Viral, Parasitic, Bacterial, and Fungal Infections: Antimicrobial, Host Defense, and Therapeutic Strategies

By Debasis Bagchi

Viral, Parasitic, Bacterial, and Fungal Infections: Antimicrobial, Host Defense, and Therapeutic Strategies highlight diverse types of infections, including viral, bacterial, parasitic, fungal, and the therapeutic efficacy of  antibiotics, antivirals, antifungals and other medications, nutraceuticals, and phytotherapeutics. This book addresses the molecular, pathophysiological, and cellular pathways involved in the process of infection. It also examines the host defense mechanisms modulated by innate and adaptive immunity. The book starts off with an introduction, which includes etiology, pathophysiology, and diagnosis of infections. It then goes on to cover a wide spectrum of salient features involved in viral, bacterial, parasitic, and fungal infections and effective therapeutic strategies. In addition, there is a complete section of eight chapters elaborating the detailed aspects of  COVID-19 infections, Mucormycosis, Omicron, and strategic vaccines and therapeutics. The book further goes on to discuss novel antibiotics, vaccines, bromhexine, boron compounds, phytotherapeutics, and aspects on boosting immune competence. Contributed by experts in the fields of viral, parasitic, bacterial, and fungal infections, the book comprehensively details the various types of infections such as herpes and COVID-19, their molecular mechanisms, and treatment strategies for those engaged in the research of infectious diseases.

• Details the pathophysiology of various classes of infections
• Examines mechanisms of pathogenesis, immunity, and therapeutics in bacterial, viral, and eukaryotic infectious diseases
• Discusses various aspects on herpes, COVID-19 infections, Mucormycosis, Omicron, vaccines, and therapeutics
• Covers the salient features on zoonosis, prion disease, and diabetic foot infections
• Provides therapeutic strategies of using new antibiotics, vaccines, bromhexine, boron compounds, structurally diverse phytotherapeutics, immune enhancers, and other modalities for treating infections

• Language: English
• Publisher: Academic Press
• Release date: Oct 15, 2022
• ISBN: 9780323898003

Book preview

Part I

Introduction

Chapter 1: Clinical applications of molecular diagnosis in infectious diseases

Lourdes Eguigurena; Shirley F. Delairb; Archana Chatterjeec    a Division of Pediatric Infectious Diseases, University of Nebraska Medical Center, Omaha, NE, United States

b Diversity, Equity, and Inclusion, Division of Pediatric Infectious Diseases, Pediatric Residency Global Health Track, University of Nebraska Medical Center, Omaha, NE, United States

c Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, United States

Abstract

Molecular diagnosis of infectious diseases is a rapidly evolving field. New technologies have been revolutionizing patient care, improving the time of diagnosis, therapeutic intervention, and subsequently, patient outcomes from many infectious diseases. These new technologies, however, do not come without challenges: selecting from the number of tests available can be daunting; an overwhelming amount of information can be obtained with unclear importance; and the cost can become prohibitive, particularly in resource constrained settings. Appropriate diagnostic and therapeutic stewardship of rapid molecular diagnostics is essential to guide their effective use, control the costs, and increase the benefits of the technology to a broader community. In this chapter, we highlight the use of molecular diagnostics in a broad but nonexhaustive array of bacterial, fungal, viral, and mycobacterial infections in clinical practice, as well as their use in therapeutic monitoring and assessing for resistance to treatment.

Keywords

Bacteria; Clinical applications; Infectious diseases; Molecular diagnosis; Resistance testing; Viruses

Introduction

When considering a particular molecular diagnostic, in addition to knowing the sensitivity, specificity, positive predictive value, the ideal specimen collecting site, the timing, and cost of the test, it is also essential to correlate these factors with the clinical scenario of the patient being tested and also whether there are intrinsic factors about the host that could affect the interpretation of test results [1]. Stewardship of diagnostic molecular tests is essential for appropriate antimicrobial stewardship as well. Once the clinician has performed the right test at the right time on the right patient, the results obtained are likely to be helpful in selecting appropriate and timely use of antimicrobials, if they are warranted. This sequence of events increases the likelihood of a good outcome for the patient [1]. This tailored and prompt approach to treating infectious diseases in patients has the potential to significantly decrease cost of care, and though the technology is not widely available to low-income regions of the world, the advances in molecular diagnostics could potentially be key to achieving global health equity more rapidly [2].

Nucleic acid amplification by polymerase chain reaction (PCR) was developed in 1987 and has revolutionized molecular diagnostic methods. It essentially is a method of extracting and purifying a nucleic acid, then using specific primers and polymerase enzymes to exponentially amplify a given target sequence [3]. Multiple modifications and improvements from this original technique led to the term nucleic acid amplification test (NAAT). NAATs allow the detection of low copies of genetic material (DNA or RNA) of an organism present in a given specimen and prepare it for postamplification analysis, leading to an increase in diagnostic sensitivity by decreasing the lower limit of detection using single and target amplification [4]. Single amplification uses branched DNA (bDNA) and hybrid capture, while target amplification uses PCR and transcription-mediated amplification (TMA) [4]. Postamplification analysis includes sequencing, reverse hybridization, and Luminex analysis, while large-scale nucleic acid analysis includes whole genome sequencing, nucleic acid analysis, and mass spectrometry [4].

