Herpes simplex virus: Resistance to acyclovir, penciclovir, foscarnet, cidofovir and adefovir( UL23 and UL30 -timidinaquinasa-DNA-polymerase- genes) - Genotypic tests (PCR and sequencing) and phenotypic assays (culture and IC50)
Herpes simplex virus (Herpes simplex virus) types 1 and 2 (HSV-1 and HSV-2) usually causes orolabial and genital infections, but can cause eye infections (herpes keratitis), meningoencephalitis, and acute mucocutaneous infections and other locations immunocompromised patients where the conditions are usually self - limiting. However, infections can spread and extended in immunocompromised patients, such as those infected with HIV (HIV), transplant recipients or solid organ transplant recipients bone marrow.
Transmission occurs through contact with secretions of an infected person, both with clinical or asymptomatic infections, which eliminate the virus. After primary infection, Herpes simplex virus establishes a long latency in the sensory nerve ganglia and may reactivate periodically. Reactivation may manifest clinically or can be silent and asymptomatic. The most common clinical manifestations are vesicular lesions affecting the mucous membranes, especially the mouth, nose or eyes for HSV-1 and anogenital region for HSV-2, although genital HSV-1 infections are increasingly found. As the symptoms caused by both generally self - limiting, treatment aims to accelerate the healing of injuries and prevent transmission in immunocompetent individuals. Both HSV-1 and HSV-2 can occasionally cause serious infections such as encephalitis and disseminated neonatal infections. HSV infections can be serious in immunocompromised patients, particularly those with impaired cellular immunity. In HIV - infected transplant recipients and solid organ or bone marrow, may cause extensive infections which tend to be longer and spread. In immunocompromised patients, and persistent mucocutaneous lesions, may cause visceral infections another location such as esophagitis, hepatitis or pneumonia.
Currently available several antiviral drugs for herpes simplex virus infections such as: acyclovir (with its prodrug valacyclovir), penciclovir (famciclovir with prodrug), foscarnet, cidofovir and adefovir. Acyclovir is the most commonly used antiviral drug, as well as treatment of Herpes simplex virus, it is used to treat infections Varicella-Zoster virus (VZV): Varicella zoster and Herpes.
Acyclovir (ACV) is a guanine nucleoside analogue, which requires phosphorylated three times to be incorporated into the viral DNA synthesis during virus replication. First phosphorylation participates in the top of stained origin (-TK- thymidine kinase), while in the second and third phosphorylation two cellular kinases are involved. Once completely phosphorylated, acyclovir triphosphate (ACV-TP) acts competitively with viral DNA polymerase and is incorporated into the viral DNA synthesis. By joining the phosphorylated analog which lacks the 3' - position hydroxyl group, it can not be continued synthesis thus acts as a chain terminator and viral DNA replication stops. Acyclovir is highly active against HSV-1, has half activity against HSV-2, a tenth of activity against Varicella zoster virus-and Epstein-Barr virus, and less activity against Cytomegalovirus. The major limitation of acyclovir is their limited oral bioavailability, so a derivative developed, the prodrug l-Valyl-ester (valaciclovir), which provides a 56% acyclovir after absorption. Valaciclovir (VACV), a prodrug that is converted in vivo to acyclovir after oral administration, and is absorbed by a human intestinal peptide carrier upon hydrolysis of the ester derivative in the small intestine. Exposed to acyclovir comments apply to valaciclovir.
Penciclovir (PCV) is a derivative of acyclic guanine with a similar spectrum of activity to acyclovir. It has a hydroxyl group 3'en the acyclic side chain derivative whose triphosphate (PCV-triphosphate) allows limited elongation (short chain terminator). Its activity is twice lower than ACV. Due to their limited absorption, prodrug famciclovir (FCV) which is a diacetylester 6-deoxipenciclovir developed. The first acetyl group is removed by esterases of the intestinal mucosa to absorb, and the second acetyl group is removed in the liver. The conversion of 6-deoxipenciclovir PCV is performed by the hepatic aldehyde oxidase.
