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CONGENITAL LYMPHOCYTIC CHORIOMENINGITIS VIRUS INFECTION: AN UNDERDIAGNOSED DISEASE

1999; Lippincott Williams & Wilkins; Volume: 18; Issue: 7 Linguagem: Inglês

10.1097/00006454-199907000-00020

1532-0987

Gisela Enders, Marita Varho-Göbel, Jürgen Löhler, Elena Terletskaia‐Ladwig, Maren Eggers,

Hepatitis B Virus Studies

Lymphocytic choriomeningitis virus (LCMV) is the prototype of the family Arenaviridae, the primary reservoir of which is rodents (e.g. mice and hamsters). The infected animals may be asymptomatic but they excrete the virus in their urine. Human LCMV infection is usually mild and nonspecific with fever, headache, nausea and myalgia. In severe cases LCMV infection can result in meningitis and meningoencephalitis. Infection wiith LCMV during the first trimester of pregnancy appears to be associated with abortions.1 Fetal infection in the second and third trimesters results in intrauterine or early neonatal death, as well as in hydrocephalus and chorioretinitis in live born infants.2-4 The diagnosis of infection with LCMV is made by isolation of the virus, by detection of the nucleic acid using PCR or by serologic tests. Assay for LCMV-specific antibodies in the indirect immunofluorescence test or enzyme-linked immunosorbent assay may be positive as early as the first day of symptoms.5, 6 The complement fixation titer generally does not rise until 10 days or longer after onset of illness.5, 7 The neutralizing titer rises late and presumably persists lifelong. Lehman-Grube and Ambrassat8 were able to demonstrate LCMV-specific neutralizing antibodies using the plaque reduction method and the neutralization test in 10 patients who had suffered clinical lymphocytic meningitis up to 30 years previously. Additionally in a later study the group of Lehman-Grube showed that LCMV-specific antibodies are detectable with the neutralization test and with the immunofluorescence method for at least 10 years postinfection.9 Recently we were also able to find LCMV-specific IgG antibodies using the immunofluorescence method in two research workers who had occupational LCMV infection as long as 30 years ago (data not shown). Therefore it appears that both methods can be used for reliable diagnosis of LCMV infection. We describe six cases of congenital LCMV infections that occurred in various parts of Western Germany between 1991 and 1997. Three of the cases were followed prospectively and three were diagnosed retrospectively (Table 1).TABLE 1: Serologic results of congenitally LCMV-infected children and their mothers Methods. Serologic investigations for LCMV infection were performed by indirect immunofluorescence on slides with monolayers of a D6-Detroit cell line persistently infected with LCMV or with monolayers of Vero cells after 48 h of inoculation with a 10−4 suspension of LCMV strain Armstrong. Suitable controls were included. Both cell lines gave similar results; however, the latter method seemed to be more sensitive for IgM detection. Only sera showing the typical granular fluorescence of the cytoplasm of the infected cells were regarded as positive. Repeated testing of follow-up sera reproduced the previous LCMV-specific titers and varied only at the range of ± one dilution step. For all cases infections by Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus, parvovirus B19 and enterovirus infections were excluded by serologic and virologic investigations. An in-house reverse transcriptase-nested PCR protocol for detection of LCMV-RNA was established at the end of 1996 and was used only in few stored serum and cerebrospinal fluid (CSF) samples. Amniotic fluid and fetal blood samples were unavailable for investigation by LCMV-PCR (Case 6). LCMV antigen was immunostained on paraffin sections by a triple step immunoenzymatic method. The deparaffinized sections were overlaid overnight at 4°C with 1/4000 dilution of a polyclonal rabbit antiserum against LCMV. Bound rabbit antibodies were stained with alkaline phosphatase-labeled anti-rabbit antibodies. Enzyme activity was revealed with naphthol AS-BI phosphate in combination with hexazotized new fuchsin (Merck, Darmstadt, Germany). Naive rabbit serum was used as a control. Results and case reports. The results of LCMV-specific serology are shown in Table 1. Case 1 (1994). A 19-year-old woman who worked in a pet shop selling hamsters, mice and other pet animals in a small town (Herten, Germany) developed a severe febrile illness between the 12th and 14th week of gestation. Routine ultrasound sonography in the 21st week of gestation disclosed an internal hydrocephalus and congestive heart failure (Dr. Giffei, St. Vincenz-Krankenhaus, Germany). Maternal and fetal blood were obtained for laboratory investigations. In the maternal blood LCMV IgG and IgM antibodies were detected, whereas only IgG antibodies were present in the fetal blood. Because of a hemoglobin value of 3.5 g/dl, the fetus received two intrauterine transfusions; however, intrauterine death occurred in the 22nd week. Histologic examination of fetal tissues revealed lymphocytic myocarditis with extensive muscle fiber calcifications and erythroblastosis with extramedullary hematopoiesis. Immunohistochemistry on fetal brain tissue using LCMV-specific antiserum documented LCMV infection. Positively labeled cells were distributed around several cerebral blood vessels. It was unclear whether the antibody-stained cells represented neural cell elements, infiltrating macrophage-like cells, or myeloid cells. In addition nucleated cells, putatively representing erythroblasts, in myocardial vascular spaces and extramedullary hematopoietic foci also showed cytoplasmic LCMV antigen immunoreactivity. The latter finding could be causally related to the anemia of the fetus. In the murine LCMV infection evidence for interferon alpha/beta-induced as well as hemolytic anemia has been well-documented.10-12 To our knowledge the immunocytochemical detection of LCMV antigen in human fetal tissue represents an unique finding. Case 2 (1992). In this case fetal internal hydrocephalus was diagnosed by second trimester routine ultrasound screening in the 26th week of gestation in a 23-year-old woman who had a hamster at home (Prenatal Diagnosis