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Year : 2007  |  Volume : 28  |  Issue : 1  |  Page : 43-47

Specific investigations in a case of sexually transmitted disease

Department of Dermatology, Venerology and Leprosy, Dr. D. Y. Patil Hospital, Nerul, Mumbai, India

Correspondence Address:
Kaleem Khan
Department of Dermatology, Venerology and Leprosy, Dr. D. Y. Patil Hospital, Sector 5, Nerul, Navi Mumbai
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0253-7184.35714

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How to cite this article:
Khan K, Gautam M, Patil S. Specific investigations in a case of sexually transmitted disease. Indian J Sex Transm Dis 2007;28:43-7

How to cite this URL:
Khan K, Gautam M, Patil S. Specific investigations in a case of sexually transmitted disease. Indian J Sex Transm Dis [serial online] 2007 [cited 2023 Sep 21];28:43-7. Available from:

   Introduction Top

Over the past few decades, there was a steady decline in the number of sexually transmitted diseases (STDs). However, with the rapid emergence of human immunodeficiency virus (HIV), which has now become a pandemic, these STDs are once again in focus. But now, the classical presentation is no longer a rule; and in such a scenario, clinical suspicion has to be confirmed by specific investigations for correct identification and thus for correct management of these diseases. [1]

This article tries to enlist the specific investigations that can be done in a case of STD attending the clinic. The list is by no means comprehensive and has to be used in conjunction with a detailed history and good clinical examination. Other laboratory tests can be done when warranted.

   Syphilis Top

Causative organism : Treponema pallidum.

Specimen collected: Serous discharge from the primary ulcer collected directly on the cover slip or slide. Site cleaned only with normal saline. Aspiration of enlarged nodes is also helpful. For serological tests, blood (serum) and cerebrospinal fluid (CSF) can be used.

  • Dark field microscopy: Bright, corkscrew-shaped, treponemes seen against a dark background. Quasi-purposeful movements noted.
  • Special stains: Levaditi's method (silver impregnation method), Fontana's method, Immunofluorescent techniques (DFA-TP).
  • VDRL (Harris test): Nonspecific slide flocculation test. Significant in secondary syphilis. Titers >16

    - suggestive. Rule out false positives.

Other Reagin tests include Wasserman's, Kahn and Rapid Plasma Reagin ( of academic interest only - Kolmer test, Automated Reagin Test, Reagin Screen Test, Toluidine Red Unheated Serum Test [TRUST] ).

  • FTA-ABS (Fluorescent Treponemal antibody - ­Absorption): Detects circulating IgG antibodies. Very high sensitivity and specificity. Remains positive for the rest of life (unless syphilis treated in early stages). Variation: FTA-ABS double stain (DS).

 Treponema pallidum Scientific Name Search magglutination (TPHA): Gradual agglutination of sensitized sheep RBCs is considered reactive. Control tubes show a compact button at the bottom. Variations: MHA-TP (Micro Hemagglutination Treponema Pallidum ), HATTS (Hemagglutination Treponemal test for Syphilis).

  • Other specific tests (rarely done): Treponema Pallidum immobilization test, Reiter Protein complement fixation test.
  • CSF: >5 lymphocytes, proteins >40 mg%.
  • PCR: Greatest value in diagnosis of neurosyphilis and congenital syphilis.
  • Colloidal gold curve (of academic interest only): On addition of CSF, color changes at various ­dilutions of gold solution are noted. Three curves seen. Paretic curve (zone 1), Leutic curve (zone 2), Meningitic curve (zone 3).

   Chancroid Top

Causative organism: Hemophilus ducreyi .

Specimen collected: Obtained from base or margins of ulcer with a cotton or calcium alginate swab and rolled over the slide to preserve morphology.

For culture, use swab or wire loop or pus aspiration from an unruptured bubo.

  • Gram stain: Gram negative intracellular/ extracellular rods arranged in a 'School-of-Fish' or 'Fingerprint swirl' or 'railroad track' appearance.
  • Culture: Requires enriched media, containing A. - Beef infusion Agar; B - Defibrinated rabbit's Blood; C - Cystine and D - Dextrose at room temperature with certain amount of moisture in the air. Optimum growth noted at 48 h as small, yellow-gray, translucent colonies.

Unique characteristic : Colonies of H. ducreyi can be pushed along the agar surface with a wire loop.

  • Biopsy (of academic interest only): From the ulcer, shows three cellular zones.

    Zone 1: Surface zone: PMNs, fibrin, RBCs, necrotic tissue.

