**3.1 ROS involvement in water stress**

Water stress is a common environmental restriction that plants frequently faced during their lives, restricting survival, reproduction, and ultimately productivity. Drought stress causes stomata closing, decrease CO2 entrance, and compromised photosynthetic rate, as well as discrepancy in the light acquire and usage and changed photochemistry in chloroplasts, triggering ROS excessive formation [49, 65]. The production of reactive oxygen species (ROS), which is assumed to lead to cellular injury, is one of the main and serious alterations due to drought stress. Though, a signaling function for ROS in activating the ROS scavenging mechanism, which might award defence or resistance to stress, has recently been discovered. This scavenging system is made up of antioxidant enzymes including SOD, catalase, and peroxidases, as well as antioxidant substances such as ascorbate and reduced glutathione; the oxidative load is largely governed by the balance between ROS formation and scavenging. Drought stress undoubtedly causes ROS production as a primary plant reaction, which might be regulated by hormones such as ABA and ethylene, which may also perform a downstream function. Unless ROS scavenging by antioxidant systems is disrupted, a high amount of ROS might exacerbate stress made harm to most cellular components [66].


**1. Introduction**

Quantum cascade (QC) photovoltaic infrared photodetectors are based on intersubband transitions of electrons [1,2]. Unlike quantum-well infrared photodetectors [3], the active regions of QC detectors can be engineered with highly flexibility, as shown for QC lasers [4]. Consequently, a variety of active region designs have been reported that expand the operational wavelength range or improve the responsivity [5–7]. Hence, photoresponses in QC detectors have been demonstrated from the near- to the far-infrared wavelengths [8,9]. QC detectors also exhibit low-noise and high-speed. Due to their biasfree operation, dark currents induced by external voltages are absent, which is important for high detectivity without an elaborate cooling system. Regarding high-speed operation, an electron transit time of less than 1 ps was substantiated in a near-infrared QC detector by a time-resolved pump–probe measurement [10]. More recently, frequency responses of several tens of gigahertz and picosecond response times have been demonstrated in the mid-infrared (MIR) QC detectors [11,12]. The intrinsic short response times are determined by high-speed electron transport via sub-picosecond intersubband scattering processes. Low-noise, high-speed QC detectors could be key devices for high-speed MIR applications.

The MIR is a molecular finger print region and, thus, is very important in fundamental science, medicine, and industry. A large number of unique and strong absorption lines that correspond to fundamental vibrational modes for many molecules are in the MIR, and they can be used to identify and quantify specific molecules. In particular, laser absorption spectroscopy (LAS) in the MIR is a powerful tool for high-precision and high-sensitivity molecular sensing because of the strong absorption of narrow-linewidth laser light [13–16]. Its most prominent application is the detection and identification of gas-phase molecules,

**Citation:** Dougakiuchi, T.; Akikusa, N. Application of High-Speed Quantum Cascade Detectors for Mid-Infrared, Broadband, High-Resolution Spectroscopy. *Sensors* **2021**, *21*, 5706. https://doi.org/10.3390/s21175706

Academic Editors: Krzysztof M. Abramski and Piotr Jaworski

Received: 30 July 2021 Accepted: 18 August 2021 Published: 24 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

because significantly high sensitivity can be obtained by long-range propagation of a collimated beam in a gas. The atmospheric windows in the MIR enable environmental measurements in free space [17,18]. As multiple absorption lines of several gases can overlap in a specific spectral region, high-resolution, high-sensitivity broadband is always required in LAS.

Heterodyne detection is an established technique to obtain high-resolution in LAS [19], and by using a high-speed detector, broadband measurements can be achieved simultaneously. In the MIR application field, HgCdTe (MCT) detectors are the most widely used photodetectors because of their high-responsivity and broad responsive wavelength covering a few micrometers [20], unlike that of QC detectors. However, in MCT detectors, a high-speed operation of several tens gigahertz is difficult in principle and not adequate for broadband heterodyne detection. On the other hand, the frequency range of a heterodyne beat signal up to 20–30 GHz, the upper limit of practical processers, corresponds to the wavelength tuning range of ~1 cm−1. In this point of view, the narrow response spectrum of QC detectors is not concern for heterodyne spectroscopy and is a preferable property to avoid background noises. Here, we demonstrate broadband heterodyne LAS by using QC lasers and a QC detector over a spectral range centered at 4.5 μm. To enhance the high speed of the QC detector, we reduced the parasitic capacitance and inductance with air-bridge wiring and a small mesa structure for the thick active region constructed from 90 cascade modules. A 3-dB cutoff frequency was measured for over 20 GHz, and the wide-band frequency response guaranteed a 0.8 cm−1 broadband spectral range for heterodyne spectroscopy. Several absorption lines of nitrous oxide (N2O) were observed over the range 2220.59–2219.76 cm−1 with 5 MHz resolution.


**2.6. Statistical analysis**

Extrasynthèse SA (Genay, France). Soybean lecithin Beakin LV3 was a gift from Archer Daniels Midland Co (Decatur, IL, USA). Ethanol, thimerosal, phenylmethanesulfonyl fluoride, and monobasic potassium phosphate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific (Pittsburg, PA, USA). Deionized water was prepared by passing distilled water over a mixed bed of cation-

Whole caseins were prepared by isoelectric precipitation of the individual skimmed milk samples obtained from a Jersey cow and French-Alpine goats. The precipitate was dissolved by the addition of NaOH to yield a solution of pH 7.0. The casein was re-precipitated, washed, and then re-suspended. The sodium caseinate was subsequently cooled to 4°C and centrifuged at 100,000 × g for 30 min to remove residual fat using a Beckman Optima XL-A (Beckman Instruments Inc., Palo Alto, CA, USA) analytical ultracentrifuge. Finally, the suspension was dialyzed against cold deionized water at 4°C for 72 h with three changes and then lyophilized. The integrity of the samples was confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and the percentages of the various component caseins estimated by densitometry as previously described [22]. The casein composition of the samples is as follows: αs2-casein (bovine casein 12.1%; caprine αs1-I-casein 9.2%; caprine αs1-II-casein 5.3%), αs1-casein (type I): (bovine casein 0%; caprine αs1-I-casein 4.0%; caprine αs1-II-casein 0%); αs1 casein (type II): (bovine casein 39.5%; caprine αs1-I-casein 21.1%; caprine αs1-II-casein 25.6%); β-casein: (bovine casein 37.2%; caprine αs1-I-casein 51.6%; caprine αs1-II-casein 60.6%); and κ-casein: (bovine casein 11.2%; caprine αs1-I-casein 13.8%; caprine αs1-II-casein 9.6%) [20].

