The introduction of in vitro nucleic acid amplification techniques, led by real-time PCR, into the clinical microbiology laboratory has transformed the laboratory detection of viruses and select bacterial pathogens. However, the progression of the molecular diagnostic revolution currently relies on the ability to efficiently and accurately offer multiplex detection and characterization for a variety of infectious disease pathogens. Microarray analysis has the capability to offer robust multiplex detection but has just started to enter the diagnostic microbiology laboratory. Multiple microarray platforms exist, including printed double-stranded DNA and oligonucleotide arrays, in situ-synthesized arrays, high-density bead arrays, electronic microarrays, and suspension bead arrays. One aim of this paper is to review microarray technology, highlighting technical differences between them and each platform's advantages and disadvantages. Although the use of microarrays to generate gene expression data has become routine, applications pertinent to clinical microbiology continue to rapidly expand. This review highlights uses of microarray technology that impact diagnostic microbiology, including the detection and identification of pathogens, determination of antimicrobial resistance, epidemiological strain typing, and analysis of microbial infections using host genomic expression and polymorphism profiles.

Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology

The present invention provides for a method of preparing a target nucleic acid fragments to produce a smaller nucleic acid which comprises the two ends of the target nucleic acid. Specifically, the invention provides cloning and DNA manipulation strategies to isolate the two ends of a large target nucleic acid into a single small DNA construct for rapid cloning, sequencing, or amplification.

Paired end sequencing

OBJECTIVE\nTo determine the cost-effectiveness and cost-benefit of an infection control program to reduce nosocomial respiratory syncytial virus (RSV) transmission in a large pediatric hospital.\n\n\nDESIGN\nRSV nosocomial infection (NI) was studied for 8 years, before and after intervention with a targeted infection control program. The cost-effectiveness of the intervention was calculated, and cost-benefit was estimated by a case-control comparison.\n\n\nSETTING\nChildren's Hospital of Philadelphia, a 304-bed pediatric hospital.\n\n\nPATIENTS\nAll inpatients with RSV infection, both community- and hospital-acquired.\n\n\nINTERVENTION\nConsisted of early recognition of patients with respiratory symptoms, confirmation of RSV infection by laboratory testing, establishing cohorts of patients and nursing staff, gown and glove barrier precautions, and monitoring and education of staff.\n\n\nOUTCOME MEASURES\nThe incidence density of RSV NI before and after the intervention was calculated as the rate per 1000 patient days-at-risk for infection. Intervention costs included laboratory testing, isolation, and administration of the program. The cost of RSV NI was estimated by comparing hospital charges for 30 cases and matched uninfected controls.\n\n\nRESULTS\nA total of 148 patients acquired NI (88 before and 60 after the intervention). The Mantel-Haenszel stratified relative risk for NI in the period before the infection control program, compared with the postintervention period, was.61 (95% confidence interval:.53-.69). By applying the preintervention stratum-specific rates of infection to the days-at-risk in the postintervention period, an estimated 100 NIs would have been expected, which in comparison to the 60 NIs observed, yielded an estimated program effectiveness of 10 RSV NIs prevented per season. The total cost of the program per season was $15 627 or $1,563/NI prevented. In comparison, the mean cost to the hospital was $9,419/case of RSV NI, resulting in a cost-benefit ratio of 1:6.\n\n\nCONCLUSIONS\nA targeted infection control intervention was cost-effective in reducing the rate of RSV NI. For every dollar spent on the program, approximately $6 was saved.

Nosocomial respiratory syncytial virus infections : The cost-effectiveness and cost-benefit of infection control.

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.

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

Method for detecting and identifying unknown bioagents, including bacteria, viruses and the like, by a combination of nucleic acid amplification and molecular weight determination using primers which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The result is a “base composition signature” (BCS) which is then matched against a database of base composition signatures, by which the bioagent is identified.

