Rapid Diagnosis Of Infection Research Articles

Infectious pleural effusions can be identified by sTREM-1 levels

Conventional methods to establish pleural infection are time-consuming and sometimes inadequate. Biomarkers may aid in making rapid diagnosis of infection. In an observational study we evaluated and compared the diagnostic value of pleural fluid levels of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), C-reactive protein and procalcitonin in intensive care patients with pleural effusions. Thirty-six patients with de novo pleural effusions were included and 20 patients with pleural effusions after cardiothoracic surgery and 20 patients with pleural effusions after esophagus surgery acted as controls. Levels of sTREM-1, C-reactive protein and procalcitonin were measured in pleural effusions. Levels of sTREM-1 were highest in empyemas, followed by infectious exudates. Levels of sTREM-1 were low in transudates and non-infectious exudates. C-reactive protein levels were highest in exudates and empyemas, while procalcitonin levels were highest in exudates. Pleural fluid with positive culture results contained higher sTREM-1 and C-reactive protein levels as compared to samples with negative culture results. A cut-off level of 50pg/mlsTREM-1 yielded a sensitivity of 93% and a specificity of 86%, while these were 87% and 67% respectively for a cut-off value of 7.5microg/ml C-reactive protein, and 60% and 64% respectively for a cut-off value of 0.15 ng/ml procalcitonin. sTREM-1 is superior to C-reactive protein and procalcitonin in detecting infection.

Procalcitonin as an early marker of bacterial infection in neutropenic febrile children with acute lymphoblastic leukemia

The aim of this study was to assess the value of procalcitonin (PCT), C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-a), interleukin (IL)-1b, IL-8, and soluble TNF receptor II (sTNFRII) in early and rapid diagnosis of infection in neutropenic children with acute lymphoblastic leukemia (ALL) and to distinguish bacterial from viral infections. The study included five groups (A, B, C, D, and E) of children with ALL undergoing intensive chemotherapy. Groups A and B consisted of neutropenic children with bacterial and viral infection, respectively. Groups C and D consisted of nonneutropenic children with bacterial and viral infection, respectively. Group E consisted of children without neutropenia and without fever. In all groups, blood samples were collected upon admission and then for 7 days on a daily basis. Levels of CRP, PCT, TNF-a, IL-1b, IL-8, and sTNFRII were determined in all blood samples. We found a highly significant difference in PCT levels between bacterial and nonbacterial episodes. Sensitivity and specificity of PCT were 94 and 96.5%, respectively. Serial measurement of PCT levels on a daily basis seems to be helpful for early prediction of severe bacterial infections, monitoring febrile episodes regarding response to antibiotic therapy, and early detection of complications in the infectious process.

Usefulness of pneumococcal antigen detection in pleural effusion for the rapid diagnosis of infection by Streptococcus pneumoniae

Pleural effusion is increasingly reported in children. Standard culture of blood or pleural fluid is frequently negative and molecular diagnosis by polymerase chain reaction is not available in all hospitals. The Binax NOW Streptococcus pneumoniae Antigen test (Binax, Portland, USA) is a rapid immunochromatographic test (ICT) for the detection of the C polysaccharide antigen. We evaluated the Binax NOW test on the pleural fluid of 73 children hospitalized with pleural effusion over a period of 4 years. In our sample, the sensitivity and specificity of ICT were high (88% and 71%, respectively), with a positive predictive value of 96%. Detection of the pneumococcal antigen in pleural fluid by ICT is easy and quick, and enables us to identify the pneumococcal origin of the effusion, thus, making the treatment of complicated pneumonia suitably and early.

Bench-to-bedside review: The promise of rapid infection diagnosis during sepsis using polymerase chain reaction-based pathogen detection

Early infection diagnosis as the cause of a patient's systemic inflammatory syndrome is an important facet of sepsis care bundles aimed at saving lives. Microbiological culture provides the main route for infection diagnosis but by its nature cannot provide time-critical results that can impact on early management. Consequently, broad-spectrum and high-potency antibiotics are essential during the immediate management of suspected sepsis in critical care but are associated with the development of drug-resistant organisms and superinfections. Established molecular laboratory techniques based on polymerase chain reaction (PCR) technology can detect pathogen DNA rapidly and have been developed for translation into a clinical diagnostic setting. In the setting of sepsis in critical care, emerging commercial systems are now available for the analysis of whole blood within hours, with the presumed aim of adoption into the current care bundles. In this review, we consider the importance of early infection diagnosis in sepsis, how this is limited by culture approaches and how the emerging PCR methods are showing promise in early clinical observational studies. The strengths and weaknesses of culture and PCR pathogen detection in whole-blood samples will be highlighted and recommendations made for urgent appropriately powered diagnostic validation studies in advance of clinical effectiveness trials before these emerging PCR pathogen detection techniques can be considered for adoption in clinical practice.

