*Review* **Telemedicine in Primary Practice in the Age of the COVID-19 Pandemic—Review**

## **Anna Romaszko-Wojtowicz 1,\*, Łukasz Ja´skiewicz 2, Paweł Jurczak 3 and Anna Doboszy ´nska 1**

**Abstract:** *Background and Objectives*: In the era of the COVID-19 pandemic, telemedicine, so far underestimated, has gained in value. Currently, telemedicine is not only a telephone or chat consultation, but also the possibility of the remote recording of signals (such as ECG, saturation, and heart rate) or even remote auscultation of the lungs. The objective of this review article is to present a potential role for, and disseminate knowledge of, telemedicine during the COVID-19 pandemic. *Material and Methods*: In order to analyze the research material in accordance with PRISMA guidelines, a systematic search of the ScienceDirect, Web of Science, and PubMed databases was conducted. Out of the total number of 363 papers identified, 22 original articles were subjected to analysis. *Results*: This article presents the possibilities of remote patient registration, which contributes to an improvement in remote diagnostics and diagnoses. *Conclusions*: Telemedicine is, although not always and not by everyone, an accepted form of providing medical services. It cannot replace direct patient–doctor contact, but it can undoubtedly contribute to accelerating diagnoses and improving their quality at a distance.

**Keywords:** COVID-19; telemedicine; primary practice

### **1. Introduction**

Telemedicine, also called telehealth, is the provision of health-related services over a distance using digital communication technologies. The origins of telemedicine date back to the 1950s, when the first mentions of the possibilities of the remote transmission of imaging tests appeared [1]. Telemedicine may take different forms, e.g., it can be in the form of communication between a patient and a doctor such as a doctor's advice via telephone, chat, or videoconference. It may also be associated with more experimental innovations, e.g., telesurgery, where a surgeon remotely manipulates surgical instruments with the aid of a robotic surgical system.

The pandemic caused by the SARS-CoV-2 virus has stimulated the more rapid development of telemedicine. Pursuant to the WHO recommendations, telemedicine was chosen as a key strategy for the provision, maintenance, and enhancement of health-related services which were disrupted by the COVID-19 outbreak [2]. There are many scientific articles dedicated to the use of telemedicine in various specialties (e.g., internal diseases, family medicine, psychiatry, oncology) [3]. According to the report presented by Omboni et al., the most common purpose of using telemedicine (49.7%) was to ensure integrated patient care, including a combination of services with the aim of providing diagnosis, treatment, observation, and rehabilitation [4]. Monitoring patients in their home environment enables doctors to gain a better insight into the social conditions of their patients' health statuses.

**Citation:** Romaszko-Wojtowicz, A.; Ja´skiewicz, Ł.; Jurczak, P.; Doboszy ´nska, A. Telemedicine in Primary Practice in the Age of the COVID-19 Pandemic—Review. *Medicina* **2023**, *59*, 1541. https:// doi.org/10.3390/medicina59091541

Academic Editors: Yusra Habib Khan, Tauqeer Hussain Mallhi, Tahir Mehmood Khan and Muhammad Salman

Received: 14 July 2023 Revised: 18 August 2023 Accepted: 22 August 2023 Published: 25 August 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/).

Of key importance for the development of telemedicine is the growing accessibility of technologies. In line with the Pew Research Centre's report of 2021, 93% of Americans use the internet [5]. Moreover, 81% of Americans have smartphones, nearly 75% have desktops or laptops, and around 50% have tablets or e-readers [6]. Nowadays, telemedicine does not have to be limited to consulting one's doctor by telephone, but can involve the use of other, more modern methods, e.g., measuring instruments which allow objectivization of symptoms reported by the patients and enable the doctor to make a correct diagnosis, which means that telemedicine can improve significantly. These are instruments such as cameras and video cameras in smartphones, digital stethoscopes, ophthalmoscopes, otoscopes, and various types of biosensors. This form of telemedicine, in which mobile medical devices and technologies are employed in order to collect health data generated by the patient (PGHD—patient-generated health data) and transmitted to health care providers, is referred to in the literature as remote patient monitoring (RPM) [7].

This starts from the simplest solutions, in which medical documentation is stored, monitored, and edited practically from anywhere in the world, to more complex ones related to artificial intelligence. In this way, it is possible, for example, to remotely supervise surgical procedures. Telemedicine is already widely used in emergency medicine systems. ECG transmission, in terms of qualification for invasive treatment, has already become a standard which is possible in most ambulances.

The purpose of this systematic review has been to evaluate telemedicine technologies that were employed during the COVID-19 pandemic, from the viewpoint of optimization of their use in situations of limited direct access to a physician. In particular, we are interested in the issue of using telemedicine techniques used in primary health care, as if creating a telemedicine primary care guide.

### **2. Material and Methods**

The study was conducted according to the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA) statement [8]. The systematic review of the literature was based on an a priori definition of inclusion and exclusion criteria, which enabled an objective selection of articles dealing with the connections between telemedicine and COVID-19. This approach allowed us to preselect the research material objectively, preventing any subjectivity in our decisions to include some or exclude other studies from the review. The search employed the following terms: "telemedicine, telehealth, lung, respiratory, COVID-19, SARS-CoV-2, diagnosis, symptoms".

The following search sequences were used in the work:

Due to the multitude of available studies and the symptomatology of COVID-19, the study was limited to searches related to the involvement of the respiratory system.

In this way, attempts were made to isolate publications enabling the assessment of the respiratory system, using methods of remote assessment of the general condition of patients, including remote registration. In particular, we wanted to present in the review solutions facilitating the work of a clinician at the level of primary care.

The review of the literature took advantage of the following databases: ScienceDirect, Web of Science, and PubMed. While searching the ScienceDirect database, a filter was activated to exclude meta-analyses, reviews (also systematic reviews), and books, in addition to which the search was limited to studies in medical sciences and that were original publications. The Web of Science database was searched for articles in medical sciences. As for PubMed, the same filters as applied to ScienceDirect were used. Some records were also retrieved via references found in published articles. No limits regarding the date of publication were set. Detailed results of the search are presented in a flow diagram (Figure 1) [9,10]. The search was carried out in the fourth and fifth weeks of March 2023.

**Figure 1.** PRISMA flow diagram.

### *Selection of Studies and Exclusion Criteria*

For further preselection of papers, the software Rayyan was employed [11]. Firstly, duplicates were eliminated and records implicated by the software as possibly being duplicates were reviewed. Next, three independent researchers analyzed the abstracts, and those dedicated to telemedicine methods used during the COVID-19 pandemic were selected. Lists of references were searched manually in order to identify further publications suitable for our analysis. Thus, articles were selected compliant with the previously established exclusion and inclusion criteria for a full-text review. Any disputes were resolved by consensus. In total, 234 publications were checked and 34 were chosen for complete analysis. Having analyzed the complete texts, 22 publications were selected for the systematic review.

The inclusion criteria were to choose original articles, published in the English language, with clearly determined measures applied to clinical results. The exclusion criteria were to discard studies with unclear measures applied to clinical results, descriptions of single cases, or series of cases with a sample size <5. The articles selected for the final analysis are collated in Table 1. A forest plot was used to visually depict publication bias (Figure 2).

**Figure 2.** Plots of the proportion of the incidence of symptomatic and asymptomatic acute respiratory tract infection in COVID-19.

**Table 1.** Detailed breakdown of work selected for analysis.

#### **Table 1.** *Cont.*

### **3. Results**

The analyzed studies were published from July 2020 to December 2022. The 22 reviewed articles deal with seven issues, presented in Figure 2: telemedicine (telephone consultations, chats, and video consultations) (n = 5); AI techniques (n = 2) and algorithms (n = 4), that is, the issues related to telehealth and remote registration of patients, and the use of USG (n = 2); stethoscopes (n = 4); mobile applications (n = 3); and the "wearable body sensor network" (n = 2). Most studies were conducted in Europe (n = 7), and the remaining papers originated from Asia (n = 7), North America (n = 6), Australia (n = 1), and South America (n = 1). The most articles about telehealth covering various types of teleconsultations was published in Europe [13–15] and one work in North America [16] and South America [12]. In Asia, all publications concerned AI techniques and remote patient monitoring. It is worth noting that all publications on the use of mobile applications [23–25] and wearables [26,27] were created there. The use of stethoscopes as a tool for remote control of the patient's health was established in Asia [29], Europe [28,30], and North America [31]. The articles on the use of ultrasonography in patient follow-up were exclusively from North America [32,33]. The creation of algorithms useful in medicine based on artificial intelligence has been described in Asia [21], Europe [19,20], North America [18,22], and Australia [17]. These divisions are presented in Figure 3.

The preselected publications focused on the following aspects: providing healthrelated consultations via telemedicine using applications designed for this purpose, as well as a remote assessment of a patient's condition with the help of the so-called wearable body sensor network, electronic stethoscopes, and a lung USG (Figure 4).

The presented review focuses on the aspects of telemedicine used in the practice of GPs. These methods focus on three main aspects, i.e., telehealth, methods of remote registration of patients, and the use of artificial intelligence methods for their assessment.

**Figure 3.** The use of telemedicine in different regions of the world. AI, artificial intelligence; RPM, remote patient monitoring.

**Figure 4.** Telemedicine architecture used in the publication. Created with BioRender.com.

### *3.1. Telehealth*

Telehealth consultations can be provided through various telemedicine channels, e.g., by telephone advice, chat, or videoconference. Remote consultations can be used both for a full patient interview, as well as to collect screening information enabling further proper triage of patients, which turned out to be particularly important during the COVID-19 pandemic [13]. The content and quality of consultations, regardless of their form (teleconsultation, chat, videoconsultation) are comparable [34].

