Diagnosis of herpes

Diagnosis of herpes is based on classical virus isolation on sensitive cell cultures, immunofluorescence and serological methods, colposcopic examination, and the use of modern molecular biological methods (PCR, dot hybridization), which allows diagnosing the entire group of herpes viruses, including HHV-6 and HHV-7 types.

Laboratory diagnostic methods for herpes infection

The main methods aimed at isolating HSV or detecting viral particles and/or their components	Auxiliary methods aimed at detecting antibodies to HSV in biological fluids of the human body
Isolation of HSV in sensitive cell and animal cultures Direct and immune electron microscopy Direct and indirect variants of the IPA IFA Molecular biological methods Latex agglutination reaction	Neutralization reaction RSC Determination of antibodies to non-structural proteins of HSV-1,2

It was shown that in 76% of patients genital herpes (GH) is caused by HSV-2, and in 24% - by HSV-1 type. Moreover, GH as a monoinfection occurred only in 22% of patients, in 78% of cases microbial associations were detected. In 46% of individuals, parasitocenosis caused by two pathogens was detected, including chlamydia were detected in 40% of cases. Gardnerella, Trichomonas, and gonococci were less frequently detected in smears.

In 27% of patients, the parasitocenosis was represented by three pathogens, in 5.2% - by four pathogens. Moreover, a combination of chlamydia with gardnerella and Candida fungi was most often noted. These data substantiate the need for a thorough bacteriological examination of patients with GH in order to identify combinations of pathogenic agents, as well as an in-depth study of the pathogenesis of mixed infections of the urogenital tract, which will allow for differentiated complex therapy of herpes infection.

Materials studied for the isolation of HSV depending on the localization of herpetic lesions

Localization of lesions	Vesicle contents	Cell scraping				CSF	Bronchial aspirate	Biopsy	Blood
		1	2	3	4
Leather	+								+
Eyes			+						+
Genitals				+					+
Anus	+				+				+
Mouth	+	+							+
CNS	+					+		+	+
Lungs		+					+		+
Liver								+	+
Congenital herpes	+	+	+				+		+

Methods of laboratory diagnostics of cytomegalovirus infection

Methods	Time required to obtain results	Notes
VIROLOGICAL
Electron microscopy	3 hours	Not very accessible
Virus isolation in cell culture (VCI)	4-20 days	Standard, Slow
Immunofluorescence staining of early AG using monoclonal antibodies	6 hours	Less specific
CYTOLOGICAL	2-3 hours	Less specific
SEROLOGICAL
RSC	2 days	Standard
RGA	1 day	Labor intensive
REEF	6 hours	Simple, specific
NRIF	6 hours	Difficult
RIMP	6 hours	Difficult
ELISA (IgM, DO)	6 hours	Fast, simple
Immunoblot	6 hours	Expensive
MOLECULAR BIOLOGICAL
MG	5-7 days	Expensive, labor-intensive
PCR	3 hours	Expensive

Methods of diagnostics of the herpes zoster virus

Diagnostic methods	Laboratory techniques
INDIRECT
Selection	Tissue culture, chicken embryos, laboratory animals, co-culture with permissive cells or helper viruses
Identification of isolates	Neutralization reaction, RSC, IF, PIEF, reaction of isolates precipitation, agglutination, IF
DIRECT
Cytology	Smears: color immunofluorescence
Histology	Pathomorphology of the cell
Structure	Embryonic microscopy, immunoelectron microscopy
Determination of antigens	IF, PIEF, RIM, IFA
Determination of local antibody production	Ig M, Ig G, Ig A: ELISA, RIA
Molecular biological approaches	Molecular hybridization, PCR

Laboratory diagnostics of infection caused by the herpes zoster virus

Diagnostic problems	Methods	Expected results
Acute primary infection	1	Detection in 2 hours
	2	Antibody levels are rising slowly
	3	Present 3 days after infection
Acute reactivated infection	1	Detection of UUU after 2 hours
	2	Antibody levels are rising slowly
	4	Present 4 days after the rash appears

1. determination of vesicles of the VEGF in liquid;
2. serology: CSC, ELISA, aimed at detection
3. serology: ELISA aimed at detecting IgM;
4. serology: ELISA aimed at detecting IgA, IgM.

