Forensic Microbiology: When, Where and How.

Forensic microbiology is a relatively new discipline, born in part thanks to the development of advanced methodologies for the detection, identification and characterization of microorganisms, and also in relation to the growing impact of infectious diseases of iatrogenic origin. Indeed, the increased application of medical practices, such as transplants, which require immunosuppressive treatments, and the growing demand for prosthetic installations, associated with an increasing threat of antimicrobial resistance, have led to a rise in the number of infections of iatrogenic origin, which entails important medico-legal issues. On the other hand, the possibility of detecting minimal amounts of microorganisms, even in the form of residual traces (e.g., their nucleic acids), and of obtaining gene and genomic sequences at contained costs, has made it possible to ask new questions of whether cases of death or illness might have a microbiological origin, with the possibility of also tracing the origin of the microorganisms involved and reconstructing the chain of contagion. In addition to the more obvious applications, such as those mentioned above related to the origin of iatrogenic infections, or to possible cases of infections not properly diagnosed and treated, a less obvious application of forensic microbiology concerns its use in cases of violence or violent death, where the characterization of the microorganisms can contribute to the reconstruction of the case. Finally, paleomicrobiology, e.g., the reconstruction and characterization of microorganisms in historical or even archaeological remnants, can be considered as a sister discipline of forensic microbiology. In this article, we will review these different aspects and applications of forensic microbiology.

A mid-term follow-up with a lung ultrasonographic score correlates with the severity of COVID-19 acute phase.

Lung ultrasound (LUS) has rapidly emerged in COVID-19 diagnosis and for the follow-up during the acute phase. LUS is not yet used routinely in lung damage follow-up after COVID-19 infection. We investigated the correlation between LUS score, and clinical and laboratory parameters of severity of SARS-COV-2 damage during hospitalization and at follow-up visit. Observational retrospective study including all the patients discharged from the COVID-19 wards, who attended the post-COVID outpatient clinic of the IRCCS Policlinico San Matteo in April-June 2020. 115 patients were enrolled. Follow-up visits with LUS score measurements were at a median of 38 days (IQR 28-48) after discharge. LUS scores were associated with the length of hospitalization (p < 0.001), patients' age (p = 0.036), use of non-invasive ventilation (CPAP p < 0.001 or HFNC p = 0.018), administration of corticosteroids therapy (p = 0.030), and laboratory parameters during the acute phase (WBC p < 0.001, LDH p < 0.001, CRP p < 0.001, D-dimer p = 0.008, IL-6 p = 0.045), and inversely correlated with lymphocyte count (p = 0.007). We found correlation between LUS score and both LDH (p = 0.001) and the antibody anti-SARS-CoV-2 titers (p value = 0.008). Most of these finding were confirmed by dichothomizing the LUS score (≤ 9 or > 9 points). We found a significantly higher LUS score at the follow-up in the patients with persistent dyspnea (7.00, IQR 3.00-11.00) when compared to eupnoeic patients (3.00, IQR 0-7.00 p < 0.001). LUS score at follow-up visit correlates with more severe lung disease. These findings support the hypothesis that ultrasound could be a valid tool in the follow-up medium-term COVID-19 lung damage.

Mid-term follow-up chest CT findings in recovered COVID-19 patients with residual symptoms.