A corn oil-in-water emulsion was prepared as follows: an organic phase was prepared by diluting 2.5% (wt/wt) of the commercial lutein in corn oil. Soybean lecithin 0.5% (wt/wt) was dissolved in the corn oil. An aqueous phase was prepared by dispersing 1.0% (wt/ wt) of lyophilized bovine casein or caprine casein into the aqueous buffer solution (5 mM phosphate, pH 7.0). A coarse emulsion of oil-in-water was prepared by mixing the organic phase (10%, wt/wt) and the aqueous phase (90%, wt/wt) using a hand-held homogenizer (Biospec Products, Inc., Bartlesville, OK, USA) at low speed. The coarse emulsion was then homogenized five times at 82.74 MPa (12,000 psi) through a high-pressure TC5 homogenizer (Stansted Fluid Power, Harlow, UK). The fine emulsion produced was then diluted (1:1, v/v) with buffer solution containing an antimicrobial agent [5 mM phosphate buffer at pH 7.0 and 1 mM (wt/v) thimerosal]. The final diluted emulsions that were used for the stability studies contained 5% (wt/wt) oil phase and 250 mg per liter of lutein. Resulting emulsions were

The particle size of the oil droplets in the lutein-enriched emulsions was measured at day 0 and day 7 after homogenization with a SALD-2101 laser diffraction particle analyzer (Shimadzu Corporation, Columbia, MD, USA). The charge of the oil droplets in the lutein-enriched

anion exchanger and was used throughout this study.

**2.2. Source of caseins**

164 Progress in Carotenoid Research

**2.3. Emulsion preparation**

stored in the dark at 5 and 15°C for 7 days.

**2.4. Physical characterization of the emulsion**

In each experiment, the results of triplicate analyses were used to test experimental variables. The data were analyzed by ANOVA using PRO GLM procedure of SAS (version 8.2, SAS Institute, Cary, NC, USA). The least significant test was used to determine significant differences among means at p < 0.05.


**4.2 Factors influencing a farmer's decision at the farm level in maize and wheat production in Uasin Gishu, county, Kenya**

In an effort to understand what influences a farmers decision to commence an activity in the maize and wheat growing calendar (when to start land preparation, planting, type of crop to grow, weeding, top dressing, spraying of weeds, fungus or bacterial infection on crops and even harvesting), the following responses were gathered from the farmers:-Those who commence their activities just because those are the dates known to them through their experience over time in maize and wheat growing were 333 (84.9%). Another group of farmers 141 (36%) rely on looking around for *"signs that rains are about to fall (wind direction from east to western side, cloud movement from eastern to western side of their farms and high sunshine intensity during the day and warm nights)".* Advice from the Ministry of Agriculture Livestock and Fisheries officials was mentioned by 29 (7.4%) of the farmers as having assisted them make decisions on the start of farming activities in their respective farms as shown in **Figure 4**.

#### **Figure 4.**

*What factors influence a farmer's decision at the farm level in maize and wheat production in Uasin Gishu, county, Kenya.*

The results reveal a farming population solely dependent on their own indigenous knowledge systems and experience gained over time in maize and wheat growing to make certain farming decisions. The fact that 333 (84.9%) farmers commence their activities just because those are the dates known to them through their experience over time and another 141 (36%) looking around at some indigenous knowledge system indicators that rains are about to fall is a clear indication that framers do base their faming decisions on their own indigenous knowledge systems and experience gained over time. However, depending solely on indigenous knowledge indicators to predict onset dates of the rains and experience gained over time practicing maize and wheat creates a big challenge to the farmer especially with the prevailing changing climate that keeps on distorting seasons known to farmers. This makes farmers vulnerable and may incur much losses related to unpredictable weather patterns brought about by climatic changes. The findings in this study correspond with findings in [24] which asserts that in different parts of the world, farmers depending on rain-fed cultivation have developed complex cultural models of weather and may be able to cite local predictors of seasonal climate. Similarly, [13] argue that even when climate information is available, incorporation of scientific climate information into local decision making may not often occur because of the way such information is communicated and disseminated. There is need to identify clear channels of information delivery and also downscale the information to make sense for the farmer for effective uptake of such information.

## **4.3 Indigenous knowledge system indicators in rainfall prediction for farmers in Uasin Gishu County, Kenya**

In an effort to understand what farmers use as their local indigenous knowledge indicators for weather prediction, farmers were asked to state what they thought were signs that rains were about to fall in their respective areas based on their experiences gained over time as maize and wheat farmers. The farmers who affirmed and believed that the real sign for rain commencement in their farms is when there are consistent lightning flashes around the Lake Region or Tindiret area in Nandi South as affirmed by 245 (62.7%). Those farmers who belief that heavy cloud cover *Integrating Local Farmers Knowledge Systems in Rainfall Prediction and Available Weather… DOI: http://dx.doi.org/10.5772/intechopen.96504*

and intense sunshine during the day was a sign that rains were about to commence were 178 (45.5%). The farmers relying on wind direction (eastwards and sometimes westwards) to alert them that the rains were about to commence were 159 (40.7%). Farmers who belief that very warm nights was a sign that rains were about to fall in their area were 195 (49.9%). Those who affirmed that in the recent years, it has been quite difficult to predict when the rains are about to fall were few farmers 42 (10.7%). The responses are as shown in **Figure 5**.

**Figure 5.**

*Indigenous knowledge system indicators for predicting rainfall in Uasin Gishu County, Kenya. (\*multiple responses - farmers were allowed to note as many reasons as were applicable).*

The findings in this study reflect a farming community that has developed its own indicators in the traditional knowledge system on rainfall prediction. The set of indicators of rainfall used by farmers include consistent lightning flashes around the Lake Region or *Tindiret* area in *Nandi* South, heavy cloud cover and intense sunshine during the day, blowing of wind towards eastern side and sometimes westwards and very warm nights. During the key informant interview to supplement and corroborate some of the response, the following were identified as other key indicators used by farmers especially among the *Nandi* community and include examining the behavior of certain plants or trees to determine rainfall near onset. Among the plant species known include the tree *Erithrina Abyssinica (Kakarwet).* The tree starts flowering red with full leaves regained, *Vernonia Auriculifera (Tepengwet)* tree starts flowering, *Flacourtia indica (Tungururwet)* start budding is an indicator for rainfall near onset and farmers prepare to commence dry planting. The Fig tree (*Ficus sycomorus*) known locally by farmers as *(Mogoiywet)* start shading leaves is an indicator for rainfall near onset. One other small plant (herb) growing in thickets or bushes *Scadoxus multiflorus* of the *Amaryllidaceae* family *(Ngotiotet)* starts flowering red around March period and found in thickets or bushes near river banks is a real indicator for rainfall near onset.

