Campylobacter sp., Shigella sp., Salmonella sp., Yersinia sp and Mycobacterium sp in synovial fluid cells or synovium from adults with recent onset undifferentiated oligoarthritis or ReA. Studies have also been able to dentifiy RNA transcripts of C. trachomatis and B. burgdorferi.
From: Infection and Autoimmunity, 2004
Related terms:
- Shigella
- Vaccine Efficacy
- Salmonella
- Bacterium
- Prevalence
- Broiler
- Escherichia coli
- Chicken
Campylobacter
Robert M. Kliegman MD, in Nelson Textbook of Pediatrics, 2020
Diagnosis
The clinical presentation ofCampylobacter enteritis can be similar to that of enteritis caused by other bacterial pathogens. The differential diagnosis includesShigella, Salmonella, Escherichia coli,Yersinia enterocolitica, Aeromonas, Vibrio parahaemolyticus, and amebiasis. Fecal leukocytes are found in as many as 75% of cases, and fecal blood is present in 50% of cases (higher in pediatric patients).Campylobacter should be considered in patients with bloody stools, fever, and abdominal pain.
The diagnosis ofCampylobacter enteritis is usually confirmed by identification of the organism in cultures of stool or rectal swabs. Isolation is most likely from selective media such as CAMPY-agar grown in microaerophilic conditions (5–10% oxygen), 1–10% carbon dioxide, with some hydrogen. SomeC. jejuni grow best at 42°C (107.6°F). Growth on solid media results in small (0.5-1.0 mm), slightly raised, smooth colonies. Organisms can be identified from stool microscopically in approximately 50% of knownCampylobacter cases. Gram stain is even less sensitive. Stool culture is >90% sensitive and is the standard method of diagnosis. Visible growth on stool culture is most often present in 1-2 days. Visible growth in blood cultures is often not apparent until 5-14 days after inoculation.
Routine culture may be adequate for isolation ofC. jejuni because of the large numbers of bacteria that are often present. However, becauseCampylobacter organisms grow more slowly under routine conditions than do other enteric bacteria, routine culture can result in failure because of overgrowth of other enteric bacteria.Campylobacter culture can be enhanced, when necessary, with selective media. However, selective culture media developed to enhance isolation ofC. jejuni may inhibit the growth of otherCampylobacter spp. Filtration methods are available and can preferentially enrich forCampylobacter by selecting for their small size. These methods allow subsequent culture of the enriched sample on antibiotic-free media, enhancing rates of isolation ofCampylobacter organisms inhibited by the antibiotics included in standard selective media. Isolation ofCampylobacter from normally sterile sites does not require enhancement procedures. Clinically, it is not necessary to speciateCampylobacter, because clinical disease is the same. Speciation can be done, when needed, and specialized laboratories can perform strain typing when required for epidemiologic purposes.
For rapid diagnosis ofCampylobacter enteritis, direct carbolfuchsin stain of fecal smear, indirect fluorescence antibody test, dark-field microscopy, or latex agglutination were used historically. Polymerase chain reaction testing is more specific and sensitive and is becoming more widely available for rapid testing, often grouped with testing for other bacterial, viral, and parasitic stool pathogens in a multiplex assay. At this time, the recommendation remains to confirm all positive rapid tests with culture, which also allows for susceptibility testing and epidemiologic investigations. Serologic diagnosis is also possible and is most helpful in patients with late-onset reactive arthritis or Guillain-Barré syndrome, since these patients may have negative stool cultures by the time of presentation with these late complications.
Examination of faeces for bacterial pathogens
S.H. Gillespie MB, BCh, BAO, MRCP(UK), MRCPath, in Medical Microbiology Illustrated, 1994
Campylobacter
Introduction
Campylobacter were first recognized in 1915 as a cause of abortion in cattle and sheep. There were later anecdotal reports of human disease caused by ‘vibrio-like organisms’. In 1972, Dekeyser developed a membrane filtration method, permitting the isolation of Campylobacter spp. from stools for the first time. However, it was only when Skirrow developed a selective medium that it became feasible to isolate Campylobacter by faecal culture in the routine laboratory and their role in human infections was firmly established.
