ASM 2016

Tox Mimics in the Critically Ill Patient

Information on Diagnosis and Take Home Points

Case #1 Discussion and more about Anti-NMDA receptor Ab encephalitis:

Background and epidemiology: Autoimmune encephalitis may be divided into several groups of diseases: those with antibodies to cell surface proteins, those with antibodies to intracellular synaptic proteins, T-cell diseases associated with antibodies to intracellular antigens, and those associated with other autoimmune disorders. Many forms of autoimmune encephalitis are paraneoplastic, often necessitating tumor screening.

Anti-N-methyl-D-aspartate (Anti-NMDA) receptor encephalitis was first described by Dalmau and colleagues in 2007. There are now more than 500 cases reported in the medical literature. Epidemiological evidence suggests that it is the most common cause of autoimmune encephalitis after acute demyelinating encephalitis. The California Encephalitis Project (between Sept 2007- Feb 2011), reviewed in detail 761 patients presenting with encephalitis. The frequency of anti-NMDA receptor encephalitis surpassed that of any single viral cause of encephalitis. There is a gender-predilection favoring women, but reports of anti-NMDA receptor encephalitis include both men and women ranging from 2 to 84 years of age.

Pathophysiology:

The NMDA receptor is a heterotetrameric assembly of an NR1 subunit usually in combination with modulatory NR2 and/or NR3 subunits. To date 7 different subunits have been identified—NR1, NR2a-d, NR3a+b. NR1 is ubiquitously distributed in the brain and is an obligate subunit of functional NMDA receptors. Patients with anti-NMDA receptor autoimmune encephalitis have IgG antibodies to the NR1 subunit of the NMDA receptor. This results in antibody mediated capping, cross-linking and internalization of the receptor, causing a decrease in NMDA receptor surface density, synaptic localization, while overall cellular structure and synaptic density is largely unchanged. So it appears that anti-NMDA receptor antibodies reversibly alter the number and distribution of glutamate receptors, which results in decreased glutamatergic synaptic function. Lack of neuronal cell death may, in part, explain why patients frequently experience recovery in this disorder. For unclear reasons, the hippocampal region is preferentially affected.

Numerous other immune mediated synaptic disorders (also known as (ASPES) Autoimmune Synaptic Protein Encephalopathy Syndrome) have been identified but due to time constraints cannot be further discussed in this forum. It is important for the clinician to recognize that analysis should include a search for additional antibodies including: Glu1/2 subunit of AMPA receptor, GABAB1 receptor, GABAA, GlyR, NR2A/NR2B subunits of NMDA receptor, mGluR5, mGluR1, DNER, GAD, DPPX, and LGI1 and Caspr2 (previously known as VGKC, Voltage-gated K+ channel).

Clinical Presentation:

There is a prodromal phase of non-specific symptoms—low-grade fever, headache, or viral-like syndrome (respiratory or GI symptoms) that often goes unnoticed and may not result in presentation to healthcare.

This is followed in several days to weeks by the onset of psychiatric symptoms or behavioral changes (including anxiety, bizarre behaviors, disinhibition, hallucinations, paranoid thoughts, grandiose or hyperreligious delusions). Erratic sleep patterns and insomnia commonly develop, as well as cognitive dysfunction such as short-term memory loss and concentration difficulties.

Progression to decreased level of consciousness, seizures, dyskinesias (including orofacial dyskinesias— grimacing or lip-smacking), choreathetoid movements or unusual postures can occur. Autonomic instability can cause cardiac dysrhythmias and hypoventilation may necessitate intubation.

Differences in adults vs. children with the disorder: adults are more likely to develop hypoventilation and autonomic instability and children experience more behavioral changes such as agitation, aggression, new-onset temper tantrums, changes in mood or personality. Speech involvement is more common in children and may include reduced speech, mutism, echolalia, or perseveration.

