Laboratory diagnosis of Syphilis caused by Treponema pallidum

Syphilis is a sexually transmitted disease (STD) caused by the spirochete Treponema pallidum. It can have various and often subtle clinical manifestations, depending on the stage of infection and the host immune status. If left untreated, syphilis can cause serious complications, such as cardiovascular disease, neurosyphilis, or congenital syphilis.

Laboratory diagnosis of syphilis is essential for confirming the infection, monitoring the treatment response, and preventing further transmission. However, laboratory diagnosis of syphilis can be challenging because of the following reasons:

• T. pallidum cannot be cultured in vitro and requires special techniques for direct detection.
• The clinical signs and symptoms of syphilis can mimic other diseases or be absent in some cases.
• The immune response to T. pallidum varies among individuals and stages of infection, and can be influenced by factors such as HIV co-infection or previous treatment.
• The interpretation of serologic tests for syphilis can be complex and requires correlation with clinical and epidemiologic data.

Therefore, laboratory diagnosis of syphilis should involve a combination of direct and indirect methods, as well as a careful evaluation of the test results in the context of the patient`s history and physical examination.

Direct methods for diagnosing syphilis include the detection of T. pallidum by microscopic examination of fluid or smears from lesions, histological examination of tissues, or nucleic acid amplification methods such as polymerase chain reaction (PCR). These methods are useful for diagnosing primary, secondary, or congenital syphilis, when active lesions are present. However, they have limitations such as low sensitivity, high cost, technical difficulty, or limited availability.

Indirect methods for diagnosing syphilis include serologic tests that measure the presence of antibodies to T. pallidum or its components. Serologic tests for syphilis fall into two categories: non-treponemal tests and treponemal tests. Non-treponemal tests measure antibodies that are produced against lipids released from damaged cells during infection. These antibodies are not specific for T. pallidum and can also be found in other conditions such as pregnancy, autoimmune diseases, or viral infections. Non-treponemal tests are useful for screening and monitoring the treatment response, as they reflect the disease activity and their titers decline after successful therapy. Examples of non-treponemal tests are the Venereal Disease Research Laboratory (VDRL) test and the Rapid Plasma Reagin (RPR) test. Treponemal tests measure antibodies that are specific for T. pallidum antigens. These antibodies persist for life after infection and do not indicate the disease activity or the treatment response. Treponemal tests are useful for confirming the diagnosis of syphilis and ruling out false-positive results from non-treponemal tests. Examples of treponemal tests are the Treponema pallidum Hemagglutination Assay (TPHA), the Fluorescent Treponemal Antibody Absorption (FTA-ABS) test, the Treponema pallidum Particle Agglutination (TPPA) test, and various enzyme-linked immunosorbent assays (ELISAs).

The recommended algorithm for laboratory diagnosis of syphilis is to perform a non-treponemal test first, followed by a treponemal test if the non-treponemal test is positive. This approach allows for a high sensitivity and specificity, as well as a cost-effective use of resources. However, some situations may require a different testing strategy, such as testing for neurosyphilis, congenital syphilis, or early syphilis. In these cases, additional tests such as cerebrospinal fluid (CSF) analysis, PCR, or dark-field microscopy may be needed.

In summary, laboratory diagnosis of syphilis is an important component of managing this disease. It requires a combination of direct and indirect methods that should be interpreted in conjunction with clinical and epidemiologic data. The choice of tests depends on the stage of infection, the availability of resources, and the patient`s characteristics.