Classification
The genus is divided into two groups depending on the presence of catalase. The catalase-negative group are usually not pathogenic to humans but are commensals and pathogens of animals. Included in this group is C. sputorum which has been found in up to 2% of faecal samples from healthy subjects, but a pathogenic role for this organism has not been found. Campylobacter mucosalis is thought to be associated with intestinal adenomatosis in pigs. Human infection has not been reported.
The catalase-positive group includes species which are pathogens and commensals of animals and humans. This group can be subdivided into thermophilic and non-thermophilic species. The non-thermophilic species C. fetus ssp. fetus and C. fetus ssp. venerealis are mainly animal pathogens. Campylobacter fetus ssp. fetus is associated with spontaneous abortion in cattle and sheep and can occasionally cause septicaemia and meningitis in patients predisposed to infection. It can also be found in the human intestine and may be responsible for some cases of diarrhoea. Campylobacter fetus ssp. venerealis is a commensal of animals, but human infection has only rarely been reported.
The thermophilic Campylobacter spp. are responsible for most human infections: the species implicated are C. jejuni, C. coli, and C. laridis. Campylobacter infection is essentially a zoonosis although person-to-person and institutional infections have been reported. These organisms are found in the intestine of poultry and cattle. Infection is transmitted to humans through eating inadequately cooked meat or drinking contaminated milk. It can also be transmitted by water if it becomes contaminated with faecal material from infected animals (Table 17.5).
Table 17.5. Campylobacter genus
| Catalase negative | C. sputorum | |
| subsp. sputorum | Mouth commensal | |
| subsp. bulbulus | Commensal bovine genital tract | |
| subsp. mucosalis | Intestinal adenomatosis of pigs | |
| Catalase positive | C. fetus | |
| Non-thermophilic | subsp. verierealis | Cattle infertility |
| subsp. fetus | Abortion in sheep and cattle; occasional opportunist | |
| Catalase positive thermophilic | C jejuni | Acute enterocolitis (see text) |
| C coli | Commensals birds and animals | |
| C. laridis | Commensal seagulls; occasional infection in man |
Clinical importance
An acute enterocolitis with prodromal symptoms of malaise, headache, myalgia, abdominal pain, and fever is typical. This is followed by acute crampy abdominal pain and diarrhoea, which may be blood stained. The illness is usually short-lived (three to five days) and long-term carriage is unusual. Serious complications are also unusual, but septicaemia may occur, and, in some cases, acute campylobacter infection may be mistaken for an acute abdomen or in infants for intussusception prompting unnecessary surgery. Campylobacter enterocolitis has also been postulated as one of the causes of reactive arthritis.
Pathogenicity determinants
Campylobacters are readily killed at low pH and therefore infection is more likely if milk is the vehicle or patients have low gastric acidity.
They possess flagella and are actively motile which may assist the organism in initiating infection. Campylobacters are thought to adhere to intestinal mucosa via L-fucose receptors. Campylobacter jejuni elaborates an enterotoxin which causes fluid accumulation in rabbit ileal loops. It has a similar mode of action to that of cholera toxin. A cytotoxin may also be implicated in the pathogenesis of enteritis. Campylobacter spp. express a low molecular weight lipopolysaccharide which is similar in structure to that of Haemophilus and Neisseria in which there is marked antigenic diversity between isolates.
Isolation
Campylobacters can be isolated from human faeces by culture on selective media and the correct conditions of atmosphere and temperature. Media are made selective for these organisms by inclusion of a mixture of antibiotics, examples of these media are shown in Table 17.6. Inoculated plates should be incubated at 43°C as this increases the selection, but if C. fetus is suspected, additional sets of plates should be incubated at 37°C as this species is inhibited by the higher temperature. A micro-aerophilic atmosphere must be provided with 5−6% oxygen, 10% carbon dioxide, and 85% nitrogen. Although candle jars may be used, this atmosphere is most usually generated by a micro-aerophilic gas generating kit, similar in principle to that employed for anaerobic isolation.