The differential depends on the stage of disease but may include new onset psychosis, schizophrenia, catatonia, viral encephalitis, lupus, multiple sclerosis, cerebral vasculitis, Creutzfeld-Jakob disease, seizure disorder or status epilepticus. The toxicologic differential may include acute drug intoxication (PCP, ketamine, 2C series, etc.), withdrawal, Wernicke encephalitis, neuroleptic malignant syndrome, or possibly serotonin syndrome. NMS is particularly difficult to sort out because patients may be treated with neuroleptics for their early psychiatric symptoms then develop autonomic instability, motor dysfunction, and hypoventilation, making it much more difficult to discern NMS from anti-NMDA receptor encephalitis.

Diagnostic Findings and Work-up:

Obviously, it is crucial to rule out alternative diagnoses such as infectious etiologies, lupus, or multiple sclerosis. Patients are often empirically covered with antimicrobials pending completion of an infectious disease work-up.

MRI of the brain is abnormal ~50-70% of the time. Cortical and subcortical T2-fluid-attenuated inversion recovery signal abnormalities and sometimes transient cortical-meningeal enhancement is seen. (In children MRI abnormalities were less common, seen in only 31% of kids in one series.)

CSF analysis is abnormal in 80-94% of cases showing lymphocytic pleocytosis or increased protein synthesis or oligoclonal bands. (In patients with initially normal CSF--cell count, protein, glucose, and oligoclonal bands should be sought. Typically, if repeat LP is performed a few days later, pleocytosis will be present).

EEG is abnormal in almost all patients. It usually shows diffuse slowing of the background in the delta- theta range. Although some patients may have focal slowing. There was a recent study that found that 30% of adults with anti-NMDA receptor encephalitis had a unique pattern on EEG called “extreme delta brush,” which is similar to a pattern seen in premature infants. Electrographic seizures during continuous EEG monitoring occur in~60% of patients. There are several reports of patients with nonconvulsive status epilepticus which seems to underscore the importance of continuous EEG monitoring.

Diagnosis is confirmed by demonstrating IgG Anti-NMDA receptor antibodies in serum or CSF. CSF being the more reliable and recommended specimen. A comparison of normalized IgG levels in serum and CSF indicates the presence of intrathecal synthesis of antibodies in virtually all patients.

Whether levels of antibodies in the CSF or serum correlate with clinical signs and symptoms or with symptomatic improvement is controversial and actively debated. The most convincing evidence suggests interpretation of Ab titers and correlation with disease severity has several limitations and potential co- founders. Consequently, decisions regarding treatment are best made based on comprehensive clinical assessment and not Ab titers.

Antibodies to cell surface or synaptic proteins are directed against conformational epitopes and reactivity is usually lost when the antigen is denatured so that these Abs cannot be detected by standard immmunoblot or ELISA. Detection of these Ab requires either an immunohistochemistry protocol adapted to cell surface antigens, live neurons cultures, or cell-based assays in which recombinant antigens are expressed in mammalian cells. Commercial and clinical laboratories use cell-based assays for determination of this type of Ab; while these assays are highly specific they are not without problems. Specificity and sensitivity of these assays vary among laboratories even when the same technique is used. Also the tests are read by visual assessment of fluorescence, making interpretation difficult if titers are low or if specimens have non-specific background reactivity.

MRI of the abdomen and pelvis should be obtained to identify ovarian teratomas. Pelvic and transvaginal U/S can also be utilized. There are estimates that ~ 56% (range of 25-56%) of women > 18 years of age with anti-NMDA receptor encephalitis have unilateral or bilateral teratomas with tumors occurring in only 30% of women <18 years of age.

Studies show positron emission tomography has variable evidence with some indicating cortical hypometabolism and others showing hypermetabolism. Currently the role of FDG-PET, 1H-MRS, SPECT, scanning in this disorder is in the experimental phase. One study showed frontal and temporal glucose hypermetabolism associated with occipital hypometabolism the authors suggest this gradient of brain glucose metabolism correlated with clinical disease severity and normalized when patients recovered.

