Multiplex real-time PCR for skin fungal infections: The diagnostic reliability in a one-year non-interventional study.

The skin fungal infection diagnostic workflow currently includes microscopic and culture-based methods as the gold standard. Recent published data described the possible limitations of these conventional techniques documenting the possibility of reducing response time intervals. The present study reports an evaluation of the DermaGenius® (DG) multiplex kit (PathoNostics) for rapid C. albicans and dermatophytes identification directly from skin samples. The investigations involved 90 specimens that underwent DNA extraction and amplification simultaneously to microscopic and culture methods. According to current guidelines, we defined a dermatophytic skin infection as the simultaneous presence of clinical evidence of skin lesions and positive results for dermatophyte elements from microscopy and/or cultures. The collected data remarked on the advantages of the molecular assay, especially in terms of sensitivity and rapidity. A statistical evaluation analysed a comparison between conventional and innovative diagnostic methods. The sensitivity, specificity, positive predictive value, and negative predictive value of DG-PCR in the cutaneous dermatophytosis were, respectively, 94.7%, 78.8%, 88.5%, and 89.6%. Based on our experience, the molecular technique could represent a diagnostic confirmation in the case of previous antifungal treatment, little biological material available, or urgent clinical conditions.

Our study aims to evaluate innovative technologies in diagnosing skin dermatophytosis. The final purpose was to describe an added diagnostic value, aiming to improve patients' outcomes. High sensitivity rates and a restricted turn-around time represent the main advantages.

Competing endogenous RNA network mediated by circ_3205 in SARS-CoV-2 infected cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a new member of the Betacoronaviridae family, responsible for the recent pandemic outbreak of COVID-19. To start exploring the molecular events that follow host cell infection, we queried VirusCircBase and identified a circular RNA (circRNA) predicted to be synthesized by SARS-CoV-2, circ_3205, which we used to probe: (i) a training cohort comprised of two pools of cells from three nasopharyngeal swabs of SARS-CoV-2 infected (positive) or uninfected (negative, UCs) individuals; (ii) a validation cohort made up of 12 positive and 3 negative samples. The expression of circRNAs, miRNAs and miRNA targets was assayed through real-time PCR. CircRNA-miRNA interactions were predicted by TarpMiR, Analysis of Common Targets for circular RNAs (ACT), and STarMir tools. Enrichment of the biological processes and the list of predicted miRNA targets were retrieved from DIANA miRPath v3.0. Our results showed that the predicted SARS-CoV-2 circ_3205 was expressed only in positive samples and its amount positively correlated with that of SARS-CoV-2 Spike (S) mRNA and the viral load (r values = 0.80952 and 0.84867, Spearman's correlation test, respectively). Human (hsa) miR-298 was predicted to interact with circ_3205 by all three predictive tools. KCNMB4 and PRKCE were predicted as hsa-miR-298 targets. Interestingly, the function of both is correlated with blood coagulation and immune response. KCNMB4 and PRKCE mRNAs were upregulated in positive samples as compared to UCs (6 and 8.1-fold, p values = 0.049 and 0.02, Student's t test, respectively) and their expression positively correlated with that of circ_3205 (r values = 0.6 and 0.25, Spearman's correlation test, respectively). We propose that our results convincingly suggest that circ_3205 is a circRNA synthesized by SARS-CoV-2 upon host cell infection and that it may behave as a competitive endogenous RNA (ceRNA), sponging hsa-miR-298 and contributing to the upregulation of KCNMB4 and PRKCE mRNAs.

Multiple HPV 16 infection with two strains: a possible marker of neoplastic progression.