The quality and reliability of laboratory diagnosis of syphilis depend largely on the proper collection and handling of specimens. Different types of specimens may be required for different diagnostic methods, such as microscopy, nucleic acid amplification tests (NAATs), culture, or serology. Here are some general guidelines for specimen collection and handling for syphilis diagnosis:

• For microscopy techniques, such as darkfield microscopy or direct fluorescent antibody test (DFA-TP), the specimen should be obtained from an active lesion, such as a chancre, mucous patch, or condyloma latum . The lesion should be cleaned with saline or sterile water to remove any blood, pus, or debris that may interfere with the visualization of T. pallidum. A sterile swab or needle should be used to gently express the serous fluid from the base of the lesion and collect it on a glass slide. The slide should be examined as soon as possible under a microscope, preferably within 20 minutes of collection. If immediate examination is not possible, the slide can be stored in a moist chamber at room temperature for up to 2 hours.
• For NAATs, such as polymerase chain reaction (PCR), the specimen should also be collected from an active lesion using a sterile swab. The swab should be placed in a cryotube containing nucleic acid transport medium or universal transport medium . The specimen should be stored at 4°C and transported to the laboratory within 24 hours. If longer storage is required, the specimen should be frozen at -20°C or -70°C.
• For culture, which is rarely performed due to the difficulty of growing T. pallidum in vitro, the specimen should be collected from an active lesion using a sterile swab or needle aspirate. The specimen should be inoculated into a suitable animal model, such as a rabbit, within 4 hours of collection. Alternatively, tissue biopsies or needle aspirates of lymph nodes can be used for culture. These specimens should be placed in 10% buffered formalin at room temperature.
• For congenital syphilis, a small section of the umbilical cord can be collected and fixed in formalin or refrigerated until processed for microscopy, NAATs, or culture. Additionally, blood samples from the infant and the mother should be collected for serology.
• For serology, which is the most widely used method for syphilis diagnosis, serum is the specimen of choice . Blood should be collected in an anticoagulant-free tube or a serum separator tube (SST) using standard venipuncture technique . Plasma can also be used for serology if collected in an EDTA tube and tested within 48 hours of collection. The tube should be labeled with the patient`s full name, date and time of collection, and a unique identifier to match the requisition form . The specimen should be allowed to clot and then centrifuged to separate the serum from the cells . The serum should be stored at 4°C and transported to the laboratory within 5 days of collection . If longer storage is required, the serum should be frozen at -20°C or -70°C .
• For cerebrospinal fluid (CSF), which is used for diagnosing neurosyphilis, a lumbar puncture should be performed using aseptic technique and collecting at least 10 mL of CSF in sterile tubes . The tubes should be labeled with the patient`s full name, date and time of collection, and a unique identifier to match the requisition form. The CSF should be stored at 4°C and transported to the laboratory within 24 hours of collection. If longer storage is required, the CSF should be frozen at -20°C or -70°C.

Specimens collected for syphilis diagnosis should be handled with care and appropriate biosafety precautions. Syphilis is a highly infectious disease that can cause serious complications if left untreated. Therefore, timely and accurate diagnosis is essential for effective treatment and prevention.

T. pallidum is a thin, spiral-shaped bacterium that cannot be seen by conventional light microscopy. Therefore, special microscopy techniques are required to visualize the spirochetes directly from clinical specimens, such as ulcer exudates or tissue biopsies. These techniques include:

• Dark-field microscopy (DFM): This method uses a dark-field condenser to illuminate the specimen from the side, creating a dark background and a bright image of the spirochetes. DFM is a simple, rapid, and inexpensive technique that can be performed at the point of care. However, it requires fresh specimens, experienced microscopists, and careful interpretation to avoid false-positive results due to artifacts or other microorganisms .
• Direct fluorescent antibody (DFA) test: This method uses fluorescent-labeled antibodies that bind specifically to T. pallidum antigens and emit light when viewed under a fluorescence microscope. DFA is more sensitive and specific than DFM and can be used on fixed specimens. However, it is more expensive, requires specialized equipment and reagents, and may be affected by background fluorescence or cross-reactivity .
• Silver staining: This method uses silver nitrate or silver proteinate to stain the spirochetes in tissue sections or smears. Silver staining can detect T. pallidum in lesions that are negative by DFM or DFA, such as gummas or cardiovascular syphilis. However, it is less sensitive and specific than other methods and requires skilled histopathologists and adequate tissue fixation .
• Immunohistochemistry (IHC): This method uses enzyme-labeled antibodies that react with T. pallidum antigens and produce a colored precipitate that can be seen under a light microscope. IHC is more sensitive and specific than silver staining and can detect T. pallidum in various tissues and stages of syphilis. However, it is also more expensive, requires specialized equipment and reagents, and may be affected by tissue processing or nonspecific staining .