Treatment:

Early diagnosis and institution of first-line immunotherapy is crucial. Although randomized controlled trials have not been done to establish the best treatment regimen, clinical experience (Dalmau et al) suggests high dose corticosteroids (methylprednisolone 1 g/daily X 5days) , intravenous immunoglobulin (IV Ig 0.4 g/kg daily X 5 days), and plasma exchange. Second line therapy is initiated for those patients not responding to first line therapy (within 10 days) and consists of rituximab (375 mg/m2 every week for 4 weeks) followed by monthly cycles of cyclophosphamide (750 mg/m2 can be given with first dose of rituximab).

Prognosis and outcomes: With the regimen described above, up to 75-80% of patients exhibit total or near-total recovery while 25% of patients exhibit persistent severe neurological deficits or die. Estimated mortality is 4-7%. Some patients will continue to experience symptomatic improvement 2 years after presentation. Autonomic instability, dyskinesias, level of consciousness, and seizures are the first to improve. Psychiatric symptoms are the last to improve and are the most commonly seen symptoms in relapse.

Many centers have limited experience with the disorder, so patients are often transferred back and forth from rehabilitation (or home) to the hospital (as was seen in our patient’s case). A multidisciplinary approach involving nurses, psychiatrists, physiatrists, and cognitive rehabilitators is needed.

Take Home Points:

1. The differential diagnosis of anti-NMDA receptor encephalitis is extensive and depends on the stage of the disease. If your differential diagnosis includes any of the following: viral encephalitis, Wernicke encephalitis, neuroleptic malignant syndrome, acute drug intoxication, withdrawal or serotonin syndrome, you should consider autoimmune encephalitis – particularly anti-NMDA receptor encephalitis.

2. Patients often receive neuroleptics for treatment of psychiatric symptoms early in the disease course, making the distinction between anti-NMDA receptor encephalitis and neuroleptic malignant syndrome more difficult.

3. CSF analysis typically shows lymphocytic pleocytosis and/or oligoclonal bands. But definitive diagnosis is made by demonstrating presence of anti-NMDA receptor antibodies (or other synaptic protein Ab) in CSF.

4. EEG monitoring is usually necessary to determine if seizures are present. Dyskinesias are a prominent clinical feature and often limits the clinician’s ability to clinically determine if seizures are occurring.

Case #2 Discussion and more about Methylmalonic Acidemias:

Background:

Methylmalonic acidemia (MMA) is one of the most common inborn errors of organic acid metabolism. Its incidence is about 1/50,000 births; all forms follow an autosomal recessive inheritance pattern. MMA encompasses a heterogeneous group of disorders with some variability in presentation, both in timing of onset, and clinical manifestations. Although this problem may present in adulthood, it usually emerges during the first year of life. Most infants appear and feed normally after birth. But due to functional impairment of methylmalonyl CoA mutase, accumulation of methylmalonic acid in body tissue and fluids occurs. The infant may develop failure to thrive, vomiting, dehydration, lethargy, respiratory distress, and severe ketoacidosis. Later complications include developmental delay, liver failure, hyperammonemia, and coma. Treatment includes dietary protein restriction (0.5-1.5 g/kg/day) and hydroxycobalamin (1 to 2 mg/day). L-carnitine and metronidazole may also be helpful. With early identification and treatment long term complications can be avoided. This is why many newborn screening programs include methylmalonic acidemia. Inborn errors of metabolism may clinically resemble numerous other diagnoses such as sepsis, DKA, seizure disorder, sudden unexpected death (“SIDS”), child abuse, and chemical or drug intoxication. Toxic alcohols are often considered in the differential diagnosis of a child presenting with unexplained severe acidosis, just as an inborn error of metabolism would present. The concern arises in the possibility of confusing the two diagnoses.