BACKGROUND: We studied the cases of single and multiple HPV infection and analyzed the correlation with negative cases, and preneoplastic and neoplastic lesions of the uterine cervix with the aim of making a contribution to the prognostic factor under discussion. METHODS: Nine hundred nine women undergoing second level screening because they had been positive at cervical cytology were enrolled. All the patients underwent colposcopy and cervical biopsy with viral genotyping. We divided mHPV infection based on the number of genotypes present: infections with 2 strains, 3 strains, 4 strains and 5 or more strains. STATISTICAL ANALYSIS: The analysis of the data was made using the χ2 test. Contingency tables were created to evaluate the correlation between single, multiple and CIN2+ infections. Values with p < 0.05 were considered statistically significant. RESULTS: The presence of genotype HPV16 in our study was associated with a 12 times greater risk of developing a high-grade lesion, OR = 12.70. The patients with single infections had the highest incidence of CIN2+ (34.1%) with respect to those with multiple infections (10.6%).When we studied in the mHPV infection the prevalence of the combinations between the genotypes, we found that in mHPV16 infections, the combinations HPV16, 18 and HPV16, 31 were the most frequent (55.5%) in CIN3 lesion. CONCLUSIONS: Our results suggest that single HPV infections have a greater risk of developing SCC with respect to multiple infections. Multiple HPV infections are relevant only in the first phase of the lesion (CIN1-CIN2), while they are absent in carcinomas, where infections are of a single genotype. In particular, among multiple infections, HPV16 infection with 2 HR genotypes is associated significantly with CIN2 / CIN3 (21/30) and has 4 times greater risk of developing a high-grade lesion. Thus, it is probable that only specific combinations of HPV (HPV16,18 - HPV 16,31) can be associated with a clinically significant impact, while other combinations can simply be correlated because of a common infection or diagnostic method used. Therefore, multiple HPV16 infections with two high-risk genotypes is a major risk of CIN2/CIN3.

Human papillomavirus and risk of prostate cancer: a systematic review and meta-analysis.

Background: There is growing evidence showing a putative association between high-risk human papillomavirus (HR-HPV) infection and an increased risk of PCa.Objective: The aim of the current meta-analysis was to evaluate the association between HPV infection and PCa risk.Methods: This analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis guidelines. We included all studies on HPV DNA or antibodies detected in biopsy tissues or sera. Available data were extracted from the article, including means and standard deviations in all case-control groups.Results: Thirty studies that investigated the link between HPV-16 and -18 were identified as eligible for this systematic review and meta-analysis, including a total of 6321 participants. The pooled OR showed increased risk of PCa (OR =1.37; p < .01) in men positive for HPV-16. There were seven studies with 2391 PCa cases and 4059 controls investigating the association between HPV-18 infection and PCa risk. Significant heterogeneity between study was found in the pooled analyzes. The pooled OR did not show increased risk of PCa (OR =0.80; p = .49) in men positive for HPV-18.Conclusions: This meta-analysis suggests that HPV-16 infection could represent a risk factor for PCa, whereas we found no such association for HPV-18. Further well-conducted studies could be useful to confirm this conclusion.

Conservative management of CIN2 p16 positive lesions in women with multiple HPV infection.

BACKGROUND: According to the 2006 American Society for Colposcopy and Cervical Pathology guidelines, positive CIN2 p16 in women over the age of 25 should be managed with excisional treatment. However, excisional treatment is associated with physical, psychological and obstetric morbidity and can have a negative impact on sexual function. In our study we sought to identify a clear management strategy, addressing the impact of routine use of p16 immunohistochemistry in this population and identify appropriate criteria for patient selection with the aim of reducing over-treatment. METHOD: We studied the medical records of 130 patients who had undergone laser therapy for CIN2. Each patient underwent colposcopy, biopsy and HPV test and were tested for p16 protein,. Patients were divided based on HPV infection into: single infections, multiple infections. All patients underwent ZTA laser therapy with follow-up (2-year follow-up). STATISTICAL ANALYSIS: Contingency tables were created to evaluate the correlation between single, multiple and CIN2+ infections. Values with p < 0.05 were considered statistically significant. RESULTS: Single infections had a histological regression of 61.8% (21/34) and a histological persistence rate of 35.3% (12/34), which was greater than the multiple infection rate. The common characteristic that the women with persistence and progression had was the dimension of the lesion and the genotype 16. Ten cases of histological persistence and the only case of progression had one lesion greater than three quarters of the cervix. CONCLUSIONS: With the progress of our understanding of the natural history of infection from human papillomavirus and the increasing use of colposcopy, thanks to the addition of HPV genotyping and the technique of immunohistochemistry, conservative management of these lesions is now possible.