### *3.2. Artificial Intelligence*

Telemedicine solutions combined with artificial intelligence allow for the creation of algorithms that facilitate decision making or guide patients or doctors through the diagnostic path. These algorithms can be used to assess the general health status and cohort of patients. They can also make the first diagnosis based on the sent image of the throat or the recorded cough sound [21].

### *3.3. Remote Patient Monitoring*

Remote patient registration methods are aimed at obtaining health parameters based on wearable body sensors, mobile apps, stethoscope, or ultrasound. Wearable body sensors, using sensors attached to the body, collect information about heart rate, saturation, respiratory rate, ECG, and body temperature [35]. It is also possible to remotely assess breath sounds and images of the lungs. Wearable body sensors are becoming an increasingly accurate diagnostic tool to help identify and treat diseases [36].

The detailed specifications of the articles, divided according to subject areas, is contained in Table 1.

### **4. Discussion**

Telemedicine, also known as telehealth, involves the use of technologies to facilitate remote patient monitoring. Scientists have developed many different forms of telemedicine systems to fight the pandemic. This literature review is supposed to emphasize the possibilities of telemedicine methods which can be used in primary health care. The COVID-19 pandemic itself led to a significant increase in the number of health-related consultations provided with the help of ICT tools. This has necessitated, in a certain manner, the development of a collaboration between IT and medicine for the best possible objectivization of the health services provided. Monitoring the health status of patients with COVID-19 remotely can take different forms, from giving advice by telephone to using custom-designed teleinformation tools via the internet. Appropriate remote monitoring of the health of patients and chronic conditions can help to reduce the number of patients who need to be hospitalized, and this lowers the costs of medical care [37]. In a paper published in 2023 in JAMA, it was shown that telemedicine advice contributes to significant time and cost savings, including transport costs and the cost of medical visits. Increasing access to a doctor results in a reduction in the number of visits, hospitalizations, and mortality [38].

In our study, we have shown the possible division of telemedicine into three categories: telehealth, AI, and remote patient monitoring. This division has been used in order to easily present the possibilities of remote medical care methods, e.g., in a family doctor's office. Thanks to the extremely rapid progress of technology in the 21st century, medical care provided through telemedicine channels does not have to differ much from direct contact with the patient. Not only that, according to the data presented in Figure 2, the recognition of respiratory tract infections by various methods of telemedicine can be just as effective.

### *4.1. Telehealth*

During the outbreak of the COVID-19 pandemic, teleconsultations served to provide adequate education to patients in order to restrain the spread of the disease. Furthermore, telemedicine gave patients emotional support [13]. However, it soon turned out that teleconsultations were on the front line in effective management and conduct during the COVID-19 pandemic. Telephone history taking in primary health care was effective in detecting pneumonia in patients diagnosed with mild SARS-CoV-2 [16]. Accorsi et al., in their randomized trial, showed that teleconsultation is comparable to an in-patient consultation for patients with a low risk of progressing to a severe course of COVID-19 infection who developed symptoms of acute respiratory tract infection [12].

Truong et al., based on their systematic review, confirmed that teleconsultations show a high level of care and satisfaction of patients [39].

### *4.2. Artificial Intelligence*

An artificial neural network (ANN), or simply a neural network, is a method of supervised learning. The learning process tries to mimic the learning that takes place in the human brain. Artificial intelligence models are employed to create appropriate groups of patients [23]. AI enables researchers to use data collected, for example, in the form of digital diagnostic tests, in order to improve telemedicine consultations.

Artificial intelligence methods based on a convolutional neural network (CNN) were used to evaluate chest X-rays of patients with COVID-19. [25] They served to help diagnose and classify COVID-19 cases, and to distinguish patients with COVID-19 from other patients with (viral or bacterial) pneumonia [40–43]. It was demonstrated that a preliminary evaluation of chest X-rays could be achieved remotely, considerably limiting of person-toperson contact. A deep learning model (CycleGAN) was used by Yoo et al. to detect severe cases of pharyngitis using a smartphone [21]. This approach was employed for screening patients in order to rapidly identify cases of pharyngitis and launch proper diagnostic and treatment procedures.

Algorithms created by artificial intelligence can support the diagnostic and therapeutic process. An example of this can be the management of community-acquired pneumonia, which can be one of the manifestations of COVID-19. Diagnosis of community-acquired pneumonia (CAP) is based on an evaluation of the signs and symptoms of a respiratory tract infection. Its clinical image varies. The manifestations of pneumonia can be divided into two groups: systemic (pyrexia, chills, malaise) and related to the respiratory system (cough, dyspnea, chest pain). The diagnosis of pneumonia in outpatients can be made on the basis of clinical manifestation not necessarily confirmed by laboratory and imaging tests. This option gained particular importance during the COVID-19 pandemic when the isolation of patients and the limitation of direct human contact were most important from the point of view of epidemiology. Porter et al. proved that the mathematical algorithm they tested enabled the accurate identification of patients with CAP of varying severity, excluding an analysis of vital signs and physical and radiological examinations, in addition to which it ensured an immediate result. These Australian researchers built an algorithm on the basis of such symptoms as pyrexia, acute cough <7 days (registered by a smartphone), and age. The algorithm was then used to make a preliminary selection of patients [17].

Coronado-Vázquez et al. carried out telemedicine monitoring of patients with COVID-19, which enabled early detection of complications as well as the monitoring and treatment of concomitant illnesses, thereby contributing to reducing the risk of hospitalization [15]. In turn, Liu et al., who used an online application specially developed for their study, enabled patients to provide in real time all new data regarding the course of the illness, which contributed to gaining a better insight into the disease itself and improved overall evaluation of the health status of a given patient. The above researchers conducted medical consultations using an online application, including a preliminary triage, with the help of a voice conversation, text messaging, photo communication, or a video call. Out of 4589 patients, 310 were referred to the hospital and 301 were recommended to see a doctor in an in-patient setting (e.g., in a hospital) for physical examination. The cited authors demonstrated that telemedicine can facilitate the initial selection of patients, particularly during health crises such as COVID-19 [23].

Telemedicine services provided via another application (nCapp) were presented by Yang et al. [24]. In this case, the mobile tool used served to synchronize and share the data concerning the diagnosis and previous treatment. The aim of this application was to enable early diagnosis of COVID-19 and classification of patients (with a focus on patients with ambiguous or false negative results of RT-PCR tests) to appropriate risk groups. Owing to this application, the remote management of new cases of COVID-19 infections was improved.

Amjad et al., in their systematic review published in January 2023, have summarized the methods of artificial intelligence currently used in medicine. They proved that telehealth based on artificial intelligence can lead to an improvement in the quality of medical practice and also contribute to its modernization [44].

### *4.3. Remote Patient Monitoring*

Monitoring of the health conditions of patients may take different forms, and one option employed nowadays is to use biosensors, which enable making non-invasive measurements. The sensors used in these devices read parameters from the skin or from the movements made by the person being monitored [45,46]. This allows the monitoring of a patient in any circumstances, including at home, remotely. The wearable body sensor network, according to Qureshi et al., is most probably the best solution for the remote monitoring of patients in health care systems. Such sensors as accelerometers, temperature sensors, or ECGs collect information about the health of a patient, acting as a monitoring network. The data are stored in a local server and can be retrieved by a clinician to aid the decision-making process [47].

Bassam et al. described a system that makes use of an online application as an external interface and an Android-based mobile application for the patient [27]. Both interfaces are synchronized with each other in order to gather data on the health of a patient. The system is composed of a device mounted like a bracelet on one of the patient's limbs. It allows recording of the following parameters: body temperature, systolic heart rate, saturation, and cough episodes. It also has a built-in GPS reader.

Similar systems used during the COVID-19 pandemic for remote registration of vital signs have been presented in the literature by Balasubramanian V., Ding X, Romaszko et al. [26,48,49].

It seems that wearable body sensors may solve the monitoring problem, as was shown by Snehi et al. [50].

Telemedicine can play a role in decreasing the costs of health care borne by the patient and by the health care system. Adequate remote monitoring of health conditions and chronic conditions can help patients to avoid expensive visits to hospital emergency wards and even hospitalization. A stethoscope is an inexpensive instrument that is easy to use, but which can considerably facilitate making a preliminary diagnosis. However, its usefulness largely depends on the user's perceptual ability and experience. In recent years, there has been a growing interest in the automation of auscultation, its standardization and digitalization. This trend gained momentum during the COVID-19 pandemic when any form of a remote physical examination helped to make a diagnosis via telemedicine. A team of researchers from Switzerland created an algorithm for the diagnosis and stratification of COVID-19 risk based on lung auscultation [28]. To this aim, an algorithm involving artificial intelligence and deep learning was developed that achieved standardization of lung auscultation. The data for their research were recorded with a digital stethoscope Littmann 3200. It was demonstrated that automated interpretation of lung auscultation can help to improve the accuracy of a physical examination. In turn, Zhu et al. employed artificial intelligence models and showed that an AI-based system can identify correctly different types of irregular murmurs detected during an auscultation examination of the lungs [29].

Pancaldi et al. created an algorithm called VECTOR (Velcro crackles detector) to evaluate Velcro crackles registered with the help of a digital stethoscope [51]. In 2022, these authors used the VECTOR algorithm to identify patients with interstitial pneumonia secondary to SARS-CoV-2 infection [30]. To this aim, a digital stethoscope Littmann 3200 was used to evaluate respiratory crackles in eight auscultation points, i.e., paravertebral lower lobes, axillary lower lobes, paravertebral middle lobes, and paravertebral upper lobes. The automated lung auscultation results proved the potential usefulness of artificial intelligence methods in the near future.

