Difference between revisions of "Team:Freiburg/Project/Diseases"
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Revision as of 17:20, 17 September 2015
Information about viruses and bacteria
One key component of our project, the DiaCHIP, are of course the antigenic peptides immobilized on the surface of the protein array. As a basis to perform cell-free expression, we did paper research to look for suitable nucleotide sequences we could use and that had been already used for serologic detection of antibodies. We decided to focus on some rather familiar or common pathogens, which will be presented in the following paragraphs.
German Measles
The Rubella Virus (RV) is a common virus that is transmitted via airborne infection with humans as the only known hosts. It causes the rubella disease, also known as German measles or Rubeola. After the first infection, the virus persists in the body for the whole life providing lifelong immunity. The diseases can therefore be referred to as typical for childhood. It goes along with exanthemas, fever, headache, rheumatic pains and swollen lymph nodes. Anyways, for some patients the infection can be asymptomatic as well. In contrast to that, in rare cases complications can occur. Those are more frequent if the patient already reached adulthood and is only then infected for the first time. In such cases arthritis, bronchitis, encephalitis and inclusion of heart damages are conceivable 1).
As RV can also cross the placenta, pregnant women getting infected in the beginning of their pregnancy might lose their child due to a spontaneous abort. If the children are born despite the infection, they might suffer from congenital rubella syndromes 2) such as deformities of the heart, cataracts or labyrinthine deafness.
Up to date, the diagnosis of RV infection is performed by serodiagnosis of patients. IgG and IgM antibodies are both tested and the presence of the virus can also be verified by Polymerase chain reaction (PCR) 3).
Therapies are only symptomatic but there is vaccination available that could prevent the disease. This is especially recommended to women planning to get pregnant.
The RV is the only member of the genus Rubiviridae and belongs to the family of Togaviridae. Its genetic information is stored as single-stranded plus-sense RNA, which encodes three structural and two non-structural proteins 4). Two of the structural proteins are the envelope proteins E1 and E2 that form heterodimers arranged in groups of three and are distributed all over the viruses surface 5). As glycoprotein E1 appears to be immunodominant, we decided to use a sequence of epitopes derived from it as RV antigen for the DiaCHIP 6).
Herpes Simplex
A lot of people might have already had little blisters in the area of the lip that disappeared after some days. In most cases those might have been provoked by the Herpes Simplex Virus Type 1 (HSV-1).
There are two different types of Herpes Simplex Virus with about 99% of identity in the gene encoding regions. Both types are transmitted via the contact of mucous membranes. Type 1 – the one we are working with – can be transmitted via kissing, type 2 is mainly transmitted via sexual contact. Below we are only referring to Herpes Simplex Virus Type 1. It remains in the body in a latent state and several outbreaks during a lifetime are possible. As about 85-90% of the world’s population is seropositive the primary infection often takes place prior to the age of five due to a transmission from parents to their child 7). The infection of newborns is dangerous to life.
After intruding the body the virus proliferates and is shed via the mucous membrane. It also infiltrates neurons in which it persists lifelong. In very severe cases the virus can cause encephalitis as well as meningitis. The reasons for sudden outbreaks of the disease are not clearly understood. A suppression of the immune system or stress are only some possible explanations.
The diagnosis of HSV-1 can be performed in different manners. Either the virus itself or antibodies against it can be identified. For the first approach it is possible to detect antigens by direct immunofluorescence, viral DNA by amplification tests (NAATs) or culture the virus. The detection of antibodies provides a good aid in identifying HSV infections. Antibody detection is based on serological tests that comprise Western Blots, enzyme-linked immunosorbent assays (ELISAs) and, rather new, multiplexed flow immunoassays (MFIs) 8).
A therapy with anti-viral drugs is possible, however, for cutaneous infections in facial or rather labial areas the application of corticoids is usually sufficient 9).