Oxytocin plasma levels in orgasmic and anorgasmic women.

The study aimed to evaluate oxytocin (Oxt) serum levels before and after sexual intercourse in women affected by anorgasmia. The sample was constituted of 15 anorgasmic women and 16 orgasmic women. The Female Sexual Function Index (FSFI, cutoff ≤26.55) and the Female Sexual Distress Scale (FSDS, cutoff ≥15) questionnaires were used to assess sexual function and sexual distress, respectively. Serum Oxt levels were measured before sexual intercourse (T0) and 5 min after coital sexual activity (T1). Anorgasmic women had an unpleasant sexual experience (FSFI total score, 20.1 ± 1.2;) and were stressed (FSDS score, 19.4 ± 1.3), whereas orgasmic women were fully satisfied with their sexual activity (FSFI total score 28.7 ± 1.3; FSDS score 11.5 ± 1.8). At T0, anorgasmic women had lower levels of Oxt than orgasmic women, 1.8 ± 0.2 pg/mL versus 2.1 ± 0.5 pg/mL, respectively, [95% CI: (-0.58, -0.01); p < .04]. At T1, Oxt levels did not change in anorgasmic women (1.8 ± 0.2 pg/mL versus 2 ± 0.4 pg/mL, p = .09). Finally, orgasmic women had higher levels of Oxt than anorgasmic women, 4.6 ± 0.7 pg/mL versus 2 ± 0.4 pg/mL, respectively [95% CI: (-3.02, -2.17); p < .001]. The repetitive processes to experience the sexual body sensations could represent a survival behavior of species by attachment to a partner.

The molecular detection of carbapenem markers with a two-levels amplification screening protocol: epidemiological and resistome insights.

Objectives: Carbapenem-resistance is a challenging healthcare concern and require specific stewardship programs. Monitoring workflows include the identification from surveillance samples, such as rectal swabs. Although culture assays represent the gold standard, data report a significant effectiveness in detecting carbapenemases genes directly from rectal swabs. The aim of this study was to evaluate the REALQUALITY Carba-Screen kit (AB ANALITICA, Padova, Italy) in detecting carbapenemases genes directly from rectal swabs, also comparing its effectiveness to culture assays results. A next-generation sequencing (NGS) was performed to investigate the positive samples about resistance markers and sequence type (ST). Methods: A number of 136 rectal swabs were collected from the University Hospital Policlinico of Catania critical wards. The samples simultaneously underwent culture and molecular assays (REALQUALITY Carba-Screen kit). The molecular method included two-steps. The first step (1 h and 6 min) rapidly excluded negative samples, while the second one (1 h and 6 min) included only positive samples for a resistance confirmation. All the positive culture samples underwent NGS analysis. Results: Statistical evaluations demonstrated high sensitivity (100%) and detection rates (92.6%) for the REALQUALITY Carba-Screen kit, which mostly correlated to the standard workflow. All the culture positive results matched the positive molecular results, which were mainly confirmed by the NGS resistome analysis. The identified ST appeared to be diversified and different from the clinically significative strains of the same setting, furnishing interesting epidemiological evidence. Conclusion: The molecular detection allowed a coordinate approach in a high-prevalence multi-drug-resistance area. The rapid identification with a multi-step procedure accelerated the infection control procedures, while the preliminary negative results reduced the overtreatment episodes. The molecular method efficacy was confirmed through the NGS. In conclusion, the molecular screening could initially lead to a more conservative approach, which may be reevaluated after a culture result about the microorganisms' identification and susceptibility profile.

Detection of serum IgA to HSV1 and its diagnostic role in sudden hearing loss.