Table 17.6. Selective agents for Campylobacter isolation
| Skirrow | Vancomycin, polymixin B, trimethoprim |
| Butzler | Bacitracin, novobiocin, cyclohexamide, colistin, cefazolin |
| Blaser | Vancomycin, polymixin B, trimethoprim, cephalothin, ampotericin B |
Identification
After 48 hours’ incubation campylobacter colonies appear as grey, flat droplets. A Gram stain will demonstrate small, curved or spiral, Gram-negative bacteria with a ‘gull wing’ morphology (Fig. 17.8). If the organism is oxidase positive it may be presumptively reported as Campylobacter spp. If species identification is required the organism should be tested for catalase production, its growth temperature requirements and its nalidixic acid sensitivity determined. Hippurate hydrolysis will distinguish between C. jejuni and C. coli. The usual reactions of campylobacter in these tests are set out in Table 17.7.
Figure 17.8. Gram stain of Campylobacter
Table 17.7. Identification of Campylobacter
| Growth 25°C | Growth 43°C | Nalidixic acid | Hippurate hydrolysis | |
|---|---|---|---|---|
| C. fetus | + | – | R | – |
| C jejuni | – | + | S | + |
| C. coli | – | + | S | – |
| C laridis | – | + | R | – |
Helicobacter pylori
This is one of the ‘new’ pathogens first described in 1983 and identified as a pathogen in gastritis and duodenal ulcer. It was originally classified as a Campylobacter but phylogenic studies have indicated that they are not related to this group and have been classified as a new genus Helicobacter.
Pathogenicity
This organism is the subject of intensive research and some of the potential pathogenicity determinants have been identified. The organism produces copious amounts of urease which may protect the organism from the effects of gastric acid. Other toxins which have been described include a mucinase and several cytotoxins, but the role of these proteins has not been established. The organism agglutinates red cells via a fibrillar haemagglutinin which may bind a glycolipid found on red cells and in the stomach.
Laboratory diagnosis
Many of the tests to diagnose patients suffering from helicobacter infection are clinical and include a 14C urea breath test. Specimens of gastric antrum can be obtained at endoscopy. The simplest diagnostic test is to place such a biopsy specimen in a urease medium and observe for a rapid colour change. Commercial examples of this approach (e.g. CLO-test) are cheap and accurate and do not require microbiological culture. Histological examination of biopsy specimens is an effective way of making the diagnosis.
Helicobacter pylori can be cultivated from biopsy specimens which have been homogenized and inoculated on fresh medium. Media which have been successfully employed include chocolate agar, Skirrow's, and Marshall's brain heart infusion agars. The antibiotic supplement should contain vancomycin 6 mg/1, nalidixic acid 20 mg/1, and amphotericin B 2 mg/1 and plates should be incubated in increased humidity in a micro-aerophilic atmosphere. Translucent colonies 1–2 mm can be seen after three days, but may be delayed until seven days. The organism can be identified on the basis of the Gram morphology, positive oxidase and catalase and rapid urease reactions. Individual isolates can be typed by chromosomal DNA restriction endonuclease digest analysis.
An ELISA can be used for serological diagnosis. The antigen used in the solid phase of this test is crude extract of a mixture of Helicobacter strains.
Antibiotic susceptibility
Helicobacter pylori is susceptible to a wide range of antibiotics including erythromycin, tetracycline, gentamicin, fluoroquinolones, and clindamycin. It is resistant to trimethoprim, sulphonamides, and nalidixic acid. The role of these agents in the treatment of disease is not established, however, and there is therefore no indication for antimicrobial susceptibility testing in routine diagnosis.
