A New Approach: Determining cyt b G143A Allele Frequency in Zymoseptoria tritici by Digital Droplet PCR

Z. tritici first appeared in Italy later than in northern-central European countries. QoIs fungicides currently play a role in STB control, used in combination with Demethylation Inhibitors (DMIs) or Succinate dehydrogenase Inhibitors (SDHIs). In this study, we set up a fast, sensitive, and accurate ddPCR protocol in order to investigate the presence and frequency of G143A substitution, causing a reduction in strobilurins’ efficacy in Z. tritici. The best PCR conditions for the clear separation of positive and negative droplets were identified. The lowest wild-type and resistant alleles frequencies were accurately determined on samples consisting of mixed DNAs from monoconidial cultures of Z. tritici and were expressed as fractional abundance. The protocol was tested by determining the copy number and frequency of alleles on gDNA purified in three Italian Z. tritici field populations representative of different fungicide management strategies. For the first time, the determination of allele concentration and the frequency of a mutation involved in Z. tritici fungicide resistance was carried out by employing digital PCR. This new approach provides a diagnostic tool that is rapid and able to detect very low G143A substitution percentages, which is very useful for fungicide resistance detection at early stages, thus, informing field management strategies for contrasting STB disease

Fatality rate and predictors of mortality in an Italian cohort of hospitalized COVID-19 patients

Clinical features and natural history of coronavirus disease 2019 (COVID-19) differ widely among different countries and during different phases of the pandemia. Here, we aimed to evaluate the case fatality rate (CFR) and to identify predictors of mortality in a cohort of COVID-19 patients admitted to three hospitals of Northern Italy between March 1 and April 28, 2020. All these patients had a confirmed diagnosis of SARS-CoV-2 infection by molecular methods. During the study period 504/1697 patients died; thus, overall CFR was 29.7%. We looked for predictors of mortality in a subgroup of 486 patients (239 males, 59%; median age 71 years) for whom sufficient clinical data were available at data cut-off. Among the demographic and clinical variables considered, age, a diagnosis of cancer, obesity and current smoking independently predicted mortality. When laboratory data were added to the model in a further subgroup of patients, age, the diagnosis of cancer, and the baseline PaO2/FiO2 ratio were identified as independent predictors of mortality. In conclusion, the CFR of hospitalized patients in Northern Italy during the ascending phase of the COVID-19 pandemic approached 30%. The identification of mortality predictors might contribute to better stratification of individual patient risk

A New Approach: Determining cyt b G143A Allele Frequency in Zymoseptoria tritici by Digital Droplet PCR

Z. tritici first appeared in Italy later than in northern-central European countries. QoIs fungicides currently play a role in STB control, used in combination with Demethylation Inhibitors (DMIs) or Succinate dehydrogenase Inhibitors (SDHIs). In this study, we set up a fast, sensitive, and accurate ddPCR protocol in order to investigate the presence and frequency of G143A substitution, causing a reduction in strobilurins’ efficacy in Z. tritici. The best PCR conditions for the clear separation of positive and negative droplets were identified. The lowest wild-type and resistant alleles frequencies were accurately determined on samples consisting of mixed DNAs from monoconidial cultures of Z. tritici and were expressed as fractional abundance. The protocol was tested by determining the copy number and frequency of alleles on gDNA purified in three Italian Z. tritici field populations representative of different fungicide management strategies. For the first time, the determination of allele concentration and the frequency of a mutation involved in Z. tritici fungicide resistance was carried out by employing digital PCR. This new approach provides a diagnostic tool that is rapid and able to detect very low G143A substitution percentages, which is very useful for fungicide resistance detection at early stages, thus, informing field management strategies for contrasting STB disease

Characterization of the aggressiveness and fungicide sensitivity of populations of Mycosphaerella graminicola (anamorph. Zymoseptoria tritici) on wheat