Undoubtedly, the COVID-19 pandemic has contributed to the search for new methods of remote diagnosis. In March 2023, *Diagnostics* published a paper comparing the types of stethoscopes, including electronic stethoscopes. The authors of this article showed many benefits of remote lung auscultation, or even the sound recording itself. They also did not omit the problem related to the use of artificial intelligence to interpret the recorded auscultation phenomena, which primarily result from their possible variability [52].

A group of 27 volunteers was submitted to a study in which a portable USG device and a teleconference on the Zoom platform were used to examine the images of the lungs of patients with COVID-19 [32]. The purpose of the study was to generate an adequate "batwing" image of the pleura interface between two rib shadows at each location on the thorax. The use of ultrasound ensures reliable monitoring and stratification of the risk of developing serious illnesses. These conclusions were confirmed by Kimura et al., in a study including 201 patients, in which the presence of a B line, which is associated with a higher risk of hospitalization of high-risk patients, was detected in the early phase of SARS-CoV-2 infection [33]. Moreover, these researchers confirmed that patients were able to perform a simple lung USG test themselves. Heldeweg et al., based on a systematic review of the literature, proved that ultrasound imaging of the lungs significantly affects clinical decision making, especially in the so-called places for quick diagnostics (e.g., emergency departments) [52].

The usefulness of a remote ultrasound exam has also been verified in cases of abdominal USG tests [53]. However, because of the topic area of this study not pertaining to the subject of our review, this paper was not included in the current systematic review.

Telemedicine is a rapidly developing field of medicine which has already been used widely owing to its usefulness and ability to provide medical services safely during the COVID-19 pandemic. It is obvious that telemedicine, nowadays, cannot replace personal patient care and that not all clinical situations can rely on video consultations alone, but it is also undeniable that in the time of medical staff shortages, telemedicine can help to improve the monitoring of the health status of patients, the functionality of the health care system, as well as the accessibility to medical advice. Not long ago, the only option for remote communication was by letter. Other remote communication channels, not to mention remote medical diagnosis methods, were unknown. The COVID-19 pandemic showed that medical care in the form of telehealth could be the not-so-distant future of medicine. Technological progress, a collaboration between IT and medicine, and the development of pro-health applications, such as 'Apple-health', remote auscultation, or imaging of internal organs, may become commonplace in everyday life.

The variety of methods used in telemedicine allows for almost unlimited possibilities for patients' remote registration. At present, it is possible to conduct a full history-taking using remote methods of communication as well as remote registration and transmission of vital measurements enriched with the possibility of recording auscultatory changes. The analysis of these data using artificial intelligence methods minimizes the risk of medical error.

### **5. Conclusions**

The COVID-19 pandemic has changed the perception of telemedicine. Conducted scientific studies have shown that telemedicine can be highly effective in recognizing upper respiratory tract infections, regardless of the type of method used.

The rapid development of 21st century technology may soon lead to even more efficient methods of telemedicine.

**Author Contributions:** Conceptualization, A.R.-W.; methodology, A.R.-W., Ł.J., P.J. and A.D.; formal analysis, A.R.-W., Ł.J. and P.J.; investigation, A.R.-W. and Ł.J.; resources, A.R.-W.; data curation, A.R.-W.; writing—original draft preparation, A.R.-W. and Ł.J.; writing—review and editing, A.R.-W., Ł.J., P.J. and A.D.; visualization, A.R.-W.; supervision, A.D.; project administration, A.R.-W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data are available in a publicly accessible repository.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

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**Diana Dueñas 1, Jorge Daza 2 and Yamil Liscano 1,\***

**Abstract:** The COVID-19 pandemic has had significant impacts on healthcare systems around the world, including in Latin America. In Colombia, there have been over 23,000 confirmed cases and 100 deaths since 2022, with the highest number of cases occurring in females and the highest number of deaths in males. The elderly and those with comorbidities, such as arterial hypertension, diabetes mellitus, and respiratory diseases, have been particularly affected. Coinfections with other microorganisms, including dengue virus, *Klebsiella pneumoniae*, and *Mycobacterium tuberculosis*, have also been a significant factor in increasing morbidity and mortality rates in COVID-19 patients. It is important for surveillance systems to be improved and protocols to be established for the early detection and management of coinfections in COVID-19. In addition to traditional treatments, alternatives such as zinc supplementation and nanomedicine may have potential in the fight against COVID-19. It is also crucial to consider the social, labor, educational, psychological, and emotional costs of the pandemic and to address issues such as poverty and limited access to potable water in order to better prepare for future pandemics.

**Keywords:** COVID-19; SARS-CoV-2; coinfections; antimicrobials; Colombia; epidemiology; coinfections; superinfections

### **1. Introduction**

In December 2019, in Wuhan, China, an unidentified coronavirus emerged, causing a major outbreak in many cities and rapidly spreading globally. This new coronavirus, known as severe acute respiratory syndrome coronavirus (SARS-CoV) 2 (SARS-CoV-2), is a virus containing a genome with 29,903 nucleotides and 29 proteins, belonging to the family Coronaviridae, subfamily Coronavirinae, and is the main cause of severe acute respiratory syndrome, also known as COVID-19 [1–3]. By the end of 2021 and early 2022, COVID-19 had infected 224 million people, and 4.6 million had died globally [4].

In addition, microbial coinfections and superinfections [5] had occurred, influenced by factors such as the potentiation of pathogenesis and the increased risk of morbidity and mortality of patients with COVID-19. Coinfection can be defined as the recovery of other pathogens in a patient with an infection within 48 h of admission [6,7], and superinfection occurs when a patient has clinical signs and symptoms of pneumonia or bacteremia combined with a positive culture of a new pathogen from a lower respiratory tract or blood specimen obtained ≥48 h after admission [8]. In other words, coinfection occurs simultaneously with the spread of the microorganism, while superinfection develops after the initial infection [9].

Latin America had been one of the most severely affected regions by the COVID-19 pandemic, accounting for 25% of global infections. Moreover, of the ten countries with the highest mortality rates worldwide, eight were from this region, including Colombia [10]. In a study conducted in Colombia, a high incidence of early mortality associated with

**Citation:** Dueñas, D.; Daza, J.; Liscano, Y. Coinfections and Superinfections Associated with COVID-19 in Colombia: A Narrative Review. *Medicina* **2023**, *59*, 1336. https://doi.org/10.3390/ medicina59071336

Academic Editors: Yusra Habib Khan, Tauqeer Hussain Mallhi, Tahir Mehmood Khan and Muhammad Salman

Received: 25 May 2023 Revised: 7 July 2023 Accepted: 17 July 2023 Published: 20 July 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/).

COVID-19 24 h after hospital admission was reported [11]. However, studies related to the incidence of coinfection and superinfection in patients with COVID-19 are generally limited, especially in Latin American countries [5,12,13]. It was observed that severely ill COVID-19 patients, especially those in the intensive care unit (ICU), were more prone to secondary infections, owing to the increased use of prophylactic or therapeutic antibiotics whose task is to ensure the successful eradication of susceptible pathogens [14]. However, antibiotic misuse poses a threat due to the increase in the number of antibiotic-resistant microorganisms; additionally, it has a negative impact on the host microbiota. Clinical evidence suggests that inappropriate empirical use of antimicrobials may be associated with increased morbidity and mortality [9,15,16]. Therefore, the objective of this review is to describe scientific evidence on SARS-CoV-2-related coinfections and superinfections and their relevance in patients with COVID-19 in Colombia.

### **2. Methodology**

A comprehensive literature search was conducted using databases such as PubMed, Scopus, SciELO, and Web of Science. The search was based on keywords related to COVID-19, SARS-CoV-2, coinfections, superinfections, Colombia, and COVID-19 treatments ((SARS-CoV-2 OR COVID-19) AND Colombia AND coinfection AND superinfection AND therapy). Relevant data corresponding to each section of the manuscript, such as definitions, pathophysiology, and treatment of SARS-CoV-2, were extracted. The extracted data were then analyzed to identify common themes, patterns, and trends related to coinfections and superinfections. The findings were organized in a narrative format, highlighting key points, similarities, and differences among the studies.

Definitions included the following:

Coinfections: "Coinfections with other microorganisms such as bacteria, fungi, and other viruses are commonly associated with respiratory viral infections. Coinfections are directly linked to increased rates of morbidity and mortality, thus requiring early diagnosis and specific treatment" [17].

Superinfection: "Superinfection is diagnosed when patients present with clinical signs and symptoms of pneumonia or bacteremia combined with a positive culture of a new pathogen from a lower respiratory tract sample (including sputum, transtracheal aspirates, or bronchoalveolar lavage fluid) or blood samples taken ≥48 h after admission" [8].