Foscarnet is an analog of pyrophosphate anion (phosphonoformic acid) that inhibits viral DNA replication by binding to the binding site pyrophosphate viral DNA polymerase. Thus pyrophosphate release terminal nucleotide triphosphate added in the growing chain of DNA, and acts as a noncompetitive inhibitor of the activity of DNA polymerase is blocked. It exerts its action at concentrations not inhibit human DNA polymerases. Unlike what happens with acyclovir, foscarnet need not be activated by viral kinases or cell, so it can be used to treat herpes simplex virus infections resistant to nucleoside analogues. Foscarnet is active against all herpesviruses, although its main indication Cytomegalovirus infections are resistant to ganciclovir, and is considered a second - line drug for the therapy of Herpes simplex virus. However, strains resistant to acyclovir virus mutations DNA polymerase may be resistant to foscarnet.
Cidofovir is an acyclic nucleotide analogue of cytidine [(S) -1- (3-hydroxy-2-phosphonyl-methoxy-propyl) -cytosine], which does not require phosphorylation by viral kinases, and becomes deoxycytidine triphosphate by cellular kinases acting as a chain terminator to be incorporated by DNA polymerase viral replication of viral DNA. This drug was introduced for the treatment of cytomegalovirus retinitis, but has been shown active against strains of herpes simplex virus resistant to acyclovir and / or foscarnet, causing progressive mucocutaneous HSV infections in immunocompromised patients, though it was not approved for this indication. It is active in vitro against other DNA viruses: Adenovirus, polyomavirus BK - virus, and poxvirus Papillomavirus.
Adefovir is an analogue of adenine [(S) -1- (3-hydroxy-2-phosphonyl-methoxy-propyl) adenine] that does not require phosphorylation by viral kinases and is active against hepatitis B virus and also against to a herpes virus infection. Cidofovir and adefovir, not requiring activation by thymidine kinase (TK), are active against herpes simplex isolates TK-TK-negative or altered. Their active forms, are competitive inhibitors of DNA polymerase and act as chain terminators.
Acyclovir resistance mutations
Changes thymidine kinase (TK), responsible for resistance to acyclovir, penciclovir with cross-resistance, they are the most common and appear in 95% of the resistant isolates. They described three types of resistances to acyclovir due to alterations of the thymidine kinase (TK):
- TK-negative mutants, which have no functional protein for phosphorylation analog acyclovir.
- Mutants TK-biased, having a concentration of TK too low to perform efficient phosphorylation of acyclovir.
- Mutants TK-altered, producing an enzyme that phosphorylates acyclovir less efficiently.
Since the introduction of acyclovir in the 80s (1980s), it has been the drug of choice for prophylaxis and treatment of herpes simplex virus infections. Despite the widespread use of acyclovir, the prevalence of resistant isolates is considered low. In immunocompetent patients it is estimated to be 0.5%. HSV infections resistant to acyclovir are an important clinical problem in immunocompromised. In these patients , the prevalence of resistance is about 5% but may reach 14 to 30% in the transplant allogeneic bone marrow. Injuries can become chronic and widespread dissemination risk. The change to a therapy with other drugs that do not require the action of thymidine kinase, such as foscarnet, cidofovir or adefovir, as soon as possible to be effective must be performed.
Thymidine kinase is a protein of 376 amino acids encoded by the UL23 gene of 1,128 bp six conserved regions including a binding site for ATP (aa 51-63), a place of attachment to nucleosides (aa 168-176), and a cysteine at codon 336, important for conformal functional active site of the enzyme. There are six highly conserved regions in the TK enzyme localized in amino acids 56-62 (site 1), 83-88 (site 2), 162-164 (spot 3), 171-173 (spot 4), 216-222 (place 5), 284-289 (location 6). The TK HSV-2 has 375 amino acids with six conserved domains in amino acids 56 to 62, 83 to 88, 163 to 165, 172 to 1q74, 217-223 and 285-290.
