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The fusion (F) protein of respiratory syncytial virus (RSV) is the major target of immunoprophylactic monoclonal antibodies (mAbs) and vaccines. Recently reported mutations in F gene antigenic sites can vary among RSV types A and B. To further understand mutations in RSV F proteins, we performed subtyping and F gene sequencing on 400 RSV-positive respiratory samples collected at four pediatric hospitals within the United States from children under 2 years old between 2018 and 2020. RSV B was predominant in 2018-2019 and RSV A in 2019-2020 (55.5% and 85.5% respectively). Compared to the reference sequence, all RSV B samples had at least one antigenic polymorphism with the most changes at sites AM14/V (100%) and Ø (93.3%) followed by II (5.8%), IV (3.9%), and p27 (2.9%). The most frequent mutations among RSV B for AM14/V site were in L172Q (100%), S173L (100%), and K191R (95.2%) while for Ø site they were in I206M (93.3%) and Q209R (93.3%). Conversely, polymorphisms were observed in only 15.3% of RSV A samples overall, specifically at antigenic sites p27 (5.9%), IV (3.0%), II (2.5%), AM14/V (2.0%), I (2.0%), and Ø (0.5%). Among RSV A cases, T122A at p27 (n = 10) and S276N at II (n = 3) were the most common substitution sites. S276N at site II was found in both RSV types. Although polymorphisms in F proteins of RSV B were more common than those in RSV A samples, changes in both subtypes were observed in key F antigenic sites which could potentially impact the efficacy of mAb therapies and vaccines.

OBJECTIVES: Diagnosing Clostridioides difficile infections in young children with high asymptomatic colonization is challenging. We compared the frequency of C difficile detection by polymerase chain reaction (PCR) in healthy control (HC) children with those with acute gastroenteritis (AGE) and evaluated fecal-lactoferrin and organism load as possible indicators of true C difficile infection disease. METHODS: Stool was collected from children RESULTS: Of 524 stools collected from 524 children (250 with AGE, 274 HCs), C difficile was detected less in children with AGE (14%, 36 of 250) than in HCs (28%, 76 of 274) stools (P < .0001). Among infants CONCLUSIONS: HC children

Acute viral respiratory infections are a major health burden in children worldwide. In recent years, rapid and sensitive multiplex nucleic acid amplification tests (NAATs) have replaced conventional methods for routine virus detection in the clinical laboratory.We compared BioFire® FilmArray® Respiratory Panel (FilmArray V1.7), Luminex NxTag® Respiratory Pathogen Panel (NxTag RPP) and Applied Biosystems TaqMan Array Card (TAC) for the detection of eight viruses in pediatric respiratory specimens. Results from the three platforms were analyzed with a single-plex real-time RT-PCR (rRT-PCR) assay for each virus.Of the 170/210 single-plex virus-positive samples, FilmArray detected a virus in 166 (97.6%), TAC in 163 (95.8%) and NxTag RPP in 160 (94.1%) samples. The Positive Percent Agreement (PPA) of FilmArray, NxTag RPP and TAC was highest for influenza B (100%, 100% and 95.2% respectively) and lowest for seasonal coronaviruses on both FilmArray (90.2%) and NxTag RPP (81.8%), and for parainfluenza viruses 1- 4 on TAC (84%). The Negative Percent Agreement (NPA) was lowest for rhinovirus/enterovirus (92.9%, 96.7% and 97.3%) on FilmArray, NxTag RPP and TAC respectively. NPA for all three platforms was highest (100%) for both parainfluenza viruses 1- 4 and influenza A and B, and 100% for human metapneumovirus with TAC as well.All three multiplex platforms displayed high overall agreement (gt;90%) and high NPA (gt;90%), while PPA was pathogen dependent and varied among platforms; high PPA (gt;90%) was observed for FilmArray for all eight viruses, TAC for six viruses and NxTag RPP for 4 viruses.

Background Despite vaccine-induced decreases in US rotavirus (RV) disease, acute gastroenteritis (AGE) remains relatively common. We evaluated AGE pathogen distribution in hospitalized US children in the post–RV vaccine era. Methods From December 2011 to June 2016, the New Vaccine Surveillance Network (NVSN) conducted prospective, active, population-based surveillance in hospitalized children with AGE. We tested stools from 2 NVSN sites (Kansas City, Houston) with Luminex x-TAG Gastrointestinal Pathogen Panels (Luminex GPP) and analyzed selected signs and symptoms. Results For 660 pediatric AGE inpatients and 624 age-matched healthy controls (HCs), overall organism detection was 51.2% and 20.6%, respectively (P 1 virus in 39% and >1 bacterium in 14% of specimens. Detection frequencies for AGE subjects vs HCs were norovirus (NoV) 18.5% vs 6.6%, RV 16.1% vs 9.8%, adenovirus 7.7% vs 1.4%, Shigella 4.8% vs 1.0%, Salmonella 3.1% vs 0.1%, and Clostridioides difficile in ≥2-year-olds 4.4% vs 2.4%. More co-detections occurred among AGE patients (37/660, 5.6%) than HCs (14/624, 2.2%; P = .0024). Per logistic regression analysis, ill contacts increased risk for NoV, RV, and Shigella (P Conclusions NoV detection was most frequent; however, RV remained important in hospitalized AGE in the post–RV vaccine era. Continued active surveillance is important to document ongoing vaccine effects, pathogen emergence, and baseline disease burden for new vaccines.

DiagnosingStool was collected from children2 years old with AGE and from HCs.Of 524 stools collected from 524 children (250 with AGE, 274 HCs),HC children2 years of age had higher rates of

BackgroundEnterovirus (EV) and parechovirus type A3 (PeV-A3) cause infections ranging from asymptomatic to life-threatening. Host immune responses in children, particularly innate responses to PeV-A3, remain largely unknown. The aim of this study was to determine aspects of the cytokine/chemokine responses to EV and PeV-A3 in cerebrospinal fluid (CSF) and plasma obtained from children with systemic/central nervous system infection.MethodsA total of 74 salvaged CSF samples (27 with EV, 23 with PeV-A3, and 24 with neither EV nor PeV-A3) and 35 paired blood samples (10 with EV, 14 with PeV-A3, and 11 with neither) were studied. Concentrations of cytokines and chemokines were measured using a customized 21-plex MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel. Additionally, clinical characteristics data for all the patients were collected from electronic medical records to evaluate the potential association between the immune response and presentations.ResultsWe demonstrate that EV and PeV-A3 infections induce different cytokine/chemokine immune responses in children. EV induces more robust responses in CSF with significantly elevated levels of fractalkine, interferon (IFN)-α2, IFN-γ, interleukin (IL)-1Rα, IL-4, IL-8, and tumor necrosis factor α; PeV-A3 induces less robust or absent responses in CSF but robust responses in plasma, with significantly higher concentrations of IFN-α2, IL-15, IL-1Rα, interferon-γ-inducible protein–10, and monocyte chemoattractant protein–1.ConclusionsHigh cytokine/chemokine concentrations in the plasma of PeV-A3 patients compared with EV patients could explain higher/more prolonged fever in PeV-A3 patients, whereas relatively low cytokine/chemokine concentrations in PeV-A3 CSF might explain the absence of CSF pleocytosis.