Bacteria

Culture-based approaches for the detection of most bacterial organisms remain the cornerstone of infectious diseases diagnostics. However, in the last two decades, molecular diagnostic tests have become increasingly important tools for the detection and identification of many bacterial pathogens [5]. There are several limitations to the use of culture, including long turnaround times, difficulty cultivating certain organisms in vitro, or need for special culture media, which can lead to low test sensitivity. Molecular assays can overcome some of these limitations and have been particularly useful in identifying uncultivable or fastidious microorganisms. Due to the high sensitivity and specificity of these tests, as well as improved turnaround time compared to cultures, there are many instances where laboratories have replaced culture-based pathogen detection with molecular methods.

Clinical applicability

Chlamydia trachomatis and Neisseria gonorrhea are among the most common bacterial causes of sexually transmitted genitourinary infections worldwide [6]. Since many patients infected with these organisms are asymptomatic but still can transmit the disease, highly sensitive and specific tests are needed for timely diagnosis and treatment to prevent their spread. In the past, cell culture was the only diagnostic method for C. trachomatis. However, cell culture was technically difficult, time demanding, and relatively insensitive [7]. Similarly, culturing N. gonorrhea is possible but not ideal because of stringent collection and transport requirements and difficulty in maintaining the viability of organisms [7,8]. Currently, NAATs are the preferred method for screening and diagnosis because of overall superior sensitivity and specificity over conventional culture. The introduction of NAATs has also facilitated the development of screening programs because these tests are highly effective in detecting bacteria even with noninvasive sampling such as self-collected vaginal swabs and urine specimens [6]. However, one limitation of these molecular methods is the inability to perform antibiotic susceptibility testing that could be clinically relevant in light of the global increase in the incidence of resistant N. gonorrhea isolates [9].

Molecular diagnostics have also become important in the diagnosis of bacterial gastroenteritis. There are several multiplex PCR assays for the detection of the main causative agents of bacterial gastroenteritis and diarrhea including pathogenic strains of Escherichia coli, Campylobacter, Salmonella, Shigella, Vibrio, and Yersinia enterocolitica [10]. The diagnosis of Clostridioides difficile (formerly Clostridium difficile) usually involves a two-step diagnostic algorithm that includes the detection of toxin producing genes by NAAT to distinguish colonization from infection [11]. Given the high sensitivity and specificity of these tests and rapid turnaround time, molecular testing has been particularly helpful to guide clinical management decisions and infection control measures [12].

One of the main advantages of molecular testing is the rapid turnaround time and the potential for rapid bacterial diagnosis. The potential impact of timely and accurate diagnostics tools in acute-care settings, especially for life-threatening infections such as bacterial meningitis or bloodstream infections is promising with these newer technologies [13]. In 2015, the first commercial multiplex NAAT for the detection of community-acquired pathogens causing meningitis and encephalitis was approved by the US Food and Drug Administration (FDA). This multiplex panel detects 14 bacteria including Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae, Escherichia coli, and Listeria monocytogenes. Although Gram stain and culture remain the test of choice for diagnosis of bacterial meningitis, NAATs can be useful in conjunction with culture in patients pretreated with antibiotics. Similarly, there are several commercial platforms to detect pathogens from blood samples. Table 1 shows selected FDA-approved NAATs for the detection of bloodstream bacteria and resistance genes. Other areas of ongoing research include the use of broad range 16 s ribosomal RNA and metagenomic next-generation sequencing (mNGS) for diagnosis of culture-negative bacterial infections [15–17]. Although the current studies are promising, more research is needed to fully understand the applicability of these techniques in clinical practice.

Table 1

aEnterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group, Proteus, Salmonella, Serratia marcescens.

bEscherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Serratia marcescens, Enterobacter cloacae complex.

cEscherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis.

Another important use for molecular techniques is the diagnosis of fastidious bacteria difficult to grow in regular cultures. This is the case for Bordetella pertussis, where PCR has replaced direct fluorescent antibody and culture as the preferred method for diagnosis early in the disease process [18]. There are two available NAATs for the detection of B. pertussis: one is a targeted PCR assay that detects multiple copies of the insertion sequence gene and the second is a multiplex respiratory panel that detects a single copy target in the toxin promoter region [19]. The targeted PCR panel is more sensitive than the multiplex assay, but the multiplex test is more specific [19]. Other fastidious respiratory pathogens that can be detected by molecular techniques include Legionella spp., Mycoplasma pneumoniae, and Chlamydia pneumoniae [20]. However, it is important to note that since multiplex bacterial panels have been developed in the last few years, their test performance and potential impact on clinical outcomes have not yet been established. As a result, it is important to use these tests cautiously and interpret the significance of a positive test taking into account the clinical context.