OBJECTIVES: More than a year has passed since the initial outbreak of SARS-CoV-2, which caused many hospitalizations worldwide due to COVID-19 pneumonia and its complications. However, there is still a lack of information detailing short- and long-term outcomes of previously hospitalized patients. The purpose of this study is to analyze the most frequent lung CT findings in recovered COVID-19 patients at mid-term follow-ups. METHODS: A total of 407 consecutive COVID-19 patients who were admitted to the Fondazione IRCCS Policlinico San Matteo, Pavia and discharged between February 27, 2020, and June 26, 2020 were recruited into this study. Out of these patients, a subset of 108 patients who presented with residual asthenia and dyspnea at discharge, altered spirometric data, positive lung ultrasound and positive chest X-ray was subsequently selected, and was scheduled to undergo a mid-term chest CT study, which was evaluated for specific lung alterations and morphological patterns. RESULTS: The most frequently observed lung CT alterations, in order of frequency, were ground-glass opacities (81%), linear opacities (74%), bronchiolectases (64.81%), and reticular opacities (63.88%). The most common morphological pattern was the non-specific interstitial pneumonia pattern (63.88%). Features consistent with pulmonary fibrosis were observed in 32 patients (29.62%). CONCLUSIONS: Our work showed that recovered COVID-19 patients who were hospitalized and who exhibited residual symptoms after discharge had a slow radiological recovery with persistent residual lung alterations. ADVANCES IN KNOWLEDGE: This slow recovery process should be kept in mind when determining the follow-up phases in order to improve the long-term management of patients affected by COVID-19.

Cerebral Toxocariasis as a Cause of Epilepsy: A Pediatric Case.

Toxocarosis is the consequence of human infection by Toxocara spp. larvae and is one of the most common ascarioses, not only in developing countries, but also in the European region, where its prevalence reaches 14%. Due to their particular behavior, children are at higher risk of this parasitic infection, whose clinical features depend on the localization of the Toxocara larvae. Neurotoxocariasis is very uncommon in children and may take different forms depending on the underlying physiopathologic process: immune reaction against the parasite antigens, vasculitis, treatment complications, or, very rarely, brain localization of Toxocara spp. larvae. The association between neurotoxocariasis and the onset of childhood epilepsy has been postulated but is still debated. Moreover, a Toxocara spp. abscess causing epileptic seizures in children has been rarely described, especially in western countries. Hereby we present a 9-year-old patient with a new diagnosis of epilepsy definitely secondary to brain abscess due to the localization of Toxocara canis larvae. Diagnosis was confirmed by neuroimaging and serological test. The successful treatment with albendazole and steroids was documented with a close and long-term clinical and neuroradiological follow-up. Our experience confirms that every case of cryptogenetic epilepsy in children deserves a neuroimaging study and, in case of cystic images, Toxocara serology is mandatory to avoid further unnecessary invasive diagnostic investigations and to set the specific drug therapy.

Competing-risk analysis of coronavirus disease 2019 in-hospital mortality in a Northern Italian centre from SMAtteo COvid19 REgistry (SMACORE).

An accurate prediction of the clinical outcomes of European patients requiring hospitalisation for Coronavirus Disease 2019 (COVID-19) is lacking. The aim of the study is to identify predictors of in-hospital mortality and discharge in a cohort of Lombardy patients with COVID-19. All consecutive hospitalised patients from February 21st to March 30th, 2020, with confirmed COVID-19 from the IRCCS Policlinico San Matteo, Pavia, Lombardy, Italy, were included. In-hospital mortality and discharge were evaluated by competing risk analysis. The Fine and Gray model was fitted in order to estimate the effect of covariates on the cumulative incidence functions (CIFs) for in-hospital mortality and discharge. 426 adult patients [median age 68 (IQR 56 to 77 years)] were admitted with confirmed COVID-19 over a 5-week period; 292 (69%) were male. By 21 April 2020, 141 (33%) of these patients had died, 239 (56%) patients had been discharged and 46 (11%) were still hospitalised. Among these 46 patients, updated as of 30 May, 2020, 5 (10.9%) had died, 8 (17.4%) were still in ICU, 12 (26.1%) were transferred to lower intensity care units and 21 (45.7%) were discharged. Regression on the CIFs for in-hospital mortality showed that older age, male sex, number of comorbidities and hospital admission after March 4th were independent risk factors associated with in-hospital mortality. Older age, male sex and number of comorbidities definitively predicted in-hospital mortality in hospitalised patients with COVID-19.

EBV DNA increase in COVID-19 patients with impaired lymphocyte subpopulation count.

