To better define the incidence and significance of cryptic chromosome lesions in acute myeloid leukemia (AML), fluorescence in situ hybridization (FISH) studies were performed in interphase cells and, when appropriate, in metaphase cells and in morphologically intact BM smears. Fifty-five adult de novo AML (group A) and 27 elderly AML or AML after myelodysplastic syndrome (AML-MDS) (group B) were tested using probes detecting the following anomalies: −5, −7, +8, deletions of 5q31, 7q31, 12p13/ETV6, 17p13/p53, 20q11. All the patients had a normal karyotype in more than 20 cells and tested negative for the common AML-associated fusion genes. No patient in group A was found to carry occult chromosome anomalies, whereas 8/27 patients in group B (P Ͻ 0.0001) showed 5q31 or 7q31 deletion (three cases each), a 17p13/p53 deletion or trisomy 8 (one case each) in 33-60% interphase cells. Metaphase cells showed only one hybridization signal at 5q31 (three cases) and 7q31 (one case), whereas two normal signals at 7q31 and chromosome 8 centromeres were seen in two patients with 7q deletion and trisomy 8 in interphase cells. The majority of blast cells (76-94%) carried the chromosome anomaly in all cases; erythroid involvement in a minority of cells was seen in three patients. In group B, the presence of occult chromosome anomalies was associated with exposure to myelotoxic agents in the workplace (5/8 cases vs 3/19, P = 0.026) and with a lower complete remission rate (0/6 patients vs 7/12, P = 0.024). We arrived at the following conclusions: (1) cryptic chromosome deletions in the order of a few hundred kb magnitude may be found in a fraction of elderly AML or MDS-related AML and not in de novo adult AML with normal karyotype; (2) these chromosome lesions are usually represented by submicroscopic rearrangements; (3) they display a specific pattern of cell-lineage involvement arguing in favor of their role in the outgrowth of the leukemic blast cells; (4) they are associated with a history of exposure to myelotoxic agents in the workplace and, possibly, with resistance to induction treatment.
Objective : When using high-throughput batched diagnostic platforms based on RT-PCR for SARS-CoV-2 detection, avoidance of the conventional nucleic acid extraction step can help to reduce the turnaround time and increase processivity. This approach can also spare reagents and plasticware, which have experienced a shortage during the initial waves of the pandemic, reducing the overall testing costs. Methods : This study evaluated the performance of extraction-free protocols based on simple dilution of the specimen in sterile RNAse free water (with or without a heating step) in comparison to standard RNA extraction protocols, using two commercial kits for molecular detection of SARS-CoV-2 (Allplex™ SARS-CoV-2 assay and Allplex™ SARS-CoV-2/FluA/FluB/RSV assay) in nasopharyngeal swabs (NPS). Results : Compared with conventional protocols, extraction-free protocols based on sample dilution without a heating step exhibited a lower analytical sensitivity: 74% and 82% with the Allplex™ SARS-CoV-2 assay (tested with 139 NPS samples) and the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay (tested with 69 NPS samples), with a mean increase of Ct values of +2.04 and +1.32, respectively. Most false negative results were observed with sampled of low viral load. Including a step of heath exposure did not improve but actually decreased the analytical sensitivity of the assay. Conclusions : Results confirmed that extraction-free protocols could be a faster and cheaper approach to SARS-CoV-2 detection in NPS samples, that could improve processivity of diagnostic platforms.
