Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid.

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.

Increased Hepatitis C Virus (HCV) Detection in Women of Childbearing Age and Potential Risk for Vertical Transmission - United States and Kentucky, 2011-2014.

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.

Genetic structure of community acquired methicillin-resistant Staphylococcus aureus USA300.

BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a significant bacterial pathogen that poses considerable clinical and public health challenges. The majority of the CA-MRSA disease burden consists of skin and soft tissue infections (SSTI) not associated with significant morbidity; however, CA-MRSA also causes severe, invasive infections resulting in significant morbidity and mortality. The broad range of disease severity may be influenced by bacterial genetic variation. RESULTS: We sequenced the complete genomes of 36 CA-MRSA clinical isolates from the predominant North American community acquired clonal type USA300 (18 SSTI and 18 severe infection-associated isolates). While all 36 isolates shared remarkable genetic similarity, we found greater overall time-dependent sequence diversity among SSTI isolates. In addition, pathway analysis of non-synonymous variations revealed increased sequence diversity in the putative virulence genes of SSTI isolates. CONCLUSIONS: Here we report the first whole genome survey of diverse clinical isolates of the USA300 lineage and describe the evolution of the pathogen over time within a defined geographic area. The results demonstrate the close relatedness of clinically independent CA-MRSA isolates, which carry implications for understanding CA-MRSA epidemiology and combating its spread.

Surveillance for invasive meningococcal disease in children, US-Mexico border, 2005-2008.

We reviewed confirmed cases of pediatric invasive meningococcal disease in Tijuana, Mexico, and San Diego County, California, USA, during 2005-2008. The overall incidence and fatality rate observed in Tijuana were similar to those found in the US, and serogroup distribution suggests that most cases in Tijuana are vaccine preventable.

Acute pancreatitis in fatal anicteric leptospirosis.

We report a fatal case of anicteric leptospirosis with pancreatitis (acute hyperglycemia and insulin requirement, elevated lipase and amylase levels), pulmonary infiltrates, and refractory shock. In disease-endemic areas, leptospirosis with pancreatitis should be considered in patients with fever and abdominal pain, and serum pancreatic enzymes, blood glucose, and serum electrolytes should be closely monitored.

West Nile virus encephalitis with thalamic involvement in an immunocompromised child.

West Nile virus has been an increasingly important pathogen in the United States since it was first reported in 1999. Neuroinvasive West Nile virus has been infrequently reported in the pediatric population. We report a case of severe West Nile virus encephalitis with cranial magnetic resonance imaging findings not yet described in children.

Prediction of neurologic sequelae in childhood tuberculous meningitis: a review of 20 cases and proposal of a novel scoring system.

BACKGROUND: Despite effective antituberculous medications, the mortality and morbidity remain high in children with tuberculous meningitis (TBM). The traditional clinical staging for TBM developed by Lincoln et al in 1960 has been widely used to predict long term neurologic sequelae (NS). In the current era of critical care medicine and corticosteroid therapy, a new scoring system is needed to predict NS more accurately in children with TBM. METHODS: We reviewed all available cases of TBM in San Diego, CA, during 1991-2001 retrospectively, and we developed a novel scoring system to predict NS in children with TBM. We assessed a tuberculous meningitis acute neurologic (TBAN) score at day 0 and on day 3 of hospitalization, to compare children who subsequently developed severe NS with those who did not. RESULTS: Among 20 children with TBM, 7 children developed severe NS and 1 child died during hospitalization. The TBAN score was higher on day 0 in those with severe NS (5.5 versus 2.0, P = 0.09), and the difference became statistically significant by day 3 of hospitalization (5.5 versus 0.0, P = 0.02). Sensitivity and specificity of the TBAN score (> or =4) on day 0 (75 and 92%) and day 3 (88 and 100%) to predict severe NS were superior to the traditional clinical staging system on day 0 (63 and 58%). CONCLUSIONS: The TBAN score is an objective marker for predicting severe NS in children with TBM.

Pediatric acute hemorrhagic leukoencephalitis: report of a surviving patient and review.

Acute hemorrhagic leukoencephalitis (AHLE) is a rare, fulminant CNS demyelinating condition usually diagnosed at autopsy. We report the clinical, laboratory, radiographic, and pathologic features of the first nonfatal case of pediatric AHLE confirmed by brain biopsy. Pathologic diagnosis of this condition may be critical to exclude more-common processes and to expedite the decision to administer high-dose corticosteroid therapy, which is potentially lifesaving.

Acute disseminated encephalomyelitis associated with enteroviral infection.