Rapid and accurate detection of influenza virus is critical for proper patient management. The Alere™ i Influenza A&B assay is an isothermal nucleic acid amplification test capable of detecting influenza A and B viruses directly from respiratory specimens. In this multicenter clinical trial conducted in the US, we evaluated the clinical performance of the Alere™ i Influenza A&B assay against that of the Prodesse ProFlu+ assay. A total of 1243 fresh, leftover nasal or nasopharyngeal swabs eluted in viral transport medium were tested by both assays. Sensitivity and specificity of the Alere™ i Influenza A&B assay were 97.8% (95% CI 94.6-99.2) and 96.6% (95% CI 95.2-97.5) for influenza A and 92.9% (95% CI 85.5-96.9) and 98.3% (95% CI 97.4-98.0) for influenza B. The Alere™ i Influenza A&B assay is an ideal molecular assay for influenza virus detection due to its high sensitivity and specificity with minimal hands-on and turn-around-time.

Highlights • Changing our respiratory panel assay decreased turn-around-time from 27.1 to 1.4 h. • The faster assay was associated with decreased empiric antibiotic use, less x-rays, and shorter LOS. • The faster assay improved optimal use of oseltamivir in influenza infected patients., Background Empiric antibiotic treatment is common among children with acute respiratory tract infections (ARTI), despite infections being predominately viral. The use of molecular respiratory panel assays has become increasingly common for medical care of patients with ARTIs. Study design This was a 6-year retrospective, single-centered study of pediatric inpatients who tested positive for an ARTI respiratory pathogen. We examined the relationship between clinical outcomes and whether the patient was tested using the Luminex Respiratory Viral Panel ([RVP]; in-use: Dec. 2009 – Jul. 2012) or Biofire Respiratory Pathogen Panel ([RP]; in-use Aug. 2012 – Jun. 2016). The prevalence and duration of pre-test empiric antibiotics, post-test oseltamivir administration to influenza patients, chest x-rays and length of stay between the two assays was compared. Results A total of 5142 patients (1264 RVP; 3878 RP) were included. The median laboratory turn-around-time for RP was significantly shorter than RVP (1.4 vs. 27.1 h, respectively; p

Background Norovirus is the leading cause of epidemic and sporadic acute gastroenteritis (AGE) in the United States. Widespread prevalence necessitates implementation of accurate norovirus detection assays in clinical diagnostic laboratories. Objective To evaluate RIDA®GENE norovirus GI/GII real-time RT-PCR assay (RGN RT-PCR) using stool samples from patients with sporadic AGE. Study design Patients between 14 days to 101 years of age with symptoms of AGE were enrolled prospectively at four sites across the United States during 2014–2015. Stool specimens were screened for the presence of norovirus RNA by the RGN RT-PCR assay. Results were compared with a reference method that included conventional RT-PCR and sequencing of a partial region of the 5′end of the norovirus ORF2 gene. Results A total of 259 (36.0%) of 719 specimens tested positive for norovirus by the reference method. The RGN RT-PCR assay detected norovirus in 244 (94%) of these 259 norovirus positive specimens. The sensitivity and specificity (95% confidence interval) of the RGN RT-PCR assay for detecting norovirus genogroup (G) I was 82.8% (63.5–93.5) and 99.1% (98.0–99.6) and for GII was 94.8% (90.8–97.2) and 98.6% (96.9–99.4), respectively. Seven specimens tested positive by the RGN-RT PCR that were negative by the reference method. The fifteen false negative samples were typed as GII.4 Sydney, GII.13, GI.3, GI.5, GI.2, GII.1, and GII.3 in the reference method. Conclusions The RGN RT-PCR assay had a high sensitivity and specificity for the detection of norovirus in stool specimens from patients with sporadic AGE.

Background Viruses are major etiological agents of childhood gastroenteritis. In recent years, several molecular platforms for the detection of viral enteric pathogens have become available. Objective/study design We evaluated the performance of three multiplex platforms including Biofire’s Gastrointestinal Panel (FilmArray), Luminex xTAG ® Gastrointestinal Pathogen Panel (GPP), and the TaqMan Array Card (TAC) for the detection of five gastroenteritis viruses using a coded panel of 300 archived stool samples. Results The FilmArray detected a virus in 199 (96.1%) and the TAC in 172 (83.1%) of the 207 samples (187 samples positive for a single virus and 20 samples positive for more than one virus) whereas the GPP detected a virus in 100 (78.7%) of the 127 (97 positive for one virus and three positive for more than one virus) samples. Overall the clinical accuracy was highest for the FilmArray (98%) followed by TAC (97.2%) and GPP (96.9%). The sensitivity of the FilmArray, GPP and TAC platforms was highest for rotavirus (100%, 95.8%, and 89.6%, respectively) and lowest for adenovirus type 40/41 (97.4%, 57.9% and 68.4%). The specificity of the three platforms ranged from 95.6% (rotavirus) to 99.6% (norovirus/sapovirus) for the FilmArray, 99.6% (norovirus) to 100% (rotavirus/adenovirus) for GPP, and 98.9% (astrovirus) to 100% (rotavirus/sapovirus) for TAC. Conclusion The FilmArray demonstrated the best analytical performance followed by TAC. In recent years, the availability of multi-enteric molecular testing platforms has increased significantly and our data highlight the strengths and weaknesses of these platforms.

Here, we report 17 nearly complete genome sequences of enterovirus D68 (EV-D68) isolated from Kansas City, MO, in 2018. Phylogenetic analysis suggests that these strains belong to subclade B3, similar to the ones that caused the 2016 epidemics in the United States but different from the 2014 outbreak B1 strains.

