Evaluation of Bacterial Viability for Fecal Microbiota Transplantation: Impact of Thawing Temperature and Storage Time.

Fecal Microbiota Transplantation (FMT) represents a promising therapeutic tool under study for several purposes and is currently applied to the treatment of recurrent Clostridioides difficile infection. However, since the use of fresh stool was affected by several issues linked to donor screening, the development of a frozen stool bank is a reliable option to standardize FMT procedures. Nevertheless, different environmental factors impact microbial viability. Herein, we report the effect of different thawing temperatures and storage conditions on bacterial suspensions in the FMT procedure. In total, 20 stool samples were divided into aliquots and tested across a combination of different storing periods (15, 30; 90 days) and thawing procedures (4 °C overnight, room temperature for 1 h; 37 °C for 5 min). Focusing on storage time, our data showed a significant reduction in viability for aerobic and anaerobic bacteria after thawing for 15 days, while no further reductions were observed until after 90 days. Instead, among the different thawing procedures, no significant differences were observed for aerobic bacteria, while for anaerobes, thawing at 37 °C for 5 min was more effective in preserving the bacterial viability. In conclusion, the frozen fecal microbiota remained viable for at least three months, with an excellent recovery rate in all three thawing conditions.

Rapid RT-PCR identification of SARS-CoV-2 in screening donors of fecal microbiota transplantation.

Since its first appearance in late 2019 in Wuhan, China, severe acute respiratory syndrome caused by Coronavirus 2 (SARS-CoV-2) has had a major impact on healthcare facilities around the world. Although in the past year, mass vaccination and the development of monoclonal antibody treatments have reduced the number of deaths and severe cases, the circulation of SARS-CoV-2 remains high. Over the past two years, diagnostics have played a crucial role in virus containment both in health care facilities and at the community level. For SARS-CoV-2 detection, the commonly used specimen type is the nasopharyngeal swab, although the virus can be identified in other matrices such as feces. Since fecal microbiota transplantation (FMT) assumes significant importance in the treatment of chronic gut infections and that feces may be a potential vehicle for transmission of SARS-CoV-2, in this study we have evaluated the performance of the rapid cartridge-based RT-PCR test STANDARD™ M10 SARS-CoV-2 (SD Biosensor Inc., Suwon, South Korea) using fecal samples. The results obtained indicates that STANDARD™ M10 SARS-CoV-2 can detect SARS-CoV-2 in stool samples even at low concentration. For this reason, STANDARD™ M10 SARS-CoV-2 could be used as reliable methods for the detection of SARS-CoV-2 in fecal samples and for the screening of FMT donors.

Bayesian estimation of post-test probability of Candida glabrata fungemia by means of serum creatinine.

Fungemia is a life-threatening condition associated with high mortality; the most frequently isolated genus is Candida. Candida glabrata is of particular concern because of its increasing resistance to azoles. We evaluated common lab tests accessible by almost all healthcare professionals to estimate the post-test probability of recovery of C. glabrata from a blood culture collected by venipuncture, positive for fungi identified by microscopic examination. Patients with blood cultures positive for C. glabrata had significantly higher median values of serum creatinine (P=0.006), and a value of ≥1.45 mg/dL was the best cut-off in discriminating C. glabrata from other Candida spp., with 0.67 [95% Confidence Interval (CI): 0.49-0.85] sensitivity and 0.75 (95% CI: 0.66-0.84) specificity; Youden's J statistic: 0.42. The receiver operator characteristic curve analysis showed an area under the curve of 0.718 (95% CI: 0.603-0.833); P=0.001. Therefore, given a pre-test probability of 24% and applying the Bayes' theorem, the post-test probability of C. glabrata fungemia with creatinine values ≥1.45 mg/dL increased to 45.8%. In conclusion, we showed how the probability of recovery of C. glabrata from blood cultures collected by venipuncture and positive for fungi can be better estimated using concurrent creatinine values.

Performance of a SARS-CoV-2 antigen rapid immunoassay in patients admitted to the emergency department.