    Zone 2: Cellular zone: Proliferating endothelial cells, lymphocytes and plasma cells.

    Zone 3: Deep zone: Marked infiltration of plasma cells.

  • ELISA: Detects antibodies to H. ducreyi.
  • PCR.
  •  Ito-Reenstierna test More Details (of academic interest only): Intradermal test. Was available as Dmelcos, a commercially produced vaccine containing 225 million organisms/ml. Intradermal injection of 0.1 ml would show as an inflammatory papule of size 0.5-1 cm after 48 h if the test was positive.
  • Auto-inoculation (of academic interest only): Producing a typical lesion by inoculating the material from a suspected sore into an incision made in a healthy area of skin.

   Granuloma Inguinale Top

Causative organism: C alymmatobacterium granulomatis .

Specimen collected : Granulation tissue near the periphery of the lesion cleaned with normal saline. A small piece of tissue taken with forceps or punch biopsy.

  • Biopsy: Epidermis shows pseudoepitheliomatous hyperplasia with acanthosis and microabscess formation. Dermis shows a dense mixed cellular infiltrate composed predominantly of mononuclear cells and diagnostic 'pathognomonic cells' (enlarged histiocytes having eccentric nucleus with single or multiple encapsulated organisms). Vascular proliferation is prominent.
  • Tissue Smear: H and E stain shows intracellular/ extracellular small dumbbell-shaped rods having deep purple/blue color surrounded by pink capsules (closed safety pin appearance).

    Special Stains: Wright, Giemsa, Pinacyanole or Warthin-Starry.
  • Culture: Yolk sac of chick embryo or on 'Dulaney slants' (coagulated egg yolk slants).

   Lymphogranuloma Venerum Top

Causative organism: Chlamydia trachomatis.

Specimen collected: Urethral exudates for gram staining.

For culture: A fiber-tipped swab with shaft made of inert material, from endocervix in females; or endo-urethal swabs (passed at least 5 cm into the urethra in males). Conjunctival swabs can also be done, especially from upper lid in neonates. Specimens need to be transported in special transport media cooled by liquid nitrogen unless processed within 2 h.

  • Gram stain: Only the absence of intracellular gram-negative diplococci is useful in microscopically distinguishing NGU from gonorrhea. Giemsa-Romanowsky stain demonstrates the presence of inclusion (elementary) bodies within infected cells.
  • Culture: McCoy cells (mouse fibroblasts) pre-treated with idoxuridine, HeLa cells are used. After culture, the cells are stained with iodine (dark brown) or Giemsa (inclusion bodies appear yellow) but best with immunofluorescence technique.
  • Complement fixation for LGV: Largely replaced by radioimmune precipitation test (RIP). Radiophosphorus is incorporated into the tissue culture of the chlamydia strain. When this is mixed with the test serum together with antiglobulin, a precipitate is produced.
  • Micro-IF antibody (Micro-IF, MIF): As specific as RIP but also demonstrates the presence of type-specific antibodies to chlamydia infection. Detectable levels of antibody may remain in the blood long after the agent disappears from the tissues.
  • DNA probes: Used for detection of non-amplified chlamydial nucleic acids. These are less sensitive than culture.
  • PCR/LCR: These are amplified DNA tests and are more sensitive than culture.
  • ELISA: For rapid detection of chlamydial components. Less sensitive than culture methods.
  • Direct fluorescent antibody detection: These make use of monoclonal antibodies (MAB) directed against species, subspecies and genus-specific antigens.
  • Frei's test (of academic interest only): Intradermal test. Was available as Lygranum (potent group-specific antigen grown in yolk sac of chick embryo). Positive test seen as a red, raised 6 mm size papule surrounded by erythema. Test is read after 48 and 72 h of injecting 0.1 ml of antigen on one forearm.

   Gonorrhea Top

Causative organism:  Neisseria More Details gonorrhoeae.

Specimen collection: Heterosexual men - urethral smears; homosexual men - urethral, rectal, pharyngeal. Women - cervical and rectal.

Disseminated infection: Blood cultures.

Septic arthritis: Synovial fluid.

