Remarks on Severity of Trauma Patients Due to Road Traffic Accidents have Been Treated at Vietduc University Hospital Assessed by RTS

Introduction
Traffic accidents are always a global problem. According to statistics of the World Health Organization - WHO, every year around the world, about 1.35 million people die, leading to 50 million people being permanently disabled due to irreversible injuries, accounting for 30-50% of total hospital admissions (3). Vietnam is the country with the highest number of traffic accident deaths in ASEAN and one of the countries with the most traffic accidents in the world. Therefore, traffic accidents are always a current topical issue in Vietnam because it’s a burden on health care and society, affecting the patient’s lives. Although the Government has been implementing many measures to reduce the number of cases and victims, Vietnam is still in the group of developing countries with high rates of morbidity and mortality caused by traffic accidents [1-4]. To improve the qualifications of trauma care, one interesting issue is updating trends in trauma care assessment. Primary trauma care requires assessment of severity to develop appropriate care strategies. The Revised Trauma Score -RTS simplifies the rapid assessment of injury based on Respiratory Rate, Maximum Blood Pressure, and traumatic brain injury severity - Glasgow Coma Scale has been widely recognized for clinical decision making. Several articles have evaluated the performance of RTS in the emergency department (ED) as a triage and prediction tool and showed the effect in clinical practice because bedside assessment tool, each of its variables can be easily and quickly calculated [5,6]. Viet Duc University Hospital, one of the leading centers of surgery in Vietnam, annually receives more than 30.000 trauma patients, most of them are serious trauma patients due to traffic accidents. Quick triage for providing proper treatment is a very important issue for health workers at the ED because it could impact the outcomes of treatment. Therefore, we have conducted this study to evaluate the effectiveness of using RTS on trauma patients at ED of Hospital.

Materials and Methods

Tool

In this study, we used the RTS including three parameters are considered for the RTS: maximum systolic blood pressure (MaxSBP, mm Hg), Respiratory Rates (RR, cycles per minute), and Glasgow Coma Scale (GCS). The RTS will range from 0 to 12, where the lower RTS is the more severe injury at the higher risk of death [5] (Table 1).

Table 1.

Setting and Participants

We prospectively analysed the clinical data of 200 patients with traffic accident acute trauma who were treated in the ED of Viet Duc University Hospital, a comprehensive tertiary surgical hospital) from December 2020 to March 2021. The patients were assessed the RTS upon admission within the first 24 hours. The data were recorded by attending nurses and doctors at the time of the patient’s presentation to the ED. Exclusion criteria were used: The patients already had airway intervention such as endotracheal intubation, mechanical ventilation. The patients died on arrival or were discharged from the ED before termination of emergency treatment, the medical records were not completed.

Data Analysis

Data were processed using SPSS 20.0 software.

Results

A total of 200 patients who met the selection criteria were analyzed. The characteristics of subjects are as follows:
*All 50 patients who dead in the group with RTS ≤ 9. The difference between survival and death rates of groups with RTS ≤ 9 and RTS ≥ 9 is statically significant with P <0.05.

Discussion

Injuries in general and traffic accidents, in particular, are still a global problem. In most developed countries, the injury classification system helps to provide appropriate care strategies, reducing complications and mortality. However, in many developing countries like Vietnam, the trauma emergency system is still incomplete and has many challenges. According to Zhejun Yu [4] each year, more than 400 000 people die in China from motor vehicle accidents or industrial accidents, among which 1%-1.8% were multiorgan/multisystem injuries. China’s regional trauma system hasn’t yet been full-fledged, and the management of trauma centers is facing great challenges. Therefore in all emergency rooms, especially in cases of overcrowding and understaffed, it is critical to rapidly screen large numbers of patients, identify the critically ill patients promptly, assess the severity of their condition and assign appropriate treatment priorities, and transfer them towards or intensive care unit are very important issues while treating the patients there [7-9].
In the past 30 years, a different trauma scoring system has been developed, most of the scales are combined with factors related to anatomy and physiology.
However, the scales are too complicated, with many variables, while the emergency needs to be done as quickly as possible. Among the commonly used scales are the Revised Trauma Score (RTS) or the T-Revised Trauma Score (T-RTS), the Severity Scale. Injury - Injury Severity Score (ISS) and Trauma Score-Injury Severity Scores (TRISS), the RTS is widely used. Many studies have evaluated the effectiveness of applying RTS to serve trauma care at the ED effectively [10-12]. In 1989 Champion HR [5] has introduced a revised scale to assess the severity of trauma based on three main indicators: Respiratory Rates - Maximum Blood Pressure – Glasgow Coma Scores abbreviated as RTS - Revised Trauma Score. According to the rating scale, the lower the RTS, the higher the risk of death. Because RTS reflects trauma severity, it is considered a useful tool to predict the patient’s survival and death. The study of R.A Lichtveld [13] of 503 trauma patients showed that when compared with non-ventilated patients with unchanged RTS, the risk of death in patients with RTS scores was 3.1 times lower (P=0.001), patients with a good initial RTS score but subsequently intubated were 2.9 times higher (P<0.001) and in patients with a low RTS, intubated were twice as likely (P<0.001) (6). According to Nguyen Huu Tu [14] if RTS ≤9 mortality rate is 78.3% compared to 3.4% of the RTS group >9 (3). Research results of Nguyen Huu Tu and Nguyen Truong Giang are similar: the higher RTS means the greater rate of survival [14,15]. In the study on the effects of T-RTS by Lam Vo Hung [6] to triage of trauma patients at the ED of An Giang hospital in the South of Vietnam in 2012 through 150 trauma patients with traffic accidents. The study has shown that RTS had a statistically significant difference in the mean value of the survival group with the death group with P = 0.000. RTS cut-off score <9 predicts mortality with a sensitivity of 88% and a specificity of 99%.
The author recommends that RTS should be widely applied in medical facilities and that the RTS scale is effective in survival prognosis. With a sensitivity of 88.2% and a specificity of 99.2%, the RTS shows an effective role in assessing the risk of death. The reports of Nguyen Huu Tu, et al. [14,15] also had similar results with sensitivity of 78.7%, 76%, and specificity of 95.1%, 84%. According to the study by Kondo Y et al. [16] about the correlation between long-term mortality and short-term mortality of RTS, T-RTS, TRISS, MGAP (mechanism, GCS, age, and arterial pressure) score, and GAP (GCS, age and arterial pressure) score. They found that T-RTS was better at predicting short-term mortality than long-term mortality. For the aging group, the study of Lam Vo Hung [6] showed that the group with the highest mortality was from 16 to 39 years old, young people who were hyperactive, disregarded traffic rules, and easy to be injured by traffic accidents (accounting for 50% of the total sample of study). In our series, the age group was the highest proportion from 21 to 60 years old, accounting for 64%, males accounted for the majority of 86.7% (Figures 1 & 2). As for the type of injury, in the study of Lam Vo Hung [6], traumatic brain injury and multiple trauma had a high mortality rate. Among the types of injuries, there was a statistically significant difference in mortality with P<0.05. Nguyen Huu Tu [14] has the same comment as us, the mortality rate due to traumatic brain injury and multiple trauma is 16.6% and 22.3%, respectively (3).

