Introduction
Key structure elements for diagnosis
Burden of genital herpes
Importance of laboratory diagnosis or testing for genital herpes
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Confirmation of clinically suspected genital herpes.
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Variable presentation of genital herpes.
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Extra-genital complications of genital herpes [20].
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Differential diagnosis with other ulcerative STIs.
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Differential diagnosis with other genital ulcerative dermatoses (Crohn’s disease, Behçet syndrome or fixed drug eruption).
Laboratory methods for direct herpes diagnosis
Collection, transport and storage of clinical specimens for herpes diagnosis
Collection site | Tools for sample collection | Collection method |
---|---|---|
Male skin or mucous membrane lesions
| • Sterile needles | • Unroof the vesicles with a sterile needle |
• Sterile cotton-tipped, Dacron or nylon flocked swab on a wooden, plastic or aluminium shaft | • Collect the content of the vesicles with a sterile swab and: | |
○ apply to a microscope slide (for immunofluorescence staining) or ○ introduce into transport media for viral culture or NAAT. | ||
• Microscope slides | ||
Male urethra
| • Sterile cotton-wool, Dacron or nylon flocked swab on a wooden, plastic or aluminium shaft | • Clean the external urethral opening region with a swab moistened in saline |
• Draw back the prepuce to avoid contamination when sampling | ||
• Insert a sterile swab carefully into the external urethral meatus (to a depth of 0.5–2 cm) and collect urethral exudates for testing | ||
Female skin or mucous membrane lesions
| • Gauze and cotton swabs,, dacron or nylon flocked swab on a wooden, plastic or aluminium shaft | • Similarly as for male skin or mucous membrane lesions |
• Microscope slides | ||
Female urethra
| • Sterile gauze swab (to remove excess discharge) | • Clean the introitus using a sterile gauze swab |
• Sterile cotton-wool, Dacron or nylon flocked swab on an aluminium shaft | • Carefully insert a sterile swab on an aluminium shaft into the urethra (to a depth of 0.5 cm) to collect exudates for testing | |
Cervix
| • Vaginal speculum | • Insert the vaginal speculum, which may be moistened in advance with warm water and |
• Sterile gauze swab | ||
• Sterile cotton-wool, Dacron or nylon flocked swab on a wooden or plastic shaft | • clean the cervical canal opening thoroughly with a sterile gauze swab | |
• Insert a cotton-wool or Dacron swab carefully into the cervical canal (to a depth of 2 cm) and collect the material from lesions. | ||
Vagina
(of prepubertal girls)
| • Sterile cotton-wool, Dacron or nylon flocked swab on an aluminium shaft | • Insert a sterile swab on an aluminium shaft carefully through the hymen into the vagina, and collect the material from the back wall of the vagina |
Urine
| • Sterile container for urine | • Ask the patient to collect the first 10–20 ml of voided urine (first catch) |
• The patients should avoid urinating for least two hours before sampling | ||
Conjunctiva
| • Sterile cotton-wool, Dacron or nylon flocked swab on an aluminium shaft | • purulent discharge must be removed before sampling with a sterile swab |
• Kimura platinum conjunctival scraper | • Move a swab over the conjunctiva of the inferior eyelid towards the interior angle of the eye (use a thin swab on an aluminium shaft for newborns) | |
• Topical ophthalmic local anaesthetic | ||
