Diabetic ketoacidosis and hyperglycemic hyperosmolar syndrome are the two commonly known life-threatening hyperglycemic emergencies of diabetes mellitus. Despite the growing hyperglycemic emergency impact among adult patients with diabetes, its incidence and predictors have not been well studied in Ethiopia. Thus, this study aimed to assess the incidence and predictors of hyperglycemic emergencies among adult patients with diabetes.p < 0.05 in the multivariable model were considered statistically significant.A retrospective follow-up study design was conducted among a randomly selected sample of 453 adult patients with diabetes. Data were entered into EPI data version 4.6 and analyzed using STATA version 14.0. A Cox-proportional hazard regression model was fitted to identify the independent predictors of hyperglycemic emergencies, and variables having a Among the total adult patients with diabetes included in the study, 147 (32.45%) developed hyperglycemic emergencies. Hence, the overall incidence of hyperglycemic emergencies was 14.6 per 100 person-years observation. The incidence of diabetic ketoacidosis was 12.5 per 100 person-years . The incidence of the hyperglycemic hyperosmolar syndrome was 2.1 per 100 person-years . The overall median free survival time was 53.85 months. Type 1 diabetes mellitus , diabetes duration of ≥ 3 years , recent acute illness , presence of comorbidity , poor glycemic control , history of medication non-compliance , follow-up frequency of 2–3 months , and without community health insurance  were significant predictors of hyperglycemic emergencies.The incidence of hyperglycemic emergencies was high. Therefore, giving greater attention to patients with identified predictors could decrease the occurrence of hyperglycemic emergencies and related public health and economic impacts. Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia associated with defects in insulin secretion, insulin action, or both . DiabeteGlobally, diabetes prevalence and its impact have increased dramatically, particularly in sub-Saharan Africa , 5. ConsAccording to a World Health Organization report 2016), there were 1.6 million diabetic-related deaths , and HGE, there wA systematic review study conducted in different regions of the world revealed that the incidence of DKA ranges from 0 to 263 per 1,000 persons per year, with the highest incidence rate in China  and the lowest incidence rate in Israel and North America  . Even thIn sub-Saharan countries, lack of funding for non-communicable diseases, poor public healthcare, lack of proper health education, poor self-glycemic control, and lack of availability of studies and guidelines specific to the population have a great impact on the rising HGEs burden , 25. ItsDespite the increasing worldwide burden of diabetes and HGEs, data on the incidence among known adult patients with diabetes are limited, particularly in Africa, and the exact incidence of HHS is not identified worldwide . PreviouIn Ethiopia, limited studies have been conducted and assessed the prevalence and associated factors of acute complications of DM , 29, 30.This study was conducted in Bahir Dar public hospitals, North West Ethiopia from 1 January 2016 to 31 December 2020, and data were collected from 16 March 2021 to 14 April 2021. Bahir Dar is the capital city of Amhara Regional State, which is 565 km away from Addis Ababa, the capital city of Ethiopia. Currently, in the city, there are three public hospitals, namely, Felege Hiwot Comprehensive Specialized Hospital (FHCSH), Tibebe Ghion Specialized Teaching Hospital (TGSTH), and Addis Alem Primary Hospital (AAPH). Chronic disease services, including diabetes mellitus treatment and follow-up services, are being delivered in all these public hospitals.Felege Hiwot Hospital is one of the compressive specialized public hospitals, which has a 500 bed capacity and around 15 adult outpatient departments (OPD) serving about 5 million people coming from more than 500 km distance. Tibebe Ghion Specialized Referral Hospital is a teaching hospital that has a 459 bed capacity and around 14 outpatient departments serving more than 5 million people. Addis Alem Hospital is a primary hospital with 47 bed occupancy and eight outpatient departments serving 100,000 people.This is a retrospective follow-up study.All adults  diagnosed with T1DM and T2DM, and who had chronic diabetes mellitus follow-up in Bahir Dar city public hospitals.All adults diagnosed with T1DM and T2DM and who have DM followed-up from 1 January 2016 to 31 December 2020.All adults whose age ≥18 years old and diagnosed with T1DM and T2DM had a DM follow-up from 1 January 2016 to 31 December 2020.All individuals who developed HGEs during the first diagnosis of DM and whose records are incomplete for important variables (date of diagnosis of DM and date of the event or last observation) and did not have at least one follow-up after the first diagnosis were excluded from the study.For sample size determination, the Stat Calc function of Epi Info software version 7 was used by applying the cohort sample size calculation technique and taking common predictor variables for HGEs with 95% confidence interval, 80% power, and a 1:1 ratio of unexposed to exposed patients. The predictor variables were glycemic control , recent All public hospitals in Bahir Dar city  were selected. There were about 1542, 500, and 800 adults diagnosed with DM in Felege Hiwot, Tibebe Ghion, and Addis Alem, respectively, with a total of 2842 adult patients with diabetes between 1 January 2016 and 31 December 2020. Then, to select the study participants from each hospital, the proportional allocation formula was used based on caseloads. After that, a list of medical record numbers of adults diagnosed with DM was prepared from diabetic follow-up logbooks for each hospital. Finally, from the prepared sample frame medical record number lists in each hospital, the computer-generated simple random sampling technique was applied to select the proportionally allocated study participants.Dependent variables include incidence rate and time to develop HGEs.Independent variables include socio-demographic variables , clinical variables , and treatment factors .Survival is defined as the time measured between the date of DM diagnosis and the date of developing HGEs or the date being censored.Adults diagnosed with DM who did not develop HGEs during the follow-up study  are considered as censored.Individuals whose age is ≥18 years old are considered adults.Diabetic ketoacidosis was defined based on a clinical diagnosis of DKA when there is RBGL of > 250 mg/dl, urine ketone body of ≥ +2, arterial pH of < 7.3, and one body .Adults having DM and other chronic medical or psychiatric diseases are considered as comorbidities.The occurrence of HGEs from diagnosis to the end of the study is considered as the event.Incomplete records are charts with incomplete information for variables of the date of DM diagnosis and the date when the event occurred or the last observation.The average 3-month FBS level ranges from 70 to 130 mg/dl or HgA1c of < 7% referred to as controlled, and uncontrolled blood glucose is defined as an average FBS of > 130 or < 70 .Age categories are young (18–44 years old), middle-aged (45–64 years old), and older age (≥ 65 years old) .We developed the data extraction checklist after reviewing different relevant literature , 26, 30.Before the data collection, a pretest was done in Felege Hiwot Comprehensive Specialized Hospital with 24 (5%) of the sample size from charts of adult patients with diabetes that were registered 1 month before the study period and charts were not included in the final sample size. Consequently, necessary modifications were made to the checklist. A 1-day training was given to data collectors and supervisors before actual data collection. The completeness of the collected data was checked on the site daily basis during data collection, with feedback from the supervisor and the investigators. In addition to this, the data were carefully entered and a double data entry was done.The data were coded, cleaned, and checked for consistency and completeness, and then the data were entered into EPI data version 4.6.0 and exported into STATA version 14.0 for cleaning, managing, and statistical analysis. Descriptive statistics were presented with frequency tables and graphs for categorical variables, and continuous variables were reported with mean [mean ± standard deviation (SD)] and median [interquartile range (IQR)]. The Kaplan–Meier survival curve was used to estimate the median survival time and the log-rank test was used to statistically test survival between different categories of independent variables. The Cox-proportional hazard model assumption was checked using the Schoenfeld residuals test, the Cox–Snell residual, and the parallel assumption test.p < 0.25 in bivariable were a candidate for multivariable analysis. A 95% CI of hazard ratio (AHR) was computed and variables having a p < 0.05 in the multivariable model were considered statistically significant with the dependent variables.The association between the independent variables and the outcome variable was assessed by the Cox-proportional hazard model. Variables with a Among the 473 reviewed charts of adult patients with diabetes, 453 of them were included in the study and analyzed. The rest 20(4.2%) charts accounted for incomplete charts and no history of follow-up after their DM diagnosis. Out of 453 adults, half (51.43%) were men and more than half (72.19%) were from urban areas . The medThe majority of patients  were patients with T2DM. Out of the total patients with diabetes, 301 (66.45%) had comorbidity and 52 (21.1%) had more than one comorbidity. Cardiovascular disorders, 202(44.59%) were found to be the most common comorbid diseases. Approximately 213 (47.02%) patients had poor 6-month glycemic control . Most ofIn total, 453 patients were followed for different periods of 5 years with 12,048 person-month observations. The overall mean follow-up length was 26.6 months (95% CI: 24.97–28.22). Out of the total patients included in the analysis, approximately 147 (32.45%) developed HGEs, of which, 92 (62.59%) were men and 108 (73.47%) had comorbidity. The overall HGEs incidence in the cohort was 14.6 per 100 person-years observation . The incidence of DKA was 12.5 per 100 person-years , which was 35.6 and 6.3 per 100 person-years among T1DM and T2DM, respectively. The incidence of HHS was 2.1 per 100 person-years , which was 0.9 and 2.4 per 100 person-years among T1DM and T2DM, correspondingly. In the entire follow-up period, a higher incidence of HGEs occurred between 0 and 12 months of follow-up interval .The overall Kaplan–Meier estimate showed that the probability of HGEs-free survival of adult patients with diabetes was high during 1.02 months of observation (99.78%). The probability of HGEs-free survival relatively falls as the follow-up month increases, with a sharp fall after 58.62 months, and a minimum HGEs-free survival probability was observed (30.72%) at 60 months of observation . The ovep-value ≤ 0.01).The log-rank test was used to check statistically for the presence of any significant differences in survival among various categorical predictors. Patients with controlled six-month glycemic control, with community health insurance, without a history of medication noncompliance, with DM duration ≥ 3 years, without comorbidities, without a history of recent acute illness, and diagnosed with T2DM had longer HGEs-free survival time than the corresponding categories with . Baseline age, sex, duration of treatment started, residency, type of drug used, and chronic diabetic complications were not statistically significant predictors associated with the incidence and time to develop HGEs  . The incIn this study, patients who had no community health insurance had a 1.63 times higher hazard of developing HGEs than those who had community health insurance. This agrees with another study . The reaPatients with a history of recent acute illness were 2.99 times more hazardous to develop HGEs compared to those without a history of recent acute illness. This is in line with studies in Ethiopia , 30 and According to this study, diabetic patients with comorbidities of psychiatric and chronic medical disorders had 2.36 times more hazard of developing HGEs compared to diabetic patients without these issues. This is supported by other studies in Ethiopia , 29, 30,In terms of diabetic duration, patients diagnosed with diabetes greater and equal to 3 years of duration had 67% less hazard of developing HGEs compared to patients with diabetes of shorter duration. Another study in Iraq  also shoRegarding the preceding 6-month glycemic control, this study noted that poor glycemic control was a strong predictor of HGEs and this is comparable with studies in the USA  and AustOur study revealed that adult patients with T1DM had 2.75 times more hazards to develop HGEs than adult patients with T2DM. This finding is supported by other studies in Ethiopia , 32. ThiThe history of medication non-compliance was a significant predictor of HGEs. Other research conducted in Ethiopia showed a similar result  . The risConcerning diabetic follow-up frequency, patients who had a diabetic follow-up every 2–3 months are 1.79 times more hazardous to develop HGEs than those who had a diabetic follow-up every month. Another study  also shoFinally, different studies in the world showed that age , 31, sexThe incidence of HGEs among adult patients with diabetes was found to be high compared to previous studies. The incidence was higher and predicted for those patients who had type 1 diabetes mellitus, a history of medication non-compliance, comorbidities, recent acute illness, poor glycemic control, follow-up frequency of 2–3 months, and who are without community health insurance. On the contrary, patients diagnosed with diabetes greater and equal to 3 years of duration had less hazard of developing HGEs. As a result, we recommend healthcare providers to monitor thoroughly and have close follow-ups for patients with the identified predictors and should also give adequate knowledge about HGEs and prevention practices for newly diagnosed patients. In addition, including patients with diabetes in community-based health insurance and making a diabetic follow-up within a month could decrease the incidence and related public health and economic impacts. Finally, we recommend conducting further prospective studies to address the missed variables and recurrent HGEs should be investigated with another study design, as this was not assessed in our study.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board (IRB) of Bahir Dar University's Ethical Review Committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. No potentially identifiable human images or data are presented in the manuscript.All authors have made substantial contributions to the study reported, whether that is in the conception of this study, design of the study, acquisition, analysis, and interpretation of data, or all these areas, took part in drafting, revising, or critically reviewing the article, read and gave the final approval for the version to be published, agreed to the journal to which the article has been submitted, and agree to be accountable for all aspects of the study.


