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When comparing oral infection v IV infection in mice, why would the CFU given be different volumes?

When comparing oral infection v IV infection in mice, why would the CFU given be different volumes?


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In the paper, orally infected mice are given 1x10^9 CFU of C. Rodentium and IV infected mice are given 5x10^7 CFU of the pathogen. Does anyone know if there is a generic reason for this? Thanks in advance.,


Intravenous delivery of pathogen produces infection more efficiently than transmucosal delivery.

10^7 is 2 orders of magnitude less than 10^9. It is not the volume that is different (although it may be), it is CFUs or "colony forming units" which is a proxy for viable pathogens in the inoculum. The oral dose is 100x that of the IV.

Getting germs directly into the bloodstream bypasses the built in protections that our mucosal surfaces have. The GI and GU tract have a lot of defenses. For example an experimental SIV infection produced intravenously in monkeys requires an order of magnitude less virus than an infection established rectally or vaginally.

I would suspect that the CFUs of a given pathogen via a given route is something established empirically as producing the desired outcome. If you used the same CFUs for an IV route as were required for the oral route, you might have a fair number of mice that just died from septic shock from the huge number of bacteria suddenly in the blood. Given the 2 order of magnitude difference of inoculum (IV vs PO) to produce infection you might conclude that the stomach kills 99% of any inoculum.


Acidification and extended storage at room temperature of mayonnaise reduce Salmonella Typhimurium virulence and viability

S. Typhimurium culturability is reduced in acidified mayonnaise and is affected by temperature.

Bacterial motility and invasive ability are inhibited in acidified mayonnaise.

S. Typhimuiurm in acidified mayonnaise preparations exhibited reduced disease capacity in mice.

S. Typhimurium in acidified mayonnaise exhibited reduced ATP production over time.


Alternative Methods of Vaccine Delivery: An Overview of Edible and Intradermal Vaccines

Vaccines are recognized worldwide as one of the most important tools for combating infectious diseases. Despite the tremendous value conferred by currently available vaccines toward public health, the implementation of additional vaccine platforms is also of key importance. In fact, currently available vaccines possess shortcomings, such as inefficient triggering of a cell-mediated immune response and the lack of protective mucosal immunity. In this regard, recent work has been focused on vaccine delivery systems, as an alternative to injectable vaccines, to increase antigen stability and improve overall immunogenicity. In particular, novel strategies based on edible or intradermal vaccine formulations have been demonstrated to trigger both a systemic and mucosal immune response. These novel vaccination delivery systems offer several advantages over the injectable preparations including self-administration, reduced cost, stability, and elimination of a cold chain. In this review, the latest findings and accomplishments regarding edible and intradermal vaccines are described in the context of the system used for immunogen expression, their molecular features and capacity to induce a protective systemic and mucosal response.

1. Introduction

One of the ten greatest public health achievements of the last century was preventative vaccination [1]. Vaccines have successfully reduced the spread of diseases and mitigated mortality associated with infectious agents such as diphtheria, tetanus, polio, measles, mumps, rubella, and hepatitis B [2]. In spite of the many successes achieved by vaccines, novel technologies and administration routes remain one of the main focuses in the vaccinology field. Although many licensed vaccines are administered by injection, in certain cases, this administration route suffers from limitations. In particular, injectable vaccines require trained personnel for the administration of the vaccine and may require multiple doses or inclusion of an adjuvant. Moreover, injectable vaccines may require specialized storage and transport conditions. From an immunological point of view, injectable vaccines are capable of eliciting robust systemic humoral responses while conferring weaker T cell-mediated immunity and mucosal protection [3, 4]. Importantly, T cell effector activity and mucosal immunity both contribute to prevention and control of infection from pathogens targeting the mucosa [5].

To improve on this limitation, alternative vaccine delivery methods coupled with novel formulations and production systems have recently been proposed. Numerous studies have focused on vaccines delivered to the mucosal interface or intradermally, demonstrating rapid and wide biodistribution of the antigen and the capacity to induce both protective mucosal (mainly mediated by secretory IgA [SIgA]) and systemic cellular and humoral responses [6–8].

In this review, we discuss current advances and advantages of edible systems based on plants, algae, yeast, insect cells, and lactic acid bacteria and of the intradermal immunization route.

1.1. The Mucosal Delivery and the Immune Response

The efficacy of the mucosal administration route is largely based on the fact that mucous membranes constitute the largest immunologic organ in the body. Moreover, this interface is endowed with well-organized lymphatic structures, termed mucosa-associated lymphoid tissue (MALT), containing both the innate and adaptive (T and B cells) arms of the immune system [9]. Furthermore, antigen-specific SIgA plays a pivotal role in protecting mucosal surfaces from both microbe adhesion and toxin activities [8]. Thus, the development of novel vaccine delivery platforms implementing the elicitation of pathogen- or toxin-specific SIgA, as well as systemic IgG, is pivotal to improve vaccine effectiveness [10].

To date, the most well-studied vaccine delivery platforms capable of eliciting both mucosal and systemic immunities are edible or intradermal vaccine formulations (Figure 1). Oral vaccines stimulate the generation of immunity in gut-associated lymphoid tissue (GALT), which includes lymph nodes, Peyer’s patches (in which lymphocytes are the major component:

20% are T cells), and isolated lymphoid follicles in the gastrointestinal tract (GIT). An effective immunization using oral vaccines is achieved when sufficient quantities of antigen are transported across the mucosal barrier by M cells into Peyer’s patches and subsequently presented to T cells by antigen-presenting cells (APCs) [11]. Briefly, professional APCs display peptide fragments of the antigen in the context of the major histocompatibility complex (MHC) on their surface, which leads to activation of CD4 + T cells [12]. Subsequently, activated CD4 + T cells support germinal center development, including B cell affinity maturation and class switching to IgA, through providing CD40/CD40 ligand interactions and cytokine secretion [13–15]. Moreover, through the expression of specific chemokine homing receptors (e.g., CXCR5 or CCR10), antigen-experienced B cells migrate to distant effector regions where they differentiate into plasma cells capable of secreting dimeric or polymeric IgA molecules that are transported into the intestinal lumen as SIgA [10, 16].

In the context of edible vaccines aimed at eliciting pathogen-specific responses, it will be necessary to overcome mucosal tolerance. Briefly, mucosal tolerance is achieved against certain foreign antigens, such as those contained in our food, and serves to prevent unnecessary and potentially detrimental immune responses in the gut mucosa. Due to this phenomenon, an erroneous mucosal vaccine formulation could induce a Treg-based tolerogenic response instead of Th17-mediated protective immunity [17]. This potential shortcoming can be circumvented using several strategies, including incorporation of an appropriate adjuvant in the vaccine formulation or using sufficiently high doses of antigen to promote induction of effector rather than regulatory cells [5, 11]. Moreover, in the context of edible-based vaccine immunizations, it will also be important to consider the characteristics of the GIT, in which several factors, including proteolytic enzymes, acidic pH, bile salts, and limited permeability, may hinder the induction of a protective immune response [10]. To this end, conjugation of the vaccine antigen with specific ligands that enhance their uptake by M cells represents a focus of ongoing studies aimed at improving immunogenicity [18]. Moreover, antigen bioencapsulation avoids degradation and conformational alterations [19].

1.2. Overview of Edible Vaccines

In the following sections, we review the various strategies underlying the development of edible vaccines. In particular, we focused on plant, algae, insect cells, whole yeast, and lactic acid bacteria-based vaccines and describe the advantages and limitations of individual systems.

1.3. Plant-Based Vaccines

Plants have been extensively used for developing novel biopharmaceutical-producing platforms in recent years, as they promote proper folding of exogenous proteins and are economically sustainable [20, 21]. This is also known in the context of “molecular farming,” in which biomolecules of commercial value are produced in genetically engineered plants. There are several ongoing clinical trials using purified antigens transiently produced in tobacco plants (Nicotiana benthamiana) for injectable vaccine formulations. For example, Medicago recently completed a phase II clinical trial using a plant-derived, virus-like particle (VLP) quadrivalent influenza vaccine and announced a phase III clinical study in the last year (ClinicalTrials.gov identifier: NCT03301051) [22].