Different viruses can be responsible for similar clinical manifestations of respiratory infections. Thus, the etiological diagnosis of respiratory viral diseases requires the detection of a large number of viruses. In this study, 6 duplex real-time PCR assays, using EvaGreen intercalating dye, were developed to detect 12 major viruses responsible for respiratory diseases: influenza A and B viruses, enteroviruses (including enterovirus spp, and rhinovirus spp), respiratory syncytial virus, human metapneumovirus, coronaviruses group I (of which CoV 229E and CoV NL63 are part) and II (including CoV OC43 and CoV HKU1), parainfluenza viruses type 1, 2, 3 and 4, human adenoviruses and human bocaviruses. The 2 target viruses of each duplex reaction were distinguishable by the melting temperatures of their amplicons. The 6 duplex real time PCR assays were applied for diagnostic purpose on 202 respiratory samples from 157 patients. One hundred fifty-seven samples were throat swabs and 45 were bronchoalveolar lavages. The results of the duplex PCR assays were confirmed by comparison with a commercial, validated, assay; in addition, the positive results were confirmed by sequencing. The analytical sensitivity of the duplex PCR assays varied from 10(3) copies/ml to 10(4) copies/ml. For parainfluenza virus 2 only it was 10(5) copies/ml. Seventy clinical samples (35%) from 55 patients (30 children and 25 adults) were positive for 1 or more viruses. In adult patients, influenza A virus was the most frequently detected respiratory virus followed by rhinoviruses. In contrast, respiratory syncytial virus was the most common virus in children, followed by enteroviruses, influenza A virus and coronavirus NL63. The small number of samples/patients does not allow us to draw any epidemiological conclusion. Altogether, the results of this study indicate that the 6 duplex PCR assays described in this study are sensitive, specific and cost-effective. Thus, this assay could be particularly useful to identify the main respiratory viruses directly from clinical samples, after nucleic acid extraction, and, also, to screen a large number of patients for epidemiological studies.
La fibrosi cistica (FC), è una malattia genetica autosomica recessiva a interessamento multiorganico causata da una mutazione nel gene CFTR che causa un'alterazione nel trasporto di sali con aumento della viscosità delle secrezioni (muco, sudore, saliva, sperma, etc.); tali secrezioni, ristagnando, occludono i dotti con progressivo danno agli organi interessati (4). L'organo maggiormente colpito è il polmone, dove queste secrezioni formano delle vere e proprie "placche" mucosali, nelle quali possono penetrare batteri in fase mobile causando infezioni respiratorie ricorrenti (7). L'infezione da parte di P. aeruginosa subentra in età adolescenziale, inizialmente ad opera di ceppi ambientali che, con caratteristiche genotipiche e fenotipiche differenti, possono colonizzare contemporaneamente il polmone FC. La persistenza di P. aeruginosa nel polmone FC è legata all'espressione da parte del germe di alcuni fattori di virulenza, tra cui diverse proteasi e pigmenti solubili e insolubili, la presenza di uno o più flagelli polari, la produzione di esopolisaccaride esterno (alginato) e la capacità di formare biofilm (2, 5). L'obiettivo del nostro lavoro è stato studiare alcuni di questi fattori di virulenza in ceppi di P. aeruginosa isolati dagli espettorati di pazienti affetti da FC, esaminando per ogni paziente coppie di isolati indistinguibili genotipicamente, ottenuti a distanza di alcuni mesi oppure che persistessero nel polmone nello stesso periodo di tempo ma con morfologia evidentemente differente, allo scopo di verificare variazioni nell'espressione dei fattori di virulenza all'interno della coppia. Lo studio è stato effettuato su 14 ceppi (7 rugosi e 7 mucoidi) di P. aeruginosa, isolati e conservati presso l'Area Funzionale di Microbiologia dell'Azienda Ospedaliera Universitaria Policlinico "Federico II" di Napoli. L'identificazione degli isolati è stata ottenuta in base a test biochimici effettuati dal sistema automatico VitekII (bioMérieux). Il DNA di ciascun ceppo è stato isolato e sottoposto ad amplificazione con primers che, amplificando regioni genomiche di lunghezza variabile comprese tra i siti di inserzione delle sequenze ERIC, evidenziassero patterns di amplificazione unici per ogni ceppo (ERIC-PCR). La funzionalità di pili e flagelli è stata evidenziata con lo studio della mobilità su agar all'1% (twitching) e 0.3% (swimming) rispettivamente. La capacità di formare bio-
In August 2018 a Moroccan man living in Tuscany developed Plasmodium falciparum malaria. The patient declared having not recently visited any endemic country, leading to diagnostic delay and severe malaria. As susceptibility to P. falciparum of Anopheles species in Tuscany is very low, and other risk factors for acquiring malaria could not be completely excluded, the case remains cryptic, similar to other P. falciparum malaria cases previously reported in African individuals living in Apulia in 2017.
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