The choice of optimum composition of a mixture of binary and ternary excipients for optimum compressional properties was investigated in this work. Excipients were chosen based on three types of excipients: plastic, elastic, and brittle fracture. Mixture compositions were selected based on a one-factor experimental design using the response surface methodology technique. Compressive properties comprising Heckel and Kawakita parameters, work of compression, and tablet hardness were measured as the main responses of this design. The one-factor RSM analysis revealed that there exist specific mass fractions that are associated with optimum responses for binary mixtures. Furthermore, the RSM analysis of the ‘mixture’ design type for the three components revealed a region of optimal responses around a specific composition. The foregoing had a mass ratio of 80:15:5 for microcrystalline cellulose: starch: magnesium silicate, respectively. Upon comparison using all RSM data, ternary mixtures were found to perform better in compression and tableting properties than binary mixtures. Finally, the finding of an optimal mixture composition has proven effective in its applicability in the context of the dissolution of model drugs (metronidazole and paracetamol). The preparation of solid dosage forms involves the use of a number of functional excipients in addition to the active pharmaceutical excipient (API) itself. Ideally, these excipients are generally not blended, either in their native/or unprocessed form(s), or without being subjected to processing under conventional techniques, such as wet/or dry granulation, spray/or freeze drying, spray/or melt granulation, etc. . The afoIn order to elucidate such an understanding, optimum compression is believed to be determined by the overall plastic/elastic and brittle-fracture contributions of a diversity of excipients comprising the whole powder blend. Each excipient undergoes a specific behavior when subjected to compression depending upon its plastic, elastic, both, or brittle-fracture extents ,5. HowevThe latter has been presented in the improvement of compressibility and compatibility for a solid dosage form comprising elastic and brittle-fracture excipients, Starch and lactose , PVP andThe work outlined herein, therefore, will elaborate on the understanding of the compression behavior of a ternary mixture composed of three components from a response surface methodology perspective (RSM). Finding an optimum mixture of the mass content and composition will be given great consideration within the context of physically mixed plastic, elastic, and brittle facture excipients. The verification of finding such an optimal mixture composition will be tested on ternary mixtures of different compositions and wet granulation preparation types.®101), lot No. 60997C, particle size 50 µm , commercial maize starch , synthetic amorphous magnesium silicate, lot No. 1771 , pharmaceutical grade metronidazole , pharmaceutical grade metronidazole , paracetamol , Flagyl® 500 mg originator product (Sanofi-Aventis), and Panadol® 500 mg originator product .The following materials, with their suppliers provided herein, were purchased; microcrystalline cellulose PH-101  in different ratios, with a total amount equal to 20 g. Then, 10 g of the foregoing was wet-granulated using distilled water. The wet granules were passed through a 2.0-mm sieve, then dried to complete dryness in a hot air oven at 40 °C for 3 h. The dried granules were passed over a mesh of 425 μm size.Bulk density (g/mL) was calculated by pouring, without tapping, 50 g of each powder into a 100-mL graduated glass cylinder. The powder volume was then recorded and used to calculate the bulk density; each sample was measured in triplicate.Next, 150 mg of the single and binary, and 100 mg of the ternary mixtures were compressed using the Gamlen Tablet Press machine . The compression forces were fixed at 100, 200, 300, 400, and 500 kg. The powders were compressed using a punch size of 6.0 mm in diameter at a speed of 60 mm/min. Force-displacement curves (work), Heckel and Kawakita analysis, hardness, and disintegration testing were all carried out upon compression of the powder samples into tablets. The test was carried out in triplicates.A number of models are available to mathematically analyze different mechanical aspects of powder compression properties. The Heckel and Kawakita analysis still remains the most commonly used models for describing such aspects ,15,16,17D is the relative density of a powder compact at pressure P. Slope k is a measure of the plasticity of a compacted material. The inverse of the slope is the yield pressure of the material (YP), which describes the tendency of the material to deform either by plastic flow or fragmentation. Accordingly, the higher the slope value, the lower the yield pressure, and the higher the ability of the material to deform plastically under pressure. Constant A is related to die filling and particle rearrangement prior to the deformation and bonding of the discrete particles.The Heckel equation (Equation (1)) describes the change in powder porosity as a function of the applied pressure upon powder densification.C.C is the degree of volume reduction of the powder column under the applied pressure P. The constant a is the minimum porosity of the material before compression, while the constant b relates to the amount of plasticity of the material. The reciprocal of b or kP defines the pressure required to reduce the powder bed by 50%. The KP values provide a direct indication of the strength of, or ability to resist, the granules [The compression behavior of the samples can be also evaluated using Kawakita analysis of powder compression data. The Kawakita equation (Equation (2)) is used to study powder compression via the degree of volume reduction granules ,19.At a certain compression pressure, the volume is reduced, and the particles’ packing reaches a minimum. These changes are associated with elastic and/or plastic deformation or with brittle fracture of the particles into smaller ones. The use of force-displacement curves has been a common evaluation technique for powder compression properties. The area under the force-displacement curve is called the work of compression .Tablet hardness was measured using a hardness tester. Ten tablets were tested. The means and standard deviations were calculated.The thickness of the compacts (tablets’ height) and the diameter were measured using a vernier caliper in (mm). The average of 10 tablets was taken.® software version 11. Analysis was carried out using a quadratic model based on a fractional factorial design of 13 runs. The response surface plots were generated to see how the responses (yield pressure (YP), KP, work of compression, tablet hardness) vary with an excipient mass content. Five lack-of-fit points and five replicate points were chosen for an optimal integrated variance (IV) design that provides a lower average prediction variance across the whole range of excipient mass contents. Binary mixtures were handled using the RSM technique  whereby For ternary components, a mixture design of three components was chosen in this work to find an optimal formulation. A quadratic polynomial option was chosen to model the response based on 10 runs. Similarly, five lack-of-fit points and five replicate points were chosen for optimal IV design. w/w Mg silicate), were blended for 10 min using an Erweka cube mixer . The powders were compressed into tablets (1 g) containing 500 mg metronidazole or 500 mg paracetamol using a single punch machine  with oblong punches (19 mm × 9.5 mm). Dissolution runs, sampling, and drug measurements were carried out according to the United States Pharmacopoeial-USP 31(2005) methods for each drug. For metronidazole tablets, the USP Apparatus 1 (basket) method was considered. A 0.1 N HCl solution was used as the dissolution medium. Dissolution was carried out at 37 ± 2 °C, 100 rpm rotational speed, and 900 mL of the medium. Samples (5 mL) were withdrawn at the time intervals of 5, 10, 15, 30, and 45 min, diluted, and then analyzed spectrophotometrically at 275 nm.Two powder mixtures, each 100 g, containing 50 g metronidazole or 50 g paracetamol, and 50 g of the ternary excipient mixture  method was considered. Then, 900 mL of phosphate buffer (pH 5.8) was used as a dissolution medium. The paddle was set to rotate at 75 rpm. Samples (5 mL) were withdrawn at the time intervals of 5, 10, 15, 30, and 45 min, then diluted using phosphate buffer (pH 7.8). The samples were filtrated through a 0.45-µm glass filter and analyzed spectrophotometrically at 243 nm.Parameters representing the compressional behavior of a powder blend were investigated in this work with reference to the influence of a combination of plastically deforming, elastic, and brittle fracture excipients for binary and ternary mixtures. MCC, maize starch, and Mg silicate were chosen to represent the aforementioned three types of excipients, respectively. Justification for this choice was tested using Heckel and Kawakita compression analysis, whose results are shown in Py), which is an inverse of the Heckel slope (1/slope), was calculated. Since the lower the yield pressure, the higher the ability of the material to deform plastically under pressure, MCC is more plastically deforming than starch, which then later deforms more plastically and to a greater extent than Mg silicate. Kawakita parameters indicate that, in terms of compressibility order, or (a), MCC > Mg silicate > starch. Thus, MCC manifested the highest volume reduction when compressed, whereas starch manifested the lowest. This is most probably attributed to the highest bulk density recorded by maize starch [KP value, or the force required to reduce the powder bed by a/2, was the highest (From the Heckel slope (K) order, MCC > starch > Mg silicate, the yield pressure , the maximum KP value at approximately 25% Mg silicate or 85% starch, maximum work of compression at approximately 25–35% Mg silicate (or 65–75% starch), and the maximum hardness at approximately 15–25% w/w mass content of Mg silicate (or 75–85% starch).For Mg silicate-starch binary mixtures, the minimum w/w, increased the KP value up to a maximum around a mass content of 70–80%, linearly increased the compression work, and finally reached a maximum tablet hardness value of approximately 75–80% w/w.Finally, for MCC-Mg silicate binary mixtures, increasing the content of MCC lowered the value of the yield pressure down to a minimum around a mass content of 80–85% YP), KP, work of compression, and tablet hardness as a function of the three mixture components. Regions in red and blue colors assign high and low values, respectively, whereas the intensity of other colors designates the high and low values of excipient mass contents. In all cases, the blue and red colors present a great deal of interest in interpreting optimum composition and tableting conditions.For the mixture design of the three components using RSM, the 2D contour plots are presented in w/w metronidazole and paracetamol showed an immediate release profile, as shown in The ternary excipient  at the contents of 10% formulated with 90% The two figures represent a typical example of immediate-release profiles. The tablet