Comparison of the EntericBio Multiplex PCR System with Routine Culture for Detection of Bacterial Enteric Pathogens

The EntericBio system uses a multiplex PCR assay for the simultaneous detection of Campylobacter spp., Salmonella enterica, Shigella spp., and Escherichia coli O157 from feces. It combines overnight broth enrichment with PCR amplification and detection by hybridization. An evaluation of this system was conducted by comparing the results obtained with the system with those obtained by routine culture, supplemented with alternative PCR detection methods. In a study of 773 samples, routine culture and the EntericBio system yielded 94.6 and 92.4% negative results, respectively. Forty-two samples had positive results by culture, and all of these were positive with the EntericBio system. This system detected an additional 17 positive samples (Campylobacter spp., n = 12; Shigella spp., n = 1; E. coli O157, n = 4), but the results for 5 samples (Campylobacter spp., n = 2; Shigella spp., n = 1; E. coli O157, n = 2) could not be confirmed. The target for Shigella spp. detected by the EntericBio system is the ipaH gene, and the molecular indication of the presence of Shigella spp. was investigated by sequence analysis, which confirmed that the ipaH gene was present in a Klebsiella pneumoniae isolate from the patient. The sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 99.3%, 91.5%, and 100%, respectively. Turnaround times were significantly reduced with the EntericBio system, and a result was available between 24 and 32 h after receipt of the sample in the laboratory. In addition, the amount of laboratory waste was significantly reduced by use of this system. In summary, the EntericBio system proved convenient to use, more sensitive than the conventional culture used in this study, and highly specific; and it generated results significantly faster than routine culture for the pathogens tested.Infectious gastroenteritis is a leading cause of morbidity and mortality worldwide (3), particularly in developing countries (2). There are also a significant economic and social costs associated with gastroenteritis (14), in addition to increased morbidity and mortality (5).Conventional culture methods remain the norm for the isolation of bacterial enteric pathogens in clinical laboratories. A major advantage of molecular methods is the reduced time to detection (13). Faster diagnostic outputs allow earlier epidemiological investigations and infection control interventions. Furthermore, the use of molecular methods highlights that conventional methods for the isolation of Campylobacter are less sensitive than PCR (15). Molecular methods suitable for use in the detection of fecal enteric pathogens have not been routinely available to clinical laboratories, until recently. Here we describe a new multiplex PCR method for clinical diagnostic use. The method combines an overnight enrichment step with PCR and hybridization detection by use of a line blot assay for the simultaneous detection of Campylobacter spp., Salmonella enterica, Shigella spp., and Escherichia coli O157 from feces. The application of the system in a clinical laboratory is also described.     

Improved Detection of Bacterial Central Nervous System Infections by Use of a Broad-Range PCR Assay

A universal PCR assay for bacteria and fungi detected meningitis pathogens in 65% of 20 cerebrospinal fluid (CSF) samples from patients with suspected central nervous system (CNS) infections compared to a 35% detection rate by culture and/or microscopy methods. Thus, the PCR assay can improve the diagnosis rate of infective meningitis when standard methods provide a negative result.     

Utility of a Commercially Available Multiplex Real-Time PCR Assay To Detect Bacterial and Fungal Pathogens in Febrile Neutropenia