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Campylobacter
Rick D. Kellerman MD, in Conn's Current Therapy 2021, 2021
Treatment
Most cases ofCampylobacter infection are mild and self-limited, and antibiotics are not required. If antibiotics are to be used, they are most effective if given early to high-risk patients, because delayed treatment (e.g., after positive stool culture results) is less likely to affect outcome. First-line choices include macrolides, such as azithromycin (<5% resistance), or fluoroquinolones, such as ciprofloxacin (Cipro). Azithromycin (Zithromax)1 is preferred for traveler’s diarrhea acquired in South and Southeast Asia, where quinolone-resistantCampylobacter is common. Azithromycin has supplanted erythromycin (Ery-Tab)1 because it is better tolerated and has fewer gastrointestinal side effects. Rarely, intravenous aminoglycosides or carbapenems are necessary in very ill patients unable to take oral medication.
Unfortunately, quinolone resistance is not limited to Asia. Resistance has also increased in the United States. The widespread practice of using antibiotics, especially fluoroquinolones (enrofloxacin [Baytril]),2 as additives to chicken feed has resulted in increasing quinolone resistance inCampylobacter strains found in poultry, the major source of infection. The Foodborne Diseases Active Surveillance Network (FoodNet) of the Centers for Disease Control and Prevention (CDC) found that 40% of US poultry products were contaminated withCampylobacter, and 10% of these strains were ciprofloxacin resistant. Although fluoroquinolones were removed from US animal feed in 2005, they continue to be used overseas. A newer strategy involves supplementing poultry feed with bacteriocins, nontoxic antimicrobial peptides, to reduceCampylobacter colonization. Irradiation of poultry meat might also be effective.
Empiric self-treatment of traveler’s diarrhea is intended to shorten the duration of symptoms and involves immediate ciprofloxacin (Cipro) or azithromycin (Zithromax)1 at the onset of diarrheal illness. One or two doses of antibiotics are usually sufficient to abort symptoms, and prolonged treatment is usually unnecessary. Loperamide (Imodium) might help control diarrhea in adults, but it should be avoided in dysenteric illness and in young children.
Prevention and management of infections
Alexandra F. Freeman, Steven M. Holland, in Stiehm's Immune Deficiencies (Second Edition), 2020
Campylobacter and related infections
Campylobacter and Helicobacter (including Helicobacter species flexispira) species are typically localized to the GI tract, but in humoral defects can cause chronic disseminated infections characterized typically by bacteremia, skin ulcerations, and osteoarticular infections.36 These infections occur typically in XLA, where the lack of IgM is thought to play a role in susceptibility. These organisms are difficult to cultivate and require selective media and atmospheres, so discussion with the microbiology laboratory is critical for effective recovery. Optimal therapy likely requires prolonged combination antibiotic therapy and even with prolonged therapy, relapse is frequent. Replacement of IgM through repeated fresh frozen plasma infusions may be considered if antimicrobial treatment alone is not sufficient.
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Acute Diarrhea
Rick D. Kellerman MD, in Conn's Current Therapy 2021, 2021
Campylobacter
Campylobacter is a common cause of diarrhea in the United States. The pathogen is often spread by consumption of undercooked meat and poultry. Symptoms may present 2 to 5days after exposure and last up to 1week. The patient presents with diarrhea (possibly bloody), abdominal pain, nausea, vomiting, and fever. Stool cultures are needed for a definitive diagnosis. Prevention includes thoroughly cooking all meat products, good hand and utensil hygiene before preparing foods, and avoiding unpasteurized milk. The treatment for severeCampylobacter diarrhea includes azithromycin (Zithromax)1 and ciprofloxacin (Cipro), although resistance to fluoroquinolones is growing. Antimicrobial susceptibility testing is required for specific resistance. Positive stool cultures should be reported to the local health department.Campylobacter infections have been linked to both rheumatoid arthritis and also Guillain-Barré syndrome.