hardness measured was relatively high (~150 N) for both drugs. Moreover, the model drugs were almost identical to the originals of the same strengths and presented a typical immediate release profile, whereby 80% of both drugs were released at 60 min.YP, KP, and work of compression values of individual components provide the worst compressional scenario for tableting. More specifically, MCC and starch recorded higher YP values, lower KP values, and higher work of compression than most compositions of the MCC-starch mixtures. The foregoing, therefore, have higher plasticity, higher resistance (hardness) of the granules, and lower energy displayed upon compression, respectively. In contrast, Mg silicate-starch binary mixtures showed better tableting behavior in the individual components than their mixtures. In this regard, starch has the lowest YP (2A), highest KP (2B), and the highest hardness (2D) of all mixtures and Mg silicate itself. Consequently, Mg silicate, as a brittle fracture material, weakens the compressional properties when added to starch. Similarly, Mg silicate, to some extent, weakens the powder/tablet properties when added to Avicel, as YP), weakening the strength of the granules (KP), and reducing the tablet hardness. It has to be further emphasized that Mg silicate reduced the work of compression when mixed with any of the plastically/or elastically deforming materials (MCC/or starch) in In the investigation of the compression properties of MCC-starch mixtures, it can be noticed that binary and ternary mixtures provide better tableting behavior than individual ones. w/w was characterized by the highest recorded plastic deformation and the lowest work of compression. Tablets created with the same composition maintained high hardness values while maintaining a reasonably high granule strength comparable to the MCC excipient when compressed alone ; the same material of the same content is covered in the desired red regions of All the aforementioned findings for binary mixtures were used to correlate compression and tableting parameters of ternary mixtures using RSM plots in Validation of such an optimum composition was checked using MCC: starch: Mg silicate ratios  other thYP, KP, work of compression, and tablet hardness compared to the dry ternary mixtures (When the same mixtures in mixtures . The topThe capability of the ternary mixture at the optimum ratio was well demonstrated upon formulation with the two high-strength model drugs (metronidazole and paracetamol in The optimal compressional behavior of physical mixtures comprising a combination of excipients is determined by a majority of plastic, a minority of brittle-fracture, and a moderate influence of elastic modes in the combination. The ideal performance of powder and tableting properties necessitates that all the three modes be present at the aforementioned distribution.


Burkholderia gladioli pose a serious threat to human health and food safety, but there is a lack of rapid and sensitive field detection methods for Burkholderia gladioli. In this study, the CRISPR/Cas12a system combined with recombinant enzyme polymerase amplification (RPA) was used to detect Burkholderia gladioli in food. The optimized RPA-CRISPR/Cas12a assay was able to specifically and stably detect Burkholderia gladioli at a constant 37 °C without the assistance of large equipment. The detection limit of the method was evaluated at two aspects, the genomic DNA (gDNA) level and bacterial quantity, of which there were 10−3 ng/μL and 101 CFU/mL, respectively. Three kinds of real food samples were tested. The detection limit for rice noodles, fresh white noodles, and glutinous rice flour samples was 101 CFU/mL, 102 CFU/mL, and 102 CFU/mL, respectively, without any enrichment steps. The whole detection process, including sample pretreatment and DNA extraction, did not exceed one hour. Compared with the qPCR method, the established RPA-CRISPR /Cas12a method was simpler and even more sensitive. Using this method, a visual detection of Burkholderia gladioli that is suitable for field detection can be achieved quickly and easily.Pathogenic variants of  Burkholderia gladioli, a kind of gram-negative bacterium, is widely distributed in nature and have been isolated from plants, the environment, and food [Burkholderia gladioli pv. gladioli, Burkholderia gladioli pv. alliicola, Burkholderia gladioli pv. agaricicola, and Burkholderia gladioli pv. cocovenenans. The first three types were phytopathogenic strains, which could cause rot symptoms in important cash crops such as rice [Burkholderia gladioli pv. cocovenenans (B. cocovenenans) is a kind of foodborne pathogen first found in Asian countries [B. cocovenenans [B. cocovenenans occurred mainly in China, Indonesia, and Mozambique [B. cocovenenans is the foodborne pathogen with the highest morbidity and mortality found so far in China [B. cocovenenans has been reported from time to time. The most famous event occurred in 2020; all nine people in one family died of acid soup poisoning during a family dinner in Heilongjiang province with a mortality rate of 100% [Burkholderia gladioli in food is urgently needed.and food ,2,3,4,5.and food ,7, Burkh as rice , gladiol as rice , and oni as rice ,10. Amonountries . It can venenans . Fermentvenenans . Food pozambique  while thzambique . B. cocoin China . Death c of 100% . It has Burkholderia gladioli mainly focuses on the individual detection of some pathogenic subspecies [Burkholderia gladioli strains are necessary. At present, a series of methods have been developed for the detection of Burkholderia gladioli, including PCR [The detection of bspecies ,18,19, abspecies ,21, univding PCR ,23,24, Pding PCR , qPCR [2ding PCR , biochemding PCR , DNA micding PCR , dPCR, ading PCR ,30. HoweThe Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system and CRISPR-associated proteins (Cas) are known for their powerful gene editing capabilities ,32. In rBurkholderia gladioli based on a conservative mitochondrial gene. Without using large instruments and a complex electrophoresis analysis, the established RPA-CRISPR/Cas12a method can be carried out easily in a very short time and has proven to be useful for the rapid and sensitive detection of Burkholderia gladioli in rice noodles, fresh white noodles, and glutinous rice flour. It can provide a technical reference for the rapid field detection of Burkholderia gladioli and can promote the development of detection technologies in food safety monitoring.In this study, technologies of CRISPR/Cas12a combined with RPA were employed to establish a rapid detection method for Burkholderia gladioli (CICC10574) were purchased from Yuweitech Technology ; Burkholderia gladioli (ATCC10248), Burkholderia gladioli pv. cocovenenans, Salmonella enteritis (CMCC50041), Vibrio parahaemolyticus, Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus were all from the Laboratory of Food Quality and Safety . TwistAmp Liquid Basic kit was purchased from TwistDx Ltd. . TIANamp Bacteria DNA Kit was purchased from Tiangen Biochemical Technology . Cas12a enzyme was provided by Editgene Co., Ltd. . The crRNA and ssDNA-FQ reporting probes (FAM-TTTTTT-BHQ) were synthesized by Genewiz Biotechnology . RNase inhibitors were purchased from Xinhai Gene Testing. NEBuffer2.1 was purchased from New England Biolabs Inc. . Rice noodles, fresh white noodles, and glutinous rice flour samples were purchased from the Triangle Market in the South China Agricultural University . 2 × TaqMan Fast qPCR Master Mix was purchased from Bioengineering .The dry powder strain or glycerol strain was cultured in liquid medium at 37 °C and centrifuged at 250 rpm for 24 h to obtain the bacterial cells in logarithmic growth phase. In order to get better activity of the strain and ensure no contamination, single colony was picked and cultured in liquid medium again. The obtained bacterial solution was centrifuged at 10,000 rpm for 1min, and the precipitate was collected for DNA extraction. In order to obtain higher purity and concentration, DNA was extracted with the TIANamp bacterial DNA extraction kit following the kit’s instructions. Burkholderia gladioli (accession ID EF552059) was selected as the target for the design of specific RPA amplification primers. According to the RPA primer design principles, a total of nine pairs of primers were designed using SnapGene and Primer Premier 5.0 software , dNTPs concentration , MgOAc concentration , and amplification time . The optimal conditions were selected according to the fluorescence intensity generated by the initial CRISPR/Cas12a system mentioned above, and a nontarget control (NTC) using sterile water instead of gDNA was set for each optimized group. All tests were repeated three times, and the standard deviation was calculated and shown in the figures via the error bar.The 16S-23S rRNA intergenic region sequences of bacteria are usually used to identify the genetic relationships among bacteria . The higsoftware . In ordeAccording to the sequence of the RPA amplification product, a 21 bp fragment with PAM site (TTTV-) was selected as the recognition target of crRNA, and a crRNA that specifically recognized the PRA amplification product was designed and synthesized. The crRNA sequence was: UAAUUUCUACUAAGUGUAGAUCAAGCAGGGGGUCGUCGGUUC . Next, tBurkholderia gladioli was established, and specific steps are as follows. After DNA extraction, RPA amplification system with a total volume of 10 μL was added in test tube and covered with 15 μL of mineral oil to avoid aerosol pollution. Then, the CRISPR/Cas12a detection system was added on the inner face of the tube cover, which remained closed during the whole detection process. The tube was first cultured in a 37 °C constant temperature water bath for 20 min, then centrifuged by a handheld centrifuge for a few seconds, and it was cultured at 37 °C for another 10 min. The results can be immediately observed with naked eye under a portable blue light glue cutter. Fluorescence signals were collected using SpectraMax i3x  at an excitation wavelength of 492 nm and an emission wavelength of 518 nm.Finally, the RPA-CRISPR/Cas12a detection method for Burkholderia gladioli and five common foodborne pathogens, including Salmonella, Vibrio parahemolyticus, Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus were used to evaluate the specificity of the established RPA-CRISPR/Cas12a method.In this study, three different strains of B. cocovenenans was diluted with sterile water to 100 ng/μL, 10−1 ng/μL, 10−2 ng/μL, 10−3 ng/μL, and 10−4 ng/μL, respectively. At the same time, the bacterial solution was also set to gradient concentration and used for detection together with the above diluted gDNA. Bacteria were cultured and counted according to the national standard of China (GB4789.2-2016) [6–101 CFU/mL. According to the significant difference of the fluorescence contrasted to the nontarget control, which was observed by naked eye, the detection limits of gDNA and bacterial concentration were determined. All experiments were repeated three times.The sensitivity of the method was determined from two levels: genomic DNA and the amount of bacteria. The detection limit of this method was determined as the lowest recognition concentration of gDNA or bacteria. The gDNA of .2-2016) . The bacBurkholderia gladioli is greatly increased [B. cocovenenans gDNA; the negative control was Salmonella gDNA, and the nontarget control was sterile water.In China, rice noodles, fresh white noodles, and glutinous rice flour are widely available and popular foods, but many of them are produced in processing places without food safety supervision, and the probability of contamination by ncreased . The samncreased , and theB. cocovenenan to obtain 6 groups of samples with the concentration of B. cocovenenan of 106 to 101 CFU/mL. Finally, DNA was extracted using the kit extraction method. The established RPA-CRISPR/Cas12a method was used to test the detection limits of these samples. All experiments were repeated three times.The rice noodle and fresh white noodle samples were directly homogenized on the ultra-clean workbench, and the glutinous rice flour was first added with sterile normal saline to make a 10-fold homogenate. Then, three samples were diluted 10-fold with sterile normal saline in the same way to make a suspension, which was allowed to stand for 2 min, and the supernatant was taken for later use. Next, 4.5 mL of the supernatant was mixed with 0.5 mL of the known concentration of Burkholderia gladioli at different concentrations in the three types of samples, and the results were verified and compared with those of the RPA-CRISPR/Cas12a method. The sample pretreatment method was consistent with the RPA-CRISPR/Cas12a method established in this study. The bacterial concentration gradients in the samples set for qPCR method was also the same with that of RPA-CRISPR/Cas12a method described above. Burkholderia gladioli was reported as “positive” when the final Ct value was less than or equal to 35. When the Ct value of the test result was greater than 35 and less than 40, the test was repeated. If the Ct value was still less than 40 and the curve had a significant logarithmic growth period, Burkholderia gladioli was also reported as “positive”; otherwise, Burkholderia gladioli was reported as “negative”. All experiments were repeated three times.The qPCR method mentioned above was used to analyze p < 0.05; ** p < 0.01; *** p < 0.001, and **** p < 0.0001.Experimental data were statistically analyzed using IBM SPSS Statistics 26 software, and all results are presented as the mean ± SD of three independent experiments. One-way ANOVA was used to analyze the difference between the experimental group and the nontarget control group (NTC). The Tukey–Kramer multiple comparison method was used for post hoc test. The specificity and sensitivity of RPA-CRISPR/Cas12a method were judged according to whether the difference was significant. Significant values were indicated by * Burkholderia gladioli. Several pathogens that are easily contaminated in food, including Salmonella, Vibrio parahemolyticus, Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus, were tested to examine the specificity of the primers. At last, a pair of primers that could specifically amplify Burkholderia gladioli and produce the highest fluorescence intensity was screened out. The sequence is 5′-CCGTCTTGATAAGGCGGGGGTCGTTGGTTCGAAT-3′ for the forward primer and 5′-CGCCAATGACAAAGACTCGAGTCAACTGACCC-3′ for the reverse primer. Using this pair of primers, the RPA amplification system was optimized  gDNA concentration and obviously different from the nontarget control. Meanwhile, B. cocovenenans colonies were cultured and diluted to different concentrations in series, and the extracted DNA was used for RPA-CRISPR/Cas12a detection. According to the results shown in 1 CFU/mL for bacterial fluid. Compared with the reported qPCR method [Burkholderia gladioli gDNA was 250 fg/μL , and for the bacterial solution, it was 102 CFU/mL. In comparison, the detection limit of this method was slightly worse than that of the qPCR method, but the detection limit of the actual bacteria was better than that of the qPCR method. This may be due to the lower tolerance of qPCR to inhibitors than RPA, resulting in the two methods showing different result trends when detecting bacterial liquid samples compared with purified DNA [The sensitivity of the RPA-CRISPR/Cas12a method was tested at both the gDNA and bacteria level. As shown in R method , the detfied DNA . This esBurkholderia gladioli, and then, they were spiked with B. cocovenenans. The final bacteria concentration in the sample ranges from 100 CFU/mL to 106 CFU/mL. In the rice noodles, fresh white noodles, and glutinous rice flour samples, the RPA-CRISPR/Cas12a assay showed a detection limit of 101 CFU/mL, 102 CFU/mL, and 102 CFU/mL against Burkholdeia gladioli, respectively (In this study, homogenized rice noodles, fresh white noodles, and glutinous rice flour samples were first proven free from contamination by ectively . The detBurkholderia gladioli in rice noodles, fresh white noodles, and glutinous rice flour obtained with qPCR was 101 CFU/mL, 102 CFU/mL, and 103 CFU/mL, respectively. The results obtained with qPCR showed a consistent trend with that of the RPA-CRISPR/Cas12a method in all three kinds of samples. In fact, the RPA-CRISPR/Cas12a method is even more sensitive to the existing qPCR methods. This may be caused by the different tolerance of qPCR and RPA to the inhibitory effects brought by the sample matrix [The accuracy of the RPA-CRISPR/Cas12a method was verified by an existing qPCR method in the three kinds of samples; as shown in e matrix . The aboBurkholderia gladioli. This method has high sensitivity and good specificity that can precisely detect Burkholderia gladioli without a cross-reaction with other common pathogens. The detection limit of gDNA was 10−3 ng/μL, and the limit of detection of the bacterial liquid was as low as 101 CFU/mL, which was better than that of the standard qPCR method. The method was then applied in food samples, and the detection limits of rice noodles, fresh white noodles, and glutinous rice flour were 101 CFU/mL, 102 CFU/mL, and 102 CFU/mL, respectively, with no need of a sample enrichment. The detection step only took 30 min, and the whole process of food sample detection with the addition of sample processing and DNA extraction takes no more than 1 h. There are few studies on the rapid detection method of Burkholderia gladioli, and most of the food contaminated samples are rice noodle products, which also have some difficulties in pre-processing. However, this method has high sensitivity, which can overcome the complex problem of DNA extraction from rice noodle products. Moreover, this rapid method can be carried out without the assistance of any large instruments, but it achieves even better sensitivity than qPCR, so it has good prospects for the efficient and rapid field detection of Burkholderia gladioli.This study established a specific RPA-CRISPR/Cas12a rapid method to detect the food-borne pathogen


Leishmania. infantum, and it is usually responsible for symptoms such as fever, pancytopenia and enlargement of the liver and spleen. Relapse is rare in immunocompetent patients as much as the mucous involvement. We present a rare case of mucosal relapse of visceral leishmaniasis in a child with SARS-CoV-2 infection and perform an extensive review of the literature about leishmaniasis relapses in children. Atypical mucosal involvement during Leishmaniasis relapse is an eventuality in pediatric patients. Clinical follow-up and periodic PCR tests must be considered essential for the early recognition and treatment of an eventual relapse.Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania and is transmitted through the bite of infected female sandflies. In the Mediterranean region, visceral leishmaniasis is caused by  Coronavirus disease 2019 (COVID-19) is a pandemic infection whose outbreak in 2020 has complicated the management of other infectious diseases worldwide [In children, COVID-19 is usually mild, and a low number of severe cases has been described when compared with adults Pediatric clinical features are usually nonspecific and include fever, cough or gastrointestinal symptoms. Laboratory findings can report a transitory lymphopenia .Coinfections represent a challenge, as they can lead to an inappropriate diagnosis and management of other life-threatening conditions, thus delaying their treatment. Leishmaniasis is a vector-borne disease caused by obligate intracellular protozoa of the genus Leishmania .Leishmania parasites are responsible for a broad variety of clinical manifestations, ranging from cutaneous and often self-healing lesions to mucocutaneous and life-threatening visceral diseases .L. donovani in Asia and Africa and by L. infantum in the Mediterranean region, where it is transmitted to humans through the bite of infected female Phlebotomine sandflies. Visceral leishmaniasis (VL) is classically caused by In Sicily, the largest island in the Mediterranean Sea, VL is endemic .If left untreated, the disease can eventually lead to death, while adequate treatment usually leads to full recovery. Even if antimonial compounds have been considered the gold standard treatment of leishmaniasis for decades ,8, lipidRecurrency, rather than reinfection, is usually the most frequent mechanism of relapse ,13,14, wL. infantum with mucosal involvement in pediatric patients. In this paper, we describe an unusual case of VL relapse with gingival localization in a child who was recently infected with SARS-CoV-2. Starting from our case, a review of the literature was carried out with the aim of analyzing the clinical characteristics, therapeutic strategies and outcomes of relapsing VL caused by L. infantum OR Leishmania infantum) AND  AND (recurrent OR relapsed OR recidiva*) AND (pediatric OR paediatric OR child* OR infant OR baby).A computerized literature review was performed by submitting the following keywords into the PubMed search engines: visceral AND  and severe anemia . Molecular and serological assays for Leishmania were found positive, and therapy with amphotericin B was started, with the schedule of 3 mg/kg/day for 5 days, followed by an end dose on the 10th day.A previously healthy 4-year-old girl was first admitted to the Pediatric Infectious Disease Department of the Children’s Hospital of Palermo in February 2022 with hyporexia, high fever, acute onset of chest pain and a positive swab test for SARS-CoV-2. Physical examination revealed mild hepatomegaly  and severe splenomegaly , subsequently confirmed by an ultrasound of the abdomen, which showed a severely enlarged spleen (14.4 cm × 6.1 cm). Blood tests revealed trilinear pancytopenia with severe neutropenia . In September 2022, 7 months after the amphotericin B therapy had ceased, she was admitted again to the Pediatric Emergency Department of our hospital because of a one-day fever (peak 38°), hyporexia and gingival hyperemia with spontaneous bleeding, in the absence of trauma history or any evident lesion, persisting over a week. The patient had been treated at home with acyclovir without any benefit. A swab test for SARS-CoV-2 was performed in a hospital with a negative result returned. She appeared to be in discreet clinical condition, slightly in pain, pale, with hyperemic pharynx without plaque or purulent secretion and hypertrophic bleeding gingiva. Mild latero-cervical lymphadenopathy, mild hepatomegaly  and severe