Other indicators include migratory birds among them the White Stock *(Kaptalaminik*) moving or flying towards the north side signifying near rainfall onset and when flight changes towards the south, this signifies rainfall cessation. In addition to the indicators, the community had a unique way or prayer asking their God for rainfall or water for their domestic use during drought manifestation. According to the key informants interviewed, the traditional prayer song *(ingoo)* was sang by mature women and those at child bearing age at night and in several groups (3 to 4 groups of women) from various locations. They could join in the prayer song at night converging at one place in the open near a watering point away from homesteads as participants sang without cloths on them. They carried with them cooking items that include cooking sticks (*mukanget ak Kurbet*) and some finger millet flour (*Beek ab Kipsongik).* This form of prayers according to key informants and other farmers participating in this study all agreed that the women prayers at night were answered almost instantly there and then because before mid-night during the singing, heavy rains would fall on the singing women as they retreat back to their homesteads happy.

The study by [18] also supports the findings in this study and explain that majority of farmers prefer indigenous forecasting knowledge more than contemporary forecasting. The reasons being that indigenous information is more compatible with local culture and it has been tested, tried and trusted. In addition, it is more specific and is in a language that can be understood better by communities. There is a clear view here that it is important to document indigenous knowledge system used by maize and wheat farmers and ultimately integrate the knowledge with science based climate forecasting. Integrating both scientific and traditional knowledge will enable uptake and ownership of climate information by communities hence helping farmers avoid losses as a result of the changing climate. This is because farmers consider the fact that their perspectives have been taken into consideration when their indicators for predicting rainfall near onset have been taken into account by the weather forecasts. This creates ownership and sustainability in the long run.

The study finding compares well with findings by [38] explaining that in Uganda, indigenous knowledge forecasters associate the onset of rainfall with the appearance of clouds. The appearance of nimbostratus and cumulonimbus clouds indicates a high probability of rainfall. Biological indicators focus on the behavior and activities of domestic and wild animals, insects and different species of plants for weather forecasting. For instance, in Uganda, the *Mvule* tree indicates onset of the rainy season. In Kenya like Ethiopia, the intestines of sheep and goats are used to forecast about the magnitude, severity, and duration of drought, drought-affected places, disease outbreak, the prospect of peace, and/or conflict. In Tanzania, the occurrence of large flocks of swallows and swans, roaming from the South to the North during the months of September to November, is an indication of onset of short rains [38]. From the afore going, to effectively mainstream access to climate and weather information in key sectors, it is important to understand the local perspectives in relation to the changing climate and coping strategies in place. Ignoring this fact might hinder uptake of weather forecasts by the farmers hence leaving them completely vulnerable to the changing climate as they would rely only on their indigenous knowledge systems to mitigate prevailing weather conditions.


**4.4 Theme 4: academic support offered by lecturers**

Minimal support from UNISA lecturers was observed. Of the six student teachers during the teaching practice, five were visited by UNISA lecturers. However, of the three lecturers who visited the student teachers, only one lecturer was helpful, and could give the student teachers advice in how to deal with lesson presentation. The following comments of the student teachers confirmed the situation:

*My lecturer once visited me when I was studying year 2. Now, No. The only person assisting is my mentor teacher. (Student Teacher 1)*

*The supervisor visited me. He had a meeting with me before the lesson and asked me how I am doing with my portfolio. He looked at it and asked when the due date is. He did not help me with my assignment. (Student Teacher 2)*

*No one came to support me, and I am left with three days to finish my five weeks teaching practice. I hope I will get support next year when studying my last year. It is frustrating to be on your own while teaching. I do not know how to handle the big class, as learners are rowdy. (Student Teacher 3)*

*Support? Sometime last year one of UNISA lecturers supported me. She was of a great help as she advised on how to prepare my lesson and how I can approach the topic I was teaching. (Student Teacher 4)*

*The lecturer who visited me showed me how to introduce a lesson. She even demonstrated how to introduce the lesson. Learners were participating and liked it. I am now trying to emulate her when I introduce the other topics of the lesson. (Student Teacher 5)*

*The other man came to observe my lesson but did not say anything concerning my teaching method. He gave me average marks and told me to improve in giving learners time to talk and not to talk alone. (Student Teacher 6)*

The support from the university staff seemed inadequate. It is customary for teacher training institutions to provide support to their student teachers when they are undertaking teaching practice. This is done by way of liaising with schools that host student teachers in their schools and arrange for the appointment of mentor teachers [47]. UNISA lecturers and external supervisors (retired teachers who assisted with the student teacher supervision) were assigned to visit student teachers at the schools where they were placed at different times during the first, second and third school terms. Most of the external supervisors were never exposed to the interactive teaching strategies during their training due to the apartheid education policy, which intended to offer poor teacher training to African teachers [26]. Therefore, the student teachers may have experienced inadequate support from the external supervisors. Hence, the student teachers felt neglected, isolated and not

supported to address challenges that they faced. The provision of academic support and presence of lecturers during teaching practice can minimise student teachers' sense of loneliness, isolation and disconnectedness in ODL.


**2. SO based diols**

The most excessively used SO based diol in PU production is fatty amide diol or fatty alkan‐ diol‐amide [FAD] (Figure 2). FAD is obtained by the base catalysed amidation of SO with diethanolamine. The structure of FAD is determined by the fatty acid composition of the starting SO.

**Figure 2.** Figure 2. FAD from (a) Linseed (35.0‐60.0 % Linolenic acid), (b) Soybean (43.0‐56.0 % Linoleic acid) and (c) Karanj (44.5–71.3 % oleic acid), Nahor (55‐66% oleic acid) , Jatropha (37‐63 % oleic), Olive (65‐80 % oleic acid)

The reaction proceeds by nucleophilic acyl substitution bimolecular (SN2) reaction mechanism. As the name suggests, FAD contains an amide group, with two hydroxyl ethyl groups directly attached to amide nitrogen along with the pendant aliphatic chain of SO. FAD is derived from different SO such as Linseed (*Linum ussitassimum*), Soybean (*Glycine max*), Karanj (*Pongamia glabra*), Nahor (*Mesua ferrea*), Jatropha (*Jatropha Curcus*), Olive (*Olea europea*), Coconut (*Cocos nucifera*) and others [20‐30] (Figure 2). FAD is used as raw material for various polymers such as PU, poly (esteramide) and poly (ether amide).