OBJECTIVES: Given the ongoing pandemic emergency, there is a need to identify SARS CoV-2 infection in various community settings. Rapid antigen testing is spreading worldwide, but diagnostic accuracy is extremely variable. Our study compared a microfluidic rapid antigen test with a reference molecular assay in patients admitted to the emergency department (ED) of a general hospital from October 2020 to January 2021. METHODS: Nasopharyngeal swabs collected in patients with suspected COVID-19 and in patients with no symptoms suggesting COVID-19, but requiring hospitalization, were obtained. RESULTS: 792 patients of median age 71 years were included. With a prevalence of 21%, the results showed: 68.7% (95% confidence interval [CI]: 60.9-75.5) sensitivity; 95.2% (95% CI: 93.1-96.7) specificity; 79.2% (95% CI: 71.4-85.3) positive predictive value (PPV); 91.9% (95% CI: 89.5-93.9) negative predictive value; 3.8 (95% CI: 2.7-5.3) positive likelihood ratio (LR+); and 0.09 (95% CI: 0.07-0.1) negative likelihood ratio (LR-). In the symptomatic subgroup, sensitivity increased to 81% (95% CI: 70.3-88.6) and PPV to 96.9% (95% CI: 88.5-99.5), along with an LR+ of 32 (95% CI: 8.2-125.4). CONCLUSIONS: The new rapid antigen test showed an overall excellent diagnostic performance in a challenging situation, such as that of an ED during the COVID-19 emergency.

Prevalence of respiratory viruses by Multiplex PCR: a four-and-a-half year retrospective study in an Italian general hospital.

Viruses are frequent causal agents of acute respiratory infections and the most common are influenza virus, respiratory syncytial virus (RSV), human parainfluenza virus (HPIV), human metapneumovirus (HMPV), rhinovirus (RV), adenovirus (AdV) and the four endemic human coronaviruses (HCoV) -229E, -NL63, -OC43, -HKU1. Multiplex real-time PCR platforms are becoming increasingly common in laboratories mostly in relation to the increased diagnostic sensitivity and reduced turnaround time. The aim of our study was to determine the prevalence of respiratory viruses in a population of patients within the S.S. Antonio e Biagio e Cesare Arrigo General Hospital catchment area of Alessandria, Italy, from January 2016 to June 2020. Therefore, we retrospectively analyzed the results of multiplex real-time PCR performed on nasopharyngeal swabs collected from consecutive patients with symptoms of respiratory infection. A total of 572 patients were included in the study subdivided as follows: pediatric 197/572 (34.4%), adults 200/572 (35%) and elderly 175/572 (30.6%). Among all samples, 235/572 (41.1%) were positive for a respiratory virus, of whom 189/235 (80.4%) were monomicrobial. The prevalence was: 15.5% (89/572) of rhinovirus/enterovirus (RV/EV); 9.4% (54/572) of RSV; 8.9% (51/572) of influenza virus; 5.4% (31/572) of AdV; 3.1% (18/572) of HCoV; 2.8% (16/572) of HPIV; and 2.3% (13/572) of HMPV. RV/EV were the pathogens most frequently involved in coinfections (34.7%, 16/46), followed by AdV (19.6%, 9/46) and influenza virus (19.6%, 9/46). Samples collected from the pediatric group were more frequently positive. The prevalence of positive pediatric samples compared to adults and elderly, respectively was: 28.4% (56/197) for RV/EV vs 10.5% (21/200) vs 6.9% (12/175), p<0.0001; 18.8% (37/197) for RSV vs 2% (4/200) vs 7.4% (13/175), p<0.0001; 13.7% (27/197) for AdV vs 1% (2/200) vs 1.1% (2/175), p<0.0001; and 6.6% (13/197) for HPIV vs 0.5% (1/200) vs 1.1% (2/175), (p<0.0001). With regard to seasonality, a significantly higher prevalence of influenza virus (p<0.0001) and RSV (p=0.029) was found during winter, with peaks in January-February. AdV peaked during winter 2018-2019 (p=0.004), while HCoV were detected with a significantly higher prevalence during winter 2019-2020 (p=0.037). With regard to HPIV, a significant peak from summer to fall 2018 was observed (p=0.016). Most viral respiratory infections have seasonal patterns and the prevalence of respiratory viruses varies according to the method, geographic area and population considered. Knowledge of local epidemiology is therefore crucial for implementation of prevention and treatment strategies.