Figure 1: Distribution by age group.

Figure 2: Distribution by sex.

In addition, Nguyen Truong Giang [15] studied 532 accident patients at 103 Hospital and found that the RTS were low in the traumatic brain injury group and the multi-traumatic group. Nguyen Duc Chinh, et al. [17] conducted a study at Viet Duc University Hospital on deaths (2016-2018) showed that the traumatic brain injury group accounted for the highest rate, especially the group with GCS from 6 to 8. Bruno Durante, et al. [18] analyzed 200 patients from December 2013 to February 2014, including trauma victims admitted to the emergency room of the Cajuru University Hospital. The patients were set up in three groups: (G1) penetrating trauma to the abdomen and chest, (G2) blunt trauma to the abdomen and chest, and (G3) traumatic brain injury. The variables we analyzed were: gender, age, day of the week, mechanism of injury, type of transportation, RTS, hospitalization time, and mortality. Regarding mortality, there were 12%, 1.35%, and 3.95% of deaths in G1, G2, and G3, respectively. The median RTS among the deaths was 5.49, 7.84, and 1.16, respectively, for the three groups. The authors concluded that RTS was effective in predicting mortality in traumatic brain injury, however failing to predict it in patients suffering from blunt and penetrating trauma. In our study, the highest proportion of combined injuries were maxillofacial injuries 44%, limb injuries 23.5%, blunt chest injuries 22% (Table 2).

Table 2: Associated lesions (N = 200).

Patients with severe traumatic brain injury according to the GCS of 6 – 8 accounted for the highest rate of 52% (Table 3). Up to 40% were operated on emergency within the first 24 hours. The rate of serious injured was discharged to die at home accounted for 24.5%, 0.5 % died in hospital, overall mortality was 25% respectively (Table 4). Regarding the RTS in our study, there were mostly in the group of RST at 10 points (50.5%) and 9 points (35.5%). There were 98 patients (49%) with RTS ≤ 9 (Table 5) of which, 91.8% with serious brain injury. All 50 fatal and critically ill patients were in this group.

Table 3: GCS, MxBP and RR (N = 200).

Table 4: Managements and outcomes at ED within first 24 hours (N = 200).

Table 5: RTS.

In a Mega-analysis of Manoochehr S [19], to compare the ability of Revised Trauma Score (RTS) and Kampala Trauma Score (KTS) in Predicting Mortality, the study was conducted by two investigations searched the Web of Science, Embase, and Medline databases and the articles in which the exact number of truepositive, true-negative, false-positive, and false-negative results could be extracted were selected. A total of 11 relevant studies (total n = 20,631) were investigated. Regarding the accuracy and performance, the author concluded that RTS was better than KTS for distinguishing between mortality and survival. Compared with the other researches of domestic and international, we find that RTS is convenient to use in a clinical emergency for trauma victims. Moreover, in addition to the GSC, RTS can also be used as a predictor of severity and mortality, helping physicians at ED to making quick decisions and providing appropriate treatment [4,6,20,21].

Conclusions
Through the study of 200 trauma patients due to traffic accidents, we found that RTS has a value in predicting survival as shown by the difference between survived group and the death group. The patients who died were in the group with scores ≤ 9 statically significant with P <0.05. Because it is easy to calculate and suitable for first aid, it is recommended to apply in clinical practice, especially in the actual conditions of Vietnam. In the difficult conditions of shortage of resources, the trauma emergency system has not been standardized, the application of RTS helps to reduce the morbidity and mortality rate.

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Molecular Detection of BCL2/IGH Rearrangement in Follicular Lymphoma in Low Resource Settings: A Phase III Diagnostic Accuracy Study

Introduction
Follicular lymphoma (FL) is the second commonest non- Hodgkin lymphoma (NHL) subtype worldwide and the commonest in certain regions like USA [1]. FL has generally an indolent clinical course, somehow influenced by the cytological grading that is not, however, of prognostic relevance [2]. Conventional chemoimmunotherapy can induce initial remissions; nonetheless, cure is still not common [3]. In fact, relapses do occur, characterized by progressive chemoresistance development. In a percentage of cases, relapsing is also associated with histological transformation to secondary DLBCL [2]. The source of relapse in patients who initially achieve complete clinical remission are residual neoplastic cells representing the so called minimal residual disease (MRD). MRD can be detected either in bone marrow and blood by molecular methods and/or in tissues (mainly lymph nodes) by PET scan [4]. The t(14;18)(q32;q21) is molecular hallmark of FL. This translocation joins the BCL2 gene located on chromosome 18q21 with the immunoglobulin heavy chain locus (IGH) on chromosome 14q32, leading to the inappropriate expression of BCL2 protein, known to be a potent apoptosis inhibitor [5,6]. Detection of the BCL2/IGH rearrangement can be clinically useful for diagnostic purposes (using fluorescence in situ hybridization on tissues), but also for staging and MRD monitoring (using molecular techniques on blood and marrow) in FL patients [3,7,8].

Different techniques can be currently applied for the molecular detection of MRD, including more conventional ones (nested-PCR and quantitative Real-Time PCR, qPCR) and more innovative like digital PCR and next generation sequencing based ones [9,10]. Despite all of them have been demonstrated to be highly effective and overall reproducible and comparable [7-10], in low resource settings it is still debated whether to routinely test, due to costs, and which technique to prefer, due to technologies availability. In this study, we performed a phase 3 diagnostic accuracy study aiming to compare the two most conventional molecular techniques for MRD detection in FL, namely nested-PCR (used as test technique) and qPCR (used as golden standard) for BCL2/IGH detection. The two approaches were chosen as the only currently available in many referral centres even with limited resources.

Material and Methods

Twenty-two FL patients for which biological samples, complete clinical information, and long-term follow up were included. All patients were at diagnosis, and samples were taken before treatment initiation as well as after CHOP-R induction therapy, and after zevalin consolidation treatment at specific time-points (+3, +6, +12, +24, +30 months) [11]. Genomic DNA was extracted from mononuclear cells of peripheral blood (PB) and bone marrow aspirate (BM) as previously described [12]. The nested-PCR and the qPCR based on TaqMan technology [ABI PRISM 7900HT Fast Real-Time PCR System (Applied Biosystem)] were performed as previously reported [3,13,14]. As for BCL2/IGH PCR assays, primers were used according to previous Italian experiences [Ladetto 2001] (Tables 1-2). GAPDH was used as control gene for qPCR. Conversely, AF4 was chosen as control gene and was amplified according to BIOMED2 protocols for nested PCR [15]. All samples were tested by both techniques in triplicate.

Table 1: Primers sequences for nested PCR (BCL2/IGH).

Table 2: Primers sequences for nested PCR (BCL2/IGH).

Calculations of sensitivity (ST), specificity (SP), positive predictive value (PPV), negative predictive value (NPV), were made by CATmaker software (Centre for Evidence Based Medicine, Oxford University, http://www.cebm.net). The limit of significance for all analyses was defined as P<0.05. The study was approved by the local Ethical Committee and was developed and conducted in respect of the Helsinki Declaration. The study was designed and conducted according to the evidence-based medicine rules, respecting the STARD requirements.

