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lab diagnosis of STD

1-neisseria gonorrhoeae

microscopy

a direct smear for gram staining may be performed as soon as the swab specimen is collected from the urethra, cervix, vagina or rectum. the swab should be rolled gently onto the slide to preserve cellular morphology and over an area less than 1 cm² (1). gram staining kits are commercially available. under oil immersion (1000x magnification), urethral smears from symptomatic males usually contain intracellular gram-negative kidney-shaped diplococci in polymorphonuclear leukocytes, the presence of which is required for the presumptive diagnosis of gonorrhea (5). the presence of extracellular gram-negative diplococci is an equivocal finding that must be confirmed by culture (1,5) or nucleic acid test. the endocervical smears from females or rectal specimens are more difficult to interpret due to the presence of other gram-negative coccobacilli, including moraxella osloensis, moraxella phenylpyruvica, kingella denitrificans and acinetobacter species (7). the sensitivity and specificity for urethral smears is 90% to 95%, respectively, versus 50% to 70% sensitivity and over 90% specificity for endocervical smears (1,8). however, the reliability of microscopic tests depends on the quality of the specimen and the experience of the microscopist (1).

gram stain of a pustular exudate.                                                                                                                                                                                    neisseria gonorrhoeaeneisseria gonorrhoeae is a gram-negative coccus, 0.6 to 1.0 µm in diameter, usually seen in pairs with adjacent flattened sides (figure 1 left). the organism is frequently found intracellularly in polymorphonuclear leukocytes (neutrophils) of the gonorrhea pustular exudates (figure 1 right). fimbriae, which play a major role in adherence, extend several micrometers from the cell surface (figure 2 below)                                                                                figure 2. neisseria gonorrhoeae (kay chernush)

culture

the current preferred laboratory method for the diagnosis of n gonorrhoeae infections is the isolation and identification of the agent (figure ?(figure1).1). culturing isolates is important for antimicrobial susceptibility testing, surveillance purposes, detecting treatment failure and characterizing outbreaks.

table 1

methods of collection of clinical specimens for the laboratory diagnosis of gonorrhea

media and cultural conditions for isolation:

the primary specimens should be inoculated onto nonselective chocolate agar and selective agar containing antimicrobial agents that inhibit the growth of commensal bacteria and fungi. the antibacterial agents in modified thayer-martin, martin lewis and new york city medium are vancomycin, colistin, trimethoprim lactate and the antifungal agents nystatin and anisomycin or amphotericin b

the inoculated plates should be incubated at 35°c to 37°c in a moist atmosphere enriched with co₂ (3% to 7%)   .

presumptive identification of n gonorrhoeae:

the presumptive identification of n gonorrhoeae rests on the isolation of an oxidase-positive, gram-negative diplococcus recovered from urogenital sites that grows on selective media (5) (figure ?(figure1).1). the gram stain of a smear of urethral exudates or endocervical secretions shows typical gram-negative intracellular diplococci. the oxidase test uses the tetramethyl derivative of the oxidase reagent (1% aqueous solution of n, n, n, n-tetramethyl-1, 4-phenylenediamine) that is commercially available (bactidroping oxidase, remel inc, usa) or prepared in-house. to perform the test, a droping of reagent is applied to filter paper or the tip of a cotton swab. culture is then applied to the filter paper or cotton swab tip using a platinum or plastic loop, wooden applicator stick or toothpick. a dark-purple colour change within 10 s indicates a positive sample (5). the catalase test (3% hydrogen peroxide) or superoxol (30% hydrogen peroxide) are other rapid tests used in the presumptive identification of n gonorrhoeae. a droping of the reagent is placed in the centre of a clean glass slide and the suspect colony is picked with a loop and emulsified in the reagent. n gonorrhoeae will produce a positive reaction with bubbling within 1 s to 2 s. weak bubbling or bubbling after 3 s indicates a negative reaction (5) (table ?(table2).2). the reagents are tested daily against reference oxidase-positive and -negative cultures to ensure quality.

table 2

supplemental tests that permit differentiation between neisseria gonorrhoeae and related species

^*+ most strains positive

^†– most strains negative. data from reference 5

confirmatory identification tests:

several methods are available to confirm the identification of n gonorrhoeae, including biochemical testing, serological testing, colourimetric testing and nucleic acid methods. more than one system may be required to confirm identification.