  • Gram stain: Gram-negative, encapsulated, diplococci (kidney-shaped cells with adjacent sides flattened) within PMN leukocytes. Direct immunofluorescent techniques are more sensitive.
  • Culture: Non-selective media - chocolate agar; Selective media - Modified Thayer-Martin medium, modified NYC medium, Neisseria clear medium, Chacko-Nair medium. Transport medium - Stuart medium. Presence of CO2-enriched atmosphere is very important. Pathogenic strains show small (0.5-1 mm diameter) translucent, nonpigmented to brownish convex colonies with finely granular surface. Neisseria species are Oxidase test positive, i.e., they stain purple upon adding oxidase reagent. Further identification is based on sugar utilization.
  • For disseminated infection:

    i. Bacteremic stage: Blood cultures are positive.

    ii. Septic joint stage: Culture of joint aspirate. Large joints, viz., shoulder or elbow, are typically involved.

  • DNA probes.
  • PCR.

   Genital Mycoplasma Top

Causative organism: Mycoplasma hominis / Ureaplasma urealyticum .

Specimen collected: Urine, prostatic secretions, blood (liquid specimen) on calcium alginate or polyester-tipped swabs.

  • Culture: Serially diluted specimen inoculated into separate agars. Shepard's 10B broth and A8 agar commonly used for inoculation. Colonies are urease-negative and have a 'fried egg' appearance.
  • Serology: Metabolism-Inhibition assays or ELISA to detect antibodies.
  • PCR.

   Trichomoniasis Top

Causative organism: Trichomonas vaginalis .

Specimen collected: Vaginal specimens - from posterior fornix. Male urethral specimens - gentle scraping with a platinum loop after urination.

  • Per speculum examination: Cervix shows punctuate hemorrhages (strawberry cervix) and a greenish-yellow foamy discharge in the vagina.
  • Wet mount: A drop of discharge and normal saline shows motile trichomonads. They are larger than PMN but smaller than epithelial cells with characteristic flagella and undulating membrane with jerky motility. Examined with light microscopy under low power.
  • Culture: Diamond's medium. Modified Feinberg-Whittington medium.
  • FA: Flourescein-labeled monoclonal antibodies.
  •  Pap smear More Details of exfoliated cervical cells may identify trichomonads.

   Gardenerella Vaginitis/Bacterial Vaginosis Top

Causative organism: Gardenerella vaginalis .

Specimen collected: Use a swab to obtain vaginal fluid and cells from the vaginal wall (not cervix), which is to be rolled across a slide and air dried.

  • Gram stain: Epithelial cells covered with gram-positive rods (clue cells).

Nugents method: Score of >7 considered diagnostic.

Spiegel method: Bacteria present or absent.

At least one clue cell in five vaginal epithelial cells.

  • Whiff test: Vaginal secretion has an unpleasant odor, which can be accentuated by adding few drops of 10% KOH. 'Dead fish' odor due to volatilization of polyamines (positive amine test).
  • pH: Using a strip of narrow-range pH paper. Significant if pH > 4.5.
  • DNA probes: Oligonucleotide probe for G. vaginalis.

   Candidiasis Top

Causative organism: Candida albicans , C. krusei , C. parapilosis , C. tropicalis .

Specimen collected: Use a swab to obtain vaginal fluid and cells from the vaginal wall.

  • Wet mount: Examined under low power, to begin with, followed by high power (40x). Addition of 10% KOH improves both sensitivity and specificity. Budding yeast cells, which can resemble hyphae (pseudohyphae).
  • Gram staining of secretions shows intensely gram-positive Candida spp.
  • Culture: Sabouraud's agar - Pinpoint white colonies which show round, thick-walled chlamydospores after 48 h. Nickerson's medium contains bismuth; so colonies turn black.
  • Latex agglutination.

   Genital Warts Top

Causative organism: Human Papilloma virus.

  • Colposcopy, Urethroscopy: Direct visualization of the verrucous growth.
  • PAP smear: For detecting asymptomatic cervical HPV infection. Presence of koilocytes is characteristic.
  • Biopsy: Papillomatosis, acanthosis with psuedoepitheliomatous hyperplasia. Characteristic cells - koilocytes (large epithelial cells containing dense nucleus with perinuclear halo and a clear cytoplasm concentrated at the periphery).
  • Southern Blot/ Dot Blot Hybridization: Identifies a specific HPV DNA type using a DNA or RNA probe made from a known type of HPV. Tissue in-situ hybridization gives a colorimetric reaction and is helpful in giving cellular detail and localization of hybridized DNA.
  • Immunohistochemistry: Detects HPV capsid ­antigen.
  • PCR.

   Genital Herpes Top

Causative organism: Herpes simplex virus Types 1 and 2.