Results and Discussion

All the enrolled patients could be studied for MRD. In total, 145 tests were performed. In fact, other than the expected 132 (22 cases by 6 timepoints), additional 13 were available from patients with longer clinical CR duration. Overall, we observed good concordance between “qualitative” nested-PCR and quantitative real-time PCR (80,86 %), in detecting MRD. The absolute sensitivity of the qPCR was in line with previously reported data [7]. Particularly, by evaluating serial dilutions of t(14;18)-positive cells into t(14;18)- negative cells, the relative sensitivity of our qPCR assay of about 10−5 resulted greater than the nested-PCR one (10-4), with an enhanced quantitative potential. This is overall in line with most studies. In terms of reproducibility, the precision of qPCR was determined by repeatability intra-assay and inter-assay; both the tests gave results of high reproducibility, above 95% considering 3 replicates. In contrast, the nested-PCR has given a lower reproducibility with discordant data and the need of additional repetitions to achieve a uniform result (three nested-PCR in mean). Overall, this is in line with previous works on qPCR. By contrast, nested PCR seemed to be “technically” more complicated and probably requiring more experienced personnel, to be consistently performed. This fact, further stress the need for adequate training and standardization processes when MRD is studied, in order to ensure the requested clinical consistency.
Consistency between the two was evaluated in terms of sensitivity and specificity. Overall, this analysis confirmed what observed in terms of reproducibility, i.e. a significantly higher efficacy of qPCR. Among 145 performed tests, 85 were concordant between the two techniques, while 59 were not (59% overall accuracy). Particularly, among 103 tests turned out to be negative by nested PCR, only 46 were instead positive by qPCR (45%). Conversely, among the 46 that resulted positive at nested PCR, only 13 were discordant and 33 consistent (72%). This was translated into remarkable specificity but low sensitivity of nested PCR (Figure 1).

Figure 1: Diagnostic accuracy analusis of qPCR vs Nested PCR (Catmaker, Oxford, UK).

Lastly, analysis of costs and practical feasibility in reduced laboratories was performed. The expenses for reagents, consumables and labor employed for the TaqMan assay was calculated about 34,00€ (4,443 KES) per sample when testing the maximum number of 5 samples in triplicate in 96 well-plates. Conversely, the analysis of 5 sample by nested-PCR has a total amount of 126,00€ (16,466 KES). This calculation was obviously optimized for running a complete TaqMan plate. By reducing the number of available samples, the cost would progressively increase. This implies that referral labs centralizing the activities are advised, particularly when resources are limited, also considering the highest initial investment for machinery. The shortest test duration of 3 hours and 14 minutes was found for the real time PCR while 18 hours and 30 minutes were needed to perform a complete nested- PCR analysis (including gene control PCR, the nested-PCR repeated for three times in mean, post PCR manipulation)

Conclusion
The present study, though based on a limited series, highlights the relevance of using a qPCR-based method to detect BCL2/ IGH rearrangements in FL patients in laboratories with limited resources. The use of TaqMan detection system was shown to be a sensitive, reproducible, and economical tool for MRD monitoring in FL. It allowed a relative sensitivity of about 10-5 providing a more accurate prognostic information [16]. Finally, the Taq Man approach in comparison with nested-PCR showed the simplest and shortest workflow sequence with a considerable gain of time and money, the average cost of 34€ per samples makes it feasible also in low resource Countries. Adequate programs of training and standardization should be then planned accordingly. 

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Anthrax Toxins and their receptors

Introduction
Under unfavorable growth conditions, B.anthracis undertake the developmental process of sporulation. B.anthracis spores are their infectious form, because contact with such spore forms under favorable conditions can lead to inhalation, skin and gastrointestinal infections [1]. For example, when spores enter the lungs, they are phagocytosed by macrophages and dendritic cells. However, some of them are able to spread throughout the body, despite the initial immune response. The spores that survive then transform into vegetative bacilli thanks to the formation of a polyɣ- D-glutamine capsule and the secretion of anthrax toxin proteins [2]. The anthrax toxin is composed of two binary combinations of three soluble proteins: 83 kDa protective antigen (PA83), 90 kDa lethal factor (LF), and 89 kDa edema factor (EF). PA forms complexes on the surface of host cells. It binds to one of two known anthrax toxin receptors, tumor endothelial marker-8 (TEM-8) or capillary morphogenesis protein-2 (CMG-2) [3]. Receptor-bound PA is proteolytically activated by a cell surface protease to generate a 63-kDa form (PA63) which oligomerizes, generating ring-shaped heptameric and octameric pore precursors [4].

These pre-channel oligomers are capable of binding up to three and four LF and/or EF molecules, respectively. The complexes are endocytosed and delivered to an acidic endosomal compartment. The PA oligomer transformed into a translocase channel, allows the transmembrane proton gradient to force lethal factor and edema factor translocation into the cytosol where they carry out their enzymatic functions, (Figure 1) [5]. Bacillus anthracis is a Grampositive, spore-forming, rod-shaped bacterium and is recognized by the presence of the pX01 and pX02 virulence plasmids, which give its unique ability to produce the anthrax toxin [6]. The plasmids pXO1 and pXO2 are very important for the virulence of B. anthracis. The pXO2 plasmid contains genes encoding the poly-D-ɣ-glutamic acid capsule, and the pXO1 plasmid contains genes encoding the toxin components: PA, EF, LF and virulence regulator anthrax toxin activator (AtxA) [7]. AtxA regulates genes encoding anthrax toxins and capsule synthesis [7]. AtxA includes the domains: two helixturn- helix (HTH), which is responsible for binding to DNA, and two phosphotransferase system (PTS) regulation domains (PRDs) and an EIIB-like domain [7]. Due to the presence of the PRDs domain in AtxA, its activity is regulated by phosphorylation strictly dependent on the presence of carbon dioxide [8,9]. These findings have important implications for developing research on the main role anthrax toxins as a major virulence factors at the initial stage of anthrax infection.

Figure 1: Schematic mechanisms of virulence B. anthracis by anthrax toxin and progression through the endocytic pathway. B. anthracis produces the three subunits of anthrax toxin: protective antigen (PA), lethal factor (LF) and edema factor (EF) encoded on plasmid pXO1 and poly-D-glutamic acid polimer capsule (CAP) ancoded on plasmid pXO2. AP-1 – activator protein 1; Cbl – E3 ubiquitin-protein ligase; CMG2 – capillary morphogenesis gene 2; E3 – ligase; EF – edema factor calmodulindependent adenylate cyclase; Fyn – tyrosine-protein kinase; LF – lethal toxin zinc metalloprotease; Src – Src-like kinase; TEM8 – tumor endothelial marker 8; VNTR – variable number tandem repeat.