OBJECTIVES: The immunologic profile and opportunistic viral DNA increase were monitored in Italian patients with COVID-19 in order to identify markers of disease severity. METHODS: A total of 104 patients infected with SARS-CoV-2 were evaluated in the study. Of them, 42/104 (40.4%) were hospitalized in an intensive care unit (ICU) and 62/104(59.6%) in a sub-intensive care unit (SICU). Human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV), Parvovirus B19 and Human Herpesvirus 6 virus reactivations were determined by real-time PCR, and lymphocyte subpopulation counts were determined by flow cytometry. RESULTS: Among opportunistic viruses, only EBV was consistently detected. EBV DNA was observed in 40/42 (95.2%) of the ICU patients and in 51/61 (83.6%) of the SICU patients. Comparing the two groups of patients, the EBV DNA median level among ICU patients was significantly higher than that observed in SICU patients. In parallel, a significant reduction of CD8 T cell and NK count in ICU patients as compared with SICU patients was observed (p<0.05). In contrast, B cell count was significantly increased in ICU patients (p=0.0172). CONCLUSIONS: A correlation between reduced CD8+ T cells and NK counts, EBV DNA levels and COVID-19 severity was observed. Other opportunistic viral infections were not observed. The relationship between EBV load and COVID-19 severity should be further evaluated in longitudinal studies.

Adaptive Natural Killer Cell Functional Recovery in Hepatitis C Virus Cured Patients.

BACKGROUND AND AIMS: Current evidence suggests that dysfunctional natural killer (NK) cell responses during hepatitis C virus (HCV) infection can be restored after viral eradication with direct acting antivirals (DAAs). However, the fate of the recently described adaptive NK cell population, endowed with increased ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), during HCV infection is poorly defined, while no study has explored the effects of DAAs on this NK subset. APPROACH AND RESULTS: We performed multicolor flow cytometry to investigate CD57+ FcεRIγneg adaptive and FcεRIγpos conventional NK cell phenotype and function before and after DAA treatment in 59 patients chronically infected with HCV, 39 with advanced liver fibrosis, and 20 with mild-moderate liver fibrosis. Moreover, bulk NK cell phenotype and function were analyzed after cytokine activation following contact with K562 target cells. The proportion of FcεRIγneg NK cells in patients with HCV was associated with increased HCV load at baseline, and it was significantly reduced after treatment. Patients with an advanced fibrosis stage displayed increased NK cell activation and exhaustion markers that normalized after therapy. Of note, adaptive NK cells from patients with HCV were characterized by increased programmed death receptor 1 expression and reduced ADCC activity at baseline. DAA treatment restored ADCC ability and reduced programmed death receptor 1 expression. CONCLUSIONS: HCV profoundly affects the frequency, phenotype, and function of adaptive NK cells. DAA therapy restores a normal adaptive NK phenotype and enhances interferon-gamma production by this cell subset.

Patients with chronic hepatitis C receiving treatment with direct acting antivirals: How is this population changing?

BACKGROUND AND AIMS: Direct acting antiviral agents (DAAs) have revolutionized the landscape of chronic hepatitis C (CHC) enabling treatment of all those infected. It remains to be determined how the characteristics of those receiving treatment are changing. MATERIALS AND METHODS: We retrospectively analysed all the patients with CHC who received treatment with DAAs in a large referral centre since 01/01/2015. We stratified their demographic, clinical and virological characteristics at baseline and the sustained virological response (SVR) rates according to the year of treatment. RESULTS: In the study were included 2565 patients. During the study period, the yearly proportion of men and cirrhotic patients decreased (p<0.001) whereas mean age increased from 59.8 to 62.2 years old (p=0.04). An increasing trend was observed in the foreign-born patients from 4.3% to 7.9%, without reaching statistical significance. The prevalence of comorbidities had also increased during the study period (p<0.001). Instead, the yearly number of experienced patients decreased significantly (p<0.001) as well as the mean MELD score of cirrhotic patients from 9 to 7.6 (p<0.001). SVR rates increased significantly, from 93.4% in 2015 to 97.1% in 2018 (P<0.05). CONCLUSIONS: The population of patients with CHC receiving DAAs is becoming older and with more comorbidities. Nevertheless, this did not impact SVR rates.

