Silvia Carletti

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

SUMMARY 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.

Molecular Diagnosis of Polymicrobial Sepsis

The recognition of sepsis is a crucial aspect in the management of critically ill patients. Recent guidelines strongly recommend the initiation of broad-spectrum antibiotic therapy within 1 hour after the recognition of a case of sepsis (1). The adjustment of antibiotic therapy is also recommended once the involved pathogen is identified (1). The current microbiological diagnosis of sepsis is based on blood culture. However, blood culture systems suffer from several limitations such as lack of rapidity and, in cases such as polymicrobial infections, low sensitivity (6). Support for cultural approaches is therefore needed, and well-designed molecular assays could improve the diagnostic flowchart (5, 6). Under this perspective, we have recently evaluated a molecular assay for the diagnosis of sepsis (LightCycler SeptiFast test; Roche Molecular Systems) (3). SeptiFast is a multiplex real-time PCR-based assay, allowing for the detection of a wide panel of bacterial and mycotic pathogens directly from blood (2). DNA is extracted by mechanical lysis with ceramic beads in a Magnalyzer instrument (Roche Molecular Systems), and after purification, it is processed in three parallel real-time PCRs (gram-positive, gram-negative, fungi). The detection of amplicons is based on dual fluorescent resonance energy transfer probes targeting the internal transcribed spacer (ITS) regions. ITS regions are species-specific multicopy noncoding sequences interspaced among conserved bacterial and fungal ribosomal DNA (4, 7). Finally, a dedicated software (SeptiFast identification software) calculates the specific melting profile of ITS-amplified products, thus allowing identification of the pathogen at the species level (2). From our experience with a cohort of neutropenic patients, SeptiFast featured sensitivity at least comparable to that observed with blood culture and a dramatically shorter turnaround time (3). For example, in a case of polymicrobial sepsis, the rapid identification of involved pathogens allowed for a prompt adjustment of therapy. Blood cultures and intravascular catheter tip culture were performed on a persistently febrile patient with multiple myeloma. Empirical intravenous antibiotic therapy with linezolid (600 mg every 12 h) and ceftriaxone (2 g once a day [QD]) was then started. In parallel, 1.5 ml of KEDTA-treated blood was also taken to perform SeptiFast. After only 6 hours, the presence of DNA belonging to Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Candida albicans was reported to the department (Fig. ​(Fig.1).1). Levofloxacin (500 mg QD) and fluconazole (400 mg QD) were then promptly added to the therapy. In the following days, the molecular results were then confirmed by culture-based approaches. Blood cultures turned positive on day 1 for gram-negative rods with Pseudomonas sp.-like morphology. Only on day 3 was the definitive identification for Pseudomonas aeruginosa and Stenotrophomonas maltophilia obtained. No growth of Candida albicans was detected in blood culture bottles after 5 days of incubation. Notwithstanding, on day 3, Candida albicans was detected on plates from catheter tip culture. The susceptibility profiles were obtained for all pathogens and confirmed the appropriateness of the therapeutic adjustment performed after the molecular results were obtained.

Isolation and genotyping of Acanthamoeba strains from corneal infections in Italy

Acanthamoeba keratitis (AK) is a corneal disease caused by members of a genus of free-living amoebae and is associated predominantly with contact lens (CL) use. This study reports 16 cases of culture-proven AK diagnosed in northern Italy. Genotype identification was carried out with a PCR assay based on sequence analysis of the 18S rRNA gene, and sensitivity and specificity were evaluated in comparison with traditional parasitological techniques. A 405 bp region of the 18S rRNA gene (ASA.S1) including diagnostic fragment 3 (DF3) was amplified using the genus-specific primers JDP1 and JDP2. Genotype assignment was based on phenetic analysis of the ASA.S1 subset of the nuclear small-subunit rRNA gene sequence excluding the highly variable DF3 region. Phylogenetic analysis was also performed on the sequences obtained. All patients complained of monolateral infection; 11 (68.75%) admitted improper CL disinfection. In 14/16 (87.5 %) subjects, corneal scrapings were stained with calcofluor white and haematoxylin and eosin and, in ten cases (62.5 %), microscopy was positive for Acanthamoeba cysts. In vitro culture on 3 % non-nutrient agar plates was obtained in all cases (100 %), whereas cloning and axenic growth were positive for 14 amoebic stocks (87.5 %). PCR analysis had 100 % sensitivity and specificity compared with in vitro axenic culture, showing positive amplification from 15 isolates. All Acanthamoeba strains belonged to the T4 genotype, the main AK-related genotype worldwide. These results confirmed the importance of a complete diagnostic protocol, including a PCR assay, for the clinical diagnosis of AK on biological samples. Genotyping allowed inclusion of all isolates in the T4 group, thus demonstrating the prevalence of this genotype in northern Italy.

Coronaviridae and SARS-associated coronavirus strain HSR1.

During the recent severe acute respiratory (SARS) outbreak, the etiologic agent was identified as a new coronavirus (CoV). We have isolated a SARS-associated CoV (SARS-CoV) strain by injecting Vero cells with a sputum specimen from an Italian patient affected by a severe pneumonia; the patient traveled from Vietnam to Italy in March 2003. Ultrastructural analysis of infected Vero cells showed the virions within cell vesicles and around the cell membrane. The full-length viral genome sequence was similar to those derived from the Hong-Kong Hotel M isolate. By using both real-time reverse transcription–polymerase chain reaction TaqMan assay and an infectivity plaque assay, we determined that approximately 360 viral genomes were required to generate a PFU. In addition, heparin (100 μg/mL) inhibited infection of Vero cells by 50%. Overall, the molecular and biologic characteristics of the strain HSR1 provide evidence that SARS-CoV forms a fourth genetic coronavirus group with distinct genomic and biologic features.

Potential role of the detection of enterobacterial DNA in blood for the management of neonatal necrotizing enterocolitis.

We present three cases of pre-term low-weight infants with suspected necrotizing enterocolitis (NEC) [one eventually recognized as a connatal cytomegalovirus (CMV) infection], microbiologically monitored using a molecular assay detecting bacterial and fungal DNA in blood. The detection of DNA from enteric pathogens in blood was interpreted as a sign of ongoing perforation, and represented a useful complement in the management of the presented cases. Moreover, these cases suggest the opportunity for larger future studies to assess the possible role of a molecular approach in the close monitoring of infants with suspected NEC or with other conditions at-risk for intestinal perforation.

Cost-effectiveness of blood culture and a multiplex real- time PCR in hematological patients with suspected sepsis: an observational propensity score-matched study

We evaluated the costs and clinical outcomes of episodes of suspected sepsis in hematological patients. A propensity score-matched study was planned, comparing a retrospective cohort managed with standard assays and a prospective cohort managed with the addition of a molecular assay. Diagnostic procedures and therapy were considered as costs variables. The primary clinical endpoint was sepsis-related mortality, whereas the length of each suspected sepsis episode was investigated as a secondary endpoint. A total of 137 and 138 episodes in the prospective and the retrospective cohorts were studied, respectively; 101 pairs of highly matched episodes were analyzed, evidencing a trend of higher mortality in the retrospective cohort. No difference in length of suspected sepsis episode was observed. Significant savings were observed in the prospective cohort, especially due to reduced costs in antifungal therapy. The apparently more expensive molecular assay favored a more rational use of economic resources without influencing, and probably improving, the clinical outcome.