HSV-1 belongs to the Human Herpesviruses and is therefore part of the family of Herpesviridae. An envelope consisting of at least 10 viral proteins surrounds the capsid, which protects the double-stranded DNA. Most of these proteins are glycoproteins, one of which is glycoprotein G that we express as HSV-1 antigen. Glycoprotein G is important for the attachment to the cell and the entry of the virus into it 10). Additionally, glycoprotein G differs significantly between the two types of Herpes Simplex Virus and therefore allows specific diagnosis 11).
Tetanus
Clostridium tetani is a little anaerobic gram-positive rod-shaped bacillus causing tetanus disease. It forms spores that can even survive in inhospitable areas and are present in soil. They can enter the human body easily via small wounds which is why injured people are at high risk of getting infected with C. tetani but cannot transmit the infection to other individuals.
In regions where people are not vaccinated and good health care is not provided, tetanus is a very common cause of death following injuries. The bacterium produces two toxins, named tetanospasmin – generally referred to as tetanustoxin – and tetanolysin. The former was found to cause tetanus by reaching the bone marrow via the nerves. There it is responsible for provoking hypersensitivity, increased reflexes and spasms 12). The latter causes damage to the heart muscle and blood components 13).
After an infection with C. tetani the first symptoms are headache, muscle and dorsal pain and a feeling of being tired. Additionally, the patient may feel some tautness in the area of the injury and reveal sensitivity to light and noise. If the patient is not taken care of, the infection is manifested by local stiffening of muscles, mainly in the area of the jaw and neck. In the following progression the patient will suffer from high fever and muscle spasms. Those will first be located in the face but spread over the whole body what causes the typical extended position. The immense tension in the muscles due to the effect of the toxins can cause lesions as well as dislocations of the joints or broken bones. As a consequence, many patients suffer from shortened muscles, ankylosis (stiffness of the joints) or spine deformity. In case of lacking appropriate health care, death due to suffocation or cardiovascular failure is common and occurs partially despite previous vaccination.
To avoid long-term effects due to an infection with C. tetani or even death, the wound is excised and surgically taken care of. Additionally, the patient will be treated with antibiotics and antibodies targeting the toxin are given.
For the DiaCHIP we expressed a part of the tetanustoxin. It consists of a heavy and a light chain that are linked by disulfide bonds 14). We worked with the carboxyl-terminal domain of the heavy chain that is able to bind to the target membrane and allows the internalization of the light chain, which is the actual toxic part 15).
Varicella Zoster
An infection going along with red and itching skin that nearly every person in the world suffers from…most might know it as chickenpox and might not even remember the first infection as this often takes place during childhood. The infection is caused by the Varicella Zoster Virus (VZV) that is transmitted via droplet infection or by having contact with blisters or mucous membranes. Following the first contact with the virus and an incubation time of about two weeks, the patient suffers from fever and exanthemas that normally heal without leaving scars.
As the virus resides in some ganglions of the body, mostly elderly or immune deficient people might again be afflicted with the disease that is then called Herpes Zoster. It manifests itself in exanthemas restricted to the area of the ganglion that is affected. Additionally, the patients are very sensitive to skin contact, have a fever and feel pain. In some cases the reactivation of the virus can lead to neuritis.
Normally infections with the virus, primary infections as well as the reactivations, are not life threatening and in most cases end without consequences for the patient. The primary infection is only a threat for newborns and immune deficient patients where a hemorrhagic development can be lethal. If adults are exposed to the virus for the first time it can also cause much more severe damage. The development of defects in the central nervous system is one of the most prominent consequences 16).
For the detection of VZV infection quantitative PCR is the method commonly used, often in combination with serological tests like ELISA. After the isolation of the virus from the patient’s blood serum antigens can be detected via immunofluorescence (IF) 17).
There is a live attenuated varicella vaccine available that is mainly applied for children and risk patients. Passive immunization with IgG antibodies is often the choice for exposed pregnant women and newborns within a time span of about 48 hours 18).