Anthrax Toxin

Anthrax toxin is comprised of three nontoxic proteins that combine on eukaryotic host cell surface to form a toxic complex. A tripartite AB-type anthrax toxin is comprised of two catalytic A moieties: lethal factor (LF) and edema factor (EF) and a single receptor-binding B moiety, designated as protective antigen (PA). Lethal factor is a zinc-dependent metalloprotease that, together with protective antigen forms lethal toxin. It is a main virulence factor and the major cause of death for the Bacillus anthracis infected organism [10]. Lethal factor specifically cleaves the N-terminal end of mitogen-activated protein kinase kinases (MAPKKs) (Pellizzari et al. 1999). Because the N-terminal domain of MAPKKs is essential for the interaction between MAPKKs and mitogen-activated protein kinases (MAPKs), the cleavage of this domain impairs the activation of MAPKs [11]. Lethal factor cleavage of MAPKKs leads to the inhibition of three major signaling pathways-ERK1/2 (extracellular signal-regulated kinase), JNK/SAPK (c-Jun N-terminal kinase) and p38 kinases [12].

They are involved in diverse cellular processes including growth, apoptosis, innate and adaptive immune responses and several responses to various forms of cellular stress. According to lethal factor crystal structure, the enzyme comprises four different domains [13]. Domain I is responsible for protective antigen binding. The catalytic site and two zinc-binding motifs are found in C-terminal of domain IV [13]. Several studies have shown that histidine residues play an important role in the catalytic activities of lethal factor. Three histidine residues, His-35, His-42, His-229 are important for lethal factor binding to protective antigen but His- 686, His-690 and specially His-669 are essential for lethal factor catalytic activity [14,15]. Edema factor (EF) is composed of two functional domains, domain I (EFN, residues 1-291) and domain II (residues 292-798) [16]. The first domain (30 kDaN-terminal PA binding domain) interacts with protective antigen whereas the second (43 kDa AC domain and 17 kDa helical domain) interacts with adenylyl cyclase [17].

A series of biochemical studies has been revealed that EF also has two conserved aspartate residues, which coordinate two magnesium ions required for adenylyl cyclase activity [17]. EF is a calmodulin-dependent adenylyl cyclase that increases intracellular cAMP concentration of infected cells. cAMP is a secondary messenger with multiple downstream effectors, including protein kinase A (PKA) and protein activated by cAMP (EPAC). High levels of cAMP generated by ET activate PKA-induced transcriptional changes including modulation of cAMP-responsive element binding (CREB) protein [18]. Study on monocyte-derived cells suggests that CREB and glycogen synthase kinase 3 (GSK-3) are important for the ET-induced expression of anthrax toxin receptor 2 [19]. In addition, cAMP as a second messenger contributes to the regulation of leukocyte chemotaxis and endothelial barrier integrity [20,21]. The results reported by Nguyen, et al. [22] demonstrated that edema toxin (ET) impedes IL-8 driven movement of neutrophils across an endothelium independent of c AMP/PKA activity.

The stability and even the formation of the EF-calmodulin complex depends on the level of calcium bound to calmodulin (CaM) [23]. The binding of calmodulin to EF is a sequential process, first the N-terminal CaM is anchored to the helical domain and next C-terminal CaM region can insert between the catalytic core and helical domains of EF [24]. It leads to a conformational change of C-terminal region to stabilize the catalytic loop of EF for enzymatic activity. According to crystallographic studies residues Leu 667, Ser 668, Arg 671, Arg 672 and Val 694 are implicated in binding of calmodulin to EF [16,25,26]. Makiya, at al. [25] identified these amino acids residues as the binding epitope of EF-neutralizing mAb EF13D, which can neutralize EF in vitro in the subnanomolar range. Other labs have also reported small molecules which inhibit EF by different mechanisms but in the micromolar range [27,28]. Nanomolar affinities are often requested for an efficient competition, which explains that antibody concentration plays a role in toxin neutralization.

A variety of other types of EF inhibitors have been proposed. Especially, various purine and pyrimidine nucleotides with unique preference for the base cytosine were studied [29]. Edema factor and lethal factor forms toxic complexes with protective antigen, edema toxin (ET), which induces tissue swelling and lethal toxin (LT), which can alter cell function and may cause death [4]. The maintenance of homeostasis of the neural microenvironment is responsible for the blood-brain barrier. It is a regulatory interface between the peripheral circulation and the central nervous system (CNS) [30]. The endothelial barrier protects the brain from microorganisms and toxins circulating in the blood. Unfortunately, pathogenic microorganisms have evolved neuroinvasiveness mechanisms to penetrate host cell barriers. In vitro and in vivo studies from several laboratories suggest a principle role for ET in modulating brain endothelial integrity by disrupting the intercellular contacts and a role for LT in promoting penetration of the blood-brain barrier and development of meningitis [31-34].

Edema toxin has been shown to alter host defense like reduced activation of antigen-presenting cells, increased release of cytokines from dendritic cells, impaired chemotaxis and differentiation of T lymphocytes [35]. ET has also been shown to play an important role in the pathogenesis of anthrax-associated shock [36]. Infection with Bacillus anthracis can be cutaneous, gastrointestinal or pulmonary (inhalational). Frequently affected organs include secondary lymph nodes, lung, spleen, kidney, liver, intestinal serosa, heart, and brain proper [37]. Destruction of the organ function is due to the secretion of LT and ET. Some labs have reported that lethal toxin can disrupt endothelial barrier function [37]. The mechanisms causing endothelial dysfunction are stimulation of endothelial apoptosis, alteration of actin fibers and cadherins and mast cell activation [36,37]. Other lab has found that endothelial permeability is under tight control system where hypoxia activates signaling through the Rho-kinase-myosin light chain phosphatase pathway which leads to increased permeability [38].

However, hypoxia can activates p38 MAP kinase signaling leading to heat shock protein 27 (hsp27) phosphorylation which decreases endothelial permeability [39]. The majority studies indicate that anthrax lethal toxin induces the apoptosis of macrophages in an activated caspase-dependent way [40,41]. The cytotoxicity of lethal toxin is related to the activation of the transcription factor- NF-κB and TNF-α (tumor necrosis factoralpha) production in bovine macrophages [42]. It was shown that in bovine macrophages lethal toxin efficiently induces inhibitor-1- κB degradation and enhances the nuclear translocation of NF-κB. Neither protective antigen nor lethal factor alone had any impact of NF-κB activation. Lethal toxin induces apoptosis and necrosis in bone marrow derived macrophages and in activated human peripheral blood monocytes [41,43].

Interestingly, human alveolar macrophages demonstrate significant resistance to all the effects of lethal toxin, including inhibition of cytokine induction, lethal toxin-mediated MEK cleavage and lethal toxin-mediated apoptosis [44]. Lethal toxin, through its effect on the p38 pathway, disrupts glucocorticoid receptor signaling [45]. In vitro study has suggested that lethal toxin may depress murine cardiomyocytes function via an NADPH oxidase-mediated superoxide production mechanism [46]. Series of histological and microbiological studies concerning the effect of LT on intestinal tissues confirm LT-induced intestinal pathology, which is marked by villous blunting, mucosal erosions and ulceration [47-49]. Protective antigen is an 83 kDa pore-forming protein that binds to the anthrax receptors on the surface of the target cells and arrange entry of lethal toxin and edema toxin into the cytosol [50]. Native form PA consists of four domains with different functions: domain 1 - proteolytic activation by furin occurs in it; domain 2 - forms a transmembrane pore to translocate edema factor and lethal factor into the cell and contributes significantly to the receptor interaction; domain 3 - mediates in self-association of nicked form of PA83; domain 4 - primarily involved in binding to anthrax toxin receptor [50,51].