Methods for indicating the immune response to herpes zoster virus infection

Approach	Method
Detection of an increase in antibody titer in the second sera	RSK, RTGA, RPGA, IF neutralization reaction, RIM, ELISA
Detection of Ig G, Ig A class-specific antibodies in the first serum sample	ELISA, IF, RIM, latex agglutination

Interpretation of the results of serological examination of patients' sera for herpesvirus infections (ELISA)

Name of infection/marker	Average threshold values for infections
	Results of the analysis	Interpretation
Cytomegaly Anti-CMV IgG (1-20 U/ml) Anti-CMV IgM (100-300%)	Positive 1-6 Positive 6-10 Positive >10 Negative Positive 100-300 Negative <90 Doubtful 90-100	Remission Exacerbation of the disease Acute phase of the disease No infection (disease) Acute phase of the disease Repeat the analysis in 2-3 weeks
Herpes simplex 1,2 serotypes Anti-HSV 1/2 total (100-900%)	Positive 100-400 Positive 400-800 Positive >800 Negative <100	Remission Exacerbation of the disease Acute phase of the disease No infection (disease)

The table presents the main methods of laboratory diagnostics of herpesvirus infections, as well as recommended biological materials that are examined when isolating HSV, taking into account the localization of herpetic lesions.

Reliable isolating herpes simplex and CMV viruses by infecting sensitive cell cultures. Thus, during virological examination of 26 patients during the relapse period, HSV was isolated on a sensitive Vero cell culture in 23 cases (88.4%). Infected cultures showed a picture of cytopathic action typical for HSV - the formation of multinucleated giant cells or an accumulation of rounded and enlarged cells in the form of clusters. In 52.1% of cases, foci of cytopathic action of the virus could be detected already by 16-24 hours after infection. By 48-72 hours of incubation of infected cultures, the percentage of materials causing specific destruction of cells increased to 87%. And only in 13% of cases were positive results detected 96 hours after infection or more or during repeated passaging.

Methods of laboratory diagnostics of generalized herpes infection

The main methods aimed at detecting (isolating) herpes viruses, their particles and their components	Auxiliary methods aimed at detecting antibodies to herpes viruses in biological fluids, detecting enzymatic shifts in blood serum
Isolation of herpes viruses on sensitive cell and animal cultures Direct and immune electron microscopy Direct and indirect variants of the immunoperoxidase method Direct and indirect variants of the fluorescent antibody method Variants of solid-phase enzyme immunoassay Variants of the molecular (DNA-DNA) hybridization method Polymerase chain reaction Latex agglutination reaction	Neutralization test Complement fixation test Latex agglutination test Indirect version of the fluorescent antibody method Indirect version of the immunoperoxidase method Variants of solid-phase enzyme immunoassay Immune blotting method Radial complement fixation method Determination of alanine and aspartate aminotransferase levels

Serological methods are used to diagnose infectious mononucleosis (an infection caused by EBV). The Paul-Bunnell reaction with ram red blood cells, a diagnostic titer of 1:28 or higher in a single blood serum test, or a 4-fold increase in antibodies when examining paired serums. The Hoff-Bauer reaction with a 4% suspension of formalinized horse red blood cells is used. The result is taken into account after 2 minutes; in infectious mononucleosis, the reaction is highly specific.

Currently, an enzyme immunoassay (EIA) method for diagnosing infectious mononucleosis is being developed. In this case, IgG and IgM antibodies in the patient's serum are determined by incubating it with lymphoblasts infected with EBV, followed by treatment with fluorescent antibodies. In the acute period of the disease, antibodies to the viral capsid antigen are determined in a titer of 1:160 and higher.

When using a number of imported commercial test systems, ELISA can detect: antibodies to EBV envelope antibodies, antibodies to EBV early antigen, total antibodies to EBV early antigen, determined in the acute phase of the disease both in the nucleus and in the cell cytoplasm, and limited antibodies to early EBV, determined in the acute phase of the disease both in the nucleus and in the cell cytoplasm, limited antibodies to EBV early antigen, determined at the height of the disease only in the cell cytoplasm, and antibodies to EBV nuclear antigen. The use of these test systems allows for differential diagnostics of a number of diseases associated with EBV.

