Swati Kumar

Acute Respiratory Infection Due to Chlamydia pneumoniae: Current Status of Diagnostic Methods

Reliable diagnosis of respiratory infection due to Chlamydia pneumoniae and investigation of its role in chronic diseases remain difficult because of the absence of well-standardized and commercially available diagnostic tests. In 2001, the US Centers for Disease Control and Prevention published recommendations for standardizing the diagnostic approach. In this review, we discuss the current state of knowledge of C. pneumoniae-associated respiratory infections in the context of epidemiological studies published during the past 5 years, with particular emphasis on the diagnostic strategies used and their impact on results. The single most likely factor underlying wide variations in data is the significant interstudy variation of the choice of diagnostic methods and criteria used. Adoption of a more unified approach, both for choices of diagnostic methods and for validation of new molecular assays, is long overdue and will be critically important for development of a standardized test for clinical laboratories.

Detection of 11 Common Viral and Bacterial Pathogens Causing Community-Acquired Pneumonia or Sepsis in Asymptomatic Patients by Using a Multiplex Reverse Transcription-PCR Assay with Manual (Enzyme Hybridization) or Automated (Electronic Microarray) Detection

ABSTRACT Community-acquired pneumonia (CAP) and sepsis are important causes of morbidity and mortality. We describe the development of two molecular assays for the detection of 11 common viral and bacterial agents of CAP and sepsis: influenza virus A, influenza virus B, respiratory syncytial virus A (RSV A), RSV B, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Legionella micdadei, Bordetella pertussis, Staphylococcus aureus, and Streptococcus pneumoniae. Further, we report the prevalence of carriage of these pathogens in respiratory, skin, and serum specimens from 243 asymptomatic children and adults. The detection of pathogens was done using both a manual enzyme hybridization assay and an automated electronic microarray following reverse transcription and PCR amplification. The analytical sensitivities ranged between 0.01 and 100 50% tissue culture infective doses, cells, or CFU per ml for both detection methods. Analytical specificity testing demonstrated no significant cross-reactivity among 19 other common respiratory organisms. One hundred spiked “surrogate” clinical specimens were all correctly identified with 100% specificity (95% confidence interval, 100%). Overall, 28 (21.7%) of 129 nasopharyngeal specimens, 11 of 100 skin specimens, and 2 of 100 serum specimens from asymptomatic subjects tested positive for one or more pathogens, with S. pneumoniae and S. aureus giving 89% of the positive results. Our data suggest that asymptomatic carriage makes the use of molecular assays problematic for the detection of S. pneumoniae or S. aureus in upper respiratory tract secretions; however, the specimens tested showed virtually no carriage of the other nine viral and bacterial pathogens, and the detection of these pathogens should not be a significant diagnostic problem. In addition, slightly less sensitive molecular assays may have better correlation with clinical disease in the case of CAP.

Update on Influenza Diagnostics: Lessons from the Novel H1N1 Influenza A Pandemic

SUMMARY The menu of diagnostic tools that can be utilized to establish a diagnosis of influenza is extensive and includes classic virology techniques as well as new and emerging methods. This review of how the various existing diagnostic methods have been utilized, first in the context of a rapidly evolving outbreak of novel influenza virus and then during the different subsequent phases and waves of the pandemic, demonstrates the unique roles, advantages, and limitations of each of these methods. Rapid antigen tests were used extensively throughout the pandemic. Recognition of the low negative predictive values of these tests is important. Private laboratories with preexisting expertise, infrastructure, and resources for rapid development, validation, and implementation of laboratory-developed assays played an unprecedented role in helping to meet the diagnostic demands during the pandemic. FDA-cleared assays remain an important element of the diagnostic armamentarium during a pandemic, and a process must be developed with the FDA to allow manufacturers to modify these assays for detection of novel strains in a timely fashion. The need and role for subtyping of influenza viruses and antiviral susceptibility testing will likely depend on qualitative (circulating subtypes and their resistance patterns) and quantitative (relative prevalence) characterization of influenza viruses circulating during future epidemics and pandemics.

Clinical and epidemiologic characteristics of children hospitalized with 2009 pandemic H1N1 influenza A infection.