Infection is the main treatment-related cause of mortality in cancer patients. Rapid and accurate diagnosis to facilitate specific therapy of febrile neutropenia is therefore urgently warranted. Here, we evaluated a commercial PCR-based kit to detect the DNA of 20 different pathogens (SeptiFast) in the setting of febrile neutropenia after chemotherapy. Seven hundred eighty-four serum samples of 119 febrile neutropenic episodes (FNEs) in 70 patients with hematological malignancies were analyzed and compared with clinical, microbiological, and biochemical findings. In the antibiotic-naïve setting, bacteremia was diagnosed in 34 FNEs and 11 of them yielded the same result in the PCR. Seventy-three FNEs were negative in both systems, leading to an overall agreement in 84 of 119 FNEs (71%). During antibiotic therapy, positivity in blood culture occurred only in 3% of cases, but the PCR yielded a positive result in 15% of cases. In six cases the PCR during antibiotic treatment detected a new pathogen repetitively; this was accompanied by a significant rise in procalcitonin levels, suggestive of a true detection of infection. All patients with probable invasive fungal infection (IFI; n = 3) according to the standards of the European Organization for Research and Treatment of Cancer had a positive PCR result for Aspergillus fumigatus; in contrast there was only one positive result for Aspergillus fumigatus in an episode without signs and symptoms of IFI. Our results demonstrate that the SeptiFast kit cannot replace blood cultures in the diagnostic workup of FNEs. However, it might be helpful in situations where blood cultures remain negative (e.g., during antimicrobial therapy or in IFI).While systemic infection is the most common cause of a febrile neutropenia episode (FNE) with significant effects on morbidity and mortality, only 30% of blood cultures taken at the onset of fever are positive (11, 15). Nonetheless, patients with FNEs are treated with broad-spectrum antimicrobial agents regardless of the result of their blood culture (7) because potentially life-threatening infections need early treatment to ensure better clinical outcome. Noninfective causes of a systemic reaction culminating in a rise in temperature such as tumor fever, drug fever, or transfusion reactions complicate the diagnostic challenge in cancer patients. In addition, the etiology of a deterioration of an FNE during antimicrobial therapy is often difficult to elucidate, since blood cultures are infrequently positive once effective antimicrobial therapy has started (4). Pathogens such as molds which are rarely found in blood cultures are not uncommon in patients with FNEs, particularly if they suffer from hematological malignancies. For these reasons, FNE is one of the conditions where new diagnostic tools to distinguish an infection from a nonmicrobial cause for fever or to identify rare pathogens are most urgently needed. In the past, raised levels of indirect markers such as procalcitonin (PCT) and interleukin 6 (3, 16) have been shown to be associated with bacteremia. Ideally, though, the cause should be identified directly and improvements in the detection of pathogens in the bloodstream should be made. In addition to refinements of the classical blood culture systems, attempts have been made to detect pathogen DNA by means of PCR. Initially, this involved conventional PCR techniques (9) detecting the gene for the 16S subunit of bacterial rRNA for the presence of bacterial DNA. Specification was then carried out by sequencing the PCR product. Later, more rapid methods were developed when real-time PCR became available (1, 14, 17). PCR results are more readily available, and the method also detects remnants of bacteria, which might make it more robust to the influence of antibiotic treatment, while potentially detecting pathogens which do not grow in the blood cultures. However, the main disadvantage, apart from higher costs, a potential for false-positive results during transient bacteremia/fungemia (e.g., during brushing of teeth), and laboratory workload, is the restriction of the spectrum of species detected. In addition, because most PCR methods use smaller sample volumes (commonly 1 to 4 ml [9, 17]), this method depends on a higher concentration of bacteria than that for the blood culture, which theoretically can reveal positive results after one living and propagating bacterial cell has been injected into the culture bottle.A new commercially available kit (SeptiFast) to detect DNA from 20 clinically relevant pathogens has recently been evaluated in a small cohort of neutropenic patients (10) with promising results. The aim of our study was to evaluate the usefulness of the SeptiFast kit in a larger cohort of patients with febrile neutropenia after chemotherapy for hematological malignancies. Also, we sought to determine a correlation between SeptiFast results and clinical findings. Altogether 784 samples from 119 FNEs in 70 patients were analyzed and compared with clinical, microbiological, and biochemical findings in the antibiotic-naïve setting and during antimicrobial therapy.     

Improved Detection of Five Major Gastrointestinal Pathogens by Use of a Molecular Screening Approach