There are several other examples where bacteria can only be detected by molecular methods where culture-based diagnosis is either extremely difficult or impossible for the routine microbiology laboratory. Tropheryma whipplei is the causative agent of Whipple’s disease, which could previously only be detected by PAS staining and electron microscopy from infected tissue [18]. Now, PCR testing from different specimens (e.g., cerebrospinal fluid, synovial fluid, lymph nodes, etc.) has facilitated the diagnosis of this potentially lethal disease [21,22]. Kingella kingae is one of the most common bacteria causing osteoarticular infections in infants and children; however, growth in traditional media is difficult and commonly missed. PCR-based assays have significantly improved the bacteriological diagnosis and time-to-detection of this organism [23]. Bartonella spp., Coxiella burnetii, Rickettsia spp., and Ehrlichia spp. are other examples of obligate intracellular bacteria in which diagnostics have significantly advanced with the use of NAATs [24].

Resistance testing

The rise in multidrug-resistant bacteria is one of the most pressing public health threats at the moment. Early antimicrobial resistance profiling is key for optimal patient management and for public health surveillance. Genotypic detection of antibiotic resistance has the potential to overcome some of the difficulties related to traditional resistance testing such as long turnaround time and variable phenotypic resistance expression. In some cases, the detection of a resistance gene associated with a certain pathogen is relatively simple. For instance, the presence of the mecA gene can be multiplexed with the nuc gene for rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) isolates [18]. In contrast, identifying β-lactam antibiotic resistance in gram negative organisms is more complex. There are thousands of β-lactamase genes responsible for resistance in gram-negative bacteria [14]. For this reason, rapid molecular assays have focused on the detection of β-lactamase genes conferring resistance to carbapenem antibiotics, also called carbapenemases. Some examples of these genes include OXA, KPC, CTX-M, NDM, VIM, and IMP β-lactamases, which have been incorporated in certain multiplex platforms for the identification of bloodstream infections (Table 1) [14].

Viruses

Annually, viral infections cause significant morbidity and mortality globally from recurrent infections, to outbreaks, to pandemics. Most of the viral pathogens involved affect mainly the respiratory and/or the gastrointestinal tract but may affect other systems as well. There exist immunizations and/or treatments for a limited number of these viruses. The need for specific, directed treatment is based on the organism and/or the host immune status and clinical course. Early detection is key to undertaking specific efforts to curtail the effects of the spread of infection such as isolation precautions to decrease transmission to susceptible hosts, immunizations in the face of an outbreak from vaccine preventable illnesses to timely directed antiviral treatment when applicable [25]. PCR, mass spectrometry, and next-generation sequencing are powerful tools in the diagnosis of viral infections, but the cost of instruments and reagents has limited their widespread use globally. However, research is ongoing to find more affordable alternatives [25].

Clinical applicability

There are several viruses that have a significant impact on global morbidity and mortality: HIV, hepatitis B, hepatitis C, and more recently, SARS-CoV-2. The latest HIV & AIDS statistics show that in 2020, 37.7 million people were living globally with HIV with 1.5 million new infections and 680,000 deaths due to AIDS annually [26]. As of 2019, there were 296 million people globally with hepatitis B and 58 million with hepatitis C [27]. There were 1.5 million people with new chronic hepatitis B and another 1.5 million new chronic hepatitis C patients diagnosed in the same period [27]. Around 1.1 million deaths occurred from complications of these chronic illnesses such as liver cancer and cirrhosis [27]. By August 1, 2021, there were almost 200 million cases of SARS-CoV-2 infection, with around 4 million deaths worldwide since the pandemic began in December 2019 [28]. From chronic diseases to emerging pathogens leading to pandemics, the importance of effectively diagnosing and instituting preventative and therapeutic measures when appropriate cannot be overstated.

NAATs have been widely used in the management of many viral infections. The use of quantitative real-time PCR in patients with HIV, hepatitis B, and/or hepatitis C infections has allowed accurate and reproducible monitoring of serum viral load [29]. In immunocompromised hosts in particular, further identification and quantification of viral load for infections due to viruses, such as CMV, EBV, VZV, BKV, may in addition to a diagnostic role, be part of preemptive monitoring to determine clinical relevance and need to make therapeutic adjustments in immunosuppression and/or antiviral therapy when appropriate [30].

Respiratory infections are a leading cause of morbidity and mortality particularly in the newborn, the elderly, the immunocompromised, and those with underlying lung disease, with ER visits and hospitalizations each year due to these infections [31]. Being able to detect viral infections rapidly with molecular testing such as PCR helps decrease the use of unnecessary antibiotics. One limitation from using the PCR test to detect viral respiratory pathogens is that it may be difficult, particularly in the immunocompromised host to detect whether there is prolonged viral shedding versus recurrence of infection [32]. Table 2 lists the current FDA-approved multiplex PCR tests that can detect common viral infections.

Table 2

With the advent in 2019 of the novel respiratory virus SARS-CoV-2, the causative agent of COVID-19, molecular methods were first used to sequence the virus. Once the pathogen was identified, the WHO recommended using real time reverse transcription polymerase chain reaction (real time RT-PCR), a highly sensitive method to help confirm suspected cases and apply infection prevention measures promptly [33]. Multiplex PCR methods have been developed to incorporate detection of SARS-CoV-2 on existing panels and are awaiting FDA approval [34].