Upon binding to receptors, PA molecules undergo furin cleavage into 20 kDa fragment and 63 kDa subunits that remain cell surface bound. Furin is a critical housekeeping enzyme involved in protoxin activation [52]. Furin is essential for introducing the anthrax toxin into macrophages in highly pathogenic strains. The PA 63 kDa molecule creates a membrane channel that allows the entry of the LF toxin into the cytoplasm of the host cell [53]. It is extremely interesting that the site of cleavage of the PA protein of anthrax toxin has homology with the S1 site of the SARS-CoV2 virus, which is also affected by furin and the transmembrane protease serine 2 (TMPRSS2) [53]. Furthermore, both the anthrax toxin and the SARS-CoV2 virus infect macrophages and respiratory epithelial cells. In both infections, furin, the infected host’s protease, is the initiating enzyme. It is a factor that activates both the anthrax toxin and the SARS-CoV2 virus protein [54]. The characteristic protein sequences affected by furin are common among influenza, measles viruses, flaviviruses and the botulinum toxin [54]. They are the socalled initiation sequences, the presence of which among bacterial or viral strains increases their pathogenicity and virulence. As mentioned earlier, furin is a key host enzyme involved in the activation of protoxins and is therefore an interesting target for the search for its inhibitors. It is highly probable that the tropism and pathogenicity of bacterial strains increases as a result of the action of furin [54,55].

Anthrax Toxin Receptors

Two different cell surface receptors mediate anthrax toxin entry to the cells: ANTXR1, tumor endothelium marker 8 (TEM- 8) and ANTXR2, capillary morphogenesis protein 2 (CMG-2) [1,3]. TEM 8 and CMG 2 are type I membrane proteins containing the domain of von Willebrand factor A, which was originally identified in the blood serum protein as a platelet adhesion factor. ANTXR1 was previously discovered as a tumor endothelium marker, which is present at very low levels in healthy tissues and significantly increased in tumor tissues. ANTXR1 shares many similarities with integrins [56]. The ANTXR1 structural domains are similar to the β1 integrin domains and interact with type I and type VI collagen which also aid in cell migration and extracellular matrix reorganization [57]. On the other hand, the cytoplasmic part of the ANTXR1 receptor, directly anchored to the cytoskeleton of the actin cell, influences cell signal transmission, similar to integrins [58,59].

Cheng at al. for the first time investigated the mechanical signal transduction pathway initiated by the mechanical stimulation of the ANTXR1 receptor and its subsequent conversion to a biological signal in bone marrow stromal cells (BMSCc) [60]. The ANTXR1-initiated mechanotransduction involving the proteins LPR6 and LPR5 (low-density lipoprotein receptor-related protein) partially activates β-catenin to transfer a mechanical signal to the cell nucleus to regulate chondrogenesis [60]. Moreover, further experiments confirmed the interaction of ANTXR1 with actin and fascin actin-bundling protein 1 (FSCN1), which may also suggest the participation of anthrax receptors in the reorganization of the cell cytoskeleton [60]. Both CMG2 and TEM8 receptors have long cytoplasmic domains of 148 and 222 amino acid residues, respectively, like many other signaling receptors, and their physiological roles are related to cell migration and extracellular matrix remodeling [61,62].

Receptors can be post-translationally modified as a result of glycosylation, palmitoylation or ubiquitination [63,64]. Glycosylation affects protein folding in the ER, movement, and function. The TEM8 receptor has putative three glycosylation sites that are necessary for the movement of this protein from the ER and reaching the cell membrane [65]. It was verified that the TEM8 receptor lacking glycosylation did not bind the anthrax toxin in HeLa cells [65]. In contrast, the CMG2 receptor in the same cells, in the absence of glycosylation, could leave the ER and reach the cell membrane where it was able to bind ligand. Both receptors can be ubiquitinated by the action of the host ubiquitin ligase, leading to endocytosis of the clathrin-dependent toxin complex [63]. This process is even necessary for the intracellular activity of the anthrax toxin. S-palmitoylation involves the attachment of a 16-carbon fatty acid to a specific cysteine to form a thioester bond. In proteins, there may be a correlation between palmitoylation and ubiquitination within the same molecule. An example of such a phenomenon is the ubiquitination of the TEM8 receptor, if it has not been palmitoylated before, which leads to its destabilization and premature degradation [66].

The cytoplasmic domain of ANTXR1 and ANTXR2 are important in regulating half-life of the receptors at the plasma membrane [67]. The palmitoylation of cysteine residues increase the half-life of these proteins by preventing its premature clearance for the cell surface [63]. In the cytoplasmic domain, both receptors contain tyrosine residues phosphorylated following binding of protective antigen by receptor which is required for efficient toxin uptake [64]. There are three isoforms of ANTXR1. The ANTXR1-sv1, the longest isoform has 564 amino acids and the medium isoform ANTXR1-sv2 has 386 amino acids [68]; ANTXR1-sv3, the short isoform does not contain the transmembrane domain, so it cannot bind of PA and probably acts as secreted protein [69]. The studies of isoforms have demonstrated that the extracellular and transmembrane domains of these receptors are essential for PA binding, oligomer formation and translocation of anthrax toxin into the cytosol [69].

Toxin Entry into Cells

Toxin entry into host cells begins when protective antigen (PA83) binds to either of two cell surface receptors, ANTXR1 or ANTXR2. Following that PA83 is proteolytically activated by furinlike protease to create an active 63kD-form (PA63). Receptor - bound PA63 has the ability to oligomerize into heptameric or octameric rings, to form a pre-pore that can bind up to three molecules of either edema factor or lethal factor. The toxin-receptor complex is then internalized preferentially via clathrin-mediated endocytosis (Figure 1). This endocytosis appears to be protein depend such as clathrin, dynamin, heterotetrameric adaptor (AP-1) and actin [70]. ANTXR1 and ANTXR2 both could interact with lipoproteinreceptor- related proteins 5 (LRP5) and lipoprotein-receptorrelated proteins 6 (LRP6) [71]. Presumably, there are required for anthrax endocytosis. The large hetero oligomeric complex is then transported to early endosomes where it is incorporated into intraluminal vesicles [69].

The acidic pH of the early endosomes induces structural changes in the PA pre-pore leading to the pore formation as well as to the partial unfolding of edema factor and lethal factor [72]. The current study found that both proteins, EF and LF undergo major conformational changes during binding to PA [69]. These are then translocated through the PA pore across the endosomal membrane. It is understood that the anthrax toxin enzymatic subunits before being released into the cytosol must be transported to late endosomes in a microtubule dependent manner which is essential to protect them from lysosomal proteases. However, many of the details of this sophisticated delivery system remain to be elucidated.