Characterization of isolates sensitive to acyclovir has demonstrated a high polymorphism in the sequence of UL23 thymidine kinase gene (TK). These mutations are not associated with resistance and are located outside the active site of the enzyme. This is important, since when the determination of resistance by genotypic testing is performed, the finding of variations (mutations) in the genomic sequence of gene URL23 not always imply the existence of drug resistance.
Half of the cases of resistance to acyclovir due to insertions (additions) or nucleotide deletions, which usually occur in homopolymeric regions repeated guanine nucleotide (Gs) or cytosine nucleotides (Cs) in the UL23 gene, where insertions (additions) or deletions result in a read offset generating a truncated nonfunctional enzyme. The two longer homopolymers, a 7 and a 6 Gs Cs, are where mutations most frequently in clinical isolates resistant to ACV are. Other mutated sequences of UL23 have a deletion in the 3 chain Cs or deletion of a sequence of G 3 Gs. Half of the other cases are due to nucleotide substitution, most of them active or preserved enzyme sites.
The main mutations described UL23 (TK) gene are:
Insertions and / or deletions Gs, Cs, As or Is, in homopolymeric regions (d: deletion; i: insertion; d / i: both) (underlined in HSV-2):
180-183 (d) 4GS; 184-187 (d) 4As; 215-217 (d) 3Cs; 219-222 (d) 4GS; 430-436 (i / d) 7gs; 433-439 (i / d) 7gs; 455-458 (d) 4Cs; 460-464 (d) 5Cs; 519-521 (d) 3Cs; 548-553 (i / d) 6CS; 551-556 (i / d) 6CS; 586-590 (i) 5Cs; 626-627 (i) 2Is; 668-669 (d) 4Cs; 808-811 (d) 4Cs; 878-880 (d) 3Gs; 896-900 (d) 5Cs; 1061-1064 (d) 4Cs.
Mutations in conserved locations (underlined in HSV-2):
- Place 1 (amino acids 56-62): R51W, Y53stop, D55N, G56S / V, P57H, H58R / L, G59R / V, G59P, G61V, K62N, T63A / I / S.
- Site 2 (amino acids 83-88): E83K, P84S,
- Location 3 (amino acids 162-164): D162A, R163H.
- Place 4 (amino acids 171-173): A68T, P173L / R, A175V, R176Q, R177W.
- Place 5 (amino acids 216-222): R216H / C / S, R217H, R220C / H, R222C, R223H.
- Place 6 (amino acids 284-289): T287M, L297S.
- Cysteine 336 or 337: C336Y, C337Y.
Mutations in non - conserved sites (underlined in HSV-2): T65N, Q104H / stop; Q105P, H105P, Q125E / L, P131S, T131P, Q144N, L158P, A167V, T245M, S182N, Q185R, V187M, A189V, G200C, T201P, E201D, R271V, P272S, D273R, R281stop, L364P.
Resistance mutations DNA polymerase
Mutations of the DNA polymerase are less common and accepted that only occur in 5% of isolates of herpes simplex virus resistant to acyclovir.
Viral DNA polymerase is a heterodimer encoded by UL30 UL42 genes and with 1235 and 1240 amino acids, HSV-1 and HSV-2, respectively. The UL30 gene encodes the catalytic subunit whereas the UL42 gene encodes a phosphoprotein activity to bind to double stranded DNA. DNA polymerase Herpes simplex virus is a multifunctional enzyme with polymerase activity to extend the DNA chain, an intrinsic activity 3'a 5'error corrector exonuclease activity and RNAse H (RNAse H) that eliminate the primers initiate the synthesis of Okazaki fragments on the replication fork. Furthermore, the catalytic subunit would interact with UL30 UL42 subunit accessory that acts as a factor processing. DNA polymerase of Herpes belongs to the family of DNA polymerases ?-like, closely related to DNA polymerases ?. In half of the carboxy terminal portion of the catalytic subunit exist conserved regions numbered I to VII as their conservation, being the most conserved region I. Also it contains a C-? region, similar to cellular polymerases ?. The order of these regions at the genomic level is: IV, ? region, II, VI, III, I, VII and V.