Clinical characteristics of coronavirus disease (COVID-19) early findings from a teaching hospital in Pavia, North Italy, 21 to 28 February 2020.

We describe clinical characteristics, treatments and outcomes of 44 Caucasian patients with coronavirus disease (COVID-19) at a single hospital in Pavia, Italy, from 21-28 February 2020, at the beginning of the outbreak in Europe. Seventeen patients developed severe disease, two died. After a median of 6 days, 14 patients were discharged from hospital. Predictors of lower odds of discharge were age > 65 years, antiviral treatment and for severe disease, lactate dehydrogenase > 300 mg/dL.

Baseline Amino Acid Substitutions in the NS5A ISDR and PKR Binding Domain of Hepatitis C and Different Fibrosis Levels and Levels of Development of Hepatocellular Carcinoma in Patients Treated with DAAs.

Variations in the interferon sensitivity-determining region (ISDR) within the NS5A region were related to the development of hepatocellular carcinoma (HCC) in patients infected with hepatitis C virus (HCV). The aim of the study was to investigate a relationship between ISDR/PKR substitutions and their association with liver fibrosis or HCC development. A total of 316 patients infected with HCV and treated with DAAs were evaluated. HCV RNA was quantified and sequenced before treatment. The liver fibrosis stage was assessed by transient elastography and equalized to METAVIR scores. Multivariate analysis showed that ≥3 substitutions in ISDR and ≥6 in PKR-bd were significantly associated with advanced fibrosis. Advanced fibrosis was observed in patients with higher substitutions in ISDR and PKR-bd. A higher correlation between advanced fibrosis and a high frequency of ≥3 substitutions in ISDR and ≥6 in PKR-bd was observed in patients infected with genotype 2c. In addition, in a higher proportion of HCC patients, advanced fibrosis (40.4% vs. 88.2%; p < 0.001) and ≥6 substitutions in PKR-bd (15.4% vs. 41.2%; p = 0.01) was observed. In conclusion, a higher number of substitutions in ISDR and PKR-bd were associated with advanced liver fibrosis, suggesting a use of like predictors for progression in the liver damage. A significantly higher number of PKR-bd substitutions was observed in HCC patients; in particular, in patients infected with HCV genotype 2c.

Viral dynamics among HCV infected patients with different genotypes treated with genotypic specific or pan-genotypic direct-acting antiviral agent combinations.

BACKGROUND: New hepatitis C virus (HCV) therapies have improved efficacy, allowed pangenotypic applications, increased barriers to drug resistance and shortened therapy duration. METHODS: Patients infected with different HCV genotypes were divided into two groups: group 1 included 169 patients receiving genotypic specific regimens (GSR), while group 2 included 186 patients receiving pan-genotypic regimens (PGR). Patient's HCV RNA was quantified and sequenced. RESULTS: Comparable sustained viral response (SVR) rates were observed in both GSR and PGR treated patients. Nevertheless, even if not significant, a greater proportion of non-detectable levels (NDL) of HCV RNA was observed in patients treated with PGR as compared with GSR. Overall, among patients in the GSR and PGR groups with residual viremia, 124/169 (73.4%) and 125/186 (67.2%) at four weeks, and 66/169 (39.1%) and 58/186 (31.2%) at eight weeks, achieved SVR. No difference was observed in the clinical outcome comparing patients in the GSR and PGR groups according to genotype. While, comparing patients between the two groups, the proportion of patients with NDL HCV RNA at four and eight weeks was higher in patients infected with genotype 1b treated with PGR (p=0.0015). A significantly higher number of patients infected with 1b had RASs at baseline (p=0.0001). In addition, the proportion of patients with treatment failure was higher in patients with RASs at baseline compared with those without (p=0.012). Overall, 2.5% patients failed to achieve SVR after DAA treatment. CONCLUSION: A sharp HCV RNA decrease was observed in patients treated with both GSR and PGR. However, even if comparable, a slightly greater number of patients treated with PGR achieved NDL HCV RNA as compared with GSR. A significant difference was observed in patients with baseline RASs, both in relation to treatment failure and genotype. In conclusion, the use of new DAA combinations helps patients achieve a more rapid virologic response.

