Laboratory diagnosis of Listeriosis caused by Listeria monocytogenes

Listeriosis is a serious infection caused by the bacterium Listeria monocytogenes. It can affect people of all ages, but it is especially dangerous for pregnant women, newborns, elderly people, and people with weakened immune systems. Listeriosis can cause septicemia, meningitis, encephalitis, abortion, stillbirth, and neonatal infection. The mortality rate of listeriosis is about 20-30%, making it one of the most lethal foodborne diseases.

Listeria monocytogenes is a gram-positive, facultative anaerobic rod that can grow at low temperatures (4°C) and high salt concentrations. It is widely distributed in the environment and can be found in soil, water, plants, animals, and food products. The main sources of human infection are contaminated dairy products, meat products, seafood, and fresh produce. Listeria can also be transmitted from mother to fetus through the placenta or from mother to newborn during delivery.

The laboratory diagnosis of listeriosis is based on the isolation and identification of Listeria monocytogenes from clinical specimens such as blood, cerebrospinal fluid (CSF), placenta, fetal tissues, or other normally sterile sites. However, the detection of Listeria monocytogenes can be challenging due to its low numbers in the specimens, its similarity to other gram-positive rods, and its slow growth on conventional media. Therefore, various techniques have been developed to enhance the sensitivity and specificity of the laboratory diagnosis of listeriosis. These techniques include microscopy, culture methods, cold enrichment technique, biochemical tests, carbohydrate fermentation tests, serology, and molecular methods. In this article, we will review these techniques and their applications in the laboratory diagnosis of listeriosis caused by Listeria monocytogenes.

The most common specimens used for the diagnosis of listeriosis are cerebrospinal fluid (CSF) and blood. These specimens are collected from patients who present with symptoms of meningitis, septicemia, or other invasive infections caused by Listeria monocytogenes. CSF is obtained by lumbar puncture and blood is drawn by venipuncture. The specimens should be transported to the laboratory as soon as possible and stored at 4°C until processing.

Other specimens that may be used for the diagnosis of listeriosis include placenta, amniotic fluid, meconium, lochia, fetal tissues, and breast milk. These specimens are collected from pregnant women or neonates who are suspected of having listeriosis or who have been exposed to a contaminated food source. Placenta and fetal tissues are obtained after delivery or abortion. Amniotic fluid and meconium are collected by amniocentesis or percutaneous umbilical blood sampling. Lochia is the vaginal discharge after childbirth. Breast milk is collected by manual expression or pumping. These specimens should also be transported to the laboratory as soon as possible and stored at 4°C until processing.

Other less common specimens that may be used for the diagnosis of listeriosis include urine, sputum, abscesses, wounds, joint fluid, peritoneal fluid, and cerebrospinal shunt fluid. These specimens are collected from patients who have localized infections or complications of listeriosis. Urine is collected by midstream clean catch or catheterization. Sputum is collected by expectoration or bronchoalveolar lavage. Abscesses, wounds, joint fluid, peritoneal fluid, and cerebrospinal shunt fluid are collected by aspiration or drainage. These specimens should be processed within 2 hours of collection or stored at 4°C until processing.

All specimens should be labeled with the patient`s name, identification number, date and time of collection, type of specimen, and clinical information. The laboratory should be notified of the suspicion of listeriosis and the relevant epidemiologic history of the patient. The laboratory should also follow the appropriate biosafety precautions when handling specimens that may contain Listeria monocytogenes.

Microscopy is a useful technique for the rapid and presumptive identification of Listeria monocytogenes in clinical specimens. However, it has some limitations and should be confirmed by culture and biochemical tests.

The most common microscopy technique used for detecting Listeria monocytogenes is the Gram stain. Gram stain is a differential staining method that distinguishes bacteria based on their cell wall structure and composition. Gram-positive bacteria have a thick layer of peptidoglycan that retains the purple dye, while gram-negative bacteria have a thin layer of peptidoglycan and an outer membrane that loses the purple dye and appears pink.

Listeria monocytogenes is a gram-positive bacterium that appears as short rods or coccobacilli under the microscope. It may be intracellular or extracellular, depending on the type of specimen and the stage of infection. For example, in cerebrospinal fluid (CSF) samples from patients with meningitis, Listeria monocytogenes may be seen inside or outside the white blood cells. In blood samples from patients with septicemia, Listeria monocytogenes may be seen in clusters or chains.

