Last data update: Jan 27, 2025. (Total: 48650 publications since 2009)
Records 1-30 (of 101 Records) |
Query Trace: Winchell J[original query] |
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Broth microdilution protocol for determining antimicrobial susceptibility of Legionella pneumophila to clinically relevant antimicrobials
Sewell M , Farley C , Portal EAR , Lindsay D , Ricci ML , Jarraud S , Scaturro M , Descours G , Krøvel AV , Barton R , Boostom I , Ure R , Kese D , Gaia V , Golob M , Paukner S , Ginevra C , Afshar B , Nadarajah S , Wybo I , Michel C , Echahdi F , González-Rubio JM , González-Camacho F , Mentasti M , Flountzi AS , Petzold M , Moran-Gilad J , Uldum S , Winchell J , Wooton M , Bernard K , Jones LC , Chalker VJ , Spiller OB . J Microbiol Methods 2024 228 107071 ![]() Currently there is no detailed, internationally agreed protocol defined to evaluate antimicrobial susceptibility testing (AST) for Legionella pneumophila (required to establish epidemiological cut-off value or "ECOFF" boundaries); therefore, antimicrobial resistance in these isolates cannot be defined. AST methods utilising media containing activated charcoal as an ingredient, to enable Legionella growth, are unreliable as noted in an internationally authored opinion paper and a new gold standard is required. Here we define a detailed protocol for broth microdilution (BMD) using defined cell culture collection-deposited control reference strains (Philadelphia-1 and Knoxville-1) as well as two accessible reference strains with moderately (lpeAB-carrying) and markedly (23S rRNA mutation-carrying) elevated azithromycin minimum inhibitory concentration (MIC). The defined protocol enables up to eight L. pneumophila strains to be set up on a single 96-well plate per antimicrobial tested. Initial ranges to routinely capture an MIC for these reference strains using clinically relevant antimicrobials azithromycin (0.01-0.25 mg/L), levofloxacin (0.008-0.03 mg/L), lefamulin (0.01-2 mg/L), rifampicin (0.0002-0.0008 mg/L) and doxycycline (0.25-16 mg/L) following incubation for 48 h at 37 °C in a shaking incubator have been empirically determined. Establishment of this internationally agreed protocol sets the scene for the next step: validation and comparison of antimicrobial ranges between international Legionella reference laboratories to establish putative resistance cut-off thresholds for these clinically relevant antimicrobials. |
Clinical and epidemiologic features of mycoplasma pneumoniae infection among adults hospitalized with community-acquired pneumonia
Kutty PK , Jain S , Diaz MH , Self WH , Williams D , Zhu Y , Grijalva CG , Edwards KM , Wunderink RG , Winchell J , Hicks LA . Int J Med Sci 2024 21 (15) 3003-3009 ![]() Background/Purpose: The burden and epidemiology of Mycoplasma pneumoniae (Mp) community-acquired pneumonia (CAP) among hospitalized U. S. adults (≥ 18 years) are poorly understood. Methods: In the Etiology of Pneumonia in the Community (EPIC) study, we prospectively enrolled 2272 adults hospitalized with radiographically-confirmed pneumonia between January 2010-June 2012 and tested nasopharyngeal/oropharyngeal swabs for Mp by real-time polymerase chain reaction (PCR). Clinical and epidemiological features of Mp-PCR-positive and -negative adults were compared using logistic regression. Macrolide susceptibility was assessed by genotyping isolates. Results: Among 2272 adults, 43 (1.8%) were Mp-PCR-positive (median age: 45 years); 52% were male, and 56% were non-Hispanic white. Only one patient had Mp macrolide resistance. Four (9%) were admitted to the intensive care unit (ICU). No in-hospital deaths were reported. Of the 9 (21%) who received an outpatient antibiotic ≤5 days pre-admission, 2 (22%) received an antibiotic with Mp activity. Variables significantly associated with higher odds of Mp detection included age {18-29 years [(adjusted odds ratio (aOR): 11.7 (95% confidence interval (CI): 5.1- 26.6) versus ≥50 years]} and radiographic lymphadenopathy [aOR: 3.5 (95% CI: 1.2- 9.3)]. Conclusions: M. pneumoniae, commonly known to cause "walking pneumonia", was detected among hospitalized adults, with the highest prevalence among young adults. Although associated with clinically non-specific symptoms, approximately one out of every ten patients were admitted to the ICU. Increasing access to M. pneumoniae point-of-care testing could facilitate targeted treatment and avoid hospitalization. |
Expanded geographic distribution for two Legionella pneumophila sequence types of clinical concern
Hamlin JAP , Kozak-Muiznieks NA , Mercante JW , Rishishwar L , Norris ET , Gaines AB , Ishaq MK , Winchell JM , Willby MJ . mSphere 2024 e0075623 ![]() ![]() Legionella pneumophila serogroup 1 sequence types (ST) 213 and 222, a single-locus variant of ST213, were first detected in the early 1990s in the Midwest United States (U.S.) and the late 1990s in the Northeast U.S. and Canada. Since 1992, these STs have increasingly been implicated in community-acquired sporadic and outbreak-associated Legionnaires' disease (LD) cases. We were interested in understanding the change in LD frequency due to these STs and identifying genetic features that differentiate these STs from one another. For the geographic area examined here (Mountain West to Northeast) and over the study period (1992-2020), ST213/222-associated LD cases identified by the Centers for Disease Control and Prevention increased by 0.15 cases per year, with ST213/222-associated LD cases concentrated in four states: Michigan (26%), New York (18%), Minnesota (16%), and Ohio (10%). Additionally, between 2002 and 2021, ST222 caused at least five LD outbreaks in the U.S.; no known outbreaks due to ST213 occurred in the U.S. during this time. We compared the genomes of 230 ST213/222 isolates and found that the mean of the average nucleotide identity (ANI) within each ST was high (99.92% for ST222 and 99.92% for ST213), with a minimum between ST ANI of 99.50% and a maximum of 99.87%, indicating low genetic diversity within and between these STs. While genomic features were identified (e.g., plasmids and CRISPR-Cas systems), no association explained the increasing geographic distribution and prevalence of ST213 and ST222. Yet, we provide evidence of the expanded geographical distribution of ST213 and ST222 in the U.S.IMPORTANCESince the 1990s, cases of Legionnaires' disease (LD) attributed to a pair of closely related Legionella pneumophila variants, ST213 and ST222, have increased in the U.S. Furthermore, between 2002 and 2021, ST222 caused at least five outbreaks of LD in the U.S., while ST213 has not been linked to any U.S. outbreak. We wanted to understand how the rate of LD cases attributed to these variants has changed over time and compare the genetic features of the two variants. Between 1992 and 2020, we determined an increase of 0.15 LD cases ascribed to ST213/222 per year in the geographic region studied. Our research shows that these STs are spreading within the U.S., yet most of the cases occurred in four states: Michigan, New York, Minnesota, and Ohio. Additionally, we found little genetic diversity within and between these STs nor could specific genetic features explain their geographic spread. |
Genomic analysis of Chlamydia psittaci from a multistate zoonotic outbreak in two chicken processing plants
Wolff BJ , Waller JL , Benitez AJ , Gaines A , Conley AB , Rishishwar L , Chande AT , Morrison SS , Jordan IK , Diaz MH , Winchell JM . J Genomics 2023 11 40-44 ![]() ![]() Four Chlamydia psittaci isolates were recovered from clinical specimens from ill workers during a multistate outbreak at two chicken processing plants. Whole genome sequencing analyses revealed high similarity to C. psittaci genotype D. The isolates differed from each other by only two single nucleotide polymorphisms, indicating a common source. |
Multiplex Real-time PCR Assay for the Detection of all Chlamydia Species and Simultaneous Differentiation of C. psittaci and C. pneumoniae in Human Clinical Specimens.
