The Effects of Sustain Loads on the EMG Activity of the Leg Muscles During Soldier’s Quasi-Static Posture Control

Introduction

During military activity, professionals are sometimes required of their physical capacity to carry out cargo transportation. This activity is performed using basic equipment for the military to last in action, such as: the combat backpack, in which the individual will carry the items needed for personal survival, and his or her weaponry that will pro-vide you with security and protection. The high military physical effort, the specific tasks performed and the great weight transported, the military are subject to a higher risk of suffering some type of injury. The increase of this transported weight reflects in the increase of the tension of the muscles of the lower limbs, which are strictly related to the injuries [1-4]. In the load carriage using packs, the military is subjected to a natural imbalance, at this point he will seek postural control to keep himself balanced. this capacity is defined as the property for maintaining an upright posture, determined by the movement of the human body’s pressure center [5]. The most important thing in this activity is the location that the individual will carry such a load in his body. It is precisely in this decision that will reflect how the muscles of the lower limbs will behave to maintain the balance already mentioned above. The way of load carriage using backpacks will also bring reflexes to the military, with regard to biomechanics and human physiology. In this case, showing that distributing the weight in the anterior and posterior part of the body, such as a double backpack, will re-quire less energy from the military; promoting lower inclination of the body, but limiting the movement of the arms and increasing body temperature [3,6].

Another analysis, the high load distribution also alters the ground reaction force, promoting the balance of the body. In its conclusion, it showed the progressive increase of this force in relation to a load of 60% of body weight and with a focus on transportation; also reporting on military weapons [7]. With all this, in our study, to measure this orthostatic equilibrium in the face of a load variation, we used surface electromyography (EMG). This choice was made taking into account that this exam will provide the motor units activated during the individual’s overload and the discharge rate produced, showing the muscular behavior [8-10]. In this part of the literature, little has been said about the effect of the support of the military backpack and the armament on the orthostatic position [11,12], analyzed by the electromyographic behavior. This gap needs to be deepened, since it is the basic military activity, it will soon bring a benefit to the Brazilian Army. Generating adaptations, or suggestions, to improve the postural control of our military. All with the purpose of reducing injuries already men-tioned above; thus, improving the combative capacity of the terrestrial force. Therefore, the aim of this study was to evaluate the effects of backpack support and military weaponry on the electromyographic behavior of the leg muscles in maintaining standing position.

Materials and Methods

It is an experimental, applied study, cross-sectional and quantitative data. The participants were composed of experienced service members and were submitted to three conditions: The first, supporting only the combat backpack (CTRL), the second condition sup-porting the combat backpack with the machine gun simulator (MAG) and the third condition supporting the combat backpack with the rifle (FZL). These moments were compared based on the electromyographic signal variables of gastrocnemius lateralis (GNL) and tibialis anterior (TBA) muscles.

Subjects

The sample was of the non-probabilistic type of voluntary character and consisted of 16 participants (male sex; age: 27.5±4.9 years; total body mass: 77.2±9.3 kg; height: 176.8±5.1 cm), experienced service members (> 6 years of service and experience in cargo support). All participants were students in the instructor course of the Brazilian Army Physical Education School. The study protocol was approved by the Ethical Committee of the Salgado de Oliveria University (file: CAEE 48000321.3.0000.9433). All participants were fully informed about the content of the study and gave their written consent.

Equipment’s and Instruments

In the reference condition, the participants wore t-shirts, shorts, boots, socks, and a military backpack. Afterward, all subjects added a rifle and a machine gun according to the test. The individual combat equipment that was used by the 16 volunteers was composed of: 01 (one) large capacity Alice campaign backpack with two liter pet bottles with sand, totaling the weight of 15kg, 01 (one) Mauser carbine model 1935 with two shin guards, one of 3kg and another of 4 kg, weighing on average 10.8kg (simulating the weight of the MAG machine gun), together with a bandolier to assist in weight control), 01 (one) Model 1935 Mauser carbine with a 1kg shin and an extra weight of 0.5kg, weighing on average 4.8kg (simulating the weight of the FAL 7.62 rifle), along with a bandolier to assist in weight control) and 01 (one) personal boot, shirt and shorts.

Procedures

Data were collected from June to September of 2021 in the Biosciences laboratory of the Brazilian Army Physical Education School. The volunteers were scheduled to only collect 4 individuals per day. At first, the ICF and anamnesis were completed. After completing the mandatory documents, each military member had his or her stature (EST) and total body mass (MCT), measured using the military physical training uniform. It was then asked that the military put the boots, for a new conference of MCT and EST. They were then instructed on the procedures to be carried out. On the days of collection the MCT was again measured, but in the control conditions with backpack (MCT_CTRL), with backpack and rifle (MCT_FZL) and with backpack and machine gun (MCT_MAG). For the acquisition of biological signals (surface electromyography - sEMG), wireless surface electrodes (Trigno Wireless System, Delsys Inc., USA) were used, amplified by a signal acquisition module ((Delsysinc., USA, 2.4GHz transmission frequency, 1kHz sam-pling frequency, common rejection mode >80dB, 10Hz high pass filter and 450Hz low pass, total gain 1000 times). The electrodes were positioned on both sides of the lower limbs in the anterior tibialis (TBA) muscle, one-third to one-fourth of the distance from the knee to the ankle, in the largest palpable muscle mass, we palpated the area while the individual performed the dorsi-flexion of the foot. In the gastrocnemius lateralis muscle (GNL), the electrodes were positioned approximately two centimeters laterally in relation to the midline of the gastrocnemius muscle [9,12,13].