VZV belongs to the family of Herpesviridae, the same family as the Herpes Simplex Virus, and contains double-stranded DNA surrounded by a capsid. A tegument fills the space between this capsid and the envelope 19). This outer layer contains different viral envelope glycoproteins, one of which is glycoprotein E. We used a sequence of immunodominant epitopes derived from this protein as VZV antigen for the DiaCHIP 20). The functional protein forms heterodimers with the glycoprotein I and was found to play an important role in cell-cell attachment as well as facilitating the entry of the virus and the assembly of the virion 21).
Syphilis
One of the most common sexually transmittable diseases nowadays, besides AIDS, is syphilis that is also a chronical disease. It is caused by the bacterium Treponema pallidum subsp. pallidum, which is a gram-negative member of the family of four Spirochaetaceae and exhibits a spiral shape 22).
The bacterium is very difficult to cultivate in vitro, partly due to the absence of some metabolic capabilities. Humans are the only natural hosts of T. pallidum and it can be passed from one individual to another not only during sex but also via kissing if there are small lesions in the mucous membrane through which they can escape and enter 23).
After infection with T. pallidum there are four stages of syphilis distinguishable. Primary syphilis is characterized by chancre at the place of infiltration. It normally heals during a time span of three to 7 weeks. Additionally, in this first phase the patient might suffer from swollen lymph nodes.
Secondary syphilis manifests itself in exanthemas and moistening areas of the skin, mainly in the genital area and between fingers and toes, which are highly infectious. Enanthemas might also show up on mucous membranes and the patient might lose body hair.
The symptoms of these first two stages of syphilis often fade after about three months and a latency period of variable time, up to years, may follow. The patient is still contagious in the early time of this period but loses this characteristic after a while.
If the infection with T. pallidum reaches the next stage, inner organs are affected what may lead to hepatitis or damages involving the aorta for example. This is also the first stage in which the neuronal system is most battered. At latest a neurosyphilis develops during quaternary syphilis and the patient suffers from psychoses and the neurons shed their myelinisation.
It is also possible that T. pallidum is transmitted via the bloodstream from mother to child causing congenital syphilis. Depending on the immune response of the fetus consequences may vary from fetal death to fetal damage or an infected newborn. The defects can also have a wide range and affect growth, internal organs like liver or spleen and the neuronal system. The onset of these effects can start years later but also immediately after birth 24).
As T. pallidum can not easily be cultured, serologic tests are the choice to diagnose an infection. There are different tests available that comprise nontreponemal tests like the toluidine red unheated serum test (TRUST) or rapid plasma reagin (RPR) as well as treponemal tests like the specific particle agglutination assay (PA), enzyme-linked immunosorbent assay (ELISA) or chemiluminescent immunoassay (CMIA). It is chosen between them according to clinical demands and possibilities 25).
If the disease is noticed early enough a treatment with penicillin can lead to a cure with, in best cases, no permanent harm 26).
T. pallidum has a rather small genome as it lacks many coding regions e.g. for metabolic enzymes. Nonetheless, its gene encodes the bacterioferritin TpF1 that was found to be immunogenic and useful for serodiagnosis of different stages of syphilis 27). This is why we used this recombinant protein as T. palldium antigen for the DiaCHIP.
Acquired Immune Deficiency Syndrome (AIDS)
Probably the best known sexually transmittable disease in the public is the Acquired Immune Deficiency Syndrome called AIDS. It is provoked by the Human Immunodeficiency Virus (HIV) that is a single-stranded positive-sense RNA retrovirus surrounded by an envelope. There are two different types of HIV, HIV-1 and HIV-2, and we focused on HIV-1 that can be divided into three distinct subgroups 28).