Wolff BJ , Gaines A , Conley AB , Norris E , Rishishwar L , Chande AT , Yang E , Diaz MH , Winchell JM . Ann Lab Med 2023 43 (4) 375-380 ![]() ![]() We developed and assessed the performance of a new multiplex real-time PCR assay for the detection of all Chlamydia species and simultaneous differentiation of Chlamydia psittaci and Chlamydia pneumoniae-two important human respiratory pathogens-in human clinical specimens. Next-generation sequencing was used to identify unique targets to design real-time PCR assays targeting all Chlamydia species, C. psittaci, and C. pneumoniae. To validate the assay, we used a panel of 49 culture isolates comprising seven C. psittaci genotypes, eight C. pneumoniae isolates, seven other Chlamydia species, and 22 near-neighbor bacterial and viral isolates, along with 22 specimens from external quality assessment (EQA) panels and 34 nasopharyngeal and oropharyngeal swabs and cerebrospinal fluid, stool, and sputum specimens previously identified as positive or negative for C. psittaci or C. pneumoniae. The assays were 100% specific, with limits of detection of 7.64- 9.02 fg/μL. The assay results matched with historical assay results for all specimens, except for one owing to the increased sensitivity of the new C. psittaci assay; the results of the EQA specimens were 100% accurate. This assay may improve the timely and accurate clinical diagnosis of Chlamydia infections and provide a greater understanding of the burden of disease caused by these agents. |
Risk factors for community-acquired bacterial infection among young infants in South Asia: a longitudinal cohort study with nested case-control analysis
Connor NE , Islam MS , Mullany LC , Shang N , Bhutta ZA , Zaidi AKM , Soofi S , Nisar I , Panigrahi P , Panigrahi K , Satpathy R , Bose A , Isaac R , Baqui AH , Mitra DK , Sadeq-Ur Rahman Q , Hossain T , Schrag SJ , Winchell JM , Arvay ML , Diaz MH , Waller JL , Weber MW , Hamer DH , Hibberd P , Nawshad Uddin Ahmed ASM , Islam M , Hossain MB , Qazi SA , El Arifeen S , Darmstadt GL , Saha SK . BMJ Glob Health 2022 7 (11) ![]() OBJECTIVE: Risk factors predisposing infants to community-acquired bacterial infections during the first 2 months of life are poorly understood in South Asia. Identifying risk factors for infection could lead to improved preventive measures and antibiotic stewardship. METHODS: Five sites in Bangladesh, India and Pakistan enrolled mother-child pairs via population-based pregnancy surveillance by community health workers. Medical, sociodemographic and epidemiological risk factor data were collected. Young infants aged 0-59 days with signs of possible serious bacterial infection (pSBI) and age-matched controls provided blood and respiratory specimens that were analysed by blood culture and real-time PCR. These tests were used to build a Bayesian partial latent class model (PLCM) capable of attributing the probable cause of each infant's infection in the ANISA study. The collected risk factors from all mother-child pairs were classified and analysed against the PLCM using bivariate and stepwise logistic multivariable regression modelling to determine risk factors of probable bacterial infection. RESULTS: Among 63 114 infants born, 14 655 were assessed and 6022 had signs of pSBI; of these, 81% (4859) provided blood samples for culture, 71% (4216) provided blood samples for quantitative PCR (qPCR) and 86% (5209) provided respiratory qPCR samples. Risk factors associated with bacterial-attributed infections included: low (relative risk (RR) 1.73, 95% credible interval (CrI) 1.42 to 2.11) and very low birth weight (RR 5.77, 95% CrI 3.73 to 8.94), male sex (RR 1.27, 95% CrI 1.07 to 1.52), breathing problems at birth (RR 2.50, 95% CrI 1.96 to 3.18), premature rupture of membranes (PROMs) (RR 1.27, 95% CrI 1.03 to 1.58) and being in the lowest three socioeconomic status quintiles (first RR 1.52, 95% CrI 1.07 to 2.16; second RR 1.41, 95% CrI 1.00 to 1.97; third RR 1.42, 95% CrI 1.01 to 1.99). CONCLUSION: Distinct risk factors: birth weight, male sex, breathing problems at birth and PROM were significantly associated with the development of bacterial sepsis across South Asian community settings, supporting refined clinical discernment and targeted use of antimicrobials. |
Histopathology Is Key to Interpreting Multiplex Molecular Test Results From Postmortem Minimally Invasive Tissue Samples
Ritter JM , Seixas JN , Walong E , Dawa J , Onyango C , Pimenta FC , da Gloria Carvalho M , Silva-Flannery L , Jenkinson T , Howard K , Bhatnagar J , Diaz M , Winchell JM , Zaki SR , Chaves SS , Martines RB . Clin Infect Dis 2021 73 S351-s359 BACKGROUND: Minimally invasive tissue sampling (MITS) is an alternative to complete autopsy for determining causes of death. Multiplex molecular testing performed on MITS specimens poses challenges of interpretation, due to high sensitivity and indiscriminate detection of pathogenic, commensal, or contaminating microorganisms. METHODS: MITS was performed on 20 deceased children with respiratory illness, at 10 timepoints up to 88 hours postmortem. Samples were evaluated by multiplex molecular testing on fresh tissues by TaqMan® Array Card (TAC) and by histopathology, special stains, immunohistochemistry (IHC), and molecular testing (PCR) on formalin-fixed, paraffin-embedded (FFPE) tissues. Results were correlated to determine overall pathologic and etiologic diagnoses and to guide interpretation of TAC results. RESULTS: MITS specimens collected up to 3 days postmortem were adequate for histopathologic evaluation and testing. Seven different etiologic agents were detected by TAC in 10 cases. Three cases had etiologic agents detected by FFPE or other methods and not TAC; 2 were agents not present on TAC, and 2 were streptococci that may have been species other than those present on TAC. Result agreement was 43% for TAC and IHC or PCR, and 69% for IHC and PCR. Extraneous TAC results were common, especially when aspiration was present. CONCLUSIONS: TAC can be performed on MITS up to 3 days after death with refrigeration and provides a sensitive method for detection of pathogens but requires careful interpretation in the context of clinicoepidemiologic and histopathologic findings. Interpretation of all diagnostic tests in aggregate to establish overall case diagnoses maximizes the utility of TAC in MITS. |
Effect of Time Since Death on Multipathogen Molecular Test Results of Postmortem Specimens Collected Using Minimally Invasive Tissue Sampling Techniques.