The detection of bacterial and parasitic gastrointestinal pathogens through culture and microscopy is laborious and time-consuming. We evaluated a molecular screening approach (MSA) for the detection of five major enteric pathogens: Salmonella enterica, Campylobacter jejuni, Giardia lamblia, Shiga toxin-producing Escherichia coli (STEC), and Shigella spp./enteroinvasive E. coli (EIEC), for use in the daily practice of a clinical microbiology laboratory. The MSA consists of prescreening of stool specimens with two real-time multiplex PCR (mPCR) assays, which give results within a single working day, followed by guided culture/microscopy of the positive or mPCR-inhibited samples. In the present 2-year overview, 28,185 stool specimens were included. The MSA was applied to 13,974 stool samples (49.6%), whereas 14,211 samples were tested by conventional methods only (50.4%). The MSA significantly increased the total detection rate compared to that of conventional methods (19.2% versus 6.4%). The detection of all included pathogens, with the exception of S. enterica, significantly improved. MSA detection frequencies were as follows: C. jejuni, 8.1%; G. lamblia, 4.7%; S. enterica, 3.0%; STEC, 1.9%; and Shigella spp./EIEC, 1.4%. The guided culture/microscopy was positive in 76.8%, 58.1%, 88.9%, 16.8%, and 18.1% of mPCR-positive specimens, respectively. Of all mPCRs, only 1.8% was inhibited. Other findings were that detection of mixed infections was increased (0.9% versus 0.02%) and threshold cycle (C_T) values for MSA guided culture/microscopy-positive samples were significantly lower than those for guided culture/microscopy-negative samples. In conclusion, an MSA for detection of gastrointestinal pathogens resulted in markedly improved detection rates and a substantial decrease in time to reporting of (preliminary) results.Infectious gastroenteritis (IG) is one of the most common diseases worldwide, killing millions of individuals each year (3, 16). In industrialized countries, IG remains a major public health burden, although mortality is low. In the Netherlands, with a population of 16.5 million, the yearly IG incidence is approximately 4.5 million (42). Although most episodes of IG are brief and do not require medical attention, the economic and social burdens of IG are significant (38).The etiology of IG includes viral, parasitic, and bacterial pathogens. Most medical microbiology laboratories use conventional diagnostic procedures, such as culture and microscopy, for routine detection of enteric pathogens. These procedures include enrichment steps, use of selective culture media, biochemical identification, serotyping, and resistance profiling. Final results are obtained after 3 to 4 days, making these procedures laborious and time-consuming. Furthermore, the detection of pathogens in stool specimens by culture is complicated. For instance, bacteria belonging to the normal gastrointestinal flora can present with the same colony mor- phology as enteric pathogens (13, 30). The resultant misidentification increases hands-on time and delay in reporting of a definite negative result. Other problems are the viable but nonculturable state of Campylobacter jejuni (24, 29) and the limited viability of shigellae outside the human body (35). These may compromise the sensitivity of culture.Conventional laboratory diagnosis of gastrointestinal parasites consists of microscopy and/or stool antigen tests. Microscopy in particular has disadvantages, as the detection and correct identification of parasites depend upon the experience and skills of the microscopist. Also, due to intermittent shedding of protozoa the sensitivity can be low, and therefore examination of multiple samples is required (8).The workload involved in examining stool samples is high. Our laboratory receives around 15,000 stool specimens annually, with daily numbers varying greatly between seasons (42). Furthermore, the majority of stool specimens do not yield a positive result (42). Therefore, methods which quickly identify the negative specimens would facilitate routine screening. Antigen detection is a fast and effective alternative for several enteric pathogens (11, 15, 23, 27, 46). However, this method has a limited sensitivity, requires one test per pathogen, and is not available for all relevant pathogens.Molecular methods provide a means for sensitive and rapid detection of enteric pathogens. However, broad application remains limited due to their assumed high costs, inhibition caused by fecal constituents (22), and the need for specialized laboratories. Due to the high throughput of stool screening and the number of possible enteric pathogens, implementation of a molecular approach which uses multiplexing of targets is mandatory (9, 10, 20, 25, 26, 36, 43, 44, 47). For the detection of enteric pathogens it has been proven feasible to use molecular methods with improved performance and turnaround time (TAT) (7, 25, 32).Since December 2006, we have implemented a molecular screening approach (MSA) for the simultaneous detection of five pathogens: Salmonella enterica, Campylobacter jejuni, Giardia lamblia, Shiga toxin-producing Escherichia coli (STEC), and Shigella spp./enteroinvasive E. coli (EIEC). This MSA, involving two internally controlled real-time multiplex PCRs (mPCRs), is now daily practice. The present report describes our experiences with the MSA over a 2-year period (2007 to 2008).(Part of this work was presented at the 19th European Congress of Clinical Microbiology and Infectious Diseases, Helsinki, Finland, 18 May 2009.)     

