In this study the detection rates of bacterial infection of hip prostheses by culture and nonculture methods were compared for 120 patients with total hip revision surgery. By use of strict anaerobic bacteriological practice during the processing of samples and without enrichment, the incidence of infection by culture of material dislodged from retrieved prostheses after ultrasonication (sonicate) was 22%. Bacteria were observed by immunofluorescence microscopy in 63% of sonicate samples with a monoclonal antibody specific for Propionibacterium acnes and polyclonal antiserum specific for Staphylococcus spp. The bacteria were present either as single cells or in aggregates of up to 300 bacterial cells. These aggregates were not observed without sonication to dislodge the biofilm. Bacteria were observed in all of the culture-positive samples, and in some cases in which only one type of bacterium was identified by culture, both coccoid and coryneform bacteria were observed by immunofluorescence microscopy. Bacteria from skin-flake contamination were readily distinguishable from infecting bacteria by immunofluorescence microscopy. Examination of skin scrapings did not reveal large aggregates of bacteria but did reveal skin cells. These were not observed in the sonicates. Bacterial DNA was detected in 72% of sonicate samples by PCR amplification of a region of the bacterial 16S rRNA gene with universal primers. All of the culture-positive samples were also positive for bacterial DNA. Evidence of high-level infiltration either of neutrophils or of lymphocytes or macrophages into associated tissue was observed in 73% of patients. Our results indicate that the incidence of prosthetic joint infection is grossly underestimated by current culture detection methods. It is therefore imperative that current clinical practice with regard to the detection and subsequent treatment of prosthetic joint infection be reassessed in the light of these results.

Detection of Prosthetic Hip Infection at Revision Arthroplasty by Immunofluorescence Microscopy and PCR Amplification of the Bacterial 16S rRNA Gene

Sepsis, a leading cause of morbidity and mortality throughout the world, is a clinical syndrome with signs and symptoms relating to an infectious event and the consequent important inflammatory response. From a clinical point of view, sepsis is a continuous process ranging from systemic inflammatory response syndrome (SIRS) to multiple-organ-dysfunction syndrome (MODS). Blood cultures are the current "gold standard" for diagnosis, and they are based on the detection of viable microorganisms present in blood. However, on some occasions, blood cultures have intrinsic limitations in terms of sensitivity and rapidity, and it is not expected that these drawbacks will be overcome by significant improvements in the near future. For these principal reasons, other approaches are therefore needed in association with blood culture to improve the overall diagnostic yield for septic patients. These considerations have represented the rationale for the development of highly sensitive and fast laboratory methods. This review addresses non-culture-based techniques for the diagnosis of sepsis, including molecular and other non-culture-based methods. In particular, the potential clinical role for the sensitive and rapid detection of bacterial and fungal DNA in the development of new diagnostic algorithms is discussed.

The Era of Molecular and Other Non-Culture-Based Methods in Diagnosis of Sepsis

A nonculture PCR-based method to characterize Neisseria meningitidis was used to test 225 clinical specimens. PCR correctly identified and predicted the serogroups of N. meningitidis of culture-proven meningococcal diseases and confirmed this diagnosis in 35% of suspected samples. This approach could be useful when culture fails to isolate N. meningitidis.

Simultaneous Approach for Nonculture PCR-Based Identification and Serogroup Prediction of Neisseria meningitidis

The rapid identification of bacteria in blood cultures and other clinical specimens is important for patient management and antimicrobial therapy. We describe a rapid (<4 h) detection and identification system that uses universal PCR primers to amplify a variable region of bacterial 23S ribosomal DNA, followed by reverse hybridization of the products to a panel of oligonucleotides. This procedure was successful in discriminating a range of bacteria in pure cultures. When this procedure was applied directly to 158 unselected positive blood culture broths on the day when growth was detected, 125 (79.7%) were correctly identified, including 4 with mixed cultures. Nine (7.2%) yielded bacteria for which no oligonucleotide targets were present in the oligonucleotide panel, and 16 culture-positive broths (10.3%) produced no PCR product. In seven of the remaining eight broths, streptococci were identified but not subsequently grown, and one isolate of Staphylococcus aureus was misidentified as a coagulase-negative staphylococcus. The accuracy, range, and discriminatory power of the assay can be continually extended by adding further oligonucleotides to the panel without significantly increasing complexity or cost.