Dawa J , Walong E , Onyango C , Mathaiya J , Muturi P , Bunei M , Ochieng W , Barake W , Seixas JN , Mayieka L , Ochieng M , Omballa V , Lidechi S , Hunsperger E , Otieno NA , Ritter JM , Widdowson MA , Diaz MH , Winchell JM , Martines RB , Zaki SR , Chaves SS . Clin Infect Dis 2021 73 S360-s367 ![]() ![]() BACKGROUND: We used postmortem minimally invasive tissue sampling (MITS) to assess the effect of time since death on molecular detection of pathogens among respiratory illness-associated deaths. METHODS: Samples were collected from 20 deceased children (aged 1-59 months) hospitalized with respiratory illness from May 2018 through February 2019. Serial lung and/or liver and blood samples were collected using MITS starting soon after death and every 6 hours thereafter for up to 72 hours. Bodies were stored in the mortuary refrigerator for the duration of the study. All specimens were analyzed using customized multipathogen TaqMan® array cards (TACs). RESULTS: We identified a median of 3 pathogens in each child's lung tissue (range, 1-8; n = 20), 3 pathogens in each child's liver tissue (range, 1-4; n = 5), and 2 pathogens in each child's blood specimen (range, 0-4; n = 5). Pathogens were not consistently detected across all collection time points; there was no association between postmortem interval and the number of pathogens detected (P = .43) and no change in TAC cycle threshold value over time for pathogens detected in lung tissue. Human ribonucleoprotein values indicated that specimens collected were suitable for testing throughout the study period. CONCLUSIONS: Results suggest that lung, liver, and blood specimens can be collected using MITS procedures up to 4 days after death in adequately preserved bodies. However, inconsistent pathogen detection in samples needs careful consideration before drawing definitive conclusions on the etiologic causes of death. |
Multiplex Real-Time Reverse Transcription PCR for Influenza A Virus, Influenza B Virus, and Severe Acute Respiratory Syndrome Coronavirus 2.
Shu B , Kirby MK , Davis WG , Warnes C , Liddell J , Liu J , Wu KH , Hassell N , Benitez AJ , Wilson MM , Keller MW , Rambo-Martin BL , Camara Y , Winter J , Kondor RJ , Zhou B , Spies S , Rose LE , Winchell JM , Limbago BM , Wentworth DE , Barnes JR . Emerg Infect Dis 2021 27 (7) 1821-1830 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019, and the outbreak rapidly evolved into the current coronavirus disease pandemic. SARS-CoV-2 is a respiratory virus that causes symptoms similar to those caused by influenza A and B viruses. On July 2, 2020, the US Food and Drug Administration granted emergency use authorization for in vitro diagnostic use of the Influenza SARS-CoV-2 Multiplex Assay. This assay detects influenza A virus at 10(2.0), influenza B virus at 10(2.2), and SARS-CoV-2 at 10(0.3) 50% tissue culture or egg infectious dose, or as few as 5 RNA copies/reaction. The simultaneous detection and differentiation of these 3 major pathogens increases overall testing capacity, conserves resources, identifies co-infections, and enables efficient surveillance of influenza viruses and SARS-CoV-2. |
Legionella antibiotic susceptibility testing: is it time for international standardization and evidence-based guidance
Portal E , Descours G , Ginevra C , Mentasti M , Afshar B , Chand M , Day J , Echahidi F , Franzin L , Gaia V , Lück C , Meghraoui A , Moran-Gilad J , Ricci ML , Lina G , Uldum S , Winchell J , Howe R , Bernard K , Spiller OB , Chalker VJ , Jarraud S . J Antimicrob Chemother 2021 76 (5) 1113-1116 Legionella pneumophila, a Gram-negative bacillus, is the causative agent of Legionnaire's disease, a form of severe community-acquired pneumonia. Infection can have high morbidity, with a high proportion of patients requiring ICU admission, and up to 10% mortality, which is exacerbated by the lack of efficacy of typical empirical antibiotic therapy against Legionella spp. The fastidious nature of the entire Legionellaceae family historically required inclusion of activated charcoal in the solid medium to remove growth inhibitors, which inherently interferes with accurate antimicrobial susceptibility determination, an acknowledged methodological shortfall, now rectified by a new solid medium that gives results comparable to those of microbroth dilution. Here, as an international Legionella community (with authors representing various international reference laboratories, countries and clinical stakeholders for diagnosis and treatment of legionellosis), we set out recommendations for the standardization of antimicrobial susceptibility testing methods, guidelines and reference strains to facilitate an improved era of antibiotic resistance determination. |
Use of Real-Time PCR for Chlamydia psittaci Detection in Human Specimens During an Outbreak of Psittacosis - Georgia and Virginia, 2018.
McGovern OL , Kobayashi M , Shaw KA , Szablewski C , Gabel J , Holsinger C , Drenzek C , Brennan S , Milucky J , Farrar JL , Wolff BJ , Benitez AJ , Thurman KA , Diaz MH , Winchell JM , Schrag S . MMWR Morb Mortal Wkly Rep 2021 70 (14) 505-509 ![]() ![]() Psittacosis is typically a mild febrile respiratory illness caused by infection with the bacterium Chlamydia psittaci and usually transmitted to humans by infected birds (1). On average, 11 psittacosis cases per year were reported in the United States during 2000-2017. During August-October 2018, the largest U.S. psittacosis outbreak in 30 years (82 cases identified*) occurred in two poultry slaughter plants, one each in Virginia and Georgia, that shared source farms (2). CDC used C. psittaci real-time polymerase chain reaction (PCR) to test 54 human specimens from this outbreak. This was the largest number of human specimens from a single outbreak ever tested for C. psittaci using real-time PCR, which is faster and more sensitive than commercially available serologic tests. This represented a rare opportunity to assess the utility of multiple specimen types for real-time PCR detection of C. psittaci. C. psittaci was detected more frequently in lower respiratory specimens (59% [10 of 17]) and stool (four of five) than in upper respiratory specimens (7% [two of 28]). Among six patients with sputum and nasopharyngeal swabs tested, C. psittaci was detected only in sputum in five patients. Cycle threshold (Ct) values suggested bacterial load was higher in lower respiratory specimens than in nasopharyngeal swabs. These findings support prioritizing lower respiratory specimens for real-time PCR detection of C. psittaci. Stool specimens might also have utility for diagnosis of psittacosis. |
The epidemiology and estimated etiology of pathogens detected from the upper respiratory tract of adults with severe acute respiratory infections in multiple countries, 2014-2015.
