Editorial

Indian Pediatrics 2001; 38: 589-595  

Isolation of Measles Virus From Cerebrospinal Fluid of Children with Acute Encephalopathy Without Rash

N.S. Wairagkar, N.J. Shaikh, R.K. Ratho+, D. Ghosh*, R.C. Mahajan+, S. Singhi* and D.A. Gadkari

From the National Institute of Virology, 20-A, Ambedkar Road, Pune 411 001, India; and Departments of Pediatrics* and Virology+, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India.
Correspondence to: Dr. N.S. Wairagkar, National Institute of Virology, 20-A, Dr. Ambedkar Road, Pune 411 001, India. E-mail: [email protected]

Manuscript received: June 2, 2000, Initial review completed: July 5, 2000,
Revision accepted: January 11, 2001.

Objective: To determine the viral agent involved in cases of acute encephalopathy in children during an outbreak in Northern India. Design: Virological and serological studies using serum and cerebrospinal fluid specimens from patients. Methods: Serum and CSF specimens were tested by IgM ELISA for IgM antibodies to variety of viruses like Japanese encephalitis, West Nile, Dengue and Measles. The specimens were inoculated into Vero cell monolayer for virus isolation. The viral strains isolated were identified by indirect immunofluorescence test and qualitative in-vitro neutralization test using polyclonal and monoclonal antibodies to measles. Identity of the isolates was reconfirmed using RT-PCR method. Results: Of the 28 specimens tested, 17 had IgM antibodies to measles. Commercial IgM ELISA kits confirmed the serological findings. Vero cell cultures yielded 4 isolates from CSF and 2 from serum specimens of six different patients. Cytopathic effect was typical of measles. Indirect imunofluorescence using polyclonal and monoclonal antibodies to measles HA protein, confirmed the measles etiology. Neutralization tests reconfirmed the measles strain isolation. RT-PCR amplified product was confirmed as measles. Conclusion: The isolation of measles virus from CSF and serum of children with acute encephalopathy without rash proved the etiological role of measles virus in this outbreak.

Key words: Acute encephalopathy, Cerebrospinal fluid, Measles, Virus isolation.

ACUTE encephalopathy outbreaks have been documented from different parts of India but often remain undiagnosed(1). These were associated with high mortality. One such outbreak involving children with acute encephalopathy was reported during October-November, 1997 from northern part of India(2). We report the investigations and findings of serological and virological stuides to identify the etiological agent.

Fifty one children (30 male, 21 female) from villages and suburban areas of plains of Haryana, adjoining Punjab and Uttar Pradesh states were referred to Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh. The age of patients ranged from 1 year to 12 years (mean age 5.69 ± 2.68 years). Twenty eight specimens (11 serum and 17 Cerebrospinal fluid) of 22 patients were investigated at National Institute of Virology, Pune for serological and virological evidence of any etiology agent.

Serum samples were tested for the presence of IgM antibodies to Japanese encephalitis, Dengue and West Nile viruses using IgM capture ELISA test(3). IgM antibodies to measles were assayed by IgM ELISA as described earlier(4). The samples were cross-checked with commercial measles IgM ELISA kit (Human, Germany, Courtesy WHO). IgM ELISA for measles was done on 16 samples for detecting presence of IgG antibodies to measles(4). Briefly, the wells of microtitre strips (NUNC, Roskilde, Denmark) coated with purified measles virus were incubated at 37°C for one hour with 1:100 dilution of serum sample (100µl). After thorough washing, goat antihuman IgM conjugated to horseradish peroxidase (Sigma Chemical Co., St. Louis, USA) at a dilution of 1:1000 was added. After five washes with PBS, chromogenic substrate (orthophenylene diamine (Sigma, USA) with 0.01% H2O2) was added in dark and kept at room temperature for 15 minutes. 4 N H2SO4 was added to stop the reaction. Strong positive and negative controls for IgM antibodies to measles virus were run in parallel. Optical density (OD) was measured at 492 nm on an ELISA reader (Labsystems, Finland). The samples were cross-checked with commercial measles IgM Kit (Himan, Germany, courtesy WHO).

Serum and CSF (200 microliters each) were inoculated into vero cell cultures, taking all precautions for avoiding cross contamination or laboratory contamination. Tissue culture fluid was passaged for three times in Vero cell monolayers for virus amplification. Adequate controls were also included in the protocol which were followed for three passages. Samples showing cytopathic effect (CPE) in three consecutive passages were taken for identification(5,6).

The virus isolates were identified by indirect immunofluorescence test. Infected and mock-infected vero cells were washed three times with PBS (pH 7.2). The cells were air dried on glass slide and fixed in chilled acetone for 20 minutes at room temperature. The fixed cells were incubated at 37°C for one hour with 1:50 dilution of rabbit anti-measles immune serum (in-house reagent). After thorough washing with PBS anti rabbit FITC conjugate (Bangalore Genei, India), 1:30 dilution was added to the cells. These were incubated at 37°C for one hour. The cells were counter-stained with 1:30,000 dilutions of Evan’s blue (Sigma, USA), mounted in glycerol buffer and observed under fluorescence microscope.