Owing to the fact that plants are edible, the notion that they could serve as a delivery vehicle, as well as biofactories, led to their use for oral vaccination in the early 1990s [23]. In recent years, additional studies have sought to overcome the limitations of conventional vaccines through development of edible formulations [24, 25]. Since the inception of the idea, it has been evident that using plants to produce vaccines would have several advantages. First, plant vaccines would likely have a low production cost and could be easily scaled-up, as has been demonstrated by the biopharmaceutical industry. Molecular farming gained visibility thanks to the success of ZMapp, the experimental drug against the Ebola virus that was produced in Nicotiana plants [26]. However, unlike biomolecule production, edible vaccine formulations do not need processing or purification steps before administration, which serves to further lower production-associated costs. Indeed, another advantage of this strategy is that plant cells would provide antigen protection due to their rigid cell wall. This is also known as the bioencapsulation effect and could increase bioavailability of antigenic molecules to the GALTs through preserving structural integrity of vaccine components through the stomach to elicit both a mucosal and a systemic immune response. Additional strategies for antigen protection can be achieved through targeting biomolecule expression inside chloroplasts or other storage organelles [27] or in the protein bodies of seeds [28, 29]. This technology also offers advantages in terms of storage and cold chain-free delivery due to the high stability of the expressed antigens bioencapsulated within the plant and seed tissues. Moreover, plant materials can be stored at elevated temperatures for longer periods and grown where needed, making this strategy even more attractive for developing countries [30]. Finally, plant-based oral vaccines are characterized by improved safety relative to traditional recombinant vaccine platforms, especially since contamination from mammalian-specific pathogens can be eliminated [30]. Indeed, some studies using lyophilized leaves have shown their advantages over fresh materials such as long-term stability, higher antigen content, and lower microbial contamination. As an example, freeze-dried CTB-EX4-expressing (CTB: cholera toxin B subunit EX4: exendin-4) leaves were shown to be stable for up to 10 months at room temperature, and lettuces expressing soluble antigen (PA protective antigen from Bacillus anthracis) were successfully stored for up to 15 months at room temperature without showing antigen degradation [31]. The antigen content in lyophilized leaf materials was also 24-fold higher than fresh leaves. An additional benefit of lyophilization was its ability to remove microbial contamination. While lyophilized lettuce had no detectable microbes, fresh leaves contained up to approximately 6000 cfu/g microbes when plated on various growing media [31].

To date, vaccine antigens have been transformed into many edible species including lettuce, tomato, potato, papaya, carrot, quinoa, and tobacco [32]. Their proper folding and enhanced expression have also been tested in animal models, proving the immunogenicity of antigens produced in these systems [24, 33].

To obtain high quantities of the protein of interest, both nuclear and chloroplast genomes have been successfully engineered. However, the latter option is preferred owing to some advantages including high levels of transgene expression (up to 70% of total soluble proteins (TSP)) [34, 35], bioencapsulation effect, and regulatory concerns since transgene containment is assured by the fact that plastids are not spread via pollen in most plants. Moreover, incorporation of vaccine antigens into the chloroplast genome would also enable the expression of multiple genes in a single operon, which would be very attractive for multivalent vaccine development. Likewise, this approach may enable the production of vaccines conferring protection against multiple infectious agents and would serve to further reduce costs associated with vaccine production and administration [36].

Unfortunately, there are some disadvantages undermining their applications. First, plastids are not suitable for production of antigens that require glycosylation for proper folding or those antigens in which a protective immune response requires glycan recognition. However, nuclear transformation represents a valid option. Secondly, antigen expression can be either transient or stable in plants, but the second is preferred in order to obtain a stable genetic resource. In fact, transgenic seeds represent a constant resource to grow the transgenic plants and to extract proteins. However, stable transformation is time-consuming [25]. Moreover, expression in stable transformed crop plants suffers from low yields, typically less than 1% of TSP [36]. On the other hand, transient expression technology using either Agrobacterium or viral vectors is robust, quick, and easy to manipulate [37]. However, this expression is typically unstable [30]. Another important challenge of plant-based oral vaccines is the lack of a proper dosing strategy because low doses may not be able to induce a sufficient immune response and high doses, as previously described, may lead to immune tolerance. To this end, freeze-drying methods were implemented to stabilize plant biomass, concentrate the antigen, and achieve an accurate dosage by quantifying the antigen in terms of dry biomass weight, as mentioned above [31, 38].

To date, there are some plant-based vaccines for the hepatitis B virus (HBV), rabies virus, Norwalk virus, enterotoxigenic E. coli, and Vibrio cholerae in phase 1 clinical trials (Table 1). Many others are still in preclinical development, including vaccines targeting a variety of pathogens such as avian influenza viruses (HPAI H5N1) [39], Helicobacter pylori [40], and coronaviruses [41].

1.4. Algae-Based Vaccines

Green microalgae, such as Chlamydomonas reinhardtii, represent another viable option for vaccine production. However, some disadvantages of plant-derived vaccines, such as low expression levels and improper glycosylation of antigen proteins, have been described [52]. Thus far, only chloroplast transformation is possible [52], and only a single organelle is present, even if it occupies half of the cell volume [53].

Stable transformed lines of green algae are easy to obtain and can lead to increased yield of expressed antigens. In fact, unicellular green algae have all the positive characteristics of plant systems, plus unique advantages over terrestrial plants. Biomass accumulation is extremely fast and can be used in its entirety. Their growth neither has seasonal constraints nor relies on soil fertility. Cross-contamination of nearby crops cannot occur, as algae can be cultured with enclosed bioreactors [54]. Furthermore, in regard to regulatory aspects, green algae, such as C. reinhardtii, are generally recognized as safe (GRAS) by the FDA. Finally, algae can be easily lyophilized and, when dried, can be stored at room temperature for up to 20 months without losing antigenic efficacy [55]. In fact, the algae cell wall assures the bioencapsulation effect, as it was proven to prevent antigen degradation by enzymes of the GIT [55].

Collectively, these characteristics indicate that algae would be an ideal host for vaccine transport without a cold chain supply. Thus, as already described for plant-derived edible vaccines, the low cost and simpler logistic in terms of manufacturing, storage, delivery, and administration of the algae-based technology make it an ideal system in the context of resource-limited settings compared to conventional vaccine formulations.

There are no algae-based vaccines currently in clinical trials however, preclinical formulations against human papillomavirus (HPV), HBV, and foot-and-mouth disease virus (FMDV) are under development [32, 52, 56] to overcome some technical problems, such as a low expression level from the nuclear genome and lack of glycosylation following chloroplast expression [52].

1.5. Insect Cell-Based Vaccines

Insect cell systems have been widely adopted because of their capacity to produce high levels of proteins and to perform cotranslational and posttranslational modifications, including glycosylation, phosphorylation, and protein processing. This expression platform allows for generation of stable transformed cell lines or transient expression driven by recombinant baculovirus infection. The baculovirus-insect cell expression system, referred to as BEVS, is one of the most well-known and used systems for large-scale production of complex proteins and, most recently, for the development of subunit vaccines [57]. To date, there are three commercially available vaccines produced in insect cells for different indications: Cervarix (GSK) for cervical cancer, Flublok (Protein Sciences, now owned by Sanofi Pasteur) for influenza, and PROVENGE (Dendreon) for prostate cancer [58].

Importantly, the baculovirus expression system is not limited only to cultured cells. Insect larvae or pupae can be used for protein production. In the context of edible vaccines using insect larvae or pupae, silkworm Bombyx mori larvae or pupae have been commercially used for the production of recombinant proteins and also as a feasible delivery system for the vaccine [59, 60]. As mentioned above, the baculovirus-silkworm expression system is able to perform cotranslational and posttranslational modifications and also able to produce large amount and multiple proteins. Moreover, since baculovirus is unable to replicate in vertebral animals, it can be considered a GRAS. Furthermore, the presence of protease inhibitors and biocapsule-like fat in the silkworms may protect recombinant proteins from enzymatic digestion in the GIT [23, 61].

Several vaccine prototypes are currently under evaluation, and strong systemic immune protective responses support the use of silkworm as a mucosal vaccine vector, as shown, for example, by high immunogenicity in mice of the urease B subunit of Helicobacter pylori produced in silkworm [60, 62]. While the data collected so far support the possible use of baculovirus-silkworm vaccines as a promising edible vaccine platform, it is only approved for food ingestion in a few Asian countries.

1.6. Whole-Cell Yeast-Based Vaccines

The industrial usage of yeasts cells for production of heterologous proteins has been well described [63, 64]. Additionally, the capability of this system to perform posttranslational modifications, the GRAS status, and the cellular wall that could protect the antigen across the GIT make engineered yeasts an attractive vaccine delivery system [23, 65]. In addition, the major drawback of this system is hyperglycosylation of recombinant proteins, but it has been already addressed by generating defective N-glycosylation strains of yeasts [66, 67].

Whole-cell yeast-based vaccines have been studied for their ability to elicit an immune response. Remarkably, some preclinical studies based on orally administrated Saccharomyces cerevisiae and developed for different infectious agents, such as HPV and Actinobacillus pleuropneumoniae, showed that this delivery system is able to induce a protective mucosal and a systemic immune response [68–70].

Moreover, the increased immunogenicity of this delivery system could be explained by the adjuvant activity of β-glucans on the yeast cell wall, which demonstrates immunomodulatory and adjuvant effects through binding of innate pathogen receptors on macrophages, DC, and neutrophils [71]. Currently, two clinical trials have been developed: GS-4774 for HBV treatment and GI-5005 for hepatitis C virus (HCV) treatment (Table 2). Regarding the clinical trial for GS-4774, despite the positive results obtained from phase 1 [72], the second phase, conducted in virally suppressed, noncirrhotic patients with chronic HBV infection did not show a clinical benefit. However, other safety and efficacy studies have been conducted on another group of patients (in particular, in treatment-naïve patients with chronic HBV) [73]. Regarding the clinical trial for GI-5005, phases I and II reported promising results [74]. In particular, in this trial, GI-5005 was used also in combination with Peg-IFN/ribavirin. However, data on efficacy have not been published yet.