**Bone Mass and Mineral Metabolism Alterations in Adult Celiac Disease: Pathophysiology and Clinical Approach**

## **Michele Di Stefano, Caterina Mengoli, Manuela Bergonzi and Gino Roberto Corazza \***

1st Department of Internal Medicine, University of Pavia, IRCCS "S.Matteo" Hospital Foundation, Pavia 27100, Italy; E-Mails<EMAIL_ADDRESS>(M.S<EMAIL_ADDRESS>(C.M<EMAIL_ADDRESS>(M.B.)

**\*** Author to whom correspondence should be addressed; E-Mail<EMAIL_ADDRESS>Tel.: +39-0382-502973; Fax: +39-0382-502618.

*Received: 9 September 2013; in revised form: 13 November 2013 / Accepted: 14 November 2013 / Published: 22 November 2013*

**Abstract:** Osteoporosis affects many patients with celiac disease (CD), representing the consequence of calcium malabsorption and persistent activation of mucosal inflammation. A slight increase of fracture risk is evident in this condition, particularly in those with overt malabsorption and in postmenopausal state. The adoption of a correct gluten-free diet (GFD) improves bone derangement, but is not able to normalize bone mass in all the patients. Biomarkers effective in the prediction of bone response to gluten-free diet are not yet available and the indications of guidelines are still imperfect and debated. In this review, the pathophysiology of bone loss is correlated to clinical aspects, defining an alternative proposal of management for this condition.

**Keywords:** celiac disease; osteoporosis; gluten-free diet; bone densitometry

## **1. Introduction**

Osteoporosis is a condition characterized by low bone mass and micro-architectural deterioration of bone tissue resulting in enhanced bone fragility and an increase in fracture risk [1]. It affects more than 75 million people in developed countries, causing 8.9 million fractures annually worldwide.  There is a general agreement in the literature that more than 75% of untreated adult

celiac disease (CD) patients with an overt malabsorption syndrome at diagnosis suffer from a loss of bone mass [3–9], and this complication also affects about half the patients with subclinical CD, presenting with minimal, transient and apparently unrelated symptoms [3,9], or asymptomatic patients diagnosed because of their first-degree kinship [4].

Moreover, an increased prevalence of celiac disease in osteoporotic patients was reported [10–12] and, even if this result was not confirmed by others [13–15], all previous results show the importance of the problem from a clinical point of view. On the contrary, the value of active screening for CD in patients with otherwise unexplained bone loss is still under debate.

Accordingly, there is no doubt that CD is a condition at high risk for secondary osteoporosis, and the evaluation of bone mass and mineral metabolism is thus very important in the clinical management of these patients. In the last few years, while the mechanisms of bone derangement in CD have been extensively studied, less attention has been paid to the clinical management of this complication: there is in fact very little information available on the timing of the first bone mineral density (BMD) measurement, on follow-up frequency, even on the best treatment options.

#### **2. Bone Damage and Mineral Metabolism Derangement in Celiac Disease**

Intestinal malabsorption and inflammation contribute to the pathophysiology of bone damage in CD. Villous atrophy is responsible for alterations of intestinal absorption, and a negative calcium balance was shown in CD patients due to several mechanisms: malabsorption of calcium in untreated patients [16], partially reversible after gluten-free diet (GFD) [17]; the reduction of calcium intake [18] also due to a secondary lactose intolerance [19]; and the reduction of intestinal calcium absorption due to its binding to intraluminal unabsorbed fatty acids [16]. Hypocalcemia can induce a compensatory increase of serum levels of parathyroid hormone (PTH), in turn responsible for an increase of bone turnover [7,20]: in untreated CD, serum PTH correlates with markers of both bone synthesis, such as osteocalcin, as well as resorption, like telopeptide of type I collagen (ICTP) [5]. Bone resorption is faster than bone neoformation, resulting in net bone loss and a high turnover osteoporosis [21]. The increase of serum PTH enhances the activity of the renal enzyme 1--hydroxylase, which converts 25-vitamin D into 1,25 vitamin D, in order to improve calcium absorption at intestinal level. However, this effort is ineffective, mainly due to the lack in immature enterocytes of celiac mucosa of calbindin [22], a vitamin D-dependent calcium-binding protein, minimizing the role of vitamin D malabsorption [23]. Finally, high levels of 1,25 vitamin D might have the paradoxical effect of increasing bone resorption, as shown in patients with chronic renal failure [4]. Accordingly, vitamin D functions are rarely impaired in untreated CD, as hyperconversion of metabolite 25-vitamin D guarantees adequate levels of the active form 1,25 vitamin D.

Intestinal malabsorption could also lead to some deficits of other minerals, fat and water soluble vitamins that could affect normal bone metabolism. In particular, low levels of zinc were described in non-treated celiac patients [24], and related to low levels of insulin-like growth factor, that are subsequently responsible for derangement in bone metabolism, growth and immune function [25].

This complex network of events is present in both overt symptomatic and subclinical CD to be a disease that is below the threshold of clinical detection without signs or symptoms sufficient to trigger CD testing in routine practice—or it is silent, equivalent to asymptomatic CD patients, even

if the extent of bone loss and the alterations of serum levels of indices of bone and mineral metabolism may be less severe than in CD patients with overt malabsorption [3].

More recently, much evidence has also suggested the role of both local and systemic inflammation in the pathophysiology of bone loss in CD, characterized by a chronic increase of both mucosal and serum pro-

{"\-1 and IL-6 [26,27]. IL-1 and {

 "\-6 has a pivotal role in bone resorption by recruiting osteoclast precursors and stimulating their differentiation [29]. In untreated CD patients, serum IL-6 levels inversely correlate with BMD [27] and directly with PTH and ICTP levels, a marker of bone resorption [30]. Recently, the existence of a complex cytokine imbalance in CD patients, affecting both osteoclast and osteoblast activity was shown: cultures of peripheral blood mononuclear cells of healthy donors with sera of untreated CD patients result in an increase in osteoclast number and IL-6 levels, together with an inhibition of IL-12 and IL-18 [31], two cytokines showing an *in vitro* inhibitory effect on osteoclastogenesis and osteoclast activity [32,33].

In the last 15 years, great attention has been given to the RANKL/RANK/osteoprotegerin pathway, that is today considered the main signaling system in bone metabolism. The receptor activator of nuclear factor -B ligand (RANKL) is expressed and secreted by osteoblasts; it binds RANK, located on the surface of osteoclast precursors, to induce the differentiation of these cells into mature osteoclasts, promoting bone resorption. Osteoprotegerin (OPG) is also secreted by osteoblasts; it acts as a decoy receptor for RANK and blocks RANK–RANKL interaction [34]. In CD patients, an increased level of OPG and RANKL was described, with an OPG/RANKL ratio significantly lower than controls. Moreover, the OPG/RANKL ratio was correlated with spine BMD [35] and with IL-6 levels [31].

