Patients with Central Nervous System Disease

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1 CLINICAL MICROBIOLOGY REVIEWS, Jan. 1989, p Vol. 2, No /89/ $02.00/0 Copyright 1989, American Society for Microbiology Role of the Virology Laboratory in Diagnosis and Management of Patients with Central Nervous System Disease TASNEE CHONMAITREE,l2* CONSTANCE D. BALDWIN,' AND HELEN L. LUCIA'2 Departments of Pediatrics' and Pathology,2 University of Texas Medical Branch at Galveston, Galveston, Texas INTRODUCTION...1 CNS DISEASES CAUSED BY VIRUSES...2 Enteroviruses...2 Normal host...2 Compromised host...3 Togaviruses, Bunyaviruses, and Reoviruses... 4 Herpesviruses...4 HSV... 4 VzV... 4 EBV... 4 CMV...4 Mumps, Measles, and Rubella...5 Lymphocytic Choriomeningitis Virus...5 Rabies... 5 HIV...5 DIAGNOSTIC METHODS FOR ENTEROVIRUSES...5 Rapid Diagnosis...5 Isolation of Virus...6 Specimen collection and transport...6 Cell culture...6 Animal inoculation...7 Identification of isolates...7 Serology...7 DIAGNOSTIC METHODS FOR NONENTEROVIRUSES...7 Togaviruses, Bunyaviruses, and Reoviruses... 7 Herpesviruses...8 Mumps, Measles, and Rubella...8 Lymphocytic Choriomeningitis Virus...8 Rabies... 8 HIV...8 MANAGEMENT OF PATIENTS WITH CNS VIRAL INFECTION...9 Aseptic Meningitis...9 Meningoencephalitis...9 Enteroviral Meningoencephalitis Associated with Agammaglobulinemia...9 INFLUENCE OF THE VIROLOGY LABORATORY ON PATIENT MANAGEMENT...9 CONCLUSION ACKNOWLEDGMENTS LITERATURE CITED INTRODUCTION Before antiviral therapy became available, viral diagnosis was used clinically primarily to identify a community outbreak of viral disease and to provide prognostic information to the patient. The delayed reports of viral culture results dictated that diagnostic information was generally not available until the patient's illness was over. Because demand for the technology was limited, diagnostic virology facilities were, until recently, available only in reference laboratories and some university hospitals. In a survey published 12 years ago, only 60% of 115 U.S. medical centers reported on-site viral diagnosis (60). Physicians avoided using these laboratories because results were delayed and often not * Corresponding author. 1 useful for patient care, costs were high, and collection and transport of specimens were difficult. Physicians were more likely to use a viral laboratory if it was located within their institution. The discovery of many types of antiviral therapy has mandated the establishment of more accessible facilities for viral diagnosis and development of more rapid diagnostic techniques. At present, increasing numbers of diagnostic facilities are becoming available at university medical centers, Veterans Administration hospitals, and even some community hospitals (81, 98, 110, 128). In addition, some central laboratories have reached out to offer viral diagnostic facilities to the surrounding community (124, 128). These laboratories vary in size and emphasis on diagnostic methods. The laboratory can now operate on a modest scale, be tailored to the needs of the patients it serves, and provide

2 2 CHONMAITREE ET AL. CLIN. MICROBIOL. REV. TABLE 1. Recognized human enterovirus serotypes Virus Types Polioviruses Group A coxsackieviruses' Group B coxsackieviruses Echoviruses" Enteroviruses' Type 23 is the same as echovirus 9. Type 10 is reclassified as a reovirus: type 28 is a rhinovirus. EnteroviruLs type 72 is hepatitis A virus. important information for patient management (81, 94, 157). This article reviews acute central nervous system (CNS) diseases caused by viruses and the usefulness of the virology laboratory in diagnosis and management of patients with these diseases. The more common enteroviral diseases are emphasized. CNS DISEASES CAUSED BY VIRUSES Viral infections of the CNS have two major clinical presentations: aseptic meningitis, which is by far the most frequent, and viral meningoencephalitis. The enteroviruses cause most acute viral infections of the CNS (35 to 83%). with mumps a distant second (1 to 40%), worldwide (9, 20, 42, 44, 116, 146). Herpesviruses, togaviruses. bunyaviruses. lymphocytic choriomeningitis virus, and measles and rubella viruses can also cause CNS disease. More recently. the role of human immunodeficiency virus (HIV) in the etiology of CNS disease has been recognized (48). Enteroviruses Enteroviruses constitute a genus of the Picornaviridae family which currently includes 69 serotypes made up of U) 110-4) Co 100 o 90 %I ) 6 E 50- Z M~~~~~~Mnh polioviruses, coxsackieviruses, echoviruses, and enterovirus types 68 to 72 (Table 1). This review will exclude from the discussion of enteroviral diseases poliomyelitis and other diseases caused by the three types of polioviruses. Enteroviruses have a worldwide distribution, with increased prevalence in temperate climates during the warm months of the year (54, ). A recent survey from the Centers for Disease Control showed that the temporal pattern of isolation of nonpolio enteroviruses differs among regions of the United States. However, a mean of 84% and a range of 65 to 93%c of virus isolations in a region were made between July and January (143). Figure 1 shows the seasonal prevalence of enteroviruses isolated in the Clinical Virology Laboratory of The University of Texas Medical Branch (UTMB) in Galveston, Tex., from 1983 to Although outbreaks of disease associated with a single serotype of enteroviruses are often reported (12. 29, 72, 144), the far more common pattern is endemic infection caused by several enterovirus types (28, ). The predominant types may vary yearly and may vary by locality even within the same year. Normal host. Enteroviral diseases in normal hosts are most often seen in young infants and children (54). Enteroviruses are mainly spread by the fecal-oral route. The incubation period ranges from 1 day to 3 weeks but is generally 3 to 5 days. CNS disease is a common manifestation of infections caused by a variety of nonpolio enteroviruses, with aseptic meningitis being the most common. Less common diseases include encephalitis, paralysis, Guillain- Barre syndrome, cerebellar ataxia, and peripheral neuritis (10, 26, 29, , 133). CNS disease sometimes occurs as a part of disseminated enteroviral infection, with viremia and involvement of heart, liver, kidneys, adrenal glands, and the blood coagulation system (14, 26, 56, 71, 75, 77). 10 Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. Months FIG. 1. Seasonal prevalence of enteroviruses as represented by number of enteroviruses (including polioviruses) isolated by month from clinical specimens submitted to the Clinical Virology Laboratory. UTMB to In two separate outbreaks in echoviruses 9 (April to July) and 11 (September to December) predominated.