Viral gastroenteritis is usually a self-limiting illness. However, in certain clinical scenarios, based on the exposure and the host immune status, they may lead to significant morbidity. In middle- to high-income countries, infectious diarrhea may be due to viruses in 75%–90% of the cases [35]. Most of the time these infections are secondary to adenovirus or norovirus, particularly in outbreak settings, and rotavirus primarily in infant and children [35]. Viral gastroenteritis may lead to prolonged shedding of viral particles in the stool and is more common in immunocompromised hosts [35]. The ability to attribute an etiology to these episodes of gastroenteritis helps decrease unnecessary antibacterial or antiparasitic treatment and focus on supportive care [32]. Table 3 lists the FDA-approved multiplex gastrointestinal pathogen panels and the viruses they detect.

Table 3

Though viral infections of the central nervous systems are not as common as respiratory and gastrointestinal viral infections, they are the most common cause of meningitis and encephalitis in many settings. The use of molecular techniques rather than viral cultures has allowed for prompt diagnosis and decrease in unnecessary use of antibiotic therapy, which has then led to decreased cost and duration of hospitalization. Viruses are the most common cause of aseptic meningitis with enteroviruses being the most common viruses isolated. Using multiplex PCR allows with one CSF sample collection to test for a panel of viruses and get a rapid turnaround, which helps to improve antimicrobial stewardship and decrease cost [37]. The Biofire FilmArray Meningitis/Encephalitis (ME) panel can detect cytomegalovirus, enterovirus, herpes simplex virus 1 and 2, human herpesvirus 6, human parechovirus, and varicella-zoster virus [37]. Prompt and reliable diagnosis of HSV 1 or 2 disease via the rapid detection of viral DNA by PCR in CSF samples in addition to blood, skin, and mucous membranes has had a major impact on timely diagnosis and treatment, particularly in neonates who are at risk for significant morbidity and mortality [38].

Resistance testing

CMV is a viral infection of particular importance in the immunocompromised and transplanted host, where there is an increased exposure to antiviral therapy in the setting of long-term treatment or recurring disease, which can lead to resistance to first line antiviral therapy. In this population, the availability of rapid and reliable resistance testing is crucial to make therapeutic adjustments to address treatment failures. In this setting, genotypic resistance testing using next-generation sequencing provides a highly sensitive and reliable way to detect mutations in the protein kinase or the DNA polymerase that confer resistance to ganciclovir, cidofovir, and foscarnet [39]. Genotypic sequencing is also used in the detection of mutations that confer reduced susceptibility to acyclovir in immunocompromised patients infected with HSV, particularly with hematopoietic stem cell transplant [40]. The standard for HIV drug resistance testing is next-generation sequencing and is an essential component of HIV treatment in settings with access to the technology. Prior to initiating therapy, in a treatment-naïve patient, or for monitoring purposes in an experienced patient whose persistently elevated viral load is concerning for virological failure, genotypic drug resistance testing is done to help tailor therapy based on the characteristics of the HIV variant [41].

Monitoring response to treatment

NAATs have been used to quantify the viral load of patients infected with HIV, hepatitis B and C and have been very useful to monitor response to treatment and to check for resistance if the viral load is not well controlled on antiviral therapy [29]. In immunocompromised and transplant patients, serially quantified viremia and/or viruria is monitored to check for response to therapy [30]. In diseases such as neonatal HSV, repeating a HSV PCR in CSF at the end of a treatment course is a useful measure to determine if therapy has been completed and that the patient can be transitioned to prophylaxis [42].

Fungi

Invasive fungal infections (IFI) are associated with high rates of morbidity and mortality, especially in immunocompromised and critically ill patients. One of the many challenges in managing fungal infections is the difficulty in diagnosis. Clinical signs and symptoms alone are often nonspecific and not reliable to make a definitive diagnosis. Studies have shown that inaccurate or delayed diagnosis are associated with poor patient outcomes [43,44]. Histopathologic identification of fungal organisms in tissue, antigen and antibody detection, and culture have been the traditional methods to diagnose IFIs. However, there are many limitations to these techniques including lack of sensitivity and specificity, need for invasive samples such as tissue biopsies, and long turnaround times [45]. Molecular diagnostics are becoming more important in clinical mycology because they offer significant advantages compared to traditional diagnostics. Firstly, molecular assays have a higher sensitivity and specificity compared to conventional testing [46,47]. Secondly, they can potentially identify organisms seen on direct microscopy but unable to grow in culture [46,47]. Thirdly, they can accurately identify organisms whose characteristics closely resemble those of other fungi [46]. Given the growing evidence of the effectiveness of molecular assays in this field, the European Organization for the Research and Treatment of Cancer/Mycoses Study Group Education and Research Consortium (EORTC/MSGERC) have revised the definitions of proven, probable, and possible fungal disease to include PCR from tissue to fulfill the definition of proven fungal infection, even in the absence of culture data [48]. Currently, the main drawback of molecular diagnostics for fungal infection is the lack of standardization of methods across laboratories, and consequently, difficulty in comparing and evaluating their clinical performance.