Baseline and Breakthrough Resistance Mutations in HCV Patients Failing DAAs.

Sustained virologic response rates have increased dramatically following direct acting antiviral (DAA) therapy in chronic HCV infection. However, resistance-associated substitutions (RASs) may occur either prior to DAA or following drug exposure. The aim of this study was to determine RASs in DAA treatment-failing patients and the role of RASs in failure treatment. Six hundred and twenty HCV patients were evaluated. Direct sequencing of HCV genes was performed at breakthrough in all 31 patients failing DAAs, and in 19 baseline patients. Deep sequencing analysis was performed in 15/19 baseline patients. RASs were detected at breakthrough in 17/31 patients and at baseline in 11/19 patients, although, only 8/19 patients carried RASs associated with the prescribed regimen. Deep sequencing analysis showed RASs at baseline in 10/15 treatment-failing patients. No significant difference was observed with the Sanger sequencing. Treatment failure in the 14/31 patients without RASs was associated with suboptimal treatment. In 54.8% of treatment-failing patients one of the causes of failure might be the presence of RASs. In the majority of patients with RASs, mutations were present at baseline. Direct resistance test is advocated before treatment and at breakthrough in order to optimize retreatment regimens.

Development and persistence of DAA resistance associated mutations in patients failing HCV treatment.

BACKGROUND: Direct-acting antiviral agents (DAAs) combined with pegylated-interferon (PegIFN) and ribavirin (RBV) are still a standard treatment in patients with genotype 1HCV infection. However, virologic response could be impaired by baseline or early selection of resistant HCV strains. OBJECTIVES: The aim of this study was to determine the onset and persistence of resistance-associated mutations (RAMs) in the NS3 and NS5B genes of DAA-naïve patients failing treatment. STUDY DESIGN: Direct sequencing of HCV NS3 was performed in 49 DAA-naïve patients with HCV genotype 1 infection. RESULTS: Eight out of 23 patients (34.7%) failed PegIFN/RBV/telaprevir during the 12-weeks of therapy. Treatment failure was associated with the development of RAMs at amino-acids 36,54,80 and 155 of the HCV protease in 6/8 patients (75%). Among patients treated with PegIFN/RBV/boceprevir treatment, 4/18 (22.2%) failed therapy. Of these, 2 (50%) carried virus strains which developed a RAM at amino-acids 54 and 155. Among HCV strains with RAMs, 7 belonged to genotype 1a and 1 to 1b. Finally, in 6/10 (60%) patients, drug-resistant variants could still be detected for up to 3-7 months after stopping therapy. CONCLUSIONS: A higher rate (p=0.49) of treatment failure was observed in patients receiving telaprevir- compared to the boceprevir-based combination. In addition, compared with genotype 1b, genotype 1a was associated with higher rates (p=0.01) of treatment failure due to virus resistant strains. Resistance testing at baseline and during DAA treatment should be taken into consideration when treating patients with new HCV combination therapies.

A rapid qPCR method to investigate the circulation of the yeast Wickerhamomyces anomalus in humans.

The yeast Wickerhamomyces anomalus has been proposed for many biotechnological applications in the food industry. However, a number of opportunistic pathogenic strains have been reported as causative agents of nosocomial fungemia. Recognition of potentially pathogenic isolates is an important challenge for the future commercialization of this yeast. The isolation of W. anomalus from different matrices and, recently, from mosquitoes, requires further investigations into its circulation in humans. Here we present a qPCR protocol for the detection of W. anomalus in human blood samples and the results of a screening of 525 donors, including different classes of patients and healthy people.

