Diagnosis of herpes

Diagnosis of herpes is based on classical viral isolation on sensitive cell cultures, immunofluorescence and serological methods, colposcopy studies, use of modern molecular biological methods (PCR, dot-hybridization), which allows to diagnose the whole group of herpesviruses, including HHV-6 and HHV-7 types.

Methods of laboratory diagnosis of herpetic infection

The main methods aimed at secreting 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 cultures of cells and animals Direct and Immune Electron Microscopy Direct and indirect MFA options ELISA Molecular biological methods Latex agglutination reaction	Neutralization reaction RSK Determination of antibodies to non-structural proteins of HSV-1,2

It is shown that 76% of patients have genital herpes (GH) caused by HSV-2, and in 24% - HSV-1 type. And GG as a monoinfection only occurred in 22% of patients, in 78% of cases, microbial associations were detected. In 46% of the patients, parasitocenosis caused by two pathogens was detected, including chlamydia were detected in 40% of cases. Less often in the smears determined gardnerelly, Trichomonas, gonococci.

In 27% of patients, parasitocenosis was represented by three, and 5.2% by four pathogens. Moreover, a combination of chlamydia with Gardnerella and fungi of the genus Candida was more often noted. These data justify the need for a thorough bacteriological examination of patients with GH with the aim of identifying combinations of pathogenic agents, as well as an in-depth study of the pathogenesis of mixed infections of the urogenital tract, which will allow a differential complex therapy of herpetic infection.

Materials to be studied in the isolation of HSV depending on the localization of herpetic lesions

Localization of lesions	Contents of vesicles	Cell scraping				CSF	Aspirate from the bronchi	Bioptate	Blood
		1	2	3	4
Leather	+								+
Eyes			+						+
Genitals				+					+
Anus	+				+				+
Mouth	+	+							+
CNS	+					+		+	+
Lungs		+					+		+
Liver								+	+
Congenital Herpes	+	+	+				+		+

Methods of laboratory diagnosis of cytomegalovirus infection

Methods	Time required to get results	Notes
VIRUSOLOGICAL
Electron microscopy	3 hours	Not widely available
Isolation of the virus in cell culture (CPD)	4-20 days	Standard, Slow
Immunofluorescent staining of early AH with monoclonal antibodies	6 hours	Less specific
CYTOLOGICAL	2-3 hours	Less specific
SEROLOGICAL
RSK	2 days	Standard
WGA	1 day	Labour intensive
REEF	6 hours	Simple, specific
NERF	6 hours	Complicated
RIMF	6 hours	Complicated
ELISA (IgM, TO)	6 hours	Fast, simple
Immunoblot	6 hours	Expensive
MOLECULAR-BIOLOGICAL
MG	5-7 days	Costly, time-consuming
PCR	3 hours	Expensive

Methods for diagnosing the herpes zoster virus

Diagnostic Methods	Laboratory techniques
INDIRECT
Allocation	Fabric culture, chicken embryos, laboratory animals, co-cultivation with permissive cells or helper viruses
Identification of isolates	Neutralization reaction, DSC, IF, IPPE, reaction of precipitate isolates, agglutination, IF
STRAIGHT
Cytology	Smears: color immunofluorescence
Histology	Pathomorphology of the cell
Structure	Embryonic microscopy, immunoelectronic microscopy
Determination of antigens	IF, PIEF, RIM, ELISA
Determination of local production of antibodies	Ig M, Ig G, Ig A: ELISA, RIA
Molecular Biological Approaches	Molecular hybridization, PCR

Laboratory diagnosis of infection caused by the herpes zoster virus

Diagnostic problems	Methods	Expected results
Acute primary infection	1	Detection after 2 hours
	2	The level of antibodies grows slowly
	3	Present 3 days after infection
Acute reactivated infection	1	Detection of ULV in 2 hours
	2	The level of antibodies grows slowly
	4	Present 4 days after the appearance of rashes

1. the determination in the fluid of vesicles of WIEF;
2. serology: DSC, ELISA, aimed at identifying
3. Serology: ELISA, aimed at detecting IgM;
4. serology: ELISA, aimed at detecting IgA, IgM.

Methods for indicating the immune response of an infection due to the herpes zoster virus

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

Interpretation of results of serological examination of patient sera on herpesvirus infections (ELISA)

Name of infection / marker	Average thresholds for infections
	Analysis results	Interpretation
Cytomegalia Anti-CMV IgG (1-20 E / ml) Anti-CMV IgM (100-300%)	Positive 1-6 Positive 6-10 Positive> 10 Negative Positive 100-300 Negative <90 Doubtful 90-100	Remission Aggravation of the disease Acute phase of the disease No infection (disease) Acute phase of the disease Repeat the analysis after 2-3 weeks
Herpes simple 1,2 serotypes Anti-HSV 1/2 total. (100-900%)	Positive 100-400 Positive 400-800 Positive> 800 Negative <100	Remission Aggravation of the disease Acute phase of the disease No infection (disease)

The table presents the main methods of laboratory diagnostics of herpesvirus infections, and also recommends biological materials that are examined for the isolation of HSV, taking into account the localization of herpetic lesions

Reliable is the allocation of herpes simplex and CMV through the infection of sensitive cell cultures. Thus, in virological examination of 26 patients in the period of relapse, HSV was isolated on a sensitive Vero cell culture in 23 cases (88.4%). In infected cultures, a pattern of cytopathic action characteristic of HSV was observed-the formation of multinucleated giant cells or the accumulation of rounded and enlarged cells in the form of bunches. In 52.1% of cases, it was possible to detect foci of the cytopathic effect of the virus by 16-24 hours after infection. By 48-72 hours of incubation of infected cultures, the percentage of materials causing specific cell destruction increased to 87%. And only in 13% of cases, positive results were detected 96 hours after infection and more or with repeated passaging.