Clinical applicability

Pneumocystis jirovecii is an opportunistic fungal pathogen that causes a severe form of pneumonia that could be life-threatening in immunocompromised hosts. As with other fungal infections in this population, establishing a diagnosis and timely treatment are critical to achieve optimal outcomes. However, Pneumocystis jirovecii cannot be cultured in vitro, and the diagnosis depends on direct visualization of cysts and/or trophozoites in lower respiratory tract specimens [49]. The problem with this method is its low sensitivity, and the performance of the test depends on the skills of the observer and the sample type [50]. PCR-based tests have been reported to have higher sensitivity and specificity for detection of Pneumocystis jirovecii in different samples including BAL fluid, sputum, and oropharyngeal wash fluid [49,51]. Another recently developed technique for diagnosis of Pneumocystis jirovecii pneumonia is the PCR detection of cell-free DNA in serum. Although, not yet commercially available, animal models have shown similar sensitivity and improved specificity compared to PCR of BAL fluid samples [52].

Invasive aspergillosis and invasive candidiasis are among the most common fungal infections in immunocompromised and critically ill patients and are associated with high mortality. Molecular methods to enhance the diagnostic yield of these pathogens continues to be an active area of research. Currently, there are several commercially available NAATs for Aspergillus spp. and Candida spp. PCR from various clinical specimens (BAL fluid, serum, plasma, and tissue) is the most broadly used molecular diagnostic test for detection of Aspergillus spp. However, studies have shown that performance of molecular assays varies depending on the collection method and specimen type; therefore, cautious interpretation by clinicians is recommended [53]. For instance, one of the major limitations of PCR testing from respiratory samples is the inability to differentiate airway colonization from invasive infection [49]. It is estimated that up to 25% of BAL samples from healthy individuals could be falsely positive by using molecular testing compared to alternative diagnostic tests [54]. A multiplex PCR panel (BioFire FilmArray) which identifies pathogens directly in positive blood culture is currently available, and includes several fungi such as Candida albicans, Candida glabrata, Candida auris, Candida parapsilosis, Candida krusei, Candia tropicalis, and Cryptococcus neoformans/gatti. Studies have validated this assay, and since it is not a technically difficult assay to perform, it has the potential of identifying fungal pathogens faster than regular culture and result in early administration of optimal antifungal therapy [55].

The Film Array multiplex meningitis/encephalitis PCR panel (BioFire Diagnostics, Salt Lake City, UT, United States) is a FDA-approved molecular assay for the diagnosis of common pathogens causing meningitis, including Cryptococcus neoformans/gatti. Although in most cases PCR assays are highly sensitive, this multiplex PCR test is less sensitive than high-performing cryptococcal antigen tests, and it can give false-negative results [56]. If there is high suspicion of these fungal pathogens, fungal stains, culture, and antigen detection remain the tests of choice and should not be replaced by molecular testing.

Resistance testing

Azole-resistant Aspergillus fumigatus has emerged in recent years [57–59]. Resistance mechanisms reported involve point mutations in the cyp51A gene, which is the target of the antifungal azoles [57]. Specifically, the TR34/L98H and TR46/Y121F/T289A mutations in the cyp51A gene and its promoter region have been associated with this pattern of resistance [57]. Two commercial multiplex real-time PCR assays have been developed in Europe (MycoGENIE (Ademtech, Pessac, France) and AsperGenius (PathoNostics, Maastricht, the Netherlands)) for the diagnosis of A. fumigatus and have incorporated the most significant cyp51A gene mutations for the detections of azole-resistant isolates [49]. These assays have been validated in BAL fluid and serum samples with promising sensitivity and specificity [60–62].

Mycobacteria

Mycobacterium tuberculosis complex and Mycobacterium leprae are the major causative agents of TB disease and leprosy. In 2019, an estimated 10 million people worldwide were diagnosed with tuberculosis disease, with 88% being adults [63]. Furthermore, TB is the leading cause of death worldwide from an infectious agent [63]. TB is a disease that primarily affects low- and middle-income countries (LMICs), and there is a need for increased screening and reliable diagnostics to initiate appropriate treatment and public health measures more promptly in order to curtail the spread of disease [63]. Leprosy is also more prevalent in LMIC and leads to significant morbidity.

Leprosy continues to burden LMIC as a result of delayed diagnosis due to a lack of gold standard diagnostic methods, which in turn leads to ongoing transmission [64]. The advent of effective multidrug therapy has not been able to break that pattern as though it helps in curtailing transmission, by the time effective treatment is initiated, significant transmission has already occurred [64]. This highlights the importance of developing and increasing access to sensitive, specific molecular diagnostic methods.

Clinical applicability

The WHO recommends using the NAATS to diagnose tuberculosis disease due to their accuracy, compared to smear microscopy; additionally, the tests are found to be particularly sensitive when there is HIV co-infection or paucibacillary TB disease [65]. There are many NAAT assays that use PCR to target a specific genetic region of the M. tuberculosis complex. Additionally, not only can the NAATs detect the MTB complex but they can also report the drug susceptibilities—a process that when done by conventional methods takes weeks. The drug susceptibilities report can reveal resistance to rifampin (RIF) and isoniazid (INH) much more promptly to help guide initial therapeutic management.