splenomegaly  were observed. No signs of cardiac or respiratory involvement were identified.3), lymphopenia (1.17 × 103/mm3), thrombocytopenia (115 × 103/mm3) and anemia . The laboratory findings also showed hyponatremia (130 mmol/dL), hypocalcemia (8.7 mmol/dL), hypoalbuminemia (3.5 g/dL), an increase of LDH (267 IU/L), C-reactive protein (1.64 mg/dL) and alkaline phosphatase levels (190 IU/L). Additional tests were performed without significant outcomes.Because of the evidence of hepatosplenomegaly, an ultrasound of the abdomen was performed and resulted in a severely enlarged spleen (15.6 cm × 6.6 cm), a mild ascitic flap and enlarged liver. Blood tests and a complete blood count revealed trilinear pancytopenia with severe neutropenia . Each clinical manifestation is typically, but not uniquely, associated with specific parasite species and depends on the immune response of the patient .L. braziliensis, L. amazonensis, L. guyanensis and L. panamensis and occur in the South American region, with a greater incidence in Peru, Bolivia, Paraguay, Ecuador, Colombia and Venezuela [The majority of mucosal leishmaniasis cases are caused by enezuela .L. infantum [L. infantum can also be responsible for a localized CL, while mucosal involvement is described in the literature almost exclusively in HIV+ or immunosuppressed patients [Sicily, as part of the Mediterranean basin, is considered to be an endemic area for VL caused by infantum , which ipatients .In the absence of treatment, VL is typically fatal within 2 years as a result of secondary bacterial infections or severe anemia; however, in immunocompetent patients, the treatment of the disease with amphotericin B guarantees a recovery rate that reaches almost 100% .A reduction in the therapeutic response and high recurrence rates are described almost exclusively in immunocompromised patients ,21, wherSeveral studies and experimental models suggest that the T helper-1 (Th1) response with IFN-g production induces a potent leishmanicidal mechanism in phagocytes, thus facilitating the resistance or resolution of the infection. Differently, the T helper-2 (Th2) response with IL-4 and IL-10 production can result in the inhibition of macrophage activation, with a consequent increase in the intracellular replication of the parasite and, therefore, an increased susceptibility to infection and the development of severe disease .Cases of recurrence with mucosal localization in patients previously treated for VL have been described mostly in adult populations with an impaired immune system  ,22,24,25In children, sporadic cases of VL relapse have recently been described, even in immunocompetent patients ; howeverOur patient represents, therefore, the first reported case of recurrence of VL with mucosal involvement in an immunocompetent pediatric patient without comorbidities.The SARS-CoV-2 infection detected during the first hospitalization represents the only risk factor specific to our case, which could have impacted the immunocompetency of the patient, thus leading to a relapse with mucosal involvement.The most described cases of coinfections with Leishmania sp. in the literature involve Plasmodium malariae and HIV. Such a trend is probably due to the insistence of these microorganisms in the same endemic regions .P. falciparum parasitemia have been registered when comparing to malaria-only infections, thus suggesting a protective effect of Plasmodium coinfections on the progress of Leishmania sp. The increased levels of pro-inflammatory cytokines (TNF-a and IFN-g) observed in the coinfected groups are probably the cause of this effect [While the effect of a cd4 decrease in patients affected by HIV is a widely recognized cause of more serious and atypical manifestations of leishmaniosis ,24,27, is effect .Regarding our case, studies have found that a few months after the SARS-CoV-2 infection, changes in the immune system can occur . Milena In our case, once we ruled out other possible causes of gingival bleeding, such as periodontal and hematological diseases, the suspicion of a leishmaniasis relapse led to the correct diagnosis by performing a PCR test.Traditionally, the gold standard for diagnosis has been a microscopic examination (after Giemsa staining) or a culture on invasive samples  . The perThe PCR test on peripheral blood represents today the most sensitive and less invasive diagnostic method for leishmaniasis. After the clinical recovery of a patient, the test still remains the best diagnostic tool for detecting an infection relapse before any clinical manifestation can occur. Qualitative and semiquantitative PCRs should thus be adopted as the standard method for monitoring the response to treatment in immunocompetent children with a periodicity of 3, 6, 9 and 12 months ,34.Liposomal amphotericin B (AmBisome) is the only drug approved by the U.S. Food and Drug Administration for the treatment of VL in immunocompetent patients, according to a dosing schedule of seven administrations on days 1–5, 14 and 21 at a dose of 3 mg/kg/day . NumerouRecommendations about therapeutic schemes for immunocompromised patients are mainly based on evidence from European studies about the HIV-coinfected population, whose treatment failure and relapse rates are particularly high. A longer period or higher dosage of treatment is needed for this group according to the WHO and international guidelines .In our case, a dose of 3 mg/kg/day of AmBisome, administered for 10 days, led to negative results of molecular tests and to the discharge of the patient. No relapse or other clinical issue has been reported during the follow-up tests.Leishmaniasis relapse with atypical mucosal involvement represents an eventuality for pediatric patients. Clinical follow-up and periodic PCR tests performed within a year of treatment must be considered essential for the early recognition and treatment of this insidious condition.


Background and Objectives: The careful selection of adequate SLNB candidates not only aims at reducing the surgical risk while identifying SLN metastasis, but also plays a crucial role in identifying the patients eligible for adjuvant therapy. Objectives: The purpose of our study was to investigate the clinical and histologic aspects of primary melanomas that correlate with the likelihood of a positive SLNB result. Materials and Methods: A total of 101 primary melanoma patients who underwent sentinel lymph node biopsies were included in the study. General patient demographics were obtained as well as localization and melanoma-specific characteristics of primary melanoma from histologic reports in addition to data derived from SLNB melanoma histopathology reports. Results: The patients with positive SLN results had a statistically significant increased Breslow thickness , higher mitotic index rate , as well as the presence of ulceration . Univariate regression analysis showed the Breslow thickness (p = 0.008), the mitotic index rate (p = 0.054), the presence of ulceration (p = 0.009), as well as the pT3-4 stage (p = 0.009) to be significant predictors of SLN positivity. The optimal cut-off values for Breslow thickness and the number of mitoses scores were determined based on ROC curve analysis. Using the Breslow thickness, mitotic index rate, presence of ulceration, and pT3-4 stage significant coefficients from the univariate regression model, a chance prediction score was developed. Conclusions: The newly developed and proposed scoring system can aid in patient selection for SLN biopsy by facilitating a more efficient risk assessment in the detection of lymph node metastases in melanoma patients. Even though melanoma accounts for 1.7% of all cancer diagnoses worldwide, it is considered to be one of the most fatal skin carcinomas, with the highest death rate among the skin tumors ,3. With The potential for the presence of metastases in the local nodal basin or elsewhere can be predicted via the biopsy and histology of the sentinel lymph node (SLN), the first lymph node involved in the lymphatic spread of melanoma. Knowing the status of regional lymph nodes carries extremely valuable prognostic information that is helpful in setting a strategy for regional disease control and in the selection of patients who would benefit from adjuvant therapy . The ideAccording to the American Joint Committee on Cancer (AJCC), eight edition, and National Comprehensive Cancer Network (NCCN) recommendations, the indications for SLNB are having intermediate-thickness melanomas, being at stages T2 and T3 (1 mm to 4 mm) with thinner melanomas (0.8–1 mm or <0.8 mm with ulceration), as well as thick melanomas (>4 mm); these should include an open discussion with the patient about potential risks and benefits of the procedure ,10,11. TSeveral clinical and histologic features have been shown to be predictive of SLN positivity. Some studies have shown that the incidence of positive SLN after SLNB mainly depends on the thickness of the primary tumor and ranges from 15 to 20% in the literature. The incidence of positive SLN after SLNB in T4 tumors is considered to be 35–40%, while for T1 lesions, the incidence is 5–7.8%, as descriptively presented ,14,15. IAdditionally, the careful selection of adequate SLNB candidates not only aims at reducing the surgical risk while identifying SLN metastasis, but also plays a crucial role in identifying the patients eligible for adjuvant therapy. The primary analysis after a median follow-up of 34 months by the German Dermatology Cooperative Oncology Group (DeCOG-SLT) did not show a benefit in terms of distant metastasis-free survival (DMFS), overall survival (OS), and recurrence-free survival (RFS) for patients undergoing CLND immediately post-SLNB, while the final analysis of the DeCOG-SLT study performed 3 years after the inclusion of the last patient also showed similar results . AdditioThe purpose of our study was to investigate the clinical and histologic aspects of primary melanomas that correlate with the likelihood of having a positive SLN result to develop and propose an easy-to-use scoring system that predicts SLN positivity based on the presence of multiple independent predictors, which is used for the more efficient selection of patients in a clinical setting.This retrospective study was conducted at the Clinic for Burns, Plastic and Reconstructive Surgery, University Clinical Center of Serbia, Belgrade. After the approval of the Institutional Review Board , data were extracted from patients’ medical records and the histopathology results. All samples were obtained from the histopathological reports from the Institute of Pathology, Faculty of Medicine, University of Belgrade, and analyzed by a pathologist with experience in the analysis of melanocytic lesions.This study included primary melanoma patients treated between 1 January 2017 and 31 March 2022. All the patients were treated according to the current recommendations of the American Joint Committee on Cancer (AJCC) 2017, 8th edition, as well as per the National Comprehensive Cancer Network (NCCN) guidelines. The inclusion criteria were all melanoma patients without clinically or radiologically verified alteration of lymph nodes and/or distant metastases. Patients with a tumor thickness of more than 0.8 mm were included, including patients with microsatelitosis