Development and Evaluation of a Molecular Assay for Detection of Nontuberculous Mycobacteria by Use of the Cobas Amplicor Platform

We have developed and evaluated a semiautomated assay for detection of nontuberculous mycobacteria (NTM) from clinical samples based on the Cobas Amplicor Mycobacterium tuberculosis test (Roche Diagnostics, Switzerland). A capture probe, specific for mycobacteria at the genus level, was linked to magnetic beads and used for the detection of amplification products obtained by the Cobas Amplicor M. tuberculosis assay. We demonstrate that the analytical sensitivity of the genus assay is similar to that of Cobas Amplicor M. tuberculosis detection. Four hundred sixteen clinical specimens were evaluated for the presence of NTM DNA. Sensitivities for smear-positive and smear-negative specimens were found to be 100% and 47.9%, respectively. Specificity was 97.7%, the positive predictive value 84.6%, and the negative predictive value 93.1%. The genus assay is easy to perform, produces reliable results, and was found to be a valuable diagnostic tool for rapid diagnosis of infections with NTM. The genus assay has the potential to detect NTM not routinely recovered by culture and to discover new mycobacterial species.

Identification of Diagnostic Biomarkers for Infection in Premature Neonates

Infection is a leading cause of neonatal morbidity and mortality worldwide. Premature neonates are particularly susceptible to infection because of physiologic immaturity, comorbidity, and extraneous medical interventions. Additionally premature infants are at higher risk of progression to sepsis or severe sepsis, adverse outcomes, and antimicrobial toxicity. Currently initial diagnosis is based upon clinical suspicion accompanied by nonspecific clinical signs and is confirmed upon positive microbiologic culture results several days after institution of empiric therapy. There exists a significant need for rapid, objective, in vitro tests for diagnosis of infection in neonates who are experiencing clinical instability. We used immunoassays multiplexed on microarrays to identify differentially expressed serum proteins in clinically infected and non-infected neonates. Immunoassay arrays were effective for measurement of more than 100 cytokines in small volumes of serum available from neonates. Our analyses revealed significant alterations in levels of eight serum proteins in infected neonates that are associated with inflammation, coagulation, and fibrinolysis. Specifically P- and E-selectins, interleukin 2 soluble receptor alpha, interleukin 18, neutrophil elastase, urokinase plasminogen activator and its cognate receptor, and C-reactive protein were observed at statistically significant increased levels. Multivariate classifiers based on combinations of serum analytes exhibited better diagnostic specificity and sensitivity than single analytes. Multiplexed immunoassays of serum cytokines may have clinical utility as an adjunct for rapid diagnosis of infection and differentiation of etiologic agent in neonates with clinical decompensation.

Assessment of Fluorescent In Situ Hybridization and PCR-Based Methods for Rapid Identification of Burkholderia cepacia Complex Organisms Directly from Sputum Samples

Several species within the Burkholderia cepacia complex (BCC) have emerged as significant opportunistic pathogens of patients with cystic fibrosis (CF). BCC infection is typically associated with a poor clinical prognosis and decreased survival. These factors, combined with the existence of highly transmissible epidemic strains, have resulted in strict segregation of BCC- and non-BCC-infected patients to minimize cross infection. Accurate and rapid diagnosis of infections is essential to enable appropriate patient management. However, the rapidly evolving taxonomy of BCC poses a considerable challenge to diagnostics. In the present study, we assessed a commercially available fluorescent in situ hybridization (FISH) assay (seaFAST Cystic Fibrosis I kit) and a novel rRNA gene-based PCR assay for the rapid identification of BCC-positive sputa, irrespective of the BCC species. We report that, while the FISH assay fails to identify all BCC species, it does identify the majority of species, including the two most clinically relevant species, B. multivorans and B. cenocepacia. The sensitivity of the assay applied to sputum was limited by nonspecific background fluorescence. While sputum processing was optimized to minimize background, the resulting sensitivity for BCC detection was 8 x 10(5) CFU/ml. In contrast, the novel PCR assay reported herein exhibits 100% sensitivity and specificity for all BCC species and can detect 10(4) CFU/ml when applied to sputum. This novel rRNA gene-based assay is currently the most sensitive BCC-specific PCR assay for the detection of BCC direct from clinical samples and as such is a valuable addition to the field of BCC diagnostics.