Infections in children
D.C. Shanson MB, FRCPath, in Microbiology in Clinical Practice (Second Edition), 1989
SOME OTHER INFECTIONS IN CHILDHOOD
Gastro-intestinal Tract Infections
Gastro-enteritis in children in Britain is often associated with rotavirus, campylobacter or salmonella infection but enteropathogenic strains of E. coli may also be the causative organisms in infants under 3 years old. Sonnei dysentery is most frequent amongst infants of pre-school age (gastro-enteritis, dysentery and other causes of diarrhoea are discussed further in Chapter 14). Worm infestations are relatively common in children in most countries and, in Britain, threadworms are especially common in young children (see Chapter 24).
Haemolytic Uraemic Syndrome
This disease mainly affects children and diarrhoea often bloody is frequently the first clinical feature which occurs prior to the triad of features characteristic of the syndrome: acute renal failure, microangiopathic haemolytic anaemia and thrombocytopenia. Organisms possibly associated with causing this syndrome include certain verotoxin producing serotypes of E. coli, Shigella dysenteriae serotype 1 (Shiga dysentery bacillus) and Salm. typhi. About 60 cases of this syndrome are reported each year in Britain (see also Chapter 14).
Urinary Tract Infections
Urinary tract infection occurs in 0·1–5·0% of children and the incidence increases with age. It mainly affects females and the majority of cases are asymptomatic, although some infants have febrile illnesses. However, the symptoms are often non-specific and the diagnosis of urinary tract infection may be missed unless urine cultures are carried out. The recognition of urinary tract infections in children is important since recurrent infections may occur, and result in decreased renal growth and impaired renal function (see Chapter 19).
Infections of the Central Nervous System
Bacterial meningitis is uncommon, but is much more frequent in infants and children under 5 years old than in adults. More than two-thirds of the cases are caused by the three primary pathogens, Neisseria meningitidis, Haemophilus influenzae and Strep. pneumoniae. Haemophilus influenzae (capsulated type b strains) causes meningitis almost entirely in infants and children. In Britain there has been a debate in recent years about the optimal timing and indications for lumbar puncture in children with suspected meningitis (see Further reading). Viral meningitis, which is far more common than bacterial meningitis, also occurs more frequently in children than adults (see Chapter 10).
Haemorrhagic Shock and Encephalopathy Syndrome
A new fatal disease in infants aged between 3 and 8 months was first reported in 1983 and since then further epidemics have occasionally been reported. Infants who were previously well are found shocked, convulsing and febrile and sometimes have vomiting and bleeding from various sites. There is probably an infective cause but the causative agent is unknown.
Skin, Bone and Joint Infections
Skin, bone and joint infections are more frequent in children than adults and are most frequently associated with Staph. aureus or Strep. pyogenes infections (see Chapters 16 and 17). A few children suffer from recurrent or persistent skin infections, because they have a congenital or acquired immunodeficiency syndrome or defect of their white blood cells.
Parvovirus Infections
In recent years parvovirus, a DNA virus, has been recognized as the cause of a common febrile disease in children which is often characterized with a facial maculopapular rash causing flushed cheeks—also known as ‘slapped cheek syndrome’ or ‘fifth disease’. The incubation period is from 5 to 20 days. Epidemics may occur and when the disease affects a patient with sickle-cell disease a haemolytic aplastic crisis can complicate infection. Sometimes there is arthralgia or arthritis (see Chapter 17). Microbiological diagnosis can be confirmed by demonstrating a rise in serum antibody titre to parvovirus or a raised IgM antibody titre to parvovirus using radioimmuno-assay or ELISA technique.
Kawasaki Disease
An acute febrile illness with mucocutaneous involvement, occurring mainly in children less than 4 years old and becoming complicated by fatal coronary arteritis, known as Kawasaki disease, was first reported in Japan but has occasionally been also reported in North America and Europe. It probably has an infective origin and various organisms including certain strains of Staph. aureus and Propionibacterium acnes have been suggested as possible causative organisms.