Clinical Utility of Platelet Count for Screening of Malaria.

Light microscopy, immunochromatographic rapid diagnostic tests and molecular methods are widely used to diagnose malaria. The aim of this study was to find variables among commonly available urgent blood tests to identify patients with low probability of having malaria in small-scale healthcare facilities in which none of the described methods is feasible within a short time. Diagnosis of malaria was made by examining both stained thick and thin blood films by light microscopy. Two hundred and eleven samples were included. Reduced platelet count and increased values of C-reactive protein (CRP) and total bilirubin were the variables most strongly associated with malaria (P<0.0001). The best screening cut-off values obtained by receiver operating characteristic curve analysis for a negative result for malaria were: platelets ≥185,000 cells/µl; CRP ≤2 mg/dl; total bilirubin ≤0.28 mg/dl. The logistic regression model of log-transformed variables showed how platelet count was the only independent variable related to the odds of having a negative blood film result for malaria (odds ratio: 2.621; 95% confidence interval: 1.441-4.768; P=0.002). A platelet count of ≥185,000 cells/µl can be considered a screening value to identify patients with high-probability of a negative blood film result for malaria.

Evaluation of a multiplex gastrointestinal PCR panel for the aetiological diagnosis of infectious diarrhoea.

Background: Infectious diarrhoea is a significant cause of morbidity worldwide. Culture and microscopy are time-consuming and have a low yield. New rapid molecular methods such as multiplex PCR, have been recently introduced for aetiological diagnosis. Aim of this study was to compare the diagnostic yield of the FilmArray gastrointestinal panel with that of standard culture for aetiological diagnosis of infectious diarrhoea.Methods: We performed a retrospective analysis of results of stool samples already processed as part of routine clinical care in the interval from March 2016 to March 2019.Results: One hundred and eighty-three stool samples from as many patients were both cultured and tested by FilmArray and the comparison of diagnostic accuracy between culture and FilmArray with respect to Campylobacter spp., Salmonella spp., Shigella spp., Yersinia enterocolitica and Shiga-like toxin producing E. coli O157 gave the following results: 100% (95% confidence interval (CI): 85-100%) sensitivity; 93.4% (95% CI: 87.9-96.6%) specificity; 74.3% (95% CI: 57.5-86.4%) positive predictive value; 100% (95% CI: 96.7-100%) negative predictive value; 2.9 (95% CI: 1.6-5.1) positive likelihood ratio; zero negative likelihood ratio. By means of FilmArray gastrointestinal (GI) panel, we could identify 34.5% more pathogens (p = .001). Bacteria were mostly detected in patients with 6 or more years of age (χ2=17.1; p = .009) during summer.Conclusions: FilmArray GI panel showed a very good diagnostic performance compared to culture for diagnosis of infectious diarrhoea and gave a more detailed picture of the spectrum of the pathogens involved.

Diagnostic accuracy of a commercial multiplex PCR for the diagnosis of meningitis and encephalitis in an Italian general hospital.

