John A. D. Leake

Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been successfully tested in proof-of-concept case studies in patients with acute illness of unknown etiology but to date has been largely confined to research settings. Here, we developed and validated a clinical mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from cerebrospinal fluid (CSF) in a licensed microbiology laboratory. A customized bioinformatics pipeline, SURPI+, was developed to rapidly analyze mNGS data, generate an automated summary of detected pathogens, and provide a graphical user interface for evaluating and interpreting results. We established quality metrics, threshold values, and limits of detection of 0.2-313 genomic copies or colony forming units per milliliter for each representative organism type. Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, spiked phages used as internal controls were reliable indicators of sensitivity loss. Diagnostic test accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% sensitivity and 99% specificity compared to original clinical test results, and 81% positive percent agreement and 99% negative percent agreement after discrepancy analysis. Subsequent mNGS challenge testing of 20 positive CSF samples prospectively collected from a cohort of pediatric patients hospitalized with meningitis, encephalitis, and/or myelitis showed 92% sensitivity and 96% specificity relative to conventional microbiological testing of CSF in identifying the causative pathogen. These results demonstrate the analytic performance of a laboratory-validated mNGS assay for pan-pathogen detection, to be used clinically for diagnosis of neurological infections from CSF.

BACKGROUND: Acute disseminated encephalomyelitis (ADEM) is a central nervous system demyelinating disease that usually follows an apparently benign infection in otherwise healthy young persons. The epidemiology, infectious antecedents and pathogenesis of ADEM are poorly characterized, and some ADEM patients are subsequently diagnosed with multiple sclerosis (MS). METHODS: We retrospectively (1991-1998) and prospectively (1998-2000) studied all persons aged < 20 years diagnosed with ADEM from the 3 principal pediatric hospitals in San Diego County, CA, during 1991-2000. Acute neurologic abnormalities and imaging evidence of demyelination were required for study inclusion. Epidemiologic variables, risk factors, clinical course, laboratory and radiographic findings, neuropathology and treatment data were analyzed. Interleukin (IL)-12, interferon-gamma (IFN-gamma) and IL-10 were assayed in blinded manner on cerebrospinal fluid (CSF) obtained prospectively from a subset of ADEM cases and compared with CSF from patients with enteroviral (EV) meningoencephalitis confirmed by polymerase chain reaction (PCR) and controls without pleocytosis. RESULTS: Data were analyzed on 42 children and adolescents diagnosed with ADEM during 1991-2000, and CSF IL-12, IFN-gamma and IL-10 levels were compared among ADEM (n = 14), EV meningoencephalitis (n = 14) and controls without pleocytosis (n = 28). Overall incidence of ADEM was 0.4/100,000/year; incidence quadrupled during 1998-2000 compared with earlier years. No gender, age stratum, ethnic group or geographic area was disproportionately affected. A total of 4 (9.5%) patients initially diagnosed with ADEM were subsequently diagnosed with MS after multiple episodes of demyelination. Although most children eventually recovered, 2 died, including 1 of the 3 ultimately diagnosed with MS. Magnetic resonance imaging was required for diagnosis among 74% of patients; computerized tomography findings were usually normal. Patients with EV had significantly higher mean CSF IFN-gamma (P = 0.005) and IL-10 (P = 0.05) than patients with ADEM and controls without CSF pleocytosis. CSF from ADEM patients had CSF cytokine values statistically similar to those of 3 patients subsequently diagnosed with MS. CONCLUSIONS: ADEM is a potentially severe demyelinating disorder likely to be increasingly diagnosed as more magnetic resonance imaging studies are performed on patients with acute encephalopathy. Further characterization of the central nervous system inflammatory response will be needed to understand ADEM pathogenesis, to improve diagnostic and treatment strategies and to distinguish ADEM from MS.

We analyzed national Haemophilus influenzae (Hi) surveillance data from 1994 and 1995 to describe the epidemiology of Hi invasive disease among persons of all ages. Serotype data were available for 376 (56%) of 669 reported Hi cases among children aged 4 years or younger; 184 (49%) were H. influenzae type b (Hib). Among children aged 4 or younger, incidence (per 100,000) of all Hi invasive disease was 1.8 in 1994 and 1.6 (p < 0.05) in 1995. Children aged 5 months or younger had the highest average annual incidence rate of Hib invasive disease (2.2 per 100,000); children aged 6 to 11 months had the next highest rate (1.2 per 100,000)(p < 0.05). Of 181 children with Hib invasive disease whose age in months was known, 85 (47%) were too young (aged 5 months or younger) to have completed a primary series with an Hib-containing vaccine. Of the 83 children with known vaccination status who were eligible to receive a primary series (aged 6 months or older), 52 (63%) were undervaccinated, and the remaining 31 (37%) had completed a primary series in which vaccine failed. Among persons aged 5 years or older with Hi invasive disease, the lowest average annual incidence was among those 20 to 39 years of age (0.15 per 100,000), and the highest was among those aged 80 years or older (2.26 per 100,000). Among persons aged 5 years or older, serotype data were available for 1,372 (71%) of the 1,940 Hi invasive disease cases; 159 (28%) of the 568 Hi cases with known serotype were due to Hib.