Rapid Diagnosis of Bacteremia by Universal Amplification of 23S Ribosomal DNA Followed by Hybridization to an Oligonucleotide Array

The PCR is the most sensitive of the existing rapid methods to detect microbial pathogens in clinical specimens. In particular, when specific pathogens that are difficult to culture in vitro or require a long cultivation period are expected to be present in specimens, the diagnostic value of PCR is known to be significant. However, the application of PCR to clinical specimens has many potential pitfalls due to the susceptibility of PCR to inhibitors, contamination and experimental conditions. For instance, it is known that the sensitivity and specificity of a PCR assay is dependent on target genes, primer sequences, PCR techniques, DNA extraction procedures, and PCR product detection methods. Even though there are many publications concerning basic protocols of a PCR assay, including DNA extraction and preparation as well as the amplification and detection of amplicons, PCR detection of bacteria in clinical specimens such as cerebrospinal fluid (CSF) has not yet been reviewed. Since a variety of clinical specimens, such as blood, urine, sputum, CSF and others, vary in regard to the nature of the content and amount available, careful design of the PCR assay for each specific specimen before a PCR application is conducted is essential. In particular, a diagnosis based on detection of a few bacteria in clinical specimens by using PCR must be carefully evaluated technically as well as microbiologically. In this regard, current studies concerning detection of Chlamydia pneumoniae in CSF obtained from patients with multiple sclerosis (MS) by using PCR provide a good example for discussion of use of the PCR assay in diagnosis. Because C. pneumoniae is difficult to culture in vitro, often low numbers of bacteria may be detected in the CSF of patients with chronic neurological diseases by PCR. Therefore, in this review general PCR protocols for detection of bacteria in clinical specimens, as well as a specific example of using PCR for detection of C. pneumoniae in CSF, will be discussed.

PCR in diagnosis of infection: detection of bacteria in cerebrospinal fluids.

Meningococcal disease is normally suspected on clinical grounds and is confirmed by isolation of Neisseria meningitidis bacteria from blood or cerebrospinal fluid or, more recently, by serology or PCR of cerebrospinal fluid. Achieving confirmation of a clinical diagnosis of meningococcal disease has become more difficult in the last few years. The pre-hospitalization administration of parenteral benzylpenicillin normally renders blood cultures sterile, and lumbar puncture is undertaken less frequently, especially in young children. We evaluated PCR for the detection of meningococcal DNA in 80 blood samples taken from patients with known or suspected meningococcal disease or from patients with other diagnoses (negative controls). Both the sensitivity and the specificity of the test were 100% for patients with confirmed cases of meningococcal disease when the blood buffy coat was used (83 to 100% sensitivity and 87 to 100% specificity with 95% confidence limits). Positive PCR results could be obtained from both blood buffy coat and serum samples. Sensitivity was unaffected by prior antibiotic treatment. PCR is a rapid, sensitive test that may be used to confirm a diagnosis of meningococcal disease by using peripheral blood samples. Introduction of this test into clinical laboratories may in some cases obviate the need for lumbar puncture to be performed on patients with suspected meningococcal disease. Our results demonstrate that a substantial number of cases of meningococcal disease are not confirmed by conventional techniques and remain undiagnosed. If the PCR test described here was widely applied, the number of cases of meningococcal disease ascertained might rise by as much as 60% greater than that recognized at present. It is likely that we are in a prevaccination era for meningococcal disease. Better case ascertainment is urgently required to assess the need for vaccines, to determine their costs and benefits, and to monitor their efficacies.

PCR of peripheral blood for diagnosis of meningococcal disease.

Subspecific typing of clinical meningococcal strains is important in the investigation of outbreaks and for disease surveillance. Serogrouping, typing, and subtyping of strains currently require isolation of a meningococcus from one or more clinical specimens. However, the increasing widespread practice of preadmission administration of parenteral antibiotics has resulted in a decrease in the frequency of positive cultures obtained from blood and cerebrospinal fluid. Confirmation of meningococcal disease can be obtained by meningococcus-specific PCR from both cerebrospinal fluid (H. Ni et al., Lancet 340:1432-1434, 1992) and peripheral blood (J. Newcombe et al., J. Clin. Microbiol. 34:1637-1640, 1996) specimens. However, current PCR protocols do not yield epidemiologically useful typing information. We report here the use of PCR-single-stranded confirmational polymorphism (PCR-SSCP) analysis to amplify and type meningococcal DNA present in clinical specimens. PCR-SSCP analysis with the VR1 region of the Neisseria meningitidis porA gene as the target produced unique banding patterns for each serosubtype. Direct PCR-SSCP of clinical specimens can therefore provide typing data that can be used to investigate the epidemiology of clusters of cases and outbreaks and for disease surveillance in situations in which culture of patient specimens proves negative.

https://jcm.asm.org/content/jcm/35/7/1809.full.pdf

PCR-single-stranded confirmational polymorphism analysis for non-culture-based subtyping of meningococcal strains in clinical specimens.

Use of pcr in the diagnosis of meningococcal meningitis

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PCR of peripheral blood for diagnosis of meningococcal disease.

PCR-single-stranded confirmational polymorphism analysis for non-culture-based subtyping of meningococcal strains in clinical specimens.

Use of pcr in the diagnosis of meningococcal meningitis