Microscopy techniques for detecting T. pallidum are useful for the diagnosis of primary, secondary, or congenital syphilis, especially when serological tests are negative or inconclusive. However, they have some limitations, such as low sensitivity in late syphilis, low specificity due to cross-reactivity or artifacts, high cost and complexity of some methods, and dependence on specimen quality and availability . Therefore, microscopy techniques should be used in conjunction with clinical evaluation and serological tests for a comprehensive diagnosis of syphilis.

One of the major challenges in the laboratory diagnosis of syphilis is the inability to culture T. pallidum in vitro. Unlike most bacteria, the organism that causes syphilis cannot be grown in artificial media or cell cultures . This limitation hinders the research on T. pallidum and syphilis, as well as the development of new diagnostic methods and vaccines .

The reasons why T. pallidum is difficult to culture are not fully understood, but some possible factors include its complex nutritional requirements, its susceptibility to oxygen and oxidative stress, its slow growth rate, and its low infectivity . Some attempts have been made to culture T. pallidum in various media, such as rabbit testicular tissue, embryonated eggs, tissue explants, and organ cultures, but none of these methods have been successful in maintaining long-term viability and multiplication of the spirochete .

Therefore, the diagnosis of syphilis relies mainly on direct detection of T. pallidum by microscopy or molecular techniques, or indirect detection of antibodies by serological tests. However, these methods also have some limitations, such as low sensitivity, specificity, availability, or cost-effectiveness. Thus, there is a need for developing new and improved methods for culturing T. pallidum in vitro, which would facilitate the understanding of its biology, pathogenesis, and immunology, as well as the discovery of novel diagnostic markers and vaccine candidates .

Nucleic acid–based tests (NATs) are molecular methods that can directly detect the DNA or RNA of T. pallidum in clinical specimens, such as genital lesions, blood, or cerebrospinal fluid (CSF). NATs can provide a rapid and specific diagnosis of syphilis, especially in cases where serology is inconclusive or microscopy is not available. However, NATs are not widely available and are mostly used in research or reference laboratories.

The most common type of NAT for syphilis is the polymerase chain reaction (PCR), which amplifies a specific target sequence of T. pallidum DNA using primers and probes. Different genes have been targeted by PCR assays for syphilis, such as polA, tpp47, bmp, tpn47, and 16S rRNA . The sensitivity and specificity of PCR tests vary depending on the gene target, the specimen type, and the PCR method (conventional or real-time). Generally, PCR tests have higher sensitivity in primary lesion exudate (75-95%) than in blood (30-70%) or CSF (20-60%). PCR tests can also be used to differentiate T. pallidum subspecies and strains by sequencing or restriction fragment length polymorphism analysis.

Other types of NATs for syphilis include transcription-mediated amplification (TMA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). These methods have similar principles as PCR but use different enzymes or mechanisms to amplify T. pallidum nucleic acids. Some of these methods have advantages over PCR, such as faster turnaround time, lower cost, or simpler equipment. However, these methods are less well-studied and validated than PCR for syphilis diagnosis.

NATs for syphilis have some limitations that should be considered before use. First, NATs may not detect all cases of syphilis, especially in late stages or after treatment, when the bacterial load is low or absent. Therefore, NATs should not replace serology as the main diagnostic tool for syphilis. Second, NATs may yield false-positive results due to contamination or cross-reaction with other treponemal species. Therefore, NATs should be performed and interpreted by trained personnel in quality-controlled laboratories. Third, NATs may not be standardized or regulated across different laboratories or regions. Therefore, NATs should be validated and calibrated with reference methods and materials before use.