When liquid extraction GC/FID is performed on serum of patients with various organic acid disorders, a false-positive result, identifying a toxic alcohol such as methanol or ethylene glycol, can occur if elution times alone are used. Headspace analysis is considered the best method for forensic analysis of volatiles avoiding the mis-identification of some volatiles based on similar retention times to other compounds.

The “Headspace” is the gas above the liquid sample in a chromatography vial. Analysis of the headspace is best for highly volatile compounds like alcohols that are partitioned into this portion of the sample. A sample of the gas phase (headspace) can then be injected into the GC column for separation and analysis of the components.

Flame ionization relies on a hydrogen flame that burns everything in the sample as it passes through the FI detector. If there is a large amount of some other organic compound present it could be volatilized and “contaminate” the sample producing spikes on the chromatogram that may have similar retention times to other volatiles (i.e. methanol in this case) and be mis-identified as such.

In the current case, GC with-FID was performed using liquid extraction (not gas from a head space) and a chromatogram based on retention times was produced, leading to identification and quantification of methanol. When a sample was taken to another laboratory for headspace analysis no methanol was detected.

In an attempt to identify the metabolite that would produce an elution time similar to methanol, methylmalonic acid was obtained from Sigma Aldrich Chemical since it is typically elevated in the serum and urine of patients with MMA. However, we were unable to generate chromatographs using various and progressively higher in vitro concentrations of pure methylmalonic acid via liquid extraction GC/FID. Consequently, it appears methylmalonic acid was not the interfering substance.

There are reports in the medical literature of parents being implicated in a child’s death and subsequently incarcerated due to such an analytical error. Shoemaker, et al., describe a 3- month-old infant who presented with lethargy, tachypnea and had profound acidosis with a pH of 7.02 and a serum bicarbonate concentration of 3 mmol/L. Additional laboratory investigation revealed serum ethylene glycol and acetone concentrations of 180 mg/L and 215 mg/L respectively. The patient recovered and was placed in foster care and parents were accused of child abuse. A second episode of severe acidosis (pH 6.9) occurred (shortly after parental visitation) and ethylene glycol was measured and found to be 911 mg/L. Despite resuscitation, hemodialysis and ethanol infusion for presumed ethylene glycol toxicity the patient ultimately died. The patient’s mother was incarcerated and accused of first-degree murder. Because the mother gave birth to a second child while incarcerated who became ill while in foster care and was ultimately diagnosed with methylmalonic acidemia, she was eventually exonerated. The authors indicate that the elution times of ethylene glycol and propionic acid are similar (using Jain and Fourney method) yet proved they were distinct by spiking the patient’s sample with ethylene glycol and generating a “double-headed” peak on gas chromatography. Woolf, et al., describe the converse of this scenario in which a patient with episodic lethargy, tachypnea and severe metabolic acidosis was extensively evaluated for an inborn error of metabolism including repeated urine and plasma amino acids, urine organic acids, serum carnitine, urine acylcarnitine profile, ammonia, and liver function tests. During the third hospitalization, ethylene glycol poisoning was suspected and confirmed.

Infants with severe metabolic acidosis and laboratory detection of volatile alcohols without a history of exposure must be evaluated for inborn errors of organic acid metabolism. Detection of volatiles using liquid extraction GC/FID must be scrutinized in order to avoid misdiagnosis. Performing headspace analysis via split column gas chromatography with flame-ionization detection is recommended to make a definitive diagnosis.

Take Home Points:

1. When infants present with unexplained metabolic acidosis, particularly if severe or recurrent, consideration must be given to both an inborn error of metabolism and intentional poisoning by a toxic alcohol. 2. This case highlights the importance of confirming the laboratory results by a second analytical method, particularly those that are used as forensic data in legal proceedings. 3. Analysis by Headspace Gas Chromatography with simultaneous Flame-Ionization and Mass Spectrometry Detection is the preferred method for forensic analysis of volatile agents (i.e., alcohols)