Histopathology Is Key to Interpreting Multiplex Molecular Test Results From Postmortem Minimally Invasive Tissue Samples.

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.

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.

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.

Legionella antibiotic susceptibility testing: is it time for international standardization and evidence-based guidance?

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.

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.

Detection and Characterization of Diphtheria Toxin Gene-Bearing Corynebacterium Species through a New Real-Time PCR Assay.

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.

Evaluation of Commercial Molecular Diagnostic Methods for Detection and Determination of Macrolide Resistance in Mycoplasma pneumoniae.

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.

Improving Detection and Response to Respiratory Events - Kenya, April 2016-April 2020.

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.

Development and Implementation of Multiplex TaqMan Array Cards for Specimen Testing at Child Health and Mortality Prevention Surveillance Site Laboratories.

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.

Mycoplasma pneumoniae Among Children Hospitalized With Community-acquired Pneumonia.

Background: The epidemiology of Mycoplasma pneumoniae (Mp) among US children (<18 years) hospitalized with community-acquired pneumonia (CAP) is poorly understood. Methods: In the Etiology of Pneumonia in the Community 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: One hundred and eighty two (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 4% (6/169) isolates. Of 178 (98%) Mp PCR-positive children tested for copathogens, 50 (28%) had ≥1 copathogen detected. Variables significantly associated with higher odds of Mp detection included age (10-17 years: adjusted odds ratio [aOR], 10.7 [95% confidence interval {CI}, 5.4-21.1] and 5-9 years: aOR, 6.4 [95% CI, 3.4-12.1] vs 2-4 years), outpatient antibiotics ≤5 days preadmission (aOR, 2.3 [95% CI, 1.5-3.5]), and copathogen detection (aOR, 2.1 [95% CI, 1.3-3.3]). Clinical characteristics were non-specific. Conclusions: Usually considered as a mild respiratory infection, Mp was the most commonly detected bacteria among children aged ≥5 years hospitalized with CAP, one-quarter of whom had codetections. Although associated with clinically nonspecific symptoms, there was a need for intensive care in some cases. Mycoplasma pneumoniae should be included in the differential diagnosis for school-aged children hospitalized with CAP.

Psittacosis Outbreak among Workers at Chicken Slaughter Plants, Virginia and Georgia, USA, 2018.

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.

Causes and incidence of community-acquired serious infections among young children in south Asia (ANISA): an observational cohort study.

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.

Epidemiology and Molecular Identification and Characterization of Mycoplasma pneumoniae, South Africa, 2012-2015.

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.

Community-Acquired Pneumonia Requiring Hospitalization among U.S. Adults.

BACKGROUND: Community-acquired pneumonia is a leading infectious cause of hospitalization and death among U.S. adults. Incidence estimates of pneumonia confirmed radiographically and with the use of current laboratory diagnostic tests are needed. METHODS: We conducted active population-based surveillance for community-acquired pneumonia requiring hospitalization among adults 18 years of age or older in five hospitals in Chicago and Nashville. Patients with recent hospitalization or severe immunosuppression were excluded. Blood, urine, and respiratory specimens were systematically collected for culture, serologic testing, antigen detection, and molecular diagnostic testing. Study radiologists independently reviewed chest radiographs. We calculated population-based incidence rates of community-acquired pneumonia requiring hospitalization according to age and pathogen. RESULTS: From January 2010 through June 2012, we enrolled 2488 of 3634 eligible adults (68%). Among 2320 adults with radiographic evidence of pneumonia (93%), the median age of the patients was 57 years (interquartile range, 46 to 71); 498 patients (21%) required intensive care, and 52 (2%) died. Among 2259 patients who had radiographic evidence of pneumonia and specimens available for both bacterial and viral testing, a pathogen was detected in 853 (38%): one or more viruses in 530 (23%), bacteria in 247 (11%), bacterial and viral pathogens in 59 (3%), and a fungal or mycobacterial pathogen in 17 (1%). The most common pathogens were human rhinovirus (in 9% of patients), influenza virus (in 6%), and Streptococcus pneumoniae (in 5%). The annual incidence of pneumonia was 24.8 cases (95% confidence interval, 23.5 to 26.1) per 10,000 adults, with the highest rates among adults 65 to 79 years of age (63.0 cases per 10,000 adults) and those 80 years of age or older (164.3 cases per 10,000 adults). For each pathogen, the incidence increased with age. CONCLUSIONS: The incidence of community-acquired pneumonia requiring hospitalization was highest among the oldest adults. Despite current diagnostic tests, no pathogen was detected in the majority of patients. Respiratory viruses were detected more frequently than bacteria. (Funded by the Influenza Division of the National Center for Immunizations and Respiratory Diseases.).