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The Gastrointestinal and Hepatobiliary Systems in HIV Infection
Donald P. Kotler, Pierre M. Gholam, in AIDS and Other Manifestations of HIV Infection (Fourth Edition), 2004
Bacterial Enteritides
Bacterial enteritides in AIDS have distinctive features. Infections with species of salmonella, shigella or campylobacter occur in HIV-infected patients, with or without AIDS (68). In one study, shigella infections tended to occur early in the disease course, while salmonella and campylobacter infections were more common in AIDS patients. It is unclear if the incidence is increased over the surrounding population, though enhanced susceptibility could be related to decreased gastric acid secretion, as noted above. The clinical presentation of salmonella infection may be reminiscent of the classical descriptions of typhoid fever (69).
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Immunodeficiency and Mucosal Immunity: An Overview
Lloyd Mayer, in Mucosal Immunology (Third Edition), 2005
Mucosal immunodeficiency does not necessarily result in an increase in common infections (with Shigella, Salmonella, and Campylobacter, for example); rather, the infections seen are with organisms that rarely cause disease in immunocompetent hosts. Many of these focus the attention on the role that the T cell plays in controlling infections. Organisms such as Cryptosporidium, Cyclospora, and Giardia require T cells for their clearance. Even in the absence of all antibodies, local mucosal infections are rare; the more common presentation is autoimmunity, chronic inflammation, or lymphoid malignancies. The common feature of these entities is that they reflect defects in immune regulation. Bacterial antigens appear to promote these responses, although direct evidence for this scenario is lacking in most immunodeficiency models (especially human models).
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General Pathology of HIV Infection
James L. Finley, ... Nancy L. Smith, in AIDS and Other Manifestations of HIV Infection (Fourth Edition), 2004
Other Bacteria
Infections with a variety of bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Neisseria meningitides, Pseudomonas aeruginosa, Salmonella, Shigella, Campylobacter, and Treponema pallidum are important causes of morbidity and mortality in HIV-infected children and adults (198–214). The multiplicity of immunologic defects of cellular and humoral types predisposes these patients to persistent infections and bacteremia (215). In addition, chemotherapeutic and antineoplastic agents used to treat opportunistic infections and neoplasms may cause neutropenia, further compromising the immune status. These infections often show atypical presentations including infections with more than one organism and a high relapse rate despite appropriate antibiotic therapy. Infections with Streptococcus pneumoniae and Haemophilus influenzae are particularly frequent in children with AIDS and the latter organism is responsible for about 10% of pneumonias in adult AIDS patients (198,202–203). Bacterial pneumonia is a very common pulmonary finding in AIDS patients at autopsy, with most cases caused by Pseudomonas aeruginosa, Staphylococcus aureus or Klebsiella pneumoniae (216). Important enteric pathogens in HIV-infected patients include Salmonella, Campylobacter, and Shigella. Salmonellosis may present initially as acute gastroenteritis or less commonly as bacteremia. The infections often have a severe clinical course with bacteremia in up to 45% of AIDS patients and show a tendency to relapse despite antibiotic therapy (200–201,203). Cultures of blood and stool usually confirm the diagnosis, but Salmonella may be recovered from many sources including brain, bone marrow, urine, and spleen. Campylobacter species have been recognized as important pathogens in the general population as well as in homosexual men and immunocompromised patients. Most infections in AIDS patients are associated with gastroenteritis while bacteremia and cholecystitis are infrequently reported (208,209). Shigella flexneri is the most common species isolated from AIDS patients with Shigella gastroenteritis (205–207). Bacteremia is infrequently seen and diagnosis is principally made by culture of stool.