1.7. Lactic Acid Bacteria-Based Vaccines

Lactic acid bacteria (LAB) are Gram-positive, nonsporulating, and nonpathogenic bacteria that have been used for decades for the production and preservation of food as well as for therapeutic heterologous gene expression, like the production of different anti-human immunodeficiency virus (anti-HIV) antibodies (scFV-m9, dAb-m36, and dAb-m36.4) by Lactobacillus jensenii and the production and functional expression of the antilisterial bacteriocin EntA in L. casei [75–77]. Given these and the ability of LAB to elicit a specific immune response against recombinant foreign antigens, these bacteria have been considered potential candidates as mucosal vaccine vectors. This delivery system can confer protection against antigen degradation and, thanks to its uptake at the GIT level, can activate both innate and adaptive immune responses [78, 79].

Many LAB, in particular, Lactobacillus spp and Bacillus subtilis, were used in preclinical studies against different infectious diseases. Different results have been obtained from these studies, but an elicited immune response was observed in all of them. As an example, the production of high levels of specific IgA and systemic IgG after immunization with bacillus spores expressing toxin A peptide repeat was reported [80], while in another paper, L. lactis expressing HEV antigen ORF2 vaccine was tested and a specific Th2-based cell-mediated immune response was revealed as well as the production of specific mucosal IgA and serum IgG [81]. Another study reported a Th1/Th2 immune response elicited after the immunization with Csenolase-expressing Bacillus subtilis [82]. Another example is the oral administration of B. subtilis spores expressing urease B of Helicobacter pylori that provide protection against Helicobacter infection [83].

An important feature of LAB is their natural adjuvanticity and their immunomodulatory effects, although the molecular mechanism of these capabilities is not completely understood [84]. Moreover, other studies reported an effect on dendritic cell maturation and an induction of cytokine secretion [85, 86]. Despite the promising characteristics of recombinant LAB as mucosal vaccine vectors and given the encouraging results from murine studies, some aspects need to be taken into consideration, namely, the fact that vaccine strains cannot be considered avirulent, even if it could be listed as GRAS, due to potential transfer of antibiotic selection markers among microbes [78, 87]. Furthermore, other factors are important for the development of LAB-based vaccines. As an example, the necessity of a suitable delivery system since different administration routes produce different immune effects. Additionally, the role of specific adjuvants and the correct localization (intracellularly or on the bacterial surface) of each expressed antigen need consideration [88]. Overall, additional studies and clinical trials are needed for the development of efficient vaccines based on LAB.

A different carrier system based on nonrecombinant Lactococcus lactis bacteria was recently developed. This system, called Gram-positive enhancer matrix (GEM), is composed of the rigid peptidoglycan (PGN) cell wall of the bacterium resulting in a nonliving particle that preserves the shape and the size as the original bacterium [89]. GEMs are used in two different ways: mixed with vaccine antigens as adjuvants or as antigen protein carriers, with the antigens bound to the surface of GEMs.

Regarding the use of GEMs as adjuvants, because of their nature, GEMs are safer adjuvants compared to others. Moreover, they retain the inflammatory properties of live bacteria and enhanced specific mucosal and systemic immune responses of the influenza subunit vaccine [90–92]. Therefore, the use of GEMs was further examined in a study investigating the immune response elicited by intranasal delivery of the influenza subunit vaccine coadministrated with GEM (FluGEM). In detail, an influenza-specific memory B cell response and the presence of long-lived antibody-secreting plasma cells were reported. Additionally, this immune response was able to confer protection from influenza infections [91]. These important results obtained in murine studies have led to a phase I clinical trial which confirmed the positive preclinical data. Systemic hemagglutination inhibition (HAI) titers and local SIgA responses were reported. Further studies will assess if this immune response confers protection against the influenza virus [93].

GEMs have also been used as antigen protein carriers. In particular, antigens are bound to GEM through the presence of a PGN-binding tag (Protan) in the antigen. Several works used this vaccination strategy, and the data support the potential of GEMs as safe vaccine delivery vehicles and their ability to elicit systemic antibodies [94–97]. Moreover, GEMs are also able to present several antigens at the same time which could be helpful for the preparation of multivalent vaccines [98]. Furthermore, the delivery of an adjuvant (GEMs) and an antigen together has been correlated with enhanced vaccine immunogenicity [97]. Lastly, as opposed to a vaccine based on LAB, the absence of recombinant DNA avoids its dissemination into the environment. However, the inability of GEMs to colonize any compartment does not allow the reduction of the number of vaccine doses.

These promising premises allowed the development of a vaccine against respiratory syncytial virus (RSV). In particular, an intranasal formulation based on the trimeric RSV fusion protein coupled with GEMs and named SynGEM was developed. Also, in this case, positive results from studies in mice and rats have been obtained, and as for FluGEM, vaccination with SynGEM resulted in the induction of a robust systemic and mucosal immune response as well as a balanced cytokine profile. These data supported further study of this vaccine in phase I clinical trial, which is currently ongoing [97]. In conclusion, GEMs represent an interesting vaccination strategy either as adjuvant or as antigen protein carriers, but as in the case of vaccine based on LAB, further studies are needed.

1.8. The Intradermal Vaccine Delivery and Its Associated Immune Response

Another vaccine delivery route capable of triggering both systemic and mucosal immunities is the intradermal route, in which the antigen is delivered through the skin using recently developed self-administrable devices. In particular, the application of microneedle technology overcomes the skin permeation barrier imposed by the stratum corneum and facilitates antigen delivery. The efficacy of this new microneedle-based immunization approach is due to the presence of several types of immune cells (such as DCs, T lymphocytes, NK cells, macrophages, and mast cells) in the epithelium [99, 100]. In fact, the cells that are responsible for triggering the inflammation cascade in the skin are the Langerhans cells (comprising 2-4% of epithelial cells). Langerhans cells are a specific DC subset that migrates into the lymph node following antigen capture and aids in the initiation of an adaptive immune response [101]. These cells are also efficiently stimulated by pathogen-associated molecular patterns (PAMPs) using an array of germline-encoded pattern recognition receptors (PRR), including toll-like receptors (TLR) and langerin (CD207) [100]. Importantly, skin resident mast cells are also key drivers of the innate immune response in the skin through the release of granules containing inflammatory mediators [102].

1.9. Intradermal Vaccination

Using conventional syringes for intramuscular or subcutaneous vaccinations, large volumes of vaccine solution can be injected (≥1 mL). Thus, the choice of the muscle or hypodermis as vaccination targets is mainly based on convenience [99]. Intradermal immunization for vaccine delivery is an upcoming strategy showing significant advantages over conventional vaccination routes. In particular, in the last few years, intradermal vaccination has gained momentum as an alternative and more effective vaccine delivery route, both from a scientific and a commercial point of view (Table 3).

PathogenFormulation/antigenIndicationClinical trial statusClinical trial IDRefs
Influenza virusSplit virusInfluenzas A and BApprovedNCT01712984, NCT02563093, NCT02258334, NCT01946438[118]
Enterotoxigenic E. colidmLT

dmLT: double mutant heat-labile enterotoxin.

Intradermal vaccination designates the delivery of an antigen directly into the dermis with a syringe, a needle, a microneedle, or a pressure injector. The standard intradermal immunization technique was invented by the French physician Charles Mantoux in 1910, while he was developing the tuberculin test. This technique allows the injection of 100-200 μL of vaccine solution. However, Mantoux’s technique requires skilled medical personnel to be performed [103]. Recent advancements have led to the development of techniques and instruments that can overcome the difficulties associated with intradermal administration [104]. In fact, different devices have been developed over the years for intradermal vaccination. Among them, solid microneedles, particle injectors, and self-administrable patches with coated microprojections or biodegradable needles have been described [105]. As previously mentioned, intradermal vaccination can induce mucosal and systemic immunities. These immunological capabilities, coupled with its ease of access, make the intradermal route an attractive vaccination delivery target [106].

Intradermal vaccination has been demonstrated to be very safe. In fact, novel devices involve the use of needles with a smaller size than the usual (25 μm and 1 mm) and make it possible to bypass the corneous layer of epidermis by creating transient micropores in the cutaneous tissues. Even if some studies have shown that intradermal vaccination can be associated with a higher incidence of local reactogenicity, including primarily mild pain, swelling, and redness, systemic side effects have not been reported. In fact, the intradermal route limits the transfer of vaccine components to the blood circulation (and the risk of septic shock) and the possible toxicity due to hepatic first-pass effect [107]. Typically, when present, local effects resolve quickly, as reported in a study comparing the safety and immunogenicity of a large number of intradermal versus intramuscular influenza vaccines [108].

Another important aspect is the possibility of improving the immunogenicity of various vaccines in immunocompromised hosts as well as during pregnancy via the intradermal route [109, 110]. As an example, it has been reported that the HBV vaccine fails to yield seroconversion in 3-5% of recipients. However, a significant improvement was observed following intradermal vaccination [111]. Additionally, it has been demonstrated that in patients on dialysis or in HIV-positive subjects, the intradermal route was more immunogenic than the standard intramuscular route [112].