3 VOL. 2, 1989 THE VIROLOGY LABORATORY AND DIAGNOSIS OF CNS DISEASE 3 TABLE 2. Age distribution of 103 children with enteroviral meningitis diagnosed at UTMB, 1983 to 1987" Age group No. of Cumulative % cases <1 mo mo mo-1 yr yr >6 yr " All had an enterovirus in the CSF. Data were collected by Cheryl Banks. Approximately 3,200 to 12,700 cases of aseptic meningitis were reported annually to the Centers for Disease Control between 1973 and 1983 (24), but its actual incidence is probably severalfold higher. Enteroviruses account for the majority of the identified agents causing aseptic meningitis, and yearly peak occurrences of the disease closely coincide with periods of most frequent enterovirus isolation (20, 24). Enteroviral aseptic meningitis caused by numerous types of coxsackieviruses, echoviruses, and enterovirus 71 (26, 29) occurs in both epidemics and isolated cases. In general, enteroviral meningitis is more common in young patients, especially those under 1 year of age. Age ranges of 103 children with enteroviral meningitis diagnosed at UTMB from 1983 to 1987 are shown in Table 2. Clinical signs and symptoms of enteroviral CNS disease in young children are mostly nonspecific and mimic those of bacterial sepsis or meningitis or both. Onset of illness can be abrupt or gradual. Fever is the most common presenting symptom, followed by irritability, lethargy, anorexia, gastrointestinal or respiratory symptoms or both, otitis media, and rash (15, 36, 137, 157, 160). In one study, apnea occurred in 9% of children with enteroviral meningitis (157). Specific signs and symptoms of CNS disease, such as nuchal rigidity and bulging fontanelle, may or may not be present. Seizures occur in <10% of patients (137, 157). Focal neurological abnormalities are rare, but transient ataxia or paralysis occasionally occurs (29, 88, 137). In older children, headache is common and CNS signs and symptoms are more prominent. Laboratory findings vary widely. Initial peripheral blood and cerebrospinal fluid (CSF) results from 103 children with enteroviral meningitis diagnosed at UTMB are reported in Table 3. Peripheral total and differential leukocyte counts usually cannot be clearly distinguished from those seen in patients with bacterial sepsis or meningitis. The CSF leukocyte count varies from none to several thousand cells per mm3, but most often is <1,000. Although monocytes-macrophages are generally the most numerous cells in CSF, the predominance of premature or segmented neutrophils is common, especially early in the disease. Performance of a second lumbar puncture 5 to 8 h after the first is generally not helpful in demonstrating a shift of CSF leukocyte composition from a neutrophil to a monocyte predominance (57). In most patients with enteroviral meningitis, CSF total protein content is within normal limits, but in a small percentage it may be slightly or even markedly elevated. CSF glucose content is usually normal but can be diminished in up to 18% of cases (137, 157). The measurement of CSF concentrations of C-reactive protein, lactic acid dehydrogenase, and alpha interferon to differentiate between bacterial and viral meningitis is not generally feasible, although some studies suggest that these tests may be helpful (1, 2, 34, 50, 62, 111). Diagnosis of enteroviral meningitis is especially difficult when CSF pleocytosis is absent, a phenomenon which has been variably reported to occur in 3 to 40% of patients (28, 58, 74, 79, 157, 159). In these cases, viral culture of the CSF is the only reliable method currently available to diagnose meningitis. Therefore, it should be kept in mind that, when clinical signs and symptoms suggest the diagnosis of enteroviral meningitis, even if the CSF contains no leukocytes, an enterovirus may still be present. Accurate diagnosis of enteroviral meningitis is important, given the potential of the disease to lead to neurological sequelae. Although data on these outcomes are conflicting (11, 46, 122, 134, 161), some evidence suggests that children with enteroviral meningitis, especially at young ages, may have long-term neurological abnormalities, including smaller head size, deficits in speech and language development, and lower intelligence. Because most of the studies involved a small number of patients and used different research methodologies, these data are not yet conclusive. These studies suggest, however, that enteroviral meningitis, especially in young infants, is not a benign disease, and accurate diagnosis may allow predictable prognosis and follow-up strategies. Compromised host. Enteroviral infections in patients with antibody deficiencies, especially X-linked agammaglobulinemia, can be severe. Two clinical syndromes of enteroviral infection in these patients include vaccine-associated paralytic poliomyelitis (162, 163) and the more commonly reported chronic enteroviral meningoencephalitis (5, 32, 45, 51, 86, 91, 92, 149, 152, 158). Enteroviral meningoencephalitis associated with agammaglobulinemia is most frequently caused by echoviruses, although group A and B coxsackieviruses have also been implicated (32, 91). Neurologic presentations may resemble those of acute onset meningoencephalitis or aseptic meningitis, with fever, headache, altered mental status, and seizures. Approximately 30% of TABLE 3. Initial peripheral blood and CSF findings for 103 patients with enteroviral meningitis diagnosed at UTMB, 1983 to 1987a Source Leukocytes/mm3 % Neutrophils % % Protein Glucose Segmented Premature Lymphocytes Monocytes (mg/dl) (mg/dl) Peripheral blood Median 11, Range 4,800-29, CSFb Median Range 0-3, a CSF culture was positive for an enterovirus in all cases. b CSF data came from 83 atraumatic taps, with <100 erythrocytes per mm3. Data were collected by Cheryl Banks.