### **3. SARS-CoV-2**

Coronaviruses are approximately 80–220-nm-diameter enveloped viruses [2]. The viral genome encodes five structural proteins which are encoded within the 3' end, namely, spike protein (S), envelope (E), membrane (M), nucleocapsid (N), and hemagglutinin esterase (HE) [1–3,18,19]. Protein S is a transmembrane glycoprotein that facilitates viral envelope binding to angiotensin-converting enzyme 2 (ACE-2) receptors expressed on the surface of host cells; it also forms protruding homotrimers on the viral surface, and this protein comprises two functional subunits: receptor binding (S1) and cell membrane fusion (S2) [1,2,18]. The E protein is the smallest protein in the SARS-CoV-2 structure, and its function is not necessary for replication. However, it plays a huge role in pathogenesis since it helps in the assembly and liberation of virions [1,18]. The M protein is the most abundant protein in the virion, and it was suggested that this protein plays a role in RNA packaging and promoting the assembly and budding of viral particles through interaction with N and accessory proteins 3a and 7a [1,2,18]. The N protein packages genetic material and binds to the viral genome in a bead-on-a-string conformation. Consequently, it modifies host-cell RNA processing, alters the TGF-β pathway by blocking apoptosis, and promotes binding of the transcription factor NF-κβ to the COX-2 promoter, leading to an inflammatory response. Notably, it is also involved in RNA replication and immune evasion [2,18]. The HE protein acts as a hemagglutinin, binds to sialic acids on surface glycoproteins, contains acetyl esterase activity, and helps the virus spread through the mucosa [1]. Among these five proteins, the most important ones are protein N and protein S. While the former helps in the

development of the capsid, the latter allows the virus to bind to target cells. Meanwhile, the most complex component is the receptor-binding domain (RBD) in the S protein because six RBD amino acids are needed to bind to the ACE-2 receptor and harbor SARS-CoV-2-like coronaviruses [1,2].

The RBDs of SARS-CoV-2 have a stronger attraction or affinity for the ACE-2 receptor compared to the RBDs of SARS-CoV. Furthermore, in the case of SARS-CoV-2, a significant portion of the RBDs is in the bound state, meaning they are attached or bound to the ACE-2 receptor. This binding state leads to a comparable or potentially lower affinity for the receptor when compared to SARS-CoV [3].

SARS-CoV-2 entry into host cells and the release of its genomes depend on a sequence of steps, but it should be noted that four of the structural proteins it possesses (S, N, M, and E) allow it to gain access to target cells. For entry, the virus requires binding of the S protein to ACE-2 on the cell's plasma membrane. However, this protein must be cleaved by the transmembrane serine protease 2 (TMPRSS2) of the cell membrane into the two subunits—S1 containing the RBD to ACE-2 and S2 facilitating viral fusion—although this cleavage can also occur by cathepsin-L in the endosomes, which helps to infect cells without TMPRSS2, but this is a slower process [18,20,21]. The literature suggests that the modified RBD residues of protein S in SARS-CoV-2 contribute to its high pathogenicity and transmissibility compared to SARS-CoV. Moreover, the presence of the polybasic furin cleavage site is not observed in other coronaviruses. Thus, this facilitates efficient cleavage of the S protein by furin and other proteases. Even the "S trimer" exists in a partially open state in highly pathogenic coronaviruses [20]. After the virus enters the target cell cytoplasm, uncoated genomic RNA is translated into polyproteins (pp1a and pp1ab), which are then assembled into replicating or virus-induced double-membrane vesicle transcription complexes. Subsequently, these complexes replicate and synthesize a nested set of genomic RNA by genome transcription, which encodes structural proteins (M, E, and N) and some accessory proteins. The endoplasmic reticulum and the Golgi complex mediate the binding of the newly formed viral particles, then finally, the new virions leave the cell by exocytosis [18].

### **4. SARS-CoV-2 Immunology**

A special feature of SARS-CoV-2 is the inhibition of receptor signaling pathways responsible for triggering antiviral immunity, mainly pattern recognition receptors (PRRs) tasked to recognize molecular patterns associated with pathogens or cellular damage (PAMPs and DAMPs). Figure 1 shows the immunology against SARS-CoV-2. PAMPs are associated with microbial pathogens [22]. The main PRRs for viral recognition are toll-like receptors 3 (TLR3) and 7 (TLR7) in the endosome or the cytosolic sensors retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Activation of these PRRs is mainly associated with IFN production [22,23]. RGI-I and MDA5 activate the mitochondrial antiviral signaling protein of the downstream adaptor in the mitochondria, followed by activation of TNF receptor-associated protein [23]. SARS-CoV-2 has the ability to suppress the production and function of type 1 IFNs, triggering IFN-stimulated genes [22,23].

Similarly, in response to coronavirus infection, humans produce TCD4+ lymphocytes, TCD8+ lymphocytes, and specific antibodies, which have protective functions against viral infections; however, these functions and their importance vary according to the viral infection. TCD4+ cell responses to primary *SARS-CoV-2* infection are more prominent than those of TCD8+, whereas the presence of specific TCD8+ cells has been associated with better outcomes in COVID-19 [24]. Humoral response to SARS-CoV-2 involves neutralizing antibodies (nAb) specific to viral epitopes. N protein epitopes are conserved among different coronaviruses, prompting the generation of cross-reactive antibodies. However, nAb targets protein S and its RBD region to neutralize the coronavirus. In turn, they protect against future infections [3,24]. Moreover, SARS-CoV-2 infection can lead to a reduction in lymphocytes [23].

**Figure 1.** (**A**) Primary mode of transmission of SARS-CoV-2, which is through respiratory droplets. (**B**) SARS-CoV-2 enters via the angiotensin-converting enzyme receptor 2 (ACE-2). It also shows tolllike receptors whose function is to recognize viral RNA in endosomes. (**C**) Activation of the antiviral innate immune response associated with IFN production and activation of proinflammatory cytokines mediated by T lymphocytes causing a cytokine storm. (**D**) Humoral response to SARS-CoV-2 with the use of neutralizing antibodies (nAb). The figure was created with https://app.biorender.com (accessed on 1 June 2023).

It is worth mentioning that the virus has an impact on the mechanisms of cellular stress activation in immunocompetent cells because it causes the activation and apoptosis of lymphocytes and macrophages as well as immunosuppression [22]. The cytokine storm correctly reflects the immune response in patients with COVID-19. The observed elevated IL-6 levels are considerably low. However, there are dynamic changes in the concentration of many cytokines, including IL-6. In addition, kynurenines, molecules related with immunosuppression, are elevated in severe COVID-19 [23].

### **5. Impact of SARS-CoV-2in Colombia**

Nearly two decades after the emergence of SARS-CoV, SARS-CoV-2spread rapidly after the first reported case in December 2019 and became a serious global health crisis [25]. Noticeably, the most affected countries were industrialized ones with strong public health systems and advanced medical facilities that have been severely strained in the course of the pandemic [26]. However, Latin America's health care systems have been significantly disrupted as SARS-CoV-2 spread around the world [27]. These nations opted for strict quarantine and the promotion of self-care, following WHO guidelines, whose objective was to separate potential carriers of the virus from uninfected individuals and thus reduce the spread of the virus, corresponding to epidemiological fences, but the social, labor, educational, psychological, and emotional costs were high [28].

In particular, the pandemic adversely affected the elderly and the disadvantaged the most. While all age groups were susceptible to SARS-CoV-2 infection, older adults suffered a higher risk of mortality [29]. Latin American countries faced an extremely protracted challenge, as factors such as poverty and limited access to potable water, among others, continued to be a critical point in the development of the region. In addition, there was a fundamental lack of preparedness to deal with a pandemic [29].

In early March 2020, the first positive cases in Colombia were reported in the city of Bogota, causing great concern among the people and leading to the implementation of weak measures to mitigate transmission. In turn, the government had to declare mandatory containment on 25 March 2020, which remained in force, totally or partially, until the beginning of 2022 [30–32]. For some authors, the health and security system, the economic sector, the social context, and people's mental health were the most critically impacted by the pandemic in the country, with the health and social security system being most affected due to the rapid chains of transmission [30].

In Colombia, from 3 January 2020 to 5 July 2023 at 4:01 p.m. CEST, there have been 6,373,599 confirmed cases of COVID-19 with 142,836 deaths reported to the WHO. As of 2 June 2023, a total of 90,506,612 vaccine doses have been administered [33].

Colombia had a case fatality rate of 2.5% with an accumulated positivity rate of 22.6%, with the highest number of cases occurring in females (53%) and the highest number of deaths in males (60.7%). Furthermore, the highest frequency of deaths was found among those between 40 and 90 years old with 389,736 deaths, peaking from 70 to 79 years old with 34,924 deaths. Among the comorbidities affecting the deceased, the most frequent were arterial hypertension (6416 deaths), diabetes mellitus (3901 deaths), respiratory diseases (2421 deaths), renal diseases (2226 deaths), obesity (1910 deaths), cardiac diseases (2083 deaths), and cancer (1115 deaths) [34].

Colombia faced six "waves," and their delimitations correlated with the restriction and relaxation of measures such as social distancing [35]. It should be noted that, according to clinical evidence, most of the cases reported in the second, third, and fourth waves were mild to moderate with lower hospital admission requirements and short hospital stays, i.e., there was a higher survival rate from COVID-19, but findings showed a large increase in the number of confirmed cases in those periods. According to the Instituto Nacional de Salud (INS), a total of 5,823,994 symptomatic cases, 488,614 asymptomatic cases, and 141828 deaths have been recorded in the six waves that have occurred in the country. The fourth and fifth waves reported higher numbers of symptomatic COVID-19 cases, with 3 January 2022 (belonging to the fifth wave) being the day with the highest number of confirmed cases (41,474). The second and fourth waves had higher numbers of asymptomatic cases reported in consultation, with 15 June 2021 (belonging to the fourth wave) being the day with the highest number of registered asymptomatic cases (5315). The fourth wave saw the highest number of COVID-19 deaths reported [34].