A viral etiology of sudden hearing loss has been hypothesized by many authors. HSV1 neurotropism and its involvement in sudden hearing loss has implicated HSV1 as one of the most accredited etiological agents. A non-invasive method such as the titration of HSV1-specific IgA was evaluated to determine the role of HSV1 as a possible cause sudden hearing loss. A prospective study was carried out by titration of serum IgA to HSV1 in 93 patients and in a control group of 50 healthy subjects and 35 subjects suffering from recent herpes labialis reactivation. Statistical analysis of the results disclosed that IgA titers to HSV1 higher than 1:80 are suggestive for the association of HSV1 infection and sudden hearing loss. Moreover, acyclovir therapy was effective in 81% of patients who showed high specific IgA titers. Overall, the titration of specific serum IgA to HSV1 can be a useful tool to determine the viral etiology of certain cases of sudden hearing loss. This method is simple to perform and minimally invasive. It can lead to a rapid presumptive diagnosis and to prompt specific therapy, reducing the need for corticosteroids.

Comparison between EUCAST Broth Microdilution and MIC Strip Test in Defining Isavuconazole In Vitro Susceptibility against Candida and Rare Yeast Clinical Isolates.

Isavuconazole is a new broad-spectrum triazole, with significant in vitro activity against yeasts. Isavuconazole in vitro susceptibility can be evaluated through broth microdilution as a reference method. Considering difficulties in equipping such methods in a laboratory routine, a commercial MIC Strip test has been designed. This study aims to implement data about isavuconazole in vitro activity and compare EUCAST broth microdilution and MIC Strip test in defining yeast isavuconazole susceptibility. The study involved 629 isolates from positive blood cultures (January 2017-December 2021). The identified species were C. albicans (283), C. glabrata (53), C. krusei (23), C. tropicalis (68), C. parapsilosis complex (151), C. guilliermondii (12), C. famata (6), S. cerevisiae (12), C. neoformans (5), S. capitata (12), and Rhodotorula species (4). All the isolates were tested with EUCAST microdilution and MIC Strip methods. The total essential agreement between these two methods was 99.3%. As a result, we can consider that both methods are useful in testing isavuconazole susceptibility. Proposed cut-off values (P-ECOFF) were calculated using ECOFFinder software. Further studies could lead to either definitive E-COFF or clinical breakpoints, which represent the most important categorization tool of the laboratory data, allowing a better insertion of an antimicrobial drug in clinical practice.

The Rapid Phenotypic Susceptibility Testing in Real-Life Experience: How the MIC Values Impact on Sepsis Fast Diagnostic Workflow.

The MIC value definition faithfully reflects antimicrobial sensitivity, profoundly impacting the infection's clinical outcome. Our study aimed to evaluate the Accelerate PhenoTM System in defining the importance of fast phenotypic susceptibility data. A number of 270 monomicrobial samples simultaneously underwent standard procedures and fast protocols after a contemporary Gram stain. Finally, we provided Turn-around Time (TAT) and statistical evaluations. The fast technology required a medium value of 7 h to complete ID and AST profiles. Although there were some spectrum limitations, it revealed an optimal success rate in microbial identification directly from positive blood cultures. The Gram-negative AST reached a 98.9% agreement between the Accelerate Pheno™ System and the standard method. In addition, the Gram-positive AST gathered a 98.7% agreement comparing the same systems. The chance to rapidly provide precise MIC values is one of the last frontiers in clinical microbiology, especially in high-prevalence antimicrobial resistance areas.

Mucorales/Fusarium Mixed Infection in Hematologic Patient with COVID-19 Complications: An Unfortunate Combination.

Hematological diseases, especially those causing severe neutropenia, represent the main factor in the development of invasive fungal infections (IFIs). Furthermore, COVID-19 has been considerably associated with IFIs due to immunological dysregulation, prolonged hospitalization in intensive care units, and immunomodulatory therapies. Opportunistic molds are correlated with elevated morbidity and mortality rates in these patients, due to immune impairment, diagnostic complexity, and therapeutic challenges. Among opportunistic fungal infections, the Mucorales and Fusarium species are considered particularly aggressive, especially during severe neutropenia. A mixed Mucorales/Fusarium infection has been rarely described in scientific literature. Herein, we report a case of Mucorales and Fusarium co-infection in a patient with acute leukemia whose clinical history was also complicated by COVID-19. Herein, we report a challenging case in order to encourage the clinical suspicion of combined fungal infections in immunosuppressed patients, performing a punctual microbiological diagnosis, and promptly administering the correct empiric and targeted antifungal therapy.