From a commercial point of view, intradermal vaccination has been primarily explored for its ability to elicit equivalent antibody responses at lower doses, a phenomenon typically described as “dose sparing” [113]. In this regard, the advantage of a low dose is most evident in high-surge situations, such as during pandemic and seasonal influenza waves, in which large populations are at an increased risk and large amounts of new antigen preparations are quickly required each year [114–116]. Above all, dose sparing is also important in a large-scale setting and in reducing the production-associated costs, especially in developing countries, where the price of the vaccine limits its use and coverage. In this regard, the U.S. Food and Drug Administration (FDA) approved the trivalent inactivated intradermal influenza vaccine for use in adults 18-64 years of age for use during the 2012-2013 season, and a quadrivalent formulation was subsequently approved in 2014. However, similar to intramuscular vaccines, the formulation of these approved intradermal vaccines is liquid and thus still dependent on the cold chain and administered through a syringe. For these reasons, novel dried solid microneedle devices, while eliciting comparable immunogenicity to intramuscular-administered vaccines, represent an innovative approach to facilitate self-administration and allow vaccine storage at room temperature [117].

2. Conclusions

Infectious diseases represent a global concern, and the most effective strategy to reduce them is vaccination. Unfortunately, not every disease can currently be prevented through vaccines. However, many strategies have been developed against infectious agents, such as the generation of neutralizing antibodies, antibiotics, and antiviral drugs [124–130], and innovative approaches are currently under development [131–133].

Many vaccines have been developed and approved against various pathogens, and countless studies have been conducted to improve their efficacy by testing new adjuvants and performing the rational identification of the antigen formulations and pathogen contaminations [134–136]. Promising results have been also achieved by changing the delivery strategy. In fact, most of the approved vaccines are administrated by injection with intrinsic limitations like those concerning the immunological aspect. Injected vaccines are able to elicit a strong humoral immunity but a weak cellular response. In addition, this type of administration is strongly associated with a systemic immunity but with a lack of mucosal response, which is helpful to block the early stages of infection since most pathogens infect through the mucosal membranes.

For these reasons, new vaccination strategies have been proposed. In particular, edible vaccines, triggering the GALT, and intradermal approaches, involving Langerhans cells, are able to elicit both a mucosal and a systemic immune response. The increased knowledge of these approaches has led to the progression of many preclinical studies and several promising clinical trials (Tables 1, 2, and 3). Moreover, these vaccine strategies are considered safe and cost-effective as no extensive antigen processing is needed [137, 138] and they are easy to administrate (Table 4). In fact, due to the opportunity of self-administration and ease of distribution compared to an injection-based approach, these two vaccination systems could improve the overall coverage.

There remain a number of obstacles and drawbacks associated with each antigen delivery platform that still need to be addressed (Table 4). Presently there are no FDA-approved compounds for edible vaccination, but new emerging technologies like nanoparticles (NPs) could help to control antigen bioavailability to avoid mucosal tolerance. NPs are particles with a mean size of 10-100 nm (up to 2000 nm), which can be used as carriers and/or adjuvants in vaccine preparation [139–141]. Moreover, NPs can be targeted to specific cell populations. As an example, NPs can be coated with antibodies recognizing a surface protein on dendritic cells [142, 143]. This approach enabled a more accurate measurement of the quantity of antigen required to elicit an immune response [144]. Finally, a more efficient immunization was demonstrated using NP-based approaches combined with an intradermal vaccine delivery [145], while oral delivery needed further investigations as they have been tested only in vitro [146, 147].

In conclusion, novel approaches eliciting a stronger mucosal response are showing promising results both in preclinical and clinical studies. Further studies are needed to implement and improve these delivery systems however, mucosal delivery is becoming the most preferred mode of vaccination.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

E.C. and V.C. contributed equally to this work.

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Copyright

Copyright © 2019 E. Criscuolo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Conclusions

The need to treat HIV and HCV infectious diseases, two epidemics of global impact, has reawakened interest in mAb-based therapy, supporting a variety of clinical studies. The results that are emerging, will help to create models for the further development of such drugs and extend their use against other viruses as well.

Although the mAb production costs are high, increasing advances of biotechnology and production systems will make them more competitive on the market, and new approaches, such as using mAb cocktails or combining mAbs with available drugs, will improve effectiveness. Treatment with mAbs as part of a drug regimen is the most likely future for mAbs that block HCV and HIV infection in order to avoid viral escape, while chronic treatment could attract further investments from pharmaceutical companies. Furthermore, broad spectrum mAbs, such as bavituximab and immunomodulatory mAbs, could be useful against a whole range of diseases, thus extending marketability and profit margins.

This review has focused on the use of intact mAbs as a novel emerging and versatile class of pharmaceuticals. It is important to note, however, that biotechnology also provides the opportunity to build various antibody formats whose improved pharmacokinetics and pharmacodynamic properties could be co-opted in the fight against infectious diseases.


Management of ACD

The anaemia observed in ACD is frequently mild, and correction may not always be necessary. There are however several reasons for attempting to correct the anaemia present. Firstly, anaemia may be deleterious in itself, with effects on the cardiovascular system needed to maintain tissue oxygen supply. Secondly, anaemia may be associated with a poorer prognosis in many chronic disease states ( Caro et al, 2001 Nissenson et al, 2003 ), although whether anaemia plays a causative role in determining prognosis is open to debate. Thirdly, treatment may improve the quality of life for patients living with chronic conditions ( Moreno et al, 2000 Littlewood et al, 2001 ).

Treatment of the underlying inflammatory or malignant process associated with ACD will often result in improvement in the degree of anaemia, examples being the use of corticosteroids in polymyalgia rheumatica, the use of TNF-α inhibitors in rheumatoid arthritis or inflammatory bowel disease (IBD) ( Moreland et al, 1997 Doyle et al, 2009 Bergamaschi et al, 2010 ), and the use of antiretroviral drugs in human immunodeficiency virus (HIV) infection ( Semba et al, 2001 ). Indeed the severity of the anaemia will frequently mirror the activity of the chronic condition causing it, for example in rheumatoid arthritis ( Vreugdenhil et al, 1990b ). However, treatment of the underlying condition may not always be possible, for example in patients with incurable cancers or chronic renal or cardiac failure and alternative strategies may be necessary. Correction of as many contributory factors as possible is also desirable, for example correction of nutritional deficiencies ( Vreugdenhil et al, 1990a ).

Blood transfusion

Blood transfusion is widely available in the developed world and is a simple means of treating patients with moderate to severe anaemia, but blood remains a precious and expensive resource, and transfusion therapy carries long-term risks of viral transmission, iron overload and alloimmunization. Transfusion should therefore be reserved for patients with severe or life-threatening anaemia in the context of ACD, and is not an appropriate treatment for patients with this form of chronic anaemia ( Cavill et al, 2006 ).

Erythropoiesis-stimulating agents

The rationale for the use of erythropoiesis-stimulating agents (ESA) in ACD is based on the blunted EPO response seen in ACD, with lower serum levels of EPO detected than would be expected for the observed degree of anaemia, together with the reduced sensitivity of erythroid progenitors to endogenous EPO seen in ACD. In addition, there is limited data to suggest that administration of EPO may reverse cytokine-mediated inhibition of erythropoiesis ( Means & Krantz, 1991 ). Recombinant human EPO (rHuEPO) and its derivatives are widely used in patients with chronic renal failure, patients with cancer undergoing chemotherapy and patients infected with HIV on myelosuppressive anti-retroviral medication. Several different rHuEPOs are currently available or in development: epoetin-α (Procrit ® Ortho Biotech, Bridgewater, NJ, USA Epogen ® Amgen, Thousand Oaks, CA, USA Eprex ® Janssen-Cilag, Cologno Monzese, Milan, Italy), epoetin-β (NeoRecormon ® , F.Hoffmann-La Roche, Basel, Switzerland) epoetin-δ, biosimilar epoetins (Retacrit ® Hospira, Alemere, the Netherlands Binocrit ® Sandoz Limited, Frimley, UK Eporatio ® Ratiopharm, Bristol, UK), darbepoietin-α (Aranesp ® Amgen), and continuous erythropoietin receptor activator (CERA) (Mircera ® F.Hoffmann-La Roche). In addition, a PEGylated synthetic dimeric peptide capable of binding to and stimulating the EPO receptor, Hematide ® (Affymax, Palo Alto, CA, USA) is undergoing clinical trials.