Cytological diagnosis of infections in renal allograft recipients

Infection is the most common cause of mortality and morbidity within the first year of transplantation. Renal allograft recipients are susceptible to various opportunistic infections because of prolonged immunosuppression. The study was carried out to evaluate the role of cytology for rapid diagnosis of infections in transplant patients. Fine needle aspiration cytology (FNAC) of superficial lesions, guided aspirations from deep seated lesions, bronchoalveolar lavage, endoscopic brushings and urine cytology was the material studied. 292 renal transplants were done during a period of nine years. 46 cases of infections were diagnosed, comprising of bacterial (26), viral (2), fungal (13) and mixed bacterial and fungal (5). Cytological examination offers a rapid and specific diagnosis of infections in these immunocompromised patients. The incidence, severity of infection and mortality in post transplant patients will decrease with early diagnosis and high index of clinical suspicion.

Current clinical management of Legionnaires’ disease

Legionella pneumophila is increasingly recognized as a cause of both sporadic and epidemic community-acquired pneumonia. Clinical manifestations of Legionnaires’ disease are not specific and current diagnostic scores are of limited use. Urinary antigen detection is an effective test for rapid diagnosis of infection caused by L. pneumophila serogroup 1. Improved outcomes regarding the time to defervescence, development of complications and length of stay, have been recently observed for patients treated with levofloxacin monotherapy. Current case-fatality rates for hospitalized patients with community-acquired Legionella pneumonia are lower than those traditionally reported for this infection. Effective preventive strategies are needed.

Rapid antibody screening by membrane chromatographic immunoassay technique

This paper describes a novel membrane chromatographic immunoassay technique suitable for rapid screening of antibodies in serum samples. This technique could potentially be utilized for antibody screening in situations where screening for exposure to one of several possible antigens is required. A synthetic microporous membrane is first selectively loaded with antibodies from the test serum sample by hydrophobic interaction. The in situ affinity membrane thus formed is sequentially pulsed with the antigens corresponding to the antibodies being screened. From the antigen chromatogram peak profile thus obtained, inferences about the antibodies present in the serum sample can then easily be made. This technique in addition to being rapid and direct is conceptually simple, and does not use any expensive media or reagents. It would potentially be useful for rapid diagnosis of infections as well as for rapid assessment of conditions such as envenomation or exposure to toxic substances.

Evaluation of the ESPLINE® INFLUENZA A&B‐N Kit for the Diagnosis of Avian and Swine Influenza

The ESPLINE INFLUENZA A&B-N kit was evaluated for its applicability to the rapid diagnosis of influenza in chickens and pigs. The kit specifically detected viral antigens in tracheal swabs and tissue homogenates of the trachea, liver, spleen, and colon of chickens inoculated with a highly pathogenic avian influenza virus strain, A/chicken/Yamaguchi/7/04 (H5N1), at 48 hr post-inoculation (p.i.) as well as in the tracheal and cloacal swabs and tissue homogenates of dead chickens. For those infected with a low pathogenic strain, A/chicken/aq-Y-55/01 (H9N2), antigens were detected only in the samples from tracheal swabs and organs 1-4 days p.i. The kit also detected viral antigens in the nasal swabs of miniature pigs infected with swine and avian influenza viruses. The kit was found to be sensitive and specific enough for the rapid diagnosis of infections of influenza A virus in chickens and pigs.

Using the polymerase chain reaction coupled with denaturing gradient gel electrophoresis to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted acute severe pancreatitis

To investigate the use of PCR and DGGE to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted severe AP. Patients with biochemical and clinical evidence of acute pancreatitis and an APACHE II score > or = 8 were enrolled. PCR and DGGE were employed to detect bacterial translocation in blood samples collected on d 1, 3, and 8 after the admission. Standard microbial blood cultures were taken when there was clinical evidence of sepsis or when felt to be clinically indicated by the supervising team. Six patients were included. Of all the patients investigated, only one developed septic complications; the others had uneventful illness. Bacteria were detected using PCR in 4 of the 17 collected blood samples. The patient with sepsis was PCR-positive in two samples (taken on d 1 and 3), despite three negative blood cultures. Using DGGE and specific primers, the bacteria in all blood specimens which tested positive for the presence of bacterial DNA were identified as E coli. Our study confirmed that unlike traditional microbiological techniques, PCR can detect the presence of bacteria in the blood of patients with severe AP. Therefore, this latter method in conjunction with DGGE is potentially an extremely useful tool in predicting septic morbidity and evaluating patients with the disease. Further research using increased numbers of patients, in particular those patients with necrosis and sepsis, is required to assess the reliability of PCR and DGGE in the rapid diagnosis of infection in AP.