A few NAAT use methods that have been approved by the WHO: the line probe assay (LPA), has been used for the detection of multiple drug-resistant TB particularly to INH and RIF [65]. Newer LPAs can now also detect mutations leading to fluoroquinolone resistance as well as other second line therapies. Another method, the loop-mediated isothermal amplification (LAMP) is a PCR amplification technique that can be used in point-of-care testing and requires limited laboratory equipment. However, the uptake of the technology is limited, with some countries trying to develop a local assay to increase use [65]. Another NAAT is the next-generation Xpert testing, using the GeneXpert platform; it detects resistance by using 4 probes that target the gene of the RNA polymerase (rpoB) of M. tuberculosis [65]. An additional test, the Xpert Ultra, can detect very low levels of bacteria and is particularly useful in cases of paucibacillary disease such as can occur in HIV infected patients, those with extrapulmonary TB and children [66]. Another advantage of the Ultra platform is that a technician with minimum training can run the tests. However, there must be continuous power that makes it less practical in areas of the world with the highest burden of TB disease [65]. There are more simple testing assays that can use tablets instead of computers and are battery operated that increases portability [65].

The detection of M. leprae by DNA PCR using samples such as nasal swab, saliva, urine, and skin biopsy can help to identify the organism [67]. This tool has the potential to serve an important public health role to curtail leprosy transmission in a timely fashion by identifying M. leprae by DNA PCR using nasal swab specimens and lead to initiation of timely treatment [67]. Furthermore, in cases of paucibacillary disease, quantitative PCR could enhance diagnosis due to its higher sensitivity, potentially leading to even timelier therapeutic interventions and improved endemic leprosy control [64].

Resistance testing

Platforms used to diagnose TB integrate resistance gene testing and are designed to be a one stop assay such as the Ultra Xpert and can be used in pulmonary and extrapulmonary TB. The turnaround time is shorter and allows the initiation of treatment based on the susceptibility of the organism isolated [66]. Molecular diagnosis with real-time PCR has helped identify drug-resistant M. leprae. Prior to this it had been difficult to identify as the bacteria does not grow well, making surveillance for resistant strains not feasible [68]. PCR sequencing for drug resistance allows the identification of gene regions associated with resistance to rifampicin, dapsone, and ofloxacin, which stresses the importance of broadening surveillance [68]. The discovery of a resistance gene for fluoroquinolone in M. leprae samples reinforces the need for better antimicrobial stewardship as this class of antibiotics is not part of the multidrug agents being used to treat leprosy [68].

Response to treatment

Being able to monitor response to therapy, particularly in the setting of multidrug-resistant TB, can help decrease morbidity and mortality from TB. Using an assay such as the molecular bacterial load assay (MBLA) uses RT-PCR to detect the rRNA of M. tuberculosis complex from sputum samples [69]. As treatment is instituted, the level of rRNA declines. MBLA is a quick, sensitive test. However, it still requires some optimization to be used more easily in the clinical setting. Furthermore, the technology is expensive and skilled professionals with adequate instrumentation that is generally found in reference laboratories are needed. Treatment for leprosy is long, and after partial treatment, bacteria can still be isolated in tissue specimens. Therefore, quantifying the amount of bacteria by RT-PCR assays has the potential of being useful to monitor response to treatment [70].

Parasites

Diagnosis of parasitic infections has traditionally relied on the morphological identification of organisms based on their life-cycle stage using microscopic methods and in fact, it is considered the gold standard. The important limitations to this approach are that it lacks sensitivity and relies heavily on a cadre of well-trained laboratory technicians [71]. In resource-limited regions of the world, which suffer from the overwhelming burden of parasitic infections, access to trained technicians and the necessary laboratory equipment is limited and the gap has been widening over time, particularly since parasitic diseases fall into the category of neglected tropical diseases (NTDs). There is less global attention and funding provided to eradicate NTDs. With the advent of molecular diagnostic methods, however, the prospect of more rapidly and efficiently diagnosing parasitic infections could potentially decrease the cost of tackling NTDs as it would require less infrastructure investment.

Clinical applicability

The most commonly used molecular diagnostics tools for diagnosis of parasitic infections are NAATs with PCR being the most widely used technique and the most commonly used sequencing targets being the 18S rRNA and the internal transcribed spacer (ITS) regions [72]. Of note, parasites, unlike bacteria and viruses, may require more than one target for identification as their gene target sequence data may be incomplete [71]. An important aspect of NAATs clinically is that the tests are very sensitive, however, for use in low-income countries, where the brunt of NTDs lie, the need for expensive reagents and instruments may be a barrier. An additional technology, the LAMP, which is an isothermal amplification method, has been found useful in the diagnosis of parasitic infections and even has potential in point of care testing [71,73–75].