4 4 CHONMAITREE ET AL. patients present with edema or dermatomyositislike syndrome, which is marked by peripheral edema, erythematous rashes, and evidence of inflammation in skin and muscle biopsy specimens (91). Regardless of the clinical manifestations at the time of onset, over the prolonged course of the disease, nearly all reported patients eventually develop some overt neurological symptoms, including headache, seizures, hearing loss, lethargy or coma, weakness, ataxia, paresthesias, diminished intellectual acuity, developmental delay, hemiparesis, cranial nerve palsies, episodic confusional states, and symptoms consistent with transient ischemic episodes (91). Other reported manifestations of this syndrome include personality changes, dysarthria, aphasia, hepatitis, and arthritis (89, 91). The diagnosis of enteroviral meningoencephalitis in these patients requires the isolation of enterovirus from the CSF. Virus may not be detected in the CSF until after the onset of symptoms and CSF abnormalities; even after detection of the virus in the CSF, negative results of viral cultures can be obtained sporadically without a change in therapy (91, 158). Togaviruses, Bunyaviruses, and Reoviruses Viral encephalitis due to togavirus, bunyavirus, and reovirus infection occurs sporadically. These viruses are transmitted by the bites of mosquitoes or ticks. The Togaviridae family contains eastern equine encephalitis, western equine encephalitis, Venezuelan equine encephalitis, and St. Louis encephalitis viruses, all of which are found in the United States (35). The group also includes Murray Valley fever (Australia), Japanese encephalitis (Japan), and several tickborne varieties of encephalitis (in Europe, especially the Soviet Union). The Bunyaviridae family contains the California group viruses, notably La Crosse virus, which is seen in the United States (113). A period of viremia precedes the onset of clinical neurologic disease. Incubation periods range from 2 to 14 days. Viral infection of brain tissue results in fever, headache, coma, and paralysis. Eastern equine encephalitis is most severe, with fatality rates of 50 to 75%; 30% or more of the survivors have neurologic sequelae severe enough to require institutionalization. Of symptomatic cases, the fatality rate of Western equine encephalitis is 10%, that of Venezuelan equine encephalitis is <1%, and that of St. Louis encephalitis is 10 to 20%. La Crosse virus infection is also mild; most manifestations are nonspecific viral syndromes rather than encephalitis, and mortality and residual morbidity occur in fewer than 2% of the patients with clinical encephalitis (35, 113). Colorado tick fever, caused by a reovirus, is endemic in the Rocky Mountain region of the United States and Canada. It is a zoonosis of rodents and is transmitted by ticks. The virus enters erythroid precursors and circulates inside mature erythrocytes, where it is protected from antibodies. Thus, the disease results in prolonged viremia. The disease is denguelike in presentation; occasionally, children will develop aseptic meningitis. The disease is self-limiting and seldom results in death (90). Herpesviruses HSV. Herpes simplex virus (HSV) is responsible for neonatal and adult encephalitis and viral meningitis. Neonatal HSV infection is acquired by passage of infants through the birth canal of infected mothers, especially those with primary infection. The frequency of occurrence is 26 of CLIN. MICROBIOL. REV. 100,000 deliveries. Infection may be limited to the skin, but more often is disseminated, with liver, lung, or CNS involvement. The CNS disease with or without dissemination is devastating. Without treatment, mortality is 50 to 85% and morbidity is 100% (6). With modern antiviral therapy using acyclovir or vidarabine, mortality has been reduced to 10 to 57%, but up to 86% of the survivors still suffer neurologic sequelae (6). HSV encephalitis in adults is usually an isolated CNS infection, localized to the temporal lobes of the brain. This viral disease is the most common fatal viral encephalitis; it occurs in 1 of 500,000 of the population. When it is left untreated, mortality is 70%, with sequelae in the majority of survivors. With acyclovir treatment, mortality is now 13 to 19% and morbidity is 44 to 62% (6). Aseptic meningitis without encephalitis also occurs, typically during primary genital infection with HSV type 2. Occasionally the patient will not have cutaneous or mucosal lesions (123, 142). VZV. Neurologic involvement is unusual during primary varicella infection (chicken pox). Isolated cerebellar ataxia is the most common CNS abnormality (50%), with nausea, vomiting, headache, and ataxia occurring 5 to 13 days after the onset of the rash and lasting from 2 to 4 weeks. The frequency has been reported to be 0.1 to 0.75% in cases of chicken pox. The mortality is low (0.5%), and the majority of children recover completely (6). A less frequent, but more severe complication is postinfectious meningoencephalitis or cerebritis, which is attributable to host response reactions. Patients present with headache, fever, and vomiting, with alerted sensorium. Seizures occur in up to 50% of the patients. The mortality is low (about 5%), and 80% of survivors have no sequelae (6, 141). A number of neurological syndromes have been noted during recurrent varicella-zoster virus (VZV) infection (zoster or shingles). Cranial and peripheral nerve palsies in the infected dermatome are seen in 1 to 6% of cases. Encephalitis, the most common CNS abnormality, occurs primarily in immunocompromised patients and is attributable to systemic dissemination of the virus from cutaneous lesions. Mortality is high (30 to 50%), but many deaths are due to concurrent VZV pneumonia, not the CNS disease. Neurological sequelae are present in as many as 30% of survivors (6). In one study, myelitis occurred as an unusual complication of VZV infection (1 of 1,210 cases) (147), as does herpes ophthalmicus with contralateral hemiplegia. Even in uncomplicated VZV infection, the CSF may show pleocytosis and increased protein concentration (112). EBV. Epstein-Barr virus (EBV) infection can present with neurologic syndromes. Serious nervous system disorders have been estimated to affect approximately 1 to 5% of hospitalized patients with infectious mononucleosis. Although neurologic symptoms may be the sole manifestation of an EBV syndrome, most appear during the course of this disease. The CNS infection may be encephalitis, aseptic meningitis, Guillain-Barre syndrome, neuritis, or subacute sclerosing panencephalitis. The encephalitis is usually mild and transient, but rarely can be fatal. Complete recovery of neurologic function occurs in almost all survivors (13, 55, 136). CMV. Although it is a common cause of neurological damage during congenital infection, cytomegalovirus (CMV) encephalitis is vanishingly rare as a complication of CMV infection acquired postnatally by hosts with normal immune function. However, this infection has become extremely common in immunocompromised patients with acquired