Community-acquired pneumonia requiring hospitalization among U.S. children.

BACKGROUND: Incidence estimates of hospitalizations for community-acquired pneumonia among children in the United States that are based on prospective data collection are limited. Updated estimates of pneumonia that has been confirmed radiographically and with the use of current laboratory diagnostic tests are needed. METHODS: We conducted active population-based surveillance for community-acquired pneumonia requiring hospitalization among children younger than 18 years of age in three hospitals in Memphis, Nashville, and Salt Lake City. We excluded children with recent hospitalization or severe immunosuppression. Blood and respiratory specimens were systematically collected for pathogen detection with the use of multiple methods. Chest radiographs were reviewed independently by study radiologists. RESULTS: From January 2010 through June 2012, we enrolled 2638 of 3803 eligible children (69%), 2358 of whom (89%) had radiographic evidence of pneumonia. The median age of the children was 2 years (interquartile range, 1 to 6); 497 of 2358 children (21%) required intensive care, and 3 (<1%) died. Among 2222 children with radiographic evidence of pneumonia and with specimens available for bacterial and viral testing, a viral or bacterial pathogen was detected in 1802 (81%), one or more viruses in 1472 (66%), bacteria in 175 (8%), and both bacterial and viral pathogens in 155 (7%). The annual incidence of pneumonia was 15.7 cases per 10,000 children (95% confidence interval [CI], 14.9 to 16.5), with the highest rate among children younger than 2 years of age (62.2 cases per 10,000 children; 95% CI, 57.6 to 67.1). Respiratory syncytial virus was more common among children younger than 5 years of age than among older children (37% vs. 8%), as were adenovirus (15% vs. 3%) and human metapneumovirus (15% vs. 8%). Mycoplasma pneumoniae was more common among children 5 years of age or older than among younger children (19% vs. 3%). CONCLUSIONS: The burden of hospitalization for children with community-acquired pneumonia was highest among the very young, with respiratory viruses the most commonly detected causes of pneumonia. (Funded by the Influenza Division of the National Center for Immunization and Respiratory Diseases.).

Mycoplasma hominis Infections Transmitted Through Amniotic Tissue Product.

Background: Mycoplasma hominis is a commensal genitourinary tract organism that can cause infections outside the genitourinary tract. We investigated a cluster of M. hominis surgical site infections in patients who underwent spine surgery, all associated with amniotic tissue linked to a common donor. Methods: Laboratory tests of tissue product from the donor, including culture, quantitative real-time polymerase chain reaction (qPCR), and whole-genome sequencing were performed. Use of this amniotic tissue product was reviewed. A multistate investigation to identify additional cases and locate any unused products was conducted. Results: Twenty-seven tissue product vials from a donor were distributed to facilities in 7 states; at least 20 vials from this donor were used in 14 patients. Of these, 4 of 14 (29%) developed surgical site infections, including 2 M. hominis infections. Mycoplasma hominis was detected by culture and qPCR in 2 unused vials from the donor. Sequencing indicated >99% similarity between patient and unopened vial isolates. For 5 of 27 (19%) vials, the final disposition could not be confirmed. Conclusions: Mycoplasma hominis was transmitted through amniotic tissue from a single donor to 2 recipients. Current routine donor screening and product testing does not detect all potential pathogens. Clinicians should be aware that M. hominis can cause surgical site infections, and may not be detected by routine clinical cultures. The lack of a standardized system to track tissue products in healthcare facilities limits the ability of public health agencies to respond to outbreaks and investigate other adverse events associated with these products.

Molecular Characterization of Mycoplasma pneumoniae Infections in Two Rural Populations of Thailand from 2009 to 2012.