Milucky J , Pondo T , Gregory CJ , Iuliano D , Chaves SS , McCracken J , Mansour A , Zhang Y , Aleem MA , Wolff B , Whitaker B , Whistler T , Onyango C , Lopez MR , Liu N , Rahman MZ , Shang N , Winchell J , Chittaganpitch M , Fields B , Maldonado H , Xie Z , Lindstrom S , Sturm-Ramirez K , Montgomery J , Wu KH , Van Beneden CA . PLoS One 2020 15 (10) e0240309 ![]() ![]() ![]() INTRODUCTION: Etiology studies of severe acute respiratory infections (SARI) in adults are limited. We studied potential etiologies of SARI among adults in six countries using multi-pathogen diagnostics. METHODS: We enrolled both adults with SARI (acute respiratory illness onset with fever and cough requiring hospitalization) and asymptomatic adults (adults hospitalized with non-infectious illnesses, non-household members accompanying SARI patients, adults enrolled from outpatient departments, and community members) in each country. Demographics, clinical data, and nasopharyngeal and oropharyngeal specimens were collected from both SARI patients and asymptomatic adults. Specimens were tested for presence of 29 pathogens utilizing the Taqman® Array Card platform. We applied a non-parametric Bayesian regression extension of a partially latent class model approach to estimate proportions of SARI caused by specific pathogens. RESULTS: We enrolled 2,388 SARI patients and 1,135 asymptomatic adults from October 2013 through October 2015. We detected ≥1 pathogen in 76% of SARI patients and 67% of asymptomatic adults. Haemophilus influenzae and Streptococcus pneumoniae were most commonly detected (≥23% of SARI patients and asymptomatic adults). Through modeling, etiology was attributed to a pathogen in most SARI patients (range among countries: 57.3-93.2%); pathogens commonly attributed to SARI etiology included influenza A (14.4-54.4%), influenza B (1.9-19.1%), rhino/enterovirus (1.8-42.6%), and RSV (3.6-14.6%). CONCLUSIONS: Use of multi-pathogen diagnostics and modeling enabled attribution of etiology in most adult SARI patients, despite frequent detection of multiple pathogens in the upper respiratory tract. Seasonal flu vaccination and development of RSV vaccine would likely reduce the burden of SARI in these populations. |
Detection and characterization of diphtheria toxin gene-bearing Corynebacterium species through a new real-time PCR assay.
Williams MM , Waller JL , Aneke JS , Weigand MR , Diaz MH , Bowden KE , Simon AK , Peng Y , Xiaoli L , Cassiday PK , Winchell J , Tondella ML . J Clin Microbiol 2020 58 (10) ![]() ![]() Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of Corynebacterium diphtheriae, with similar illness produced occasionally by toxigenic Corynebacterium ulcerans or, rarely, Corynebacterium pseudotuberculosis While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene (tox). Nontoxigenic tox-bearing (NTTB) Corynebacterium isolates have been described, but impact of these isolates on the accuracy of molecular diagnostics is not well characterized. Here, we describe a new triplex RT-PCR assay to detect tox and distinguish C. diphtheriae from the closely related species C. ulcerans and C. pseudotuberculosis Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized Corynebacterium isolates accurately, with 100% analytical sensitivity for all targets. Analytical specificity with isolates was 94.1%, 100%, and 99.5% for tox, Diph_rpoB, and CUP_rpoB targets, respectively. Twenty-nine NTTB Corynebacterium isolates, representing 5.9% of 494 nontoxigenic isolates tested, were detected by RT-PCR. Whole-genome sequencing of NTTB isolates revealed varied mutations putatively underlying their lack of toxin production, as well as eight isolates with no mutation in tox or the promoter region. This new Corynebacterium RT-PCR method provides a rapid tool to screen isolates and identify probable diphtheria cases directly from specimens. However, the sporadic occurrence of NTTB isolates reinforces the viewpoint that diphtheria culture diagnostics continue to provide the most accurate case confirmation. |
Causes of fever in primary care in Southeast Asia and the performance of C-reactive protein in discriminating bacterial from viral pathogens
Althaus T , Thaipadungpanit J , Greer RC , Swe MMM , Dittrich S , Peerawaranun P , Smit PW , Wangrangsimakul T , Blacksell S , Winchell JM , Diaz MH , Day NPJ , Smithuis F , Turner P , Lubell Y . Int J Infect Dis 2020 96 334-342 OBJECTIVES: We investigated causes of fever in the primary levels of care in Southeast Asia, and evaluated whether C-reactive protein (CRP) could distinguish bacterial from viral pathogens. METHODS: Blood and nasopharyngeal swab specimens were taken from children and adults with fever (>37.5 C) or history of fever (<14 days) in Thailand and Myanmar. RESULTS: Of 773 patients with at least one blood or nasopharyngeal swab specimen collected, 227 (29.4%) had a target organism detected. Influenza virus type A was detected in 85/227 cases (37.5%), followed by dengue virus (30 cases, 13.2%), respiratory syncytial virus (24 cases, 10.6%) and Leptospira spp. (9 cases, 4.0%). Clinical outcome was similar between patients with a bacterial or a viral organism, regardless of antibiotic prescription. CRP was higher among patients with a bacterial organism compared to those with a viral organism (median 18mg/L, interquartile range [10-49] versus 10mg/L [</=8-22], p-value 0.003), with an area under the curve of 0.65, 95% confidence interval (0.55-0.75). CONCLUSIONS: Serious bacterial infections requiring antibiotics are exceptions rather than the rule in the first lines of care. CRP-testing could assist in ruling out such cases in settings where diagnostic uncertainty is high and routine antibiotic prescription is common. The original CRP randomised-controlled trial (RCT) was registered with ClinicalTrials.gov, number NCT02758821. |
Improving detection and response to respiratory events - Kenya, April 2016-April 2020
Idubor OI , Kobayashi M , Ndegwa L , Okeyo M , Galgalo T , Kalani R , Githii S , Hunsperger E , Balajee A , Verani JR , da Gloria Carvalho M , Winchell J , Van Beneden CA , Widdowson MA , Makayotto L , Chaves SS . MMWR Morb Mortal Wkly Rep 2020 69 (18) 540-544 Respiratory pathogens, such as novel influenza A viruses, Middle East respiratory syndrome coronavirus (MERS-CoV), and now, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are of particular concern because of their high transmissibility and history of global spread (1). Clusters of severe respiratory disease are challenging to investigate, especially in resource-limited settings, and disease etiology often is not well understood. In 2014, endorsed by the Group of Seven (G7),* the Global Health Security Agenda (GHSA) was established to help build country capacity to prevent, detect, and respond to infectious disease threats.(dagger) GHSA is a multinational, multisectoral collaboration to support countries towards full implementation of the World Health Organization's International Health Regulations (IHR).( section sign) Initially, 11 technical areas for collaborator participation were identified to meet GHSA goals. CDC developed the Detection and Response to Respiratory Events (DaRRE) strategy in 2014 to enhance country capacity to identify and control respiratory disease outbreaks. DaRRE initiatives support the four of 11 GHSA technical areas that CDC focuses on: surveillance, laboratory capacity, emergency operations, and workforce development.( paragraph sign) In 2016, Kenya was selected to pilot DaRRE because of its existing respiratory disease surveillance and laboratory platforms and well-developed Field Epidemiology and Laboratory Training Program (FELTP) (2). During 2016-2020, Kenya's DaRRE partners (CDC, the Kenya Ministry of Health [MoH], and Kenya's county public health officials) conceptualized, planned, and implemented key components of DaRRE. Activities were selected based on existing capacity and determined by the Kenya MoH and included 1) expansion of severe acute respiratory illness (SARI) surveillance sites; 2) piloting of community event-based surveillance; 3) expansion of laboratory diagnostic capacity; 4) training of public health practitioners in detection, investigation, and response to respiratory threats; and 5) improvement of response capacity by the national emergency operations center (EOC). Progress on DaRRE activity implementation was assessed throughout the process. This pilot in Kenya demonstrated that DaRRE can support IHR requirements and can capitalize on a country's existing resources by tailoring tools to improve public health preparedness based on countries' needs. |
Evaluation of Commercial Molecular Diagnostic Methods for the Detection and Determination of Macrolide Resistance in Mycoplasma pneumoniae .