Myoelectric activity recorded from: gastrocnemius lateralis (GNL) and anterior tibialis (TBA) normalized for the maximum amplitude of myoelectric activity obtained during the maximum isometric voluntary contraction test (MIVC). The CIVM was performed with the individual seated on a stretcher with the trunk at 80º flexion in relation to the hip and with the knee positioned at 90º flexion and suspend-ed on the side of the table. After positioning, the participants performed the dorsi-flexion and plantar flexion against resistance imposed by the evaluator. This resistance was maintained for 5 seconds for each movement in both lower segments. After that, they climbed onto the power platform and looked for the upright and static posture, staring at a target on the wall in front of them, at a distance of 3 meters. The positioning of the feet, on the platform, was standardized, using a plastic wedge with an angle of 30º, making the heels stick together and the tips of the feet distant. Each participant was submitted to three conditions of estabilometric evaluation, being sustaining the combat backpack, sustaining the combat backpack and the Mauser carbine and sustaining the combat backpack and the MAG machine gun, in each of these positions were made 3 collections. Each measurement lasted 90 seconds, with 30 seconds (15 initial seconds and 15 final seconds) being discarded where sEMG analyses occurred in 60 seconds. The sEMG signals of the muscles and the platform of force were sincronized by means of an accelerometer positioned in the dorsal region of the boot (Table 1).

Table 1: Descriptive statistics with mean, standard deviation (SD), minimum (Min.) and maximum (Max.) of the total body mass (kg) in the conditions with backpack, backpack and rifle and with backpack and machine gun.

Note:

1Significant difference between MCT_CTRL and MCT_MAG condition.

2Significant differences between MCT_CTRL and MCT_FZL condition.

The signals were analyzed in specific software in which the test parameters consisted of continuous collection and thus initially stored in files on the computer hard drive for the processing of digital signals in MATLAB environment (R2015a) version 8.5.0 (The Math works Inc Natick, Massachusetts, USA) that provided root Mean square (RMS) and full-time analysis (iEMG) related time domain data. The RMS values are summed on each side according to the analyzed muscle. This sum generated a unique value for the TBA and GNL muscle in the respective conditions (CTRL, FZL and MAG). The same calculation was made for iEMG. After these values were normalized by the peak of the RMS and iEMG of the MIVC of each muscle.

Statistical Analysis

All data were stored and analyzed using the statistical program Statistical Package for the Social Sciences for Windows (SPSS) version 20.0 (SPSS Inc. Chicago, Illinois, USA). Shapiro Wilk’s normality test rejected the hypothesis of equality of EMG variables for different load conditions, and Friedman’s two-way test was performed to analyze the variance of related samples. All significance values (p value) were determined as <0.05. Descriptive statistics (mean, standard deviation, maximum and minimum) were calculated for each data set if presented in graphic form using Graphpad Prism software version 8.0.1 (Graphpad Software Inc. San Diego, California, USA).

Results

The total body mass (MCT) obtained initially was that of the control condition (93.56±9.16 kg). Then we obtained the MCT of the FZL condition, with the military carrying the simulacrum of the Rifle (98.36±9.16 kg), in which already presented a statistically significant difference between the CTRL condition (p=0.014). Finally, the MCT of the MAG condition, with the military carrying the MAG machine gun weight (104.36±9.16 kg), finally, this last condition was demonstrated with a statistically significant difference, both from the CTRL condition (p=0.0001), and from the FZL condition (p=0.014).When performing the maximum voluntary isometric contraction (CIVM), we obtained the measure adopted as 100% of the RMS (1.0 in u.a.), represented in the ordinal axis of Figures 1 & 2. When performing the sEMG analysis in the time domain, In Figure 1, we used the square root of the mean signal obtained (RMS) to quantify our muscle activation in relation to the maximum military activation, all of the two muscles under analysis, anterior tibialis (TBA) and gastrocnemiuslateralis (LNG). With this, we found that the CTRL condition (TBA: 0,204 [0,062 - 0,366]; LNG: 0,209 [0,067 - 0,371]) did not present a significant difference. The RMS values in the FZL condition (TBA: 0,195 [0,008 - 0,381]; LNG: 0,204 [0,013 - 0,385]) and in the GAM condition (TBA: 0,221 [0,072 - 0,293]; LNG: 0,225 [0,077 - 0,298]) were not statistically significant differences. However, there is a low-er maximum variation of this muscle activation in the latter condition. Another form of sEMG quantification is the integral of the entire area, in the frequency spectrum filled by the signal [7]. In this condition of Figure 2, we obtained for CTRL condition the result of 0.225 (0.064 - 0.368) for TBA and 0.231 (0.069 - 0.373) for GNL, both muscles without significant differences. For FZL condition, the results were 0.213 (0.012 - 0.382) for TBA and 0.218 (0.015 - 0.387) for LNG, also without significant differences for both muscles. Finally, in the GAM condition, the results were 0.219 (0.074 - 0.295) for TBA and 0.224 (0.079 - 0.300) for GNL, as well as the previous ones without significant difference. Results similar to Figure 1 with the treatment of MRH, corroborating the reliability of the data obtained in the time domain.