5 VOL. 2, 1989 THE VIROLOGY LABORATORY AND DIAGNOSIS OF CNS DISEASE 5 immunodeficiency syndrome (AIDS). It is characterized by relentless progression of dementia and eventual death (105). Mumps, Measles, and Rubella Clinically apparent CNS involvement occurs in about 15% of mumps cases, although CSF abnormalities are much more common (19, 21). Although on a worldwide basis mumps remains the second leading cause of aseptic meningitis and encephalomyelitis (20, 42, 44, 116, 146), the incidence has decreased dramatically in the United States with the introduction of large-scale immunization. Mumps has decreased 98% in incidence, and in 1983, the last year for which the Centers for Disease Control collected statistics on mumps encephalomyelitis, seven cases were reported in the United States (23). The symptoms range from headache and listlessness to seizures with altered states of consciousness. Complications include meningitis, encephalitis, and meningoencephalitis. In the absence of parotitis, the meningoencephalitis is clinically indistinguishable from enteroviral infection (70). Measles encephalitis complicates approximately 1 per 1,000 cases of measles (22, 65, 78). Since the introduction of measles vaccine, the incidence has declined in parallel to the decline of measles. The risk of encephalitis rises with the age of the patient. CNS symptoms usually appear after the eruption of the rash, with abrupt recurrence of fever, headache, lethargy, irritability, and confusion. The majority of patients improve and return to normal after 2 to 3 days, but approximately 30% progress to coma, which may persist for days or weeks. Attempts to recover infectious measles virus from the CSF are seldom successful; this syndrome is probably caused by a host response reaction. Some 10 to 15% of the cases are fatal, and 25% have severe permanent CNS sequelae. Rubella can also cause postinfectious encephalitis (1 of 6,000 cases). The symptoms are similar to but milder than those seen with measles, and persisting morbidity occurs in <10% of cases; mortality is very low (141, 151). Lymphocytic Choriomeningitis Virus Lymphocytic choriomeningitis virus is an arenavirus which is endemic in mice and can be transmitted to humans. Infections with lymphocytic choriomeningitis virus have an influenzalike prodromal illness, with arthralgias, anorexia, nausea, light-headedness, and pneumonitis. CNS symptoms include headache, nuchal rigidity, and, rarely, coma and death (2 of 79 cases) (3, 4, 97). Rabies Rabies infection of the CNS is an encephalitis. The virus is a zoonosis; humans usually acquire the infection from the bite of infected animals, although infection through the respiratory route may also occur. This infection is extremely rare in the United States, where most disease is associated with wild-animal infection. However, in many parts of the world where domestic dogs are not vaccinated routinely, rabies remains a feared disease (151). Mortality is 100%. HIV HIV has recently been recognized as a significant CNS viral pathogen. The disease can present as a transient aseptic meningitis early in infection. An insidious progressive encephalitis ultimately resulting in dementia and death can also occur. As we begin to control the secondary infections of patients with AIDS, this chronic encephalitis is assuming greater clinical significance. Clinical evidence of this disease has been reported in 65% of patients with AIDS. Postmortem studies suggest that CNS infection is always present, even when it is not clinically apparent (48). DIAGNOSTIC METHODS FOR ENTEROVIRUSES Rapid Diagnosis Methodologies for rapid diagnosis of enteroviral meningitis and encephalitis by means other than cell culture are still under development. Detection of CNS enteroviral infection is technically difficult for two reasons. Enteroviruses are frequently present in body fluids in low concentrations; CSF may contain only 10 to % tissue culture infective doses per ml during infection (160). In addition, different enteroviruses express relatively few common antigenic determinants (R. H. Yolken and J. F. Modlin, Pan Am. Gr. Rapid Viral Diagn. Newsl. 12:1-2, 1986), so any detection method which depends on recognizing viral antigen must include a large number of reagents and multiple tests. Despite these difficulties, development of rapid diagnostic methods for these pathogens has made significant recent progress. Immunofluorescent (IF) staining of CSF leukocytes during enteroviral meningitis has been demonstrated (145), with detection and identification of the infecting enterovirus in 36 of 40 specimens within 2 h. By this method, the virus can be identified on days 6 through 10 of infection, when growth in cell culture is no longer possible. The disadvantages of the method are the need for maintaining a large number of antisera, the low sensitivity of the reagents under the required dilutions, and, in some samples, the lack of a sufficient number of leukocytes in the CSF for measurement (145). In addition, frequent nonspecific fluorescence of CSF lymphocytes has been reported by subsequent workers (52). Counterimmunoelectrophoresis has been used to detect bacterial antigens in CSF. However, attempts to detect enteroviral antigen by this method were unsuccessful, probably because of the small quantity of antigen present (17). Complementary deoxyribonucleic acid probes have been used in more recent attempts to detect enterovirus (67, 68, 125, 126, 148). These probes have identified enteroviruses, which were artificially added to CSF, and subgenomic probes detected a number of different serotypes of enteroviruses, suggesting that a small number of probes, perhaps as few as two, might be able to detect the wide range of enterovirus serotypes (126). However, the limit of sensitivity of the probes was % tissue culture infective doses per ml, which is not enough to be clinically useful. It has been suggested that nucleic acid hybridization methods could be used after concentration of the virus by immunoprecipitation, but the large number of antisera needed would limit the practicality of this procedure (148). Another possible approach would be to search for enteroviruses in specimens other than those from the CNS. There have been reports of rapid detection of enteroviruses in serum, using monoclonal antibodies against epitopes of VPI peptide which are shared by all enteroviruses except hepatitis A (166, 167). Although the test is highly sensitive, the monoclonal antibody is not widely available. Since the majority of children with enteroviral meningitis have culturable virus in their stool, probably at a higher titer than in CSF, searching for enteroviruses in stool may provide some clue to the diagnosis. By using an enzyme-linked immuno-

6 6 CHONMAITREE ET AL. sorbent assay (ELISA), virus was detected in 8 of 11 stools from which coxsackievirus was grown (165). The large number of serotypes makes this method cumbersome, with 24 microdilution tray wells required for each specimen, but it is especially useful for detecting group A coxsackieviruses, which grow poorly in culture (164). With a complementary deoxyribonucleic acid probe, an enterovirus was detected in seven of eight culture-positive stools (67). The single probe, which copied about two-thirds of the coxsackievirus B3 genome, was able to detect a number of different enteroviruses, suggesting the possibility that relatively few reagents would be required to detect this group. An additional method for detection of enteroviruses uses protein A-gold immunoelectron microscopy. This method is reported to be 200- to 1,000-fold more sensitive than direct electron microscopy and 2- to 40-fold more sensitive than immunoelectron microscopy. However, it requires specific antisera, which limits the practical utility of the method (63). Unfortunately, all techniques which detect enteroviruses in stool have limited value because asymptomatic children commonly shed enteroviruses, including vaccine polioviruses, in stool. CNS infection would, therefore, have to be confirmed by some other means. Isolation of Virus Isolation in cell culture is currently the most sensitive and reliable method for detecting enteroviruses in clinical specimens, except for most serotypes of group A coxsackieviruses. These viruses do not grow in cell culture and must be inoculated into newborn mice or guinea pig embryos (61, 93, 94). In general, the cytopathic effect (CPE) produced by enteroviruses in standard cell culture is quite distinctive and can be recognized fairly early and with accuracy by experienced technicians (59, 61, 94). A rapid report of a presumptive diagnosis of enterovirus infection based on CPE in cell culture, without waiting for the specific typing of the isolate, can have an impact on patient management (28, 157). If the laboratory aims to provide such rapid reporting, the cell culture should be observed for enterovirus CPE daily and at least once during the weekend for the first 4 to 5 days of the culture and then less frequently for 10 to 14 days thereafter. Use of a multiple cell culture system to grow enteroviruses can also increase the sensitivity as well as the speed of enterovirus isolation (38; T. Chonmaitree, C. Ford, C. Sanders, and H. Lucia, Abstr. Annu. Meet. Am. Soc. Microbiol. 