Infectious meningitis and encephalitis are potentially life-threatening conditions caused mostly by bacterial and viral agents. Rapid diagnosis and prompt treatment are associated with a more favorable outcome. In recent years nucleic acid amplification tests have been developed to speed detection and identification of pathogens directly from cerebrospinal fluid (CSF). The aim of this study was to compare the diagnostic accuracy of a commercially available multiplex PCR assay for etiological diagnosis of infectious meningitis directly from CSF samples with culture. A secondary endpoint was to look for a possible screening threshold based on main CSF indices and urgent blood test results, to define CSF samples with low pre-test probability of PCR and/or culture-positive result. We performed a secondary analysis of results of CSF samples already processed as part of routine clinical care from February 2016 to December 2018. In all, 109 CSF samples were included in the study and a total of 14 bacteria were identified by either PCR, culture or both methods, along with nine samples positive for viruses. The comparison of PCR results with culture showed no significant difference: 7/109 (6.4%) vs 13/109 (11.9%) respectively, p=0.07. After exclusion of the isolates not detectable by the multiplex PCR panel, the diagnostic accuracy was: 100% (95% confidence interval (CI): 54.1% to 100%) sensitivity; 98.9% (95% CI: 93.5% to 99.9%) specificity; 85.7% (95% CI: 42% to 99.2%) positive predictive value; 100% (95% CI: 95.1% to 100%) negative predictive value; 96 (95% CI: 13.6 to 674.6) LR+; Zero LR-; Cohen's kappa: 0.918, p<0.0001. CSF protein value ≤ 28 mg/dl and CSF glucose/blood glucose ratio ≥0.78 were associated with both PCR-negative result for bacteria or viruses and culture-negative result. The multiplex PCR evaluated in this study showed a very good diagnostic performance compared to culture, and the thresholds found can be a useful tool to best choose which samples to test.

Epstein-Barr virus reactivation in allogeneic stem cell transplantation is highly related to cytomegalovirus reactivation.

Monitoring of Epstein-Barr virus (EBV) load and pre-emptive rituximab is an appropriate approach to prevent post-transplant lymphoproliferative disease (PTLD) occurring after hematopoietic stem cell transplantation (HSCT). This pre-emptive approach, based on EBV-DNA monitoring through a quantitative polymerase chain reaction, was applied to 101 consecutive patients who underwent allo HSCT at our Institute (median age 50). A single infusion of rituximab was administered to 11 of 16 patients who were at high risk for progression to PTLD, defined as a DNA value >10 000 copies/mL. All patients cleared EBV DNAemia, without any recurrences. Main factors significantly associated with high risk for PTLD were as follows: (i) unrelated vs. sibling (26% vs. 7%; p = 0.011); (ii) T-cell depletion (29% vs. 6%; p = 0.001); (iii) graft versus host disease (GVHD; 30% vs. 7%; p = 0.002); and (iv) cytomegalovirus (CMV) reactivation (29% vs. 4%; p = 0.001). Multivariate analysis showed that CMV reactivation was the only independent variable associated with EBV reactivation. We conclude that: (i) a single infusion of rituximab is able to prevent the risk of progression into EBV-related PTLD; and (ii) CMV reactivation is strongly associated with EBV reactivation; therefore, an intensive EBV monitoring strategy could be advisable only in case of CMV reactivation.

Evaluation of a rapid antigen and antibody combination test in acute HIV infection.

BACKGROUND: New strategies at implementing HIV testing including rapid HIV assays are highly recommended to avoid late diagnosis. To shorten the diagnostic window period, the first point-of-care HIV assay, Determine HIV ½ Ag/Ab Combo (D4G, Alere, I) for the combined detection of p24 and anti-HIV antibody has been recently marketed and mainly tested in high prevalence setting. OBJECTIVES: To establish D4G performances in acute HIV infection (AHI) in a setting at low HIV-1 prevalence. STUDY DESIGN: D4G performances were compared with HIV-1 RNA levels in a panel of well-characterized serum specimens from 17 patients with AHI. For specificity, 124 anti-HIV negative serum specimens from patients seeking HIV testing were studied. RESULTS: D4G detected HIV infection in 15/17 patients. D4G antigen was positive in only 5 patients (29.4%), 4 of them with a viral load >10 million copies/mL. D4G antibody was reactive in other 10 patients (sensitivity: 58.8%, viral load from 70,161 to 8,120,000 copies/mL). Combined D4G sensitivity for acute HIV-1 infection was 88.2%; no false positive or invalid result was recorded (100% specificity, positive and negative predictive values: 100% and 98.4%, respectively). CONCLUSION: In spite of a poor antigen sensitivity with optimal performances only for viral load >10 million copies/mL, D4G performances in acute HIV-1 infection were enhanced by the addition of p24 testing to the antibody. Improved HIV rapid testing to shorten the window period is important as rapid tests play a major role in expanding access to HIV testing and preventing HIV transmission.