Figure 1: Root mean square (RMS) of the electromyographic signal in arbitrary unit (u.a.) comparing the conditions with backpack (CTRL), backpack and rifle (FZL) and backpack and machine gun (MAG) of the anterior tibialis (TBA) and gastrocnemius lateralis (GNL) muscles.

Figure 2: Electromyographic signal integral (iEMG) in arbitrary unit (u.a.) comparing the conditions with backpack (C-TRL), backpack and rifle (FZL) and backpack and machine gun (GAM) of the anterior tibialis (TBA) and gastrocnemius lateralis (GNL) muscles.

Discussion

Military activity requires the transportation of heavy cargo, whether backpack, equipment, or weaponry, for extended periods of time. We have shown in our study that the addition of the rifle and the MAG machine gun were statistically significant for the difference in total body mass (MCT), this is a potential risk factor for the occurrence of lesions in the locomotor system [14]. However much this difference in weight, when we add the MAG and compare it with the CTRL situation, it is significant, it is an increase of less than 20% of the MCT. The backpacks with less than 20% of body weight were not sufficient to activate the lower limbs muscles in the static position, noting a significant difference only in the rectus abdominis [15-17]. With the results of our sEMG, we observed that there was no statistically significant difference when comparing the three situations tested. All of them had about 20% of the muscular activation when compared to the total maximum voluntary isometric contraction. Several may be the reasons for this event, first we performed the measurement in the static position, while Simpson, et al. [18] collected these variables in displacement and found an increase in gastrocnemius lateralis activity, suggesting that a possible collection in displacement be more reliable the real employment situation of military troops [19].

In our research we analyzed the tibialis anterior and gastrocnemius lateralis muscles, in both results they were similar and did not present significant statistical differences. Corroborating with Lindner, et al. [1], in his study, found that these muscles, already mentioned above, also did not demonstrate significant muscle activation when transporting military equipment, however, in this same research, the greatest increase in electromyographic activity was in the rectus femoris muscle after adding the backpack. Birrel, et al. [7] presented in their research that there are statistically significant differences in the ground reaction force when walking with a rifle, but when dealing with muscle activation measured by sEMG. We are talking about the recruitment of muscle fibers to perform a certain activity, that is, the greater the difficulty of the task required, the greater the recruitment of muscle fibers. In our research, when we added the rifle or the MAG machine gun, the activation was similar to the CTRL moment, which would be the individual with only the backpack, it is worth noting that both the rifle and the MAG were inserted with the bandolier wrapping behind the neck with the armament resting in front of the body which suggests a greater demand of the upper back muscles due to the location closest to which weight was supported, thus requiring little muscle recruitment of the lower extremities, the previous statement is confirmed by the study by Lindner, et al. [1] which showed that the weight of the rifle showed no significant difference in muscle activation in the lower limbs, when the weight of the armament is carried by the upper limbs. Confirming our thesis Thuresson, et al. [18] showed that the weight of a helmet placed on the head did not reflect on the lower limbs, but on the muscles of the neck and upper back due to its proximity to the place of weight inserted.

Contributing to the findings of our research, Majumdar, et al. [20] in his study on the transport of loads in military personnel, found that the body adopts some postural changes to decrease muscle overload, but that if this load is carried is low, between 6.5% and 27.2% of body weight will not cause orthostatic changes, confirming our results, since the load we added in the military was well below 25% of the MCT. Our sample was composed of experienced service members, all with at least seven years of military service, with good physical fitness, being a very restricted and specific sample. Therefore, also attributed this factor to one of the causes of low muscle activation and concluded that experienced service members supported larger loads due to better training, physical condition and greater strength, supporting loads between 47% and 64% of your body weight while maintaining a normal gait pattern [21,22]. The present study has limitations such as: the small sample collected; the evaluation only in the static position; the weight of the additional transported load is relatively low; the collection time is small. The study did not evaluate other possible factors that contributed to the low electromyographic activity found in the research, such as: muscle strength of the lower limbs, and a strength test of this muscle group may have been performed previously; activity of other synergetic muscles, which may be collected in later studies, adding sEMG in the biceps femoris for example; finally, postural changes of individuals, and photogrammetry software may be used to quantify these changes. It is suggested to carry out more studies in the area, a crosssectional survey with military personnel during a march, with the continuous monitoring of the electromyographic activity of the lower limbs. Another suggestion would be a longitudinal study, analyzing the sample with the manipulation of the transported load variable and verifying the behavior of the military with the increase of this variable.