Method for rapid detection and identification of bioagents

We report on the use of an electronic microarray to simultaneously type influenza A and B viruses and to distinguish influenza A virus subtypes H1N1 and H3N2 from the potentially pandemic avian virus subtype H5N1. The assay targets seven genes: the H1, H3, H5, N1, and N2 genes of influenza A virus; the matrix protein M1 gene of influenza A virus; and the nonstructural protein (NS) gene of influenza B virus. By combining a two-step reverse transcription-multiplex PCR with typing and subtyping on the electronic microarray, the assay achieved an analytical sensitivity of 102 to 103 copies of transcripts per reaction for each of the genes. The assay correctly typed and subtyped 15 different influenza virus isolates, including two influenza B virus, five A/H1N1, six A/H3N2, and two A/H5N1 isolates. In addition, the assay correctly identified 8 out of 10 diluted, archived avian influenza virus specimens with complete typing and subtyping information and 2 specimens with partial subtyping information. In a study of 146 human clinical specimens that had previously been shown to be positive for influenza virus or another respiratory virus, the assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The assay is a rapid, accurate, user-friendly method for simultaneously typing and subtyping influenza viruses.

Multiplex Assay for Simultaneously Typing and Subtyping Influenza Viruses by Use of an Electronic Microarray

The invention includes compositions and methods of detection of Bacillus anthracis that use oligonucleotide probes specific for genetic material contained in the pXO1 and pX02 plasmids in nucleic acid hybridization reactions. Embodiments of the method may include additional probes specific for other gene sequences to distinguish B. anthracis from other bacterial species present in a sample or to provide an indication that the assay was performed properly even when no Bacillus sequence is detected.

Assay and compositions for detection of Bacillus anthracis nucleic acid

Nucleic acid oligonucleotide sequences are disclosed which include amplification oligomers and probe oligomers which are useful for detecting multiple types of human papillomaviruses (HPV) associated with cervical cancer. Methods for detecting multiple HPV types in biological specimens by amplifying HPV nucleic acid sequences in vitro and detecting the amplified products are disclosed.

Detection of nucleic acids from multiple types of human papillomaviruses

The NanoChip400 system uses multiplex PCR chemistry and electronic microarray detection of influenza A and B viruses; respiratory syncytial viruses A and B; and human parainfluenza virus types 1, 2, and 3. The results obtained with the NanoChip 400 system were compared with those obtained by direct fluorescent-antibody staining (DFA) and real-time PCR with 122 and 130 specimens, respectively. Concordance between DFA and NanoChip 400 system was obtained for 106 of 122 (86.9%) specimens. On the basis of discrepancy analysis with specimens available for confirmatory real-time PCR testing, the sensitivity and specificity of the NanoChip 400 were 98.6% and 100%, respectively. With respect to specimens previously tested by real-time PCR, the NanoChip 400 system demonstrated a sensitivity of 91.1% and a specificity of 100%. The NanoChip 400 system provides clinical laboratories with a practical, rapid, and sensitive method for the detection of common respiratory viruses.

Evaluation of the NanoChip 400 System for Detection of Influenza A and B, Respiratory Syncytial, and Parainfluenza Viruses

Methods for detecting influenza virus A and influenza virus B nucleic acids in biological samples by using in vitro amplification and detection are disclosed. Compositions that are target-specific nucleic acid sequences and kits comprising target-specific nucleic acid oligomers for amplifying in vitro influenza virus A or influenza virus B nucleic acid and detecting amplified nucleic acid sequences are disclosed.

Sylvia A. Norman

Papers

Multiplex Assay for Simultaneously Typing and Subtyping Influenza Viruses by Use of an Electronic Microarray

Assay and compositions for detection of Bacillus anthracis nucleic acid

Detection of nucleic acids from multiple types of human papillomaviruses

Evaluation of the NanoChip 400 System for Detection of Influenza A and B, Respiratory Syncytial, and Parainfluenza Viruses

Compositions and assays to detect influenza virus A and B nucleic acids