Anaphylaxis is an acute, potential life-threatening systemic allergic reaction that may have a wide range of clinical manifestations. Severe anaphylaxis and/or the need for repeated doses of epinephrine to treat anaphylaxis are risk factors for biphasic anaphylaxis. Antihistamines and/or glucocorticoids are not reliable interventions to prevent biphasic anaphylaxis, although evidence supports a role for antihistamine and/or glucocorticoid premedication in specific chemotherapy protocols and rush aeroallergen immunotherapy. Evidence is lacking to support the role of antihistamines and/or glucocorticoid routine premedication in patients receiving low- or iso-osmolar contrast material to prevent recurrent radiocontrast media anaphylaxis. Epinephrine is the first-line pharmacotherapy for uniphasic and/or biphasic anaphylaxis. After diagnosis and treatment of anaphylaxis, all patients should be kept under observation until symptoms have fully resolved. All patients with anaphylaxis should receive education on anaphylaxis and risk of recurrence, trigger avoidance, self-injectable epinephrine education, referral to an allergist, and be educated about thresholds for further care.

BACKGROUND: In 2014, enterovirus D68 (EV-D68) was responsible for an outbreak of severe respiratory illness in children, with 1,153 EV-D68 cases reported across 49 states. Despite this, there is no commercial assay for its detection in routine clinical care. BioFire® Syndromic Trends (Trend) is an epidemiological network that collects, in near real-time, deidentified. BioFire test results worldwide, including data from the BioFire® Respiratory Panel (RP).OBJECTIVES: Using the RP version 1.7 (which was not explicitly designed to differentiate EV-D68 from other picornaviruses), we formulate a model, Pathogen Extended Resolution (PER), to distinguish EV-D68 from other human rhinoviruses/enteroviruses (RV/EV) tested for in the panel. Using PER in conjunction with Trend, we survey for historical evidence of EVD68 positivity and demonstrate a method for prospective real-time outbreak monitoring within the network.STUDY DESIGN: PER incorporates real-time polymerase chain reaction metrics from the RPRV/EV assays. Six institutions in the United States and Europe contributed to the model creation, providing data from 1,619 samples spanning two years, confirmed by EV-D68 gold-standard molecular methods. We estimate outbreak periods by applying PER to over 600,000 historical Trend RP tests since 2014. Additionally, we used PER as a prospective monitoring tool during the 2018 outbreak.RESULTS: The final PER algorithm demonstrated an overall sensitivity and specificity of 87.1% and 86.1%, respectively, among the gold-standard dataset. During the 2018 outbreak monitoring period, PER alerted the research network of EV-D68 emergence in July. One of the first sites to experience a significant increase, Nationwide Children's Hospital, confirmed the outbreak and implemented EV-D68 testing at the institution in response. Applying PER to the historical Trend dataset to determine rates among RP tests, we find three potential outbreaks with predicted regional EV-D68 rates as high as 37% in 2014, 16% in 2016, and 29% in 2018.CONCLUSIONS: Using PER within the Trend network was shown to both accurately predict outbreaks of EV-D68 and to provide timely notifications of its circulation to participating clinical laboratories.

Urinalysis (UA) has routinely been used as a screening tool prior to urine culture set up. BacterioScan 216Dx is an FDA-cleared semiautomated system to detect bacterial growth in urine. The aim of this study was to evaluate 216Dx in comparison to UA for diagnosis of urinary tract infection (UTI) in children. Clean-catch, unpreserved urine samples from children aged

Background The use of Sample-to-answer (STA) platforms for the detection of influenza A/B and respiratory syncytial virus (RSV) have greatly improved patient care. These diagnostic assays based on nucleic acid amplification are rapid, accurate and relatively easy to perform. Objectives We compared four such platforms for detecting FluA, FluB, and RSV from adult respiratory specimens: Hologic Panther Fusion® Flu A/B/RSV (Fusion), Cobas® Influenza A/B & RSV (Liat), Luminex Aries® Flu A/B & RSV (Aries), and Diasorin SimplexaTM Flu A/B & RSV (Simplexa). Study Design Nasopharyngeal (NP) swabs (n = 224) from adults were tested on these platforms and results were compared to Center for Disease Control and Prevention recommended real-time RT-PCR assay for influenza A/B and RSV. Subtyping for FluA and FluB was performed for discrepant analysis where applicable. Results Of the 82 FluA, 26 FluB, 15 RSV-positive specimens tested, the positive and negative percentage agreements (PPA and NPA respectively) for FluA detection were 100/100 (Fusion), 95.1/100 (Liat), 92.5/100 (Aries), and 84.1/99.3 (Simplexa); PPA and NPA for FluB detection were 92.3/99.5 (Fusion), 96/99.5 (Liat), 100/99.5 (Aries), and 80.8/100 (Simplexa); and for RSV detection were 100/100 (Fusion), 100/100 (Liat), 88.6/99.5 (Aries), and 73.3/100 (Simplexa). 82 confirmed FluA included 23 pH1N1 and 57 H3N2 strains with 2 strains remaining untyped. Of the 26 confirmed FluB, 25 were of the Yamagata lineage and 1 of unknown lineage. Conclusion Only 2 STA platforms demonstrated >95% PPA for the detection of all three targets while all the 4 platforms demonstrated >95% NPA for FluA, FluB and RSV.

To measure the impact of rapid influenza real-time qualitative reverse transcriptase polymerase chain reaction (RT-PCR) on patient management in busy pediatric emergency department (ED) and urgent care clinic settings.We developed a brief, elective survey that clinicians completed when an influenza RT-PCR order was placed in the ED or urgent care clinic between February 18, 2019, and March 13, 2019. We captured the clinical suspicion for influenza, intended management plans, and actual management plans once influenza RT-PCR results were available.We evaluated 339 encounters, of which 164 (48.4%) had a positive influenza RT-PCR. Clinical suspicion for influenza was a nonsignificant predictor for influenza PT-PCR positivity (P = .126). After rapid influenza RT-PCR results were available, clinicians changed their original plans in 44.5% of influenza RT-PCR positive vs 92.6% of influenza RT-PCR negative cases (P .0001). Change in plans for antiviral use was observed in 26% of influenza positive vs 77% of influenza negative cases (P .0001). A total of 135 antiviral prescriptions were avoided in patients with negative influenza RT-PCR.Implementation of a rapid and accurate influenza RT-PCR in the acute care setting is important to systematically diagnose influenza in children and improve outpatient management decisions, because clinical suspicion for influenza is inaccurate. A negative influenza RT-PCR decreases unnecessary antiviral use and has the potential for significant cost savings.