However, Gram stain alone is not sufficient to identify Listeria monocytogenes, as there are other gram-positive rods or coccobacilli that may resemble it, such as Corynebacterium, Bacillus, or Enterococcus. Therefore, additional microscopy techniques are needed to differentiate Listeria monocytogenes from other bacteria.

One such technique is the modified acid-fast stain. This stain is based on the ability of some bacteria to resist decolorization by acid-alcohol after being stained with a red dye. Listeria monocytogenes is partially acid-fast, meaning that it retains some of the red dye but appears lighter than fully acid-fast bacteria such as Mycobacterium. This can help to distinguish Listeria monocytogenes from non-acid-fast gram-positive rods or coccobacilli.

Another technique is the immunofluorescence assay. This assay uses fluorescent antibodies that bind specifically to antigens on the surface of Listeria monocytogenes. When viewed under a fluorescence microscope, Listeria monocytogenes appears as bright green dots against a dark background. This can help to confirm the presence of Listeria monocytogenes in specimens that are positive by Gram stain or modified acid-fast stain.

Microscopy techniques for detecting Listeria monocytogenes are useful for providing rapid and preliminary results, but they have some drawbacks. They require skilled personnel and equipment, they may have low sensitivity and specificity, and they may not detect low numbers of bacteria or mixed infections. Therefore, microscopy techniques should always be followed by culture and biochemical tests for definitive diagnosis of Listeriosis caused by Listeria monocytogenes.

Listeria monocytogenes is a facultative anaerobe that can grow on most conventional laboratory media, such as 5% sheep blood agar, chocolate agar, and brain-heart infusion broth. However, it may be difficult to isolate from specimens that are heavily contaminated with other bacteria, such as stool, food, or environmental samples. Therefore, selective and differential media are often used to enhance the recovery and identification of Listeria monocytogenes.

Some of the commonly used selective media for Listeria monocytogenes are:

• PALCAM agar: This medium contains polymyxin B, acriflavine, and lithium chloride to inhibit the growth of most gram-negative bacteria and some gram-positive bacteria. It also contains esculin and ferric ammonium citrate to differentiate Listeria monocytogenes from other Listeria species based on the production of black colonies due to esculin hydrolysis.
• Oxford agar: This medium contains colistin and nalidixic acid to inhibit the growth of most gram-negative bacteria and some gram-positive bacteria. It also contains lithium chloride and mannitol to differentiate Listeria monocytogenes from other Listeria species based on the production of yellow colonies due to mannitol fermentation.
• Modified Oxford agar: This medium is similar to Oxford agar but contains moxalactam instead of colistin to inhibit the growth of most gram-negative bacteria and some gram-positive bacteria. It also contains phenol red as a pH indicator to enhance the detection of mannitol fermentation by Listeria monocytogenes.
• Rapid`L.mono agar: This medium contains cefsulodin and cycloheximide to inhibit the growth of most gram-negative bacteria and fungi. It also contains esculin and ferric ammonium citrate to differentiate Listeria monocytogenes from other Listeria species based on the production of black colonies due to esculin hydrolysis. Additionally, it contains pyruvate and bromocresol purple to differentiate Listeria monocytogenes from other esculin-positive bacteria based on the production of turquoise colonies due to pyruvate utilization.

The selective media for Listeria monocytogenes are usually incubated at 35°C for 24 to 48 hours. However, some strains may require longer incubation or lower temperature (25°C) for optimal growth. Therefore, it is recommended to subculture any suspicious colonies onto non-selective media for further identification.

The identification of Listeria monocytogenes from culture can be confirmed by various biochemical tests, such as catalase, oxidase, indole, methyl red, Voges-Proskauer, citrate, urease, carbohydrate fermentation, CAMP test, motility, and arylesterase activity. Alternatively, molecular methods such as PCR or MALDI-TOF can be used for rapid and accurate identification of Listeria monocytogenes from culture.

Cold enrichment is a technique that enhances the recovery of Listeria monocytogenes from specimens that are heavily contaminated with other bacteria. It is based on the ability of Listeria to grow at low temperatures (4°C) while most other bacteria are inhibited.

The procedure involves inoculating the specimen into a selective broth medium, such as Fraser broth or University of Vermont (UVM) broth, and incubating it at 4°C for 1 to 4 weeks. The broth is then subcultured onto selective agar media, such as Oxford agar or PALCAM agar, and incubated at 35°C for 24 to 48 hours. The colonies that grow on the agar are examined for typical morphology and hemolysis of Listeria monocytogenes.