Conclusion

The body mass of the total FZL and MAG condition were significantly different. The sEMG of the TBA and GNL muscles in the CTRL condition showed about 20% of muscle activation in relation to VSD, the other two conditions resembled the control condition and did not present a statistically significant difference for both muscles measured. A similar result was observed in iEMG, in which the CTRL condition was not significantly different from the FZL and MAG condition, both of TBA and GNL, serving to corroborate with sEMG. These results indicate that the addition of the rifle load and the MAG machine gun, were not sufficient to significantly activate the muscles of the lower extremities, during the maintenance of the quasi-static posture with combat backpack.

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Persian Medicine Surgical Knowledge can Impress Modern Surgery Improvements

Introduction

Millions of people undergo surgery worldwide each year [1,2]. Surgery is an occupation defined by its influence on remedy by means of bodily invasion. Although harshness and risks of opening a living person’s body have long been obvious, the benefits slowly and gradually worked out [3]. Patient satisfaction as an indicator of health care quality may provide information about a hospital’s ability to provide good service as a part of the patient experience [4]. We could help practitioners to better encompass patient perspectives in service delivery and improve patient satisfaction through assessing patient experience [5]. The Bravewell describes integrative medicine like employing a personalized strategy as “an approach to care that puts the patient at the center and addresses the full range of physical, emotional, mental, social, spiritual, and environmental influences that affect a person’s health.” [6] Thus, Complementary and alternative medicine (CAM) can play an important role in patients’ satisfaction [7]. Choosing a treatment method could be affected by cultural and social differences. These kinds of medicine are used for different aims such as therapy, disease prevention and preservation of well-being [8]. This issue has been considered for thousands of years due to historical evidence from Shiraz Azodi Hospital [9].

Lack of enough previous research studies on this topic highly motivates us for more research. The ability of healing many different medical conditions made surgery as a vital field within the history of medical science. It has been an important portion of medicine from antiquity to the modern era. Therefore, the ancient Persian civilization has a great contribution in the development of medical practice in the history of surgery [10]. Our inherited knowledge will be an incentive for us to continue to strive to improve the patient’s quality of life [11]. Nowadays, traditional ways of curing sicknesses originating in ancient societies are called complementary medicine. Many of the traditional medical systems are based on essential principles and centuries of practices by healers [12]. CAM is a general term containing various therapeutic methods, which are not considered as a part of conventional medicine. Popular and widely known examples are acupuncture as a part of traditional Chinese medicine (TCM), naturopathy and homeopathy, phytotherapy and anthroposophical medicine [13]. The development rate of CAM is growing these days. In 2012, approximately one-third of the US population used CAM [14], but its usage in the hospital setting is little known. Furthermore, its risks or benefits in practice is ignored [15]. Yearly visits to alternative practitioners have been assessed at 629 million, which is higher than the number of primary care visits. Most of this high usage rate belongs to college-educated individuals and those with annual income more than $35,000 [7].

Current Findings

According to an article which is published in CDC, Complementary and alternative medicine (CAM) covers a diverse range of ancient to new-age approaches that assert to prevent or cure illness. Literally CAM methods are not part of conventional medicine, because there is inadequate proof that they are harmless and effective. Practitioners use Complementary interventions combined with conventional treatments; however alternative interventions are used as a replacement for conventional medicine” [16]. Although there are remarkable articles on noninvasive CAM, a few research about interventional treatments is accessible. Some researchers believe that Traditional Chinese Medicine (TCM) is a consummate medical system that has developed over millennia to embrace practices and procedures like nutrition, acupuncture, herbal medicine, manual therapies, and mind-body therapies such as qi gong [17]. But it was noted in another article, that according to Confucian teaching medicine in ancient China has been traditionally noninvasive. The human body was considered sacred, and therefore it cannot be dissected. In the Chinese medical canon, the Nei Ching, surgery was chosen under two conditions: once as a last way when all other methods fail, and another time concerning the healing of sores.

The best treatment is to weigh and to contemplate careful removal, besides cutting and scooping out exposed and spoiled particles. The Japanese made no developments in surgery until about the 14th century [18]. But in ancient India and Persia, surgeons were extremely respected, and their operating ability with nose plastic and cataract couching is known. Surgery was categorized in medieval Europe as a handicraft profession and belonged to the barbers guild with the soap cup as symbol, much inferior to the academic trained medical doctors [19]. Persian medicine inspects disease and health through a much wider lens and presents various strategies for the treatment of diseases such as preventive health, food therapy, herbal medicine, hands-on physical manipulations, etc [20]. Persians have an advanced system of medicine founded on a humoral paradigm that existed before Islam. In the early Islamic era (9-12th century AD), Persian medicine boomed and became the main model of medicine in the world [21]. Numerous Iranian medical books such as the Canon of Avicenna (Al-Qanun fit-teb or Canon of Medicine, 1025 AD), were textbooks in medical schools until the 16th century in Europe and were applied in the 19th century in the Middle East [22]. Persian scientists contributed to the improvements of modern medical knowledge particularly anatomy. For example, in the Persian book “Tashrih-e badan-ensan” (human body anatomy), also known as Tashrih-e Mansuri, many tips about muscles and their functions were told [23].