Specimens from different sources can be used to help diagnose parasitic infections. Because of its sensitivity, NAAT testing is used to detect parasitemia via PCR from organisms such as Plasmodium spp., Babesia spp., Leishmania spp., and Trypanosoma cruzi in the blood [76–79]. In the case of Plasmodium spp., low level of parasitemia (though useful when screening blood to prevent transfusion-related malaria) [80] has a major limitation in that it may not indicate ongoing infection, since low levels of parasitemia can be observed even after effective antimalarial treatment has been administered [81]. Another important use of the PCR testing in malaria is using multiplex platforms to allow for simultaneous detection of plasmodium species that cause malaria in a high throughput manner that can allow screening of large populations in epidemiological and surveillance studies; this is particularly important in regions of the world where malaria is endemic and prompt diagnosis and treatment are essential [82]. The NAAT is also a useful adjunct diagnostic tool for congenital infections such as toxoplasmosis and Chagas diseases, as they allow the use of maternal and/or newborn blood and placenta, though amniotic fluid is only useful to detect Toxoplasmosis spp. and not Trypanosoma cruzi [83–85]. Finally, a key additional specimen for which NAAT has been useful is for the diagnosis of parasitic infections is stool. When compared to microscopy and antigen detection, the NAAT is more sensitive, and there are several commercially available multiplex platforms that allow the detection of several parasites (Table 4) [87]. Using three different samples of liquid or semisolid (preferably fresh) stool, transferred promptly to a container with preservatives and not contaminated with toilet water or urine may increase diagnostic yield [87].

Table 4

Resistance testing

Antiparasitic therapy resistance testing is not routinely available and in fact, except for Plasmodium spp. resistance testing, is rarely done. Overall, parasites, aside from Plasmodium spp., are rarely resistant to treatment. Additionally, it is not feasible to cultivate them in vitro [71]. When dealing with Plasmodium spp., resistance testing is crucial to determine the optimal therapeutic choice. Globally, Plasmodium falciparum is resistant to chloroquine and sulfadoxine-pyrimethamine. In certain parts of Southeast Asia, artemisinin-resistant Plasmodium falciparum is prevalent and there may be a delayed decrease of parasitemia when artesunate monotherapy or artemisinin-based combination therapy are used. A mutation in the Kelch13 gene is noted in these resistant cases [88]. Chloroquine-resistant Plasmodium vivax is widespread in certain parts of Southeast Asia and Oceania but estimates of the true extent of resistance may be masked by the coadministration of primaquine in the region [88].

Monitoring response to treatment

The response to antimalarial treatment is measured by the level of parasitemia post treatment. It was initially used to assess the response specifically to chloroquine. Antimalarials with long half-lives may complicate the interpretation of this level [89,90]. In the setting of concurrent HIV infection and visceral leishmaniasis, there is poorer outcome. Using PCR testing to monitor parasitemia due to Leishmania spp. has been useful as a marker of response to therapy and a sensitive and specific method to diagnose relapse of disease [91].

Molecular diagnostics in special circumstances

Infection control

Molecular diagnostics are being increasingly used in the field of infection prevention and control, both in the healthcare and community settings. This technology has been effectively used in the identification, investigation, and control of nosocomial pathogens. Genotypic methods are now widely used for epidemiologic typing and outbreak investigation. Molecular typing is generally performed to determine the relatedness between isolates. Epidemiologically related isolates share the same DNA profile, whereas unrelated isolates have different patterns [92]. If isolates from different patients share the same DNA profile, it is likely that there was patient-to-patient transmission or by a common source [92]. Molecular typing methods are helpful in documenting healthcare-associated transmission, differentiating between different strains, determining reinfection versus relapse, and identifying and tracking antimicrobial-resistant isolates [92,93].

Another significant contribution of molecular diagnostics is the rapid detection of bacterial pathogens of public health significance. For instance, early diagnosis of highly transmissible diseases like M. tuberculosis, B. pertussis, N. meningitidis is crucial for determining adequate isolation precautions, starting early treatment, decreasing the likelihood of transmission and identifying potential exposures that may require antimicrobial prophylaxis [18]. Molecular assays are also used to decrease the rates of healthcare-associated infections through surveillance screening for pathogens such as methicillin-resistant S. aureus (MRSA) or resistant Enterobacteriaceae. Since patients colonized with these organisms are important reservoirs for disease transmission and self-infection, early detection, and isolation of carriers can reduce the risk of transmission [94].

The use of next-generation sequencing (NGS) is transforming the practice of infection prevention and control. High-throughput NGS allows detailed large-scale analyses of entire pathogen genomes [95]. This new technology has contributed to important progress in phylogenetics, epidemiology, understanding the evolution of antimicrobial resistance patterns and virulence gene detection. Currently, NGS technology has been successfully used for outbreak investigation in hospitals and public health laboratories. Although NGS is a promising technology in the field, there are several barriers to its widespread implementation. A major limitation is the need for rigorous quality control and standardization processes; benchmarks for quality control are yet to be determined [95]. Other problems include the need for specialized infrastructure and resources, personnel with expertise in the area, and costs.

Antimicrobial stewardship

Antimicrobial stewardship is an important and validated tool to decrease the use of unnecessary antimicrobials and optimize antimicrobial dosing, duration of therapy, and route of administration with the ultimate goal of decreasing the emergence of resistant pathogens and minimizing drug-related toxicities [96]. The microbiology laboratory plays a fundamental role in the development and implementation of Antimicrobial Stewardship Program (ASP) activities. The Infectious Diseases Society of America and the Society for Healthcare Epidemiology published updated guidelines in 2016 for the implementation of ASPs which recommend the active collaboration between ASPs and clinical microbiology to improve patient outcomes [97]. Diagnostic stewardship refers to the appropriate use of laboratory testing to guide patient management and treatment in real time, with the goal of enhancing clinical outcomes and limiting the spread of antimicrobial resistance [98]. New molecular technologies and rapid diagnostics provide several opportunities for partnership between ASPs and the microbiology laboratory.