Conclusion
Bacillus anthracis has two virulence factors, a poly-ɣ-Dglutamine capsule and bipartite toxins. The capsule of B. anthracis contributes to pathogenesis by blocking phagocytosis. The lethal toxin (LT) and edema toxin (ET) play a significant role in the pathogenesis of the disease. The study of the mechanisms by which these toxins modulate host defense has tremendously improved. The discovery of anthrax toxin receptors is a relevant for anthrax pathogenesis. Anthrax toxin receptors can regulate ligand-binding after conformational changes. A better understanding of anthrax pathogenesis may allow design of effective inhibitors. Future studies of anthrax toxins and their receptors promises to yield more information concerning toxin entry into the cells and therapeutic applications.

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The New and Effective Methods for Removing Sulfur Compounds from Liquid Fuels: Challenges Ahead- Advantages and Disadvantages

Introduction
Combustion of liquid fuels with organosulfur compounds such as sulfides, disulfides, thiophenes and the corresponding derivatives emits harmful gases SOx and NOx. HDS is main methods used for desulfurization, but this process is inefficient in removing organo sulfur compounds [1]. So recently, former techniques such as adsorption desulfurization (ADS) and oxidation desulfurization (ODS) were considered [2]. The main challenge of the ADS method is the selection of adsorbents with high adsorption capacity and selectivity [3]. Vafaee, et al. [4], synthesized nanosorbents of (A: Ni, CO & Mg) AFe2O4-SiO2 by an auto-combustion sol-gel method and used them in the ADS process. Also, Vafaee, et al. [5] used NiFe2O4- Polyethylene glycol catalyst for ultrasound assisted oxidative desulfurization (UAOD) process using central composite design (CCD) under response surface methodology (RSM). Consequently, ferrites in the adsorbent and phase transfer catalyst were easily separated and recycled via magnetic field for desulfurization process.

Conclusion
In this study, efficiency of ADS and UAOD methods with the AFe2O4-SiO2 (A: Ni, Co & Mg) nanoadsorbent and NiFe2O4-PEG phase transfer nanocatalysts were reviewed. In the UAOD process, increasing the temperature and oxidant amount had the greatest effect on increasing the percentage of DBT conversion. In addition, one of the main challenges of ADS and UAOD methods is the use of adsorbents and phase transfer catalysts with easy separation and recovery capabilities. Therefore, using the magnetic field caused by ferrites in the adsorbent and phase transfer catalyst structure, they were easily separated and recycled after desulfurization.

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Vitamin D Supplementation for the Treatment of Depression in Females in a Private Practice Clinic

Introduction
During the last decade there is a strong interest regarding the effect that vitamin D plasma levels can have in depression [1]. There are also studies that suggest the use of vitamin D supplementation either by mouth or through light therapy as an add-on therapy for depression [2]. Influenced by this evidence, during the winter season of 2019, we used Vitamin D3 supplementation mainly as an add-on therapy for the treatment of patients suffering from depression in our private practice. In order to better assess the results of this intervention and also to communicate our experience to other practitioners, we concluded a small case series study with all our depressed patients that received vitamin d supplementation, during a certain time frame.

Material and Methods

Subjects

During autom of 2018 and witner 2018-2019 Every patient that was treated for depression that was presented with residual depressive symptoms was assessed for Vitamin 25(OH) blood levels. Subjects with Vitamin D blood levels below 30ngr/ml where assessed for this study. In total ten patients were assessed. Out of them eight where included in the study since two of the patients were suffering from psychosis and not depression and were excluded. All patients were Caucasian women with a mean age of 54,29 (S.D 16,75). (Table 1)

Table 1: Various numerical parameters of the study, initial with final vitamin d levels were compared with paired T test and had a statistically significant difference with p= 0,014.

Method

All subjects were prescribed with Vitamin D3 oral supplementation oral dose ranging between 2000 and 5000 UI of Vitamin D3 per day was administered. One of them was treated with Vitamin D3 as monotherapy while in the rest; Vitamin D3 was used as an add-on therapy. Levels of Vitamin 25(OH) were assessed again within two months’ time frame. Patients with major changes in their treatment such as addition of another antidepressant were to be excluded from this study but in our sample nothing like this occurred during the time frame of this study. Qualitive analysis was used on the psychiatric records that were kept in our private practice in order to assess the symptoms that are more likely correlated with low vitamin D levels. Qualitive analysis was also used in the follow up assessment of the patient in order to detect the symptoms that might have responded to vitamin D supplementation. Final assessment took place three months after the first assessment for each individual. Paired T test was used to compare changes in Vitamin 25(OH) blood levels. SPSS for windows in version 15.0 was used for this comparison.

Results

All assessed patients had some type of Vitamin D deficiency mean level of Vitamin D(OH) was 12,04 ngr/ml (S.D. 7,995) with levels ranging from 4,5ngr/ml to 26,5ngr/ml. Seven out of eight having vitamin d blood levels below 20ngr/ml. The mean dose of Vitamin D(OH) supplementation was 4142,88units/per day (S.D 1399,75), with most of them taking a dose of 4000 units/per day. Final assessment of Vitamin D (OH) levels took place within two months period, mean time of 52,67 days (S.D. 30,651) while it was still in winter. There was a significant increase of Vitamin D (OH) blood levels 25,65ngr/ml (S.D. 11,559) p=, 014 (Table 1). Qualitive analysis showed that the main complain that all patients had in common was psychomotor retardation less common but significant was also morning depression. These symptoms and especially psychomotor retardation tend to improve in various degrees two months or more after the beginning of Vitamin D supplementation (Figure 1).

Figure 1:

Discussion

This is a prospective case series; its results are hopeful. It seems that there is a strong possibility that depressed female patients with a certain residual symptom profile might also suffer from vitamin d deficiency. More specifically symptoms such as psychomotor retardation or morning depression seem to be more correlated with vitamin d deficiency [3]. This study was conducted in total in winter time. This happened since we wanted to reduce confounders such as sun exposure, which is much more likely to happen during summer, and can change the levels of vitamin d in blood regardless of our supplementation [4]. It is important to understand though that in this case psychiatrists should change their treatment culture. While in almost all their care tend to treat almost completely without biological markers, vitamin d supplementation for treatment of depression requires a different approach. In our study first we check and if there was a vitamin d deficiency and then we prescribed supplementation of vitamin D [5]. Vitamin D3 was prescribed since it seems to be a better alternative in comparison with other vitamin D supplements such as Vitamin D2 [6].
A significant improvement in depressive symptoms, that was correlated time wise at least with the increased of Vitamin D blood levels, was observed. This is in accordance with patient’s satisfactions, which do not consider Vitamin D as another ‘Psychiatric drug’. Caution should be placed thought to the regular follow up of Vitamin D levels since high above normal Vitamin D levels can also be toxic [7]. So if Vitamin D levels are raised above limits Vitamin D supplementation should be stopped. Furthermore, during summer period Vitamin D levels are raised since our body composes it to higher degree due to increased sun light exposure, thus vitamin D levels must be more thoroughly checked during summer time. This brings us to another point. The aim of supplementation is the increase of vitamin D levels in the body. If we can achieve that with other means except prescribing a supplement such as sun exposure it is also good practice to try. The knowledge of the effect that vitamin d can have in the mood and the benefits of sun exposure related to it, might motivate a significant proportion of depressed patients to increase their outdoors activities [8].
This study has significant limitations. Firstly, it is a small study. There are very few patients included. The researchers present this as a case series and not a cohort or other more powerful type of study. Furthermore, the fact that all patient were Caucasian women increases somehow the power of this study. The other limitation is the fact that no standardized assessment of patients initial status or treatment progress, with the use of questionnaires occurred. This makes more difficult to interpret study’s findings. To our view since the aim of our approach was to treat residuals symptoms it was difficult for these symptoms to be detected through formal questionnaires that asses overall depression, furthermore the observation and consensus of two specialized psychiatrists has its value when we asses depression and gives some addition credibility to the results. Also, qualitive analysis that we used is an acceptable measure of outcome [9].

