Date Published: March 13, 2019
Publisher: Public Library of Science
Author(s): Sara Guiducci, Maria Moriondo, Francesco Nieddu, Silvia Ricci, Elisa De Vitis, Arianna Casini, Giovanni Maria Poggi, Giuseppe Indolfi, Massimo Resti, Chiara Azzari, Adriana Calderaro.
Invasive meningococcal disease (IMD) is a highly lethal disease. Diagnosis is commonly performed by culture or Realtime-PCR (qPCR).
Our aim was to evaluate, retrospectively, whether culture positivity correlates with higher bacterial load and fatal outcome. Our secondary aim was to compare culture and qPCR sensitivity.
The National Register for Molecular Surveillance was used as data source. Cycle threshold (CT), known to be inversely correlated with bacterial load, was used to compare bacterial load in different samples.
Three-hundred-thirteen patients were found positive for Neisseria meningitidis by qPCR, or culture, or both; 41 died (case fatality rate 13.1%); 128/143 (89.5%) blood samples and 138/144 (95.8%) CSF were positive by qPCR, 37/143 (25.9%) blood samples and 45/144 (31.2%) CSF were also positive in culture. qPCR was 3.5 times (blood) or 3.1 times (CSF) more sensitive than culture in achieving a laboratory diagnosis of IMD (OR 24.4; 95% CI 12.2–49.8; p < .10−4; Cohen’s κ 0.08 for blood and OR 49.0; 95% CI 19.1–133.4; p<10−4; Cohen’s κ 0.02; for CSF). Positivity of culture did not correlate with higher bacterial loads in blood (mean CT 27.7±5.71, and CT 28.1±6.03, p = 0.739 respectively in culture positive or negative samples) or in CSF (mean CT 23.1±4.9 and 24.7±5.4 respectively in positive or negative CSF samples, p = 0.11).CT values in blood from patients who died were significantly lower than in patients who survived (respectively mean 18.0, range 14–23 and mean 29.6, range 16–39; p<10−17). No deaths occurred in patients with CT in blood over 23. Positive blood cultures were found in 10/25 (40%) patients who died and in 32/163 (19.6%) patients who survived, p = 0.036, OR 2.73; 95% CL 1.025–7.215), however 60% of deaths would have remained undiagnosed with the use of culture only. In conclusion our study demonstrated that qPCR is significantly (at least 3 times) more sensitive than culture in the laboratory confirmation of IMD. The study also demonstrated that culture negativity is not associated with lower bacterial loads and with less severe cases. On the other side, in patients with sepsis, qPCR can predict fatal outcome since higher bacterial load, evaluated by qPCR, appears strictly associated with most severe cases and fatal outcome. The study also showed that molecular techniques such as qPCR can provide a valuable addition to the proportion of diagnosed and serotyped cases of IMD.
Neisseria meningitidis is a major etiologic agent of bacterial meningitis and one of the most important causes of sepsis, meningitis and other invasive bacterial diseases [1–2]. Invasive meningococcal disease (IMD) is a serious infectious disease with high morbidity and mortality rates. Diagnosis is commonly performed by culture methods or by the Real-time Polymerase Chain Reaction (qPCR).
One-hundred and eighteen hospital from 18/20 Italian Regions participated in the molecular surveillance project (S1 and S2 Figs). The two regions that did not participate in the surveillance represent, together, 0.7% of the Italian population. Patients with invasive meningococcal infections were recruited from 85 hospitals. Clinical and demographic data are shown in Table 1.
Several factors are known to be associated with IMD outcome: among these meningococcal serotypes and bacterial load (measured with qPCR) or host-related factors such as IL-1 and IL-1RN polymorphisms .
In conclusion our study demonstrated that qPCR is significantly (at least 3 times) more sensitive than culture in the laboratory confirmation of IMD. We also demonstrated that culture negativity is not associated with lower bacterial loads and with less severe cases. Actually, even among the group of patients who died about 60% of patients would not have had laboratory confirmation without the use of qPCR. Bacterial load, evaluated by qPCR, appears strictly associated with disease outcome, since higher bacterial load were strongly associated with most severe cases and fatal outcome. Moreover our study highlighted the importance of PCR testing in confirming the diagnosis of IMD and in identifying meningococcal serotype even in culture-negative samples. Therefore, in settings with high rates of culture-negative results [5,23], molecular techniques such as qPCR can provide a valuable addition to the proportion of diagnosed cases of IMD.