Directly after the infection the patient suffers from unspecific flu-like symptoms like fever, diarrhea, exhaustion and swollen lymph nodes. A period free of symptoms follows, which is why the contagion is often misinterpreted and not linked to the HI Virus. During this latency period the immune system of the patient is damaged as the Virus infiltrates T cells and destroys them 29).
Following the latency period that lasts about 10 years on average most people enter a third period of infection where they are more sensible to infectious diseases, as the immune system is debilitated. In the course of time, this stadium develops into full-blown AIDS, which is composed of pneumonia, neurological diseases and cancer - mostly Kaposi’s sarcoma.
HIV is assured detected by different approaches of Nucleic acid testing (NAT) like real
time PCR or transcription mediated amplification. First hints at a possible infection are
anyways mostly given by serological detection of antibodies for example via ELISA
30).
There is no vaccination available and no possibility of healing the patient yet. The only possibility to improve the standard of living of infected people are medications that try to prevent the reproduction of the virus, which can be achieved via different approaches but they often exhibit severe side effects 31).
The structure of HIV is quite well known due to the great importance in medical treatment and care. On its envelope the virus exhibits many different proteins that provoke an immunogenic response. For our purposes we used a multi-epitopic recombinant protein that is constructed with six different epitopes 32).
Salmonellosis
Often, people with a general malaise and diarrhea blame “bad food” for it. The rather mild food poisoning is in many cases elicited by an infection with Salmonella enterica subsp. enterica serovar Typhimurium. This gram-negative rod-shaped bacterium mainly appears in eggs, poultry or beef and can infect humans as well as other mammals. After taking up a sufficient amount of bacteria via contaminated food, this always leads to salmonellosis.
Due to an enterotoxin released by S. Typhimurium intestinal inflammation is induced and the patient suffers from diarrhea, vomiting and sometimes fever. In contrast to the rather dangerous infection with Salmonella enterica subsp. enterica serovar Typhi that provokes the systemic illness typhoid fever, the infection with S. Typhimurium normally ebbs off within few days. Other organs are normally not infected. 33).
The diagnosis of a S. Typhimurium infection results from bacteriological culturing with subsequent PCR based detection or via serological tests, mainly ELISAs 34).
For our purposes concerning a S. Typhimurium antigen for the DiaCHIP we were provided with a plasmid containing an immunogenic part of the dihydroxyacid dehydratase and a corresponding single chain antibody, which were both discovered via phage display 35).
3)
Alter et al., 2015. Common Childhood Viral Infections. Current Problems in Pediatric and Adolescent Health Care.
4)
Abernathy et al., 2013. Analysis of whole genome sequences of 16 strains of rubella virus from the United States, 1961-2009. Virology Journal.
6)
Best et al., 1995. Use of rubella virus E1 fusion proteins for detection of rubella virus antibodies. Journal of Clinical Microbiology.
7)
Steiner & Benninger, 2013. Update on Herpes Virus Infections of the Nervous System. Current Neurology and Neuroscience Reports.
8)
Ledeboer et al., 2014. Light Microscopy, Culture, Molecular, and Serologic Methods for Detection of Herpes Simplex Virus. Journal of Clinical Microbiology.
10)
Montgomery et al., 1996. Herpes Simplex Virus-1 Entry into Cells Mediated by a Novel Member of the TNF/NGF Receptor Family. Cell.
11)
Han et al., 2013. Development of Recombinant Antigen Array for Simultaneous Detection of Viral Antibodies. PLOS ONE.
12)
Afshar et al., 2011. Narrative Review: Tetanus – A Health Threat After Natural Disasters in Developing Countries. Annals of Internal Medicine.
14)
Bohner & Schiavo, 2005. Tetanus Toxin Is Transported in a Novel Neuronal Compartment Characterized by a Specialized pH Regulation. Journal of Biological Chemistry.
15)
Rui et al., 2011. Enhanced expression of soluble recombinant tetanus neurotoxin Hc in Escherichia coli as a tetanus vaccine candidate. Immunobiology.