NATs for syphilis are promising techniques that can enhance the diagnosis and management of this infection. However, more research and development are needed to improve their availability, performance, and utility in clinical practice.

Serology tests are blood tests that measure the presence of antibodies against T. pallidum, the bacteria that causes syphilis. There are two types of serology tests: non-treponemal and treponemal.

Non-treponemal tests detect antibodies that are produced in response to lipids released from damaged cells during syphilis infection. These antibodies are not specific to T. pallidum and can also be found in other conditions, such as viral infections, autoimmune diseases, or pregnancy. Therefore, non-treponemal tests are used as screening tests for syphilis and to monitor the response to treatment. However, they cannot distinguish between active and past infection, and they may give false-negative results in early or late stages of syphilis.

The most common non-treponemal tests are the Venereal Disease Research Laboratory (VDRL) test and the Rapid Plasma Reagin (RPR) test. Both tests use cardiolipin, a lipid derived from beef heart, as the antigen to detect antibodies in the serum. The VDRL test is performed on a slide or in a tube, and the RPR test is performed on a card. Both tests rely on visual observation of flocculation (clumping) of the antigen-antibody complex. A positive result indicates the presence of non-treponemal antibodies, but it does not confirm syphilis infection. A negative result may indicate the absence of infection or a false-negative due to low antibody levels or prozone effect (high antibody levels that interfere with flocculation).

Other non-treponemal tests include the Toluidine Red Unheated Serum Test (TRUST) and the Unheated Serum Reagin (USR) test. These tests are similar to the VDRL test but use different stabilizers and inhibitors to enhance sensitivity and specificity.

Treponemal tests detect antibodies that are specific to T. pallidum antigens. These antibodies persist for life even after successful treatment of syphilis, so they cannot be used to monitor treatment response or to differentiate between active and past infection. However, they can confirm syphilis infection in patients who have positive non-treponemal tests or who have clinical signs of syphilis.

The most common treponemal tests are the Treponema pallidum hemagglutination assay (TPHA), the Treponema pallidum particle agglutination (TPPA) assay, and the fluorescent treponemal antibody absorption (FTA-ABS) test. The TPHA and TPPA tests use red blood cells or gelatin particles coated with T. pallidum antigens to detect antibodies in the serum by hemagglutination (clumping of red blood cells) or particle agglutination (clumping of gelatin particles). The FTA-ABS test uses killed T. pallidum organisms fixed on a slide as antigens and fluorescein-labeled anti-human antibodies as markers to detect antibodies in the serum by fluorescence microscopy. A positive result in any of these tests indicates the presence of treponemal antibodies and confirms syphilis infection.

Other treponemal tests include the microhemagglutination Treponema pallidum (MHA-TP) test, which uses sheep erythrocytes coated with T. pallidum antigens; the enzyme immunoassays (EIA), which use synthetic or recombinant T. pallidum antigens; and the Western blot, which uses purified T. pallidum proteins separated by electrophoresis.

A group-specific test that detects antibodies against a protein antigen common to all treponemes is the Reiter`s protein complement fixation test (RP-CFT). However, this test has low sensitivity and is rarely used.

The interpretation of serology tests for syphilis depends on several factors, such as the stage of infection, the history of previous infection or treatment, and the presence of co-infections or cross-reacting conditions. In general, a positive non-treponemal test followed by a positive treponemal test is considered diagnostic of syphilis infection. A negative non-treponemal test followed by a negative treponemal test is considered indicative of no infection or very early infection. A negative non-treponemal test followed by a positive treponemal test is considered indicative of past treated infection or latent infection. A positive non-treponemal test followed by a negative treponemal test is considered indicative of a false-positive non-treponemal test due to other causes or a prozone effect.