The pathophysiological role of autoantibodies against OPG is also debated, as in a recent paper the presence of these antibodies was detected in a man with CD, high bone turnover and severe osteoporosis not responsive to GFD and to calcium and vitamin D supplementation [36]. This observation was not confirmed by a subsequent study on a large cohort of CD patients on GFD [37].

Further factors are linked to endocrine and reproductive disorders, commonly part of CD clinical presentation. In particular, early menopause and periods of amenorrhea could occur in women, due partly to malnutrition and partly to hormonal imbalance, and could worsen the severity of osteoporosis [38]. In men, hypogonadism was described, due to a reversible androgen resistance [39] and to hyperprolactinemia [40], and considered a possible adjunctive factor risk for osteoporosis [41]. Finally, CD is frequently associated with autoimmune thyroiditis and type I diabetes mellitus [42]: both these disorders are at high risk for osteoporosis [43,44].

#### *2.1. Effect of GFD*

Strict adherence to GFD allows BMD improvement but it is not able to normalize it in all cases. Mucosal recovery does not appear to be the only determining variable: in fact, with the same histological response, bone mass normalization is present in celiacs on GFD since early infancy [45] but not always in patients on GFD for the same length of time but diagnosed at a later age. In particular, normalization of BMD levels in childhood CD may be complete as early as after two years of GFD [46]. On the contrary, in adults, many cross-sectional studies demonstrated higher BMD levels in treated *vs.* untreated CD patients but still lower than in healthy volunteers [3–9,47–50]; also

the prevalence of alterations of indices of bone and mineral metabolism is lower in treated *vs.* untreated patients [47,51,52]. These results were shown in patients on GFD for a median duration of 28.5 months [5], in patients on GFD from a mean of 3.6 years [47] and in a group of patients treated for a mean of 16 years, a very long period of GFD [52]. It is therefore evident that the early onset of bone damage, probably before achieving bone mass peak, is an important time point determining GFD-induced bone mass gain. Even longitudinal studies are of little help here, as the longest period of GFD evaluated was 5 years [53]. On the other hand, these studies have provided important information on the kinetic of bone mass recovery with the start of GFD and correlations with the modifications of bone-mineral metabolism parameters. Following a GFD with optimal compliance for a period of one year allows a significant improvement of BMD values, ranging from 5% [6] to 8% [3] according to different studies. These results were confirmed in a larger cohort of patients enrolled at diagnosis and restudied after one year of GFD [8]. In a two-year study, GFD improved not only bone mass but also serum levels of indices of bone and mineral metabolism. BMD improvement was more evident after two years than after one year of GFD, suggesting that a period longer than one year was necessary to point out intrinsic capacities of an individual patient to recover bone mass. Serum levels of propeptide of type I procollagen (PICP) at diagnosis proved to be a strong predictor of bone mass gain after two years, suggesting the possibility of selecting the group of patients with high levels of bone matrix formation activity that is more likely to readily respond to GFD [54]. In a three-year study, BMD increased in 92% of CD patients in GFD with a mean bone mass gain around 3%–4% per year. However, only 12% of patients showed a normalization of BMD. In particular, in a small group of patients, it was evident that relatively good bone mass gain was present during the first year, but was negligible in the subsequent study period [55]. These observations agree with a five-year study showing femoral and lumbar BMD values at five years similar to BMD values at one-year follow-up both in men and women, with the exception of trochanter values, which proved to be higher at five-year measurement than one-year values [53].

In summary, BMD values normalize only in children, when diagnosed early in infancy and if they follow a long-term GFD with optimal adherence. On the contrary, BMD values in adults show a good improvement in the first period, generally around two years, after the institution of a GFD; the improvement is then generally unsatisfactory and treatment with a mineral-active drug should probably be considered. Nevertheless, CD patients show a wide range of response to GFD and risk factors for osteoporosis include old age at diagnosis and the degree of osteopenia in late diagnosis, compliance to GFD, menstrual status, *i.e.*, late age at menarche, early menopause, periods of amenorrhea, low body mass index (BMI), low dietary calcium intake, inadequate physical activity and use of glucocorticoids [4,52,56,57]. What appears to emerge is that as age progresses and, in women as menopause approaches, the ability to recover bone mass seems to diminish, being greatest in childhood and lowest in peri- and postmenopausal women. In this latter subgroup of patients, waiting two or three years to determine the extent of GFD-induced bone mass gain could thus be incorrect and the start of treatment with a mineral-active drug should be earlier, probably at diagnosis.

The availability of predictive markers of GFD-induced bone mass gain could be a solution for this problem, but the mechanism responsible for the unsatisfactory improvement is not completely clear. A persistent reduction of fractional calcium absorption was shown in patients on GFD, besides the improvement of intestinal mucosa architecture [17], and in a subgroup of patients the persistence of a secondary hyperparathyroidism and a significant correlation between serum PTH levels and femoral BMD were shown [17]. The possible role of secondary hyperparathyroidism was suggested by other papers [20,58] but also disproved [59], and the proposed pathophysiological mechanisms for the persistent raise of serum PTH were residual villous atrophy leading to calcium malabsorption [20], a reduction of calcium intake [18], but also a slow reversal of parathyroid hyperplasia [60]. Partial adherence to GFD [55] and incomplete mucosal recovery [57] could also have a role in subgroups of treated patients.

Circulating factors secondary to persistent activation of the mucosal immune system could directly interfere with osteoclastogenesis and osteoblast activity. It was shown that in patients following GFD for a mean period of 40 months [31] the prevalence of bone damage is around 40%, and circulating levels of cytokines (IL-6, IL-1beta, TNF-alfa, TNF-beta, IL-12, IL-18, RANK-L, OPG) are significantly lower than in untreated patients, but significantly higher than in healthy volunteers. In particular, the osteoclastogenic activity of sera from patients on long-term GFD proved to be still significantly higher than sera of healthy volunteers and serum cytokine levels were not correlated to PTH levels [31]. An altered ratio between RANKL and OPG in untreated patients normalizes to healthy volunteer levels in patients on GFD [31,35]. Accordingly, the dietary treatment with GFD alone is not able to completely control the increased osteoclast differentiation and activity present in CD, as confirmed by a strong correlation between OPG/RANKL ratio and BMD [35], and the mechanism responsible for bone damage does not involve PTH. Finally, while a three-year period of GFD determines a significant decrease of IL 6, which is significantly inversely correlated at diagnosis with lumbar BMD, it cannot normalize IL-
 "\-1 receptor antagonist serum levels [27].

