WEBVTT 1 00:00:08.233 --> 00:00:10.500 Hello, my name is Miranda Chimchar. 2 00:00:10.500 --> 00:00:13.933 I am a Pgy two pathology resident 3 00:00:13.933 --> 00:00:17.300 at air U P laboratories in the University of Utah. 4 00:00:17.833 --> 00:00:22.666 Today I'm going to be talking to you about the herpes simplex viruses one and two. 5 00:00:23.066 --> 00:00:24.933 And I'm going to start with the basics. 6 00:00:24.933 --> 00:00:29.000 So it should be approachable to all levels of learning. 7 00:00:29.700 --> 00:00:32.700 And so we'll go on to my objectives. 8 00:00:33.433 --> 00:00:36.266 First I'm going to be talking about the evolution 9 00:00:36.266 --> 00:00:39.800 and classification of the herpes simplex viruses one and two. 10 00:00:40.366 --> 00:00:43.166 And I'll call them HSV one. And HSV two. 11 00:00:43.166 --> 00:00:46.933 Periodically throughout the talk I'll explain the symptoms 12 00:00:46.933 --> 00:00:50.266 transmission management and prevention of infection. 13 00:00:50.800 --> 00:00:54.800 And I'll lastly describe the testing modalities we have for herpes 14 00:00:54.800 --> 00:00:58.733 simplex viruses and their indications and limitations. 15 00:01:01.366 --> 00:01:03.500 First I'm going to start very broadly 16 00:01:03.500 --> 00:01:07.833 and talk about how the herpes simplex viruses are classified. 17 00:01:08.233 --> 00:01:11.533 They're part of the herpes verde family which includes 18 00:01:11.533 --> 00:01:14.933 envelope viruses with linear double stranded DNA. 19 00:01:15.733 --> 00:01:19.500 This, family of viruses not only includes the herpes 20 00:01:19.500 --> 00:01:24.133 simplex viruses one and two, but also varicella zoster virus, 21 00:01:24.133 --> 00:01:27.733 which is responsible for chicken pox and shingles. 22 00:01:28.033 --> 00:01:33.866 The Epstein-Barr virus, which is responsible for mono cytomegalovirus. 23 00:01:33.866 --> 00:01:38.600 Human herpes virus six seven, Kaposi's sarcoma associated herpes 24 00:01:38.600 --> 00:01:42.066 virus, and most recently herpes B virus 25 00:01:42.600 --> 00:01:46.266 which was found in other primates but has been shown to jump 26 00:01:46.266 --> 00:01:50.300 to humans and cause inflammation around the brain or encephalitis. 27 00:01:51.700 --> 00:01:54.700 The families further divided into subfamilies. 28 00:01:54.733 --> 00:01:57.733 I've bolded the alpha herpes viruses. 29 00:01:57.766 --> 00:02:00.766 That is where the herpes simplex viruses fall. 30 00:02:00.766 --> 00:02:02.166 first I'm going to talk a little bit 31 00:02:02.166 --> 00:02:05.166 about the structure of the herpes simplex viruses. 32 00:02:05.433 --> 00:02:10.433 Once again it has a linear double stranded DNA depicted here. 33 00:02:10.933 --> 00:02:14.666 The capsid depicted in this orange red 34 00:02:14.666 --> 00:02:18.900 color is about 125 nanometers in diameter, 35 00:02:19.133 --> 00:02:22.866 and forms a 20 sided polygon or icosahedral. 36 00:02:23.733 --> 00:02:26.200 The envelope depicted here 37 00:02:26.200 --> 00:02:29.566 is actually made up from the host cells, 38 00:02:30.000 --> 00:02:33.200 and it is studded with the viral membrane proteins. 39 00:02:33.566 --> 00:02:36.966 Most importantly would be the glycoproteins B and D, 40 00:02:36.966 --> 00:02:40.333 which help the virus bind to host cells and infect them. 41 00:02:41.000 --> 00:02:46.033 And then lastly, between this envelope and capsid, we have this space 42 00:02:46.033 --> 00:02:50.366 called the argument that's filled with complex multi subunit proteins. 43 00:02:50.866 --> 00:02:55.100 For herpes simplex virus one, it's primarily orally transmitted. 44 00:02:55.200 --> 00:02:58.266 But it can affect the genitals as well. 45 00:02:58.600 --> 00:03:02.266 Increasingly we're seeing in certain populations 46 00:03:02.333 --> 00:03:05.533 more infections of HSV with genital lesions, 47 00:03:05.966 --> 00:03:09.966 especially for those, young women and men who have sex with men. 48 00:03:10.966 --> 00:03:11.333 This, 49 00:03:11.333 --> 00:03:14.433 though, primarily is going to be orally transmitted. 50 00:03:14.600 --> 00:03:19.500 So affecting the mucosa of the mouth, nose and eyes. 51 00:03:19.766 --> 00:03:24.200 And because it is primarily orally transmitted, it's, most commonly 52 00:03:24.500 --> 00:03:27.600 an infection that occurs first in childhood. 53 00:03:28.233 --> 00:03:32.100 It's estimated that 3.7 billion people worldwide 54 00:03:32.100 --> 00:03:34.566 are affected under the age of 50. 