Much of the literature relating to the use of ESAs in ACD comes from renal medicine, but there is also evidence that these agents have useful activity in other forms of ACD, for example that seen in rheumatoid arthritis ( Pincus et al, 1990 Peeters et al, 1996 ), IBD ( Schreiber et al, 1996 ), HIV infection ( Henry et al, 1992 ) and cancer ( Ludwig et al, 1995 ). Only relatively small studies of EPO usage have been performed in patients with ACD secondary to inflammatory conditions, for example the study by Pincus et al (1990) in which four of 13 patients treated with rHuEPO at doses ranging from 50–150 iu/kg thrice weekly showed haematological responses, whereas none of four patients in the placebo arm responded. In another study ( Schreiber et al, 1996 ), 34 patients with IBD refractory to iron therapy were randomly assigned to receive oral iron plus EPO or oral iron and placebo: after 12 weeks, haemoglobin levels had increased by more than 10·0 g/l in 82% of the patients in the erythropoietin group, as compared with 24% of those in the placebo group (P = 0·002). However the improvement in treatment of inflammatory conditions, such as rheumatoid arthritis or IBD, with anti-inflammatory and disease modifying agents, such as TNF-α inhibitors, with associated improvements in haemoglobin levels, means that there is only a limited place for ESAs in their treatment.

There are many more studies of the use of EPO in patients with both solid tumour and haematological malignancies ( Littlewood et al, 2001 Henke et al, 2003 Witzig et al, 2005 ) with response rates of 40–80% being seen. Many of these studies describe patients receiving anti-cancer treatment, so the anaemia observed may be partly due to the myelosuppressive effects of chemotherapy or radiotherapy, rather than to the inflammatory effects of malignancy alone. However, early studies indicate that, although relative EPO deficiency contributes to the anaemia of cancer in patients who are untreated, this effect is increased by the effects of chemotherapy ( Miller et al, 1990 ). Smith et al (2003) performed a dose- and schedule determining study in 188 patients with cancer not currently receiving chemotherapy, showing responses in the majority of patients. A recent large systematic review of 46 randomized controlled trials of ESA therapy in patients with cancer concluded that patients receiving EPO had a mean 16·3 g/l higher haemoglobin level than controls, were 18% less likely to require blood transfusions, and had improved health-related quality of life, but survival benefits could not be established ( Wilson et al, 2007 ).

Responses may be reduced in ACD patients with more marked inflammation ( Nordström et al, 1997 ) or where there is associated iron deficiency, especially in patients with IBD ( Gasche et al, 2004 ), highlighting both the importance of aiming treatment at the underlying condition and of ensuring replenishment of iron stores in patients who are iron deficient. As discussed above, it may not always be easy to determine whether patients have ACD alone or ACD/IDA, and evidence is accumulating that iron supplementation may be desirable in many patients treated with ESAs to ensure optimal response (see below).

Predicting which patients with ACD will respond to exogenous EPO would be useful, but although various algorithms incorporating baseline endogenous EPO level, early response indicators and other factors, none of these will reliably predict response, at least in the setting of cancer-related anaemia ( Littlewood et al, 2003 ).

There has recently been mounting concern at possible detrimental effects of EPO administration in ACD, both in terms of cardiovascular risk and thrombosis, and relating to possible risks of tumour recurrence in patients with ACD related to malignancy. The CHOIR (Correction of Haemoglobin and Outcomes in Renal Insufficiency) study showed that trying to achieve a target Hb level of 135 g/l (compared with 113 g/l) increased the risk of cardiovascular events and did not improve quality of life ( Singh et al, 2006 ) and the TREAT (Trial to Reduce Cardiovascular Events With Aranesp Therapy) study by Pfeffer et al (2009) , demonstrated that patients with diabetes and chronic kidney disease were at greater risk of stroke following ESA administration and no clear benefit was observed. A randomized study of EPO in patients with non-small cell lung cancer (NSCLC) who were not receiving chemotherapy was terminated prematurely when a higher mortality rate was observed in the group receiving EPO ( Wright et al, 2007 ).

These, and other, studies, together with suggestions that some tumour cells might express EPO receptors, raising the possibility that EPO might modulate tumour growth via cytoprotective effects, led the Food and Drug Administration (FDA) in the United States to recommend that (i) prescribers should use the lowest dose of ESAs that would gradually increase Hb concentration to a level that would avoid the need for transfusion and (ii) treatment with ESAs might increase the risk of serious cardiovascular events and death when administered to produce Hb levels >120 g/l ( Jenkins, 2007 ). In addition, the FDA recommends that (iii) ESAs should not be used in specific tumour types (breast, head and neck, NSCLC), nor be administered to patients with active malignancy not receiving chemo- or radiotherapy. Similar conclusions are reached in the updated guidelines published recently by the American Society for Hematology (ASH) and the American Society for Clinical Oncology (ASCO) ( Rizzo et al, 2010 ). However, a recent large meta-analysis of over 15 000 patients in 60 studies of ESAs in patients with cancer has shown no evidence that ESAs reduce survival or increase tumour progression in patients with cancer, although some increase in venous thrombembolism was observed ( Glaspy et al, 2010 ). In addition, two recent studies have cast doubt on the idea that EPO receptors may be expressed at significant and clinically relevant levels on non-haematopoietic cells, including tumour cell lines ( Sinclair et al, 2010 Swift et al, 2010 ), and it is clear that further, well-designed, clinical trials are necessary to define the role of ESAs in the anaemia of malignancy. In the meantime, blood transfusion remains an option for treatment of anaemia in patients with contraindicated cancers or those at high risk of venous thromboembolism.

Iron therapy

The recognition of the role of functional iron deficiency in the pathogenesis of ACD, together with the development of new formulations of parenteral iron, have led to a re-evaluation of iron supplementation in the management of this anaemia. As already discussed, IDA frequently co-exists with ACD, and it is clearly important that true deficiency of iron is corrected. However, even in patients with ‘pure’ ACD, iron supplementation may theoretically be beneficial ( Goodnough et al, 2010 ). Iron deficiency may also develop during the treatment of ACD with ESAs and limit the haematological response to these agents ( Kaltwasser et al, 2001 Cavill et al, 2006 ).

Oral iron supplements are often poorly tolerated, and patients frequently exhibit poor compliance: in addition, patients with ACD will usually have raised hepcidin levels, which would be expected to inhibit intestinal iron absorption. However, oral iron is cheap, widely available, and easy to administer, and given the difficulties in ruling out concomitant IDA in many patients with ACD, a therapeutic trial of oral iron will be undertaken by many clinicians treating ACD. It must however be recognized that failure to respond to oral iron rules out neither true, nor functional iron deficiency.

There is little literature on the use of intravenous iron supplementation alone in the treatment of ACD. Cazzola et al (1996) reported on the beneficial effects of intravenous (IV) iron in 20 consecutive patients with juvenile chronic arthritis, although it is likely that a significant proportion of these patients also had true iron deficiency. Studies in patients with gynaecological cancer ( Kim et al, 2007 Dangsuwan & Manchana, 2010 ) also showed a benefit in terms of reduced transfusion requirements for those receiving intravenous iron supplementation. However baseline iron status was not reported in either of these papers, and clearly larger studies are needed.

Much of the literature concerning intravenous iron supplementation has come from the field of renal medicine, where the superiority of parenteral over oral iron supplementation is now well established, and not only improves the responses to ESAs but can also lead to reduced doses of ESAs being used ( Locatelli et al, 2009 ). The DRIVE (Dialysis Patients’ Response to IV iron and with Elevated Ferritin) trial ( Coyne et al, 2007 ) randomized selected haemodialysis patients with elevated ferritin and reduced transferrin saturation to receive or not receive intravenous ferric gluconate together with EPO. The patients who received IV iron showed more rapid and better responses in Hb level than the controls, and similar responses were seen in patients with transferrin saturations above and below 19%, leading the authors to conclude that functional iron deficiency was a significant contributor to anaemia in this setting, and that this could be overcome by intravenous iron supplementation.

There is now evidence that intravenous iron can enhance the effects of ESAs in patients with other forms of ACD, particularly cancer-related anaemia (reviewed by Littlewood & Alikhan, 2008 ). Auerbach et al (2004) randomized 155 patients being treated with ESAs for chemotherapy-related anaemia to no iron, oral iron or intravenous iron: there were significant improvements in haematological responses in patients receiving intravenous iron compared with those receiving either no iron or oral iron. These observations have been confirmed in several subsequent studies ( Hedenus et al, 2007 Henry et al, 2007 Bastit et al, 2008 Pedrazzoli et al, 2008 ). Criteria for exclusion of co-existent IDA varied between these trials, and it is possible that significant numbers of patients included were in fact iron deficient, but the study by Hedenus et al (2007) is of particular interest as it enrolled only patients with lymphoproliferative malignancies not receiving chemotherapy, and all patients had detectable bone marrow iron stores.

In contrast, a recent study by Steensma et al (2011) randomized patients with chemotherapy-associated anaemia to no iron, oral iron or intravenous iron plus darbepoietin: all had serum ferritin >20 μg/l and transferrin saturations <60%. There was no difference in erythropoietic response between the three groups. The mean pre-treatment ferritin levels in this study were higher than in the other studies, suggesting this population was less likely to have co-existent IDA, and the doses and scheduling of iron infusions were lower. Both these observations may partly explain the different results observed, but it is clear that further prospective studies, with better characterization of baseline iron stores are needed to define the role of intravenous iron supplementation in this setting. The ASH/ASCO guidelines ( Rizzo et al, 2010 ) recommend periodic monitoring of iron status in patients receiving treatment with ESAs but fall short of recommending intravenous supplementation to augment responses.