Quantitative Real-Time PCR Assay Panel for Detection and Type-Specific Identification of Epidemic Respiratory Human Adenoviruses

Outbreaks of human adenovirus (HAdV) acute respiratory illness (ARI) have been well documented among civilians and unvaccinated military recruits. Among the 7 recognized HAdV species (A to G), species B (particularly serotypes 3, 7, 11, 14, and 21) and E (serotype 4) have more often been associated with epidemic ARI. Rapid detection and type-specific identification of these viruses would enhance outbreak response and help guide prevention and control measures. To this end, we developed type-specific real-time quantitative PCR (qPCR) assays for HAdV types 3, 4, 7, 11, 14, 16, and 21 targeting the HAdV hexon gene. All type-specific qPCR assays reproducibly detected as few as 10 copies/reaction of quantified hexon recombinant plasmids with a linear dynamic range of 8 log units (10¹ to 10⁸ copies); in contrast, a generic qPCR assay that detects all HAdV types run concurrently detected between 10 and 100 copies/reaction, depending on the virus type. No nonspecific amplifications were observed with concentrated nucleic acid from 51 HAdV prototype strains or other common respiratory pathogens. All members of a panel of 137 previously typed HAdV field isolates and positive clinical specimens were correctly characterized by the type-specific qPCR assays; two different HAdV types were detected in three of the clinical specimens and confirmed by amplicon sequencing. The qPCR assays permit sensitive, specific, and quantitative detection and identification of seven clinically important respiratory HAdVs and should provide a convenient adjunct to classical typing methods for a rapid response to HAdV outbreaks.     

Detection of Colonization by Carbapenemase-Producing Gram-Negative Bacilli in Patients by Use of the Xpert MDRO Assay

Detecting colonization of patients with carbapenemase-producing bacteria can be difficult. This study compared the sensitivity and specificity of a PCR-based method (Xpert MDRO) for detecting bla_KPC, bla_NDM, and bla_VIM carbapenem resistance genes using GeneXpert cartridges to the results of culture with and without a broth enrichment step on 328 rectal, perirectal, and stool samples. The culture method included direct inoculation of a MacConkey agar plate on which a 10-μg meropenem disk was placed and plating on MacConkey agar after overnight enrichment of the sample in MacConkey broth containing 1 μg/ml of meropenem. Forty-three (13.1%) samples were positive by PCR for bla_KPC and 11 (3.4%) were positive for bla_VIM; none were positive for bla_NDM. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the PCR assay for bla_KPC were 100%, 99.0%, 93.0%, and 100%, respectively, compared to broth enrichment culture and sequencing of target genes. The sensitivity, specificity, PPV, and NPV of the assay for bla_VIM were 100%, 99.4%, 81.8%, and 100%, respectively. Since none of the clinical samples contained organisms with bla_NDM, 66 contrived stool samples were prepared at various dilutions using three Klebsiella pneumoniae isolates containing bla_NDM. The PCR assay showed 100% positivity at dilutions from 300 to 1,800 CFU/ml and 93.3% at 150 CFU/ml. The Xpert MDRO PCR assay required 2 min of hands-on time and 47 min to complete. Rapid identification of patients colonized with carbapenemase-producing organisms using multiplex PCR may help hospitals to improve infection control activities.     

Detection of Bacterial Pathogens in Purulent Clinical Specimens by Immunofluorescence Techniques

A technique is described which may be used to identify Haemophilus influenzae type b, Streptococcus pneumoniae, group A and B streptococci, and Staphylococcus aureus in smears of clinical specimens.     