The rapid and accurate detection of influenza A virus (FluA), influenza B virus (FluB), and respiratory syncytial virus (RSV) improves patient care. Sample-to-answer (STA) platforms based on nucleic acid amplification and detection of these viruses are simple, automated, and accurate. We compared six such platforms for the detection of FluA, FluB, and RSV: Cepheid GeneXpert Xpress Flu/RSV (Xpert), Hologic Panther Fusion Flu A/B/RSV (Fusion), Cobas influenza A/B & RSV (Liat), Luminex Aries Flu A/B & RSV (Aries), BioFire FilmArray respiratory panel (RP), and Diasorin Simplexa Flu A/B & RSV (Simplexa). Nasopharyngeal (NP) swab specimens (n = 225) from children previously tested by RP were assessed on these platforms. The results were compared to those of the Centers for Disease Control and Prevention (CDC)-developed real-time reverse transcription-PCR (rRT-PCR) assay for influenza A/B viruses and RSV. Subtyping for FluA and FluB was performed for discrepant analysis where applicable. The percent sensitivities/specificities for FluA detection were 100/100 (Fusion), 98.6/99.3 (Xpert), 100/100 (Liat), 98.6/100 (Aries), 98.6/100 (Simplexa), and 100/100 (RP). The percent sensitivities/specificities for FluB detection were 100/100 (Fusion), 97.9/99.4 (Xpert), 97.9/98.3 (Liat), 93.7/99.4 (Aries), 85.4/99.4 (Simplexa), and 95.8/97.7 (RP); and those for RSV detection were 98.1/99.4 (Xpert), 98.1/99.4 (Liat), 96.3/100 (Fusion), 94.4/100 (Aries), 87/94.4 (Simplexa), and 94.4/100 (RP). The 75 strains confirmed to be FluA included 29 pH1N1, 39 H3N2, 4 sH1N1, and 3 untyped strains. The 48 strains confirmed to be FluB included 33 strains of the Yamagata lineage, 13 of the Victoria lineage, 1 of both the Yamagata and Victoria lineages, and 1 of an unknown lineage. All six STA platforms demonstrated >95% sensitivity for FluA detection, while three platforms (Fusion, Xpert, and Liat) demonstrated >95% sensitivity for FluB and RSV detection.

Background Enterovirus 68 (EV-D68) causes acute respiratory tract illness in epidemic cycles, most recently in Fall 2014, but clinical characteristics of severe disease are not well reported. Objectives Children with EV-D68 severe respiratory disease requiring pediatric intensive care unit (PICU) management were compared with children with severe respiratory disease from other enteroviruses/rhinoviruses. Study design A retrospective review was performed of all children admitted to Children’s Mercy Hospital PICU from August 1-September 15, 2014 with positive PCR testing for enterovirus/rhinovirus. Specimens were subsequently tested for the presence of EV-D68. We evaluated baseline characteristics, symptomatology, lab values, therapeutics, and outcomes of children with EV-D68 viral infection compared with enterovirus/rhinovirus-positive, EV-D68-negative children. Results A total of 86 children with positive enterovirus/rhinovirus testing associated with respiratory symptoms were admitted to the PICU. Children with EV-D68 were older than their EV-D68-negative counterparts (7.1 vs. 3.5 years, P = 0.01). They were more likely to have a history of asthma or recurrent wheeze (68% vs. 42%, P = 0.03) and to present with cough (90% vs. 63%, P = 0.009). EV-D68 children were significantly more likely to receive albuterol (95% vs. 79%, P = 0.04), magnesium (75% vs. 42%, P = 0.004), and aminophylline (25% vs. 4%, P = 0.03). Other adjunctive medications used in EV-D68 children included corticosteroids, epinephrine, and heliox; 44% of EV-D68-positive children required non-invasive ventilatory support. Conclusions EV-D68 causes severe disease in the pediatric population, particularly in children with asthma and recurrent wheeze; children may require multiple adjunctive respiratory therapies.

Background The rotavirus disease burden has declined substantially since rotavirus vaccine was introduced in the United States in 2006. The aim of this study was to determine the viral etiology of acute gastroenteritis (AGE) in US children aged Methods The New Vaccine Surveillance Network (NVSN) of geographically diverse US sites conducts active pediatric population-based surveillance in hospitals and emergency departments. Stool samples were collected from children aged Results Detection rates of pathogens in children with AGE versus those of HCs were, respectively, 23.0% versus 6.6% for norovirus (P < .01), 23.0% versus 16.0% for adenovirus (P = .08), 11.0% versus 16.0% for parechovirus A (P = .09), 11.0% versus 9.0% for enterovirus (P = .34), 7.0% versus 3.0% for sapovirus (P = .07), 3.0% versus 0.3% for astrovirus (P = .01), and 3.0% versus 0.4% for rotavirus (P = .01). A high prevalence of adenovirus was detected at 1 surveillance site (49.0% for children with AGE and 43.0% for HCs). Norovirus GII.4 New Orleans was the most frequently detected (33.0%) norovirus genotype. Codetection of >1 virus was more common in children with AGE (16.0%) than in HCs (10.0%) (P = .03). Conclusions Norovirus, astrovirus, sapovirus, and rotavirus were detected significantly more in children with AGE than in HCs, and norovirus was the leading AGE-causing pathogen in US children aged

BACKGROUND Enterovirus D68 (EV-D68) has been sporadically reported as a cause of respiratory tract infections. In 2014, an international outbreak of EV-D68 occurred and caused severe respiratory disease in the pediatric population. METHODS A retrospective chart review was performed of children admitted to Children's Mercy Hospital from August 1, 2014, to September 15, 2014, with positive multiplex polymerase chain reaction testing for EV/rhinovirus (RV). Specimens were subsequently tested for EV-D68, and clinical data were obtained from the medical records. Patients with EV-D68 were compared with children presenting simultaneously with other EV/RV. RESULTS Of 542 eligible specimens, children with EV-D68 were significantly older than children with other EV/RV (4.6 vs. 2.2 years, P < 0.001). Children with EV-D68 were more likely to have a history of asthma (38.6% vs. 30.0%, P = 0.04) or recurrent wheezing (22.1% vs. 14.8%, P = 0.04). EV-D68-positive children more commonly received supplemental oxygen (86.7% vs. 65.0%, P < 0.001), albuterol (91.2% vs. 65.5%, P < 0.001) and corticosteroids (82.9% vs. 58.6%, P < 0.001). Age ≥5 years was an independent risk factor for intensive care unit management in EV-D68-infected children. Children with a history of asthma or recurrent wheezing and EV-D68 received supplemental oxygen (92.7% vs. 82.4%, P = 0.007) and magnesium (42.7% vs. 29.7%, P = 0.03) at higher rates and more continuous albuterol (3 vs. 2 hours, P = 0.03) than those with other EV/RV. CONCLUSIONS EV-D68 causes severe disease in the pediatric population, particularly in children with a history of asthma or recurrent wheezing. EV-D68-positive children are more likely to require therapy for refractory bronchospasm and may need intensive care unit- level care.