Non-treponemal tests are blood tests that measure the presence of antibodies that are produced by the host in response to the cellular damage caused by T. pallidum infection. These antibodies are not specific for T. pallidum, but react with lipids (cardiolipin) that are released from damaged host cells or from the surface of the bacteria.

Non-treponemal tests are used as screening tests for syphilis, as they are rapid, simple, and inexpensive. However, they have some limitations, such as:

• Decreased sensitivity in early primary syphilis and late syphilis
• False-positive reactions due to other infections or conditions
• False-negative reactions due to high antibody titers (prozone effect)
• Need for laboratory equipment and trained personnel

Non-treponemal tests should always be confirmed by a treponemal test, which is more specific for T. pallidum antibodies.

The main non-treponemal tests are:

• VDRL (Venereal Disease Research Laboratory) test: This test uses a cardiolipin antigen that is mixed with heat-inactivated serum in a tube or slide. If antibodies are present, they form visible clumps (flocculation) with the antigen. The test can be performed on serum or cerebrospinal fluid (CSF).
• RPR (Rapid Plasma Reagin) test: This test is similar to the VDRL test, but uses a modified cardiolipin antigen that contains choline chloride (to prevent complement interference) and charcoal particles (to enhance visibility). The test is performed on unheated serum on a slide or card.
• TRUST (Toluidine Red Unheated Serum Test): This test is identical to the VDRL test, but uses a toluidine red pigment instead of charcoal to improve readability. The test is performed on unheated serum on a card.
• USR (Unheated Serum Reagin) test: This test is similar to the VDRL test, but uses EDTA as an antigen stabilizer and choline chloride as an inhibitor of non-specific reactions. The test is performed on unheated serum on a slide or card.

Non-treponemal tests can also provide a quantitative measure of antibody titers, which can be used to monitor the disease activity and treatment response. A fourfold change in titer is considered significant. However, non-treponemal titers can remain positive for months or years after successful treatment, or become negative spontaneously in untreated patients. Therefore, non-treponemal tests cannot be used to diagnose reinfection or relapse.

Treponemal antibody tests detect antibodies that are specific for syphilis. These tests include TP-PA, various EIAs, chemiluminescence immunoassays, immunoblots, and rapid treponemal assays. Treponemal antibodies appear earlier than nontreponemal antibodies. They usually remain detectable for life, even after successful treatment. Therefore, treponemal antibody tests are useful for confirming a syphilis diagnosis, but not for monitoring the response to therapy or detecting reinfection.

Some of the commonly used treponemal antibody tests are:

• Microhemagglutination Treponema pallidum (MHA-TP) test: In this test, heated serum is mixed with a sonicated non-pathogenic Reiter strain and incubated with sheep erythrocytes coated with sonicated Treponema pallidum Nichols strain in a microtiter plate. If antibodies to Treponema pallidum are present, they react with the sensitized erythrocytes to produce a uniformly thin mat of agglutinated cells that covers the entire bottom of the well.
• Treponema pallidum hemagglutination assay (TPHA): This test is similar to MHA-TP, but uses unheated serum and unsonicated T. pallidum antigen. TPHA is more sensitive and specific than MHA-TP.
• Treponema pallidum–particle agglutination (TPPA): This test works on the same principle as TPHA, but uses colored gelatin particles instead of red blood cells as the carrier of T. pallidum antigen. TPPA is more stable and easier to read than TPHA.
• Fluorescent treponemal antibody absorbed (FTA-ABS): This test uses patient serum that has been absorbed with a non-pathogenic treponeme to remove non-specific antibodies. The absorbed serum is then layered on a slide coated with killed T. pallidum and incubated with fluorescein-conjugated antihuman antibody reagent. The slide is examined under a fluorescent microscope for the presence of apple-green fluorescent spirochetes.
• Enzyme immunoassays (EIAs): These tests use enzyme-labeled antigens or antibodies to detect treponemal antibodies in serum or plasma samples. EIAs are highly sensitive and specific, and can be automated and standardized.
• Western blot: This test separates the antigens of T. pallidum by electrophoresis and transfers them to a nitrocellulose membrane. The membrane is then incubated with patient serum and enzyme-labeled antihuman antibody reagent. The presence of treponemal antibodies is indicated by bands corresponding to specific antigens on the membrane.
• Rapid treponemal assays: These tests are point-of-care tests that use immunochromatographic methods to detect treponemal antibodies in whole blood, serum, or plasma samples. They are simple, rapid, and inexpensive, but less sensitive and specific than laboratory-based tests.