It is not yet known how intravenous iron might overcome the reticuloendothelial blockade on iron utilization thought to be fundamental to the pathogenesis of ACD, but it is possible that the infused iron may become bound directly to transferrin rather than being taken up by macrophages, and is thus available to the erythron. There are however no in vitro data to support this hypothesis.

Safety issues also need to be considered when using intravenous iron, particularly as older preparations were associated with significant adverse events, including anaphylaxis ( Auerbach & Ballard, 2010 ). Recent pharmacological developments have led to the release of several new iron formulations including low molecular weight iron dextran (Cosmofer ® Pharmacosmos, Holbaek, Denmark), iron sucrose (Venofer ® Vifor Pharma, Glattbrugg, Switzerland), ferric carboxymaltose (Ferinject ® Syner-Med Ltd, Purley, UK) and sodium ferric gluconate (Ferrlecit ® Watson Laboratories, Morristown, NJ, USA). In the trials above, no excess of adverse effects was observed with these newer intravenous iron preparations. One hypothesis for the hypoferraemia seen in ACD is that low iron levels might inhibit bacterial growth, as iron is essential for the growth and survival of intracellular bacteria, but there is no evidence to date that supplemental iron increases the risk of infections. However the long-term effects of intravenous iron administration on other parameters, for example tumour growth and cardiovascular disease, have not been studied.


I. Bloodstream Infections and Infections of the Cardiovascular System

A. Bloodstream Infections and Infective Endocarditis

The diagnosis of bloodstream infections (BSIs) is one of the most critical functions of clinical microbiology laboratories. For the great majority of etiologic agents of BSIs, conventional blood culture methods provide positive results within 48 hours incubation for >5 days seldom is required when modern automated continuous-monitoring blood culture systems and media are used [1, 2]. This includes recovery of historically fastidious organisms such as HACEK [1] (Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and Kingella) bacteria and Brucella species (spp) [3, 4]. Some microorganisms, such as mycobacteria and dimorphic fungi, require longer incubation periods others may require special culture media or non-culture-based methods. Although filamentous fungi often require special broth media or lysis-centrifugation vials for detection, most Candida spp grow very well in standard blood culture broths unless the patient has been on antifungal therapy. Unfortunately, blood cultures from patients with suspected candidemia do not yield positive results in almost half of patients. Table 2 provides a summary of diagnostic methods for most BSIs.

For most etiologic agents of infective endocarditis, conventional blood culture methods will suffice [3&ndash5]. However, some less common etiologic agents cannot be detected with current blood culture methods. The most common etiologic agents of culture-negative endocarditis, Bartonella spp and Coxiella burnetii, often can be detected by conventional serologic testing. However, molecular amplification methods may be needed for detection of these organisms as well as others (eg, Tropheryma whipplei, Bartonella spp). In rare instances of culture-negative endocarditis, 16S polymerase chain reaction (PCR) and DNA sequencing of valve tissue may help determine an etiologic agent.

The volume of blood that is obtained for each blood culture request (also known as a blood culture set, consisting of all bottles procured from a single venipuncture or during one catheter draw) is the most important variable in recovering bacteria and fungi from adult and pediatric patients with bloodstream infections [1, 2, 5, 6]. For adults, 20&ndash30 mL of blood per culture set (depending on the manufacturer of the instrument) is recommended and may require >2 culture bottles depending on the system. For neonates and adolescents, an age- and weight- appropriate volume of blood should be cultured (see Table 3 below for recommended volumes). A second important determinant is the number of blood culture sets performed during a given septic episode. Generally, in adults with a suspicion of BSI, 2&ndash4 blood culture sets should be obtained in the evaluation of each septic episode [5, 7].

The timing of blood culture orders should be dictated by patient acuity. In urgent situations, 2 or more blood culture sets can be obtained sequentially over a short time interval (minutes), after which empiric therapy can be initiated. In less urgent situations, obtaining blood culture sets may be spaced over several hours or more.

Skin contaminants in blood culture bottles are common, very costly to the healthcare system, and frequently confusing to clinicians. To minimize the risk of contamination of the blood culture with commensal skin microbiota, meticulous care should be taken in skin preparation prior to venipuncture. In addition, new products are now available that allow diversion and discard of the first few milliliters of blood that are most likely to contain skin contaminants. Consensus guidelines [2] and expert panels [1] recommend peripheral venipuncture as the preferred technique for obtaining blood for culture based on data showing that blood obtained in this fashion is less likely to be contaminated than blood obtained from an intravascular catheter or other device. Several studies have documented that iodine tincture, chlorine peroxide, and chlorhexidine gluconate (CHG) are superior to povidone-iodine preparations as skin disinfectants for blood culture [1, 2]. Iodine tincture and CHG require about 30 seconds to exert an antiseptic effect compared with 1.5&ndash2 minutes for povidone-iodine preparations [2]. Two recent studies have documented equivalent contamination rates with iodine tincture and CHG [8, 9]. CHG is not recommended for use in infants <2 months of age but povidone-iodine followed by alcohol is recommended.

Blood cultures contaminated with skin flora during collection are common but contamination rates should not exceed 3%. Laboratories should have policies and procedures for abbreviating the workup and reporting of common blood culture contaminants (eg, coagulase-negative staphylococci, viridans group streptococci, diphtheroids, Bacillus spp other than B. anthracis). These procedures may include abbreviated identification of the organism, absence of susceptibility testing, and a comment that instructs the clinician to contact the laboratory if the culture result is thought to be clinically significant and requires additional workup and susceptibility results.

Physicians should expect to be called and notified by the laboratory every time a blood culture becomes positive since these specimens often represent life-threatening infections. If the physician wishes not to be notified during specific times, arrangements must be made by the physician for a delegated healthcare professional to receive the call and relay the report.

Key points for the laboratory diagnosis of bacteremia/fungemia:

  • Volume of blood collected, not timing, is most critical.
  • Disinfect the venipuncture site with chlorhexidine or 2% iodine tincture in adults and children >2 months old (chlorhexidine NOT recommended for children <2 months old), using povidone-iodine and alcohol).
  • Draw blood for culture before initiating antimicrobial therapy.
  • Catheter-drawn blood cultures have a higher risk of contamination (false positives).
  • Do not submit catheter tips for culture without an accompanying blood culture obtained by venipuncture.
  • Never refrigerate blood prior to incubation.
  • Use a 2- to 3-bottle blood culture set for adults, at least 1 aerobic and 1 anaerobic use 1&ndash2 aerobic bottles for children and consider aerobic and anaerobic when clinically relevant.
  • Streptococcus pneumoniae and other gram-positive organisms and facultatively anaerobic organisms may grow best in the anaerobic bottle (faster time to detection).

B. Infections Associated with Vascular Catheters

The diagnosis of catheter-associated BSIs is often one of exclusion, and a microbiologic gold standard for diagnosis does not exist. Although a number of different microbiologic methods have been described, the available data do not allow firm conclusions to be made about the relative merits of these various diagnostic techniques [10&ndash12]. Fundamental to the diagnosis of catheter-associated BSI is documentation of bacteremia. The clinical significance of a positive culture from an indwelling catheter segment or tip in the absence of positive blood cultures is unknown. The next essential diagnostic component is demonstrating that the infection is caused by the catheter. This usually requires exclusion of other potential primary foci for the BSI. Some investigators have concluded that catheter tip cultures have such poor predictive value that they should not be performed [13].

Numerous diagnostic techniques for catheter cultures have been described and may provide adjunctive evidence of catheter-associated BSI however, all have potential pitfalls that make interpretation of results problematic. Routine culture of intravenous catheter tips at the time of catheter removal has no clinical value and should not be done [13]. Although not performed in most laboratories, the methods described include the following:

  • Time to positivity (not performed routinely in most laboratories): Standard blood cultures (BCs) obtained at the same time, one from the catheter or port and one from peripheral venipuncture, processed in a continuous-monitoring BC system. If both BCs grow the same organism and the BC drawn from the device becomes positive >2 hours before the BC drawn by venipuncture, there is a high probability of catheter-associated BSI [14].
  • Quantitative BCs (not performed routinely in most laboratories): one from catheter or port and one from peripheral venipuncture obtained at the same time using lysis-centrifugation (Isolator) or pour plate method. If both BCs grow the same organism and the BC drawn from the device has 5-fold more organisms than the BC drawn by venipuncture, there is a high probability of catheter-associated BSI [15, 16].
  • Catheter tip or segment cultures: The semi-quantitative method of Maki et al [12] is used most commonly interpretation requires an accompanying peripheral BC. However, meticulous technique is needed to reduce contamination and to obtain the correct length (5 cm) of the distal catheter tip. This method only detects organisms colonizing the outside of the catheter, which is rolled onto an agar plate, after which the number of colonies is counted organisms that may be intraluminal are missed. Modifications of the Maki method have been described as have methods that utilize vortexing of the catheter tip or an endoluminal brush (not performed routinely in most laboratories). Biofilm formation on catheter tips prevents antimicrobial therapy from clearing agents within the biofilm, thus requiring removal of the catheter to eliminate the organisms.