Leal SMJr , Totten AH , Xiao L , Crabb DM , Ratliff A , Duffy LB , Fowler KB , Mixon E , Winchell JM , Diaz MH , Benitez AJ , Wolff BJ , Qin X , Tang YW , Gonzalez M , Selvarangan R , Hong T , Brooks E , Dallas S , Atkinson TP , Zheng X , Dien Bard J , Waites KB . J Clin Microbiol 2020 58 (6) ![]() ![]() We evaluated six commercial molecular tests targeting M. pneumoniae: the BioFire FilmArray Respiratory Panel (RP), the Meridian Alethia Mycoplasma Direct, the GenMark ePlex Respiratory Pathogen Panel (RPP), the Luminex NxTAG RPP, the ELITech ELITe InGenius Mycoplasma MGB Research Use Only Polymerase Chain Reaction (PCR), and the SpeeDx Resistance Plus MP. Laboratory-developed PCR assays at the University of Alabama at Birmingham and the Centers for Disease Control and Prevention were used as reference standards. Among 428 specimens, 212 were designated confirmed-positives for M. pneumoniae The highest clinical sensitivities were found with the InGenius (99.5%) and the FilmArray RP (98.1%). The Resistance Plus MP identified 93.3% of the confirmed-positive specimens, whereas 83.6%, 64.6%, and 55.7% were identified by the ePlex RPP, NxTAG RPP, and Mycoplasma Direct assays, respectively. There was no significant difference between the sensitivity of the reference methods and that of the FilmArray RP and InGenius assays, but the remaining four assays detected significantly fewer positive specimens (p < 0.05). Specificities of all assays were 99.5 - 100%. The Resistance Plus MP detected macrolide resistance in 27/33 specimens resulting in a sensitivity of 81.8%. This study provides the first large scale comparison of commercial molecular assays for detection of M. pneumoniae in the United States and identified clear differences among their performance. Additional studies are necessary to explore the impact of variable test performance on patient outcome. |
Psittacosis outbreak among workers at chicken slaughter plants, Virginia and Georgia, USA, 2018
Shaw KA , Szablewski CM , Kellner S , Kornegay L , Bair P , Brennan S , Kunkes A , Davis M , McGovern OL , Winchell J , Kobayashi M , Burton N , de Perio MA , Gabel J , Drenzek C , Murphy J , Holsinger C , Forlano L . Emerg Infect Dis 2019 25 (11) 2143-2145 During August-October, 2018, an outbreak of severe respiratory illness was reported among poultry slaughter plant workers in Virginia and Georgia, USA. A multiorganizational team investigated the cause and extent of illness, determined that the illness was psittacosis, and evaluated and recommended controls for health hazards in the workplace to prevent additional cases. |
Development and Implementation of Multiplex TaqMan Array Cards for Specimen Testing at Child Health and Mortality Prevention Surveillance Site Laboratories.
Diaz MH , Waller JL , Theodore MJ , Patel N , Wolff BJ , Benitez AJ , Morris T , Raghunathan PL , Breiman RF , Whitney CG , Blau DM , Winchell JM . Clin Infect Dis 2019 69 S311-s321 ![]() ![]() Child Health and Mortality Prevention Surveillance (CHAMPS) laboratories are employing a variety of laboratory methods to identify infectious agents contributing to deaths of children <5 years old and stillbirths in sub-Saharan Africa and South Asia. In support of this long-term objective, our team developed TaqMan Array Cards (TACs) for testing postmortem specimens (blood, cerebrospinal fluid, lung tissue, respiratory tract swabs, and rectal swabs) for >100 real-time polymerase chain reaction (PCR) targets in total (30-45 per card depending on configuration). Multipathogen panels were configured by syndrome and customized to include pathogens of significance in young children within the regions where CHAMPS is conducted, including bacteria (57 targets covering 30 genera), viruses (48 targets covering 40 viruses), parasites (8 targets covering 8 organisms), and fungi (3 targets covering 3 organisms). The development and application of multiplex real-time PCR reactions to the TAC microfluidic platform increased the number of targets in each panel while maintaining assay efficiency and replicates for heightened sensitivity. These advances represent a substantial improvement in the utility of this technology for infectious disease diagnostics and surveillance. We optimized all aspects of the CHAMPS molecular laboratory testing workflow including nucleic acid extraction, quality assurance, and data management to ensure comprehensive molecular testing of specimens and high-quality data. Here we describe the development and implementation of multiplex TACs and associated laboratory protocols for specimen processing, testing, and data management at CHAMPS site laboratories. |
Use of TaqMan Array card for the detection of respiratory viral pathogens in children under 5 years old hospitalised with acute medical illness in Ballabgarh, Haryana, India.