Manipulation and physical therapies (a’amal-e-yadavee) is a significant part of Persian medicine which believed can remove waste materials from body. Himayat is an intervention to remove the excess blood, which was prescribed by a traditional Persian medicine physician and executed by a person (Hijama) experienced in a’amal-e-yadavee [24]. Doctors during the 3rd-9th centuries of Hurghada, besides prescribing medications for the treatment of uterine diseases and another disease, used surgical techniques such as cutting, fissuring, veining, cupping, and burning to cure diseases. They tried to minimize the consequence for the patient [25]. Surgeons like Jan Yzerman in the early 14th and Thomas Fijens in the late 16th century makes it obvious that previous physicians and surgeons had an enduring effect on the surgical practitioners in the world [26]. The official history of surgery shows the beginning of anesthesia back to the 18th century [27]. Ancient Persians had applied various anesthetic agents, from which Wine (Alcohol) alone or together with Hashish and other herbal medications [28]. Maybe for the first-time anesthesia was used in surgical procedures in ancient Iran. The general anesthesia had at least been defined in ancient Persian texts. The proof of this fact is the text of Shahnameh. [29]. In Persian medicine and anesthetic drugs, there are combinations used after surgery or trauma like sesame oil to decrease pain and bruising and prevent skin discoloration [30] and Portulaca oleracea, to improve post-operational-induced Peritoneal adhesion [31].

Future Perspective

Knowledge of medicinal plants usage is the consequence of many years of struggling against diseases due to which man learned to track drugs in seeds, fruit bodies, barks and other parts of the plants [32,33]. Creating novel drugs for old diseases is becoming more and more significant. Pursuiting new methods to document medicinal plants and herbs will decrease the price and the time required to process the medicine [34]. Nevertheless, we probably missed some useful information about drugs or surgery methods from ancient medicine. Thus, we can resurrect them from ancient book resources to improve our knowledge for help humankind more than now. More data are needed to assess the potential benefits of other modalities of CAM. Indeed, the Education of healthcare providers should be encouraged [35]. According to present challenges and the tendency of society to take traditional medicine facilities and the prosperous history of Persian medicine in Iran, providing fair access to traditional medical services should be more concerned [36]. Also, we think researchers should be supported to have continued research on surgical tips and methods in ancient texts to improve current methods in surgery and wellbeing.

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Clinical Relevance of Red Cell Distribution Width and Reticulocytes Hemoglobin Content in Children with Fever without Source

Introduction

The main reason to consult in a pediatric emergency service is fever [1]. Up to 20% of these fever episodes have no apparent source in children under 3 years old and represent a specific nosological entity called fever without source (FWS) [2]. FWS represents a frequent and challenging situation, because both the timely distinction between a viral and bacterial etiology, as well as the early identification of individual with benign and selflimiting disease prone to be treated in an ambulatory settings, still remain partially met clinical needs [2,3]. FWS stratification tools include several clinical prediction rules, biological parameters, such as leukocytosis, C-reactive protein (CRP), and procalcitonin (PCT). So far these risk stratifications have been mostly dedicated to distinguish between viral and severe bacterial infections (SBI) with suboptimal discriminant accuracy [4]. If PCT values below 0.3 ng/ml have shown some promise to effectively rule-out SBI [5,6], knowing whether this single biomarker would outperform clinical prediction rules, enhance their discriminate accuracy for SBI or display sufficient negative predictive value (NPV) is still uncertain [3,6]. Considering these limitations, the integration of multiple biomarkers into a biomarker-based score showed some promise. To this respect the Lab-Score combining CRP, PCT, and urine analysis results probably represent the most discriminatory algorithm available so far [2,7-9], especially when used in a step by step approach in combination with clinical presentation, age and absolute neutrophils count, with optimal NPV for SBI [10]. Nevertheless, because of the lack of specific markers for viral infection, approximately 50% of children with FWS of viral etiology are currently exposed to unnecessary antibiotic treatment and are hospitalized [11].

Therefore, the identification of a biomarker highly specific for viral infections or allowing the early distinction between FWS patients with self-limited disease from those requiring hospitalization could be of considerable interest to optimize patient triage at the emergency room. Among emerging biomarkers of possible interest in FWS, several new hematological parameters automatically provided by SysmexTM analyzers could represent appealing candidates [12-16]. Among them, the red blood cell distribution width (RDW-CV) measuring the degree of heterogeneity of erythrocyte volume, and the reticulocytes hemoglobin content (Ret-He) indicating the iron availability for erythropoiesis, may be promising. In adults suffering from Influenza infections or septic shock, RDW-CV elevation was found to be associated with a worse prognosis [12,13]. On the other hand, in community acquired pneumonia, Ret-He has been shown to be decreased transiently in response to the Interleukin6–dependent hepcidin production leading to iron sequestration in other compartments than those involved in red blood cells maturation [14,15]. Whether Ret-He changes could reflect the infection severity or be of clinical relevance especially in infectious settings is still elusive. Therefore, in this pilot study we investigated whether RDW-CV and Ret-He, already available at no additional costs, could provide meaningful diagnostic and prognostic information in FWS when compared to the Lab-score, and whether these parameters would improve the discriminant accuracy of the Lab-score, both in term of hospitalization duration prediction and ability to confirm the presence of a viral infection

Materials and Methods

The research ethics committee of Geneva University Hospitals approved the study protocol (CER 15-082), and Informed consent given by parent or legal representative before enrolment. The study was performed in accordance with the declaration of Helsinki.