• The Kimura scraper is used to sample the bases of lesions (either ulcers or the bases of burst vesicles). Before collecting the sample, the spatula is sterilised by heating in a flame and allowed to cool | ||
Rectum
a
| • Rectal speculum or proctoscope | • Rectal material is taken under direct vision, with the aid of a proctoscope or rectal speculum. Use of a blind technique results in considerable loss of sensitivity |
• Sterile cotton-wool, Dacron or nylon flocked swab on a wooden or aluminium shaft | ||
• Insert a swab on a wooden or plastic shaft to a depth of 3 cm and collect the material from all rectal walls by circular motions for 10 seconds | ||
• If faecal material is impacted, the swab should be discarded and the sampling procedure repeated. |
Test method | Conditions | Comments |
---|---|---|
Viral culture
| • Immediately after sampling the material must be placed in appropriate transport medium, such as Eagle’s medium with addition of antibiotics | • Herpes simplex virus is sensitive to both the temperature and to drying out |
• The material should preferably be transported to the laboratory on ice, and kept at °4°C for up to 48 hours | ||
• Material should not be kept for more than 4 hours at room temperature | ||
• Accurately marked test tubes must be placed in a hermetic reservoir and transported to the laboratory accompanied by the relevant documentation including the investigation method requested | ||
Antigen detection and nucleic acid amplification tests
| Transport medium is usually provided by the manufacturer of the diagnostic commercial assay | • The material is generallly delivered in special test tubes with transport medium according to the manufacturer’s instructions for each test |
• If the sample transportation procedure is not described in the manufacturer’s instructions or in-house test systems are used, transportation is performed as follows: | ||
o Clinical material placed in univesal transport medium should be transported in a cool bag at 4 ± 2°C | ||
o Urine should be delivered to the laboratory within three hours of collection, at ambient temperature | ||
• Test tubes containing clinical material should be transported to the laboratory accompanied by the relevant documentation including the investigation method requested | ||
Microscopy (direct examination or immunofluorescence)
| • If there is a need to save the material for more than 24 hours, the smear should be fixed with 96% ethyl alcohol for three minutes | • If the rules of sampling and conditions of transportation of the biological material are not followed (e.g. slides are broken, unmarked or stuck together or there is no material on the slide), microscopy examination should not be carried out |
• Each smear on a microscope slide should be placed in the transportation container and transported to the laboratory accompanied by the relevant documentation including the investigation method requested | ||
• Method rarely used now |
Laboratory methods for direct herpes diagnosis
Method | Principle | Sample | Sensitivity | Specificity | Advantages | Disadvantages |
---|---|---|---|---|---|---|
Viral antigen detection
| Immunopreoxidase staining | Swab | Middle (80%) | High (90%) | Reagent cost | Fresh vesicles |
Smears from lesions | ||||||
Rapid (<4 hours possible) | Suboptimal sensitivity | |||||
Smear or vesicular fluid of exudate from base of vesicle | ||||||
Does no require the integrity of the specimen | ||||||
Typing possible | ||||||
Capture ELISA | Swab | High (Genital ulcer: >95%) | High (62-100%) | Fresh vesicles | ||
Vesicular fluid or exudate from base of vesicle | No viral typing | |||||
Rapid test device | Swab | Unknown | Unknown | Point-of-care testing | Not yet evaluated | |
Vesicular fluid or exudate from base of vesicle | ||||||