Center, Munster, Germany). [In Germany antenal screening (prenatal care) includes at least three obligatory ultrasound examinations at various stages of pregnancy]. Specific LCMV IgG and IgM antibodies were detectable in the first maternal blood sample (IgG 1/128, IgM 1/16), whereas in the later samples LCMV-specific IgM antibodies were not detected. Only elevated IgG antibodies were found In fetal, cord and newborn blood samples. The IgG titer rise between the second and third maternal sera may be not significant because the samples were not evaluated in the same test run. The newborn girl had hydrocephalus. At 9 months she developed therapy-resistant four extremity spasms and severe psychomotor retardation. In 1994 chorioretinitis was diagnosed leading to scarred retinae. The girl who is now 7 years old is mute; unable to sit, focus and swallow; and therefore needs tube feeding. Case 3 (1996). Internal hydrocephalus was diagnosed by routine ultrasound in the 29th week of gestation in a 23-year-old pregnant woman who immigrated from Russia. Specific LCMV IgG and IgM antibodies were detected in the maternal blood and only IgG antibody in somewhat lower titer was detected in the fetal serum. No clinical and serologic data were available for newborn infant. Case 4 (1994). This case concerns a 28-year-old woman with a twin pregnancy, living under very low socioeconomic and housing conditions in a village near Heidelberg. She was exposed to mice in the first and second trimester. The twins were delivered by cesarean section in the 36th week of gestation because of dystrophy of one of the fetuses. After delivery both twins showed dystrophy, hydrocephalus, chorioretinitis and microcephaly. Serologic studies in the maternal sera and blood of both infants obtained 2 weeks and 5 years after birth showed only LCMV-specific IgG antibodies with declining titer at the age of 4 months. The infants were hospitalized at the age of 7 months because of spasms, severe retarded psychomotor development and blindness. Case 5 (1992). A 3-month-old Turkish boy (Prof. Dominik, children's clinic, Ludwigshafen, Germany) was evaluated for recurrent signs of meningitis and conjunctivitis. Chorioretinitis could not be observed and cranial ultrasound examinations were unremarkable. Sera taken at 3 and 6 months showed specific LCMV IgG but no IgM antibodies, whereas maternal serum sample at 3 months postpartum showed the same IgG titer as the 3-month-old infant. A low IgG titer (1/2) was detected in the infant's CSF at 3 months age and a weakly positive reverse transcriptase-PCR. The clinician reported that the child was asymptomatic at the age of 15 months, and no further specimens were obtained. Infection was considered to have been acquired postnatally. Case 6 (1991). Congenital virus infection was suspected by the Department of Pediatrics of the University of Kiel in a 5-month-old girl because of chorioretinitis with intracranial calcifications, hydrocephalus, microcephaly and almost complete blindness. Serologic studies in serum and CSF sample at 5 months of age excluded T. gondii, cytomegalovirus, rubella virus, herpes simplex virus, Treponema pallidum and Listeria. No testing was done for LCMV. A second serum sample taken at the age of 9 months revealed LCMV IgG antibodies in high titer in the serum (IgM-negative) and in low titer in CSF. The IgG antibodies had increased to an even higher titer at 2 years of age. The rise in LCMV-IgG-titer could be reproduced in the same test run. Discussion. In recent years a number of proven cases of perinatal LCMV infection have been reported by American pediatricians.3, 4 Although LCMV was first recognized in England13 and documented in Germany1, 2 and Lithuania,6 this congenital infection received little attention in Europe as illustrated in Case 6. Previous serologic studies in Germany indicate that between 1.2 and 9.1% of the adult rural population are seropositive.14 The six cases documented in our laboratory during 1991 through 1997 confirm the relevance of congenital LCMV infection. It is of interest to know the sources of infection and living conditions in considering the diagnosis of LCMV infection. In three of the six cases contact with rodents was established, and Case 6 lived in an agriculturally dominated part of Western Germany. Infected pets, house mice and rats pose a risk to pregnant women. Serologic assays should be able to detect antibodies in patients infected with all wild-type strains of LCMV because the immune sera of mice infected with various laboratory strains revealed broad cross-neutralization, demonstrating a variable but close relationship between LCMV strains.15 Our case reports support the findings of American investigators that LCM virus infection is an underrecognized cause of congenital disease with features similar to those of cytomegalovirus or T. gondii. When there is ultrasonographic evidence of hydrocephalus in the fetus or newborn including properly signs of chorioretinitis, diagnostic attempts should include tests for LCMV serology (IgG and IgM antibodies) and LCMV RNA detection. Even though there is not yet a specific drug treatment available for LCMV, an agent-specific diagnosis is helpful for in further management of the child. Acknowledgments. We thank Professor Dr. R. Ackermann for providing the LCMV-carrying D6-Detroit cell line, for encouraging us to publish our case reports and for critical reading of the manuscript. We thank our colleagues of the molecular biology laboratory for performing the PCR. We are grateful to obstetricians, gynecologists, pathologists, pediatricians, general practitioners and patient's mothers for sending samples of maternal and fetal tissue and blood of infants at birth and during follow-up. We also thank Dr. Wiectorek (Department of Pediatrics, University of Munster) for providing retrospective information on the clinical condition of the infant of Case 2. Gisela Enders, M.D. Marita Varho-Göbel, Ph.D. Jürgen Löhler, M.D. Elena Terletskaia-Ladwig, M.D. Maren Eggers, Ph.D. Medizisch-Diagnostik Gemeinschaftslabor und Kollegen and Institut für Virologie, Infektiologie und Epidemiologie e.V.; Stuttgart, Germany (GE, MVG, ETL, ME) Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie; University of Hamburg; Hamburg, Germany (JL)