In addition, the INS reports 22,968 complete genomes sequenced with 267 lineages identified from 25,066 tests. Among the lineages found in the first sampling on 5 September 2021, there was a similar prevalence between the Mu (55%) and Delta (45%) variants. Patiño et al. [36] analyzed the effective reproduction number (Rt) of *SARS-CoV-2*, the virus that causes COVID-19, in Cali, Colombia between April and July 2021. The study found that Rt values were higher during the period of frequent protests compared to the preceding and following months. Genomic analysis revealed the circulation of 16 different lineages of SARS-CoV-2, including variants of concern (VOCs) and variants of interest (VOIs). The study suggests that the spread of highly virulent strains of SARS-CoV-2 in Cali and other parts of Colombia was facilitated by the limited biosecurity strategies during the period of political turmoil and social demonstrations, as well as the movement of large numbers of people into and out of the city. The Mu variant is thought to have been introduced to Cali on two separate occasions, and may have contributed to the 55% increase in the number of reported cases during the protests.

By 2 January 2022, 62% belonged to the delta variant and 38% to the Omicron variant, which later became dominant with 100% in circulation on 13 March 2022. On 8 May 2022, two sublineages of the Omicron strain, BA.2.12.1 (20%) and BA.2.x (80%), were presented. Finally, on the last sampling date of 7 August 2022, two sublineages of the Omicron variant, BA.4 (40%) and BA.5 (60%), were revealed [34]. Finally, until 18 December 2022, the omicron BQ.1.x subvariant was reported with 90% prevalence in Colombia and the XBB variant with 10%, the latter being the most transmissible variant [37].

### **6. Coinfections Associated with SARS-CoV-2 in Colombia**

Coinfections with microbial pathogens have played an important role in increasing the morbidity and mortality rate in pandemics, and SARS-CoV-2 is no exception [38]. Coinfection between other microorganisms and this new coronavirus is an important factor to take into account in COVID-19, as it may increase the difficulties in diagnosis, management, and prognosis, and even worsen symptoms and mortality [12]. It should be noted that a wide spectrum of atypical presentations were found to be associated with COVID-19, which complicated critically ill immunocompromised patients [39]. Table 1 lists the most frequent coinfections; however, bacterial and viral coinfections were most frequently reported in patients with COVID-19 and had proportions as high as 50% among non-survivors. However, severe cases of COVID-19 related to fungal infections were documented, especially representing a major threat to life in ICU patients [16,38,40,41]. Mechanical ventilators and catheters are risk factors for nosocomial infections [14,40].

The source and specific nature of these infections have not yet been fully explored, but there is evidence to suggest that multidrug-resistant bacteria, such as *Staphylococcus aureus*, *Streptococcus pneumoniae*, *Klebsiella pneumoniae*, and *Escherichia coli*, are among the pathogens believed to be causative agents. *Acinetobacter* spp., *Enterobacter* spp., *Enterococcus* spp., and *Pseudomonas* spp. are also associated with hospital-acquired infections. Similarly, several studies have indicated that coinfection with fungi such as *Aspergillus* spp. and *Candida* spp. can increase mortality rates [14,41,42]. In addition, coinfections with SARS-CoV-2 and other respiratory viruses are unusual. They have been observed from 3.2% to 22.4% of the time, with rhinovirus–enterovirus (6.9%) and respiratory syncytial virus (5.2%) being the most commonly reported [43].

**Table 1.** Coinfections with *SARS-CoV-2* worldwide. HIV: Human Immunodeficiency Virus; MRSA: Methicillin-Resistant Staphylococcus Aureus.

#### **Table 1.** *Cont.*

Molina et al. [63], who conducted a study in eight hospitals in Colombia, reported the most frequently seen microorganisms in coinfections in the ICU as follows: *Staphylococcus aureus*, *Streptococcus agalactiae*, and *Klebsiella pneumoniae* [63].

The most common coinfections with SARS-CoV2 in Colombia, as shown in Table 2, were dengue virus (DENV), *Klebsiella pneumoniae*, *Mycobacterium tuberculosis* (MTB), *Pneumocystis jirovecii* (*P. jirovecii*), *Cryptococcus neoformans*, *rhinovirus–enterovirus*, *adenovirus*, *human immunodeficiency virus* (HIV), and *Trypanosoma cruzi.*

**Table 2.** Coinfections with *SARS-CoV-2* in Colombia.

### **7. SARS-CoV-2, and Dengue Coinfection**

Initial differentiation between dengue and COVID-19 is a challenge as both infections have similar symptoms, such as fever, diarrhea, myalgia, and headache, and these overlap [64,65]. However, studies suggested that SARS-CoV-2 and DENV coinfection had less severe symptoms compared to isolated monoinfection [13]. Tropical countries, especially those with endemic DENV, approached a syndemic state, because multiple patients were coinfected with both SARS-CoV-2 and DENV. COVID-19 can be misdiagnosed with dengue. This situation complicates matters, so it can be difficult to distinguish between early infections and coinfections, generating an important risk for the population and demanding greater attention from health systems, because both viruses can cause serious complications, mainly through the cytokine storm in lung tissue caused by macrophage hyperactivation [13,27,64].

Diagnosing coinfection requires a combination of tests for the direct detection of the virus and indirect techniques that measure the immune response [65]. It is important to take into account the clinical and epidemiological particularities of both infections [13,65].

In 2020, 28,068 cases of dengue were reported in the department of Valle del Cauca, of which a case fatality rate of 6.6% was reported for severe dengue. It should be noted that from weeks 1 to 36, the cases were higher than expected, but from weeks 37 to 52, they were within the limit according to their historical behavior between 2013 and 2017 [63]. Likewise, in 2021, 8141 cases of dengue were reported, with 23 probable deaths due to severe dengue, although the number of cases was within the expected range [75]. However, a rebound peak of dengue was observed due to the replenishment of susceptible individuals with low exposure to infection. In addition, the number of patients requiring intensive care and mechanical ventilation increased. Therefore, regions such as Valle del Cauca should consider intensified preparedness for such scenarios, and further studies should be conducted to address this critical issue promptly to reduce the potential overload on the national health system [64].

### **8. Coinfections with Bacteria**

*Klebsiella pneumoniae* is a Gram-negative species that can reside in the gastrointestinal tract [76]. In immunocompromised patients, it can cause serious infections, including urinary tract infections, respiratory infections, soft tissue infections, peritonitis, and sepsis [45,68]. Lipopolysaccharide and cell wall protein receptors are responsible for pathogenicity and determine the process of binding to host cells and provide protection against the human immune system response [76]. Mechanical ventilation, exposure to carbapenems and β-lactamase/β-lactamase inhibitors, renal replacement therapy, transfusions, and prolonged hospital stay are risk factors for coinfection with *Klebsiella pneumoniae* [77]. Patients with COVID-19, in whom immune mechanisms appear to be weakened by this viral infection, should follow rational antibiotic therapy with the aim of preventing bacterial resistance [76].

*Mycobacterium tuberculosis* (MTB) is an acid-fast bacterium and is the main causative agent of human tuberculosis. Evidence suggests a twofold increased mortality risk in patients with COVID-19 and tuberculosis [27]. For example, a systematic review reported an increased risk of mortality in patients with coinfection of these two microorganisms, although MTB and SARS-CoV-2 coinfections are poorly understood. On the other hand, a meta-analysis and systematic case study of SARS-CoV-2 coinfection with drug-resistant tuberculosis failed to show the same. In both cases, the evidence was inconsistent, and more high-quality studies are needed to better understand the causal association [51]. However, another study reported that active or latent tuberculosis increased susceptibility to *SARS-CoV-2* and disease severity [27].

### **9. Coinfections with Fungi**

One of the underestimated microorganisms in patients with coronavirus disease 2019 is *Pneumocystis jirovecii*, an opportunistic infection that mainly affects immunosuppressed patients [52]. It occurs due to an imbalance between T lymphocyte subtypes, mainly due to the absence of CD4+ T cells, causing a deficiency in the immune response and generating a predominance of TCD8+, causing epithelial damage secondary to an excessive inflammatory state. In severe infections, 50% of patients may require hospitalization in the ICU, with a mortality rate of up to 40–60% [39,52]. SARS-CoV-2 infection can cause a state of immunodeficiency that may allow the appearance of this opportunistic fungus. At the same time, *P. jirovecii* and SARS-CoV-2 infections presented as joint processes, primarily in immunocompromised patients. It presents acutely with severe hypoxemia and the rapid deterioration of respiratory function, requiring invasive mechanical ventilation. It was found that the (1,3)-β-D-glucan detection technique is of potential use for the detection of *P. jirovecii* in patients with acute SARS-CoV-2 coinfection [39].

*Cryptococcus neoformans* is an encapsulated yeast-like fungus that is considered an opportunistic and rare pathogen in transplant recipients [50] and can cause cryptococcosis in immunocompromised patients. The most frequent and severe form of presentation is infection of the central nervous system, manifesting as subacute or chronic meningitis and characterized by headache, nausea, vomiting, fever, and altered consciousness; pulmonary, skin, lymph node, or other organ involvement may also occur to a lesser extent [66]. COVID-19 has been observed to increase infection by other rare pathogens in immunocompetent patients, such as that caused by *Aspergillus* spp., and studies have reported that patients required ICU stay and invasive mechanical ventilation secondary to infection by COVID-19, without a history of immunosuppression. Therefore, it can be said that coinfection with SARS-CoV-2 and Cryptococcus is rare. For example, in 2021, among 293 patients in a case study conducted in China, there was only one case reported [50,66]. However, it should be taken into account that SARS-CoV-2 infection may be an etiology for fungal infection by *Cryptococcus neoformans* due to the great multisystemic impact and the multiorgan dysfunction established by the viral agent, as the main cause of immunosuppression predisposing to infections by this microorganism. At the same time, management with glucocorticoids (dexamethasone) may favor immunological compromise [66].