Therefore, persistent inflammation in treated CD patients could have a role in the persistence of bone mass derangement. In particular, the predominant mechanism responsible for bone derangement seems different between short-term and long-term treated CD patients: in the period immediately after diagnosis, the malabsorption of calcium and the consequent hormonal and vitamin D alterations appear to be the prevalent pathophysiological mechanism, their correction allowing a satisfactory bone mass gain, comparable to the effect of administering mineral-active drugs in postmenopausal osteoporosis [61]. Unfortunately, the extent of the loss of bone mass in untreated CD is very often higher than the extent of the recovery induced just by GFD in the early stages of treatment and, once the GFD-induced metabolic surge that occurs in this phase is over, persistent bone loss seems due to the persistent activation of a local mechanism, related to chronic inflammation. Preliminary data from our group confirm this hypothesis, as an in-depth evaluation of hormonal and local factors suggests that high levels of OPG and low levels of PICP select the subgroup of CD patients with a persistent reduction of bone mass, despite strict adherence to GFD and architectural villi reconstitution [62]. If confirmed, these markers might be used to identify those patients who need mineral-active treatment associated with gluten-free diet.

#### **3. Fracture Risk**

BMD is only one of the factors that contribute to establishing the extent of fracture risk in osteoporotic patients. Other factors are related to bone mechanical characteristics, such as stiffness of cortical bone, but also to inadequacy of protectors from trauma (body mass, fat and muscle compartments) and to neuromuscular dysfunction [63].

Several studies pointed out the prevalence of fracture in celiac population, but with a very important heterogeneity in methods (study design, sample selection, fracture data collection) and cohorts studied (treated/untreated CD), making available data often inconsistent and difficult to interpret [64]. Most authors agree on the increased prevalence of fracture in CD patients [63,65–71] and a recent meta-analysis evaluating a total of 20,955 CD patients and 96,777 controls described a risk of fracture 43% greater in CD [72]. Data on fractures were collected by mailed questionnaires, by personal interviews or by medical records; consequently, results on peripheral fractures might be more easily estimated and axial fractures underestimated. Only a cross-sectional study explored the existence of asymptomatic vertebral fractures by spinal X-ray and did not find an increase of vertebral fractures in CD patients [68]. However, if CD patients are subdivided according to the clinical presentation, peripheral fracture risk proves to be higher than controls in patients with overt malabsorption symptom, while it is similar to the general population in subclinical and silent presentation [71]. These data were confirmed in a more recent case-control study, pointing out a higher peripheral fracture risk also in men and underlining again the importance of adherence to GFD [63]. However, to confirm what was said above with respect to pathophysiology, in a population-based study in Olmsted County, CD patients showed a fracture risk twice that of controls, and this figure persisted unchanged during GFD [66].

In general, however, large population-based studies should be interpreted with care, since, for example, in one study on the fracture risk in CD patients on a cohort of 1021 celiac patients, a possible misclassification of patients could have accounted for the negativity of results, as data were extracted from the National Patient Discharge Register, known for a low estimated validity of diagnosis of CD (78%) [73].

It is, finally, likely that, in addition to just BMD measurement, assessment of the physical characteristics of bone, such as its elasticity, can add something to our understanding of the mechanisms that favor fractures [74,75]. Unfortunately, no studies are available that correlate bone ultrasound densitometry parameters with fracture risk in CD patients.

#### **4. Clinical Management**

Only a limited number of international recommendations are available on the clinical management of osteoporosis in CD, probably due to the lack of sufficient data on patient follow-up and the role of the menopause. As already stated, there is no clinical or biochemical marker to select the subgroup of patients not responding to GFD alone with an improvement of bone mass and which will be characterized by a high risk of fractures; accordingly, we are not able, as yet, to optimize both treatment and timing of BMD follow-up measurement.

In 2000, the British Society of Gastroenterology published the guidelines for osteoporosis in CD [76]. General advice aimed at modifying lifestyle factor risk was provided, such as enhancing physical activity, stopping smoking, avoiding alcohol excess. Moreover, a daily calcium intake of 1500 mg, even by pharmacological supplementation, and vitamin D supplementation, if inadequate serum levels were evident, was suggested. Bone densitometry was recommended at diagnosis for all patients, to detect osteoporosis early and to obtain the greatest possible benefit from treatment, or at least at menopausal age for women and at the age of 55 years for men. Postmenopausal women with normal BMD should repeat densitometry after two years. Osteoporotic postmenopausal women and men over 55 years should be offered treatment and bone densitometry yearly to monitor treatment efficacy.

The subsequent awareness of an ultimately low absolute risk of fracture, even if higher than in the general population [77], determined strong criticism of the extensive use of bone densitometry at diagnosis, suggesting that BMD measurement should be restricted to patients with high short-term fracture risk, such as patients non-compliant with GFD or who failed to respond to dietary treatment, on glucocorticoid therapy, with untreated hypogonadism, older age, low BMI, and previous fragility fracture [78]. This led to reconsideration of the guidelines and the proposal of BMD measurement only in clinically non-responder patients, especially those with low BMI, in menopausal women and after 55 years for men [79]. In 2003, the American Gastroenterological Association guidelines on osteoporosis in gastrointestinal disease suggested that bone densitometry should be performed in adults with newly diagnosed celiac disease after one year of GFD, to allow for stabilization of bone density, implementation of GFD, calcium and vitamin D supplementation as needed, and, if necessary, bisphosphonates and hormonal therapy were strongly encouraged in osteoporotic patients [80].

A Canadian Position Statement on evaluation and management of skeletal health in CD was recently published [81]. BMD measurement was suggested at diagnosis only in adults with classic CD, and after one year of GFD in adults with asymptomatic or silent CD. The latter group of patients should be considered for earlier BMD evaluation in the presence of risk factors such as menopause, older age, history of fragility fracture, unexplained iron deficiency anemia, vitamin D deficiency/insufficiency, and high titers for CD serological markers. Indications for follow-up were also given: BMD should be re-evaluated after one year of GFD in the presence of osteopenia/osteoporosis at diagnosis, and after two years in cases of documentation of normal bone mass. The assessment of bone and mineral metabolism by dosing serum calcium, albumin, 1,25 dihydoxicolecalciferol and PTH levels should be repeated every six months until normalization.

