RESUMO
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.
RESUMO
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.
Assuntos
Infecções por Chlamydia , Chlamydia , Chlamydophila psittaci , Humanos , Chlamydophila psittaci/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Chlamydia/genética , Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/microbiologiaRESUMO
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.
Assuntos
Infecções Bacterianas , Infecções Comunitárias Adquiridas , Lactente , Recém-Nascido , Gravidez , Feminino , Humanos , Masculino , Estudos Longitudinais , Teorema de Bayes , Infecções Comunitárias Adquiridas/complicações , Infecções Comunitárias Adquiridas/epidemiologia , Fatores de Risco , Estudos de Coortes , Estudos de Casos e Controles , Índia/epidemiologiaRESUMO
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.
Assuntos
Manejo de Espécimes , Autopsia , Criança , Humanos , Imuno-HistoquímicaRESUMO
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; nâ =â 20), 3 pathogens in each child's liver tissue (range, 1-4; nâ =â 5), and 2 pathogens in each child's blood specimen (range, 0-4; nâ =â 5). Pathogens were not consistently detected across all collection time points; there was no association between postmortem interval and the number of pathogens detected (Pâ =â .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.
Assuntos
Pulmão , Manejo de Espécimes , Autopsia/métodos , Causas de Morte , Criança , Pré-Escolar , Coleta de Dados , Humanos , Lactente , Manejo de Espécimes/métodosRESUMO
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 102.0, influenza B virus at 102.2, and SARS-CoV-2 at 100.3 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.
Assuntos
COVID-19 , Vírus da Influenza A , Humanos , Vírus da Influenza A/genética , Vírus da Influenza B/genética , Reação em Cadeia da Polimerase Multiplex , Transcrição Reversa , SARS-CoV-2RESUMO
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.
Assuntos
Chlamydophila psittaci/isolamento & purificação , Surtos de Doenças , Programas de Rastreamento/métodos , Psitacose/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Adulto , Chlamydophila psittaci/genética , Fezes/microbiologia , Feminino , Georgia/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Psitacose/epidemiologia , Escarro/microbiologia , Virginia/epidemiologia , Adulto JovemRESUMO
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.
Assuntos
Legionella pneumophila , Legionella , Doença dos Legionários , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Doença dos Legionários/diagnóstico , Doença dos Legionários/tratamento farmacológico , Padrões de ReferênciaRESUMO
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.
Assuntos
Bactérias/classificação , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/microbiologia , Vírus/classificação , Adulto , Idoso , Doenças Assintomáticas/epidemiologia , Bactérias/genética , Bactérias/isolamento & purificação , Bangladesh , Teorema de Bayes , Feminino , Guatemala , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Epidemiologia Molecular , Nasofaringe/microbiologia , Orofaringe/microbiologia , Reação em Cadeia da Polimerase , Vírus/genética , Vírus/isolamento & purificação , Adulto JovemRESUMO
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.
Assuntos
Corynebacterium diphtheriae , Difteria , Corynebacterium/genética , Corynebacterium diphtheriae/genética , Toxina Diftérica/genética , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
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. GHSA is a multinational, multisectoral collaboration to support countries towards full implementation of the World Health Organization's International Health Regulations (IHR).§ 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.¶ 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.
Assuntos
Surtos de Doenças/prevenção & controle , Programas de Rastreamento/organização & administração , Vigilância em Saúde Pública , Doenças Respiratórias/diagnóstico , Doenças Respiratórias/prevenção & controle , Fortalecimento Institucional , Humanos , Quênia/epidemiologia , Projetos Piloto , Doenças Respiratórias/epidemiologiaRESUMO
OBJECTIVES: This study 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. (nine cases, 4.0%). Clinical outcomes were similar between patients with a bacterial or a viral organism, regardless of antibiotic prescription. CRP was higher among patients with a bacterial organism compared with those with a viral organism (median 18 mg/L, interquartile range [10-49] versus 10 mg/L [≤8-22], p = 0.003), with an area under the curve of 0.65 (95% CI 0.55-0.75). CONCLUSIONS: Serious bacterial infections requiring antibiotics are an exception rather than the rule in the first line 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 was registered with ClinicalTrials.gov, number NCT02758821.
Assuntos
Infecções Bacterianas/complicações , Proteína C-Reativa/metabolismo , Febre/etiologia , Atenção Primária à Saúde , Adulto , Antibacterianos/uso terapêutico , Sudeste Asiático , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/tratamento farmacológico , Criança , Pré-Escolar , Feminino , Febre/diagnóstico , Febre/microbiologia , Febre/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Mianmar , TailândiaRESUMO
We evaluated six commercial molecular tests targeting Mycoplasma pneumoniae, namely, 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 (RUO) PCR, and the SpeeDx Resistance Plus MP assays. 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 PCR (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 to 100%. The Resistance Plus MP assay 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 various test performances on patient outcome.
Assuntos
Mycoplasma pneumoniae , Pneumonia por Mycoplasma , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Humanos , Macrolídeos/farmacologia , Mycoplasma pneumoniae/genética , Patologia Molecular , Pneumonia por Mycoplasma/diagnósticoRESUMO
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.
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Matadouros , Psitacose/epidemiologia , Adulto , Georgia/epidemiologia , História do Século XXI , Humanos , Pessoa de Meia-Idade , Psitacose/história , Psitacose/microbiologia , Vigilância em Saúde Pública , Virginia/epidemiologia , Adulto JovemRESUMO
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.
Assuntos
Vigilância da População/métodos , Manejo de Espécimes/métodos , África Subsaariana , Ásia , Bactérias/genética , Criança , Saúde da Criança , Mortalidade da Criança , Doenças Transmissíveis/diagnóstico , Fungos/genética , Humanos , Laboratórios , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Vírus/genéticaRESUMO
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.
Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Pré-Escolar , Humanos , Índia , Lactente , Recém-Nascido , Técnicas de Diagnóstico Molecular/métodos , Sensibilidade e Especificidade , Vírus/isolamento & purificaçãoRESUMO
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.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Sistema Respiratório/microbiologia , Infecções Respiratórias/diagnóstico , Adolescente , Adulto , Feminino , Hospitalização , Humanos , Quênia , Klebsiella pneumoniae/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Nasofaringe/microbiologia , Nasofaringe/virologia , Orofaringe/microbiologia , Orofaringe/virologia , Escarro/microbiologiaRESUMO
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.
Assuntos
Doenças Transmissíveis/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Sistemas Automatizados de Assistência Junto ao Leito/economia , Chlamydia/genética , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/virologia , Análise Custo-Benefício , HIV-1/genética , Recursos em Saúde/economia , Humanos , Neisseria gonorrhoeae/genética , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Reprodutibilidade dos Testes , Zika virus/genéticaRESUMO
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.
Assuntos
Sepse Neonatal/sangue , Complicações Infecciosas na Gravidez/sangue , Infecções Estreptocócicas/sangue , Streptococcus agalactiae/isolamento & purificação , Idade de Início , Hemocultura , Feminino , Humanos , Recém-Nascido , Sepse Neonatal/epidemiologia , Sepse Neonatal/microbiologia , Sepse Neonatal/patologia , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Complicações Infecciosas na Gravidez/microbiologia , Complicações Infecciosas na Gravidez/patologia , África do Sul , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , Streptococcus agalactiae/genética , Streptococcus agalactiae/patogenicidade , Ureaplasma/isolamento & purificação , Ureaplasma/patogenicidadeRESUMO
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.