### **10. Coinfection with Virus**

*Rhinoviruses* (*RVH*) and *enteroviruses* (*EVH*) belong to the *Picornaviridae* family and are the main cause of infections worldwide. They are characterized as small with a singlestranded RNA genome in an icosahedral capsid. In a study by Kim et al. [78], it was observed that 9.5% of 1217 patients with respiratory symptoms tested positive for SARS-CoV-2 and 318 for another microorganism. Of the group positive for SARS-CoV-2, 20.7% were positive for one or more pathogens, with *RVH* and EVH being the most frequent [78,79]. Regarding concomitant SARS-CoV-2 and *rhinovirus*–*enterovirus* infection, it is recommended that the multiplex PCR respiratory panel be performed only for severe patients and those in whom a positive result requires modification of treatment to prevent disease progression and even death [79].

*Adenoviruses* are icosahedral viruses that possess a double-stranded DNA genome, belonging to the *Adenoviridae* family. They cause only respiratory, ocular, and enteric disease in humans. Cases of coinfection with SARS-CoV-2 and respiratory viruses are poorly documented; for example, by 2020, only two patients with *adenovirus* coinfection had been documented. The pathogenesis of coinfection is not clearly explained, although there are hypotheses about the low presentation of SARS-CoV-2 without a predisposing risk factor. In addition, it has been documented that there is a higher proportion of coinfection in patients with acute respiratory distress syndrome and septic shock and requiring ICU admission [43].

HIV, which belongs to the *Retroviridae* family, is characterized by attacking the immune system and thus generating a state of immunodeficiency. People living with HIV (PLHIV) should not consider themselves protected against SARS-CoV-2, as people with low CD4+ Tcell counts may have worse outcomes than people with normal immunity [27,51]. Moreover, poorer COVID-related outcomes were observed in patients with HIV than those without the infection, especially in those with multimorbidity and advanced age [51]. A recent prospective cohort study reported that 8% of PLHIV infected with *SARS-CoV-2* required admission to the ICU. It should be noted that clinical manifestations, disease severity, and mortality are independent of HIV or antiretroviral-related factors.

There are possible similarities between HIV*-1* and SARS-CoV-2 proteins. At the same time, it has not been identified as a common comorbidity in patients with COVID-19. In addition, there are differences between the receptors through which HIV and other pathogenic *coronaviruses* enter target cells, the ways of assembly, and their encapsulation. In the case of HIV, it does so near the cytoplasmic membrane, and in the case of *coronavirus*, the process takes place in the endoplasmic reticulum, which may suggest that there is no synergistic or cooperative pathogenesis [53].

### **11. Coinfections with Parasites**

The protozoan parasite, *Trypanosoma cruzi*, is the main infectious agent of Chagas disease. This disease causes cardiac and gastrointestinal complications, among others, and is endemic in Latin America [80]. The intense inflammatory process of COVID-19 in immunocompromised patients could potentially influence the evolution of the disease and latently trigger reactivation of Chagas disease due to viral interference of the infection. It is important to highlight that different clinical and epidemiological scenarios may increase susceptibility to SARS-CoV-2 infection, because the new *coronavirus* disease has a significant impact on the heart. Likewise, the pandemic influences access to treatment for people with acute and chronic indeterminate Chagas disease [68].

In patients with SARS-CoV-2 pneumonia and immunosuppression, antibiotic treatment should be initiated in order to address opportunistic pulmonary infections. However, there are infections that are not covered by first-line antibiotics [39]. An example is *P. jirovecii*, where the first choice is the use of trimethoprim–sulfamethoxazole, a broad-spectrum antibiotic for bacterial and fungal germs [39,52]. Another case is *Klebsiella pneumoniae*, which has a very low sensitivity profile to most categories of antibiotics [14,38,45,76,77] and is very difficult to treat, but the drug combinations aztreonam and ceftazidime/avibactam or meropenem/vaborbactam show universal coverage against beta-lactamase-producing *Enterobacteriaceae*, including those with extensive drug resistance [76].

However, concerns about coinfections have led to significant antimicrobial use in up to 80% of critically ill patients with COVID-19, and their overuse could lead to antimicrobial resistance [38,63,81]. The rate of antimicrobial resistance ranged from 33.3% to 90.0%, depending on the infecting species [45]. The use of antibiotics over the last 50 years has exerted selective pressure on susceptible bacteria and may have favored the survival of resistant strains [15]. Of concern is that there is clinical evidence suggesting that inappropriate empirical use of antibiotics and other broad-spectrum antimicrobials may be associated with increased mortality [16]. One of the main aspects that needs to be evaluated in the prevalence of coinfections is the application of empirical antimicrobial treatment in patients with SARS-CoV-2 infection [38]. In addition, it is important to know more information about the prevalence of coinfections in the community [82], and hospital-acquired or healthcare-associated infections need to be continuously monitored and controlled. These should not only focus on minimizing the spread of SARS-CoV-2 infection but also on reducing bacterial cross-transmission, particularly of multidrug-resistant organisms [76]. Therefore, strategies should be established to improve antimicrobial stewardship in patients with COVID-19.

### **12. SARS-CoV-2—Associated Superinfections in Colombia**

SARS-CoV-2 superinfections increase difficulties in the prognosis, diagnosis, and treatment of patients [83]. According to the U.S. Centers for Disease Control and Prevention, "a superinfection is an infection that follows a previous infection, especially when caused by pathogens that are resistant or have become resistant to previously used antibiotics" [6,84]. The mechanisms of superinfections include virus-induced damage to the respiratory system, decreased mucociliary clearance, and damage to the immune system. The decrease in lymphocytes and host immune function is the main reason that facilitates superinfection [83]. It should also be noted that clinical deterioration, elevated inflammatory markers, and bilateral radiological infiltrates may lead to misperception regarding the presence of a co-pathogen and should be used as an impetus to initiate comprehensive diagnostic workup with sampling, rather than as an indicator of underlying superinfection [84].

Superinfection in hospitalized patients with COVID-19 is associated with disease progression and poor prognosis. This situation increases antimicrobial treatment and mortality. They have even been related mainly to ICU admission, especially with the use of mechanical ventilation and catheters, and patients with comorbidities. In addition to having a higher prevalence, they also have a higher risk of death than in other patients [5,6,83]. The study by Clancy et al., (2021) [85] described several common risk factors, including being older than 60 years, male, ICU admission, mechanical ventilation, renal failure requiring hemodialysis, arterial hypertension, diabetes mellitus, and cancer [5,8,83].

Similarly, Paparoupa et al. [84] reported that 45% of invasively mechanically ventilated patients with COVID-19 pneumonia had bacterial, viral, or fungal respiratory superinfection in at least one of the sequential study periods. Therefore, the frequency of hospital-acquired superinfections remained low despite the fact that many patients received treatment that resulted in severe immunosuppression.

Factors such as the empirical use of antibiotics, isolation measures, or the host macrophage activation explain this. At the same time, the lack of additional microbiological testing after SARS-CoV-2 was detected may have also contributed. Further studies will be needed to elucidate the role of each measure in reducing superinfections [5]. Table 3 lists the most frequent superinfections. *Acinetobacter* spp. has been identified as a common infection in ventilated patients. It is more frequent in patients with superinfection [6].

**Table 3.** Superinfections with SARS-CoV-2.

The superinfections with SARS-CoV-2 documented so far in Colombia, shown in Table 4, are bacterial in nature, with *Raoultella planticola* and *Pandoraea pnomenusa* standing out.

 Superinfections with SARS-CoV-2 in Colombia.

*R. planticola* is a bacterium of the *Enterobacteriaceae* family that can be found in soil and water, and is associated with seafood consumption, biliary tract diseases, malignancy, diabetes mellitus, trauma, immunosuppression, and nosocomial infection. Initially considered harmless, the number of cases has increased in recent years, mainly consisting of cystitis, bacteremia, and pneumonia. Most strains of *R. planticola* are usually multisensitive and treatment is effective with second and third generation cephalosporins, aminoglycosides, and fluoroquinolones. With respect to *R. planticola* infection as a complication of SARS-CoV-2 infection, there is a paucity of cases reported in the literature. However, an infection by this microorganism has been reported as a complication of pulmonary bulla, a rare complication of COVID-19 affecting only 1% of patients, secondary to SARS-CoV-2 infection [94].

*Pandoraea pnomenusa* is a bacterium belonging to the *Pandoraea* genus. It should be noted that its usual presentation is the colonization of structurally abnormal airways. Infection generated by this microorganism occurs rarely, but its mortality rate is high, reaching up to 60%. It usually presents with multiple antimicrobial resistance. It presents an intrinsic ß-lactamase of the OXA type and a gyrB gene [95].

Bacterial superinfection in hospitalized patients with COVID-19 is associated with disease progression and poor prognosis. For example, in March 2020, a case of pneumonia caused by *Staphylococcus aureus* secreting leukocidin toxin in a man with mechanical ventilation was reported and treated with piperacillin–tazobactam, linezolid, meropenem, and gentamicin; however, the patient died 17 days after admission [8].

Antimicrobial stewardship will continue to be a priority because antimicrobial use in SARS-CoV-2 -infected patients remains higher than in superinfections [14]. Antimicrobial stewardship principles help guide the appropriate use of antibiotics [8]. Paparoupa et al. [84] demonstrated extensive use of broad-spectrum antibacterials in more than 70% of COVID-19 cases.