Conclusion
Although results are quite preliminary, there is a strong feeling, that Vitamin D supplementation is effective in treating certain depressive symptoms. Of course, much further study is needed for any firm conclusions.

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The Diagnostic Value of Galactomannan Testing in Bronchoalveolar Lavage Fluid on the Diagnosis of Pulmonary Aspergillosis in Patients with Chronic Respiratory Diseases

Introduction
According to the definition of chronic respiratory diseases (CRD) by the World Health Organization, CRD is a group of diseases that affect the airways and other structures of the lungs, the most common include COPD, bronchial asthma, bronchiectasis, etc. [1]. In-depth studies in recent years have found that pulmonary aspergillosis can also occur in patients with chronic respiratory diseases (CRD) [2,3]. As delayed treatment of pulmonary aspergillosis always leads to high mortality rate, early recognition of CRD with pulmonary aspergillosis is extremely important. Galactomannan (GM) is a thermally stable polysaccharide on the cell wall of aspergillus filaments, which is released into the blood from the tip of the mycelium during aspergillus growth [2]. GM can be detected in the blood in the early stages of infection. Nevertheless, various factors have been found in clinical practice to cause false positives and false negatives in galactomannan testing. Bronchoalveolar lavage fluid (BALF) can be applied to detect pathogens on lung lesions in the early stage of aspergillus infection.

Although BALF has been recommended for GM testing by domestic and foreign guidelines, there is no unified standard for BALF-GM testing cut-off value [2,3]. In this study, bronchoalveolar lavage fluid (BALF) was collected from 100 patients with suspected clinical pulmonary Aspergillus infections by means of bronchoscopy. BALF GM test and serum GM test were compared to assess the diagnostic value of galactomannan testing in bronchoalveolar lavage fluid on the diagnosis of pulmonary aspergillosis in patients with chronic respiratory diseases.

Patients and Methods

Patient Selection

Between June 2019 and December 2019,100 patients with suspected clinical pulmonary aspergillus infections from three different hospitals (50 patients from the Guangzhou Thoracic Hospital,45 patients from the Guangdong Province People’s Hospital, and 5 patients from the First Affiliated Hospital of Sun Yat-Sen University, respectively) were enrolled in this retrospective analysis. They all suffered from chronic respiratory diseases include COPD, bronchial asthma, bronchiectasis, etc. Data of all patients were collected, including age, sex, smoking history, past medical history and medication history, length of stay, laboratory tests, chest imaging examination, pathogen examination, lung pathology and bronchoscopy results. Serum and BALF GM tests were performed during their hospitalization. Factors that might cause false positives in the GM test such as piperacillin/tazobactam were excluded. Hematological malignancies, hematopoietic stem cell transplantation, solid organ transplantation, HIV infection, and patients with incomplete clinical data were excluded from the study.

Statistical Analysis

a. Koimogorov-smirnov test (K-S test) was used by SPSS 25 software to determine whether the target variables were normally distributed. If the measurement data conformed to normal distribution at the same time, it was represented by mean ± standard deviation (`X±s). For the measurement data that did not conform to normal distribution, it was represented by M(P25-P75). The counting data was expressed by percentage or constituent ratio. Independent sample T test was used for BALF GM values of the case group and the control group, paired sample T test was used for BALF GM values and serum GM values of the case group, non-parametric rank-sum test and Mann-Whitney U test were used for samples that did not conform to normal distribution.
b. SPSS 25 software was used to draw ROC curves of the diagnostic efficacy of BALF and serum GM test in the case group and the control group, and the optimal cut-off value of BALF and serum GM test for pulmonary aspergillosis was calculated.
c. The data of baseline features, clinical features and imaging examination of the subjects were analyzed with the independent sample T test or chi-square test for normal distribution, and non-parametric rank-sum test for nonnormal distribution. The differences were considered to be statistically significant when p<0.05.
d. According to several guidelines, the cut-off value of GM was between 0.5 and 1.5, and the cut-off value of 0.5, 0.8, 0.9, 1.0, 1.2 and 1.5 have been reported in many guidelines and metaanalyses. Sen, spe, positive predictive value (PPV), and negative predictive value (NPV) of BALF GM were calculated.

Results

Patient characteristics and data.4 patients with incomplete data and follow-up loss were excluded, and a total of 96 patients were included in this study. According to the diagnosis standards of IDSA (2016) [2], 43 patients were diagnosed by pathological data (proven diagnosis), and 3 cases were diagnosed by radiology, etiology, and other clinical examinations (probable diagnosis). Both of them were included in the case group. The control group included 6 cases of possible pulmonary aspergillosis and 44 cases of nonpulmonary aspergillosis. Clinical data of patients were collected (Table 1). The most common clinical symptoms in the case group were cough (41cases,89.1%), hemoptysis (30 cases,65.2%) and expectoration (27 cases,58.7%). Whereas in the control group were cough (36 cases,72.0%), expectoration (27 cases,54.0%) and fever (16 cases,32.0%). The clinical symptoms of hemoptysis and cough were statistically different between the two groups.
The imaging findings of patients in the two groups included nodular shadow, patchy shadow, consolidation shadow, air crescent sign, cavity and aspergillus balls. Nodular shadow (27 cases,58.7%) and cavity (22 cases,47.8%) were dominant in the case group, while patchy shadow (14 cases,28.0%) and nodular shadow (13 cases,26.0%) were dominant in the control group. The imaging manifestations of nodular shadow, cavity and aspergillus bulb were statistically different between the two groups. Microbiological examination results. In the case group there were 21 cases (45.7%) of positive aspergillus in BALF culture and 3 cases (6.50%) of positive aspergillus in BALF smear microscopy. The serum GM value was 0.18(0.12-0.34) in the case group and 0.12(0.07-0.21) in the control group, showing no statistical difference. BALF GM value was 1.93(0.61-5.78) in the case group and 0.51(0.25-0.82) in the control group, Z value =-4.709. BALF GM value in the case group was higher than that in the control group, P<0.05 (Table 2).

Table 1: Baseline characteristics of patients in case group and control group(%).

Note: *P value < 0.05, the difference was statistically significant

Table 2: Comparison of microbiological examination results between the case group and the control group.