Background Highly multiplexed molecular assays are popular in clinical laboratories due their high sensitivity, specificity and relatively rapid turn-around time (TAT) for results. Luminex™ respiratory viral panel (RVP) detects 12 respiratory viruses, while BioFire™ respiratory panel (RP) detects 20 respiratory pathogens (17 viruses, 3 bacteria). The aim of the current study was to compare the impact of RVP and RP assay on management of hospitalized children aged ≤24 months. Methods Retrospective data were collected to compare the clinical impact from two multiplex molecular assays (RVP, December 2008–May 2012; RP August 2012–June 2015) on management and outcomes of hospitalized patients. Patients aged ≤24 months and positive for at least one respiratory virus were included. Patients who were (1) receiving immune suppressive therapy, (2) neonates requiring intensive care, or (3) hospitalized for >7 days were excluded. Results A total of 810 patients in RVP and 2,095 patients in RP group were included. The median TAT for RVP and RP assay were 29 hours (IQR 26–58 hours) and 4 hours (IQR 2–8 hours), respectively (P Conclusion Rapid availability of test results from RP assay was associated with reduced antibiotic use, timely antiviral therapy and decreased LOH. The implementation of a more comprehensive respiratory multiplex molecular assay with rapid reporting of test results has the potential to improve management of hospitalized children, decrease unnecessary antibiotic therapy and reduce overall costs. Disclosures R. Selvarangan, BioFire Diagnostics: Board Member and Investigator, Consulting fee and Research grant; Luminex Diagnostics: Investigator, Research grant

Background There are many FDA cleared multiplexed respiratory assays available in the United States, ranging from 3 to 20 targets per assay. FilmArray Respiratory Panel (RP) is one of the mega-multiplexed assay, includes 20 targets and results are available in 1 hour. Recently Hologic has received FDA-clearance for several smaller respiratory “Panther Fusion assays” (3–4 targets/assay) and results are available in

Background Urinalysis (UA) has been routinely used as a screening tool prior to microbial culture set-up in many laboratories. BacterioScan 216Dx instrument utilizes laser light scattering technology to detect bacterial growth in urine and results are available in 3 hours. The aim of this study was to compare the performance of 216 DX and UA against culture as gold standard. Methods Clean-catch, unpreserved, either UA positive (leukocyte esterase > trace, or nitrite positive or white blood cells >5/hpf) or UA negative samples from children aged ≤18 years were tested by 216Dx within 24 hours of sample collection. “ Likely positive” samples by 216Dx were tested by MALDI-TOF for direct bacterial identification. Sensitivity and specificity of 216Dx and UA was determined against urine culture. Results Total of 205 urine samples were included in this study, of which 48.0% (98/205) and 52.0% (107/205) were UA positive and negative, respectively. 77.0% of samples were collected from female and median age was 108 months. Overall sensitivity, specificity, positive, and negative predictive value (PPV and NPV) of 216Dx and UA are shown in table below. Of 27 true positive (TP) samples by 216Dx, 77.0% (21/27) were successfully identified by MALDI-TOF. There were a total of 96 samples identified as contamination/normal flora by culture. Among these, 63 samples (65.0%) were detected as true negative (TN) by 216Dx vs. 50 samples (53.1%) as TN by UA. Two false negative (FN) samples by 216Dx and one FN by UA were K. oxytoca, S. epidermidis (both >100 K cfu/ml) and E. coli (>100 K cfu/mL), respectively. Conclusion Although sensitivity of both 216Dx and UA is comparable, specificity of 216Dx was higher than UA. Also, 216Dx showed better performance in detecting urine contamination, thus reducing laboratory reagent and labor cost. Faster turn-around-time of 216Dx coupled with rapid identification of uropathogen by MALDI-TOF has the potential to reduce unnecessary antibiotic use, improve patient management and reduce overall healthcare-related cost. Assay TP FP TN FN %Sensitivity (95% CI) %Specificity (95% CI) %PPV (95% CI) %NPV (95% CI) 216Dx 27 36 140 2 93.1 (75.7–98.8) 79.5 (72.6.0-85.1) 42.8 (30.6–55.9) 98.6 (94.4–99.7) UA 28 70 106 1 96.5 (80.3–99.8) 60.2 (52.5–67.4) 28.5 (20.1–38.7) 99.0 (94.1–99.9) Disclosures All authors: No reported disclosures.

Summary U18666A is a cholesterol transport-inhibiting agent that is used widely to mimic Niemann–Pick type C disease. The effect of U18666A on tumour necrosis factor (TNF)-α production in mouse macrophage cell line, RAW 264·7 cells and peritoneal macrophages was examined. U18666A induced TNF-α mRNA expression 48 h after the treatment, and TNF-α production 48 and 72 h after stimulation in RAW 264·7 cells. U18666A accumulated intracellular free cholesterol in the culture of normal medium but not cholesterol-free medium. U18666A also induced reactive oxygen species (ROS) generation in normal medium but much less in cholesterol-free medium. Anti-oxidant N-acetyl-L-cysteine (NAC) abolished U18666A-induced TNF-α production. U18666A led to the phosphorylation of p38 mitogen-activated protein kinase 24 and 48 h after the stimulation and the p38 activation was inhibited in presence of cholesterol-free medium or NAC. A p38 inhibitor reduced U18666A-induced TNF-α production. Taken together, U18666A was suggested to induce TNF-α production in RAW 264·7 cells via free cholesterol accumulation-mediated ROS generation.