It is important that centers collect and publish their clinical, microbiological, and antimicrobial prescribing data. Further research is also needed on current infection control guidelines [8].

### **13. Therapeutic against COVID-19 in Colombia**

In the context of COVID-19, antibiotics are primarily used to treat secondary bacterial infections that may occur as complications of the viral disease. These bacterial infections can manifest as secondary bacterial pneumonia, respiratory tract infections, or urinary tract infections, among others. The use of antibiotics in these cases aims to treat concurrent bacterial infections or prevent their occurrence in patients with compromised immune systems, particularly those who are in a severe condition [83]. In their study on antibiotic resistance during COVID-19 in Valle del Cauca, Colombia, Hurtado et al. [4] analyzed data from 31 hospitals and compared antibiotic resistance and consumption before (March 2018 to July 2019) and during (March 2020 to July 2021) the pandemic. The results showed an increase in the total number of bacterial isolates during the pandemic, accompanied by a significant decrease in resistance for four bug–drug combinations. However, there was a noticeable rise in vancomycin resistance among *Enterococcus faecium*. Overall, antibiotic consumption increased, except for meropenem in ICU settings. These findings suggest that the COVID-19 pandemic contributed to an increase in community-acquired infections, resulting in changes in antibiotic resistance patterns. Monitoring the increasing resistance of *E. faecium* to vancomycin and implementing effective infection control measures is crucial.

In a study conducted by Valladales-Restrepo et al., (2023) in Colombia, a descriptive cross-sectional study was carried out to examine the utilization of systemic antibiotics among patients diagnosed with COVID-19 between 2020 and 2022. The study involved eight clinics and included a total of 10,916 predominantly male patients with a median age of 57 years. Approximately 57.5% of the patients received antibiotics, with ampicillin/sulbactam and clarithromycin being the most frequently prescribed ones. Based on the WHO AWaRe classification, the majority of prescribed antibiotics belonged to the Watch category, followed by access and reserve categories. Several factors were found to be associated with a higher likelihood of receiving systemic antibiotics, such as male gender, older age, presence of dyspnea, rheumatoid arthritis, high blood pressure, in-hospital treatment, or ICU admission, and the use of systemic glucocorticoids, vasopressors, or invasive mechanical ventilation. Despite the low prevalence of bacterial coinfections, a significant proportion of COVID-19 patients received antibiotics, with a noticeable dominance of Watch antibiotics, which deviates from the recommendations provided by the World Health Organization [96].

In Colombia, various treatments have been used to combat COVID-19, including Remdesivir, Molnupiravir, Tocilizumab, and Convalescent Plasma Therapy. Remdesivir works by inhibiting the replication of the virus in the body and has been used in hospitalized patients with severe illness. Its emergency use authorization has made it available for those in need during the pandemic [97–99]. Molnupiravir is an antiviral medication that has shown activity against SARS-CoV-2 and has been authorized for emergency use in the treatment of COVID-19 by the Colombian regulatory agency, the National Institute for Surveillance of Drugs and Food (INVIMA) [100]. Preclinical studies have shown that Molnupiravir has the potential to significantly reduce viral load and decrease virus transmission in animal models. Additionally, early findings from clinical trials and systematic reviews have demonstrated promising results, indicating a reduction in hospitalization and mortality rates among patients with mild to moderate COVID-19. Molnupiravir presents a new ray of hope in combating the pandemic and could play a crucial role in the treatment of this disease [101–103].

Tocilizumab, on the other hand, is an immunosuppressive medication used in severe cases of COVID-19. This drug works by blocking interleukin-6 (IL-6), an inflammatory protein involved in the exaggerated immune response that can lead to severe complications in COVID-19 patients. Tocilizumab is administered via intravenous infusion and has been used in patients exhibiting excessive inflammatory response, such as cytokine release syndrome [104–106].

Convalescent Plasma Therapy is another treatment option used in Colombia for patients with COVID-19. It involves transfusing blood plasma from individuals who have recovered from the disease and have developed antibodies against the virus. It is believed that these antibodies can help fight the infection and improve symptoms in sick patients. However, it is important to note that the effectiveness of this therapy is still being investigated, and more scientific evidence is needed to support its widespread use [107,108].

### **14. Therapeutic Alternatives against COVID-19**

The COVID-19 pandemic predisposes patients to potentially life-threatening infections in the ICU, hindering proper diagnosis and treatment [109]. Thus, specific therapy for COVID-19 should take into account coverage of local endemic pathogens that may occur in a similar manner, particularly while confirmation of SARS-CoV-2 infection is pending [17].

On the other hand, the WHO treatment guideline recommends empirically prescribed broad-spectrum antibiotics to treat possible coinfections. However, the effect of this respiratory disease on antimicrobial resistance is a dimension that requires necessary attention, since 15% to 50% of bacterial isolates are resistant to at least one antimicrobial group [109]. Therefore, antimicrobial therapy should be evaluated against a patient's host factors and local epidemiology on a daily basis [17].

It has been documented that systemic glucocorticoids improve survival when administered to moderate or severe COVID patients. Its use is associated with reduced oxygen therapy and decreased risk of invasive mechanical ventilation among patients receiving supplemental oxygen [98,110]. However, treatment with corticosteroids was associated with a higher risk of progression of hospital stay and it is not clear if there is an increased risk of superinfection in non-severe COVID patients [110]. There are currently drugs or vaccines available to inhibit the new *coronavirus*. Although current vaccines effectively prevent serious complications and deaths, treatment options are still under validation, especially for immunocompromised patients [111]. Therefore, to avoid viral exposure, it is important to adhere to the following measures: maintaining social distancing, wearing face coverings, practicing frequent hand washing, using alcohol-based hand sanitizers when necessary, and avoiding touching the face. However, it is important to note that not all of these measures should be grouped together. While the effectiveness of alcohol-based hand sanitizers has been highlighted [17], it is also worth mentioning that the effectiveness of face coverings, such as masks, has been extensively studied. Additionally, it is crucial to be mindful of the potential contribution of these practices to the threat of antimicrobial resistance. Therefore, alternatives such as supplementation and the development of nanomedicine should be considered.

Zinc (Zn) is involved in several biological processes as a cofactor and signaling molecule in the immune system. Additionally, it is an important component of the hormone thymulin, whose function is in T-cell differentiation, maturation, and NK actions [17]. Zn deficiency causes IL-10 dysregulation that alters Th1 cell response and macrophage functions [112]. Zn deficiency is associated with the risk and extreme progression of COVID-19 [17]. On the other hand, it has been shown that Zn ions can inhibit *coronavirus* RNA polymerase activity by reducing replication [112]. Thus, Zn supplementation may be associated with a lower mortality rate in patients with COVID-19, although more research is needed to understand the intimate mechanisms of antiviral activity [17].

Nanotechnology allows the manipulation and evaluation of individual molecules, and nanotechnology applied to medicine, also called nanomedicine, has been used to improve care in neurological, cardiovascular, and infectious diseases, and cancer [17]. Nanotechnology-based targets should be harnessed to aid in the fight against COVID-19, as well as any future pandemics, including the use of biosensors, virus inhibition by nanosystems, new vaccines and drugs, superfine filters for face masks or blood filters, and improvements to contact-tracing instruments, as it provides significant benefits and early-stage disease detection. In addition, established methods do not require specialized instrumentation, which provides a pathway to simple integral answers [113,114].

Another alternative is the use of REGEN-COV for patients who are at high risk of progressing to severe COVID-19 and who are not fully vaccinated or are not expected to develop a complete immune response [98]. This combination of monoclonal antibodies has been shown to reduce the viral load in symptomatic outpatient patients and the number of medical visits. It also has in vitro activity against current strains and reduces the risk of hospitalization or death. However, it may be associated with worse outcomes when given to patients who require high-flow oxygen or mechanical ventilation [98,115].

### **15. Conclusions**

The COVID-19 pandemic has had a significant impact on Latin America, particularly on countries with weaker healthcare systems. The disease has disproportionately affected the elderly and disadvantaged, and has been exacerbated by comorbidities such as hypertension, diabetes, and respiratory diseases. Coinfections with other microorganisms, such as bacteria, viruses, and fungi, have also contributed to the morbidity and mortality rates of COVID-19 in the region. In Colombia, dengue virus, *Klebsiella pneumoniae*, *Mycobacterium tuberculosis*, *Pneumocystis jirovecii*, *Cryptococcus neoformans*, and *rhinovirus–enterovirus* have been reported as common coinfections with SARS-CoV-2. There is a need to improve surveillance systems and establish protocols for the early detection and management of coinfections in COVID-19 to reduce the burden on healthcare systems. While current vaccines have been effective in preventing serious complications and deaths, there is a lack of specific treatment options for COVID-19, especially for immunocompromised patients. Alternative approaches, such as zinc supplementation and the use of nanomedicine, have been proposed as potential therapies, but more research is needed to fully understand their effectiveness.

**Author Contributions:** D.D.; investigation, D.D.; writing—original draft preparation, Y.L. and J.D.; writing—review and editing, D.D., J.D. and Y.L.; visualization, Y.L.; supervision, Y.L.; project administration. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. All authors have read and agreed to the published version of the manuscript.