16)
Grahn & Studahl, 2015. Varicella-zoster virus infections of the central nervous system - Prognosis, diagnostics and treatment. Journal of Infection.
17)
Grahn & Studahl, 2015. Varicella-zoster virus infections of the central nervous system – Prognosis, diagnostics and treatment. Journal of Infection.
18)
Gershon & Gershon, 2013. Pathogenesis and Current Approaches to Control of Varicella-Zoster Virus Infections. Clinical Microbiology Reviews.
19)
Xia et al., 2015. Insights into the function of tegument proteins from the varicella zoster virus. Science China Life Sciences.
20)
Bäckström et al., 2011. Recombinant glycoprotein E produced in mammalian cells in large-scale as an antigen for varicella-zoster-virus serology. Journal of Virological Methods.
21)
Mo et al., 2000. Glycoprotein E of Varicella-Zoster Virus Enhances Cell-Cell Contact in Polarized Epithelial Cells. Journal of Virology.
23)
Ho & Lukehart, 2011. Syphilis: using modern approaches to understand an old disease. Journal of Clinical Investigation.
25)
Yang et al., 2014. The Diagnostic Value and Performance Evaluation of Five Serological Tests for the Detection of Treponema pallidum. Journal of Clinical Laboratory Analysis.
27)
Wu et al., 2013. Evaluation of the Recombinant Protein TpF1 of //Treponema pallidum// for Serodiagnosis of Syphilis. Clinical and Vaccine Immunology.
30)
Albertoni et al.,
2014. Mini review: Current molecular methods for the detection and quantification of
hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1.
International Journal of Infectious Diseases.
32)
Jafarpour et al., 2014. Clustered epitopes within a new poly-epitopic HIV-1 DNA vaccine shows immunogenicity in BALB/c mice. Molecular Biology Reports.
33)
Monack et al., 2012. Innate immune response to Salmonella typhimurium, a model enteric pathogen. Gut Microbes.
One key component of our project, the DiaCHIP, are of course the antigenic peptides immobilized on the surface of the protein array. As a basis to perform cell-free expression, we did paper research to look for suitable nucleotide sequences we could use and that had been already used for serologic detection of antibodies. We decided to focus on some rather familiar or common pathogens, which will be presented in the following paragraphs.
The Rubella Virus (RV) is a common virus that is transmitted via airborne infection with humans as the only known hosts. It causes the rubella disease, also known as German measles or Rubeola. After the first infection, the virus persists in the body for the whole life providing lifelong immunity. The diseases can therefore be referred to as typical for childhood. It goes along with exanthemas, fever, headache, rheumatic pains and swollen lymph nodes. Anyways, for some patients the infection can be asymptomatic as well. In contrast to that, in rare cases complications can occur. Those are more frequent if the patient already reached adulthood and is only then infected for the first time. In such cases arthritis, bronchitis, encephalitis and inclusion of heart damages are conceivable 1).
As RV can also cross the placenta, pregnant women getting infected in the beginning of their pregnancy might lose their child due to a spontaneous abort. If the children are born despite the infection, they might suffer from congenital rubella syndromes 2) such as deformities of the heart, cataracts or labyrinthine deafness.
Up to date, the diagnosis of RV infection is performed by serodiagnosis of patients. IgG and IgM antibodies are both tested and the presence of the virus can also be verified by Polymerase chain reaction (PCR) 3).
Therapies are only symptomatic but there is vaccination available that could prevent the disease. This is especially recommended to women planning to get pregnant.
The RV is the only member of the genus Rubiviridae and belongs to the family of Togaviridae. Its genetic information is stored as single-stranded plus-sense RNA, which encodes three structural and two non-structural proteins 4). Two of the structural proteins are the envelope proteins E1 and E2 that form heterodimers arranged in groups of three and are distributed all over the viruses surface 5). As glycoprotein E1 appears to be immunodominant, we decided to use a sequence of epitopes derived from it as RV antigen for the DiaCHIP 6).