Patient Population and Study Design

This ancillary study was derived from a soon published cohort [17]. Participants for this prospective, single-center, and epidemiological diagnostic study were enrolled in the emergency room (ER) division of the Geneva University Hospitals between November 2015 and December 2017. Briefly, 241 patients aged <3 years-old were admitted to the pediatric ER of Geneva University Hospitals (a tertiary care hospital) with a diagnosis of FWS. FWS was defined as a febrile episode of less than 7 days with no cause determined by the history or the physical exam. The exclusion criterias for this study were unavailable blood samples or unavailable SysmexTM datas, comorbidities predisposing to infections such as cancer, primary or secondary immunodeficiency, and iatrogenic immunosuppression. From the initial 241 patients, 170 had to be excluded because of missing RDW-CV and Ret-He data, leaving 71 patients available for this exploratory study (Figure 1). Besides usual blood investigations for the normal care of children presenting with FWS, blood and urine culture were obtained for all patients. Real-time PCR was used for Adenovirus (AdV, quantitative assay, Argene commercial kit) and Herpes Human Virus- 6 (HHV- 6, qualitative assay, in-house assay followed by quantitative assay, Genesig commercial kit) [18], whereas quantitative and semiquantitative, real-time, reverse- transcription (RT)-PCR were used for Hepatitis E virus (HEV) [19] and Human Parechovirus (HPeV) respectively [20]. Semi-quantitative results were reported as cycle threshold (CT) values; samples with CT values <40 were considered positive. Quantitative results were reported in copies/ml (17). Medical history and the Lab-score, were obtained at admission and relevant information was recorded on an individual case report form [17].

Study Endpoints

Two predetermined endpoints were considered for this explorative study. The primary endpoint consisted in hospitalization duration >24h (HD>24h). The secondary endpoint consisted in a final diagnosis of viral infection defined by the identification of aforementioned viral pathogens or in presence of a highly suggestive clinical presentation in absence of documented bacterial infection. Purely bacterial, mixed infections or undefined etiologies were considered as other etiologies. The endpoints adjudication was performed by one senior physician blinded to the participant’s biological data.

Biological Analyses

Venous blood samples were collected in heparinate lithium and Ethylene Di-amino Tetra Acetate (EDTA) vials on patient admission to the pediatric ER, prior to treatment initiation. Samples were immediately processed for routine requirement. PCT and CRP measurements were performed on Cobas 8000 instruments (Roche, module c801 and module c702, respectively).

Generic and Specialized Hematological Parameters

Blood samples were collected in pediatric tubes containing EDTA and then analyzed for CBC-DIFF and reticulocytes count on a Sysmex XN-10 instrument. Besides classical hematological parameters, such as the leucocytes count, hemoglobin concentration and the neutrophils count, the XN-10 provides new parameters delivering complementary information for the granulocytic lineage and the red lineage [16]. We studied 6 of these new parameters. Three for the granulocytic lineage: the Neutrophil Reactive Intensity (NEUT–RI), the Neutrophil Granularity Intensity (NEUTGI) and Neutrophils Width on y axis (NEUT-WY); and 3 for the red lineage: the hemoglobin content of reticulocytes (Ret-He), the difference in cellular hemoglobin content between reticulocytes and erythrocytes (Delta-He) and the Red Cell Distribution Width CV (RDW-CV). Further details regarding these specialized hematological parameters can be found in Figures 1-3.

Figure 1: White blood cells scattergram in the WBC channel. The scattergram in the WBC channel represents on the x axis the structure of the cells (SSC) and on the y axis the fluorescence (SFL). The NEUT-RI represents the mean fluorescence of neutrophils and is related to the activation and the immaturity of the cells. The NEUT-GI represents the mean value of high angle diffraction and represents the complexity of the neutrophils (nucleus, granulations, …) [16]. The red cloud named EO represents eosinophils, the blue cloud named NEUT + BASO represents neutrophils and basophils, the green cloud named MONO represents monocytes, the purple cloud named LYMPH represents lymphocyts.

Figure 2: Red blood cells scattergram in the RET channel. The scattergram in the RET channel represents on the x axis the fluorescence and on the y axis the size of the red blood cells. The Ret– He is calculated from the Ret–Y (mean value of the red blood cell size on the y axis) and represents the mean hemoglobin content of red blood cell precursors (Reticulocytes). The Delta-He is the difference in cellular hemoglobin content between reticulocytes and erythrocytes.

Figure 3: Red Cell Distribution Width Standard Deviation (RDW-SD) and Red Cell Distribution Width Coefficient of Variation (RDW-CV) derived from the red blood cells curve in impedance. The RDW-CV is calculated from the RDW-SD which is the width of the impedance curve of the red blood cells 20% above the base line.

Statistical Analyses

Table 1: Determinant of the Lab-score.

Note: *Positive urine dipstick: positive leukocytes esterase, or nitrite test result. LE: leucocytes; NI: Nitrites.