A case of acute disseminated encephalomyelitis in 13-year-old boy associated with enterovirus is described. The patient had symptoms of severe headache and photophobia for 2 days. Diagnosis was made on the basis of diffuse high intensity white matter lesions in the left frontoparietal region seen on magnetic resonance imaging, and positive enterovirus polymerase chain reaction in cerebrospinal fluid. His symptoms improved substantially without specific therapy, and he recovered without neurologic sequelae.

Acute disseminated encephalomyelitis in childhood: epidemiologic, clinical and laboratory features.

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.

Parasitic disease in the pediatric intensive care unit - A review of severe manifestations of pediatric parasitic disease.

Parasitic diseases, previously thought to be the domain of developing nations only, are being increasingly recognized as a significant source of morbidity/mortality in developed and developing nations alike. While many parasites cause long-term low-level disease, some parasitic diseases can have severe manifestations necessitating intensive care unit admission. In this review, we focus on severe malaria (including cerebral malaria), neurocysticercosis, Strongyloides hyperinfection syndrome, American trypanosomiasis (Chagas disease), baylisascariasis and amoebic meningoencephalitis.

Altered neutrophil counts at diagnosis of invasive meningococcal infection in children.

BACKGROUND: Invasive meningococcal infections can be devastating. Substantial endotoxemia releases mature and immature neutrophils. Endothelial margination of mature neutrophils may increase the immature-to-total neutrophil ratio (ITR). These changes have not been previously well-described in invasive meningococcal disease. METHODS: Using 2001 to 2011 data from the US Multicenter Meningococcal Surveillance Study, the diagnostic sensitivity and clinical correlates of white blood cell count, absolute neutrophil count (ANC), immature neutrophil count (INC) and ITR were evaluated alone and in combination at the time of diagnosis of invasive meningococcal disease. RESULTS: Two hundred sixteen patients were evaluated: meningococcemia (65), meningitis (145) and other foci (6). ANC ≤1000/mm(3) or ≥10,000/mm(3) was present in 137 (63%), INC ≥500/mm(3) in 170 (79%) and ITR ≥0.20 in 139 (64%). One or more of these 3 criteria were met in 204 of the 216 (94%). Results were similar for meningococcemia and meningitis subgroups. All 13 cases with mildest disease met 1 or more of the 3 criteria. Eight children presented with ANCs <1000/mm(3): 3 of them died and a fourth required partial amputation in all 4 limbs. CONCLUSIONS: Invasive meningococcal disease is characterized by striking abnormalities in ANC, INC and/or ITR. Neutropenia was associated with a poor prognosis. Notably, without INCs, 37% of cases would have been missed. Automated methods not measuring immature white blood cells should be avoided when assessing febrile children. Serious infection should be considered when counts meet any of the 3 criteria.

Comparative analysis of severe pediatric and adult leptospirosis in Sao Paulo, Brazil.

Although leptospirosis may be fatal in childhood, the experience of many clinicians working in disease-endemic areas is that classic Weil's disease and death are less common among pediatric patients. The aim of the study was to ascertain disease spectrum and outcome differences in severe pediatric and adult leptospirosis in a large at-risk population. Epidemiologic, clinical, and laboratory data were obtained on hospitalized cases from São Paulo during 2004-2006. A total of 42 case-patients < 18 years of age and 328 case-patients ≥ 18 years of age were tested during the study. Compared with children, adults had higher rates of jaundice (P = 0.01), elevated serum bilirubin levels (P < 0.01), oliguria (P = 0.02), and elevated creatinine levels (P = 0.01) but not for thrombocytopenia or pulmonary involvement. The overall case-fatality rate was 27% (adult) versus 5% (pediatric) (P < 0.01). Severe pediatric leptospirosis may be less likely to show all classic features of Weil's disease and may be less fatal than in adults.

Multicenter surveillance of invasive meningococcal infections in children.

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.

Mycobacterial peritonitis in pediatric peritoneal dialysis patients.

Peritonitis is the most common complication and the leading cause of death in pediatric peritoneal dialysis (PD) patients. According to the most recent data available from the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS), approximately 25% of pediatric PD patients who die succumb to infection. There are no reported cases of Mycobacterium tuberculosis (MTB) or Mycobacterium avium-intracellulare peritonitis in the NAPRTCS registry. With an increasing incidence of MTB worldwide and the impairment of cellular immunity in chronic renal failure patients, it is not surprising that mycobacterium peritonitis can occur in PD patients. We report two pediatric PD patients with mycobacterial peritoneal infection diagnosed over an 11-year period at our institution. One patient presented with a malfunctioning Tenckhoff catheter and again 3 years later with hyponatremia and ascites. The other presented with recurrent culture-negative peritonitis. These cases illustrate the importance of more extensive evaluation of PD complications, to include evaluation for mycobacterium with special media or peritoneal biopsy, in the above clinical settings if the routine work-up is unrevealing.