Virus culture
| HSV isolation susceptible cells | Swab | Low to high depending of the clinical context | High (≈100%) | Allows virus isolation | Less sensitive than PCR |
Skin lesions | ||||||
Sample storage and transport conditions influence sensitivity | ||||||
Classically, “gold standard” method | ||||||
Vesicular fluid or exudate from base of vesicle | Vesicular content :>90% | |||||
(➜ Rapid transport, cooled, protected from light in virus transport medium) | ||||||
Currently, “preferred” test (CDC 2010) | ||||||
Ulcer : 95% | ||||||
Swab : 70%-80% | ||||||
Labor-intensive | ||||||
Mucosal sample without lesions Biopsies | Mucosa without lesion: 30% | Simplicity of sampling | ||||
Expensive | ||||||
Virus typing | Specialized laboratories | |||||
Resistance | Results in 2/7 days | |||||
Phenotype testing* | Arrangement with laboratory necessary | |||||
Conjunctival/corneal smear | ||||||
Neonates | ||||||
Molecular biology
| HSV DNA detection and/or quantitation by NAAT, including in-house classical PCR, real-time PCR and commercial assays | Swab | Highest | High. | High sensitivity. | Only in specialized laboratories |
Skin lesions | (98%) | (≈100%) | ||||
Vesicular fluid or exudate from base of vesicle | Containment of potential cross-contamination important | Currently, “preferred” test (CDC 2010) | Not standardized | |||
Allows virus detection and typing in the same test | Not validated for all samples | |||||
Mucosal sample without lesions | ||||||
Risk of contamination (PCR) | ||||||
May be relatively expensive (real-time PCR) | ||||||
Rapid | ||||||
Aqueous/vitreous humor | May be automated. | |||||
Labor efficient | Routine resistance genotyping not available | |||||
Cortico-spinal fluid | ||||||
Result within 24–48 h, possibly in <3 hours | ||||||
Blood | ||||||
Resistance genotyping | ||||||
Method of choice for CSF | ||||||
Real-time PCR:
| ||||||
Rapid amplification | ||||||
Quantitative analysis | ||||||
Reduced risk of contamination | ||||||
Method of choice for skin lesions | ||||||
Cytological examination
| Tzanck smears | Skin/mucosal lesions | Low | Low | Inexpensive | Fresh lesions |
Papanicolaou or Romanovsky stain | low sensitivity and no distinction between HSV-1 and HSV-2, nor between HSV and varicella zoster virus infection | |||||
Biopsies | ||||||
Conjunctival/corneal smears | ||||||
Detection of infected cells by direct immunoflorescence | Smears, Tissue section Smear from base of vesicle | Middle | High | Inexpensive | Fresh vesicles | |
(Genital ulcer: 70-90% | (>95%) | Rapid (<4 hours possible) | Suboptimal sensitivity | |||
Asymptomatic : < 40-50%) | ||||||
Typing possible | Time-consuming | |||||
Labor-intensive | ||||||
Not standardized |
Sampling site or type of sample | Preferred diagnostic method |
---|---|
Vesicule on skin and mucous membranes Ulcer | NAAT; viral culture; antigen detection* |
Urethra (male) | NAAT; antigen detection* |
Cervix/urethra (female) | NAAT; antigen detection* |
Urine (men and women) | NAAT; viral culture |
Vulva/vagina (prepubertal girls) | NAAT |
Vagina (women after hysterectomy) |
Virus isolation and typing in cell culture
Antigen detection
Virus detection and quantification by molecular biology
Indirect serological diagnosis of herpetic infections
Method | Principle | Sample | Sensitivity | Specificity | Advantages | Disadvantages |
---|---|---|---|---|---|---|
Western blot
| Western blot HSV-1 | Serum | ≈100% | ≈100% | Reference (“gold standard”) test proposed by University of Washington (USA) | Not commercially available |
Expensive | ||||||
[UW-WB] | ||||||
Specific of HSV-1 and HSV-2 | 2–3 days for results | |||||
Western blot HSV-2 | ||||||