A lot of people might have already had little blisters in the area of the lip that disappeared after some days. In most cases those might have been provoked by the Herpes Simplex Virus Type 1 (HSV-1).
There are two different types of Herpes Simplex Virus with about 99% of identity in the gene encoding regions. Both types are transmitted via the contact of mucous membranes. Type 1 – the one we are working with – can be transmitted via kissing, type 2 is mainly transmitted via sexual contact. Below we are only referring to Herpes Simplex Virus Type 1. It remains in the body in a latent state and several outbreaks during a lifetime are possible. As about 85-90% of the world’s population is seropositive the primary infection often takes place prior to the age of five due to a transmission from parents to their child 7). The infection of newborns is dangerous to life.
After intruding the body the virus proliferates and is shed via the mucous membrane. It also infiltrates neurons in which it persists lifelong. In very severe cases the virus can cause encephalitis as well as meningitis. The reasons for sudden outbreaks of the disease are not clearly understood. A suppression of the immune system or stress are only some possible explanations.
The diagnosis of HSV-1 can be performed in different manners. Either the virus itself or antibodies against it can be identified. For the first approach it is possible to detect antigens by direct immunofluorescence, viral DNA by amplification tests (NAATs) or culture the virus. The detection of antibodies provides a good aid in identifying HSV infections. Antibody detection is based on serological tests that comprise Western Blots, enzyme-linked immunosorbent assays (ELISAs) and, rather new, multiplexed flow immunoassays (MFIs) 8).
A therapy with anti-viral drugs is possible, however, for cutaneous infections in facial or rather labial areas the application of corticoids is usually sufficient 9).
HSV-1 belongs to the Human Herpesviruses and is therefore part of the family of Herpesviridae. An envelope consisting of at least 10 viral proteins surrounds the capsid, which protects the double-stranded DNA. Most of these proteins are glycoproteins, one of which is glycoprotein G that we express as HSV-1 antigen. Glycoprotein G is important for the attachment to the cell and the entry of the virus into it 10). Additionally, glycoprotein G differs significantly between the two types of Herpes Simplex Virus and therefore allows specific diagnosis 11).
Clostridium tetani is a little anaerobic gram-positive rod-shaped bacillus causing tetanus disease. It forms spores that can even survive in inhospitable areas and are present in soil. They can enter the human body easily via small wounds which is why injured people are at high risk of getting infected with C. tetani but cannot transmit the infection to other individuals.
In regions where people are not vaccinated and good health care is not provided, tetanus is a very common cause of death following injuries. The bacterium produces two toxins, named tetanospasmin – generally referred to as tetanustoxin – and tetanolysin. The former was found to cause tetanus by reaching the bone marrow via the nerves. There it is responsible for provoking hypersensitivity, increased reflexes and spasms 12). The latter causes damage to the heart muscle and blood components 13).
After an infection with C. tetani the first symptoms are headache, muscle and dorsal pain and a feeling of being tired. Additionally, the patient may feel some tautness in the area of the injury and reveal sensitivity to light and noise. If the patient is not taken care of, the infection is manifested by local stiffening of muscles, mainly in the area of the jaw and neck. In the following progression the patient will suffer from high fever and muscle spasms. Those will first be located in the face but spread over the whole body what causes the typical extended position. The immense tension in the muscles due to the effect of the toxins can cause lesions as well as dislocations of the joints or broken bones. As a consequence, many patients suffer from shortened muscles, ankylosis (stiffness of the joints) or spine deformity. In case of lacking appropriate health care, death due to suffocation or cardiovascular failure is common and occurs partially despite previous vaccination.
To avoid long-term effects due to an infection with C. tetani or even death, the wound is excised and surgically taken care of. Additionally, the patient will be treated with antibiotics and antibodies targeting the toxin are given.