Gaur B , Saha S , Iuliano AD , Rai SK , Krishnan A , Jain S , Whitaker B , Winchell J , Lal RB , Broor S . Indian J Med Microbiol 2019 37 (1) 105-108 ![]() Historical specimens collected from hospitalized children were tested for the following 13 viruses: influenza A and B; respiratory syncytial virus (RSV); parainfluenza viruses 1-3; human metapneumovirus; rhinovirus; coronaviruses 229E, OC43, NL63 and HKU1 and Adenovirus using monoplex real-time reverse transcriptase polymerase chain reaction (rRT-PCR). They were retested using TaqMan Array Card (TAC), a micro-fluidic system, capable of simultaneous multi-pathogen testing, to evaluate its sensitivity and specificity against monoplex rRT-PCR. TAC showed high sensitivity (71%-100%) and specificity (98%-100%) for these viruses in comparison to monoplex rRT-PCR. Multi-specimen detection with high sensitivity and specificity makes TAC a potentially useful tool for both surveillance and outbreak investigations. |
Comparison of respiratory pathogen yields from Nasopharyngeal/Oropharyngeal swabs and sputum specimens collected from hospitalized adults in rural Western Kenya
Nyawanda BO , Njuguna HN , Onyango CO , Makokha C , Lidechi S , Fields B , Winchell JM , Katieno JS , Nyaundi J , Ade F , Emukule GO , Mott JA , Otieno N , Widdowson MA , Chaves SS . Sci Rep 2019 9 (1) 11237 ![]() Molecular diagnostic methods are becoming increasingly available for assessment of acute lower respiratory illnesses (ALRI). However, nasopharyngeal/oropharyngeal (NP/OP) swabs may not accurately reflect etiologic agents from the lower respiratory tract where sputum specimens are considered as a more representative sample. The pathogen yields from NP/OP against sputum specimens have not been extensively explored, especially in tropical countries. We compared pathogen yields from NP/OP swabs and sputum specimens from patients ≥18 years hospitalized with ALRI in rural Western Kenya. Specimens were tested for 30 pathogens using TaqMan Array Cards (TAC) and results compared using McNemar’s test. The agreement for pathogen detection between NP/OP and sputum specimens ranged between 85–100%. More viruses were detected from NP/OP specimens whereas Klebsiella pneumoniae and Mycobacterium tuberculosis were more common in sputum specimens. There was no clear advantage in using sputum over NP/OP specimens to detect pathogens of ALRI in adults using TAC in the context of this tropical setting. |
Assessment of eight nucleic acid amplification technologies for potential use to detect infectious agents in low-resource settings
Cantera JL , White H , Diaz MH , Beall SG , Winchell JM , Lillis L , Kalnoky M , Gallarda J , Boyle DS . PLoS One 2019 14 (4) e0215756 Nucleic acid amplification technologies (NAATs) are high-performance tools for rapidly and accurately detecting infectious agents. They are widely used in high-income countries to diagnose disease and improve patient care. The complexities associated with test methods, reagents, equipment, quality control and assurance require dedicated laboratories with trained staff, which can exclude their use in low-resource and decentralized healthcare settings. For certain diseases, fully integrated NAAT devices and assays are available for use in environmentally-controlled clinics or emergency rooms where relatively untrained staff can perform testing. However, decentralized settings in many low- and middle-income countries with large burdens of infectious disease are challenged by extreme environments, poor infrastructure, few trained staff and limited financial resources. Therefore, there is an urgent need for low-cost, integrated NAAT tools specifically designed for use in low-resource settings (LRS). Two essential components of integrated NAAT tools are: 1) efficient nucleic acid extraction technologies for diverse and complex sample types; and 2) robust and sensitive nucleic acid amplification and detection technologies. In prior work we reported the performance and workflow capacity for the nucleic acid extraction component. In the current study we evaluated performance of eight novel nucleic acid amplification and detection technologies from seven developers using blinded panels of RNA and/or DNA from three pathogens to assess both diagnostic accuracy and suitability as an essential component for low-cost NAAT in LRS. In this exercise, we noted significant differences in performance among these technologies and identified those most promising for potential further development. |
Surveillance for incidence and etiology of early-onset neonatal sepsis in Soweto, South Africa.
Velaphi SC , Westercamp M , Moleleki M , Pondo T , Dangor Z , Wolter N , von Gottberg A , Shang N , Demirjian A , Winchell JM , Diaz MH , Nakwa F , Okudo G , Wadula J , Cutland C , Schrag SJ , Madhi SA . PLoS One 2019 14 (4) e0214077 ![]() BACKGROUND: Globally, over 400,000 neonatal deaths in 2015 were attributed to sepsis, however, the incidence and etiologies of these infections are largely unknown in low-middle income countries. We aimed to determine incidence and etiology of community-acquired early-onset (<72 hours age) sepsis (EOS) using culture and molecular diagnostics. METHODS: This was a prospective observational study, in which we conducted a surveillance for pathogens using a combination of blood culture and a polymerase chain reaction (PCR)-based test. Blood culture was performed on all neonates with suspected EOS. Among the subset fulfilling criteria for protocol-defined EOS, blood and nasopharyngeal (NP) respiratory swabs were tested by quantitative real-time reverse-transcriptase PCR using a Taqman Array Card (TAC) with 15 bacterial and 12 viral targets. Blood and NP samples from 312 healthy newborns were also tested by TAC to estimate background positivity rates. We used variant latent-class methods to attribute etiologies and calculate pathogen-specific proportions and incidence rates. RESULTS: We enrolled 2,624 neonates with suspected EOS and from these 1,231 newborns met criteria for protocol-defined EOS (incidence- 39.3/1,000 live-births). Using the partially latent-class modelling, only 26.7% cases with protocol-defined EOS had attributable etiology, and the largest pathogen proportion were Ureaplasma spp. (5.4%; 95%CI: 3.6-8.0) and group B Streptococcus (GBS) (4.8%; 95%CI: 4.1-5.8), and no etiology was attributable for 73.3% of cases. Blood cultures were positive in 99/1,231 (8.0%) with protocol-defined EOS (incidence- 3.2/1,000 live-births). Leading pathogens on blood culture included GBS (35%) and viridans streptococci (24%). Ureaplasma spp. was the most common organism identified on TAC among cases with protocol-defined EOS. CONCLUSION: Using a combination of blood culture and a PCR-based test the common pathogens isolated in neonates with sepsis were Ureaplasma spp. and GBS. Despite documenting higher rates of protocol-defined EOS and using a combination of tests, the etiology for EOS remains elusive. |
Culture of Clinical Specimens Reveals Extensive Diversity of Legionella pneumophila Strains in Arizona.
Raphael BH , Huynh T , Brown E , Smith JC , Ruberto I , Getsinger L , White S , Winchell JM . mSphere 2019 4 (1) ![]() Between 2000 and 2017, a total of 236 Legionella species isolates from Arizona were submitted to the CDC for reference testing. Most of these isolates were recovered from bronchoalveolar lavage specimens. Although the incidence of legionellosis in Arizona is less than the overall U.S. incidence, Arizona submits the largest number of isolates to the CDC for testing compared to those from other states. In addition to a higher proportion of culture confirmation of legionellosis cases in Arizona than in other states, all Legionella pneumophila isolates are forwarded to the CDC for confirmatory testing. Compared to that from other states, a higher proportion of isolates from Arizona were identified as belonging to L. pneumophila serogroups 6 (28.2%) and 8 (8.9%). Genome sequencing was conducted on 113 L. pneumophila clinical isolates not known to be associated with outbreaks in order to understand the genomic diversity of strains causing legionellosis in Arizona. Whole-genome multilocus sequence typing (wgMLST) revealed 17 clusters of isolates sharing at least 99% identical allele content. Only two of these clusters contained isolates from more than one individual with exposure at the same facility. Additionally, wgMLST analysis revealed a group of 31 isolates predominantly belonging to serogroup 6 and containing isolates from three separate clusters. Single nucleotide polymorphism (SNP) and pangenome analysis were used to further resolve genome sequences belonging to a subset of isolates. This study demonstrates that culture of clinical specimens for Legionella spp. reveals a highly diverse population of strains causing legionellosis in Arizona which could be underappreciated using other diagnostic approaches.IMPORTANCE Culture of clinical specimens from patients with Legionnaires' disease is rarely performed, restricting our understanding of the diversity and ecology of Legionella Culture of Legionella from patient specimens in Arizona revealed a greater proportion of non-serogroup 1 Legionella pneumophila isolates than in other U.S. isolates examined. Disease caused by such isolates may go undetected using other diagnostic methods. Moreover, genome sequence analysis revealed that these isolates were genetically diverse, and understanding these populations may help in future environmental source attribution studies. |
Performance and workflow assessment of six nucleic acid extraction technologies for use in resource limited settings
Beall SG , Cantera J , Diaz MH , Winchell JM , Lillis L , White H , Kalnoky M , Gallarda J , Boyle DS . PLoS One 2019 14 (4) e0215753 Infectious disease nucleic acid amplification technologies (NAAT) have superior sensitivity, specificity, and rapid time to result compared to traditional microbiological methods. Recovery of concentrated, high quality pathogen nucleic acid (NA) from complex specimen matrices is required for optimal performance of several NA amplification/detection technologies such as polymerase chain reaction (PCR). Fully integrated NAAT platforms that enable rapid sample-to-result workflows with minimal user input are generally restricted to larger reference lab settings, and their complexity and cost are prohibitive to widespread implementation in resource limited settings (RLS). Identification of component technologies for incorporation of reliable and affordable sample preparation with pathogen NA amplification/detection into an integrated platform suitable for RLS, is a necessary first step toward achieving the overarching goal of reducing infectious disease-associated morbidity and mortality globally. In the current study, we evaluate the performance of six novel NA extraction technologies from different developers using blinded panels of stool, sputum and blood spiked with variable amounts of quality-controlled DNA- and/or RNA-based microbes. The extraction efficiencies were semi-quantitatively assessed using validated real-time reverse transcription (RT)-PCR assays specific for each microbe and comparing target-specific RT-PCR results to those obtained with reference NA extraction methods. The technologies were ranked based on overall diagnostic accuracy (analytical sensitivity and specificity). Sample input and output volumes, total processing time, user-required manual steps and cost estimates were also examined for suitability in RLS. Together with the performance analysis, these metrics were used to select the more suitable candidate technologies for further optimization of integrated NA amplification and detection technologies for RLS. |
Genomic heterogeneity differentiates clinical and environmental subgroups of Legionella pneumophila sequence type 1.