The effect of toll-like receptor (TLR) 7 ligand pretreatment on the production of tumor necrosis factor (TNF)-α in response to TLR7 or TLR2 ligand was examined in order to establish a new TLR-mediated tolerance. RAW 264.7 macrophage-like cells were treated with imiquimod R837 as a TLR7 ligand for 18 h, washed and incubated in fresh culture medium 6 h. The second challenge with imiquimod R837 as a TLR7 ligand or Pam3CysSK4 as a TLR2 ligand resulted in reduced TNF-α production in TLR7 ligand-pretreated cells. There was impaired activation of NF-κB, p38 and stress-activated protein kinase (SAPK) in the tolerant cells. The expression of IRAK-M as a negative regulator of TLR signaling was markedly augmented in the tolerant cells while the interleukin-1 receptor-associated kinase (IRAK)-1 functioned normally. The involvement of IRAK-M in the TLR7-mediated tolerance is discussed.

The mechanism of interleukin (IL)-10-mediated inhibition of tumor necrosis factor (TNF)-α production was studied by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. IL-10 inhibited TNF-α production transiently at an early stage after LPS stimulation. IL-10 inhibited the activation of nuclear factor (NF)-κB, p38 and stress-activated protein kinase (SAPK) in LPS-stimulated RAW 264.7 cells. Although the level of MyD88 protein increased in response to LPS, IL-10 prevented the LPS-induced MyD88 augmentation. There was no significant difference in the MyD88 mRNA expression between the cells pretreated with or without IL-10 in response to LPS. Therefore, IL-10 was suggested to inhibit LPS-induced TNF-α production via reduced MyD88 expression.

The mechanism underlying acute lung injury in lethal endotoxic shock induced by administration of lipopolysaccharide (LPS) into alpha-galactosylceramide (alpha-GalCer)-sensitized mice was studied. Sensitization with alpha-GalCer resulted in the increase of natural killer T (NK T) cells and the production of interferon (IFN)-gamma in the lung. The IFN-gamma that was produced induced expression of adhesion molecules, especially vascular cell adhesion molecule-1 (VCAM-1), on vascular endothelial cells in the lung. Anti-IFN-gamma antibody inhibited significantly the VCAM-1 expression in alpha-GalCer-sensitized mice. Very late activating antigen-4-positive cells, as the counterpart of VCAM-1, accumulated in the lung. Anti-VCAM-1 antibody prevented LPS-mediated lethal shock in alpha-GalCer-sensitized mice. The administration of LPS into alpha-GalCer-sensitized mice caused local production of excessive proinflammatory mediators, such as tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and nitric oxide. LPS caused microvascular leakage of proteins and cells into bronchoalveolar lavage fluid. Taken together, sensitization with alpha-GalCer was suggested to induce the expression of VCAM-1 via IFN-gamma produced by NK T cells and recruit a number of inflammatory cells into the lung. Further, LPS was suggested to lead to the production of excessive proinflammatory mediators, the elevation of pulmonary permeability and cell death. The putative mechanism of acute lung injury in LPS-mediated lethal shock using alpha-GalCer sensitization is discussed.

Summary The effect of interferon (IFN)-γ and/or lipopolysaccharide (LPS) on Fas-mediated cell death with anti-Fas agonistic antibody in vascular endothelial cells was examined using a mouse END-D cell line. Anti-Fas agonistic antibody exhibited cytotoxic actions on END-D cells. Fas-mediated cell death was enhanced by LPS or IFN-γ. The combination of IFN-γ and LPS significantly enhanced cell death compared to IFN-γ or LPS alone. IFN-γ and LPS augmented cell surface expression of Fas, but not tumour necrosis factor (TNF) receptor 1. Inhibitors of p38 mitogen-activated protein kinase (MAPK) prevented augmentation of Fas expression in IFN-γ and LPS-treated END-D cells. IFN-γ and LPS-treated END-D cells did not become susceptible to TNF-α or nitric oxide-mediated cytotoxicity. IFN-γ and LPS thus appear to augment selectively Fas expression via activation of p38 MAPK and enhance Fas-mediated cell death in END-D cells. Furthermore, administration of IFN-γ and LPS into mice induced in vivo expression of Fas on vascular endothelial cells and Fas ligand (FasL) on peripheral blood leucocytes. The relationship between enhancement of Fas-mediated cell death by IFN-γ and LPS and the development of vascular endothelial injury is discussed.

Lipopolysaccharide (LPS) enhances the production of nitric oxide (NO) in interferon (IFN)-gamma-stimulated vascular endothelial cells. We studied the mechanism by which LPS enhances IFN-gamma-induced NO production by using the murine vascular endothelial cell line, END-D. LPS enhanced IFN-gamma-induced NO production via augmented expression of inducible type NO synthase (iNOS) mRNA. LPS significantly augmented the activation of interferon regulatory factor (IRF)-1 in IFN-gamma-stimulated END-D cells, although it did not affect the activation of either MyD88-dependent nuclear factor (NF)-kappaB or MyD88-independent IRF-3. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), prevented the nuclear translocation of IRF-1 in LPS and IFN-gamma-stimulated END-D cells, and inhibited the iNOS expression and NO production in those cells. Therefore, it is proposed that LPS enhanced NO production in IFN-gamma-stimulated END-D cells via augmenting p38 MAPKmediated IRF-1 activation.

Antioxidants are able to inhibit inflammatory gene expression in response to lipopolysaccharide via down-regulating generation of intracellular reactive oxygen species (ROS) as second messengers. The effect of manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a synthetic metalloporphyrin with antioxidant activity, on tumor necrosis factor (TNF)-alpha production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells was examined. MnTBAP prevented the generation of intracellular ROS in lipopolysaccharide-stimulated RAW 264.7 cells and further inhibited lipopolysaccharide-induced TNF-alpha production. MnTBAP exclusively prevented the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK/JNK) whereas it did not affect the phosphorylation and activation of nuclear factor-kappaB and extracellular signal regulated kinase 1/2. MnTBAP was suggested to inhibit lipopolysaccharide-induced TNF-alpha production by the prevention of intracellular ROS generation and subsequent inactivation of p38 MAPK and SAPK/JNK.