The same procedure was followed when the cells were tested with mouse monoclonal antibodies to measles virus HA protein (Institut Pasture de Lyon, France) for the confirmation of isolates(6).

The virus isolates were also confirmed by qualitative in-vitro neutralization test. The neutralization tests were done in 24 well plate using vero cell monolayers following standard protocols. 100TCID50 (Tissue culture Infec-tious Dose) of isolates and 20 units of equine antimeasles anti serum (supplied by Center for Biological Evaluation and Research, FDA, USA) and/or rabbit anti measles serum (in-house reagent) were incubated at 37°C for one hour and added on vero cells. Normal rabbit serum served as negative control. The plates were incubated at 37°C with 5% CO2 and cytopathic effect was observed every day for six days. After the appearance of CPE, the cells were fixed and stained with Amido black. CPE inhibition was used as indicator for neutralization(6,7).

At Centers for Disease Control and Prevention, Atlanta, USA, confirmation of identity of isolate was sought by RT-PCR method. RNA was extracted from two representative isolates from this outbreak by guanidinium acid-phenol method(8). cDNA was synthesized from these RNA using Superscript One Step RT-PCR with Platinum Taq System (Life Technologies, GIBCO BRL, USA). Briefly, RT-Platinum mix, 2x reaction mix, 5 mM Magnesium Sulfate, Rnase inhibi-tor, Rnase free water were mixed according to the standardized protocol. Measles primers for the C terminus of N gene (590 bp) MV60 (GCT ATG CCA TGG GAG TAG GAG TGG) and MV63 (ggc ctc tcg cac cta gtc tag) were used in the reaction. The PCR conditions were 55°C for 30 minutes followed by 94°C for 2 minutes, then 40 cycles of 94°C for15 seconds followed by 55°C for 30 sec followed by 72°C for 30 seconds; after 40 cycles incubation at 72°C for 7 minutes and cooled to 4°C. The amplified product was analyzed by 1.5% agarose gel electrophoresis with ethidium bromide staining.

The children presented with history of fever without any rash as prodromal symptom. Onset of encephalopathy was within three days of fever. All, except 2 cases turned afebrile at the onset of encephalopathy. Thereafter children went through stages of vomiting, agitation with abnormal behavior, rapidly progressing to coma with abnormal posturing. Seizures were seen in 29.4%. Twenty seven (52.9%) children died, 7 left the hospital against medical advice in moribund state and only 17 survived. Death occurred within 2 days of onset of altered sensorium. The detailed clinical features are described elsewhere(2).

The serum and CSF samples of patients tested, did not have IgM antibodies to Japanese encephalitis (JE). West Nile (WN) and Dengue-2 (Den-2) when tested by IgM Capture ELISA. CSF and serum samples when tested for hemagglutination inhibition (HI) antibodies against Influenza Type A and B strains showed presence of HI antibodies to A (H3N2) strain (titers 1:40 and 1:80) in 2 sera, indicating past exposure to these strains. The results of measles IgM and IgG ELISA are tabulated in Table I. Of 28 samples tested, 17 (8/11 serum and 9/17 CSF) were positive for anti-measles IgM antibodies which included five pairs of serum/CSF collected from same patient. A total of 12 children had measles specific IgM in either serum or CSF. The status of six serum samples was reconfirmed with commercial IgM ELISA kit with one discrepant result.

Eight out of 16 samples did not have IgG antibodies to measles when tested by IgG ELISA. Measles specific IgG antibodies were not detected in three patients yielding the virus strains.

Of the 28 samples inoculated into Vero cell cultures, cytopathic effect (CPE) was observed on day 3 post infection, in cultures with four CSF and 2 serum samples of 6 different individuals. CPE was typical of measles with syncytia formation and vacuolar appearance. All the isolates obtained were confirmed as measles with indirect immunofluorescence assay (IFA) using rabbit anti measles immune serum (NIV reagent) and rabbit anti measles immune serum (Serum Institute of India, Pune). Two isolates were reconfirmed by IFA using mouse monoclonal antibodies against measles HA protein (HA C155, supplied by Institut Paseur de Lyon, France). The qualitative in vitro neutralization tests (NT) done in 24 well Vero cell culture plate revealed that all the six isolates were neutralized by equine anti-measles immune serum (supplied by Center for Biological Evaluation and Research, FDA, USA) and rabbit anti-measles immune serum (in-house reagent), thus reconfirming the identity of isolates as measles. RT-PCR amplified product under ultra-violet illumination revealed positive bands for two strains thus confirming the identity.

Thus, the serological and virological and molecular studies provided strong evidence for the measles etiology of this outbreak of encephalopathy in children without typical measles rash.

This outbreak had many unusual features. The outbreak was very severe with significant mortality of the children affected. Rapid death within 48 hours of onset of encephalopathy was another feature. The characteristic maculo-papular rash was not observed in these patients. The exposure to classic measles case could not be traced. There was no classic measles outbreak in this region during the same time. Wide geographic area was involved with scattered cases. However, geographic cluster-ing was not observed. The affection with similar illness was limited to siblings of 7 patients.