1988, C84, p. 346). This technique is discussed below. Specimen collection and transport. Clinical specimens should be collected as soon after the onset of clinical symptoms as possible. For enteroviral CNS infection, CSF is the most specific specimen used, although some types of enteroviruses such as enterovirus 71 are infrequently isolated from CSF (29, 131). The isolation rate of enteroviruses from CSF of patients with presumptive enteroviral meningitis varies from 43 to 77% in different geographic areas and with different predominant enterovirus serotypes (28, 100, 157). CSFs containing a few or no cells have also been reported to yield enteroviruses in a significant number of cases (28, 74, 79, 157, 159); therefore, these CSFs should also be cultured for the viruses when clinical signs and symptoms of the patient suggest the diagnosis of enteroviral CNS disease, especially during an epidemic period. Throat swab and rectal swab or stool specimens, although not from CNS-specific sites, can give an early (28, 157) and sometimes the only clue to diagnosis. Because infants and children with CLIN. MICROBIOL. REV. enteroviral meningitis may also have viremia (37), blood culture for detection of virus in mononuclear leukocytes or serum or both (117) from a febrile child may also be useful in making a definitive diagnosis. Enteroviruses are relatively stable and survive well at temperatures of 4 to -70'C, but measurable loss of activity occurs when the specimens are kept at room temperature for many hours or allowed to dry (93, 95). Throat and rectal swab specimens should be placed in viral transport medium, which is provided by most virology laboratories, and stored refrigerated or frozen if necessary. Fresh stool specimens can be transported in conventional containers. Cell culture. The cell types used most widely to grow enteroviruses include primary monkey (rhesus, cynomolgus, or African green) kidney cells (PMK) and human diploid fibroblast cells such as WI-38 or MRC-5 (66, 93, 95). Other cell types include continuous human heteroploid cells (HeLa and HEp-2), primary or diploid human embryonic kidney, human fetal kidney, human amnion, human rhabdomyosarcoma (RD), chicken embryo, and the BGM line of African green monkey cells (66, 93, 95). The growth requirements of different groups of enteroviruses vary considerably. All three types of polioviruses grow well in PMK, human embryonic kidney, human diploid fibroblast, HeLa, BGM, and RD cells (66, 93, 95). The CPE develops quickly and often destroys the monolayer within 3 days. While many serotypes of group A coxsackieviruses require animal inoculation, some serotypes (A7 and A9) grow readily in PMK cells. Coxsackievirus A21 grows best in HeLa and HEp-2 cells. Other group A coxsackieviruses, including A2, A4, All, A13, A15, A16, A18, A20, and A24, can also be isolated in human amnion, chicken embryo, or hamster kidney cells (84, 93, 135, 153). Group B coxsackieviruses do not replicate well in human diploid fibroblasts and RD cells but grow well in PMK and BGM cells (95). No single cell culture system is suitable for recovery of all common human enteroviruses. The more cell types used, the more serotypes of enteroviruses will be detected. For practical purposes, however, three or four cell types are probably the maximum that most clinical laboratories can handle. Combined use of three or four cell types can significantly increase yield and speed of virus recovery from clinical specimens (38, 61, 79, 137) compared with use of only standard culture. Use of BGM, a continuous African green monkey kidney cell line, increases speed and efficiency of recovery for group B coxsackieviruses (39, 96, 130). RD cells are particularly useful for echoviruses and some serotypes of group A coxsackieviruses (7, 38, 130, 150). Use of subpassages of primary human embryonic kidney cells increases the isolation rate of both echoviruses and group B coxsackieviruses (Chonmaitree et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1988). In a study during two consecutive enterovirus seasons (38), use of BGM and RD, in addition to the standard cell culture repertoire of PMK and human diploid fibroblast cells, increased the isolation rate of enteroviruses from clinical specimens by 18%. Furthermore, 35% of specimens which were positive in BGM, RD, or both were recognized 1 day earlier than in the standard cell lines. In our experience, the speed of recovery and the recovery rate of enteroviruses are increased by 11% when BGM and human embryonic kidney cells, in addition to PMK and human diploid fibroblast cells, are used (Chonmaitree et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1988). Table 4 demonstrates the speed of recovery of several enteroviruses; the data are from studies published between 1971 and Cumulative percentages of positive cultures ranged from 37

7 VOL. 2, 1989 THE VIROLOGY LABORATORY AND DIAGNOSIS OF CNS DISEASE 7 TABLE 4. Speed of enterovirus recovery by observation of CPE in cell culture No.of ~~~~Cumulative % posiitive at day: Study (reference). yr published Cell lines used" Nso. 1fC2 4 7 isolates I Herrmann (59) Standard, HeLa Chonmaitree et al. (28) Standard. BGM Rubin (128), 1984 Standard NA" Dagan and Menegus (38), 1986 Standard. BGM. RD Wildin and Chonmaitree (157), 1987 Standard, BGM " Standard cell culture was PMK and human diploid fibroblast cells. BGM is a continuous cell line of African green monkey kidney cells, and RD is human rhabdomyosarcoma cells. "NA. Not available. to 59% by day 3 and from 82 to 97% by day 7, depending on cell types used. Animal inoculation. Inoculation into newborn mice is the method of choice for primary isolation of most group A coxsackieviruses (66, 93, 95). Although group B coxsackieviruses and some newer enterovirus serotypes are also pathogenic in newborn mice, it is easier to grow them in cell culture. Group A coxsackieviruses cause generalized myositis accompanied by a flaccid type of paralysis. Group B viruses cause focal myositis and typical lesions in the interscapular fat pad and brain and occasionally produce myocarditis, endocarditis, hepatitis, and necrosis of acinar tissue of the pancreas (93). Identification of isolates. An isolate can be identified as an enterovirus with reasonable certainty on the basis of characteristic CPE in cell culture, combined with other information such as specimen source, time of year, and clinical history of the patient (61, 95). When an enterovirus is isolated from CSF or blood from a patient with no recent history of poliovirus vaccination, it is safe to assume that the isolate is a nonpolio enterovirus and is the cause of the disease. If the isolate grows in cell cultures inoculated with a throat swab, rectal swab, or stool specimen, both nonpolio enterovirus, which is probably causing the disease, and shedding of vaccine polioviruses must be considered. A specific identification of the serotype can be accomplished by virus neutralization (NT), using intersecting virus antiserum pools (85). If only vaccine polioviruses need to be separated from nonpolio enteroviruses, a simple NT test can be performed with pooled antisera to the three types of poliovirus (95). Serology Serologic diagnosis of enteroviral infection classically relies upon comparison of neutralizing antibody activity (25) in acute- and convalescent-phase sera. A fourfold increase in neutralizing antibody titer against the suspected strain of virus is considered consistent with infection. The titer of antibody against other strains may also rise because of cross-reactivity. Use of an ELISA method to detect the presence of immunoglobulin M (IgM) antibody during acute infection has been tested. At 2 weeks after infection, 16 of 19 patients had detectable IgM titers against a pooled enterovirus antigen. Of 16 patients, 10 had IgM specific for the enterovirus causing the infection, while 6 had a heterotypic response (25). Similar heterotypic responses have been noted by workers who used counterimmunoelectrophoresis (99) and immunodiffusion (132). During an outbreak of echovirus type 33 infection