Summary Background Enterovirus D68 (EV-D68) has been infrequently reported historically, and is typically associated with isolated cases or small clusters of respiratory illness. Beginning in August, 2014, increases in severe respiratory illness associated with EV-D68 were reported across the USA. We aimed to describe the clinical, epidemiological, and laboratory features of this outbreak, and to better understand the role of EV-D68 in severe respiratory illness. Methods We collected regional syndromic surveillance data for epidemiological weeks 23 to 44, 2014, (June 1 to Nov 1, 2014) and hospital admissions data for epidemiological weeks 27 to 44, 2014, (June 29 to Nov 1, 2014) from three states: Missouri, Illinois and Colorado. Data were also collected for the same time period of 2013 and 2012. Respiratory specimens from severely ill patients nationwide, who were rhinovirus-positive or enterovirus-positive in hospital testing, were submitted between Aug 1, and Oct 31, 2014, and typed by molecular sequencing. We collected basic clinical and epidemiological characteristics of EV-D68 cases with a standard data collection form submitted with each specimen. We compared patients requiring intensive care with those who did not, and patients requiring ventilator support with those who did not. Mantel-Haenszel χ 2 tests were used to test for statistical significance. Findings Regional and hospital-level data from Missouri, Illinois, and Colorado showed increases in respiratory illness between August and September, 2014, compared with in 2013 and 2012. Nationwide, 699 (46%) of 1529 patients tested were confirmed as EV-D68. Among the 614 EV-D68-positive patients admitted to hospital, age ranged from 3 days to 92 years (median 5 years). Common symptoms included dyspnoea (n=513 [84%]), cough (n=500 [81%]), and wheezing (n=427 [70%]); 294 (48%) patients had fever. 338 [59%] of 574 were admitted to intensive care units, and 145 (28%) of 511 received ventilator support; 322 (52%) of 614 had a history of asthma or reactive airway disease; 200 (66%) of 304 patients with a history of asthma or reactive airway disease required intensive care compared with 138 (51%) of 270 with no history of asthma or reactive airway disease (p=0·0004). Similarly, 89 (32%) of 276 patients with a history of asthma or reactive airway disease required ventilator support compared with 56 (24%) of 235 patients with no history of asthma or reactive airway disease (p=0·039). Interpretation In 2014, EV-D68 caused widespread severe respiratory illness across the USA, disproportionately affecting those with asthma. This unexpected event underscores the need for robust surveillance of enterovirus types, enabling improved understanding of virus circulation and disease burden. Funding None.

In the letter [“New rapid diagnostic tests: a real improvement for clinical use?”][1] by Fernandez-Santos et al. ([1][2]), concern is raised regarding use of rapid influenza detection tests (RIDTs) for detection of influenza A and B viruses in patients due to their varied sensitivities. A

Previously, we found that mouse TH2.52 cells possess the characteristic of CD5(+) B1 cells and proliferate in response to lipopolysaccharide (LPS). The effect of LPS on cytokine production by TH2.52 B1 cells was studied. TH2.52 cells constitutively produced a small amount of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and TNF-alpha and IL-6 production was markedly enhanced by LPS stimulation. Although interferon (IFN)-gamma caused the production of various cytokines, such as IL-2, IL-4, IL-6 and TNF-alpha in TH2.52 cells, LPS did not cause the production of such cytokines. LPS did not induce IFN-beta production in TH2.52 cells and TH2.52 cells lacked the expression of several molecules participating in the MyD88-independent pathway in LPS signaling. Defective responsiveness of TH2.52 B1 cells to LPS in cytokine production might be responsible for the failure of IFN-beta production due to the lack of molecules participating in the MyD88-independent pathway.

The effect of 5-fluorouracil (5-FU) on the production of nitric oxide (NO) in macrophages was examined by using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. 5-FU at non-toxic concentrations significantly inhibited NO production in LPS-stimulated RAW 264.7 cells. The inhibition by 5-FU was mediated by attenuated expression of an inducible NO synthase protein and mRNA. 5-FU inhibited the activation of nuclear factor (NF)-kappaB and the subsequent nuclear translocation. Furthermore, 5-FU inhibited the phosphorylation of Akt, an upstream molecule of NF-kappaB signaling. 5-FU did not affect a series of mitogen-activated protein kinases. Therefore, 5-FU was suggested to inhibit the LPS-induced NO production in activated macrophages through preventing Akt-dependent NF-kappaB activation.

The effect of lipopolysaccharide (LPS) on the cell death induced by endoplasmic reticulum (ER) stress agents in RAW 264.7 cells was studied. LPS prevented the cell death by brefeldin A, but not thapsigargin and tunicamycin. CpG DNA as well as LPS prevented brefeldin A-induced cell death whereas tumor necrosis factor-alpha or interferon-gamma did not. Brefeldin A-induced cell death was mediated with apoptotic cell death and it was significantly inhibited by LPS. LPS abolished the activation of ER stress-related caspases, such as caspases 1, 3, and 4. LPS prevented brefeldin A-induced morphological changes in RAW 264.7 cells. Further, LPS prevented brefeldin A-induced Golgi dispersion. Therefore, LPS was suggested to diminish the stress of ER/Golgi complexes induced by brefeldin A and inhibit apoptosis. The preventive action of LPS on brefeldin A-induced apoptosis is discussed.

The effect of alpha-galactosylceramide (alpha-GalCer) on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal cells was studied. alpha-GalCer augmented LPS-induced NO production in mouse peritoneal cells, but not in RAW 264.7 macrophage cells. alpha-GalCer augmented NO production, but not tumor necrosis factor (TNF)-alpha production in LPS-stimulated peritoneal cells. Peritoneal cells produced a significant level of interferon (IFN)-gamma in response to alpha-GalCer and anti-IFN-gamma antibody abolished the augmentation of LPS-induced NO production by alpha-GalCer. Moreover, anti-IFN-gamma antibody prevented the enhanced expression of an inducible type of NO synthase mRNA by alpha-GalCer. alpha-GalCer did not augment LPS-induced NO production in peritoneal cells from natural killer T (NKT)-deficient mice. Therefore, it was suggested that alpha-GalCer might augment LPS-induced NO production in peritoneal cells through release of IFN-gamma from NKT cells.

The effect of lipopolysaccharide on doxorubicin-induced cell death was studied by using mouse RAW 264.7 macrophage cells. Pretreatment with lipopolysaccharide at 10 ng/mL prevented doxorubicin-induced cell death and the inhibition was roughly dependent on the concentration of lipopolysaccharide. Posttreatment with lipopolysaccharide for 1 hour also prevented doxorubicin-induced cell death. Lipopolysaccharide inhibited DNA fragmentation and caspase-3 activation in doxorubicin-treated RAW 264.7 cells, suggesting the prevention of doxorubicin-induced apoptosis. Lipopolysaccharide did not significantly inhibit doxorubicin-induced DNA damage detected by single-cell gel electrophoresis (comet) assay. Lipopolysaccharide definitely inhibited the stabilization and nuclear translocation of p53 in doxorubicin-treated RAW 264.7 cells. Lipopolysaccharide, as well as being an inhibitor of p53, abolished doxorubicin-induced apoptosis. Therefore, p53 was suggested to play a pivotal role in the prevention of doxorubicin-induced apoptosis in RAW 264.7 cells by lipopolysaccharide.