Primary HIV type 1 infection.

Emerging evidence suggests that early events in human immunodeficiency virus type 1 (HIV-1) infection may play a critical role in determining disease progression. Although there is limited evidence on which to base medical decisions, the diagnosis and treatment of acute HIV-1 infection may have virologic, immunologic, and clinical benefits. In addition, rapid diagnosis of infection may prevent unknowing transmission of HIV-1 during a period of high-level viremia. We review the basic principles of primary HIV-1 infection, clinical and diagnostic markers of acute seroconversion, approaches to management, and new therapeutic strategies.

Molecular diagnostic techniques

The impetus for the development of new diagnostic techniques comes primarily from three sources: • the need for more rapid diagnosis of infections caused by micro-organisms that are slow or difficult to grow (e.g. myco-bacteria) • a search for highly sensitive methods that can detect low pathogen numbers (e.g. as found in early meningitis) • the increasing importance of old, or discovery of new, pathogenic micro-organisms (e.g. hepatitis C virus, HCV). Despite initial optimism, many new diagnostic techniques are never accepted into routine practice, usually because they are labour intensive and/or expensive. Also, though new tests may in theory be more sensitive than traditional culture-based methods, problems with specificity and reproducibility between centres may be significant. Quality-control surveys of laboratories which have introduced polymerase chain reaction (PCR)-based methods for detection of mycobacterial or HCV DNA/RNA found that false-positive results were widespread. In response to similar findings, an independent quality-assurance programme has been established by the Virology Committee of the AIDS Clinical Trials Group in the USA. For continuing certification, each participating laboratory must correctly test eight whole blood samples, and must include HIV-1 DNA copy standards with each PCR assay performed. As increasing efforts are made to standardize methodology and to identify and eliminate factors responsible for reductions in sensitivity and specificity, new techniques are gaining widespread acceptance. Another problem has been how to validate the accuracy of nucleic acid tests that may be more sensitive than any previous technology (i.e. better than the ‘gold-standard’ test). In practice, this situation is resolved by comparison of the new test with results from multiple different alternative testing methods (discrepant analysis).

Diagnosis of Helicobacter pylori infection.

A number of invasive and non-invasive techniques are available for the diagnosis of infection with Helicobacter pylori. These can be used to establish whether infection is present and to confirm that eradication has been achieved after treatment. Invasive techniques require biopsy of the gastric mucosa; such techniques include histology, bacterial culture, the rapid urease test and the polymerase chain reaction (PCR) technique. The principal non-invasive techniques are the 13C-urea breath test and serological assays in serum or saliva samples. Histological examination of gastric biopsy samples should be mandatory at the initial presentation of the patient because it also gives insight on the status of the gastric mucosa. The rapid urease test can be used to give a rapid diagnosis of infection. Eradication should also be confirmed by histological examination, though other sensitive tests such as bacterial culture, PCR or the 13C-urea breath test may also be used.

PCR-based diagnosis of Helicobacter pylori infection and real-time determination of clarithromycin resistance directly from human gastric biopsy samples.

A novel PCR detection assay that amplifies the Helicobacter pylori-specific vacuolating cytotoxin gene (vacA) and thus enables rapid diagnosis of infection is described. Additionally, a real-time probe hybridization melting point analysis assay to detect all three mutations in the 23S rRNA gene associated with clarithromycin resistance was applied directly to antral gastric biopsy samples. Comparison with culture and an alternative PCR assay targeting the 16S rrn gene showed that the vacA assay was sensitive and specific when tested on biopsy samples from 121 patients. Clarithromycin susceptibilities could be determined in the majority (92.3%) of culture-positive gastric biopsy samples analyzed, four of which generated melting peaks indicative of clarithromycin resistance by either an A-->G or A-->C mutation. The presence of the mutations correlated with the clarithromycin disk diffusion sensitivities of matched cultures. This PCR-based system was simple to perform and could be completed in 3 to 4 h, thereby overcoming the delays associated with conventional culture methods for H. pylori identification and susceptibility testing.