Hepatitis C virus (HCV) infection is a leading cause of liver-related morbidity and mortality (1). Transmission of HCV is primarily via parenteral blood exposure, and HCV can be transmitted vertically from mother to child. Vertical transmission occurs in 5.8% (95% confidence interval = 4.2%-7.8%) of infants born to women who are infected only with HCV and in up to twice as many infants born to women who are also infected with human immunodeficiency virus (HIV) (2) or who have high HCV viral loads (3,4); there is currently no recommended intervention to prevent transmission of infection from mother to child (3). Increased reported incidence of HCV infection among persons aged ≤30 years (5,6) with similar increases among women and men in this age group (6), raises concern about increases in the number of pregnant women with HCV infection, and in the number of infants who could be exposed to HCV at birth. Data from one large commercial laboratory and birth certificate data were used to investigate trends in HCV detection among women of childbearing age,* HCV testing among children aged ≤2 years, and the proportions of infants born to HCV-infected women nationally and in Kentucky, the state with the highest incidence of acute HCV infection during 2011-2014 (6). During 2011-2014, commercial laboratory data indicated that national rates of HCV detection (antibody or RNA positivity(†)) among women of childbearing age increased 22%, and HCV testing (antibody or RNA) among children aged ≤2 years increased 14%; birth certificate data indicated that the proportion of infants born to HCV-infected mothers increased 68%, from 0.19% to 0.32%. During the same time in Kentucky, the HCV detection rate among women of childbearing age increased >200%, HCV testing among children aged ≤2 years increased 151%, and the proportion of infants born to HCV-infected women increased 124%, from 0.71% to 1.59%. Increases in the rate of HCV detection among women of childbearing age suggest a potential risk for vertical transmission of HCV. These findings highlight the importance of following current CDC recommendations to identify, counsel, and test persons at risk for HCV infection (1,7), including pregnant women, as well as consider developing public health policies for routine HCV testing of pregnant women, and expanding current policies for testing and monitoring children born to HCV-infected women. Expansion of HCV reporting and surveillance requirements will enhance case identification and prevention strategies.

OBJECTIVES: Meningococcal disease continues to result in substantial morbidity and mortality in children, but there is limited recent surveillance information regarding serogroup distribution and outcome in children in the United States. The objective of this study was to collect demographic, clinical, laboratory, and outcome information for infants and children who had Neisseria meningitidis infections of various serogroups and were cared for in 10 pediatric hospitals. METHODS: Investigators at each of the participating hospitals identified children with meningococcal infections and collected demographic and clinical information using a standard data form. Meningococcal isolates were sent to a central laboratory for serogrouping by slide agglutination and penicillin susceptibility. RESULTS: From January 1, 2001, through March 15, 2005, 159 episodes of systemic meningococcal infections were detected. The greatest numbers of children were younger than 12 months (n = 41) or were 12 to 24 months of age (n = 22). Meningitis was the most common clinical manifestation of disease accounting for 112 (70%) cases; 43 (27%) children had bacteremia only. Children who were younger than 5 years (17 of 102) were significantly less likely to require mechanical ventilation than children who were 5 to 10 years of age (12 of 24) or children who were older than 10 years (13 of 33). Overall, 55 (44%) isolates were serogroup B, 32 (26%) were serogroup C, and 27 (22%) were serogroup Y. All but 1 isolate (intermediate) were susceptible to penicillin. The overall mortality rate was 8% (13 of 159) but was greater for children who were > or = 11 years of age (7 [21.2%] of 33) than for children who were younger than 11 years (6 [4.8%] of 126). Unilateral or bilateral hearing loss occurred in 14 (12.5%) of 112 children with meningitis. CONCLUSIONS: The morbidity and the mortality of meningococcal infections are substantial. With the recent licensure of meningococcal conjugate vaccines, our baseline trends in meningococcal disease can be compared with those seen after widespread vaccination to assess the success of routine immunization.