in France, after no reports of the disease for more than 15 years, tests for IgM detected 85% (33 of 39) of patients with viral infection. The unusual circumstances made use of a single reagent feasible (27). Because 3 to 21 days are needed for a patient to generate either an 1gM or an IgG response (25, 47, 99, 132), serology is useful for retrospective studies rather than routine diagnoses. One group of workers took advantage of the cross-reactivity between enteroviral strains and developed a generalscreening ELISA for enteroviral infection, using coxsackievirus B1-5 antigens with specimens from 45 patients with aseptic meningitis (8). Twenty-one patients had an enterovirus recovered from CSF, feces, or sera, while 30 had positive ELISAs. The method was useful for both detecting infection in culture-negative patients and confirming systemic infection in patients with virus recovered only from feces. The viruses isolated were coxsackievirus B5 and echoviruses 5, 6, 7, and 11. The disadvantage of the test was the need to use all five antigens, which made the procedure expensive. The authors stressed the need for a single, cross-reactive reagent that could be used in these tests. DIAGNOSTIC METHODS FOR NONENTEROVIRUSES Togaviruses, Bunyaviruses, and Reoviruses No rapid diagnostic methods have been developed for togavirus, bunyavirus. and reovirus infections (52). Because of the relatively long period of time between infection and the development of neurologic disease (5 to 14 days), virus is no longer present in blood when the patient is symptomatic (35). Venezuelan and western equine encephalitis viruses have relatively shorter incubation periods (2 to 10 days), and for the first 2 to 3 days after onset of illness, viremia may still be present. Virus has also been isolated from nasopharyngeal secretions in Venezuelan equine encephalitis. In fatal cases, virus can be isolated from brain tissue. The specimens can be inoculated intracranially into suckling mice or cell cultures of primary duck embryo, Vero, LLC-MK2, or BHK cells (35, ). Arthropod cell cultures have also been used (138). Serologic testing is the routine method of diagnosis. Complement fixation. hemagglutination inhibition, or plaque reduction NT are performed on acute and convalescent (2 to 5 weeks) sera. Because of the long incubation periods, hemagglutination inhibition and NT are frequently positive early in the disease. Complement-fixing antibody appears later or may fail to develop. The complement fixation test also shows cross-reaction with other arboviruses. so that for people from endemic areas, or those who have received yellow fever vaccine, hemagglutination inhibition and NT are more useful (35).

8 8 CHONMAITREE ET AL. Herpesviruses In disseminated neonatal HSV infection, several diagnostic procedures are possible. When skin lesions are present, infection with HSV can be confirmed by detecting viral antigen in smears of lesion scrapings or biopsies, using IF, immunoperoxidase, or ELISA procedures (80, 83, 104, 108). Sensitivity of these methods varies from 50 to 96% and specificity varies from 91 to 100%. Detection of viral antigens is much easier in vesicular fluid than in swabs of cervix or other contaminated sites. A modification of the simple ELISA, the enzyme immunofiltration staining assay, is reportedly more sensitive with both vesicular fluid and contaminated specimens. However, this procedure is not yet commercially available (30, 31). Virus isolation is possible by using lesion, throat, eye, and umbilical swabs, blood, and CSF. Over 90% of HSV grow in human diploid fibroblast cells within 4 days. Use of rabbit kidney or guinea pig embryo cells enhances rapidity of isolation by 1 to 2 days (18, 82, 129). Rapid detection by use of human fetal fibroblast cell lines, combined with IF or immunoperoxidase staining of inoculated cells, usually permits recognition of the virus after an overnight incubation (106, 109). A method combining cell culture and staining is the shell vial centrifugation method. This technique detects HSV within 16 to 48 h after inoculation (49, 114, 120, 154). Serologic examination is of no assistance is diagnosing neonatal infection due to HSV (6, 119). Adult HSV meningoencephalitis is not usually associated with the peripheral presence of virus. Fever of any etiology can cause reactivation of mucocutaneous HSV lesions; thus, patients with encephalitis caused by other viruses may have peripheral HSV lesions. Brain biopsy is recommended for the diagnosis of encephalitis before therapy is started, both to confirm the diagnosis and to exclude other treatable infections which often mimic signs and symptoms of HSV encephalitis. The biopsy should be of the involved area (usually the temporal lobe) recognized by either electroencephalography or computerized tomographic scan. The tissue should be divided, with part being sent to the histology laboratory for immunoperoxidase staining and the rest inoculated into cell culture as described above. CSF is not usually positive for virus (52). Although development of methods to detect specific viral glycoprotein in CSF is in progress (127), no such procedures are currently available. Serology is not useful for diagnosis (6, 33). The characteristic lesions of chicken pox or herpes zoster are frequently sufficient for diagnosis of VZV infection. However, HSV will occasionally cause vesicles in an apparent dermatomal distribution, with lesions which are indistinguishable from those of VZV infection. If the vesicular lesions are not recognizable, scrapings of the lesions may be stained by using IF procedures. Isolation of VZV from the vesicles can be performed in human fetal fibroblasts. The virus is relatively slow growing (4 to 7 days) and may be difficult to isolate because of the presence of antibody or interferon in the vesicle, especially in the case of herpes zoster. Virus has also been isolated from the CSF in some cases (123). Serologic recognition of the presence of specific IgM may be useful for diagnosing primary chicken pox but is not useful with herpes zoster (52). Infection with EBV can be rapidly recognized in serum by using the Monospot procedure, if the heterophile antibody has been induced. This test can be confirmed by IF procedures for specific IgM to EBV capsid antigen. In children, the Monospot procedure is frequently negative, and the IF CLIN. MICROBIOL. REV. procedure is necessary to recognize infection with EBV (52). Although it is possible to isolate virus from blood or CSF, the procedure is slow and requires cultivation of the virus in lymphocytes. It is used only for research purposes. Recognition of CNS infection due to CMV in the immunocompromised host is extremely difficult. Toxoplasma and HIV encephalitis present with similar symptoms. Neither positive serology nor isolation of CMV from peripheral sites is sufficient for differential diagnosis. In one study, the virus was isolated from CSF in two of nine patients with AIDS, so this approach may provide some useful information for AIDS patients (105). Therapy with gancyclovir is sometimes begun without proof of current active CMV infection, if antibody to CMV is present in the serum. Mumps, Measles, and Rubella No rapid procedures are available for diagnosing mumps (52). Isolation of the virus from CSF can be done in PMK, human diploid fibroblast, or HeLa cells. Cytopathology can be confirmed by a hemadsorption test within 6 to 7 days. Serologic diagnosis can be made by testing for complementfixing, hemagglutination-inhibiting, or NT antibodies (64). Rapid diagnosis of measles infection during the exanthematous period is achieved by detection of measles antigen by ELISA or IF in the nasopharyngeal secretions prior to and at the onset of rash and in skin biopsy up to a few days after onset of the rash. The virus is not recoverable from CSF or brain biopsy (101). Specific IgM to measles appears during the first days of the illness, peaks at 10 days, and is detectable for 4 weeks or more (52). Similar procedures are available for rubella. Lymphocytic Choriomeningitis Virus No rapid methods have been developed to detect infection with