Analyses were performed using STATISTICA™ software (StatSoft, Tulsa, OK, USA). Fisher’s bilateral exact test and Mann– Whitney U-test were used where appropriate. Associations between biomarkers and study endpoints are presented as the odds ratio (OR) and corresponding 95% confidence interval (95% CI). Multivariable analyses with logistic regression were used to assess associations between continuous variables. In this model, endpoints were set as dependent variables, and the Lab-Score (Table 1) was set as the unique confounder because of the limited sample size. Adjusted analyses were performed only in case of significant univariate analyses. ROC analyses were performed using ANALYSE-IT™ software for Excel (Microsoft, Redmond, WA, USA) to identify the biomarker with the best area under the curve (AUC). AUC comparisons were performed according to the nonparametric approach proposed by DeLong, et al. [21]. The optimal cut-off was determined in a post-hoc based upon ROC curve results. Corresponding sensitivity (SE), specificity (SP), positive predictive value (PPV), negative predictive value (NPV) with the respective 95% CIs are given. A value of p<0.05 was considered statistically significant.

Results

Baseline Clinical Demographic and Biological Characteristics

The clinical features of our population are summarized in Table 2. A total of 71 patients were included in the cohort. Among them 19 were discharged <24 hours, and 38 had a final diagnosis of viral infection. Among the latter, 11 had an enteroviral meningitis (15.5%), 12 a viral upper respiratory tract infections (16.9 %), 12 a viral gastroenteritis (16.9%), 1 a hand-foot-and-mouth disease (1.41%), 1 a viral rash (1.41%), and 1 a viral meningitis (1.41%). The remaining patients included had either bacterial infections only, mixed infections (bacterial and viral) (n=7), or undefined etiologies (n=3). A total of 52 patients had a hospital stay superior or equal to 24 hours (73.24%).

RDW-CV as an Independent Predictor of the Hospitalization Duration >24h (HD >24)

Table 2 shows that patients with HD>24h had higher median levels of RDW-CV upon inclusion when compared to those with HD<24h (14.1% versus 13.1% p<0.0001). The results were further confirmed by the ROC curves analyses (Table 3) showing that the AUC of the RDW-CV for an HD>24h was 0.79 (95%CI:0.67-0.92, p<0.0001), which was the highest for all parameters tested. In comparison, the ROC curve of the Lab-score displayed an AUC of 0.66 (95%CI: 0.53-0.79, p=0.0068). The AUC difference between RDW-CV and the Lab-score was nevertheless not found to be significant according to the Delong method (delta: 0.13, p= 0.11; Table 3). Adding RDW-CV to the Lab-score significantly increased the latter AUC from 0.66 to 0.84 (95CI%: 0.72-0.95; delta: 0.18; p=0.001, Table 3). Furthermore, logistic regression analyses indicated that for each percent of RDW-CV

increase, there was a concomitant 3.28-fold increase in the risk of HD>24h (OR: 3.28, 95%CI:1.57-6.87, p=0.0015) which remained unchanged after the adjustment for the Lab- score (OR: 3.76, 95%CI: 1.11-12.67, p=0.03) (Table 4). Conversely, the risk association for the Lab-score was independent of RDW-CV (adjusted OR: 1.68,95% CI: 1.07 - 2.65; p=0.03). Taken together, these results indicate that both RDW-CV and the Lab-score are independently associated with HD>24h, with an apparent superior strength of association Privileging the PPV, the optimal cut-off of RDW-CV was found to be of 15.2 % with a PPV of 100 % (95%CI: 63-100), a NPV of 31% (95%CI: 20-44), a SN of 17% (95%CI:9-31), and a SP of 100% (95%CI:79-100; Table 5).

Table 2: Patient Baseline Characteristics.

Note: *Correspond to significant AUC differences.

GB: WBC: White Blood Cells; PNN: Neutrophils; IG#: Immature Granulocytes; NRBC: Nucleated Red Blood Cells; GR: RBC : Red blood cells

Table 3: Discriminant accuracies of hematological parameters for hospitalization duration ≥ 24h and infections of viral etiology.

Note: *Correspond to significant AUC differences.

Table 4: Risk associations for hospitalization duration ≥24h.

Note: *Adjusted for the Lab-score, except when **. Adjusted analyses were performed only in case of significant univariate analyses. ** adjusted for RDW-CV. *** adjusted for Labscore and age

Table 5: Optimal cut-off values.

Note: *Based upon ROC curves analyses. **Set in order to maximize PPV.