Background: In 2009, pandemic H1N1 influenza caused significant morbidity and mortality worldwide. We describe the clinical and epidemiologic characteristics of children and adolescents hospitalized for 2009 H1N1 infections in Milwaukee, Wisconsin from April 2009 to August 2009. Methods: We conducted retrospective chart reviews of hospitalized patients with laboratory-confirmed 2009 H1N1 infections. Data on financial burden associated with these infections were obtained and analyzed. Results: A total of 75 children hospitalized for 2009 pandemic H1N1 infections were identified; the median age was 5 years (range, 2 months–19.2 years); 56% were males; 56% were Non-Hispanic Blacks; and 75% had at least one underlying medical condition. Twenty-four percent had only upper respiratory symptoms. Bacterial coinfections occurred in 1.3%. All but one patient received antivirals, 80% of patients received antibacterials, 18.6% were admitted to the intensive care unit, 6% required mechanical ventilation, 2.6% required extracorporeal membrane oxygenation, and 2.6% died. The total charges incurred for H1N1 influenza hospitalizations were

Phylogeography of the Spring and Fall Waves of the H1N1/09 Pandemic Influenza Virus in the United States

4,454,191, with individual charges being highest for children >12 years of age. Conclusions: The majority of children with pandemic H1N1 influenza-associated hospitalizations had uncomplicated illness despite the frequent presence of high-risk conditions in our patient population. Laboratory-confirmed 2009 pandemic H1N1 influenza hospitalizations resulted in substantial health care and economic burden during the first wave of the pandemic in spring 2009.

Whole Genome Sequencing and Evolutionary Analysis of Human Respiratory Syncytial Virus A and B from Milwaukee, WI 1998-2010

ABSTRACT Spatial variation in the epidemiological patterns of successive waves of pandemic influenza virus in humans has been documented throughout the 20th century but never understood at a molecular level. However, the unprecedented intensity of sampling and whole-genome sequencing of the H1N1/09 pandemic virus now makes such an approach possible. To determine whether the spring and fall waves of the H1N1/09 influenza pandemic were associated with different epidemiological patterns, we undertook a large-scale phylogeographic analysis of viruses sampled from three localities in the United States. Analysis of genomic and epidemiological data reveals distinct spatial heterogeneities associated with the first pandemic wave, March to July 2009, in Houston, TX, Milwaukee, WI, and New York State. In Houston, no specific H1N1/09 viral lineage dominated during the spring of 2009, a period when little epidemiological activity was observed in Texas. In contrast, major pandemic outbreaks occurred at this time in Milwaukee and New York State, each dominated by a different viral lineage and resulting from strong founder effects. During the second pandemic wave, beginning in August 2009, all three U.S. localities were dominated by a single viral lineage, that which had been dominant in New York during wave 1. Hence, during this second phase of the pandemic, extensive viral migration and mixing diffused the spatially defined population structure that had characterized wave 1, amplifying the one viral lineage that had dominated early on in one of the worlds largest international travel centers.

Development of a Rapid Automated Influenza A, Influenza B, and Respiratory Syncytial Virus A/B Multiplex Real-Time RT-PCR Assay and Its Use during the 2009 H1N1 Swine-Origin Influenza Virus Epidemic in Milwaukee, Wisconsin

Background Respiratory Syncytial Virus (RSV) is the leading cause of lower respiratory-tract infections in infants and young children worldwide. Despite this, only six complete genome sequences of original strains have been previously published, the most recent of which dates back 35 and 26 years for RSV group A and group B respectively. Methodology/Principal Findings We present a semi-automated sequencing method allowing for the sequencing of four RSV whole genomes simultaneously. We were able to sequence the complete coding sequences of 13 RSV A and 4 RSV B strains from Milwaukee collected from 1998–2010. Another 12 RSV A and 5 RSV B strains sequenced in this study cover the majority of the genome. All RSV A and RSV B sequences were analyzed by neighbor-joining, maximum parsimony and Bayesian phylogeny methods. Genetic diversity was high among RSV A viruses in Milwaukee including the circulation of multiple genotypes (GA1, GA2, GA5, GA7) with GA2 persisting throughout the 13 years of the study. However, RSV B genomes showed little variation with all belonging to the BA genotype. For RSV A, the same evolutionary patterns and clades were seen consistently across the whole genome including all intergenic, coding, and non-coding regions sequences. Conclusions/Significance The sequencing strategy presented in this work allows for RSV A and B genomes to be sequenced simultaneously in two working days and with a low cost. We have significantly increased the amount of genomic data that is available for both RSV A and B, providing the basic molecular characteristics of RSV strains circulating in Milwaukee over the last 13 years. This information can be used for comparative analysis with strains circulating in other communities around the world which should also help with the development of new strategies for control of RSV, specifically vaccine development and improvement of RSV diagnostics.