biochemical tests:

n gonorrhoeae can be differentiated from other neisseria species, moraxella species, kingella species and other commensals based on its ability to grow on appropriate selective and nonselective media, produce acid from glucose, maltose, lactose, sucrose and fructose, reduce nitrate, produce polysaccharide from sucrose and exhibit dnase production (table ?(table2).

chromogenic enzyme substrate :

these tests are based on the presence of preformed chromogenic enzyme in the culture and, thus, require a heavy inoculum of the organism grown on selective medium to permit rapid speciation of isolates. the enzymes that are detected by these systems include beta-galactosidase, gamma-glutamylaminopeptidase and prolyl-hydroxyprolyl aminopeptidase. many of these methods are commercially available

fluorescent antibody test: the fluorescein isothiocyanate-labelled monoclonal antibody in the microtrak n gonorrhoeae culture confirmation test (trinity biotech plc, ireland) reacts specifically with n gonorrhoeae. the antibody binds specifically to both proteins ia and ib (porins, or outer membrane proteins). when viewed under a fluorescence microscope, cultures positive for n gonorrhoeae show apple-green fluorescent staining of the kidney-shaped diplococci. although this test has been reported to be 100% specific for n gonorrhoeae (11), nonspecific fc binding to unrelated organisms such as staphylococcus aureus has been observed. therefore, only gram-negative, oxidase-positive diplococci should be tested. because test sensitivity is 99% (11), an alternative confirmation method should be used when this test gives an unexpected negative result

coagglutination tests: coagglutination tests can be performed on primary culture and, therefore, confirmed results can be obtained one day earlier than tests that require subculturing from primary culture plates. some of these tests do not require fresh or viable cells. the cell suspensions are boiled and combined with monoclonal antibodies that detect heat-stable epitopes on the pori outer membrane protein and detection reagent. coagglutination with test reagents indicates a positive result.

both false-positive results (cross-reactions with other neisseria species such as neisseria meningitidis, neisseria lactamica, n cinerea and k denitrificans) and false-negative results have been reported for the coagglutination tests (10,12-14). therefore, another test should be performed if the following conditions occur: the isolates suspected of being gonococci do not react with appropriate reagents or give equivocal results positive results are obtained on isolates from extragenital sites in low-risk patients and finally, positive results are obtained on isolates from children

nucleic acid methods are suitable for detecting n gonorrhoeae in specimens that may not contain viable organisms due to long transportation time or exposure to extreme temperature conditions. unlike the experience with c trachomatis, the performance of nucleic acid methods for the detection of n gonorrhoeae has not been appreciably better than that of a proficient specimen transport and culture system (5,19-21). dna amplification tests may not be suitable for test-of-cure because gonococcal dna may be present for weeks after successful treatment of an infection (5). however, the pace 2 (gen-probe, usa) has been reported to be a suitable test-of-cure assay for gonorrhea as early as six days after the treatment of genital infections

amplification methods

pcr:

table 3

nucleic acid detection tests (including dna probe tests, direct probe and amplified probe detection)

typing of n gonorrhoeae

strain differentiation has been used in epidemiological and medicolegal cases, outbreak investigations, and to monitor the distribution of antimicrobial-resistant strains and track the transmission of specific strains in a population.

antimicrobial susceptibility testing

when cultures are available, susceptibility testing should be performed on n gonorrhoeae in the laboratory or sent to a reference laboratory where such tests are available these results are useful for investigating treatment failure and monitoring the efficacy of currently recommended therapies.

table 4

minimal inhibitory concentration (mic) ranges (mg/l) of neisseria gonorrhoeae reference strains *^†

*mics for the world health organization (who) strains were determined using gc medium base (difco, usa) supplemented with 1% kellogg s defined supplement. ranges established by the national microbiology laboratory in winnipeg, manitoba. ^†acceptable ranges of mics as per national committee for clinical laboratory standards document m100-s13 (m7-a6), january 2003 (45)

all people who test positive for gonorrhea should be tested for other sexually transmitted diseases such as chlamydia, syphilis and human immunodeficiency virus.^[8]

herpes simplex – diagnosis

virologic tests

viral culture tests are made by taking a fluid sample, or culture, from the lesions as early as possible, ideally within the first 3 days of appearance. the viruses, if present, will reproduce in this fluid sample but may take 1 - 10 days to do so. if infection is severe, testing technology can shorten this period to 24 hours, but speeding up the timeframe during this test may make the results even less accurate. viral cultures are very accurate if lesions are still in the clear blister stage, but they do not work as well for older ulcerated sores, recurrent lesions, or latency. at these stages the virus may not be active enough to reproduce sufficiently to produce a visible culture.