Mycosphaerella graminicola, anamorfo Zymoseptoria tritici, è l’agente causale della septoriosi. Questo patogeno, diffuso in Europa, in Italia solo in tempi recenti è causa di notevoli perdite di produzione. L’impiego di cultivar tolleranti è un importante mezzo di controllo ma il più diffuso è il trattamento con fungicidi. L’obiettivo principale dello studio è stato valutare la sensibilità di isolati italiani di Z. tritici alle famiglie di fungicidi impiegate nella difesa (QoI, DMI e SDHI). Questo ha portato anche alla messa a punto di un protocollo in droplets digital PCR (ddPCR) per valutare la frequenza della G143A, sostituzione responsabile della resistenza alle strobilurine. Un altro obiettivo ha invece riguardato la sensibilità di varietà di frumento coltivate in Italia a Z. tritici approfondendo la specificità di infezione di isolati del patogeno al frumento tenero e duro. Dalla valutazione della sensibilità a QoI, DMI e SDHI e dall’analisi delle sequenze cytb, cyp51 e Sdhb-c-d si è potuto concludere come le tre famiglie possono essere impiegate nella difesa in Italia. A causa della presenza di cali di efficacia riscontrati nei confronti di QoI e DMI appare importante seguire le indicazioni anti-resistenza proposte dal Fungicide Resistance Action Committee. L’ utilizzo della ddPCR nello studio della frequenza della G143A ha permesso di misurare in maniera sensibile e accurata la sostituzione. Protocolli simili potrebbero essere sviluppati nei confronti di altre mutazioni causanti resistenza ai fungicidi, sia in Z. tritici che in altri patogeni. La valutazione della sensibilità delle varietà di frumento al patogeno, è avvenuta attraverso l’inoculazione delle piante con isolati provenienti da frumento duro e tenero. I risultati hanno mostrato l’emergere della specializzazione degli isolati saggiati alle due specie di frumento ma studi più approfonditi, potrebbero confermare il fenomeno. Le informazioni apportate da questa tesi possono contribuire sostanzialmente alla gestione di Z. tritici in campo.Mycosphaerella graminicola anamorph. Zymoseptoria tritici is the casual agent of Septoria Tritici Blotch (STB). The pathogen is widespread in Europe, but in Italy only recently is causing heavy losses of yield. Against STB genetic resistance is an important approach, but disease control mostly rely to fungicide applications. The main aim of the study was test the fungicide sensitivity (QoI, DMI e SDHI) of Italian Z. tritici istrains. This aim led to set up of protocol in droplets digital PCR (ddPCR) to investigate the frequency of G143A substitution causing a reduction of strobilurins’ efficacy. Another aim was test the sensitivity of Italian wheat variety to Z. tritici investigating the specificity of pathogen strains to bread and durum wheat. From the sensitivity test to Qoi, DMI and SDHI and from the analysis of cytb, cyp51 e Sdhb-c-d genes this study showed a reduction of sensitivity of STB strains to QoI and DMI. However all the three family of fungicide can be used in field management of the pathogen but is important follow the anti-resistance strategy proposed by Fungicide Resistance Action Committee. The use of ddPCR made it possible to obtain a sensitive and accurate measurement of the frequencies of G143A substitution. Similar protocols could be developed to investigate other mutations involved in fungicide resistance in Z. tritici as well as in other plant pathogens. The sensitivity of wheat variety to STB was evaluated inoculating the plants with strains collected from bead and durum wheat. The results showed the emergence of specialization of isolates tested to bread and durum wheat but another test could confirm the supposition. The information make from this study could improve the management of Z. tritici in field

PRELIMINARY RESULTS OF STROBILURIN SENSITIVITY OF ZYMOSEPTORIA TRITICI ITALIAN STRAINS

Mycosphaerella graminicola (anamorph: Zymoseptoria tritici) is the causal agent of leaf blotch, the most important foliar disease of wheat in Northern and Central Europe. In Italy only during the last few years, the incidence of the disease has been increased. The most common strategies for the control of the disease is the use of fungicides in particular QoIs, DMIs, and more recently were introduced the SDHIs. The resistance to QoIs and DMIs was already common in the main wheat cultivated areas in the world (Cools and Fraaije, 2008; Estep et al., 2015; Fraaije et al., 2005; Gisi et al., 2005; Leroux et al., 2005; Stammler and Semar, 2011; Stewart et al., 2014) while in Italy no results coming from wide and specific sensitivity monitoring are available. The aim of this study was to test the sensitivity of Z. tritici strains to QoI in order to obtain the first data about the Italian scenario. Leaves of bread and durum wheat were collected during 2015 from 10 fields with different use of fungicides (wild type, experimental centers and commercial ones) located in the North of Italy. The sensitivity of 60 isolates to azoxystrobin at different concentrations (0-0.001-0.01-0.1-1-2-10-20 mg/L of active ingredient) have been determined in vitro by microtiter assay (Stammler and Semar, 2011). The EC50 values of the wild types ranged from 0.02 to 0.12 mg/L of azoxystrobin, while the first analyzed isolates collected from experimental plots and commercial fields showed EC50s variable from 1.92 to 5.1 mg/L. Only one isolate pointed out an EC50 of 20.6 mg/L of active ingredient. On the base of these first results, we can suppose the presence of a slight decrease of sensitivity of Z. tritici isolates collected from Italian fields (according to Gisi et al., 2005). Molecular analysis on the presence of G143A substitution will be carried out