Studies on Mycoplasma pneumoniae in Thailand have focused on urban centers and have not included molecular characterization. In an attempt to provide a more comprehensive understanding of this organism, we conducted a systematic random sampling to identify 3,000 nasopharyngeal swab specimens collected from January 2009 through July 2012 during population-based surveillance for influenza-like illness in two rural provinces. M. pneumoniae was detected by real-time PCR in 175 (5.8%) specimens. Genotyping was performed using the major adhesion protein (P1) and multilocus variable-number tandem-repeat analysis (MLVA). Of the 157 specimens typed, 97 were P1 type 1 and 60 were P1 type 2. Six different MLVA profiles were identified in 149 specimens, with 4/5/7/2 (40%) and 3/5/6/2 (26%) predominating. There was no discrete seasonality to M. pneumoniae infections. Examination of the 23S rRNA sequence for known polymorphisms conferring macrolide resistance revealed that all 141 tested to possess the genotype associated with macrolide susceptibility.

Improved Detection of Respiratory Pathogens by Use of High-Quality Sputum with TaqMan Array Card Technology.

New diagnostic platforms often use nasopharyngeal or oropharyngeal (NP/OP) swabs for pathogen detection for patients hospitalized with community-acquired pneumonia (CAP). We applied multipathogen testing to high-quality sputum specimens to determine if more pathogens can be identified relative to NP/OP swabs. Children (<18 years old) and adults hospitalized with CAP were enrolled over 2.5 years through the Etiology of Pneumonia in the Community (EPIC) study. NP/OP specimens with matching high-quality sputum (defined as ≤10 epithelial cells/low-power field [lpf] and ≥25 white blood cells/lpf or a quality score [q-score] definition of 2+) were tested by TaqMan array card (TAC), a multipathogen real-time PCR detection platform. Among 236 patients with matched specimens, a higher proportion of sputum specimens had ≥1 pathogen detected compared with NP/OP specimens in children (93% versus 68%; P < 0.0001) and adults (88% versus 61%; P < 0.0001); for each pathogen targeted, crossing threshold (CT) values were earlier in sputum. Both bacterial (361 versus 294) and viral detections (245 versus 140) were more common in sputum versus NP/OP specimens, respectively, in both children and adults. When available, high-quality sputum may be useful for testing in hospitalized CAP patients.

Rapid Laboratory Identification of Neisseria meningitidis Serogroup C as the Cause of an Outbreak - Liberia, 2017.

On April 25, 2017, a cluster of unexplained illness and deaths among persons who had attended a funeral during April 21-22 was reported in Sinoe County, Liberia (1). Using a broad initial case definition, 31 cases were identified, including 13 (42%) deaths. Twenty-seven cases were from Sinoe County (1), and two cases each were from Grand Bassa and Monsterrado counties, respectively. On May 5, 2017, initial multipathogen testing of specimens from four fatal cases using the Taqman Array Card (TAC) assay identified Neisseria meningitidis in all specimens. Subsequent testing using direct real-time polymerase chain reaction (PCR) confirmed N. meningitidis in 14 (58%) of 24 patients with available specimens and identified N. meningitidis serogroup C (NmC) in 13 (54%) patients. N. meningitidis was detected in specimens from 11 of the 13 patients who died; no specimens were available from the other two fatal cases. On May 16, 2017, the National Public Health Institute of Liberia and the Ministry of Health of Liberia issued a press release confirming serogroup C meningococcal disease as the cause of this outbreak in Liberia.

Current and emerging Legionella diagnostics for laboratory and outbreak investigations.

Legionnaires' disease (LD) is an often severe and potentially fatal form of bacterial pneumonia caused by an extensive list of Legionella species. These ubiquitous freshwater and soil inhabitants cause human respiratory disease when amplified in man-made water or cooling systems and their aerosols expose a susceptible population. Treatment of sporadic cases and rapid control of LD outbreaks benefit from swift diagnosis in concert with discriminatory bacterial typing for immediate epidemiological responses. Traditional culture and serology were instrumental in describing disease incidence early in its history; currently, diagnosis of LD relies almost solely on the urinary antigen test, which captures only the dominant species and serogroup, Legionella pneumophila serogroup 1 (Lp1). This has created a diagnostic "blind spot" for LD caused by non-Lp1 strains. This review focuses on historic, current, and emerging technologies that hold promise for increasing LD diagnostic efficiency and detection rates as part of a coherent testing regimen. The importance of cooperation between epidemiologists and laboratorians for a rapid outbreak response is also illustrated in field investigations conducted by the CDC with state and local authorities. Finally, challenges facing health care professionals, building managers, and the public health community in combating LD are highlighted, and potential solutions are discussed.