  • Tzanck smear: Presence of multinucleate giant cells.
  • Histopathology: Ballooning degeneration with intra-epidermal vesicles. Intracellular inclusion bodies (Cowdry type A) and giant cells seen.
  • Culture: Human diploid fibroblast cells or HeLa cells. Viral growth evident by its cytopathic effect on culture cells.
  • Immunofluorescence: Direct better than indirect fluorescent technique. Requires frozen sections of tissue or lesion scrapings where HSV-infected cells show bright apple-green fluorescence.
  • Electron Microscopy: Only if viral load in the specimen > 106-107 particles/ml.
  • DNA probes: Nucleic acid probes are available for detecting HSV-1 and HSV-2 DNA sequences.
  • ELISA: For detecting viral antigens.
  • PCR.

   Genital Molluscum Top

Causative organism: Pox virus.

  • Light cryotherapy makes central umbilication prominent.
  • Gram stain: Crush smear of white pasty material shows cells containing inclusion bodies (molluscum bodies or Henderson-Paterson bodies). Other stains include Wright or Giemsa.
  • Histopathology: Acanthotic epidermis with central crater. Keratinocytes in spinous layer show intracytoplasmic inclusion bodies which are initially eosinophilic and later (near the top) basophilic.
  • Electron microscopy: Brick-shaped pox virus particles seen.

   Genital Scabies Top

Causative organism: Sarcoptes scabiei var-hominis.

Specimen collected: Skin scrapings or a punch biopsy of a burrow.

Burrow ink test: Suspect burrow rubbed with ink pen and immediately wiped off with alcohol. The burrow gets stained as a thin thread-like line. Variation: Tetracycline solution can be used instead, and the burrow will fluoresce yellow-green under Wood's Lamp.

Skin scraping: With a no. 15 blade, the skin is scraped and the contents spread over a slide. After adding a drop of mineral oil, the mite or its eggs can be visualized under the microscope. Variation: 10% KOH can be used, but if kept for long, it can dissolve the mite itself.

  • Biopsy: A punch or shave biopsy will show the mite in a burrow or its eggs or fecal matter (scybala) within the stratum corneum.
  • Dissecting microscope: For direct visualization of the mite in the burrow with its eggs, giving a 'peas in a pod' appearance.
  • Epiluminescence microscopy (Dermascope): Does not differentiate between living and dead mite.

   References Top

1.Lewis JS. Selection and Evaluation of Test and Quality Control. In: Stephen Holmes, Holmes KK. Atlas of Sexually Transmitted Diseases and AIDS. 2 nd ed. Mosbe-Wolfe: Spain; 1996. p. 320-4.  Back to cited text no. 1    
2.Larsen SA, Steiner BM, Rudolp AH. Laboratory diagnosis and interpretation of tests for syphilis. Clin Microbiol Rev 1995;8:1-21.  Back to cited text no. 2    
3.Lewis DA. Chancroid: Clinical manifestation, diagnosis and management. Sex Transm Inf 2003;79:68-71.  Back to cited text no. 3    
4.Nayar M, Chandra M, Saxena HM, Bhargava NC, Sehgal VN. Donovanosis: A histopathological study. Indian J Pathol Microbiol 1981;24:71-6.  Back to cited text no. 4  [PUBMED]  
5.Black CM. Current methods of laboratory diagnosis of Chlamydia trachomatis infections. Clin Microbiol Rev 1997;10:160-84.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Johnson RE, Newhall WJ, Papp JR, Knapp JS, Black CM, Gift TL, et al . Screening tests to detect Chlamydia trachomatos and Nisseria gonorrhoeae infections-2002. MMWR Recomm Rep 2002;51: 1-38.  Back to cited text no. 6    
7.Morse SA, Holmes KK. Gonococcal infections. In: Hoeprich PD, Jordan MC, Ronald AR, editors. Infectious diseases. 5 th ed. Harper and Row: New York; 1994. p. 670-85.  Back to cited text no. 7    
8.Stary A. Urethritis-Diagnosis of non-gonococcal urethritis. Dermatol Clin 1998;16:723-6.  Back to cited text no. 8  [PUBMED]  
9.Spiegal CA, Amsel R, Holmes KK. Diagnosis of bacterial vaginosis by direct Gram stain of vaginal fluid. J Clin Microbiol 1983;18:170-7.  Back to cited text no. 9    
10.Bryan JA. Laboratory diagnosis of viral infections. In: Conn RBed: Current diagnosis, 7 th ed. WB Saunders: Philadelphia; 1995. p. 174-82.  Back to cited text no. 10    


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