Ret-He as a Non-Meaningful Marker of Infections of Viral Etiologies

Table 2 shows that patients with a final diagnosis of viral infection had higher median levels of Ret-He upon study inclusion when compared to those with infections of other etiologies (29.9 pg vs 26.25 pg, p=0.004). ROC curves analyses (Table 3) indicated that Ret-He had an AUC of 0.70 (95%CI: 0.57-0.84, p=0.002), whereas the Lab- score (cut-off: 3 points (22)) (displayed the highest diagnostic accuracy with an AUC of 0.88 (95%CI: 0.79- 0.96, p<0.0001) to detect an infection of viral etiology. The AUC comparison between Lab-score and Ret-He, indicated that the AUC difference was significant with a delta of 0.18 and a p-value of 0.047. Logistic regression analyses indicated that if Ret-He was significantly associated with a final diagnosis of viral infection in unadjusted analysis (OR:1.31;95%CI: 1.09-1.57, p=0.004), the association was lost after adjusting for the Lab-score. Furthermore, none of the parameters tested remained significant when adjusted for the Lab-score (Table 6). Privileging the PPV value, the optimal cut-off value found was 29.8 pg with a PPV of 76% (95% CI: 0.53- 0.89), a SP of 81% (95% CI:0.63-0.92), a SN of 53% (95% CI:0.35- 0.70), and NPV of 60% (95% CI: 0.46-0.76; Table 5).

Table 6: Risk associations for infections of viral etiology.

Note: *Adjusted for the Lab-score. Adjusted analyses were performed only in case of significant univariate analyses.

Discussion

The key finding of this pilot study is that both RDW-CV and the Lab-score were independent predictors of a HD>24h in children with FWS, whereas the remaining hematological parameters tested were not, after the adjustment for the Lab-score. To the best of our knowledge, this is the first report of the Lab-score as predictor of hospital duration. Indeed, so far most of the studies performed analyzed the capacity to distinguish patients with SBI [2,7,10,22]. Nevertheless, despite being significant, the AUC was rather modest (0.66) and whether it would be enough to influence patient management remains to be tested in other larger studies. On the other hand, RDW-CV tended to have a better AUC (0.79) and displayed an optimal positive predictive value of 100% at the 15.2% cut-off. Although derived in a post-hoc manner in order to optimize positive predictive value, this cut-off is very close to previously reported RDW-CV cut- offs (between 14.5 % and 15.5%) predicting mortality in patients with SBI or septic shock [12,23- 25]. Furthermore, if the AUC difference between RDW-CV and the Lab- score (0.79 vs 0.66; p=0.13) was not found to be significant according to the Delong method [21], adding RDW-CV to the Labscore nevertheless substantially increased the latter AUC from 0.66 to 0.84 (p=0.001) (Table 6). These results indicate that RDWCV values above 15.2% in FWS would allow the clinicians to early identify patients requiring prolonged hospitalization regardless of infection etiology, and accordingly to improve patients triage in the emergency room.

Furthermore, being automated and available 24h/24h with a turn-around time around 1 minute, RDW-CV results would meet most of the requirements needed for an emergency test. Nevertheless, these appealing preliminary results need to be replicated and validated at a larger scale before any clinical recommendation can be made. Also, knowing whether RDW-CV should be introduced into the Lab-score or considered separately to optimally identify patients requiring hospitalization awaits clarifications. The other principal findings concerning the Ret-He is that this parameter was not significant when adjusted with the Labscore to predict the viral etiology in FWS, as the other parameters tested. However, it is interesting to mention that the median value of Ret-He for viral infections (29.9 pg vs 26.25 pg) was higher than the one retrieved in other etiologies (including bacterial, mixed infections and undefined etiologies), which can be explained by the fact that iron sequestration is more severe in case of septic conditions (26,27). The Ret-He AUC to predict a uniquely viral infection was not found to be optimal (AUC: 0.70), especially as it was not found to be independently associated with this diagnosis, when adjusted for the Lab-score. Furthermore, privileging the PPV, the optimal post-hoc retrieved cut-off (29.8 pg) only displayed a PPV of 76%, which is too low to be considered for rule-in purposes, especially given the lower end of the 95%CI (53%). There are several limitations in this study. Firstly, due to the limited sample size of this pilot study, we could not provide a proper interpretation of non-significant findings reported. Nevertheless, given the strength and independent nature of the association between RDWCV and HD>24h, those preliminary results clearly indicate that RDW-CV could represent an appealing biomarker to early identify FWS patients requiring hospitalization.

A second important limitation resided in the fact that the optimal cut-off for RDW-CV (and Ret-He) was determined in a post-hoc manner. Therefore, the current proposed cut-off would require further independent validation in other larger prospective studies. Finally, the fact that our population exclusively included children where reference intervals for RDW-CV and Ret-He are still undetermined, we could not further extrapolate on the relevance of the proposed cut-offs from adult populations. However, this pilot study opens some new perspectives in the research of new but readily automatically available biomarkers to optimize patient management flux presenting to the ER with FWS.

Conclusion

In conclusion, it appears that the RDW-CV is a good independent predictor of the hospitalization duration superior or equal to 24 hours with an optimal PPV of 100%. Moreover, when added to the Lab-score, the RDW-CV was found to increase the prognostic capacity of the Lab-score, one of the best available risk stratification tools in FWS. When above 15.2% RDW-CV has the potential to help the clinician to early identify FWS patients requiring hospitalization, and as such could facilitate patient management flux in the emergency room. On the other hand, none of the biomarker tested was found to outcompete the Lab-score in distinguishing between fevers of purely viral origin from fevers of other etiologies. Those preliminary findings need to be replicated and validated at a larger and multicenter scale before any clinical recommendation can be done.

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