For the DiaCHIP we expressed a part of the tetanustoxin. It consists of a heavy and a light chain that are linked by disulfide bonds 14). We worked with the carboxyl-terminal domain of the heavy chain that is able to bind to the target membrane and allows the internalization of the light chain, which is the actual toxic part 15).
An infection going along with red and itching skin that nearly every person in the world suffers from…most might know it as chickenpox and might not even remember the first infection as this often takes place during childhood. The infection is caused by the Varicella Zoster Virus (VZV) that is transmitted via droplet infection or by having contact with blisters or mucous membranes. Following the first contact with the virus and an incubation time of about two weeks, the patient suffers from fever and exanthemas that normally heal without leaving scars.
As the virus resides in some ganglions of the body, mostly elderly or immune deficient people might again be afflicted with the disease that is then called Herpes Zoster. It manifests itself in exanthemas restricted to the area of the ganglion that is affected. Additionally, the patients are very sensitive to skin contact, have a fever and feel pain. In some cases the reactivation of the virus can lead to neuritis.
Normally infections with the virus, primary infections as well as the reactivations, are not life threatening and in most cases end without consequences for the patient. The primary infection is only a threat for newborns and immune deficient patients where a hemorrhagic development can be lethal. If adults are exposed to the virus for the first time it can also cause much more severe damage. The development of defects in the central nervous system is one of the most prominent consequences 16).
For the detection of VZV infection quantitative PCR is the method commonly used, often in combination with serological tests like ELISA. After the isolation of the virus from the patient’s blood serum antigens can be detected via immunofluorescence (IF) 17).
There is a live attenuated varicella vaccine available that is mainly applied for children and risk patients. Passive immunization with IgG antibodies is often the choice for exposed pregnant women and newborns within a time span of about 48 hours 18).
VZV belongs to the family of Herpesviridae, the same family as the Herpes Simplex Virus, and contains double-stranded DNA surrounded by a capsid. A tegument fills the space between this capsid and the envelope 19). This outer layer contains different viral envelope glycoproteins, one of which is glycoprotein E. We used a sequence of immunodominant epitopes derived from this protein as VZV antigen for the DiaCHIP 20). The functional protein forms heterodimers with the glycoprotein I and was found to play an important role in cell-cell attachment as well as facilitating the entry of the virus and the assembly of the virion 21).
One of the most common sexually transmittable diseases nowadays, besides AIDS, is syphilis that is also a chronical disease. It is caused by the bacterium Treponema pallidum subsp. pallidum, which is a gram-negative member of the family of four Spirochaetaceae and exhibits a spiral shape 22).
The bacterium is very difficult to cultivate in vitro, partly due to the absence of some metabolic capabilities. Humans are the only natural hosts of T. pallidum and it can be passed from one individual to another not only during sex but also via kissing if there are small lesions in the mucous membrane through which they can escape and enter 23).
After infection with T. pallidum there are four stages of syphilis distinguishable. Primary syphilis is characterized by chancre at the place of infiltration. It normally heals during a time span of three to 7 weeks. Additionally, in this first phase the patient might suffer from swollen lymph nodes.
Secondary syphilis manifests itself in exanthemas and moistening areas of the skin, mainly in the genital area and between fingers and toes, which are highly infectious. Enanthemas might also show up on mucous membranes and the patient might lose body hair.
The symptoms of these first two stages of syphilis often fade after about three months and a latency period of variable time, up to years, may follow. The patient is still contagious in the early time of this period but loses this characteristic after a while.
If the infection with T. pallidum reaches the next stage, inner organs are affected what may lead to hepatitis or damages involving the aorta for example. This is also the first stage in which the neuronal system is most battered. At latest a neurosyphilis develops during quaternary syphilis and the patient suffers from psychoses and the neurons shed their myelinisation.