polymerase chain reaction (pcr) tests are much more accurate than viral cultures, and the cdc recommends this test for detecting herpes in spinal fluid when diagnosing herpes encephalitis (see below). pcr can make many copies of the virus? dna so that even small amounts of dna in the sample can be detected. pcr is much more expensive than viral cultures and is not fda-approved for testing genital specimens. however, because pcr is highly accurate, many labs have used it for herpes testing.

an older type of virologic testing, the tzanck smear test, uses scrapings from herpes lesions. the scrapings are stained and microscopically examined for the virus. findings of specific giant cells with many nuclei or distinctive particles that carry the virus (called inclusion bodies) indicate herpes infection. the test is quick but accurate 50 - 70% of the time. it cannot distinguish between virus types or between herpes simplex and herpes zoster. the tzanck test is not reliable for providing a conclusive diagnosis of herpes infection and is not recommended by the cdc.

serologic tests

serologic (blood) tests can identify antibodies that are specific to the virus and its type, herpes virus simplex 1 (hsv-1) or herpes virus simplex 2 (hsv-2). when the herpes virus infects someone, their body? s immune system produces specific antibodies to fight off the infection. if a blood test detects antibodies to herpes, it? s evidence that you have been infected with the virus, even if the virus is in a non-active (dormant) state. the presence of antibodies to herpes also indicates that you are a carrier of the virus and might transmit it to others.

newer “type-specific” assays test for antibodies to two different proteins that are associated with the herpes virus:

·                glycoprotein gg-1 is associated with hsv-1

·                glycoprotein gg-2 is associated with hsv-2

although glycoprotein (gg) type-specific tests have been available since 1999, many of the older nontype-specific tests are still on the market. the cdc recommends only type-specific glycoprotein (gg) tests for herpes diagnosis.

serologic tests are most accurate when administered 12 - 16 weeks after exposure to the virus. recommended tests include:

·                herpeselect. this includes two tests: elisa (enzyme-linked immunosorbent assay) or immunoblot. they are both highly accurate in detecting both types of herpes simplex virus. samples need to be sent to a lab, so results take longer than the in-office biokit test.

·                biokit hsv-2 (also marketed as surevue hsv-2). this test detects hsv-2 only. its major advantages are that it requires only a finger prick and results are provided in less than 10 minutes. it is very accurate, although slightly less so than the other tests. it is also less expensive.

·                western blot test. this is the gold standard for researchers with accuracy rates of 99%. it is costly and time consuming, however, and is not as widely available as the other tests.

false-negative (testing negative when herpes infection is actually present) results can occur if tests are done in the early stages of infection. false-positive results (testing positive when herpes infection is not actually present) can also occur, although more rarely than false-negative. your doctor may recommend that you have the test repeated.

doctors recommend serologic herpes tests especially for:

·                people who have had recurrent genital symptoms but no negative herpes viral cultures

·                confirming infection in people who have visible symptoms of genital herpes

·                determining if the partner of someone diagnosed with genital herpes has acquired herpes

·                people who have multiple sex partners and who need to be tested for different types of stds

at this time, doctors do not recommend screening for hsv-1 or hsv-2 in the general population.

genital warts

dark-field examination of scrapings from wart cells shows marked vascularization of epidermal cells, which helps to differentiate genital warts from condylomata lata

  associated with second-stage syphilis. applying 5% acetic acid (white vinegar) to the warts turns them white. warts usually are diagnosed early by visual inspection biopsy is indicated only when neoplasia is strongly suspected.

molluscum contagiosum (mc) is a viral infection of the skin or occasionally of the mucous membranes. it is caused by a dna poxvirus called the molluscum contagiosum virus (mcv). mcv has no animal reservoir, infecting only humans. there are four types of mcv, mcv-1 to -4 mcv-1 is the most prevalent and mcv-2

low magnification micrograph of molluscum contagiosum. h& e stain.

high magnification micrograph of molluscum contagiosum, showing the characteristic molluscum bodies. h& e stain.

diagnosis is made on the clinical appearance the virus cannot routinely be cultured. the diagnosis can be confirmed by excisional biopsy.

histologically, molluscum contagiosum is characterized by molluscum bodies in the epidermis above the stratum basale, which consist of large cells with:

• abundant granular eosinophilic cytoplasm (accumulated virons), and

   

• a small peripheral nucleus.