Rapid diagnostic technologies have the potential of decreasing time to appropriate therapy and timely identification of antimicrobial resistance. The implementation of molecular testing in consultation with clinicians and ASP teams who can provide antibiotic guidance at the time results are available have been proven to improve patient care [99,100]. For instance, the use of molecular blood culture identification panels and MALDI-TOF technology are important tools to decrease the time to optimal therapy and time to de-escalation [100]. Another example is the use of the respiratory PCR panel to assist in the decision making and de-escalation of antibiotic use for lower respiratory tract infections [101]. The development of new rapid and more precise diagnostics will continue to greatly benefit the field of ASP in the future.

Global health

LMIC often face a high burden of infectious diseases that without timely identification and treatment are life threatening. At the same time, resources for diagnostics are limited and clinical decisions are often made empirically that can lead to many problems such as antimicrobial overuse, suboptimal treatment, increasing rates of antimicrobial resistance, and ultimately, increased morbidity and mortality. Therefore, building diagnostic and laboratory capacity in these countries is fundamental. Despite all of the advantages of molecular testing, this technology remains mostly inaccessible in developing countries. The term 10/90 gap has been used to highlight the disparities between industrialized and developing countries; it refers to the idea that 90% of the research invested in genomics and related biotechnologies addresses the health needs of only 10% of the world’s population [2].

The majority of the current platforms require specialized infrastructure, reliable electricity and cold storage, sterile environments, and highly trained personnel. However, many of these are not available in most developing nations. In general, the ideal characteristics of a high-impact diagnostic test in LMIC include being affordable and sustainable, sensitive and specific, simple to perform by persons with minimal training, minimal equipment requirements (e.g., electricity-independent, battery operated, portable), having rapid turnaround time, and accessible to a large population [2]. In 2003, the World Health Organization Special Programme for Research and Training in Tropical Diseases (WHO/TDR) published a set of criteria for the ideal diagnostic test in developing countries. These criteria are referred to by the acronym ASSURED (Table 5) and have been used as the benchmark for the development of new point of care diagnostics in limited-resource settings [36,102].

Table 5

Currently, there are a few portable molecular technologies designed specifically for developing countries such as portable nucleic acid thermocyclers [103]. These machines are battery powered and have the capability of performing PCR, RT-PCR, and isothermal amplification [103]. This technology has been applied in the diagnosis of HPV, N. gonorrhea, C. trachomatis, Treponema pallidum, and Trichomonas vaginalis. Molecular point-of-care testing for infectious diseases are also becoming increasingly important in limited-resource countries. Further research is needed to develop newer and adaptable technologies to ultimately continue to expand molecular diagnosis in the developing world.

Conclusion

Though significant advances have been made in molecular diagnostic methods, there is still room for substantial improvement in reducing costs more significantly to impact the care of populations that bear the brunt of infections, particularly in resource-limited settings. A major goal with more rapid, efficient, and accurate diagnostic tests is to achieve global health equity by increasing access to innovative diagnostics that will help decrease the burden of infectious diseases across the globe.

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Chapter 2: Airway mucus, infection, and therapeutic strategies

Monali NandyMazumdar    Department of Dermatology and Laboratory of Inflammatory Skin Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, United States

Abstract

The respiratory tract epithelium is hydrated by a mucous layer, which forms a protective barrier for the defense against sometimes life-threatening foreign particles, including bacteria and viruses. The mucous layer is fluid in nature and facilitates ciliary beating, enabling mucociliary clearance. The mucus consists of a complex array of macromolecules called mucins, which are linked to complex long carbohydrate chains called O-glycans. Certain debilitating disorders such as cystic fibrosis, and even the recent COVID-19 can lead to the impairment of this layer by affecting its transport properties. In turn or individually, pathogens of the respiratory tract can evade this barrier by directly interacting with the intact mucus and bypassing it or by surviving in the already damaged layer. Therefore, understanding the specificities of mucin dysfunction and the mucin-pathogen relationship in immunocompromised infections is important for the development of therapeutics targeting mucin-associated mechanisms.

Keywords

Mucous layer; Mucins; Immunocompromised; Respiratory disorders; Infections

Acknowledgment

I would like to thank Dr. Shih-Hsing Leir for helpful discussions regarding the content of the chapter and his help in proofreading the manuscript.

Introduction

The surface of the respiratory milieu consists of epithelial cells, which are the first cellular network encountered by invading microbes. Therefore, the epithelial surface has developed several mechanisms to act as a defense to prevent infection by foreign invaders.

Several mechanisms are prevalent in the airway epithelium that include the maintenance of the tightness of the barrier, and secretion of mucus and several antimicrobials and their interactions with the function of the immune system through cytokines and other signaling molecules [1]. The mucosal surface (mucosa) plays an important role in guarding the lung against external harmful insults. Situated on the epithelial surface, the mucosa makes the epithelium wet and forms an integral protective mechanism in