Evaluation of Luminex xTAG Gastrointestinal Pathogen Panel Assay for Detection of Multiple Diarrheal Pathogens in Fecal Samples in Vietnam

Diarrheal disease is a complex syndrome that remains a leading cause of global childhood morbidity and mortality. The diagnosis of enteric pathogens in a timely and precise manner is important for making treatment decisions and informing public health policy, but accurate diagnosis is a major challenge in industrializing countries. Multiplex molecular diagnostic techniques may represent a significant improvement over classical approaches. We evaluated the Luminex xTAG gastrointestinal pathogen panel (GPP) assay for the detection of common enteric bacterial and viral pathogens in Vietnam. Microbiological culture and real-time PCR were used as gold standards. The tests were performed on 479 stool samples collected from people admitted to the hospital for diarrheal disease throughout Vietnam. Sensitivity and specificity were calculated for the xTAG GPP for the seven principal diarrheal etiologies. The sensitivity and specificity for the xTAG GPP were >88% for Shigella spp., Campylobacter spp., rotavirus, norovirus genotype 1/2 (GI/GII), and adenovirus compared to those of microbiological culture and/or real-time PCR. However, the specificity was low (∼60%) for Salmonella species. Additionally, a number of important pathogens that are not identified in routine hospital procedures in this setting, such as Cryptosporidium spp. and Clostridium difficile, were detected with the GPP. The use of the Luminex xTAG GPP for the detection of enteric pathogens in settings, like Vietnam, would dramatically improve the diagnostic accuracy and capacity of hospital laboratories, allowing for timely and appropriate therapy decisions and a wider understanding of the epidemiology of pathogens associated with severe diarrheal disease in low-resource settings.     

Application of Alternative Nucleic Acid Extraction Protocols to ProGastro SSCS Assay for Detection of Bacterial Enteric Pathogens

As an alternative to automated extraction, fecal specimens were processed by investigational lysis/heating (i.e., manual) and by chromatography/centrifugation (i.e., column) methods. ProGastro SSC and Shiga toxin-producing Escherichia coli (i.e., STEC) indeterminate rates for 101 specimens were 1.0% to 3.0% for automated, 11.9% for manual, and 24.8% to 37.6% for column methods. Following freeze-thaw of 247 specimens, indeterminate rates were 1.6% to 2.4% for manual and 0.8 to 5.3% for column methods. Mean processing times for manual and column methods were 30.5 and 69.2 min, respectively. Concordance of investigational methods with automated extraction was ≥98.8%.     

Rapid and Direct Detection of Herpes Simplex Virus in Cerebrospinal Fluid by Use of a Commercial Real-Time PCR Assay

Central nervous system infection due to herpes simplex virus (HSV) is a medical emergency and requires rapid diagnosis and initiation of therapy. In this study, we compared a routine real-time PCR assay for HSV types 1 (HSV-1) and 2 (HSV-2) to a recently FDA-approved direct PCR assay (Simplexa HSV-1/2 Direct; Focus Diagnostics, Cypress, CA) using cerebrospinal fluid samples (n = 100). The Simplexa HSV-1/2 assays demonstrated a combined sensitivity and specificity of 96.2% (50/52) and 97.9% (47/48), respectively. In addition, the Simplexa assay does not require nucleic acid extraction, and the results are available in 60 min.     

Potential for Use of the Seegene Anyplex MTB/NTM Real-Time Detection Assay in a Regional Reference Laboratory

Requests for direct molecular diagnosis of mycobacterial disease are increasingly warranted. The Anyplex MTB/NTM assay demonstrates sensitivities, specificities, and positive and negative predictive values of 1.00, 0.96, 0.93, and 1.00 for Mycobacterium tuberculosis complex (MTBC) and 1.00, 0.97, 0.75, and 1.00 for nontuberculous mycobacteria (NTM) detection, respectively, making it a suitable screening test for mycobacterial detection.     