AN ITALIAN POPULATION OF ZYMOSEPTORIA TRITICI: PATHOGENICITY AND STROBILURINS SENSITIVITY

Mycosphaerella graminicola (anamorph: Zymoseptoria tritici) is the causal agent of leaf blotch, the most important foliar disease of wheat in northern and Central Europe. In Italy only during the last few years, the incidence of the disease has been increased. During 2015 a Z. tritici population was collected by different cultivar of bread and durum wheat cultivated in experimental plots and commercial fields of Emilia-Romagna region (North Italy). The aim of the study was to test the pathogenicity of each Z. tritici strains on both (bread and durum) wheat species in greenhouse. In fact, some researchers observed that the majority of strains isolated from durum wheat are lowly virulent on bread wheat cultivars, whereas the majority of strains isolated from bread wheat are lowly virulent on durum wheat (Gohari et al., 2015 Mol. Plant Pathol.,16: 931–945). Another aim was to test the sensitivity of Z. tritici population to fungicides as strobilurins by in vitro assays. The preliminary data about pathogenicity confirm the cultivar specificity in the infection process. Further data are required in order to confirm the hypothesis in an Italian Z. tritici population too. The results about sensitivity test showed an EC50 value of the wild types ranged from 0.1 to 2.88 mg/l of azoxystrobin, while the isolates collected from experimental plots and commercial fields showed EC50 value ranged from 0.01 to 5.1 mg/l. These first results show a slight decrease of sensitivity of Z. tritici isolates collected from Emilia- Romagna region (according to Gisi et al., 2005 Modern Fungicides and Antifungal Compounds IV, pp 89-101)

Simple Parameters from Complete Blood Count Predict In-Hospital Mortality in COVID-19

Introduction. The clinical course of Coronavirus Disease 2019 (COVID-19) is highly heterogenous, ranging from asymptomatic to fatal forms. The identification of clinical and laboratory predictors of poor prognosis may assist clinicians in monitoring strategies and therapeutic decisions. Materials and Methods. In this study, we retrospectively assessed the prognostic value of a simple tool, the complete blood count, on a cohort of 664 patients (F 260; 39%, median age 70 (56-81) years) hospitalized for COVID-19 in Northern Italy. We collected demographic data along with complete blood cell count; moreover, the outcome of the hospital in-stay was recorded. Results. At data cut-off, 221/664 patients (33.3%) had died and 453/664 (66.7%) had been discharged. Red cell distribution width (RDW) (χ2 10.4; p4.68 was characterized by an odds ratio for in-hospital mortality OR=3.40 (2.40-4.82), while the OR for a RDW>13.7% was 4.09 (2.87-5.83); a platelet count>166,000/μL was, conversely, protective (OR: 0.45 (0.32-0.63)). Conclusion. Our findings arise the opportunity of stratifying COVID-19 severity according to simple lab parameters, which may drive clinical decisions about monitoring and treatment

Tocilizumab for patients with COVID-19 pneumonia. The single-arm TOCIVID-19 prospective trial

BackgroundTocilizumab blocks pro-inflammatory activity of interleukin-6 (IL-6), involved in pathogenesis of pneumonia the most frequent cause of death in COVID-19 patients.MethodsA multicenter, single-arm, hypothesis-driven trial was planned, according to a phase 2 design, to study the effect of tocilizumab on lethality rates at 14 and 30 days (co-primary endpoints, a priori expected rates being 20 and 35%, respectively). A further prospective cohort of patients, consecutively enrolled after the first cohort was accomplished, was used as a secondary validation dataset. The two cohorts were evaluated jointly in an exploratory multivariable logistic regression model to assess prognostic variables on survival.ResultsIn the primary intention-to-treat (ITT) phase 2 population, 180/301 (59.8%) subjects received tocilizumab, and 67 deaths were observed overall. Lethality rates were equal to 18.4% (97.5% CI: 13.6-24.0, P=0.52) and 22.4% (97.5% CI: 17.2-28.3, P<0.001) at 14 and 30 days, respectively. Lethality rates were lower in the validation dataset, that included 920 patients. No signal of specific drug toxicity was reported. In the exploratory multivariable logistic regression analysis, older age and lower PaO2/FiO2 ratio negatively affected survival, while the concurrent use of steroids was associated with greater survival. A statistically significant interaction was found between tocilizumab and respiratory support, suggesting that tocilizumab might be more effective in patients not requiring mechanical respiratory support at baseline.ConclusionsTocilizumab reduced lethality rate at 30 days compared with null hypothesis, without significant toxicity. Possibly, this effect could be limited to patients not requiring mechanical respiratory support at baseline.Registration EudraCT (2020-001110-38); clinicaltrials.gov (NCT04317092)