in the histopathological reports. Patients treated for primary melanomas of less than 0.8 mm thickness were also included if ulceration or other risk factors were present. Patients treated for melanoma with satellite, in-transit, or distant metastases, as well as previously treated patients for melanoma or other non-skin related malignancies, patients on immunosuppressive therapy, patients who have already undergone extensive targeted lymph basin surgery, and patients who have undergone primary wide excision were excluded from the study.2 method, while ulceration status was marked as present versus absent.General patient demographics were obtained, such as age and sex, as well as date of biopsy, localization, and melanoma-specific characteristics of primary melanoma, e.g., subtype, Breslow thickness, Clark grade, mitotic index rate, and ulceration status. According to the primary distribution, all the melanomas were divided into 4 localizations: (a) head and neck, (b) trunk, (c) upper extremities, and (d) lower extremities. All the samples were histologically divided into 4 large groups: (a) superficial spreading melanoma (SSM), (b) nodular (NOD), (c) lentigo maligna (LMM), and (d) other , nevoid, spitzoid, dermal, desmoplastic, meltump, malignant blue nevus, polypoid, and Reed nevus-like melanoma). The Breslow thickness was divided into four categories, according to the guidelines of the 8th edition of the AJCC from 2017: <1 mm, 1.01–2 mm, 2.01–4 mm, and >4 mm. The Clark depth was categorized into five levels (1–5). The mitotic index rate was calculated using the hot spot/mmmTc-sulfur radiocolloid. Data derived from SLNB melanoma histopathology reports included the size of the largest metastatic deposit, the number of positive sentinel nodes, and the localization of metastasis in the sample. The localization of the metastatic deposit in the sentinel lymph node was categorized as parenchymatous, capsular, subcapsular, extensive, or parenchymal-subcapsular. The sentinel lymph node tumor burden was classified according to the Rotterdam criteria related to the diameter of the largest metastatic deposit . The clinical stage of the disease was determined based on the thickness of the tumor according to Breslow (T), the status of the lymph nodes (N), and the presence or absence of distant metastases (M).Sentinel lymph nodes were mapped via lymphoscintigraphy using technetium 99t-test or the Mann–Whitney U test was used to evaluate the differences between the positive and negative sentinel biopsy numerical data. Univariate logistic regression analysis was used to determine factors related the prediction rate of positive sentinel biopsy. The results are expressed as relative risk and corresponding 95% confidence intervals (CI). Model discrimination performance was tested using sensitivity, specificity, positive, and negative predictive values. The C statistic, representing the area under the receiver operating characteristic curve (ROC), was used for the overall assessment of the predictive model. In all analyses, the level of statistical significance was set at p ≤ 0.05. For the statistical analysis, the SPSS version 25 statistical software  was used.Descriptive statistics, including means, medians, standard deviations and percentiles for numerical variables, and numbers and percentages for categorical variables, were used to characterize the study sample. No imputation methods were used in the analysis. Associations between the categorical data were evaluated using Pearson’s chi-square test. Student’s A total of 101 patients underwent a sentinel lymph node biopsy. A total of 59.4% were male, while 40.6% were female. The mean patient age was 56.26 ± 15.09 years . All the patients were divided into three age groups: younger than 40, 41–60, and older than 60, with the majority of patients being in the older-than-60 group . The characteristics of the patients included in the study, as well as the histopathological characteristics of primary melanoma, are presented in p = 0.002), higher mitotic index rate , and a higher percentage of ulcerated lesions  than the patients with negative SLN results did, aligning with a higher proportion of pT3-T4 cases among the patients with positive SLN results . Conversely, sex, age, the anatomic site of primary melanoma, and Clark levels were not significantly associated with SLN positivity (p > 0.05). Nodular melanoma was the most prevalent histopathological subtype in the SLN-positive group (47.4% vs. 26.6%), although this finding did not reach statistical significance (p = 0.78). The comparison between the SLN-positive and SLN-negative groups is shown in Patients with positive SLN results had a statistically significant increased Breslow thickness . p = 0.01). The optimal cut-off values for Breslow thickness > 2 and mitotic index rate scores > 3 were determined based on the ROC curve analysis.The diagnostic performance of the Breslow thickness, mitotic index, presence of ulcerations, as well as pT3-4 stage in predicting SLN positivity was tested, and the sensitivity, positive predictive value (PPV), specificity, and negative predictive value (NPV) are shown in p = 0.008), mitotic index rate (p = 0.054), presence of ulcerations (p = 0.009), and pT3-4 stage (p = 0.009) to be significant predictors of SLN positivity. In a multivariate regression model, the most significant independent predictor was the pT3-4 stage (p = 0.009)  .p = 0.008). The distribution of the SLN positivity scores in our study population is shown in Using the significant Breslow thickness, mitotic index rate, presence of ulceration, and pT3-4 stage coefficients from the univariate regression model, the chance prediction score was developed. The newly developed SLN positivity score was determined by assigning the chance of SLN positivity according to the presence of the predictors. A score of 0 presents an unlikely chance of SLN positivity, while there is a low chance (score 1) if one predictor is present, a medium chance (score 2) if two predictors are identified, a high chance (score 3) if there are three predictors, and an extremely high chance (score 4) with all four predictors present. In our research, we found that patients exhibited varying rates of positive SLNB results based on their scores: 10.5% of those with a score of 0, 5.3% for patients with scores of 1 and 2, 31.6% for patients with scores of 3, and 47.4% for those with a score of 4 , thick melanomas (>4 mm), SLNB in senior patients, as well as some histologic subtypes of melanoma. All the protocols agree on offering SLNB to patients with more than a 5% chance of having a positive SLNB result . Many st2 vs. ≥1 mitosis/mm2) [p = 0.009), with the mitotic rate index being a significant predictor of SLNB positivity (p = 0.05). Santos et al. reported a statistically significant association between the mitotic rate index and SLN positivity in a group of people with melanomas ranging from 1 to 4 mm in thickness (p = 0.034), while Rodriguez et al. identified hazard ratios statistically significant for the association of a mitotic rate index >3 mm2 and SLNB positivity [2 to be a statistically significant predictor of positive SLNB (adjusted OR 1.63 (95%CI 1.13–2.36)) [2, as it presents an important predictor of SLNB positivity per their recommendations as well as per our scoring system [While the AJCC, seventh edition, included the mitotic rate index as an important predictor of SLNB positivity, the AJCC, eighth edition, did not find the mitotic rate index as a valuable predictor when presented as a dichotomous variable (<1 mitosis/mmsis/mm2) . Still, sitivity ,24. Addig system .The presence of ulcerations has been widely accepted as a predictor of SLNB positivity found in all guidelines and by most authors in independent, single-center studies, as well as in our study ,22,24. FA Breslow thickness >2 mm was found to be a significant predictor of positive SLN results in our study. While the AJCC, eighth edition, and the NCCN recommendations based on a fairly large population sample advise SLNB to patients with a Breslow thickness >0.8 mm with ulceration and a Breslow >1 mm with or without ulceration, other authors report different thresholds for Breslow thickness as an independent risk factor . NeverthThe inclusion of pT staging enhances the scoring system by consolidating histopathological features, such as Breslow thickness and ulceration status, into a single variable. This facilitates a more structured categorization and assists in identifying patients who would derive the greatest benefit from SLNB.The choice of patients eligible for SLNB should be based on the existing globally accepted protocols, such as the AJCC or the NCCN, which include the most common, evidence-based features of melanoma patients. Our scoring system is proposed for the quicker and more efficient evaluation of patients in unconventional circumstances, such as patients with desmoplastic, acral melanoma, senior population, or in facilities with limited resource settings . Certain histologic subtypes show a variable predisposition for SLN metastasis. In pure desmoplastic melanomas, SLNB positivity was found in 5.4% of cases, while in mixed desmoplastic melanomas, the rate of SLNB positivity was 13.8% . ConversThe score developed based on our results aims at simplifying decision making in a clinical setting, though it is in need of further refinement.This study is mainly limited by its retrospective design. Additionally, the study population may be susceptible to a selection bias, as it relied mostly on patient referrals to a tertiary institution. The selection of patients for SLNB was conducted at a single center, based on AJCC, eighth edition, criteria and further clinical judgement, limiting the population sample of thin as well as thick melanomas and omitting the further stratification of melanoma characteristics. The above-mentioned reasons could have attributed to our discovery of the 2 mm threshold for Breslow thickness as a predictor of SLN positivity. The small sample size resulted in only 14 patients with melanomas < 1 mm, rendering further statistical analysis using a 1 mm Breslow cut-off unfeasible. This limitation may also partly be due to the significant proportion of our cohort including patients treated over the past three years, including the period during and after the COVID-19 pandemic, which could have influenced the data distribution of our sample. One of the main limitations of this study is the results for score 0, which still identifies 10.5% of patients with a positive SLNB, meaning there are still patients invisible to this scoring system. As previously stated, these results are likely influenced by the small population sample as well as the distribution of the population in the sample. Further validation of the proposed score is necessary on a larger population in a multicentric design, as is the refinement of each individual component of the score and its thresholds.This newly developed and proposed scoring system can aid in patient selection for SLN biopsies by facilitating the more efficient risk assessment for the detection of lymph node metastases in melanoma patients. Further research is needed to refine the thresholds of SLN positivity predictors as well as validate this scoring system’s effectiveness.