BACKGROUND Recent evidence suggests that the vitamin A equivalency of beta-carotene from plant sources is lower than previously estimated. OBJECTIVE We assessed the effect of 60 d of daily supplementation with 750 microg retinol equivalents (RE) of either cooked, pureed sweet potatoes; cooked, pureed Indian spinach (Basella alba); or synthetic sources of vitamin A or beta-carotene on total-body vitamin A stores in Bangladeshi men. DESIGN Total-body vitamin A stores in Bangladeshi men (n = 14/group) were estimated by using the deuterated-retinol-dilution technique before and after 60 d of supplementation with either 0 microg RE/d (white vegetables) or 750 microg RE/d as sweet potatoes, Indian spinach, retinyl palmitate, or beta-carotene (RE = 1 microg retinol or 6 microg beta-carotene) in addition to a low-vitamin A diet providing approximately 200 microg RE/d. Mean changes in vitamin A stores in the vegetable and beta-carotene groups were compared with the mean change in the retinyl palmitate group to estimate the relative equivalency of these vitamin A sources. RESULTS Overall geometric mean (+/-SD) initial vitamin A stores were 0.108 +/- 0.067 mmol. Relative to the low-vitamin A control group, the estimated mean changes in vitamin A stores were 0.029 mmol for sweet potato (P = 0.21), 0.041 mmol for Indian spinach (P = 0.033), 0.065 mmol for retinyl palmitate (P < 0.001), and 0.062 mmol for beta-carotene (P < 0.002). Vitamin A equivalency factors (beta-carotene:retinol, wt:wt) were estimated as approximately 13:1 for sweet potato, approximately 10:1 for Indian spinach, and approximately 6:1 for synthetic beta-carotene. CONCLUSION Daily consumption of cooked, pureed green leafy vegetables or sweet potatoes has a positive effect on vitamin A stores in populations at risk of vitamin A deficiency.

The role of p38 mitogen-activated protein kinase (MAPK) on vacuole formation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was examined. LPS definitely induced the formation of vacuoles in RAW 264.7 cells and SB202190 as a p38 specific inhibitor also induced slight vacuole formation. The simultaneous treatment with LPS and SB202190 induced many more vacuoles in RAW 264.7 cells than the treatment with LPS or SB202190 alone, and the vacuoles were extraordinarily large in size. On the other hand, an inactive inhibitor of p38 MAPK did not augment LPS-induced vacuole formation. Further, the inhibitors of other MAPKs and nuclear factor (NF)-kappaB pathways did not affect it. The extraordinarily large vacuoles in RAW 264.7 cells treated with LPS and SB202190 were possibly formed via fusion of small vacuoles. However, SB202190 did not augment vacuole formation in CpG DNA or interferon (IFN)-gamma-stimulated RAW 264.7 cells. The role of p38 MAPK in the vacuole formation in LPS-stimulated macrophages is discussed.

The effect of piceatannol on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was examined. Piceatannol significantly inhibited NO production in LPS-stimulated RAW 264.7 cells. The inhibition was due to the reduced expression of an inducible isoform of NO synthase (iNOS). The inhibitory effect of piceatannol was mediated by down-regulation of LPS-induced nuclear factor (NF)-kappaB activation, but not by its cytotoxic action. Piceatannol inhibited IkappaB kinase (IKK)-alpha and beta phosphorylation, and subsequently IkappaB-alpha phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, piceatannol did not affect activation of mitogen-activated protein (MAP) kinases including extracellular signal regulated kinase 1/2 (Erk1/2), p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Piceatannol inhibited the phosphorylation of Akt and Raf-1 molecules, which regulated the activation of IKK-alpha and beta phosphorylation. The detailed mechanism of the inhibition of LPS-induced NO production by piceatannol is discussed.

The level of circulating endotoxin was determined in the sera from children hospitalized for treatment of diarrhea at the International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh. A significant level of endotoxin was detected in the sera from 23 (54%) of 42 children. On the other hand, in 32 normal controls, endotoxin was below the limits of detection of the assay. Antibiotic and fluid therapy markedly reduced the level of serum endotoxin and improved the general condition of most patients. Non-survivors ( n = 5) had higher levels of circulating endotoxin before treatment than survivors ( n = 37), suggesting a significant correlation between the serum endotoxin level before treatment and mortality. Malnutrition did not affect the serum endotoxin level in the patients with diarrhea. It was suggested that infection of Gram-negative bacteria might be involved in a significant number of patients with diarrhea in Bangladesh and that endotoxin might play a pathogenic role in those patients.

Twenty-one atypicalShigella flexneritype 4 strains isolated from patients attending the Dhaka treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh, were extensively characterized and compared withS. flexneriserotypes 4a and 4b. The atypical strains agglutinated only with the type antigen factor 4 and did not agglutinate with any group factors, thereby excluding their characterization into serotype 4a or 4b. Of the 21 strains, 85.7% did not ferment mannitol but were able to ferment most of the sugars, whereas the remaining 14.3% strains fermented mannitol but were unable to ferment most of the sugars. Most of the strains were resistant to ampicillin, tetracycline, and trimethoprim-sulfomethoxazole. All of the strains harbored the 140-MDa plasmid, had theipaHgene, had thesengene (encodingShigellaenterotoxin 2), had the ability to bind Congo red, and were positive for keratoconjunctivitis in the guinea pig eye, attesting their invasive properties. All of the strains contained a middle-range plasmid (35 to 62 MDa) as well as a number of stable small plasmids, yielding mainly two plasmid profiles which were different from those of 4a and 4b strains. Conjugation and curing experiments suggested that the middle-range plasmids harbored a self-transferable multiple antibiotic resistance marker. Pulsed-field gel electrophoresis analysis of all of the tested strains yielded two types with numerous subtypes, whereas ribotyping yielded only two types which were completely different from those of types 4a and 4b. This study concluded that two different clones of atypicalS. flexneritype 4 exist and strongly suggests that these are new subserotypes ofS. flexnerithat await further serological classification.