Methods of laboratory diagnosis of generalized herpetic infection

The main methods aimed at the detection (isolation) of herpesviruses, 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 the solid-phase enzyme-linked immunosorbent assay Variants of the molecular (DNA-DNA) hybridization method Polymerase chain reaction Latex agglutination reaction

Neutralization reaction Complement fixation reaction Latex agglutination reaction Indirect variant of fluorescent antibody method Indirect variant of immunoperoxidase method Variants of solid phase enzyme immunoassay Immune blotting method Radial complement fixation method Determination of levels of alanine and aspartate aminotransferases

Serological methods are used to diagnose infectious mononucleosis (infection caused by VEB). The reaction of Paul Bunnel with erythrocytes of a ram, a diagnostic titer of 1:28 and higher with a single study of serum, or a 4-fold increase in antibodies in the examination of paired sera. Use the Goff-Bauer reaction with a suspension of 4% formalized red blood cells of the horse. The result is taken into account after 2 minutes, with infectious mononucleosis the reaction is highly specific.

Currently, an enzyme immunoassay (ELISA) is being developed to diagnose infectious mononucleosis. In this case, IgG and IgM antibodies in the serum of the patient 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 above.

When using a number of imported commercial test systems in ELISA, antibodies to antigenes of the EBV membrane, antibodies to the early VEB antigen, general antibodies to the early VEB antigen, determined in the acute phase of the disease and in the nucleus and in the cytoplasm of the cell, and limited antibodies to early VEB, determined in the acute phase of the disease and in the nucleus and cytoplasm of the cell, limited antibodies to the early VEB antigen, determined at the height of the disease only in the cytoplasm of the cell, and antibodies to the nuclear antigen of EBV. The use of these test systems allows differential diagnosis of a number of diseases associated with EBV.

After a positive ELISA that showed antibodies to EBV, a confirmatory immunoblotting reaction is made, in which the presence of antibodies to individual marker proteins of EBV (p-proteins) is determined: p23, p54, p72 (the presence of this protein indicates the possibility of EBV multiplication), p 138. These above laboratory methods are used to control the effectiveness of treatment.

The sensitivity of the virological methods is 85-100%, the specificity is 100%, the study time is 2-5 days. Often in practice, the method of direct immunofluorescence (PIF) with polyclonal or monoclonal antibodies against HSV-1 and HSV-2 is used. The UIF method can be easily reproduced in a conventional clinical laboratory, it is not costly, the sensitivity is above 80%, the specificity is 90-95%. Immunofluorescence microscopy revealed the presence of cytoplasmic inclusions, morphological features, percentage of infected cells in smears-scrapings from the urethra, cervical canal, cervix, rectum.

The UIF method gives 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 herpetic infection according to the UIF data:

• aggregation of nuclear matter, exfoliation of karyoolma;
• presence of the so-called. Nuclei "holes", when only one karyolemma remains from the nucleus of the cell;
• the presence of intranuclear inclusions - Caudry's calf.

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

The content of infected cells in smears (PIF reaction) of patients with genital herpes, treated with acyclovir

Disease Periods	Percentage in smears
	Infected cells					Normal cells
	More than 75%	50-75%	40-50%	10%	Single cells in n / sp
Relapse (before treatment)	25%	63%	12%
	(20)	(50)	(10)
Remission (after treatment)				25%	31%	44%
				(20)	(25)	(35)

Using many years of UIF and the method of dot-hybridization, almost 100% of the cases coincided with the results of the study. It should be noted that in order to increase the reliability of GH diagnosis, especially in cases of subclinical and low-manifest forms of herpes, it is recommended to use 2-3 methods of laboratory diagnosis, especially when examining pregnant women, women with unsuccessful obstetric anamnesis, persons with unspecified gynecological diagnosis.

So, when PCR diagnosis of viral-bacterial infections of the urogenital tract, it is necessary to evaluate the positive results obtained taking into account anamnesis, the presence (or absence) of specific clinical symptoms of the disease. If chlamydia is detected with PCR, then in this case it is possible to talk about infection and solve the problems of therapy accordingly. In the case of detection of mycoplasmas (ureaplasmas), which are opportunistic microorganisms, additional culture studies are required to confirm the diagnosis, that is, the sowing of the material from the patient to sensitive cell cultures. Only when positive results are obtained in the culture analysis can we speak 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 medicinal forms (antibiotics, fluoroquinolones, etc.).

Perhaps a one-stage infection with several viruses of the family Nepresviridae. Often we detected infection of one patient with HSV-1, HSV-2 and CMV viruses. Much more often infected with several herpes viruses were patients with clinical and laboratory manifestations of secondary IDS (patients with oncohematological, oncological, HIV-infected). Thus, it is shown that clinical and immunological disorders progressing in HIV infection are accompanied by an increase in the number of herpesviruses detected by the molecular hybridization method. In this prognostically most significant can be considered a complex one-stage detection of DNA of HSV-1, CMV and HHV-6 type.

[1], [2], [3]