Strong evidence suggests a correlation between degeneration and mitochondrial deficiency. Typical cases of degeneration can be observed in physiological phenomena  as well as in neurological neurodegenerative diseases and cancer. All these pathologies have the dyshomeostasis of mitochondrial bioenergy as a common denominator. Neurodegenerative diseases show bioenergetic imbalances in their pathogenesis or progression. Huntington’s chorea and Parkinson’s disease are both neurodegenerative diseases, but while Huntington’s disease is genetic and progressive with early manifestation and severe penetrance, Parkinson’s disease is a pathology with multifactorial aspects. Indeed, there are different types of Parkinson/Parkinsonism. Many forms are early-onset diseases linked to gene mutations, while others could be idiopathic, appear in young adults, or be post-injury senescence conditions. Although Huntington’s is defined as a hyperkinetic disorder, Parkinson’s is a hypokinetic disorder. However, they both share a lot of similarities, such as neuronal excitability, the loss of striatal function, psychiatric comorbidity, etc. In this review, we will describe the start and development of both diseases in relation to mitochondrial dysfunction. These dysfunctions act on energy metabolism and reduce the vitality of neurons in many different brain areas. The mitochondrion is an evolutionary organelle that originated from the established symbiotic relationship between alpha-proteobacteria and eukaryotic cells . This orThere is abundant evidence in the literature associating mitochondrial disruption with neurological and neurodegenerative diseases ,5,6. The2), that aid energy transfer to the electron transport chain (ETC), embedded within the extensive inner mitochondrial membrane [2 to O2. Ultimately, the energy released by the transfer of electrons is used for the thermodynamically unfavorable pumping of protons against their concentration gradient from the matrix to the intermembrane space to generate an electrochemical gradient known as mitochondrial membrane potential (ΔΨm) [Mitochondria are organelles responsible for several critical processes in neuronal function and dysfunction, including energy metabolism, calcium homeostasis, and signal transduction . The bramembrane ,14. The al (ΔΨm) . Proper al (ΔΨm)   in the mitochondria [2+ homeostasis plays a key role in cellular bioenergetics and signaling [2+ passage across the outer mitochondrial membrane (OMM) is mediated by the VDAC channel, whose different selectivity for cations or anions is voltage-dependent [2+ flux. Conversely, increases in the membrane potential (20–30 mV) result in a conformational change that allows a 4- to 10-fold increase in Ca2+ influx [2+ permeability: the mitochondrial calcium uniporter (MCU) contributes to the potential-dependent Ca2+ influx into the mitochondrial matrix, while the mitochondrial Na+/Ca2+ exchanger (NCLX) is one of the main units involved in Ca2+ extrusion [2+ concentrations [2+ is compartmentalized, and the average resting cytosolic concentration is remarkably low [2+ homeostasis through its interaction with the mitochondria [2+ uptake in the mitochondria probably occurs only at sites in close proximity between the ER and mitochondria [2+ uptake. In addition, ER-mitochondria contacts are critical for Ca2+ uptake in dendritic mitochondria but not in axonal mitochondria [2+ storage. In the absence of MICU3, synaptic function is impaired [Indeed, mitochondrial functions go beyond the primary role of ATP production because these organelles are intimately involved in numerous processes operating within the cell, including calcium homeostasis, the generation of free radical species (ROS), steroid synthesis, apoptosis, and cell signaling pathways . Particuchondria . Mitochoignaling . Ca2+ paependent . For insotential –30 mV rextrusion . In genetrations ,23  [2O2, which is in turn scavenged by the enzyme catalase to produce water [Mitochondrial calcium-signaling in neurons regulates metabolism and energy production, which are crucial for neurotransmission and sustaining synaptic plasticity . In addies (ROS) . Mitochoes (ROS)   is an emerging therapeutic target, since it is involved in cellular resistance to oxidants. In detail, Nrf2 controls the basal expression of genes coding for enzymes and proteins involved in antioxidant and detoxifying action, repair and removal of damaged proteins and organelles, the inflammatory response, and mitochondrial bioenergetics ,51. AddiIt is therefore understood that mitochondrial integrity and homeostasis are prerequisites for proper cellular functioning, especially for neurons, which are polarized, complex cells with high energy demands . Not surFirstly, the mitochondria provide the energy needed to undertake a wide range of neuronal functions, such as the maintenance of resting membrane potential, the restoration of ionic balance after depolarization, the cycling of synaptic vesicles, and the transport of proteins and organelles from the soma to distal sites. Interestingly, the mitochondria in axons and dendrites have different morphologies, some are small and sparsely distributed in the former, whereas others are elongated and densely distributed in the latter. In addition, axonal and dendritic mitochondria differ in movement, metabolism, and responses to neuronal activity .Secondly, several lines of evidence support the role of mitochondria in the mobilization and recycling of synaptic vesicles . NeurotrOverall, the importance of the mitochondria in neurons is unequivocal. Therefore, mitochondrial dysfunction leads to a plethora of severe conditions, from impaired neuronal development to various neurodegenerative diseases.A great deal of evidence shows the effects of mitochondrial dysfunction on degenerative diseases . DiseaseA degenerative disease is regarded as a type of non-physiological condition whose origins in a tissue or organ worsen over time. Energy failure has been linked to degenerative processes such as cancer, aging, neuroendocrine disease, neurodegenerative disease, and inflammatory diseases. Likewise, results from many studies support the involvement of the mitochondria in neurodegenerative diseases, particularly PD ,70,71,72Huntington disease is an incurable degenerative disorder caused by a mutation in the huntingtin gene, where the CAG sequence is excessively repeated. This mutation alters numerous cellular processes and leads to cell apoptosis. One important alteration is caused by the impairment of the mitochondrial metabolism. In a study conducted on the fly model, the formation of an abnormal ring-shaped mitochondria was observed; this particular shape was previously identified in mitophagy-blocked cells in which PINK1 overexpression was able to rescue the regular shape and function of the mitochondria. PINK1 over-expression was able to improve bioenergetic efficiency (increasing ATP levels) and rescue neuronal integrity in the adult drosophila model of HD . HD is aParkinson’s and Huntington’s diseases are both neurodegenerative diseases with multiple mitochondrial bioenergy alterations related to metabolism, oxidative stress, dynamics of biogenesis transport, and mitophagy . As a reParkinson’s disease animal models are associated with multiple mitochondrial defects for review ,124,125 Chemically, the neurotoxin agent is widely used to recapitulate Parkinsonian features in various animal models ,128. In Experimental studies in A53T mutant mice, a transgenic animal model that expresses human α-synuclein, show that they develop mitochondrial DNA damage and degeneration with an apoptotic-like death of neocortical, brainstem, and motor neurons . In the 2+ transfer in the mitochondria, while its silencing impairs mitochondrial function by loosening the ER-mitochondria interface [The neurons are high-energy consumers and employ most of the energy at the level of the synapse to preserve and restore ionic gradients and for the uptake and recycling of neurotransmitters . Variousnterface . Howevernterface . The wilnterface . Caspasenterface ,151,152 N-terminal ubiquitin-like domain with a function as E3 ubiquitin-protein ligase [The discovery of many genes related directly or indirectly to the mitochondria in PD underlines the implication of mitochondrial bioenergy in its pathogenesis. For instance, genes linked to early-onset recessive PD include: Parkin , PINK1 , and DJ-n ligase ,159,160.n ligase . Parkin n ligase ,162,163.n ligase . In a mon ligase . Other sn ligase . Deficitn ligase . Function ligase . The dopn ligase ,166 may n ligase . Furthern ligase . On the n ligase . Calciumn ligase . Recent n ligase .Another gene linked to early-onset recessive PD is PINK1, which encodes a protein with a serine/threonine kinase catalytic domain, whether cytosolic or mitochondrial-associated . PINK KO2O2 consumption, mitochondrial Trx activity, total glutathione  levels, mitochondrial glutaredoxin (GRX) activity, and a decrease in mitochondrial glutathione reductase (GR) activity [DJ-1 gene mutations cause autosomal recessive early-onset PD. DJ1 encodes a protein of the superfamily that belongs to the ThiJ/PfpI-like. Under normal conditions, DJ-1 is confined throughout the cytoplasm of neurons, with a small amount localized to the mitochondrial matrix and intermembrane space , indicatactivity . Furtheractivity  H fluorescence and a loss in mitochondrial membrane potential (ΔΨm) [N-acetyl aspartate in R6/2 mice, similar to symptomatic HD patients [Both R6/1 and R6/2 strains express a single copy of a human genomic fragment that contains 116 and 144 CAG repeats of HTT under the control of the human HTT promoter . These mal (ΔΨm) . Severalpatients ,205,206,patients . 2O2 levels in YAC128 mice and defects in Ca2+ handling [N-acetylcysteine administration delayed the onset and progression of motor deficits in R6/1 mice [Another interesting contribution to assessing the deficits of mitochondrial function and redox deregulation is the experimental data performed with PET in YAC128 transgenic mice . PET anahandling  support 6/1 mice , which m6/1 mice . At the 6/1 mice ,192. Int6/1 mice ,209,210.6/1 mice . Moreove6/1 mice   in the CNS of living patients is not easy. However, analyzing peripheral tissues and fluids can offer a reliable measure. Although the core pathology in PD and HD is central, a number of substantial abnormalities occur at the systemic or peripheral level. Peripheral blood mononucleate cells (PBMCs) are one of the most reliable “in vivo PD models”. Peripheral blood mononucleate cells present several alterations in critical metabolic pathways and accumulate α- synuclein in pathological forms, well recapitulating PD-related neuropathology ,213. In Degenerative diseases commonly entail mitochondrial impairment. The mitochondria represent the major source of bioenergy for the cell, and as a consequence, their putative failure leads to a lack of energy. Daily energy metabolism for both humans and organisms is the sum of daily energy expenditure and daily energy intake. Daily energy expenditure may be divided into different categories: (1) energy spent on basal metabolism; (2) energy spent by thermic increment following food intake; (3) energy spent on thermoregulation; and (4) energy spent during daily activities . In partMitochondrial biogenesis is a process that continuously balances the degradation of dysfunctional mitochondria inside the cells. The disruption of this homeostasis, which may be guaranteed in all functioning cells, represents an additional point that focalizes the loss of energetic function, which may precede cellular loss. This critical balance is called mitochondrial dynamics. During these processes, mitochondrial fusion and fission regulate the number and size of the mitochondria, while mitochondrial biogenesis increases the mitochondrial mass inside the cells by producing ATP as a response to greater energy demand. Mitochondrial biogenesis requires a very multifaceted process that starts with the coordination of the nuclear and mitochondrial expression programs. The regulator for the cited process is the protein PGC-1α, which is able to interact with many transcription factors, such as myocyte enhancer factor 2C (MEF2C), nuclear respiratory factors 1 and 2 (NRF1 and NRF2), peroxisome proliferator-activated receptors (PPAR), estrogen-related receptor alpha (ERRα) and many others involved in coordinating mitochondrial biogenesis and oxidative metabolism ,244,245.A consistent number of mitochondria are localized at the presynaptic terminals, to provide ATP for intense synaptic activity . In factPreviously, we have referred to the role of the PINK1 gene in early inherited parkinsonism, and these findings are in support of the theory that links mitochondrial dysfunction to PD.In addition, the mitochondria in aging people show different features ; they swIt was demonstrated in both PD and HD that the presence of environmental stress can increase the production of oxidative species and can also modify the structure of proteins and DNA inside the mitochondria and its nuclei . UsuallyIn rodent models of HD, only the progression of the disease leads to neuronal loss. Cell dysfunctions may generate symptoms, and in particular, abnormal synaptic statements due to a lack of ATP . In contIn PD models, dysfunctions in striatal plasticity are described in detail  and are These areas are linked to movement and cognitive ability. For this reason, it is not a surprise that both diseases can affect cognitive or thinking abilities and motor functions. Studies on rodent models of PD show impairment in the bioenergetic pathway and a block in function in mitochondrial complexes I and III. Additionally in genetics, phenotypic models were induced by toxin exposure in the early stages of the disease as well as in the developmental stage ,125,238.Interestingly, this evidence has been confirmed by translational studies in men, in which the association between mitochondrial deficit and biomarkers of PD and HD was shown . ActuallNumerous evidences show the role of mitochondria in neurodegenerative diseases. Approximately 1209 papers described the mitochondria’s involvement in non-neurological degenerative processes  A,B. AmonConversely, many questions remain unresolved, especially about how mitochondrial failures progress and the loss of energy efficiency triggered by environmental events. However, it is clear that in these two illnesses, the role of mitochondria in providing the energetic functions of the neuron is still lacking . Therefore, in-depth studies in this field could provide more insight and knowledge about movement disorders and could offer new therapeutic solutions for pathologies, such as PD and HD, for which only symptomatic cures currently exist.