LCM (52). The virus can be isolated from CSF or blood with Vero cells, but detection of positive cultures requires IF techniques because CPE is not apparent. Therefore, infection is usually diagnosed by a fourfold rise in complementfixing antibodies 1 to 2 weeks after infection (123). Rabies Rapid diagnosis of rabies is possible by using biopsy of the skin of the posterior neck, or conjunctival smears, and IF procedures to recognize viral antigen (151). Serologic diagnosis, using mouse infection NT or the rapid fluorescentfocus inhibition test, can also be performed. Antibody in either serum or CSF indicates infection in the absence of past immunization. These procedures are generally available in reference laboratories, but not in most clinical virology laboratories. HIV Infection with HIV is recognizable by serologic tests. A positive screening ELISA for antibody, followed by a confirming procedure, usually Western blot (immunoblot) examination, is indicative of systemic HIV infection. Although isolation of virus is possible, this method is generally reserved for research use because of the time and difficulties involved. There is currently no good means of antemortem diagnosis of CNS disease. Postmortem, viral antigen can be detected in brain tissue by immunoperoxidase techniques, but positive results have been noted in patients without CNS

9 VOL. 2, 1989 THE VIROLOGY LABORATORY AND DIAGNOSIS OF CNS DISEASE 9 symptoms. It is possible that all people infected with HIV have CNS infection (16, 140). MANAGEMENT OF PATIENTS WITH CNS VIRAL INFECTION Aseptic Meningitis Because clinical signs, symptoms, and initial laboratory findings do not always distinguish bacterial from viral meningitis, the main practical problem for the clinician is whether to hospitalize the patient and administer intravenous (i.v.) antibiotics. In some instances, the diagnosis of viral disease may be suggested by evidence of rash or parotitis accompanying aseptic meningitis, especially when the disease occurs during an outbreak of aseptic meningitis (76). When a lumbar puncture is traumatic and in consequence the CSF results are not interpretable or when the patient is very young, the physician may choose to initiate antibiotic therapy and continue it until the diagnosis of bacterial meningitis can be excluded or the diagnosis of viral meningitis can be confirmed. However, when the patient has been partially treated with antibiotics, positive viral culture results are needed to support termination of antibiotic therapy, since negative bacterial culture results are no longer meaningful. Rapid antigen detection of bacteria which commonly cause meningitis, although widely used, makes little impact on patient management when the results are positive (53). When the result is negative, however, it may in some cases suggest the diagnosis of viral meningitis. Misdiagnosis of bacterial as viral meningitis can have devastating consequences (137), and every effort should be made to prevent this error. One approach is to hospitalize and initiate i.v. antibiotic therapy in every patient with the diagnosis of meningitis, especially young infants, until a bacterial diagnosis is excluded or a viral diagnosis is confirmed. This practice, although very popular, is also very costly. Furthermore, the patient carries all the risks of hospital-related morbidity, complications of parenteral fluid therapy and antibiotics, iatrogenic diagnostic and therapeutic mishaps, and family stress caused by separation from the young patient and loss of work productivity (36, 40). Rapid confirmation of the viral etiology of the disease will therefore greatly benefit the patient. The proven benefits will be reviewed in the following section. There is no specific treatment for viral meningitis except when it is caused by HSV. Only supportive and symptomatic treatment such as rest, analgesics, and antipyretics, if necessary, is recommended. Meningoencephalitis Specific drug therapy is available only for HSV encephalitis; treatment of other forms of viral meningoencephalitis is largely supportive (121). Vidarabine was the first antiviral drug shown to be effective in treatment of HSV encephalitis (156). Subsequently, two independent groups, one from Sweden (139) and the other from the NIAID Collaborative Antiviral Study Group (155), demonstrated the superiority of acyclovir over vidarabine. The current recommendation is to give acyclovir i.v. three times a day for at least 10 days. The efficacy of acyclovir in CNS disease caused by VZV, EBV, or CMV has not been demonstrated. Supportive therapy for viral meningoencephalitis consists of control of increased intracranial pressure, control of seizures, and general measures such as maintenance of fluid and electrolyte balance, oxygenation, pulmonary toilet, skin care, and ventilatory support if necessary. In most instances, appropriate antibiotics for bacterial meningitis are administered until CSF culture is negative for bacteria, after the usual 48- to 72-h incubation period. Enteroviral Meningoencephalitis Associated with Agammaglobulinemia The treatment for enteroviral meningoencephalitis associated with agammaglobulinemia is empiric; there is no current consensus on the best therapy. Because no specific drug against enteroviruses is available, the only therapeutic modality with efficacy is the administration of specific neutralizing antibody, usually as part of a mixed immunoglobulin preparation. Some reports suggest that administration of i.v. immunoglobulin, plasma, or immune serum with a high titer against the causative virus could improve outcome (91, 118, 158). Others have been successful in using intrathecal or intraventricular immunoglobulin in combination with i.v. immunoglobulin (45, 73, 91). INFLUENCE OF THE VIROLOGY LABORATORY ON PATIENT MANAGEMENT For a diagnostic virology laboratory to make an impact on patient management, ample communication between the physicians and the laboratory is required. The laboratory faces the challenge of educating physicians to order appropriate tests, carry out appropriate specimen collection and transportation, and interpret the results correctly. Physicians, in turn, can provide information about the diseases they commonly deal with, so the laboratory can emphasize tests that optimize the physician's management of the patient. An on-site virology laboratory, if available, is the most useful because it enhances physician-laboratory communication. In addition, specimen transport time is reduced to a minimum, allowing early inoculation with specimens that have the highest virus titer. When an on-site laboratory is not available, however, appropriate specimen collection and rapid transport can provide similar yields (124). The usefulness of a diagnostic test in patient management depends on the sensitivity and specificity of the test, its ease in execution, and the cost and timely availability of the results (53). In general, the sooner the results are provided, the more helpful they can be in patient management. Rapid diagnosis such as microbial antigen detection from clinical specimens is therefore preferred for initial patient management. However, because of the problems with sensitivity, specificity, and availability of rapid antigen detection tests, isolation of the microbial organism remains the standard for diagnosis in most instances. Rapid antigen detection, when available, is generally performed in parallel with culture, and a definite diagnosis is generally not made until culture results are available. To ensure that viral culture results are most effective in patient management, the laboratory, with the physician's cooperation, can maximize the speed of virus isolation by increasing the amount of virus in the specimen (appropriate collection and transport), using a combination of cell types appropriate for the virus to be recovered, increasing the frequency with which cultures are examined, and reporting results rapidly to the physicians as soon as they are available. Many clinical virology laboratories are capable of providing diagnostic information on diseases caused by enterovi-