Note: *P value < 0.05, the difference was statistically significant

Diagnostic effificacy of the BALF GM test. When the GM cutoff value was 0.5,0.8,0.9,1.0,1.2,1.5, the sensitivity of BALF GM test decreased with the increase of GM cut-off value, and the specificity increased with the increase of GM cut-off value. When the diagnostic threshold of serum GM test was 0.5 and 1.0, the sensitivity decreased with the increase of the threshold, but the specificity did not change. BALF GM test had higher sensitivity but lower specificity than serum GM test (Table 3). The area under ROC curve of BALF-GM was 0.779(95%CI: 0.684-0.874),standard error was 0.0487,Z value was 5.727,P =0.001, Youden index was 0.4939,when thre hold>0.96,the sensitivity and specificity were 67.4%,82.0% respectively (Figure 1, Table 4). The area under ROC curve of serum-GM was 0.638(95%CI: 0.439-0.807), standard error was 0.121, Z value was 1.147, P=0.255, Youden index was 0.3116, when threshold> 0.18, Sen was 47.8%, Spe was 83.3%; When serum-GM threshold ≥0.18, AUROC was the highest, for which the sensitivity and specificity were 45.5%,83.3% respectively (Figure 2, Table 4).

Figure 1: ROC curves of BALF-GM test in two groups.

Figure 2: ROC curves of serum-GM test in two groups.

Table 3: Diagnostic value of different GM test limits for BALF GM test.

Table 4: Comparison of ROC curve analysis parameters between BALF-GM test and serum GM test.

Discussion

Structural lung disease is a major cause of pulmonary aspergillosis, including bronchiectasis, PTB, bronchial asthma, COPD, etc. Long-term and chronic diseases lead to the destruction of the normal anatomical and physiological structure of the lungs, the destruction of the mucosal barrier of respiratory epithelial cells, and increase the ability of aspergillosis to adhere to airway epithelium. In addition, cilium lodging and degeneration of airway epithelium and obstruction of clearance of respiratory secretion increase the chance of aspergillus infection [4,5]. This study also confirmed that patients with pulmonary aspergillosis had more chronic respiratory diseases in the case group than in the control group (bronchiectasis 58.7% vs.12.0%, P=0.001),PTB (82.6 vs.20.0%,P=0.001),COPD (43.5% vs.22.0%,P=0.025),which was consistent with the results reported in literature [6].The early clinical manifestations of pulmonary aspergillosis are not specific, and the typical chest CT findings are often related to the time of disease occurrence and the severity of lesion development, and the imaging findings cannot lead to a definite etiological diagnosis.
Traditional methods such as smear microscopy and fungal culture have long cycle, low positive rate and are susceptible to environmental pollution. Therefore,a variety of auxiliary examination methods are used to achieve the purpose of early diagnosis. Galactomannan (GM) is a specific polysaccharide of aspergillus cell wall. At present, GM can be detected clinically by blood, BALF, pleural effusion, cerebrospinal fluid and lung tissue, and it is one of the common antigens for the diagnosis of aspergillosis. A large number of existing studies have proved that the sen, spe, ppv and diagnostic coincidence rates of BALF were higher than those of serum GM. The results of this study showed that the cutoff values of BALF GM test were all higher than serum GM, which was consistent with the results of previous studies. The uniform diagnostic threshold of BALF GM has been disputed at home and abroad. The IDSA 2016 guidelines again recommended BALF GM and serum GM tests as laboratory tests for pulmonary aspergillosis. However, they did not specify a BALF GM value, but the diagnostic threshold of serum GM test was ≥0.5[2]. In 2019, EORTC/MSGERC scholars updated the definition of IFD, which clearly indicated for the first time that the clinical diagnostic threshold of BALF GM as pulmonary aspergillosis was: serum GM≥1.0,2 BALF GM≥1.0; or a single serum GM≥0.7+a single BALF GM≥0.8 [7,8].
In this study, through ROC curve analysis, the AUROC of BALF GM test was 0.779(95%CI:0.684-0.874).When BALF GM test limit> 0.96,Sen was 67.4%,Spe was 78.0%,PPV was 73.8%, NPV was 72.2%, PLR was 3.06.When serum GM limit was greater than 0.18,AUROC was the highest, Sen was 45.5%,Spe was 83.3%,and P=0.255.The purpose of this study was to understand the value of BALF GM in the early diagnosis of pulmonary aspergillosis in patients with nonneutropenia complicated with pulmonary underlying diseases. Our results showed that the sensitivity of serum GM test was lower than BALF GM test regardless of setting GM≥0.5, ≥0.8, or ≥1.0 as the diagnostic threshold of BALF GM. When GM threshold was≥0.5, Sen,Spe,PPV of BALF GM were 80.43%,48.0%,58.73% respectively. When the BALF-GM threshold was increased to ≥1.0, the PPV was significantly increased. Compared with previous studies [9,10], BALF GM values of patients with chronic respiratory diseases were different from those of patients with traditional diseases such as neutropenia, hematological malignancies, parenchymal organ transplantation, hematopoietic stem cell transplantation, and immunosuppressant use.
At present, some scholars have proposed that different optimal diagnostic boundaries should be set for patients with different underlying diseases and different immune states, such as neutropenia and non-neutropenia [10], organ transplantation (including hematopoietic stem cell transplantation) and non-solid organ transplantation [11,12], hematological malignancies [13,14], etc. Similarly, the interpretation of BALF GM test results should also be based on the full assessment of the underlying diseases and immune status of patients to determine the optimal BALF GM diagnostic threshold for various patients, so as to improve the diagnostic efficacy of BALF GM in the diagnosis of pulmonary aspergillosis in different populations. Research and clinical practice at home and abroad have found that many factors affecting GM tests cause false positives and false negatives in GM tests, which often confuses clinical work and even leads to misdiagnosis, missed diagnosis and excessive antifungal treatment.
In this study, it was found that BALF GM value was higher in some patients without aspergillus infection in the control group, while BALF GM value was lower in a small number of patients with aspergillus infection in the case group, resulting in false negative in addition to sample dilution during BALF collection, which might also be related to the use of antifungal drugs. A recent review suggested that false negatives in GM tests were associated with the use of antifungal active agents and myxolytic agents [15]. Using beta lactam classes of antibiotics (especially piperacillin/ he azole temple, amoxicillin/clavulanic acid potassium, etc.), intravenous use of parenteral nutrition, blood product containers containing glucose acid, severe gastrointestinal mucous membrane inflammation, multiple myeloma will lead to GM false positive [15]. Clinical cases have also reported that contamination of sterile containers could lead to false positives of GM [16]. According to previous studies and the results of this study, the early diagnosis of pulmonary aspergillosis requires combining imaging examination, histopathology, smear microscopy, fungal culture, aspergillosis antigen detection, aspergillosis antibody detection, and molecular biological examination.

Conclusion
In this study, BALF GM test is more valuable than serum GM test for diagnosis. BALF GM test is more significant for the diagnosis of pulmonary aspergillosis. The best limit, sensitivity and specificity of BALF GM test are 0.96,67.4% and 82.0%(P=0.01). The optimal threshold of BALF GM may vary with host-based diseases and even with different species of Aspergillus. BALF GM value of pulmonary aspergillosis under different immune states needs more clinical data. At the same time, when serum GM and BALF GM are used in clinical practice, it is necessary to fully understand and identify the false positive and false negative of GM, and to diagnose pulmonary aspergillosis by integrating patient factors, clinical manifestations, imaging examination and pathogenic microbial examination.

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