**Funding:** This review was funded by the General Direction of Research of the Universidad Santiago de Cali convocatoria Interna No. 02–2023.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** To the General Direction of Research of the Universidad Santiago de Cali.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **Abbreviations**

### **References**

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**7.8. Repair of metro railway (Budapest, Hungary)**

#### **Task:**

Metro line East-West in Budapest has been operated since 1970. During the operation the railway must withstand a great load. Daily operation represent passing over of 7000 wheels with axis pressure of 7,9 tons. Despite everyday maintenance, signs of damage occurred and repair works were launched.

#### **Solution:**

332 Polyurethane

**Figure 29.** Dilatation joint before the grouting

**Figure 30.** Dilatation joint after the grouting

**Task:**

**7.8. Repair of metro railway (Budapest, Hungary)**

Metro line East-West in Budapest has been operated since 1970. During the operation the railway must withstand a great load. Daily operation represent passing over of 7000 wheels A new method of repair of the concrete sleepers was proposed based on anchoring and use of PU resin. The performance of the works concurrently led also to stopping of water inflows from the subsoil of the railway. Concrete sleepers were stabilized and anchored without the necessity to replace them (Figs 31,32).

Grouting works were performed between 2005 and 2006, mainly during night time and without necessity of putting the metro out of operation. Total of 2842 anchors were made of the following parameters: length of the anchor 500 mm, diameter of the anchor 18 mm, diameter of borehole 25 mm, grouting material Supermin® with reaction start of 2 minutes. Further, total of 2430 grouting boreholes were drilled of the following parameters: borehole diameter 14 mm, length up to 1 m, length of inserted steel bars up to 600 mm, diameter of the bars 8 mm, grouting material Geopur® 082/1000 with reaction time 2 minutes.

**Figure 31.** Detail of the grouting works

**Figure 32.** View of the railway after repair works

In addition, water infiltration from below of the railway was stopped in a total length of 58 m of the metro tunnel using material Geopur® 082/350.

Applied methods proved to be very effective and did not disturb the regular operation of the metro line.

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**3. Results**

## *3.1. Patient Information*

Patient characteristics can be seen in Table 1. The patient was a male with a history significant for a clinical diagnosis of neurofibromatosis type 1—patient had a plexiform neurofibroma, spinal neurofibromas, café au lait macules, and multiple first-degree relatives with neurofibromatosis type 1—and was 40 years old at the time of diagnosis of MPNST. He presented with a large tumor located in the left neck. Resection showed a high-grade malignant peripheral nerve sheath tumor, 10.2 cm in the largest dimension, with negative margins. The patient did not receive any adjuvant therapy for his MPNST following initial resection due to poor performance status. He recurred 21 months after the initial diagnosis and ultimately died secondary to complications from metastatic disease (33 months after initial diagnosis). Samples were taken in three di fferent locations within the primary tumor immediately following the inititial resection for the purpose of this study.

#### **Table 1.** Patient Characteristics.

 By French Federation of Cancer Centers Sarcoma Group Grading System (FNCLCC) [46]; OS = Overall Survival-time from diagnosis of MPNST to death.

#### *3.2. Histology of Biopsy Sites*

We first reviewed the H&E images of the tumor to correlate histology to the gross images of the tumor. H&E stained sections in Figure 2 show representative images of the three sampled areas. Area #1 demonstrates tissue of a spindle cell neoplasm of neural di fferentiation arranged in fascicles with elongated hyperchromatic nuclei and a mild to moderate amount of cytoplasm. The tumor purity of this sample was >95%. Area #2 shows spindled cells in a background of hemorrhage, a finding commonly seen in these high-grade tumors with a tumor purity of >95%. Area #3 represents an area of necrosis, another characteristic finding for MPNST. This sample showed >95% tumor purity.

**Figure 2.** H&E stained sections of the biopsy sites. H&E stained sections (20X) show areas (#1) of relatively uniform, spindled cells with fascicular growth pattern, characteristic for MPNST. Sampled area #2 shows evidence of hemorrhage within the tumor, a feature commonly seen in MPNST. Area #3 shows abundant tumor necrosis.

#### *3.3. Whole Exome Sequencing (WES), RNA Sequencing (RNA-Seq), and Copy Number Analysis*

We first interrogated the sequencing data to identify the germline NF1 variant within this tumor. Figure 3 shows a lollipop plot identifying the patient's likely NF1 germline variant based on exclusion of any variants with minor allele frequency >0.05 in the 1000 genomes database. Next, to

investigate intra-tumoral heterogeneity within the sample, RNA sequencing of the three sample sites was performed and is shown in Figure 4.

**Figure 3.** Location of NF1 germline variant. One intronic germline variant, NC\_000017.11:g.31296270C>T (rs11080149). was identified and is depicted in this figure.

**Figure 4.** RNA-Seq Heatmap. Normalized read counts by counts per million (CPM) in differentially expressed genes are depicted here. Distinct gene expression profiles can be appreciated in each biopsied area. Each column is depicted as list of genes.

Distinct gene expression profiles were observed in each of the areas sampled. The top 16 differentially expressed genes are listed in Table 2 and include a number of genes involved in transcription and translation. We next performed a copy number analysis of the three biopsy sites to determine whether or not different copy number alterations were observed in each area (Figure 5). Distinct copy number signatures can be appreciated in each of the three samples further illustrating intra-tumoral heterogeneity. Additionally, we evaluated the single nucleotide variants found in each of the samples. This broad overview of all somatic variants is depicted in the waterfall plot in Figure 6. Again, distinct somatic variants can be appreciated across different areas. We next explored the potential significance of these variants through further bioinformatics analysis. While the biological significance of each of these variants is uncertain, there is evidence that some of these variants may play a role in the pathogenesis. For each variant in a coding region, CBioPortal [47] was queried for each gene to determine if the somatic variant was in a functional domain. Additionally, the RNAseq data was queried to determine if the variant in a specific area of the tumor influenced the gene expression of that gene in a specific area. Finally, SIFT and Polyphen were used to predict pathogenicity. Table 3a,b list the somatic variants in the coding region that may play a role in the pathogenesis of this tumor based on the above criteria. For those mutaions in non-coding regions, the Ensembl Variant E ffect Predictor [33] was used to determine whether or not the variant would be predicted to a ffect gene expression. All of the identified variants were classified as modifiers, indicating that pathogenicity prediction is di fficult, thus the e ffects of these variants are unclear. (Table 3c). Further details of the somatic variants can be found in Supplemental Table S1. Next, a gene ontology analysis was performed. To do this, a list of genes in copy number aberrant (CNA) regions was extracted. For each area, the list of genes located in the CNA regions intersected with the di fferentially expressed gene list reported in the RNA di fferential expression analysis, and PantherDB [45] was utilized to identify pathways that may be a ffected by these genes. Table 4 displays the unique genes in each area with copy number aberrations and alterations in gene expression. Genes depicted in Area 1 have been reported in the literature to serve a myriad of functions in tumorigenesis, including base excision repair, nucleotide excision repair, and alternative splicing [48–55]. Those in Area 2 are involved in several di fferent pathways, including transcriptional regulation in addition to ribosomal and proteasomal function [56–60]. Finally, the genes in Area 3 consist of several ribosomal subunits and small nucleolar RNAs, suggesting that both translation and transcription are uniquely a ffected compared to other areas [61–63]. This analysis suggests that there may be di fferent functional programs at play across the three areas. Next, we manually reviewed the data to look for changes in other known drivers of MPNST including TP53, ATRX, EED, SUZ12, and CDKN2A. There were no copy number changes or somatic mutions in any of these genes. Finally, we performed a careful manual review of all of the shared and unique somatic variants and copy number alterations in each area in order to develop a predicted clonal evolution. Figure 7 depicts the predicted phylogenetic tree of the subclones from each area, representing the likely clonal evolution of the tumor.

**Table 2.** Top Differentially Expressed Genes. The gene-specific analysis was used to test for differential expression of genes or transcript between sample regions in Partek ® Flow ®. Statistical cutoff are made by these following parameters: *p*-value <= 0.05; FDR step up <= 0.05; Fold Change <−2 or >2.

**Figure 5.** Copy Number Variation Plot. Copy number variation plots for each biopsied site demonstrate distinct copy number signatures.

**Figure 6.** Somatic Variant Waterfall Plot. All somatic variants displayed on a waterfall plot. Each row represents a gene. Distinct somatic variant signatures are appreciated.

*CSK* 2 Chr15:74798671 frameshift p.(Glu25fs) Y *TSPAN9* 2 Chr12:3283047 frameshift p.(Leu218fs) Y

**Figure 7.** Phylogenetic Tree. A predicted phylogenetic tree of the tumor subclones.


**5. Panton-Valentine leukocidin**

The identification of Panton-Valentine leukocidin (PVL) genes in the isolates of *Staphylococcus* aureus in children with DVT has been reported in several studies.3,6,8,10,11 These reports suggest that PVL may be associated with enhanced inflammatory response in cases of musculoskeletal infection including pneumonia, multifocal osteomyelitis, bacteremia, and concurrent pyomyositis, subperiosteal abscesses or intra-osseous abscesses requiring surgical debridment.3,6,8,10,11 The advent of rapid PCR technology as a tool to identify the presence of PVL may prove valuable in identifying potentially "silent" cases of DVT that would otherwise go unrecognized. To this point, however, the technology to perform rapid testing to identify the bacterial genetics of the *S*. *aureus* isolates is not readily available at most institutions. Our meta-analysis identified 20 children out of the 58 reported cases with DVT who were found to be PVL positive (34.5%).3,6,8,10,11


# **Using Vis-NIR Spectroscopy for Predicting Quality Compounds in Foods**

Edited by Mercedes Del Río Celestino and Rafael Font Villa Printed Edition of the Special Issue Published in *Sensors*

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