BNP对急性呼吸困难患者的应用价值

目的：通过观察急性呼吸困难患者的血浆B型钠尿肽（BNP）含量变化,探讨BNP在急性呼吸困难患者确定或排除心力衰竭中的诊断价值。方法：收集2006年1月～2007年12月来本院急诊室就诊的急性呼吸困难患者（236人）的资料,分成心源性呼吸困难组和肺源性呼吸困难组进行整理、统计和分析。结果：①心源性呼吸困难组BNP含量426.1（75.9～687.0）ng/L,显著高于肺源性呼吸困难组BNP含量47.2（22.1～101.2）ng/L（P〈0.01）。②由受试者工作特征曲线（ROC）分析显示,BNP在ROC曲线下面积为0.854,95%的可信区间为0.806～0.884;确定诊断临界值（cut-off）为100ng/L;敏感度80.5%,特异度79.3%,正确诊断指数为0.66。结论：BNP可作为急性呼吸困难患者的常规检查项目,便于快速确定心源性或肺源性呼吸困难,及时采取治疗措施。     

Parallel Detection of Bacterial Pathogens in Cerebrospinal Fluid with 16S rRNA Probe Microarray

The most commonly used method for detection ofpathogenic bacteria in cerebrospinal fluid (CSF) speci mens of clinical laboratories is isolation and identificationof the causative agents by cultural method, biochemicaland serological t…     

Comparison of the Luminex Respiratory Virus Panel Fast Assay with In-House Real-Time PCR for Respiratory Viral Infection Diagnosis

The Luminex xTAG Respiratory Virus Panel (RVP) assay has been shown to offer improved diagnostic sensitivity over traditional viral culture methods and to have a sensitivity comparable to those of individual real-time nucleic acid tests for respiratory viruses. The objective of this retrospective study was to test a new, streamlined version of this assay, the RVP Fast assay, which requires considerably less run time and operator involvement. The study compared the performance of the RVP Fast assay with those of viral culture, a direct fluorescent assay (DFA), and a panel of single and multiplex real-time PCRs in the testing of 286 respiratory specimens submitted to the Edinburgh Specialist Virology Centre for routine diagnosis of viral infection between December 2007 and February 2009. At least one respiratory viral infection was detected in 13.6% of specimens by culture and DFA combined, in 49.7% by real-time PCR, and in 46.2% by the RVP Fast assay. The sensitivity and specificity of the RVP Fast assay compared to the results of real-time PCR as the gold standard were 78.8% and 99.6%, respectively. Real-time PCR-positive specimens missed by the RVP Fast assay generally had low viral loads or were positive for adenovirus. Additionally, a small number of specimens were positive by the RVP Fast assay but were not detected by real-time PCR. For some viral targets, only a small number of positive results were found in our sample set using either method; therefore, the sensitivity of detection of the RVP Fast assay for individual targets could be investigated further with a greater number of virus-positive specimens.Viral infections of the respiratory tract have traditionally been diagnosed in the laboratory by culture of respiratory specimens and direct fluorescent assay (DFA). However, the availability of real-time PCR has allowed us to detect respiratory viruses with greater sensitivity and shorter turnaround times (12). In recent years, a number of new respiratory viruses have been identified, so we must now consider a wider range of viruses in our diagnoses (see, e.g., references 1 and 14). However, the number of fluorophores that can be differentiated in a multiplex real-time PCR assay limits the number of viral targets that can be detected.One solution is to screen each specimen with several different multiplex real-time PCRs to cover a large number of viruses (4). An alternative, the xTAG respiratory virus panel (RVP) assay (Luminex Molecular Diagnostics Inc., Toronto, Canada), is based on suspension microarray technology, which enables the detection of a large number of targets in a single reaction (6, 9). The xTAG RVP assay has been shown to offer results comparable or superior to those of culture/DFA and nucleic acid tests for the diagnosis of respiratory viral infections (7, 10). Recently, the RVP assay has been used successfully for the detection of etiological agents in outbreaks of respiratory illness (3, 15).The latest version of this test, the RVP Fast assay, has a simpler protocol and a shorter turnaround time than the original assay but still detects 19 different viral and subtype targets: influenza A virus (with additional subtyping: H1, H3, and H5), influenza B virus, respiratory syncytial virus A (RSV-A), RSV-B, parainfluenza virus 1 (PIV-1), PIV-2, PIV-3, PIV-4, adenovirus, human metapneumovirus, coronaviruses 229E, NL63, OC43, and HKU1, enterovirus/rhinovirus (EV/RhV), and human bocavirus. Here we compare the performance of the RVP Fast assay with those of culture/DFA and in-house real-time PCR assays, using respiratory specimens collected for routine viral testing.