Mercante JW , Caravas JA , Ishaq MK , Kozak-Muiznieks NA , Raphael BH , Winchell JM . PLoS One 2018 13 (10) e0206110 ![]() ![]() Legionella spp. are the cause of a severe bacterial pneumonia known as Legionnaires' disease (LD). In some cases, current genetic subtyping methods cannot resolve LD outbreaks caused by common, potentially endemic L. pneumophila (Lp) sequence types (ST), which complicates laboratory investigations and environmental source attribution. In the United States (US), ST1 is the most prevalent clinical and environmental Lp sequence type. In order to characterize the ST1 population, we sequenced 289 outbreak and non-outbreak associated clinical and environmental ST1 and ST1-variant Lp strains from the US and, together with international isolate sequences, explored their genetic and geographic diversity. The ST1 population was highly conserved at the nucleotide level; 98% of core nucleotide positions were invariant and environmental isolates unassociated with human disease (n = 99) contained ~65% more nucleotide diversity compared to clinical-sporadic (n = 139) or outbreak-associated (n = 28) ST1 subgroups. The accessory pangenome of environmental isolates was also ~30-60% larger than other subgroups and was enriched for transposition and conjugative transfer-associated elements. Up to ~10% of US ST1 genetic variation could be explained by geographic origin, but considerable genetic conservation existed among strains isolated from geographically distant states and from different decades. These findings provide new insight into the ST1 population structure and establish a foundation for interpreting genetic relationships among ST1 strains; these data may also inform future analyses for improved outbreak investigations. |
Causes and incidence of community-acquired serious infections among young children in south Asia (ANISA): an observational cohort study
Saha SK , Schrag SJ , El Arifeen S , Mullany LC , Shahidul Islam M , Shang N , Qazi SA , Zaidi AKM , Bhutta ZA , Bose A , Panigrahi P , Soofi SB , Connor NE , Mitra DK , Isaac R , Winchell JM , Arvay ML , Islam M , Shafiq Y , Nisar I , Baloch B , Kabir F , Ali M , Diaz MH , Satpathy R , Nanda P , Padhi BK , Parida S , Hotwani A , Hasanuzzaman M , Ahmed S , Belal Hossain M , Ariff S , Ahmed I , Ibne Moin SM , Mahmud A , Waller JL , Rafiqullah I , Quaiyum MA , Begum N , Balaji V , Halen J , Nawshad Uddin Ahmed ASM , Weber MW , Hamer DH , Hibberd PL , Sadeq-Ur Rahman Q , Mogan VR , Hossain T , McGee L , Anandan S , Liu A , Panigrahi K , Abraham AM , Baqui AH . Lancet 2018 392 (10142) 145-159 BACKGROUND: More than 500 000 neonatal deaths per year result from possible serious bacterial infections (pSBIs), but the causes are largely unknown. We investigated the incidence of community-acquired infections caused by specific organisms among neonates in south Asia. METHODS: From 2011 to 2014, we identified babies through population-based pregnancy surveillance at five sites in Bangladesh, India, and Pakistan. Babies were visited at home by community health workers up to ten times from age 0 to 59 days. Illness meeting the WHO definition of pSBI and randomly selected healthy babies were referred to study physicians. The primary objective was to estimate proportions of specific infectious causes by blood culture and Custom TaqMan Array Cards molecular assay (Thermo Fisher, Bartlesville, OK, USA) of blood and respiratory samples. FINDINGS: 6022 pSBI episodes were identified among 63 114 babies (95.4 per 1000 livebirths). Causes were attributed in 28% of episodes (16% bacterial and 12% viral). Mean incidence of bacterial infections was 13.2 (95% credible interval [CrI] 11.2-15.6) per 1000 livebirths and of viral infections was 10.1 (9.4-11.6) per 1000 livebirths. The leading pathogen was respiratory syncytial virus (5.4, 95% CrI 4.8-6.3 episodes per 1000 livebirths), followed by Ureaplasma spp (2.4, 1.6-3.2 episodes per 1000 livebirths). Among babies who died, causes were attributed to 46% of pSBI episodes, among which 92% were bacterial. 85 (83%) of 102 blood culture isolates were susceptible to penicillin, ampicillin, gentamicin, or a combination of these drugs. INTERPRETATION: Non-attribution of a cause in a high proportion of patients suggests that a substantial proportion of pSBI episodes might not have been due to infection. The predominance of bacterial causes among babies who died, however, indicates that appropriate prevention measures and management could substantially affect neonatal mortality. Susceptibility of bacterial isolates to first-line antibiotics emphasises the need for prudent and limited use of newer-generation antibiotics. Furthermore, the predominance of atypical bacteria we found and high incidence of respiratory syncytial virus indicated that changes in management strategies for treatment and prevention are needed. Given the burden of disease, prevention of respiratory syncytial virus would have a notable effect on the overall health system and achievement of Sustainable Development Goal. FUNDING: Bill & Melinda Gates Foundation. |
Mycoplasma pneumoniae among children hospitalized with community-acquired pneumonia
Kutty PK , Jain S , Taylor TH , Bramley AM , Diaz MH , Ampofo K , Arnold SR , Williams DJ , Edwards KM , McCullers JA , Pavia AT , Winchell JM , Schrag SJ , Hicks LA . Clin Infect Dis 2018 68 (1) 5-12 Background: The burden and epidemiology of Mycoplasma pneumoniae (Mp) among U.S. children (<18 years) hospitalized with community-acquired pneumonia (CAP) are poorly understood. Methods: In the Etiology of Pneumonia in the Community (EPIC) study, we prospectively enrolled 2254 children hospitalized with radiographically-confirmed pneumonia from January 2010-June 2012 and tested nasopharyngeal/oropharyngeal swabs for Mp using real-time polymerase chain reaction (PCR). Clinical and epidemiological features of Mp-PCR-positive and -negative children were compared using logistic regression. Macrolide susceptibility was assessed by genotyping isolates. Results: In the EPIC study, 182(8%) children were Mp-PCR-positive (median age: 7 years); 12% required intensive care and 26% had pleural effusion. No in-hospital deaths occurred. Macrolide resistance was found in 6/169(4%) isolates. Of 178(98%) Mp-PCR-positive children tested for co-pathogens, 50(28%) had >/=1 co-pathogen detected. Variables significantly associated with higher odds of Mp detection included age {10-17 years [adjusted odds ratio (aOR): 7.9 (95% confidence interval (CI): 4.5-13.6)] and 5-9 years [aOR: 4.8 (CI: 2.9-7.8)] vs. 2-4 years}, outpatient antibiotics </=5 days pre-admission [aOR: 2.3 (CI: 1.5-3.4)], and co-pathogen detection [aOR: 2.1 (CI: 1.3-3.1)]. Clinical characteristics often seen included hilar lymphadenopathy, rales, headache, sore throat, and decreased breath sounds. Conclusions: Usually considered as a mild respiratory infection, M. pneumoniae was the most commonly detected bacteria among children >/=5 years hospitalized with CAP; one-quarter of whom had co-detections. Although associated with clinically non-specific symptoms, there was a need for intensive care support in some cases. M. pneumoniae should be included in the differential diagnosis for school-aged children hospitalized with CAP. |
Epidemiology and Molecular Identification and Characterization of Mycoplasma pneumoniae, South Africa, 2012-2015.