There was no detectable epidemiological linkage between the children from whom virus strains were isolated. Although, history of previous measles immunization was not available, the absence of measles specific IgG antibodies in few patients indicated lack of previous exposure to measles or measles immunization.

Care was taken during virus isolation procedures for preventing laboratory con-tamination. No standard reference strain was handled during this period. The presence of serological evidence (IgM antibodies) in children who yielded isolates and isolation in only 6 samples of the 28 samples processed, ruled out the possibility of laboratory contamination.

Isolation of measles virus from CSF and serum of patients and serological backup of presence of IgM antibodies to measles and molecular confirmation at the global reference laboratory proved the etiology of this outbreak as measles. More information about the strains involved in this outbreak would only be revealed on sequencing. Earlier also we have isolated measles virus from serum of children with encephalitis without rash, from southern and western India outbreaks (National Institute of Virology, unpublished data). Isolation of similar etiological agent from three outbreaks from different parts of India 1000 km apart and with similar clinico-epidemiological features provide additional strength to the linkage of measles virus strains with these outbreaks.

The clinical and histopathological features of these cases satisfy the diagnostic criteria of Reye’s syndrome(2,9). Reye’s syndrome has been associated with many viral agents(10). But recovery of measles virus from CSF of patients proved a strong linkage. Association of Reye’s syndrome and measles has been reported(11,12). Earlier also we have isolated measles virus from CSF of patients without features of encephalitis(6). Thus, it can be said that in this episode of encephalopathy, measles virus had an etiological role. Although, presence of antibodies to varicella zoster were reported(2) in some cases, we could confirm all the isolates as measles only. Varicella zoster might be the co-factor in some cases.

Though, we have proved the etiology of measles in this outbreak, there were certain missing links and missing factors which were difficult to explain. Non-clustering of cases, absence of rash, no detectable exposure to classical measles, incidence in non-seasons of measles are all missing links. Only further studies including sequencing of these strains and animal pathogenecity would unravel the full nature of the strains isolated.

Isolation of measles virus strains from CSF is traditionally reported to be difficult with very few reports available in the literature(13,14). In the present study, measles virus isolation from CSF was relatively easier and cytopathic effect was observed in 72 hours of inoculation. We have found Vero cell line maintained in our laboratory sufficiently susceptible to Indian measles strains(6,15).

Measles may manifest without rash in immunosuppressive state(16,17). However, in our patients there was no history of immuno-suppression and patients were HIV antibody negative. We have earlier isolated measles virus from patients without immuno-suppression(6).

The important neurological manifestations associated with measles include subacute sclerosing panencephalitis(18), measles inclusion body encephalitis and post measles encephalitis(19). With the present study, acute encephalopathy without rash can be added to the long list of neurological manifestations. Measles virus infection manifesting as mild fever without rash as prodrome, producing, acute encephalopathy in the present outbreak indicates unusual presentation in vaccine era and warrants increased surveillance for such manifestations.

Authors are grateful to Dr. William Bellini, Dr. Paul Rota, Ms Stephanie Liffick, Mr Russel Katz of WHO Global Measles Strain Bank Laboratory at Center for Disease Control and Prevention, Atlanta, USA for help with their technical expertise for molecular confirmation. Aurhors are also grateful to Dr. Fabian WILD, Institute Pasteur de Lyon, France, for supply of mouse monoclonal to measles proteins, Dr. Judy Beeler, Centre for Biological Evaluation and Research, FDA, USA and Serum Institute of India, Pune for supply of anti measles immune sera; Dr. V.S. Padbidri, Officer-in-charge NIV, Pune for guidance, review and critical comments; Dr. B.L. Rao for HI tests for Influenza, Dr. J.P. Thakare for IgM capture ELISA for arboviruses; and Dr. M.M. Gore for critical comments.

Contributors: NSW suspected viral etiology, planned and carried out virological and molecular investiga-tions and drafted the manuscript. He will act as the guarantor for the paper. NJS conducted virological and serological investigations. RKR collected speci-mens and carried out initial investigations. DG and SS were clinicians in-charge of patients who investi-gated the outbreak and provided clinical data. RCM supervised initial investigations and reviewed the manuscript. DAG supervised the virological investi-gations and reviewed the manuscript.

Funding: RT-PCR studies were supported by Centers for Disease Control, Atlanta, USA. NSW received support from Department of Biochemistry, Government of India, for molecular studies. Virus isolation and identification studies were carried out as in-house project of National Institute of Virology, Pune. Measles monoclonal antibodies were supplied to us under WHO-UNDP Program of Vaccine Development.

Competing interests: None stated.

Table I Serological and Virological Results     

ND: Not done, *: IgM results correlate with commercial IgM ELISA.
# : Does not correlate with commercial IgM ELISA; - : no isolate.
$ : Isolates confirmed by IFA with monoclonal antibodies.

All isolates were confirmed by IFA and NT using antimeasles immune sera.

 

Key Messages

IgM antibodies to measles virus were detected in the serum and CSF samples. The isolation of measles virus from CSF and serum of children with acute encephalopathy without rash proved the etiologIcal role of measles virus in this outbreak.

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