Syphilis is a curable bacterial infection that can be treated with antibiotics. The type and duration of treatment depend on the stage and severity of the infection, as well as the patient`s medical history and allergies .

The first-line recommended treatment for syphilis is penicillin, an antibiotic that can kill the bacteria that cause syphilis. Penicillin can be given as a single injection of long-acting benzathine penicillin G for primary, secondary, or early latent syphilis (infection within the last year), or as three doses of long-acting benzathine penicillin G at weekly intervals for late latent syphilis or syphilis of unknown duration. Penicillin is also the only effective treatment for pregnant women and babies with congenital syphilis, and for patients with neurosyphilis (infection of the central nervous system) .

Patients who are allergic to penicillin can be treated with alternative antibiotics, such as doxycycline, tetracycline, erythromycin, or ceftriaxone . However, these antibiotics are not recommended for pregnant women or patients with neurosyphilis, who should undergo penicillin desensitization if necessary .

Treatment will cure the infection and prevent further damage, but it will not repair damage already done by the bacteria. Therefore, it is important to diagnose and treat syphilis as early as possible. Patients should also avoid sexual contact with new partners until their sores completely heal, and notify their previous sexual partners so they can receive testing and treatment if needed. Patients should also have regular follow-up blood tests to monitor their response to treatment and check for possible reinfection .

Syphilis is a sexually transmitted infection (STI) that can cause serious health problems without treatment. The best way to prevent syphilis is to practice safer sex and get tested regularly for syphilis and other STIs. Some of the prevention and control measures for syphilis are:

• Avoid sexual contact with infected persons. Syphilis is spread by direct contact with a syphilis sore during vaginal, anal, or oral sex. Syphilis can also be passed from a mother with syphilis to her unborn baby during pregnancy or childbirth.
• Use a dental dam or condom in every sexual encounter. Condoms prevent the spread of syphilis by preventing contact with a sore. Sometimes sores occur in areas not covered by a condom. Contact with these sores can still transmit syphilis.
• Avoid multiple sexual partners. Having more than one sexual partner increases the risk of getting syphilis and other STIs.
• Avoid sharing needles. Sharing needles with someone who has syphilis can also transmit the infection.
• Get tested regularly for syphilis and other STIs. Testing is the only way to know for sure if you have syphilis or not. You should get tested regularly if you are sexually active and are a gay or bisexual man; have HIV; are taking pre-exposure prophylaxis (PrEP) for HIV prevention; or have partner(s) who have tested positive for syphilis. All pregnant people should receive syphilis testing at their first prenatal visit and again during the third trimester and at delivery.
• Get treated right away if you test positive for syphilis. Syphilis is easily cured with antibiotics, usually penicillin. Treatment can stop the infection from progressing and prevent complications. You should also inform your past and present sexual partners that you have syphilis, so they can get tested and treated too. Do not have sex with anyone until you have completed your treatment and your syphilis sores are completely healed.
• Educate yourself and others about syphilis. Learn about the signs and symptoms of syphilis, how it is transmitted, how it can be prevented, and how it can be treated. Share this information with your sexual partners, friends, family, and community. You can also find reliable sources of information online, such as the World Health Organization (WHO), the Centers for Disease Control and Prevention (CDC), or Planned Parenthood .

Syphilis is an old disease that can still cause serious health problems today. However, it can be prevented and controlled with safer sex practices, regular testing, and prompt treatment. By following these measures, you can protect yourself and others from this infection.

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