Clinical application of the Canadian guidelines does not, however, seem to allow substantial resource savings, and is thus very similar to the earlier British proposal. In patients with asymptomatic or silent CD with the aforementioned risk factors, early prescription of bone densitometry is indicated, since anemia and vitamin D alterations show a very high prevalence also in this subgroup. Considering the prevalence of bone loss in patients with and without overt malabsorption symptoms, BMD measurement could provide more important information in asymptomatic than in clinically overt malabsorber patients: bone loss is highly prevalent in overt malabsorption, and these patients could undergo mineral-active therapy as of diagnosis; conversely, asymptomatic/silent patients should be screened for bone loss presence. Moreover, the most important risk factor at diagnosis seems to be the age of the patient, and patients well over the age of peak bone mass could be treated without measuring BMD, while those patients below or shortly after the age of the peak should undergo BMD measurement. This approach seems the most correct one in optimizing the use of bone densitometry, but the problem remains for the free dispensing of the mineral-active drug, which in Italy depends on the presence of a pathological fracture. Optimization of this phase could be achieved by performing a radiological study of the lumbar spine in patients most at risk, such as symptomatic subjects, peri- and postmenopausal women and men over the age of 55, in order to detect vertebral fractures, together with a complete case history for previous fractures. In Italy (Lombardia Region), the cost of lumbar and femoral densitometry is €88.66 (€44.33 for each segment), while the cost of a lumbar spine X-ray is only €34.80.

In conclusion, there is no general agreement on the correct timing of bone densitometry in celiac patients; screening at diagnosis seems to be not justified in all patients and the proposed alternative approach is explained in Figure 1.

**Figure 1.** Diagnostic and therapeutic approach to CD patients without a previous fracture.

#### Repeat Densitometry every two years

Treatment with a mineral-active drug in association to GFD, even without BMD measurement, should be prescribed to patients at high short-term risk of fracture, including symptomatic patients, asymptomatic patients in peri and postmenopausal period, men older than 55 years, low calcium intake, low BMI, poor compliance to GFD, or unresponsiveness to GFD following steroid therapy; in these cases, measure BMD after two years of treatment. To allow the free dispensing of the drug, patients should be screened for the presence of fracture and X-ray of the spine could be adequate to this aim. Young asymptomatic patients with a normal BMD should be re-evaluated at peri-menopausal period (female patients) or over 55 years (male patients).

As regards the choice of the drug for the treatment of osteoporosis in CD, there are no longitudinal studies dealing with this topic and we have no information on which to base our choice in the individual subgroups of patients. The current approach is clearly based on post-menopausal osteoporosis treatment with a choice between a weekly administration of alendronate and a monthly administration of ibandronate. The use of denosumab, a decoy receptor for RANKL able to reduce the activation of the osteoclast system, seems very interesting, also on the basis of the data obtained on the RANK/RANKL/OPG system. The results of the first studies are awaited in order to define the best strategy for the different types of CD patients.

#### **5. Conclusions**

In conclusion, in the pathophysiology of bone derangement in CD patients, both malabsorption and the persistent activation of inflammation at intestinal level are important, in a two-step model. Biomarkers with a predictive role of the normalization of BMD levels are needed and the evaluation of the RANK/RANKL/OPG system could offer some inputs on this topic.

#### **Conflicts of Interest**

The authors declare no conflict of interest.

#### **References**

Reprinted from *Nutrients*. Cite as: Pellicano, R.; de Angelis, C.; Ribaldone, D.G.; Fagoonee, S.; Astegiano, M. 2013 Update on Celiac Disease and Eosinophilic Esophagitis. *Nutrients* **2013**, *5*, 3329-3336.

*Review*


**5. Effects of EOs on milk fatty acids profile**

Milk fat content and FA composition are primarily determined by lipid metabolism in the rumen and the mammary gland [39]. EO supplementation can reduce the levels of C8 and C12 fatty acids. This occurs because EOs can produce potent inhibitors that hinder the synthesis of de novo FA in the mammary gland [40, 41].

The rate and extent of FA biohydrogenation could also impact variations among animal species [42]. In small ruminants, supplementation of EOs is more effective in the biohydrogenation process. For instance, Juniper EO addition enhanced the presence of n-3 fatty acid in goat milk, while anise, clove, and juniper had minimal impact on milk components [43]. Cinnamon oil elevated the concentration of unsaturated FA, n3 linolenic acid, and conjugated linoleic acid [44]. The inclusion of garlic or juniper EO in the diet of dairy cows resulted in an increased proportion of conjugated linoleic acid in milk [45]. Clove and thyme EO were found to be more effective in decreasing the biohydrogenation process and increasing the concentration of C18:3 n-3, C18:4 n-3, and n-3 FA in milk fat and decreased the concentrations of C18:3 n-6, C20:4 n-6, and n-6 FA in the milk of lactating goats [6].

Dairy cattle fed a blend of EOs (capsaicin, carvacrol, cinnamaldehyde, and eugenol) secreted more CLA, suggesting that EO influenced the ruminal biohydrogenation of PUFA [46]. Supplementation with 750 mg of a mixture of EOs had no change in milk FA profile [47]. However, supplementing 2 g/d of the same mixture increased the concentration of CLA in milk fat [48].


**9. Plural drivers of biodiversity losses**

To acquaint with what biodiversity is and why it should be important is the major step toward pluralism; however, it is not sufficient on its own. It is also important to recognize why bio-diversity in its diverse forms is being lost and what are different

#### *Biodiversity Conservation of Western Himalayas: A Pluralistic Approach DOI: http://dx.doi.org/10.5772/intechopen.107075*

combinations of activities at various labels might reduce or repeal the damage to nature meticulously. Specifically, one has to take out what is generally called the causes of biodiversity loss and degradation of nature [50, 51] or—drawing on our plural characterization above—what kinds of human actions and social processes are leading to the undermining of facets of nature and what makes those actions and processes persist.

Firstly, there should be a strong tendency to cast explanation in universal or globalized terms. Even though it is useful to categorize the biggest drivers of biodiversity decline as a resource over and land-cover change for agriculture purposes at a large scale, these analyses have often been conducted in a collective way without distinguishing these processes in terms of localities or actors [52]. Thus, the driver-based studies should go further to find out what sectors are responsible for detrimental activities and who benefited from them.

Secondly, scientific analysis of drivers normally hazards reducing bio-diversity, to a set of the singular index, reflecting a desire to let science drive policy at the cost of opening space for other means to know the natural world and as a result for consideration. Adding to that, biodiversity cannot be just lessened to a singular index, but the issue itself is much more complex than, for instance, the conventional drivers—pressures—state—impacts—responses structure can be handled [53, 54]. There are manifold clarifications for several causes behind the continued reduction of biodiversity. Economists thought that mankind as mostly independent realistic drivers and thus, motivate the utilization of biodiversity to observe win-win solutions. Conversely, ecologists should give priority to dependent and post-colonial structures of authority that relieve local communities of land rights which leads to state community differences and as a result, may advocate restitution of these human rights, and chiefly respect to the world views of native local communities as the first step toward sustainable management of nature. Others may emphasize macro-level institutional failure based on ever-expanding capital accumulation as the overarching single cause of the ongoing ecological crisis [55].