After a positive ELISA test that reveals antibodies to EBV, a confirmatory immunoblotting reaction is performed, which determines the presence of antibodies to individual EBV marker proteins (p-proteins): p23, p54, p72 (the presence of this protein indicates the possibility of EBV reproduction), p 138. The above laboratory methods are also used to monitor the effectiveness of treatment.

The sensitivity of virological methods is 85-100%, specificity is 100%, the study time is 2-5 days. The direct immunofluorescence method (DIF) with polyclonal or monoclonal antibodies against HSV-1 and HSV-2 is often used in practical work. The DIF method is quite easily reproducible in a regular clinical laboratory, is not expensive, the sensitivity is above 80%, the specificity is 90-95%. Immunofluorescence microscopy revealed the presence of cytoplasmic inclusions, morphological features, the percentage of infected cells in smears-scrapings from the urethra, cervical canal, cervix, rectum.

The PIF method provides an idea of the morphological properties of cells and changes in the localization of HSV antigens. In addition to direct signs of cell damage by herpes viruses (detection of specific luminescence), there are indirect signs of herpes infection according to PIF data:

• aggregation of nuclear matter, detachment of the karyolemma;
• the presence of so-called “hole” nuclei, when only one karyolemma remains from the cell nucleus;
• the presence of intranuclear inclusions - Cowdry bodies.

When performing PIF, the physician receives not only a qualitative but also a quantitative assessment of the state of infected cells, which we used to assess the effectiveness of the antiviral therapy with acyclovir (AC). Thus, 80 patients with simple genital herpes (GH) were examined using the PIF method in dynamics. It was shown that if before treatment with acyclovir, 88% of patients had a high percentage of infected cells (50-75% and higher) in smears, then after one course of acyclovir, healthy cells were detected in smears of 44% of patients, in 31% of cases, single infected cells were noted, and in 25% of patients there were up to 10% infected cells.

Content of infected cells in smears (PIF reaction) of patients with genital herpes treated with acyclovir

Periods of illness	Percentage content in smears
	Infected cells					Normal cells
	More than 75%	50-75%	40-50%	10%	Single cells in the field of view
Relapse (before treatment)	25%	63%	12%
	(20)	(50)	(10)
Remission (after treatment)				25%	31%	44%
				(20)	(25)	(35)

Using PIF and the dot hybridization method for many years, it has been noted that the results of the study coincide in almost 100% of cases. It should be noted that in order to increase the reliability of the diagnosis of herpes, especially in cases of subclinical and low-manifest forms of herpes, it is recommended to use 2-3 methods of laboratory diagnostics in the work, especially when examining pregnant women, women with an unfavorable obstetric history, and persons with an unspecified gynecological diagnosis.

Thus, in PCR diagnostics of viral and bacterial infections of the urogenital tract, it is necessary to evaluate the positive results obtained taking into account the anamnesis, the presence (or absence) of specific clinical symptoms of the disease. If chlamydia is detected using PCR, then in this case there is a high probability of infection and the issues of therapy can be resolved accordingly. In case of detection of mycoplasmas (ureaplasmas), which are opportunistic microorganisms, additional cultural studies are required to confirm the diagnosis, i.e., sowing material from the patient on sensitive cell cultures. Only if positive results are obtained in the cultural analysis can we talk about laboratory confirmation of the diagnosis of mycoplasmosis. The same method will allow, if necessary, to determine the sensitivity of the isolated mycoplasmas to frequently used dosage forms (antibiotics, fluoroquinolones, etc.).

Simultaneous infection with several viruses of the Herpesviridae family is possible. We often detected infection of one patient with HSV-1, HSV-2 and CMV viruses. Patients with clinical and laboratory manifestations of secondary IDS (oncohematological, oncological, HIV-infected patients) were significantly more often infected with several herpes viruses. Thus, it has been shown that clinical and immunological disorders progressing in HIV infection are accompanied by an increase in the number of herpes viruses detected by the molecular hybridization method. In this case, the most prognostically significant can be considered the complex simultaneous detection of HSV-1, CMV and HHV-6 type DNA.

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