Papillomavirus Infection as Potential Cause of Miscarriage in the Early Gestational Age: A Prospective Study.

The possible association between human papillomavirus (HPV) infection and negative pregnancy outcomes has been debated in the literature, with conflicting results from clinical trials. While some authors support a link between HPV and miscarriage, others argue that the mere detection of the virus does not necessarily indicate a causal relationship with negative pregnancy outcomes. In this study, we conducted a prospective, controlled investigation of the potential association between HPV infection and miscarriage. Our study included 59 women who had experienced a miscarriage and 57 women who had undergone voluntary termination of pregnancy (TOP) within the 12th week of gestation. We assessed HPV prevalence, maternal age, and HPV genotype in both groups and evaluated the relationship between these factors and pregnancy outcome. Unlike previous studies that only identified HPV in cases of abortion, we also correlated the positivity of chorionic villi with gestational age in both groups. We found a close correlation between positive chorionic villi and very early gestational age, with all 13 cases of virus-positive chorionic villi in the miscarriage group occurring in gestational periods of less than 8 + 5 weeks (<60 days) (RR = 28.6). Our analysis showed no correlation between HPV infection and maternal age or viral genotypes. The results suggest that the presence of HPV alone is not enough to cause spontaneous abortion, but a high viral load in early pregnancy may increase the risk of negative outcomes. These findings have important implications for the management of HPV infection during pregnancy and may provide a rationale for the use of HPV vaccines to reduce the incidence of spontaneous abortion and infertility due to preclinical spontaneous abortions.

Prevalence of Onychomycosis in Diabetic Patients: A Case-Control Study Performed at University Hospital Policlinico in Catania.

Diabetes is characterized by an increased rate of serum glucose due to defects in insulin secretion, insulin action or both conditions. Glucose excesses can lead to extended cellular damage, with the consequence of several infectious and non-infectious skin disorders. The aim of the present study was to evaluate the toenail onychomycosis incidence in diabetic patients and healthy ones. The non-interventional, retrospective study was performed at the mycology laboratory of the University hospital "Policlinico-San Marco" in Catania, Italy, for over one year. Nail clippings were collected to perform microscopic and cultural exams, which allowed for the identification of fungal aetiological agents. A total of 715 patients (47 diabetic and 668 non-diabetic patients) were enrolled. In diabetic patients, dermatophytes were the most common cultural isolates (50%), followed by yeasts and moulds in 30.8% and 19.2%, respectively. In non-diabetic patients, the distribution of dermatophytes, yeasts and non-dermatophytic moulds was 67.4%, 5.3% and 27.3%, respectively. According to our results, diabetic patients are more predisposed to nail fungal infection. Our data suggest that dermatological follow-ups should always be performed for diabetic patients. All skin and nail disorders should be carefully monitored to perform a diagnostic confirmation and correct management of diabetic patients.

Use of real time multiplex PCR for the diagnosis of dermatophytes onychomycosis in patients with empirical antifungal treatments.

BACKGROUND: Onychomycosis is a nail fungal infection mainly caused by dermatophytes. Diagnostic confirmation is conventionally made by direct microscopy and culture, which suffer from low or moderate sensitivity. Several molecular methods have been used for dermatophytes detection and identification directly from nail samples. The aim of this study was the evaluation of the DermaGenius®(DG) multiplex kit in detecting and identifying dermatophytes from nail samples of untreated and treated patients with a clinical suspicion of onychomycosis. METHODS: All the patients underwent a nail scarification, performed with a sterile scalpel to collect small nail fragments from the suspected site of infection. All nail clippings were first analysed by microscopic and culture methods to define a diagnostic confirmation. DG PCR assays were retrospectively applied to the same samples. RESULTS: A total of 109 toenails were collected for the microscopic, culture and DG PCR assays. The sensitivity, specificity, positive and negative predictive values of DG in the onychomycosis diagnosis in all 109 patients were respectively 78.5%, 100%, 100%, and 75.9%. Only for cultural exams the rate of positive results was significantly different in the two groups of patients with a percentage of 73.7% in untreated patients versus a 40.7% value in treated patients (P < 0.05). CONCLUSIONS: Our results suggest that the use of DG kit could be useful to confirm the diagnosis of onychomycosis, implementing sensitivity especially in patients who underwent antifungal treatments without any clinical improvement.