Carrim M , Wolter N , Benitez AJ , Tempia S , du Plessis M , Walaza S , Moosa F , Diaz MH , Wolff BJ , Treurnicht FK , Hellferscee O , Dawood H , Variava E , Cohen C , Winchell JM , von Gottberg A . Emerg Infect Dis 2018 24 (3) 506-513 ![]() During 2012-2015, we tested respiratory specimens from patients with severe respiratory illness (SRI), patients with influenza-like illness (ILI), and controls in South Africa by real-time PCR for Mycoplasma pneumoniae, followed by culture and molecular characterization of positive samples. M. pneumoniae prevalence was 1.6% among SRI patients, 0.7% among ILI patients, and 0.2% among controls (p<0.001). Age <5 years (adjusted odd ratio 7.1; 95% CI 1.7-28.7) and HIV infection (adjusted odds ratio 23.8; 95% CI 4.1-138.2) among M. pneumonia-positive persons were associated with severe disease. The detection rate attributable to illness was 93.9% (95% CI 74.4%-98.5%) in SRI patients and 80.7% (95% CI 16.7%-95.6%) in ILI patients. The hospitalization rate was 28 cases/100,000 population. We observed the macrolide-susceptible M. pneumoniae genotype in all cases and found P1 types 1, 2, and a type 2 variant with multilocus variable number tandem repeat types 3/6/6/2, 3/5/6/2, and 4/5/7/2. |
Comparative genome analysis reveals a complex population structure of Legionella pneumophila subspecies.
Kozak-Muiznieks NA , Morrison SS , Mercante JW , Ishaq MK , Johnson T , Caravas J , Lucas CE , Brown E , Raphael BH , Winchell JM . Infect Genet Evol 2018 59 172-185 ![]() ![]() The majority of Legionnaires' disease (LD) cases are caused by Legionella pneumophila, a genetically heterogeneous species composed of at least 17 serogroups. Previously, it was demonstrated that L. pneumophila consists of three subspecies: pneumophila, fraseri and pascullei. During an LD outbreak investigation in 2012, we detected that representatives of both subspecies fraseri and pascullei colonized the same water system and that the outbreak-causing strain was a new member of the least represented subspecies pascullei. We used partial sequence based typing consensus patterns to mine an international database for additional representatives of fraseri and pascullei subspecies. As a result, we identified 46 sequence types (STs) belonging to subspecies fraseri and two STs belonging to subspecies pascullei. Moreover, a recent retrospective whole genome sequencing analysis of isolates from New York State LD clusters revealed the presence of a fourth L. pneumophila subspecies that we have termed raphaeli. This subspecies consists of 15 STs. Comparative analysis was conducted using the genomes of multiple members of all four L. pneumophila subspecies. Whereas each subspecies forms a distinct phylogenetic clade within the L. pneumophila species, they share more average nucleotide identity with each other than with other Legionella species. Unique genes for each subspecies were identified and could be used for rapid subspecies detection. Improved taxonomic classification of L. pneumophila strains may help identify environmental niches and virulence attributes associated with these genetically distinct subspecies. |
Simultaneous quantitation of multiple contraceptive hormones in human serum by LC-MS/MS
Blue SW , Winchell AJ , Kaucher AV , Lieberman RA , Gilles CT , Pyra MN , Heffron R , Hou X , Coombs RW , Nanda K , Davis NL , Kourtis AP , Herbeck JT , Baeten JM , Lingappa JR , Erikson DW . Contraception 2018 97 (4) 363-369 OBJECTIVE: To develop a method to simultaneously quantify five commonly used hormonal contraceptives (HC) and 2 endogenous sex steroids by liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS) and apply this method to human serum samples. STUDY DESIGN: We developed a method to simultaneously analyze ethinyl estradiol (EE2), etonogestrel (ENG), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), and norethisterone (NET), along with estradiol (E2) and progesterone (P4) in human serum for a Shimadzu Nexera-LCMS-8050 LC-MS/MS platform. We analyzed serum collected from women self-reporting use of oral contraceptives, contraceptive implants or injectable contraceptives (n=14) and normally cycling women using no HC (n=15) as well as pooled samples from women administered various HC (ENG, n=6; LNG, n=14; MPA, n=7; NET, n=5). RESULTS: Limits of quantitation were 0.010ng/ml for E2, EE2, and P4; 0.020ng/ml for ENG, LNG, and MPA; and 0.040ng/ml for NET. Precisions for all assays, as indicated by coefficient of variation (CV) were less than or equal to 12.1%. Accuracies for all assays were in the range of 95-108%. Endogenous hormone values obtained from analysis of human serum samples are in agreement with levels previously reported in the literature for normally cycling women as well as for women taking the appropriate HC. CONCLUSIONS: We have developed a robust, accurate, and sensitive method for simultaneously analyzing commonly used contraceptive steroids and endogenous sex steroids in human serum. IMPLICATIONS: This analytical method can be used for quantitating contraceptive steroid levels in women for monitoring systemic exposure to determine drug interactions, nonadherence, misreporting, and proper dosing. |
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