Mutations of the DNA polymerase found in clinical isolates Amino acid substitutions are located in the region II, III, VI and VII, directly or indirectly involved in the recognition nucleotide binding or pyrophosphate, and in catalysis. The largest number of mutations is grouped in regions II and III. Mutations in the conserved regions II and VII are often associated with resistance to acyclovir and foscarnet. Some mutations have been described in other conserved regions or outside these regions.
Gene mutations in UL30 (DNA polymerase) (Underlined the HSV-2)
- Region IV (amino acids 437-479; 438-480): -
- ? C region (amino acids 577-637; 578-638): Y577H; D581A; E597K / D; A605V;
- Region II (amino acids 694-736; 699-741): R700G; V715G / M; A719T / V; A724T; S724N; S729N.
- Region VI (amino acids 772-791; 777-796): L774F; L778M; D780N; D785N, L782I.
- Region III (amino acids 805-845; 810-850): V813M; N815S; T821M; G841S; R842S.
- Region I (amino acids 881-896; 886-901): G885A / R; D886N; T887K; D888A; S889A; F891C / Y; V892M;
- Region VII (amino acids 938-946; 943-951): Y941H.
- Region V (amino acids 953-963; 954-968): N961K.
- In other areas: D368A; E370A; E460D; V462A; G464V; K522E; P561S; V573M; P797T; D907V; D912V / A.
Foscarnet resistance mutations
Most foscarnet resistance mutations have nucleotide substitutions in the conserved regions II, III, VI or VII and a non - conserved region between the regions I and VII of the UL30 gene.
Some isolated retain their sensitivity to acyclovir and cidofovir. However, mutations in regions II and VII may confer resistance to acyclovir and foscarnet.
- Region II: V715G; S724N; S729N
- Region VII: Y941H.
Cidofovir resistance mutations
Mutations associated with resistance to cidofovir are located in the following regions of the UL30 gene (DNA polymerase):
- Region II: R700M.
- Region III: G841C; G850I.
- Region VI: L773M.
- Region VII: Y941H
- C ? region: V573M.
Adefovir resistance mutations
Although adefovir was introduced for therapeutic of chronic infections with hepatitis B, it is active against herpes simplex virus, including resistant acyclovir and foscarnet isolated. Resistant foscarnet isolated, with mutations in regions II and VI, and between regions I and VII of DNA polymerase show a decreased susceptibility to adefovir, suggesting that could be selected isolated herpes simplex virus resistant adefovir in patients treated with foscarnet. The S724N and L778M mutations that confer cross - resistance to acyclovir and foscarnet, cause decreased sensitivity to both cidofovir and adefovir.
Phenotypic tests for IC50
Phenotypic resistance testing with exposure to infected with various concentrations of antiviral cell culture are reference tests (Gold Standard) and serving to demonstrate elevation of inhibitory concentrations against each of the drugs. These tests are not subject to the interpretation of the findings of genomic mutations that can not imply resistance, but only one gene polymorphism thymidine kinase or DNA polymerase. The problem with these tests is slow and therefore have been replaced by evidence of genotypic resistance that can provide results in a period much shorter time, and can also be made directly with clinical samples without isolating previously virus in cell culture.
Tests in IVAMI:
- Genotypic resistance tests by molecular detection of resistance mutations in UL23 (thymidine kinase) gene and the UL30 gene (DNA polymerase) (PCR and sequencing).
- Phenotypic resistance tests by virus isolation and determination of inhibitory concentrations IC50 in cell culture.
- Herpetic lesion sample. This sample can be used for genotypic resistance testing and is essential to isolate the virus and perform phenotypic resistance testing (IC50 concentrations).
- Cepa (isolation) Herpes simplex virus isolated in the laboratory. This strain can be used for testing and genotypic resistance tests for phenotypic resistance.
- Extracted DNA sample that has provided a positive molecular test (PCR) for Herpes simplex virus. The extracted DNA can only be used for genotypic resistance tests.
Preservation and shipment of sample:
- Refrigerated (preferred) for less than 2 days.
- Frozen for more than 2 days.
Cost of the test:
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