It is also possible that T. pallidum is transmitted via the bloodstream from mother to child causing congenital syphilis. Depending on the immune response of the fetus consequences may vary from fetal death to fetal damage or an infected newborn. The defects can also have a wide range and affect growth, internal organs like liver or spleen and the neuronal system. The onset of these effects can start years later but also immediately after birth 24).
As T. pallidum can not easily be cultured, serologic tests are the choice to diagnose an infection. There are different tests available that comprise nontreponemal tests like the toluidine red unheated serum test (TRUST) or rapid plasma reagin (RPR) as well as treponemal tests like the specific particle agglutination assay (PA), enzyme-linked immunosorbent assay (ELISA) or chemiluminescent immunoassay (CMIA). It is chosen between them according to clinical demands and possibilities 25).
If the disease is noticed early enough a treatment with penicillin can lead to a cure with, in best cases, no permanent harm 26).
T. pallidum has a rather small genome as it lacks many coding regions e.g. for metabolic enzymes. Nonetheless, its gene encodes the bacterioferritin TpF1 that was found to be immunogenic and useful for serodiagnosis of different stages of syphilis 27). This is why we used this recombinant protein as T. palldium antigen for the DiaCHIP.
Probably the best known sexually transmittable disease in the public is the Acquired Immune Deficiency Syndrome called AIDS. It is provoked by the Human Immunodeficiency Virus (HIV) that is a single-stranded positive-sense RNA retrovirus surrounded by an envelope. There are two different types of HIV, HIV-1 and HIV-2, and we focused on HIV-1 that can be divided into three distinct subgroups 28).
Directly after the infection the patient suffers from unspecific flu-like symptoms like fever, diarrhea, exhaustion and swollen lymph nodes. A period free of symptoms follows, which is why the contagion is often misinterpreted and not linked to the HI Virus. During this latency period the immune system of the patient is damaged as the Virus infiltrates T cells and destroys them 29).
Following the latency period that lasts about 10 years on average most people enter a third period of infection where they are more sensible to infectious diseases, as the immune system is debilitated. In the course of time, this stadium develops into full-blown AIDS, which is composed of pneumonia, neurological diseases and cancer - mostly Kaposi’s sarcoma.
HIV is assured detected by different approaches of Nucleic acid testing (NAT) like real
time PCR or transcription mediated amplification. First hints at a possible infection are
anyways mostly given by serological detection of antibodies for example via ELISA
30).
There is no vaccination available and no possibility of healing the patient yet. The only possibility to improve the standard of living of infected people are medications that try to prevent the reproduction of the virus, which can be achieved via different approaches but they often exhibit severe side effects 31).
The structure of HIV is quite well known due to the great importance in medical treatment and care. On its envelope the virus exhibits many different proteins that provoke an immunogenic response. For our purposes we used a multi-epitopic recombinant protein that is constructed with six different epitopes 32).
Often, people with a general malaise and diarrhea blame “bad food” for it. The rather mild food poisoning is in many cases elicited by an infection with Salmonella enterica subsp. enterica serovar Typhimurium. This gram-negative rod-shaped bacterium mainly appears in eggs, poultry or beef and can infect humans as well as other mammals. After taking up a sufficient amount of bacteria via contaminated food, this always leads to salmonellosis.
Due to an enterotoxin released by S. Typhimurium intestinal inflammation is induced and the patient suffers from diarrhea, vomiting and sometimes fever. In contrast to the rather dangerous infection with Salmonella enterica subsp. enterica serovar Typhi that provokes the systemic illness typhoid fever, the infection with S. Typhimurium normally ebbs off within few days. Other organs are normally not infected. 33).
The diagnosis of a S. Typhimurium infection results from bacteriological culturing with subsequent PCR based detection or via serological tests, mainly ELISAs 34).
For our purposes concerning a S. Typhimurium antigen for the DiaCHIP we were provided with a plasmid containing an immunogenic part of the dihydroxyacid dehydratase and a corresponding single chain antibody, which were both discovered via phage display 35).