Finally, communal analytic effect on biodiversity change has been assembled into 'a political' clarification that directly emphasizes population pressure-based descriptions for the loss of interpreted pristine environment and more political descriptions that unite concern for communal justice, and acceptance of racially co-established concept about the natural world, with other justifications like widespread property conjecture placed in the middle. This divergence allows conservation groups to accentuate what looks doable and gives the existence of leading economic-political structures rather than what desires to be done.


**Acknowledgements**

The authors extend their heartfelt gratitude to their affiliation institute for encouraging publishing of such works for the readers of the journal. Authors also acknowledge and thank the editors for the opportunity to publish their work.


**1. Introduction**

Healthcare-associated infections (HAIs) are among the most serious preventable complications in neonatal intensive care units (NICUs) [1]. Preterm infants are susceptible to HAIs because of their immature immune systems and prolonged need for indwelling catheters [2]; the risk of these diseases is inversely associated with birth weight and gestational age and increases with time spent in care [3]. The most common type of HAI in NICUs is bloodstream infection (BSI), which can occur in isolation or in association with urinary tract infections and meningitis [3]. The main microorganisms responsible for HAIs include *Staphylococcus aureus*, coagulase-negative *Staphylococci*, and *Enterococci*. In addition, recent years have recorded a considerable increase in HAIs sustained by Gramnegative bacteria and fungi, especially *Candida* spp., which are mainly responsible for ventilator-associated pneumonia (VAP) and urinary tract infections, but also for peritonitis and meningitis [3,4].

According to a study conducted in 2016/2017 [5] using the European Centre for Disease Prevention and Control protocol, the prevalence of HAIs in Italian NICUs was around 5%. However, the organizational challenges experienced during the COVID-19 pandemic may have limited the effectiveness of traditional HAI prevention and control efforts, resulting in an increase in their incidence, as already reported for the adult intensive

**Citation:** Ceparano, M.; Sciurti, A.; Isonne, C.; Baccolini, V.; Migliara, G.; Marzuillo, C.; Natale, F.; Terrin, G.; Villari, P.; The Collaborating Group. Incidence of Healthcare-Associated Infections in a Neonatal Intensive Care Unit before and during the COVID-19 Pandemic: A Four-Year Retrospective Cohort Study. *J. Clin. Med.* **2023**, *12*, 2621. https://doi.org/10.3390/ jcm12072621

Academic Editors: Olivier Mimoz, Luca Brazzi and Giorgia Montrucchio

Received: 2 March 2023 Revised: 24 March 2023 Accepted: 29 March 2023 Published: 30 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

care unit (ICU) of Umberto I teaching hospital of Rome [6] or in a recent systematic review that investigated *Pseudomonas aeruginosa* bacteremia [7]. Indeed, despite NICUs having one of the lowest COVID-related caseloads among all ICUs, they are still vulnerable to indirect adverse effects of the COVID-19 pandemic [8]. During the pandemic, NICUs faced challenges that were different in nature from those in pediatric and adult ICUs; there were particular concerns relating to clinical workflows and parent–child interactions [9,10], including uncertainty in how to address the risk of exposure for mothers and their babies, reorganization of processes and operations aimed at minimizing risks to staff and patients, and frequent changes in clinical scenarios [8].

While the impact of the pandemic on incidence rates of nosocomial infections in adult ICUs has been investigated [6,11–13], data from the NICU setting are scarce. A few indirect effects of the pandemic on NICUs have been described, such as psychological distress or obstacles in implementing family-centered care [14]. In addition, a reduction in hospitalwide availability of alcohol-based hand rubs was reported to be associated with an increase in the rate of central line-associated BSIs in a single-center study [8]. However, few studies have investigated the impact of pandemic-related measures on the incidence of HAIs in preterm infants admitted to NICUs [8,15]. Therefore, the aim of this study was to analyze the occurrence of HAIs in neonates admitted to the NICU of Umberto I teaching hospital of Rome before and during the COVID-19 pandemic and to identify key factors associated with HAI onset.

#### **2. Materials and Methods**

#### *2.1. Setting*

In this cohort study, we retrospectively analyzed patients hospitalized in the NICU of Umberto I teaching hospital of Rome from 1 March 2018 to 28 February 2022. Patients were followed until discharge or 23 March 2022. The NICU has a total of six beds in which healthcare providers take care of critically ill babies born in this hospital or coming from other hospitals in Rome and the Lazio region via the Neonatal Emergency Transport Service. We followed the STROBE guidelines to report our findings. Microorganisms' antimicrobial susceptibility profiles were defined according to the classification proposed by Magiorakos et al. [16] (if applicable), whereas coagulase-negative *Staphylococci* were considered as susceptible or resistant to oxacillin and/or glycopeptides [17]. The institutional ethics board of the Umberto I teaching hospital of Rome approved this study (protocol no. 888/2022).

#### *2.2. Data Collection*

Data about patients hospitalized in the NICU were retrieved from the prospective patient-based HAI surveillance system that has been conducted in the unit since March 2014 by the Department of Public Health and Infectious Diseases of Sapienza University of Rome. The surveillance personnel routinely review and collect data from patients' medical records, including clinical data and microbiological findings, on a weekly basis using a standardized form. All neonates hospitalized in the NICU for at least 48 h are included and followed until their discharge from the NICU. Data on date of birth, date of admission and discharge, sex, gestational age, birth weight (BW), type of delivery, admission diagnosis (preterm birth, twin pregnancy, or respiratory distress syndrome), exposure to invasive devices (days of central line catheterization, including umbilical catheter, central venous catheter and peripherally inserted catheter, and days of mechanical ventilation), use of antimicrobial agents (days), site of infection, date of infection onset, and microorganism isolated are routinely collected. An infection is considered to be healthcare-associated if it occurs 48 h after birth or admission. The surveillance system records central/umbilical lineassociated bloodstream infections (CLABSIs) and ventilator-associated pneumonia (VAP) and any other type of infection that occurs during hospitalization, the diagnosis of which is determined by an infectious disease specialist. All infections are defined according to the

standard diagnostic criteria published by the Center for Disease Control and Prevention (CDC), adapted to neonatal pathology [18].