Detect early sero-conversion to HSV-2 in patient with prior HSV-1 infection | ||||||
Earliest sero-conversion : 13 days | ||||||
Enzyme immune-assay
| Monoclonal antibody-blocking EIA | Serum’ | ≈100% | ≈100% | Reference (“gold standard”) test proposed by the Central Public Health Laboratory in the United Kingdom; 98% concordance with WU-WB | Not commercially available |
(African sera : 98%) | (African sera : 97%) | |||||
Distinguish between HSV-1 and HSV-2 | ||||||
Enzyme immune-assay
| ELISA | Serum | 93–98% | 93–99% | Commercially available | May lack of sensitivity and specificity |
Distinguish between HSV-1 and HSV-2 | Lack of specific on African sera | |||||
Point of care tests
| Immuno-filtration | Serum Capillaryblood | 96% | 87–98% | Less expensive than Western blot | Commercially available only for HSV-2 |
Accurate results rapidly (6 min.) | Expensive | |||||
Not for large volume screening | ||||||
Easily to carry out | ||||||
Detects seroconversion within 4 weeks of presentation of 80% of patients with HSV-2 episodes | Complexity nonwaived (moderate) |
HSV-1 | HSV-2 | ||||||
---|---|---|---|---|---|---|---|
Assay | Manufacturer | Format | Collection method | Sensitivity | Specificity | Sensitivity | Specificity |
Biokit HSV-2 Rapid Test
| Biokit | Point of care | Heparinized capillary whole blood, serum | NA | NA | 93%-96% | 95%-98% |
HerpeSelect HSV-1 and HSV-2 Immunoblot
| Focus Diagnostics | Western blot with recombinant proteins | Serum | 99.3% | 95.1% | 97.3% | 93.7% |
HerpeSelect HSV-1 ELISA, HerpeSelect HSV-2 ELISA
| Focus Diagnostics | ELISA | Serum | 91.2%-96% | 92. 3%-95.2% | 96.1% -100% | 97.0%-96.1% |
CaptiaHsv 1 IgG Type Specific Elisa Kit&CaptiaHsv 2 IgG Type Specific Elisa Kit
| Trinity Biotech | ELISA | Serum | 87.9%-87.7% | 100%-98.2% | 96.7%-100% | 90.3%-91.5% |
Liaison HSV-1 & Liaison HSV-2 Type SpecificIgGAssay
| Diasorin | ELISA | Serum | 96.9%-98.7% | 91.3%-96.8% | 98.1%-94.8% | 98.0%-97.3% |
Zeus ELISA HSV GG-2 IgG Test System & Zeus ELISA HSV GG-1 IgG Test System
| Zeus Scientific | ELISA | Serum | 96.8% | 97.1% | 98.8% | 100% |
BioPlex HSV-1 & HSV-2 IgG panel
| Biorad | Luminex | Serum, lithium heparini plasma, EDTA plasma | 100%-100% | 98. 3%-97.4% | 99.4%-100% | 100%-100% |
Elecsys HSV-1 IgG and HSV-2 IgG assays
| Roche Diagnostics | Chemiluminescence | Serum, lithium heparin plasma, EDTA plasma | 94.2%-91.0% | 90. 3%-95.7% | 93.6%-97.8% | 98.7%-98.7% |
HSV-2 detection by direct method | HSV-1-specific IgG | HSV-2-specific IgG | Interpretation | |
---|---|---|---|---|
First assessment of genital lesions
| Positive | Positive or negative | Negative | Acute HSV-2 infection |
Repeat HSV-2-specific serology within 15-30 days | ||||
Positive | Positive or negative | Positive | Recurrent HSV-2 infection with HSV-2 infection acquired at least 6 weeks ago | |
No lesions
| NA | Negative | Negative | Patients at risk for acquiring orolabial or genital HSV-1 infection and/or HSV-2 infections |
NA | Positive | Negative | Patients at risk for acquiring orolabial or genital HSV-2 infections | |
NA | Positive | Positive | HSV-1 and HSV-2 past-infections | |
Recurrentgenitallesions
| Positive | Positive or negative | Positive | Recurrent HSV-2 infection |
Negative | Negative | Positive | Possible recurrent HSV-2 infection Other potential causes of genital ulcerative disease should be considered |
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Recurrent genital symptoms or atypical symptoms with negative HSV cultures;
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Clinical diagnosis of genital herpes without laboratory confirmation;
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Partner with genital herpes.