Rapid Semiautomated Subtyping of Influenza Virus Species during the 2009 Swine Origin Influenza A H1N1 Virus Epidemic in Milwaukee, Wisconsin

Rapid, semiautomated, and fully automated multiplex real-time RT-PCR assays were developed and validated for the detection of influenza (Flu) A, Flu B, and respiratory syncytial virus (RSV) from nasopharyngeal specimens. The assays can detect human H1N1, H3N2, and swine-origin (S-OIV) H1N1 Flu A viruses and were effectively used to distinguish Flu A infections (of all subtypes) from Flu B and RSV infections during the current S-OIV outbreak in Milwaukee, WI. The analytical limits of detection were 10−2 to 101 TCID50/ml depending on the platform and analyte and showed only one minor cross-reaction among 23 common respiratory pathogens (intermittent cross-reaction to adenovirus at >107 TCID50/ml). A total of 100 clinical samples were tested by tissue culture, both automated assays, and the US Food and Drug Administration-approved ProFlu+ assay. Both the semiautomated and fully automated assays exhibited greater overall (Flu A, Flu B, and RSV combined) clinical sensitivities (93 and 96%, respectively) and individual Flu A sensitivities (100%) than the Food and Drug Administration-approved test (89% overall sensitivity and 93% Flu A sensitivity). All assays were 99% specific. During the S-OIV outbreak in Milwaukee, WI, the fully automated assay was used to test 1232 samples in 2 weeks. Flu A was detected in 134 clinical samples (126 H1N1 S-OIV, 5 H1N1 [human], and 1 untyped) with 100% positive agreement compared with other “in-house” validated molecular assays, with only 2 false-positive results. Such accurate testing using automated high-throughput molecule systems should allow clinicians and public health officials to react quickly and effectively during viral outbreaks.

Utilization of Nucleic Acid Amplification Assays for the Detection of Respiratory Viruses

ABSTRACT In the spring of 2009, a novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) emerged and began causing a large outbreak of illness in Milwaukee, WI. Our group at the Midwest Respiratory Virus Program laboratory developed a semiautomated real-time multiplex reverse transcription-PCR assay (Seasonal), employing the NucliSENS easyMAG system (bioMérieux, Durham, NC) and a Raider thermocycler (HandyLab Inc., Ann Arbor, MI), that typed influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) and subtyped influenza A virus into the currently circulating H1 and H3 subtypes, as well as a similar assay that identified H1 of S-OIV. The Seasonal and H1 S-OIV assays demonstrated analytical limits of detection of <50 50% tissue culture infective doses/ml and 3 to 30 input copies, respectively. Testing of the analytical specificities revealed no cross-reactivity with 41 and 26 different common organisms and demonstrated outstanding reproducibility of results. Clinical testing showed 95% sensitivity for influenza A virus and influenza B virus and 95 and 97% specificity compared to tissue culture. Comparisons of results from other molecular tests showed levels of positive agreement with the Seasonal and H1 S-OIV assay results of 99 and 100% and levels of negative agreement of 98 and 100%. This study has demonstrated the use of a semiautomated system for sensitive, specific, and rapid detection of influenza A virus, influenza B virus, and RSV and subtyping of influenza A virus into human H1 and H3 and S-OIV strains. This assay/system performed well in clinical testing of regular seasonal influenza virus subtypes and was outstanding during the 2009 Milwaukee S-OIV infection outbreak. This recent outbreak of infection with a novel influenza A (H1N1) virus also demonstrates the importance of quickly distributing information on new agents and of having rapid influenza virus subtyping assays widely available for clinical and public health decisions.

Infection with Simkania negevensis in Brooklyn, New York.

Viruses are major contributors to morbidity and mortality from acute respiratory infections in all age groups worldwide. Accurate identification of the etiologic agent of respiratory tract infections is important for proper patient management. Diagnosis can be problematic, because a range of potential pathogens can cause similar clinical symptoms. Nucleic acid amplification testing is emerging as the preferred method of diagnostic testing. Real-time technology and the ability to perform multiplex testing have facilitated this emergence. Commercial platforms for nucleic acid amplification testing of respiratory viruses include real-time polymerase chain reaction (PCR), nucleic acid sequence-based amplification, and loop-mediated isothermal amplification. Multiplex PCR with fluidic microarrays or DNA chips are the most recent diagnostic advance. These assays offer significant advantages in sensitivity over antigen detection methods and in most cases also over traditional culture methods. A limited number of assays, however, are commercially available, thus laboratory developed assays frequently are used. This article reviews the performance of commercially available assays and discusses issues relevant to the development of in-house assays.