L1 gene sequence of a putative novel type human papillomavirus in an immunocompetent patient with glans lichen sclerosus.

The identification of a putative novel type human papillomaviruses (HPV) strain related to HPV-RTRX3 in a subject with penile skin warts and glans lichen sclerosus is reported. A beta-HPV-RTRX3-like strain was detected in a immunocompetent patient with glans lichen sclerosus. HPV screening was performed by PCR in L1 gene. The MY fragment showed 99% nt identity with HPV-RTRX3 and 64.5% nt identity with HPV-37. The remaining part of the L1 gene showed similarity with HPV 80, 15, 17, and 37. Based on the presence of penile lichen sclerosus and the HPV-RTRX3-like strain found in our patient, a potential correlation was hypothesized.

Liver fibrosis, microbial translocation and immune activation markers in HIV and HCV infections and in HIV/HCV co-infection.

BACKGROUND: Liver fibrosis is accelerated in patients co-infected with human immunodeficiency virus and hepatitis C viruses. AIMS: We investigated the correlation between liver fibrosis, immune activation and microbial translocation. METHODS: This cross-sectional study included patients with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) mono-infections, HIV/HCV co-infection, and healthy controls (20 subjects/group). Peripheral blood was analysed to determine the levels of Forkhead box 3 (Foxp3) T cells, TGF-ß1, CD14 (soluble and surface isoforms), IL-17 and bacterial translocation products. These measurements were correlated to the severity of liver fibrosis, measured with the FIB-4 score and transient elastography. RESULTS: Foxp3T cell levels were significantly elevated in HIV mono-infected and co-infected groups (p<0.0005). FIB-4 and liver stiffness values inversely correlated with TGF-ß1 (p=0.0155 and p=0.0498). Bacterial DNA differed significantly in the HIV-positive compared to the other groups: HIV/HCV co-infected subjects had significantly higher serum levels of bacterial translocation products, CD14, and IL-17 levels (p<0.001). CONCLUSIONS: Fibrosis stage in HIV/HCV co-infection may be influenced by immune activation due either by viral infections or to bacterial translocation.

Phylogenetic analysis of HIV type 1 CRF02_AG in multiple genes in Italian and African patients living in Italy.

Human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) 02_AG is a major recombinant variant in different geographic areas and is predominant in West and Central Africa. Of particular interest is the increased frequency of CRF02_AG in patients living in Italy. In the present study, phylogenetic analyses were performed on gag, pol (integrase), and env (gp120 and gp41) gene sequences from 34 CRF02_AG-infected patients living in Italy. Thirty out of 34 (89.4%) patients were from western Africa, 3/34 (8.8%) were born in Italy, and 1/34 (2.9%) was from Cuba. Phylogenetic analysis revealed the presence of a well-supported clade (aLRT score>0.75) of sequences only in gp120 and gp41 trees. Evolutionary rate estimation showed a faster evolution for the gp120 gene with respect to the gag, integrase, and gp41 genes. This finding was confirmed by the analysis of interpatient variability. Intrapatient variability was greater in gp120 gene sequences; 10/19 (52.6%; p<0.001) patients had a ratio of dN/dS>1 as compared with gag, integrase, and gp41 gene sequences with dN/dS ratios<1. In summary, phylogenetic analyses of CRF02_AG strains offer a perspective on intrapatient and interpatient variability among CRF02_AG-infected patients living in Italy. In addition, divergent phylogenetic relationships were observed among different genomic regions.

Failure of repeated treatment with praziquantel and arthemeter in four patients with acute schistosomiasis.

Schistosomiasis is on the rise but still difficult to treat in international travelers; it should be suspected in patients returning from endemic areas. Praziquantel (PZQ) is not effective and may aggravate symptoms. More recently, combination treatment with artemisinin derivatives have shown promising results. We report four cases of acute schistosomiasis (AS) in which several courses of combined therapy had been necessary to obtain negative serology.