Context | Indication and interpretation |
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Asymptomatic patients | Not routinely recommended |
Confirmation of clinical diagnosis | HSV-2 antibodies are supportive of a diagnosis of genital herpes. |
History of recurrent or atypical genital disease with direct virus detection negative | HSV-1 antibodies do not differentiate between genital and oropharyngeal infection. |
Counseling of HSV-2 IgG-negative, HSV-1 IgG-positive patients should take into account that HSV-1 is an uncommon cause of recurrent genital disease. | |
First-episode genital herpes | Differentiation between primary and established infection guides counseling and management. |
At the onset of symptoms, the absence of HSV IgG against the virus type detected in the genital lesion is consistent with a primary infection. | |
Seroconversion should be demonstrated at follow-up. | |
Partner with genital herpes | Knowledge of infection status can guide patient education and counseling if the partnership is discordant. |
Pregnant women | Not routinely recommended. |
HSV-1 and/or HSV-2 seronegative women should be counseled about strategies to prevent a new infection with either virus type during pregnancy. | |
HIV infected patients | Not routinely recommended. |
Although HSV-2 seropositivity increases the risk of HIV transmission and frequent HSV recurrences augment HIV replication, there is limited evidence to inform the management of HSV-2 co-infection in HIV-infected patients without symptoms of genital herpes. | |
Limited data suggest an increased risk of perinatal HIV transmission among HSV-2 seropositive HIV-infected women. As the evidence is not consistent, testing of HIV-positive pregnant women is not routinely recommended. |
Therapeutic monitoring: drug resistance testing
Gene | Drug | Aminoacidmutationsa | Stop codona | Nucleotide insertion/deletionb | Association of mutationsa |
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HSV-1 TK
|
ACV
| R51W, Y53P/D/H, D55N, G56S/V, P57H, K62N, H58R/L, G59R/Y/W, G61V, K62N, T63A/I/S, T64A/S, T65N, E83K, P84S, V87H, T103P, Q104H, H105P, Q125E/L, M128A/F, G129D, G144N/R, A156V, D162A, R163H/C, A167V, A168T, L170P, Y172C/F, P173L/R, A174P, A175V, R176Q, L178R, S181N, Q185R, V187M, A189V, G200C/D, T201P, G206R, L208H, R216C/H/S, R220C/H, R222C/H, L227F, Y239S, T245M/P, T287M, L297S, L315S, C336Y, L364P | Y53, S74, E95, T103, Q104, R176, Q250, Q261, R281, L341, C336, Q342, L364, A375 | 133-136, 153-155, 180-183, 184-187 430-436, 437-438,455-458, 460-464, 464-465, 548-553,615-619, 666-669, 853-856, 878-880, 896-900, 1061-1064 | |
HSV-2 TK
|
ACV
| R34C, R51W, G56E, G59P, P85S, N100H, Q105P, T131P, R177W, S182D, S182N, V192M, T202A, R217H, R221H, R221C, R223H, L228I,D229H, R272V, P273S, D274R, T288M,C337Y | A28, L69, D137, Q222, Y240, T264 | 215-217, 219-222222, 439-440, 452, 467, 519-521, 551-556, 586-591, 626-628, 808-812 | R272V + P273S + D74R |
P85S + N100H + V192M | |||||
HSV-1 DNA pol
|
ACV
| D368A, E370A, V462A, K532T, Y557S, Q570R, D581A, G597K/D, A605V, Q618H, Y696H, R700G, L702H, V714M, V715M, F716L, A719V/T, S724N,E771Q, L774F, L778M, D780N, L782I, P797T, E798K, L802F, V183M, N815L/S/T/V/Y/E, Y818C, T821M, G841S/C, R842S, S889A, F891C/Y, V892S, D907V, I922N/T, Y941H, V958L, R959H, N961K, D1070N | A719V + V904M | ||
A327T + A605V | |||||
T566A + A605V | |||||
FCV
| N494S, A605V, F716L, A719V,A719T, S724N, L778M, D780N, L782I, E798K, F891C, D907V, V958L | A719V + V904M | |||
A327T + A605V | |||||
S724N + A916V | |||||
CDV
| A136T, R700H, R700M, S724N, T821M, L1007H, I1028T | ||||
ACV + FCV
| A605V, F716L, A719V, A719T, S724N, L778M, D780N, L782I, E798K, F891C, D907V, V958L | A719V + V904M | |||
A327T + A605V | |||||
ACV + CDV
| T821M | ||||
HSV-2 DNA pol
|
ACV
| E250Q, R628C, E678G, A724V, S725G, D785N, D912N/V | |||
FCV
| S725G, S729N, L783M, D912V | ||||
ACV + FCV
| S725G, D912V |