C. Infected (Mycotic) Aneurysms and Vascular Grafts

Infected (mycotic) aneurysms and infections of vascular grafts may result in positive blood cultures. Definitive diagnosis requires microscopic visualization and/or culture recovery of etiologic agents from representative biopsy or graft material (Table 4).

D. Pericarditis and Myocarditis

Numerous viruses, bacteria, rickettsiae, fungi, and parasites have been implicated as etiologic agents of pericarditis and myocarditis. In many patients with pericarditis and in the overwhelming majority of patients with myocarditis, an etiologic diagnosis is never made and patients are treated empirically. In selected instances when it is important clinically to define the specific cause of infection, a microbiologic diagnosis should be pursued aggressively. Unfortunately, however, the available diagnostic resources are quite limited, and there are no firm diagnostic guidelines that can be given. Some of the more common and clinically important pathogens are listed in Table 5 below. When a microbiologic diagnosis of less common etiologic agents is required, especially when specialized techniques or methods are necessary, consultation with the laboratory director should be undertaken. There is considerable overlap between pericarditis and myocarditis with respect to both etiologic agents and disease manifestations.


OP06 Time is Money and Radiation Burden - a carbon-11 ‘two-in-one-pot’ production system

C. Vraka 1 , C. Philippe 1 , T. Zenz 1 , M. Mitterhauser 1,2 , M. Hacker 1 , W. Wadsak 1,3 , V. Pichler 1

1 Medical University of Vienna, Department of Biomedical Imaging and Image-guided Therapy, Vienna, Austria 2 Ludwig Boltzmann Institute Applied Diagnostics, Vienna, Austria 3 CBmed, Graz, Austria

Correspondence: C. Philippe

Aim: The use of positron emission tomography (PET) for specific molecular examinations is increasing steadily and therefore the demand for selective and specific PET-tracers is rising accordingly. Currently, only one tracer per synthesizer can be produced (non-cassette based) within a time frame of approximately 2 h and a radiation burden of approximately 1 h. A minimum decay time of 6 half-lives (around 2 h) between two carbon-11 productions within the same hot-cell is essential. Therefore, in clinical routine (8 h day) only two syntheses of carbon-11 labeled compounds per day are possible. Consequently, the number of examinations with 11 C-labeled tracers is extremely limited (number of productions high synthesis costs and few production runs due to number of hot cells and synthesizers). To improve this situation, the aim of this study was the simultaneous production of two 11 C-PET-tracers using a ‘two-in-one-pot’ reaction reducing time, cost, and radiation burden. Exemplarily, this simultaneous production was successfully performed for two commonly used brain PET-tracers, [ 11 C]Harmine and [ 11 C]DASB.

Methods: Production runs were performed using a commercially available GE Tracerlab FX C Pro. 1 mg of the precursors, MASB and Harmol, were dissolved in DMSO and 5 M NaOH was added to the solution. [ 11 C]CH3I was subsequently bubbled through the precursor solution. After a reaction time of 2 min at 100°C, the crude dual-tracer mixture was purified by means of semi-preparative HPLC. The synthesis module was expanded with a self-constructed semi-automated formulation unit (Fig. 1) to ensure parallel SPE-purification and formulation of both tracers after HPLC (Fig. 1).

Scheme of the synthesizer including the self-constructed unit for the formulation of the second tracer

Exemplary RP-HPLC chromatogram for the separation of [ 11 C]Harmine and [ 11 C]DASB in a single run

Results: Both PET-tracers were prepared simultaneously in a ‘two-in-one-pot’ reaction (n = 3) and successfully purified using one single HPLC run. Radiochemical yield was 2.0 ± 0.3 GBq (2.3 ±0.5% not corrected for decay based on [ 11 C]CO 2 @EOB) for [ 11 C]DASB and 2.0 ± 0.7 GBq (2.2 ± 0.8%) for [ 11 C]Harmine, respectively. Hence, both products were received in the same amount (ratio 1:1). The qualities of both tracers complied with the European Pharmacopoeia monographs.

Conclusion: We herewith describe the first simultaneous production of two 11 C-PET-tracers in a ‘two-in-one-pot’ reaction. Both products were in full accordance with quality control parameters fulfilling the standards for parenteral human application. This simultaneous radiopharmaceutical preparation lead to a significant reduction of radiation burden, reduction of amount of operator time (-50%), cost reduction (-46.2%) and, subsequently, to considerable gain in overall efficiency of the production process.


FliZ Regulates Expression of the Salmonella Pathogenicity Island 1 Invasion Locus by Controlling HilD Protein Activity in Salmonella enterica Serovar Typhimurium

FIG. 1 . Model for the Salmonella pathogenicity island 1 (SPI1) regulatory network. The expression of hilA, the master regulator for SPI1, is controlled by HilD, HilC, and RtsA, which act in a complex feed-forward loop. Each can independently activate expression of their own gene as well as each other and hilA. Signals are integrated by HilD HilC and RtsA act as amplifiers of those signals. For clarity, the genes encoding HilD, HilC, RtsA, and HilA are not shown. The solid arrows indicate direct gene activation. T3SS, type three secretion system. FIG. 2 . FliZ activates hilA through hilD. β-Galactosidase (β-Gal) activity was examined in strains containing hilA-lacZ transcriptional fusions and the indicated plasmids and/or mutations. The strains were grown under SPI1-inducing conditions. β-Galactosidase activity units are defined as (micromoles of ONP formed per minute × 10 3 )/(OD600 × milliliter of cell suspension) and are reported as means ± standard deviations (error bars) for four replicate samples relative to the results for the wild-type (WT) strain. The strains used were JS749, JS778, JS946, JS798 to JS807, JS947, and JS948. FIG. 3 . FliZ activation of hilA is dependent on HilD. (A) β-Galactosidase activity in strains containing a hilA-lacZ transcriptional fusion and the indicated mutations after growth under SPI1-inducing conditions. (B) β-Galactosidase activity of strains containing a hilA-lacZ transcriptional fusion and indicated mutations with rtsA under the control of a tetracycline-regulated promoter. The strains were grown under SPI1-inducing conditions with the indicated concentrations of tetracycline (Tet). The strains used were JS749 and JS950 to JS956. β-Galactosidase activity units are defined as (μmol of ONP formed per min × 10 3 )/(OD600 × ml of cell suspension) and are reported as means ± standard deviations (n = 4). FIG. 4 . FliZ acts at the level of HilD protein. (A) β-Galactosidase activity in strains containing either a hilD-lacZ transcriptional or translational fusion and the indicated plasmids. The fusion joints of the two constructs are identical (14). The strains were grown under SPI1-inducing conditions with 10 mM arabinose. Arabinose is required for induction of pHilC but was included in all cultures. The strains used were JS883, JS957, JS958, JS892, JS959, and JS960. (B) β-Galactosidase activity in strains containing a hilA-lacZ transcriptional fusion and the indicated mutations. The strains were grown under SPI1-inducing conditions (left panel) or in LB medium (0.5% NaCl) with the indicated tetracycline concentrations and with shaking (right panel). The strains used were JS749, JS778, JS633, JS961, JS962, and JS963. β-Galactosidase activity units are defined as (μmol of ONP formed per min × 10 3 )/(OD600 × ml of cell suspension) and are reported as means ± standard deviations (n = 4). FIG. 5 . FliZ regulates hilA independently of HilE and RpoS. (A) β-Galactosidase activity in strains containing a hilA-lacZ transcriptional fusion and the indicated mutations after growth under SPI1-inducing conditions. The strains used were JS749, JS576, JS577, JS579, JS633 to JS636, and JS964 to JS967. (B) β-Galactosidase activity of strains containing sodCII, katE, or hilA transcriptional fusions in otherwise wild-type or rpoS backgrounds with or without pFliZ. The strains were grown under SPI1-inducing conditions. The strains used were JS749, JS968, JS969, JS970, JS909, JS910, JS971, JS972, JS531, JS541, JS973, and JS974. β-Galactosidase activity units are defined as (μmol of ONP formed min −1 ) × 10 3 /(OD600 × ml of cell suspension) and are reported as means ± standard deviations (n = 4). FIG. 6 . HilD protein levels in relation to FliZ and HilE. The hilD-3×FLAG construct is under tetRA control, and all strains contained a hilA-lacZ transcriptional fusion and the indicated mutations or plasmids. (A) HilD protein levels in stationary-phase cells. The strains were grown under SPI1-inducing conditions with 0.4 μg/ml tetracycline. The cultures were divided to determine β-galactosidase activity and to perform the Western blot analysis to detect FLAG-tagged HilD. Extracts from equal concentrations of cells were loaded on the gel. The intensities of the bands were quantified using ImageJ and are presented above the gel relative to the wild-type strain (set at 1). Note that the doublets seen are artifacts of this particular gel. The strains used were JS975 to JS979. (B) HilD protein stability in cells in late log phase. The genotypes for lon and fliZ strains are indicated to the left of the gels (++ indicates overproduction [pFliZ]). The cells were induced with 0.8 μg/ml tetracycline and grown in LB medium (0.5% NaCl) with shaking to late log phase, and antibiotics were added to stop transcription and translation. β-Galactosidase activity produced from the hilA-lacZ fusion in the samples shown on these gels was determined from each sample taken at time zero. ImageJ was used for half-life analysis. The half-life was calculated from 2 (lon) or 3 replicates of the experiments. The mean half-life ± SEM is listed for each background. The strains used were JS975, JS976, JS977, JS980, and JS981. FIG. 7 . FliZ regulates HilD in the absence of Lon protease. (A) β-Galactosidase activity in strains containing a hilA-lacZ transcriptional fusion and various mutations or pFliZ as indicated. The strains were grown under SPI1-inducing conditions. The strains used were JS749 and JS982 to JS985. (B) Immunoprecipitation of FliZ-3×FLAG. Strains produced either wild-type FliZ or 3×FLAG-tagged FliZ as indicated in lon + or lon mutant backgrounds. The cultures were grown under SPI1-inducing conditions. FLAG-tagged protein was immunoprecipitated from lysates from equal concentrations and numbers of cells. The proteins were separated by SDS-PAGE and subjected to Western blot analysis to detect FLAG-tagged protein. The strains used were 14028, JS987, JS988, and JS989.

