 h preceding illness onset were associated with illness. The absence of an evening meal significantly modified the effect of eating litchis on illness (odds ratio [OR] 7·8 [95% CI 3·3–18·8], without evening meal; OR 3·6 [1·1–11·1] with an evening meal). Tests for infectious agents and pesticides were negative. Metabolites of hypoglycin A, MCPG, or both were detected in 48 [66%] of 73 urine specimens from case-patients and none from 15 controls; 72 (90%) of 80 case-patient specimens had abnormal plasma acylcarnitine profiles, consistent with severe disruption of fatty acid metabolism. In 36 litchi arils tested from Muzaffarpur, hypoglycin A concentrations ranged from 12·4 μg/g to 152·0 μg/g and MCPG ranged from 44·9 μg/g to 220·0 μg/g. Interpretation Our investigation suggests an outbreak of acute encephalopathy in Muzaffarpur associated with both hypoglycin A and MCPG toxicity. To prevent illness and reduce mortality in the region, we recommended minimising litchi consumption, ensuring receipt of an evening meal and implementing rapid glucose correction for suspected illness. A comprehensive investigative approach in Muzaffarpur led to timely public health recommendations, underscoring the importance of using systematic methods in other unexplained illness outbreaks

Assessment of genetic fidelity of <i>in vitro</i> raised plants of <i>Pittosporum eriocarpum</i>.

<p>(A) SCoT fragments obtained with primer SCoT 35. (B) ISSR fragments obtained from primer number 860. (C)RAPD fragments obtained with primer OPL10. L- 1Kb ladder; MP- mother plant (Control); A1-A9- <i>in vitro</i> raised plants.</p

<i>Pittosporum eriocarpum</i>.

<p>(A) Nodal explant. (B) Shoot induction in MS medium supplemented with BA (mg/l). (C) Root induction in MS medium supplemented with IBA (mg/l). (D) <i>In vitro</i> grown plantlets. (E) <i>In vitro</i> grown plantlets in soil rite. (F) <i>In vitro</i> raised plantlets in natural conditions.</p

Dendrogram illustrating coefficient similarity among <i>in vitro</i> raised plants with mother plant of <i>P</i>. <i>eriocarpum</i> by UPGMA cluster analysis from the RAPD data set showing genetic relationship.

<p>MP- mother plant; A1-A9- <i>in vitro</i> raised plants.</p

ANOVA of the effect of BA and IBA and combined BA and IBA on shoot length.

<p>ANOVA of the effect of BA and IBA and combined BA and IBA on shoot length.</p

Effect of various concentrations of BA and IBA on shoot induction from nodal explants of <i>P</i>. <i>eriocarpum</i>.

<p>Effect of various concentrations of BA and IBA on shoot induction from nodal explants of <i>P</i>. <i>eriocarpum</i>.</p

Effect of various concentrations of BA and IBA (mg/l) on shoot length in <i>P</i>. <i>eriocarpum</i>.

<p>Effect of various concentrations of BA and IBA (mg/l) on shoot length in <i>P</i>. <i>eriocarpum</i>.</p

Effect of types of auxins on root induction in <i>P</i>. <i>eriocarpum</i>.

<p>(A)Number of roots produced per regenerated shoot. (B)Root length. (C) Basal callus formation per explant.</p

Genetic Homogeneity Revealed Using SCoT, ISSR and RAPD Markers in Micropropagated <i>Pittosporum eriocarpum</i> Royle- An Endemic and Endangered Medicinal Plant

<div><p><i>Pittosporum eriocarpum</i> Royle, a medicinally important taxon, is endemic to Uttarakhand region of Himalaya. It has become endangered due to over-collection and the loss of habitats. As raising plants through seeds in this plant is problematic, a reliable protocol for micropropagation using nodal explants has been developed. High shoot regeneration (95%) occurred in MS medium augmented with BA 0.4mg/l in combination IBA 0.6mg/l. <i>In vitro</i> regenerated shoots were rooted in MS medium supplemented with three auxins, of which 0.6 mg/l indole butyric acid proved to be the best for rooting (90%) with maximum number of roots per shoot. Thereafter, rooted plants were hardened and nearly 73% of rooted shoots were successfully acclimatized and established in the field. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity amongst nine <i>in vitro</i> raised plantlets with mother plant. DNA fingerprints of <i>in vitro</i> regenerated plantlets displayed monomorphic bands similar to mother plant, indicating homogeneity among the micropropagated plants with donor mother plant. The similarity values were calculated based on SCoT, ISSR and RAPD profiles which ranged from 0.89 to 1.00, 0.91 to 1.00 and 0.95 to 1.00 respectively. The dendrograms generated through Unweighted Pair Group Method with arithmetic mean (UPGMA) analysis revealed 97% similarity amongst micropropagated plants with donor mother plant, thus confirming genetic homogeneity of micropropagated clones. This is the first report on micropropagation and genetic homogeneity assessment of <i>P</i>. <i>eriocarpum</i>. The protocol would be useful for the conservation and large scale production of <i>P</i>. <i>eriocarpum</i> to meet the demand for medicinal formulations and also for the re-introduction of <i>in vitro</i> grown plants in the suitable natural habitats to restore the populations.</p></div

Amplification products generated with SCoT, ISSR and RAPD markers among mother plant and <i>in vitro</i> raised plants.

<p>Amplification products generated with SCoT, ISSR and RAPD markers among mother plant and <i>in vitro</i> raised plants.</p