10 10 CHONMAITREE ET AL. ruses, herpesviruses, and HIV. For viral diagnosis of CNS infections caused by togaviruses, bunyaviruses, reoviruses, mumps, measles, rubella, lymphocytic choriomeningitis virus, and rabies, reference laboratories are generally consulted. While laboratory diagnosis of many CNS viral infections is not specifically required for supportive and symptomatic treatment of the diseases, direct benefits to the patient can result from a timely diagnosis of HSV CNS infection, a treatable disease, and enteroviral infection, the most common viral infection of the CNS. The virology laboratory plays a direct role in management of patients with herpes simplex encephalitis, the same way a bacteriology laboratory directs the management of patients with bacterial meningitis. When a patient is considered to have HSV CNS disease, specimens are collected from biopsied brain tissue or CSF or both. Direct IF or ELISA staining of brain tissue, using specific HSV antibody, can be performed, and results are available within a few hours (80, 83, 104, 108, 128). Culture results for confirmation are generally available in 1 to 5 days (94). Although there is no specific treatment for enteroviral disease, immunocompromised patients with CNS disease benefit from immunoglobulin therapy. The virology laboratory can play a direct role in making a diagnosis of enteroviral infection. If NT is available, the laboratory can help to identify which high-titer immunoglobulins are needed for the specific virus serotype. More commonly, the laboratory can make a diagnosis of enteroviral meningitis, a disease encountered by most physicians dealing with children, especially during the summer and fall months. Early evidence suggesting that a diagnosis of enteroviral meningitis might affect patient management was first reported by Pearson et al. in 1972 (110). During a 1970 epidemic of aseptic meningitis attributed primarily to echovirus 3, the epidemiologic diagnosis provided by the virology laboratory caused physicians to change their practice style. During the first half of the epidemic, all of the patients with meningitis were hospitalized and 60% of them were given antibiotics. During the second half of the epidemic, after many cases of enteroviral meningitis had been identified, less than half of meningitis patients were hospitalized and only a few of these were given i.v. antibiotics. Singer et al. (137) subsequently used viral culture results to develop a protocol for management of CNS infections during an epidemic of enteroviral aseptic meningitis. These reports demonstrated the usefulness of the virology laboratory in general management of patients during epidemics of enteroviral disease, although they did not directly affect management of the individual patient. Enteroviruses are by far the most common pathogens causing hospitalization for suspected sepsis in young infants during the summer and fall in the United States (37, 74). Making a viral diagnosis in these infants early in the course of hospitalization provides tremendous benefits to the patients themselves, as well as helping to identify a community outbreak, guiding in infection control, and educating physicians regarding the diseases they are dealing with. In Rochester, N.Y., during the summer and fall months of 1979 and 1980, 111 patients were diagnosed as having aseptic menin- *gitis or meningoencephalitis or both (28). Enteroviruses were identified as the cause of meningitis in 46 cases (41%). The diagnosis of enteroviral meningitis was based on positive viral culture of CSF, throat swab, and/or rectal swab or stool samples. By using standard cell cultures and BGM, the viruses were isolated from these specimens within 1 to 14 days. The isolation of enteroviruses from these 46 patients directly influenced the diagnosis and management of 22 CLIN. MICROBIOL. REV. cases (48%) and confirmed the diagnosis as well as specified the etiologic agent in the other 24 cases (52%). The effects of virus isolation on the diagnosis, management, and prognosis of patients with enteroviral meningitis are as follows. * Early withdrawal of antibiotics * Early hospital discharge * Avoidance of i.v. administration and complications * Avoidance of unnecessary tests and their complications * Diagnosis of meningitis in patients without CSF pleocytosis * Specification of etiologic agent of meningitis * Indication for prognosis * Ability to diagnose and guide high-titer immunoglobulin therapy for immunocompromised patients Neonates, patients who had received oral antibiotics before a lumbar puncture was performed, and patients with questionable CSF findings were all hospitalized and treated with i.v. antibiotics until an enterovirus was isolated. Virus isolation resulted in withdrawal of antibiotics and early discharge of these patients, saving thousands of dollars and helping to avoid the potential complications of i.v. drug administration and unnecessary tests. In addition, positive enteroviral culture results helped identify young infants who would not otherwise have been diagnosed as having a CNS disease, such as infants with no CSF pleocytosis or those whose spinal taps were uninterpretable. The prognosis in these cases was altered because of the possible sequelae of the disease in this age group (11, 46, 122, 134, 161). At UTMB in Galveston, the virology laboratory has been shown to have a similar influence on patient management (157). From 1983 to 1985, 69 patients under 18 years of age were diagnosed and treated for enteroviral meningitis. With three cell types, including BGM, cultures of CSF, throat swab, and/or rectal swab or stool specimens were found positive for an enterovirus within 1 to 11 days. Half of the 49 patients in whom the diagnosis was made, based on positive CSF culture, benefited directly from the viral culture results. From the point of view of cost-effectiveness, viral diagnosis is highly advantageous. The cost of viral culture at UTMB is comparable to that of bacterial blood culture or rapid bacterial antigen detection. Although the laboratory tests required for making or excluding the diagnosis of bacterial sepsis, with or without meningitis, include only a CSF examination and blood and CSF bacterial culture, numerous other tests are often ordered. These include nonspecific tests such as complete leukocyte count and differential, platelet count, urinalysis, urine bacterial culture, and serum electrolytes. Rapid bacterial antigen detection for three to four common bacteria from CSF or urine or both is also frequently performed, although results rarely change patient management (53). If the patient continues to have fever while on antibiotic therapy, tests such as chest X rays, computerized tomographic scan, and repeat examination of CSF may be obtained. All of these tests are costly and put the patient at risk for untoward effects. Positive enteroviral culture results, especially early in the course of the disease, provide a definitive diagnosis which can save the patient from unnecessary investigations and treatment and sometimes indicate a different prognosis. CONCLUSION Numerous types of viruses cause CNS infection. Among these, enteroviruses are by far the most common cause of acute CNS infection, a disease that mimics sepsis and

11 VOL. 2, 1989 THE VIROLOGY LABORATORY AND DIAGNOSIS OF CNS DISEASE 11 bacterial meningitis, especially in infants and young children. CNS infection caused by HSV, although not common, is now treatable. Provided that results can be obtained expeditiously, the virology laboratory can play an important role in the diagnosis and management of both enteroviral and HSV CNS infection, while it provides diagnosis and guides in supportive therapy for other CNS viral diseases. It is hoped that knowledge of the impact of the virology laboratory on management of patients as presented here will stimulate an increased and more intelligent use of virology laboratories, improved communication and cooperation between laboratory and physician, and an increased demand for the establishment of these laboratories at institutions where none are currently available. 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