Discriminatory Weight of SNPs in Spike SARS-CoV-2 Variants: A Technically Rapid, Unambiguous, and Bioinformatically Validated Laboratory Approach.

BACKGROUND: The SARS-CoV-2 virus has assumed considerable importance during the COVID-19 pandemic. Its mutation rate is high, involving the spike (S) gene and thus there has been a rapid spread of new variants. Herein, we describe a rapid, easy, adaptable, and affordable workflow to uniquely identify all currently known variants through as few analyses. Our method only requires two conventional PCRs of the S gene and two Sanger sequencing reactions, and possibly another PCR/sequencing assay on a N gene portion to identify the B.1.160 lineage. METHODS: We selected an S gene 1312 bp portion containing a set of SNPs useful for discriminating all variants. Mathematical, statistical, and bioinformatic analyses demonstrated that our choice allowed us to identify all variants even without looking for all related mutations, as some of them are shared by different variants (e.g., N501Y is found in the Alpha, Beta, and Gamma variants) whereas others, that are more informative, are unique (e.g., A57 distinctive to the Alpha variant). RESULTS: A "weight" could be assigned to each mutation that may be present in the selected portion of the S gene. The method's robustness was confirmed by analyzing 80 SARS-CoV-2-positive samples. CONCLUSIONS: Our workflow identified the variants without the need for whole-genome sequencing and with greater reliability than with commercial kits.

Synergy of molecular and serological methods in minimally invasive diagnosis of enteroviral cardiac infection.

Treatment of myocarditis and pericarditis can differ on the basis of aetiology: systemic or auto-immune disease can be positively influenced by corticoid therapy, whereas this kind of treatment can worsen the course of virus-induced disease. Therefore, the aetiological diagnosis is extremely important. The synergistic use of minimally invasive serological, IgG, IgM, IgA, and neutralizing titres, and RNA detection was evaluated on representative patients out of 238 suffering from cardiopathies. The results obtained for each case can yield reliable guidelines that rapidly highlight the presence of a viral aetiology so that an endomyocardial biopsy can be performed thus eliminating incorrect therapies. Thus, not only is this technique rapid, minimally invasive providing the clinician with decisive data, but it is cost effective for the health system.

Resistance to Echinocandins Complicates a Case of Candida albicans Bloodstream Infection: A Case Report.

Invasive candidiasis is known to be one of the most common healthcare-associated complications and is caused by several Candida species. First-line drugs, particularly echinocandins, are effective, but there are increasing reports of resistance to these molecules, though rarely related to C. albicans. Even though the rate of echinocandins resistance remains low (<3%), sporadic cases are emerging. Here, we present a case of bloodstream infection by a pan-echinocandin-resistant Candida albicans affecting a critically ill patient, who died in an intensive care unit following therapeutic failure and multiple organ dysfunction syndrome. This case highlights the need to suspect pan-echinocandin resistance in patients with prolonged echinocandin exposure, particularly in the presence of urinary tract colonization. Our study shows the importance of sequencing to predict therapeutic failure in patients treated with echinocandins and persistent candidemia.

In vitro fosfomycin study on concordance of susceptibility testing methods against ESBL and carbapenem-resistant Enterobacteriaceae.