BCR::ABL1-negative myeloproliferative neoplasms such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF) are clonal diseases with the presence of characteristic “driver mutations” in one of the genes: JAK2, CALR, or MPL. The search for mutations in these three genes is required for the diagnosis of MPNs. Nevertheless, the progress that has been made in the field of molecular genetics has opened a new era in medicine. The search for additional mutations in MPNs is helpful in assessing the risk stratification, disease progression, transformation to acute myeloid leukemia (AML), or choosing the right treatment. In some cases, advanced technologies are needed to find a clonal marker of the disease and establish a diagnosis. This review focuses on how the use of new technologies like next-generation sequencing (NGS) helps in the diagnosis of BCR::ABL1-negative myeloproliferative neoplasms.The classical  BCR::ABL1-negative myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders with the clonal proliferation of one or more types of cells of the myeloid lineage. MPNs are associated with an increased risk of thrombosis and a tendency to transform into acute myeloid leukemia. According to the fifth edition of the World Health Organization (WHO) criteria, MPNs include chronic myeloid leukemia (CML), which is defined by the presence of the Philadelphia chromosome and BCR::ABL1 fusion gene, polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia (CEL), juvenile myelomonocytic leukemia, and myeloproliferative neoplasms, not otherwise specified. PV, ET, and PMF are referred to as classical BCR::ABL1-negative MPNs. CNL, CEL, juvenile myelomonocytic leukemia, and myeloproliferative neoplasms, not otherwise specified, are less common and called non-classical or atypical MPNs. The current criteria for the diagnosis of myeloproliferative neoplasms are provided by the fifth edition of the World Health Organization Classification of Haematolymphoid Tumours: Myeloid and Histiocytic/Dendritic Neoplasms, and the International Consensus Classification (ICC) of Myeloid Neoplasms and Acute Leukemias. The diagnosis still depends on the clinical picture, laboratory tests, evaluation of bone marrow biopsy, and molecular tests for the presence of mutations in the JAK2, CALR, or MPL genes. These mutations, called “driver mutations,” are crucial for diagnosis and have prognostic and therapeutic significance  patients. Nevertheless, driver mutations are not the only mutations identified in patients with MPNs. The use of advanced techniques of molecular genetics, such as NGS, plays a very important role in hematooncology diagnosis. The development of high-throughput NGS methods diversified the mutational landscape and improved our understanding of pathophysiology. These molecular details also made it possible to improve the diagnosis, assign a better prognosis score, and track the effectiveness of treatments. Comprehensive gene sequencing of blood cancer patients is becoming more widely available. We wanted to share a graphical flowchart of stepwise molecular testing ending with NGS   . Additiogression . In ET pc events . High JAMF cases . The qua disease .CALR is a highly conserved protein that is localized in the endoplasmic reticulum. It is involved in the quality control of N-glycosylated proteins and the modulation of calcium ion homeostasis. Mutations in this gene are the second most common genetic abnormality in myeloproliferative neoplasms after JAK2V617F gene mutations. This type of mutation is not found in patients with PV. CALR mutations are detected in about 20%–25% ET cases and 35% PMF  in patients with myelofibrosis showed an association with a higher VAF of CARL, a lower hemoglobin level and platelet count, higher peripheral blood CD34+ cell counts, the need for cytoreduction therapy, and shorter leukocytosis-free survival. In addition, the authors emphasize that high-molecular risk mutations (HMR) were more frequent in CALR patients with high VAF  analysis. Sanger sequencing can be useful in confirming the type of mutation; however, this technique has limited sensitivity. Next-generation sequencing (NGS) has the lowest limit of detection (LOD), but it is quite expensive for routine diagnostics  encodes a receptor for thrombopoietin that regulates megakaryopoiesis and platelet production, and is also crucial in the self-renewal of hematopoietic stem cells. Mutations in the ery rare . PatientF or AML . Mutatioffective .JAK2, CALR, and MPL can be seen in about 90% of MPN cases; nevertheless, in 2% of PV, 15% of ET patients, and approximately 8%–10% of MF cases, driver mutations are not detected; such patients are termed TN patients. Some patients may have rare mutations in the JAK2, MPL, and CALR genes that are not detected by routine diagnostics. In other cases, the mutation may have been at a very low mutant allele burden and can be detected only by applying a more sensitive methodology. Implementation of more advanced techniques of molecular biology, like next-generation sequencing, allowed to improve the understanding of triple-negative patients and revealed the presence of non-driver mutations. NGS helps in finding a clonal marker to confirm the diagnosis. Triple-negative PMF patients were associated with poor survival and increased leukemic transformation. These patients tend to have lower Hb levels, platelet and leukocyte counts, and a higher IPSS  risk. TN-ET patients are associated with a low vascular event rate and a lower risk of leukemic transformation compared to TN-MF patients , mRNA splicing , signaling pathways , and transcription factors (RUNX1 and TP53)  in less well-studied genes, the need to distinguish between germline and somatic mutations, and the lack of established guidelines regarding which patients and which genes should be tested are all challenges to clinical testing for these neoplasms. The most important aspect is data analysis, where bioinformatic tools are needed for mapping the reads to the human reference genome. Clarifying the somatic vs. germline origin of the mutations is one of the biggest problems in using and interpreting NGS data. Filtering NGS results would require a reliable method to distinguish between somatic and germline mutations. Variant categorization and annotation, including classifying variants into distinct “tiers” based on pathogenicity or clinical importance, may differ between laboratories in addition to interlaboratory variations in VAFs and the assignment of somatic status to variants. In general, it is recommended that interpretations follow specialist guidelines, such as those provided by the College of American Pathologists, the American Society for Clinical Oncology, and the Association for Molecular Pathology . Despitend TP53) . The appnd TP53) .TET2 and ASXL1 genes. In addition, mutations in TET2 and SF3B1 were associated with an older age, SF3B1 with higher platelet counts, and ASXL1 with palpable splenomegaly. Mutations in the IDH2, EZH2, and SH2B3 genes were crucial as risk factors for survival in ET patients. Leukemia-free survival was associated with mutations in TP53, EZH2, SRSF2, and IDH2, while myelofibrosis-free survival was associated with SF3B1 and U2AF1 mutations. This study identified SH2B3, IDH2, U2AF1, SF3B1, EZH2, and TP53 variants as being “adverse” in patients with ET. The most common mutations among PV patients were also TET2 and ASXL1 genes. A phenotypic correlation in PV was observed only between SH2B3 and palpable splenomegaly. In PV patients, mutations in SRSF2, ASXL1, and IDH2 were associated with inferior survival. Significant association was also noted between SRSF2, IDH2, and RUNX1 for leukemia-free survival, and SRSF2 and RUNX1 for myelofibrosis-free survival. Considering overall, leukemia-free or myelofibrosis-free survival, ASXL1, SRSF2, and IDH1 mutations were related as “adverse.” Through the use of next-generation sequencing, this work has defined mutations that were associated with poorer overall survival  occurs in 20%–30% of PV patients, and this has been correlated with an increased risk of thrombosis, disease progression, and shorter survival. The possibility of evolving resistance to HU treatment has been estimated at 64% in patients with the utations . Some ofutations . Similar therapy . The preon alpha . Anotherutations .TET2 mutations were associated with an increased risk of thrombosis, which was independent of driver mutation status and age. Nevertheless, such an association was not noticed in a cohort of Italian patients with ET , identify patients who have a higher risk of transformation to AML or a higher risk of death  had decreased OS and LFS compared to patients with only one additional mutation or those without any  have been associated with poorer prognosis and disease progression. In these groups of patients, leukemic transformation was detected only in patients with high-risk mutations . They also divided the patients into four categories: TP53 group, high-risk group, ASXL1 only, and “others.” Patients in the TP53 and ASXL1-only groups were more frequently diagnosed with SMF. Patients with TP53 and the high-risk group had the worst OS. Among patients with PMF, a negative prognostic effect of ASXL1-only was noted in comparison with the “others” category. In PMF, ASXL1 mutations among patients from the high-risk group mutation were associated with shorter OS. In PMF, VAF for ASXL1 in the TP53 and high-risk group was greater than those for ASXL1-only. Unlike SMF, VAF was lower in those groups, which may suggest that ASXL1 mutations are early driving events in PMF, but in SMF, these mutations might be acquired later. In SMF, the worst OS had the TP53 group. They noted that there were no differences between the OS of the ASXL1-only group and “other” or high-risk groups. In SMF, ASXL1 mutations in the high-risk group had no impact on OS. These recent studies have emphasized the concept that PMF and SMF are different biological entities; furthermore, they also confirmed the adverse effect of the ASXL1 mutation on PMF not in SMF  uses mutations, karyotype, and clinical data; GIPSS  is based solely on mutations and karyotype. The use of the NGS technique to assess the risk of patients with PMF is very helpful, especially in a model that is based on genetic changes [ and the possibility of incorporating them into prognostic scoring systems highlight the importance of genetic testing in hematooncology.