BIOLOGICAL THEMES EXPLORED WITH THE USE OF MODELS

Sections 2 and 3 have introduced necessary conceptual tools to appreciate the insight that mathematical models can offer when used in conjunction with experimental data. We hope that we have also provided a rough guide of how different formats of data can be explored mathematically. The purpose of the present section is to present biological themes which customarily come up in the study of within-host dynamics of infectious disease and how they have been addressed through the use of mathematical models. As modeling examples remain limited in the area of bacterial dynamics, we include conceptually similar studies in other pathogenic classes including viruses and parasites. These case studies are by no means an exhaustive representation of the entirety of the literature body but have been selected to serve as illustrations of the concepts.

Quantification of the relative contribution of different immune system components to the progression of infection

Within-host models of parasitic, viral and bacterial infection often seek to determine the relative contributions of different components of the immune system in regulating the dynamics of infection. In the current treatment paradigm, the role of the host's immune response is often neglected, and therapeutic agents are administered for fixed periods of time usually in the form of monotherapy and regardless of the infectious load. It is now becoming increasingly recognized that a first step toward optimization of existing therapies is the induction of synergistic effects between the host immunity and the standalone effect of the therapy (Gjini and Brito 2016). To thoroughly understand this interaction, mechanistic mathematical models can be used in two main ways. First, one can use a series of nested models prospectively, starting from simpler models and adding features of the immune response while quantifying the impact of each new addition in the process. Second, by enabling the segregation between unobserved processes of replication and killing, mathematical models begin to shed light on the black-box of host–pathogen interactions and inform further biological experiments. For example, if rapid killing is identified as the main driver of an observed decline in bacterial numbers, it is reasonable to first look in the direction of known cidal branches of the host immunity.

One such approach was taken by Grant et al. in 2008 where following the identification of early bactericidal activity in a Salmonella mouse model, infection progression in wild type mice was compared to that in NADPH oxidase deficient mice to unravel an important role of that immunological component in inducing the inferred bactericidal effect. In other pathogen classes, mathematical models addressing similar questions were successfully used much earlier.

With regards to host immune system-bacterial interactions, mathematical modeling of the Mycoplasma species has been ongoing. The Kirschner group have developed a series of increasingly complex mathematical models to describe the role of different cell types and chemokines of the immune system in the progression of early tuberculosis (TB) infection. Their compartmental model based on ordinary differential equations including the lung and the draining lymph node (DNL) has been used to study the dynamics of early infection, particularly the role of dendritic cells in T-cell priming (Marino and Kirschner 2004) and, later, the roles of dendritic cell trafficking to and from the DNL (Marino et al. 2004), cytotoxic T-cell-mediated Mycobacteria killing (Sud et al. 2006), TNF-α and anti-inflammatory IL-10 (Cilfone et al. 2013) in host defence. Their contributions identified the macrophage infection rate and T-cell-mediated immunity as the two key elements in determining the trajectory of an infection into one of (i) primary TB, (ii) primary TB with clearance, (iii) latency and (iv) reactivation (Marino and Kirschner 2004).

Comparison of the effect of different strains on infection dynamics

Fitting within-host models to samples of different strains of the infectious pathogen can also facilitate our understanding of how the within-host dynamics of infection vary across different strains of the same species. Different strains of pathogens are responsible for differences in seasonal and local outbreaks of contagious and deadly infections such as influenza (Du et al. 2017), cholera (Weill et al. 2019), community-acquired pneumonia (Zhang et al. 2019) and others. These pathogens, albeit very closely related, can show extreme differences in transmission rates (e.g. in Mycobacterium tuberculosis in Verma et al. 2019), response to therapeutic agents (e.g. in Vibrio cholerae in Weill et al. 2019) and virulence (e.g. in swine fever virus in Portugal et al. 2014). In this context, mathematical models allow for sensitivity analyzes to identify which parameter(s) have the greatest impact on a given outcome this can be helpful in highlighting potential causes that drive inter-strain differences.

For instance, Hur et al. ( 2013) fit models of influenza infection to experimental data on seasonal and pandemic strains of flu. They found that the only parameter that varied between the pandemic and seasonal strains was the viral replication rate, indicating that intracellular viral replication may affect pathogenicity.

Comparison of the effects of different therapeutic interventions on infection dynamics

Within-host models of infection (both theoretical data-driven) can also reveal important insights into the effect of different drugs at the level of the host–pathogen interaction and identify effective treatment strategies (e.g. decide whether it is more efficient to prevent replication or increase killing). For example, Rong and Perelson ( 2010) evaluated the effect of different Hepatitis C (HCV) treatment strategies. Protease inhibitors are being increasingly used in combination with pegylated interferon and ribavirin to treat HCV-1 infection, but there remain concerns of relapse after treatment. They developed a deterministic mathematical model to examine viral load dynamics before and after treatment with a protease inhibitor. Banerjee, Keval and Gakkhar ( 2013) considered the effect of ribavirin being used in combination with interferon therapy for HCV infection. Although the study was theoretical in nature, it found that – provided a certain threshold of drug efficacy – a triphasic response of viral load could be observed, leading to eradication of the virus.

Comparison of the effects of different inoculum size on infection dynamics

Infections can take off with inocula of variable sizes. However, the inoculum size affects the population composition of the infectious agents and how they respond to therapy. Formulating a deterministic mechanistic model, Meredith et al. ( 2015) reported that inoculum size determines the efficacy of β-lactam antibiotics when administered to bacterial populations of which at least some members harbor extended β-lactamase activity. If β-lactam antibiotics were administered in high-density populations, then some members would survive and re-establish the infection. They reported that the population was sensitive when its initial density was sufficiently low or examined in a short time window. Given these properties, they reasoned that optimal antibiotic dosing may remain effective in bacterial populations even when they harbor resistance genes.

Studying the dynamics of infection across different scales

Mathematical models can be employed to study host–pathogen interactions at multiple levels, from cellular to whole-organism and even population level. A solid knowledge of the versatility of mathematical techniques allows the use of the same tools to study questions on different scales. With judicious use, mathematical models can also combine insight acquired at different levels e.g. the single cell and organ levels and use this to gain novel insights about disease progression (Gog et al. 2015). For example, a stochastic mathematical model generated by the Perelson lab showed that early HIV dynamics differ depending on whether infected target cells produce virions continuously or do so in a single burst (Pearson, Krapivsky and Perelson 2011). This study shows how events at the single-cell level can have a profound impact on infection dynamics at the whole-organism level ultimately affecting clinically important quantities used for diagnosis and as guides for therapeutic intervention.

Furthermore, it is possible to use the predictions from modeling the host–pathogen interactions to inform models at higher scales. In 2009, Heffernan and Keeling ( 2009) took advantage of well-founded predictions about immunity in a measles-infected host (Heffernan and Keeling 2008) to predict the effect of vaccination at the population level.

Investigating the evolutionary dynamics of infectious disease within the host

Finally, mathematical models have been used to characterize and quantify the evolutionary dynamics of infectious agents within a host. For instance, Chisholm and Tanaka ( 2016) developed a mechanistic mathematical model to examine the evolution of M. tuberculosis within its host. M. tuberculosis is observed to enter a latent, dormant state, but, at first glance, a state of dormancy is not advantageous for the pathogen as it does not permit replication. However, the study demonstrated that latency can be an evolutionarily desirable state.

Furthermore, Fabre et al. ( 2012) formulated a deterministic mechanistic model of competing viral populations within host plants. They parameterized it according to the carrying capacity of the plant, the intrinsic rate of increase of each variant and the competition strength each genotype exerts on the others. They determined the forms of selection processes occurring between competing viruses within a host plant, and the intensity and temporal variation of genetic drift experienced by viruses during host plant colonization. Parameters were statistically inferred by model fitting to high-throughput sequence data of the viral counts obtained from the plants over time, and model selection was performed (after testing several models reflecting different mechanisms of competition).


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