Cold enrichment has several advantages over direct plating of specimens onto selective agar media. It increases the sensitivity of detection by allowing the growth of small numbers of Listeria in the presence of competing flora. It also reduces the number of false-positive results by suppressing the growth of non-Listeria bacteria that may mimic Listeria on selective agar media.

However, cold enrichment also has some limitations and drawbacks. It is a time-consuming and labor-intensive technique that requires careful monitoring and subculturing of the broth. It may also increase the risk of contamination and cross-contamination during handling and storage of the specimens. Moreover, it may not be suitable for all types of specimens, such as those containing psychrotrophic bacteria that can also grow at low temperatures and interfere with the isolation of Listeria.

Therefore, cold enrichment should be used as a complementary method to direct plating, rather than as a substitute. It should be applied to specimens that are likely to contain low numbers of Listeria or high numbers of competing flora, such as food samples, environmental samples, or fecal samples. It should not be used for specimens that are expected to contain high numbers of Listeria or low numbers of competing flora, such as blood or cerebrospinal fluid. Cold enrichment should also be performed in conjunction with other methods, such as microscopy, culture, biochemical tests, and molecular methods, to confirm the identification and characterization of Listeria monocytogenes.

Biochemical tests are used to confirm the identity of Listeria monocytogenes after preliminary screening by microscopy and culture methods. These tests are based on the metabolic and enzymatic characteristics of the bacteria. Some of the commonly used biochemical tests for Listeria monocytogenes are:

• Catalase test: This test detects the presence of catalase enzyme, which breaks down hydrogen peroxide into water and oxygen. Listeria monocytogenes is catalase-positive, meaning that it produces bubbles when exposed to hydrogen peroxide. This test helps to differentiate Listeria from other gram-positive bacteria that are catalase-negative, such as Streptococcus and Enterococcus.
• Oxidase test: This test detects the presence of cytochrome c oxidase enzyme, which transfers electrons from a donor to oxygen. Listeria monocytogenes is oxidase-negative, meaning that it does not change the color of a paper strip impregnated with a chemical indicator when exposed to oxygen. This test helps to differentiate Listeria from other gram-negative bacteria that are oxidase-positive, such as Pseudomonas and Neisseria.
• Indole test: This test detects the presence of indole, which is a breakdown product of tryptophan by tryptophanase enzyme. Listeria monocytogenes is indole-negative, meaning that it does not produce a red color when mixed with Kovac`s reagent. This test helps to differentiate Listeria from other gram-positive rods that are indole-positive, such as Erysipelothrix and Lactobacillus.
• Methyl red test: This test detects the presence of mixed acid fermentation, which lowers the pH of the medium. Listeria monocytogenes is methyl red-positive, meaning that it turns the medium red when added with methyl red indicator. This test helps to differentiate Listeria from other gram-positive rods that are methyl red-negative, such as Corynebacterium and Bacillus.
• Voges-Proskauer test: This test detects the presence of 2,3-butanediol fermentation, which produces acetoin as an intermediate. Listeria monocytogenes is Voges-Proskauer-negative, meaning that it does not turn the medium pink when added with Barritt`s reagent. This test helps to differentiate Listeria from other gram-positive rods that are Voges-Proskauer-positive, such as Enterobacter and Klebsiella.
• Citrate test: This test detects the ability of bacteria to use citrate as a sole carbon source. Listeria monocytogenes is citrate-negative, meaning that it does not grow on a medium containing sodium citrate and bromothymol blue indicator. This test helps to differentiate Listeria from other gram-positive rods that are citrate-positive, such as Aerococcus and Gardnerella.
• Urease test: This test detects the presence of urease enzyme, which hydrolyzes urea into ammonia and carbon dioxide. Listeria monocytogenes is urease-negative, meaning that it does not change the color of a medium containing urea and phenol red indicator. This test helps to differentiate Listeria from other gram-positive rods that are urease-positive, such as Proteus and Staphylococcus.

These biochemical tests can be performed individually or in combination using commercial kits or automated systems. The results can be interpreted by comparing them with standard reference charts or databases. Biochemical tests can provide rapid and reliable identification of Listeria monocytogenes in clinical and food samples.

Another way to identify L. monocytogenes from other Listeria species is to perform carbohydrate fermentation tests. These tests are based on the ability of bacteria to ferment different sugars and produce acid or gas as a result. The fermentation of sugars can be detected by using specific media that change color or pH when acid is produced, or by using Durham tubes that trap gas bubbles inside.