Rapid diagnosis of infection in the immunocompromised patient

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Detection of Mycobacterium tuberculosis by PCR amplification with pan-Mycobacterium primers and hybridization to an M. tuberculosis-specific probe

Nucleic acid amplification techniques such as the PCR are very useful in the rapid diagnosis of infections by Mycobacterium tuberculosis. However, recent studies have shown that the accuracy of results can vary widely when tests are performed with nonstandardized reagents. We have developed a PCR assay for the detection of M. tuberculosis that is both rapid and accurate. The assay reagents are standardized and quality controlled. False-positive results due to carryover contamination are prevented by the incorporation of dUTP coupled with uracil-N-glycosylase restriction. This assay also employs pan-Mycobacterium amplification primers, allowing for flexibility in the mycobacterial species that can be identified from a single amplification reaction. The amplification is very sensitive; amplification products generated from as few as three bacteria can be detected by agarose gel electrophoresis. DNAs isolated from 33 of 34 mycobacterial species tested were amplified efficiently. Only DNA from Mycobacterium simiae did not amplify. The amplification is also very specific. Amplification products were generated only from the DNAs of bacteria in closely related genera such as Corynebacterium. The nonmycobacterial amplicons do not pose a problem, as they do not hybridize to mycobacterium-specific probes. Hybridization of amplicons to an M. tuberculosis-specific probe allows for the unambiguous identification of M. tuberculosis complex organisms. The clinical performance of this PCR assay was evaluated against that of culture in 662 respiratory specimens. Sensitivities of 100 and 73.1% were obtained from smear-positive and -negative respiratory specimens, respectively. The corresponding specificities were 100 and 99.8%. The high sensitivity and specificity, coupled with the potential for detecting a wide range of mycobacteria, make this assay a useful tool in the clinical management of mycobacterial infections.

Diagnosis ofHelicobacter pyloriInfection

A number of invasive and non-invasive techniques are available for the diagnosis of infection with Helicobacter pylori. These can be used to establish whether infection is present and to confirm that eradication has been achieved after treatment. Invasive techniques require biopsy of the gastric mucosa; such techniques include histology, bacterial culture, the rapid urease test and the polymerase chain reaction (PCR) technique. The principal non-invasive techniques are the 13C-urea breath test and serological assays in serum or saliva samples. Histological examination of gastric biopsy samples should be mandatory at the initial presentation of the patient because it also gives insight on the status of the gastric mucosa. The rapid urease test can be used to give a rapid diagnosis of infection. Eradication should also be confirmed by histological examination, though other sensitive tests such as bacterial culture, PCR or the 13C-urea breath test may also be used.

Value of Liver Biopsy for the Rapid Diagnosis of Infection in Human Immunodeficiency Virus-Infected Patients Who Have Unexplained Fever and Elevated Serum Levels of Alkaline Phosphatase or -Glutamyl Transferase

We prospectively determined the value of liver biopsy for microbiological diagnosis of infection in patients infected with the human immunodeficiency virus (HIV) who had unexplained fever and whose serum levels of alkaline phosphatase or gamma-glutamyl transferase were at least 1.5 times the upper limit of normal. From December 1989 to December 1991, 108 HIV-infected patients were referred to the Liver Unit at Hôpital Laënnec (Paris) with liver abnormalities related to viral hepatitis (generally chronic), AIDS-related sclerosing cholangitis, or nonspecific lesions (detected on histologic examination). Twenty-four patients had unexplained fever and increased levels of alkaline phosphatase or gamma-glutamyl transferase, and none had evidence of hepatobiliary disease. All 24 patients had undergone routine microbiological tests to determine the cause of their chronic fever. The results of all microbiological tests were negative. We performed liver biopsies for these 24 patients and examined the specimens by means of standard direct microbiological techniques; in addition, the specimens were cultured, the specimens were analyzed by standard histopathologic methods, and specific histologic studies for fungi, mycobacteria, and viruses were performed. A microbiological diagnosis was made in 13 (54%) of the 24 cases within 12 hours to 3 days of the liver biopsy. In conclusion, liver biopsy is a powerful diagnostic tool for rapid diagnosis of infection in HIV-infected patients who have unexplained fever and abnormal liver function test results.