A number of studies have suggested that HIV infection may be associated with rapid progression of secondary syphilis or tertiary neurosyphilis, even following appropriate antibiotic therapy (210,211). Unusual clinical presentations, atypical serologic responses after appropriate treatment and false-negative serologic tests, make the diagnosis of syphilis in HIV-infected patients difficult (212,214,217). Darkfield examination or direct fluorescent antibody staining of scrapings or exudates from suspicious primary lesions will help to establish the diagnosis even with negative rapid plasma reagin (RPR) or Venereal Disease Research Laboratory (VDRL) serologic studies. Silver stains such as the Steiner or Warthin-Starry can be performed on tissue biopsies but in the author's experience are difficult to interpret (218). Overall, the diagnosis of syphilis can be problematic in HIV-infected patients, and many clinicians will presumptively treat patients for early syphilis and closely monitor serial serologic tests to detect a delayed antibody response.
Bacterial infections are seen more commonly at autopsy than in biopsy specimens. Few pathologic studies, however, describe the microscopic features of pure bacterial enteric infections uncomplicated by the more common pathogens seen in AIDS patients. In typical enteric infections by most of these organisms, the initial phase is an invasion of the epithelium with propagation of the bacteria in epithelial cells followed by entry into the lamina propria. The subsequent reaction is acute and pyogenic with superficial mucosal necrosis and pseudomembrane formation beginning as a focal process and in severe infections becoming confluent. Organisms can be demonstrated within epithelial cells, lamina propria, and adherent membranes by Gram, Giemsa, and silver stains. Microbiologic culture will confirm the species of the enteropathic bacteria. This typical reaction pattern is modified by the host's ability to mount an immunologic response based on the degree of immune suppression. Dissemination to other organs will result in necrotizing lesions associated with scan inflammatory infiltrates and an abundance of organisms, particularly in neutropenic patients.
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Foodborne Pathogen Detection, Using Rapid Technologies
S.F. Al-Khaldi, ... S. Sharma, in Encyclopedia of Microbiology (Fourth Edition), 2014
Enzyme-Linked Immunosorbent Assay
In the past 30 years, ELISA is a commonly applied in vitro method for reliable and quantitative detection of bacteria (e.g., E. coli, Listeria, Staphylococcus aureus, Campylobacter, Vibrio cholerae, Bacillus cereus, Salmonella spp., and C. botulinum) and their toxins in food, water, and environmental and clinical samples (Singh et al., 2013; Banada and Bhunia, 2008). ELISA is a rapid, high-throughput, quantitative immunoassay that is based on an antigen–antibody interaction and detected by an enzyme producing a chromogenic or fluorescent signal. ELISAs are performed in different formats depending on the location of antigen or the antibody on the solid surface. In general, ELISAs are performed in three formats: competitive inhibition, indirect, and sandwich ELISAs (Figure 14).
Figure 14. Diagrammatic representation of ELISA formats: (a) competitive inhibition ELISA, (b) indirect ELISA, and (c) sandwich ELISA. S, substrate; CR, color reaction; E, enzyme; PA, primary antibody; SA, secondary antibody; CA, capture antibody; DA, detection antibody; Ag–Ab, antigen–antibody.
Competitive ELISA: the primary antibody is mixed in a separate tube with various dilutions of bacteria (or antigen/toxin) and added to the wells containing immobilized antigen. This allows only the free unbound antibody to bind to the immobilized antigen. A secondary antibody–enzyme conjugate and substrate system is added for color development. The highest dilution of cells showing the minimum reaction or equivalent to a background control is considered positive.
Indirect ELISA: the antigen is first immobilized in the wells of a microtiter plate and then incubated with the primary antibody solution. A secondary enzyme-tagged antibody is added to bind to the primary antibody and then the reaction is developed with an enzyme-specific substrate to produce color for the measurement. For a horseradish peroxidase (HRP)-tagged antibody, tetramethylbenzidine (TMB) is used to develop color. If a fluorescent molecule (e.g., fluorescein isothiocyanate, Cy5, Alexa-Fluor) is used, the reaction is quantified by the amount of fluorescence emitted.
Sandwich ELISA: this works similarly to indirect ELISA except that the microtiter plate is first coated with a capture antibody and then the antigen is added into the wells. The reaction is developed using the labeled detection or tracer antibody in either single step or in two steps with the help of an additional secondary conjugated antibody.
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