L. monocytogenes can be differentiated from other Listeria species by its ability to ferment rhamnose and methyl α-D-mannopyranoside, but not mannitol and xylose. These sugars can be tested individually or in combination using commercially available media such as RAPID`L.mono (Bio-Rad), which contains 10 different sugars and allows the identification of L. monocytogenes within 48 hours.

The carbohydrate fermentation tests are simple and inexpensive, but they may not be very reliable for some strains of L. monocytogenes that show atypical or variable reactions. Therefore, it is recommended to confirm the results with other biochemical or molecular tests.

Serology and molecular methods are used for epidemiologic investigations of listeriosis outbreaks and sources. Serology is based on the detection of specific antibodies against Listeria antigens in serum samples from infected patients or animals. Molecular methods are based on the analysis of DNA or RNA sequences from Listeria isolates or samples.

Serologic classification is done only in reference laboratories and is primarily used for epidemiologic studies. There are 13 known serovars based on O (somatic) and H (flagellar) antigens. Serotypes 1/2a, 1/2b, and 4b make up more than 95% of the isolates from humans and are responsible for most infections in neonates and adults. Serotype 4b causes most of the foodborne outbreaks.

Molecular methods can provide more accurate and detailed information on the genetic relationships between Listeria strains and their sources. These methods include pulsed-field gel electrophoresis (PFGE), which is the most commonly used technique for epidemiologic investigations of suspected outbreaks. PFGE generates DNA fingerprints by cutting the bacterial genome with restriction enzymes and separating the fragments by electrophoresis. Other molecular methods include multilocus sequence typing (MLST), which compares the sequences of several housekeeping genes; multilocus variable-number tandem-repeat analysis (MLVA), which measures the number of repeats in specific genomic regions; and whole-genome sequencing (WGS), which determines the complete DNA sequence of an isolate. These methods can reveal the genetic diversity, evolution, and transmission patterns of Listeria strains and help to identify outbreak clusters, sources, and risk factors.

Serology and molecular methods are complementary tools for the surveillance and control of listeriosis. They can help to improve the diagnosis, prevention, and treatment of this serious infection.

Listeriosis is a serious infection that requires prompt medical attention. The treatment of listeriosis depends on the severity of the symptoms and the risk factors of the patient.

Most people with mild symptoms of listeriosis, such as fever, headache, and diarrhea, do not need antibiotic treatment and recover on their own. However, they should drink plenty of fluids to prevent dehydration and monitor their symptoms closely.

People with severe symptoms of listeriosis, such as confusion, loss of balance, convulsions, or signs of infection in the blood or brain, need urgent antibiotic treatment. The most commonly used antibiotics for listeriosis are ampicillin and gentamicin, which are given intravenously (through a vein) in a hospital setting. These antibiotics can kill the bacteria and clear the infection. Sometimes, other antibiotics such as trimethoprim-sulfamethoxazole, penicillin, or vancomycin may be used if the patient is allergic to ampicillin or gentamicin or if the bacteria are resistant to these drugs.

People who have a greater chance of developing severe listeriosis, such as pregnant people, older adults, and people with weakened immune systems, should also receive antibiotic treatment as soon as possible, even if they have mild symptoms. This is because listeriosis can cause serious complications for these groups, such as miscarriage, stillbirth, premature delivery, neonatal infection, meningitis, encephalitis, endocarditis, or death. Antibiotic treatment can reduce the risk of these outcomes and improve the prognosis of listeriosis.

The duration of antibiotic treatment for listeriosis varies depending on the type and extent of the infection. Generally, it ranges from 10 to 21 days for non-pregnant adults and 4 to 6 weeks for pregnant people and newborns. The doctor will monitor the patient`s response to the treatment and adjust it accordingly.

In addition to antibiotic treatment, supportive care may be needed for some patients with listeriosis. This may include fluid and electrolyte replacement, pain relief, anti-inflammatory drugs, anticonvulsants, or surgery.

Listeriosis is a preventable disease that can be avoided by following proper food safety practices. These include washing hands and utensils before handling food, cooking food thoroughly, avoiding cross-contamination between raw and cooked foods, refrigerating food promptly, discarding expired or spoiled food, and avoiding high-risk foods such as unpasteurized dairy products, deli meats, hot dogs, fresh vegetables, and fresh fruits. By taking these precautions, one can reduce the exposure to Listeria monocytogenes and prevent listeriosis.

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