OBJECTIVES: The increasing emergence and diffusion of multidrug-resistant (MDR) pathogenic bacteria, both in hospital and community settings, is inducing clinicians to reconsider old antibiotics, such as fosfomycin, to overcome the difficulties posed by these microorganisms. Recent studies have reported good in vitro activity of fosfomycin against extended spectrum ß-lactamases (ESBL) and carbapenem-resistant Enterobacteriaceae. The aim of this study was to assess thein vitro activity of fosfomycin by different methods against 120 clinical MDR isolates. METHODS: Fosfomycin minimum inhibitory concentrations were determined using the agar dilution reference method (AD), gradient test (GT), broth microdilution method (BMD), according to CLSI recommendations, and automated systems (VITEK 2 and BD Phoenix) against 85 carbapenem-resistant Klebsiella pneumoniae and 35 ESBL-producing Escherichia coli. Agreement and discrepancies between the evaluated methods and the reference method were calculated. RESULTS: Fosfomycin showed very good activity against ESBL-producing E. coli (88.6%). Excellent agreement (100%) between the three (AD, BMD and GT) susceptibility methods was found for E. coli. No major errors were observed. The fosfomycin resistance rate ranged from 24% (KPC-producing) to 100% (NDM-OXA-48 co-producing) K. pneumoniae. For all carbapenem-resistant K. pneumoniae strains, categorical agreement was >90% for all methods except for VITEK 2, which was 84%. CONCLUSIONS: When ESBL E. coli isolates are found to be susceptible to fosfomycin with automated systems, it is not necessary to verify these results with the AD reference method; while for resistant strains, the GT can be used. In cases of KPC K. pneumoniae resistant to fosfomycin, the AD method is the only reference method.

Diagnostic surveillance by Candida albicans germ tube antibody in intensive care unit patients.

BACKGROUND: The diagnosis of Invasive Candidiasis (IC) presents serious problems, mainly associated with the absence of pathognomonic symptoms of the disease and the difficulty of isolating the fungus in blood culture. Candida albicans germ tube antibody (CAGTA) provides a rapid and simple test for diagnosis of IC. The aim of this study was to evaluate the diagnostic role of the CAGTA in the monitoring of critically-ill patients at risk of developing IC. METHODS: During diagnostic surveillance in the intensive care units (ICU) CAGTA was performed twice a week if predetermined risk factors were present and a positive result was considered when a serum titer ≥1/160 was detected in at least one sample. RESULTS: Seventy critically ill patients were included in the study. Twenty-three patients with proven/probable IC were identified. The sensitivity, specificity, PPV, and NPV of CAGTA for the diagnosis of proven/probable IC in all 70 patients were 91.3%, 68.1%, 58.3%, and 94.1%, respectively. Statistically significant highest titers were found in patients with proven/probable IC as well as increasing titers more than 1/160. CONCLUSIONS: Our results suggest that detection of CAGTA could be a useful biomarker for the diagnosis of proven and probable IC in critical patients during prolonged ICU stay. During the monitoring it is opportune to evaluate the titers kinetics since the clinical diagnosis of proven/probable IC coincided with increase titer from negative (<1/160) to more than 1/160.

Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection.

Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) is the gold standard method for the diagnosis of COVID­19 infection. Due to pre­analytical and technical limitations, samples with low viral load are often misdiagnosed as false­negative samples. Therefore, it is important to evaluate other strategies able to overcome the limits of RT­qPCR. Blinded swab samples from two individuals diagnosed positive and negative for COVID­19 were analyzed by droplet digital PCR (ddPCR) and RT­qPCR in order to assess the sensitivity of both methods. Intercalation chemistries and a World Health Organization (WHO)/Center for Disease Control and Prevention (CDC)­approved probe for the SARS­CoV­2 N gene were used. SYBR­Green RT­qPCR is not able to diagnose as positive samples with low viral load, while, TaqMan Probe RT­qPCR gave positive signals at very late Ct values. On the contrary, ddPCR showed higher sensitivity rate compared to RT­qPCR and both EvaGreen and probe ddPCR were able to recognize the sample with low viral load as positive even at 10­fold diluted concentration. In conclusion, ddPCR shows higher sensitivity and specificity compared to RT­qPCR for the diagnosis of COVID­19 infection in false­negative samples with low viral load. Therefore, ddPCR is strongly recommended in clinical practice for the diagnosis of COVID­19 and the follow­up of positive patients until complete remission.