55 00:03:34.566 --> 00:03:39.100 And that number is going to be highest in low to middle income countries, 56 00:03:39.100 --> 00:03:44.133 such, such as sub-Saharan Africa and Latin America, where serology, 57 00:03:44.300 --> 00:03:49.000 meaning those that have antibodies against the virus are greater than 90%. 58 00:03:49.766 --> 00:03:54.500 Looking at the United States, it's about 50 to 70% of healthy adults 59 00:03:54.500 --> 00:03:57.500 that have positive antibodies for this virus 60 00:03:59.266 --> 00:04:02.266 compared to herpes simplex virus two, 61 00:04:02.300 --> 00:04:05.966 which is normally sexually transmitted with genital lesions. 62 00:04:06.466 --> 00:04:10.033 Though this virus can infect the oral mucosa as well. 63 00:04:10.233 --> 00:04:13.566 We see it a lot less frequently than we do 64 00:04:13.566 --> 00:04:16.733 see HSV one crossing over to the genitals. 65 00:04:17.766 --> 00:04:19.900 It is estimated that 491 66 00:04:19.900 --> 00:04:25.333 million people between the ages of 15 and 49 are infected worldwide, 67 00:04:25.833 --> 00:04:29.500 and an estimated 11.9% of people from again 68 00:04:29.566 --> 00:04:33.233 the age of 14 to 49, in the United States are infected. 69 00:04:34.633 --> 00:04:39.466 That starting age is a little bit higher than for HSV one, 70 00:04:39.700 --> 00:04:43.233 because, again, this is primarily a sexually transmitted disease. 71 00:04:43.500 --> 00:04:45.333 And so we don't see it. 72 00:04:45.333 --> 00:04:49.533 People see people becoming infected until they're sexually active. 73 00:04:50.300 --> 00:04:53.700 Women are two times more likely than men to be infected, 74 00:04:53.800 --> 00:04:56.700 and it's hypothesized that men have an easier time 75 00:04:56.700 --> 00:05:00.433 transmitting it to women than women have transmitting it to men. 76 00:05:02.100 --> 00:05:04.833 Compared to HSV one, there's more 77 00:05:04.833 --> 00:05:08.466 frequent recurrence or outbreaks of, the lesions. 78 00:05:08.766 --> 00:05:11.866 And there's more subclinical shedding, meaning that 79 00:05:11.866 --> 00:05:16.100 they may be asymptomatic but still shedding viral particles. 80 00:05:17.100 --> 00:05:19.400 And then those that are infected with HSV 81 00:05:19.400 --> 00:05:24.633 two are 2 to 3 times more likely to acquire an HIV infection. 82 00:05:25.266 --> 00:05:27.966 And that's because they'll have these 83 00:05:27.966 --> 00:05:31.966 frequent sores, and outbreaks of the HSV two, 84 00:05:32.200 --> 00:05:35.866 which allows, a break in the skin barrier 85 00:05:35.866 --> 00:05:38.866 and susceptibility to HIV. 86 00:05:41.300 --> 00:05:43.100 This is just reiterating 87 00:05:43.100 --> 00:05:48.200 what I said on the past two slides and wanting to kind of side 88 00:05:48.200 --> 00:05:51.666 by side, compare HSV one to HSV two. 89 00:05:52.133 --> 00:05:56.500 So again, the site of the lesions for HSV one is primarily oral 90 00:05:56.500 --> 00:06:00.366 but increasingly genital, especially in those, particular 91 00:06:00.366 --> 00:06:04.966 population populations for HSV two primarily genital. 92 00:06:05.233 --> 00:06:07.966 Again, can get a little crossover, 93 00:06:07.966 --> 00:06:10.966 but not as much as HSV one. 94 00:06:10.966 --> 00:06:14.000 The prevalence is going to be higher for HSV 95 00:06:14.000 --> 00:06:17.900 one than it is HSV two, and that's true worldwide. 96 00:06:17.900 --> 00:06:23.533 And in the United States, the associated risk for HSV two would be, 97 00:06:23.666 --> 00:06:27.666 you have an increased risk of contracting HIV 98 00:06:27.933 --> 00:06:31.433 and that women are two times more likely than men to contract it. 99 00:06:31.866 --> 00:06:34.000 And then clinical considerations, 100 00:06:35.000 --> 00:06:38.400 if someone is infected with HSV two, 101 00:06:38.866 --> 00:06:41.866 they are three times more likely to have 102 00:06:41.866 --> 00:06:47.000 a subclinical infection by HSV one if they are infected by HSV one, 103 00:06:47.333 --> 00:06:51.766 meaning that if they're infected, they likely will not have any lesions 104 00:06:51.766 --> 00:06:54.766 or sign of that HSV one infection. 105 00:06:54.800 --> 00:06:58.800 And then for HSV two, again, there's going to be more subclinical shedding. 106 00:06:59.033 --> 00:07:02.166 There's going to be more frequent recurrences of the lesions. 107 00:07:05.000 --> 00:07:06.400 For both of them the 108 00:07:06.400 --> 00:07:10.000 the symptoms include with the initial infection kind of a flu 109 00:07:10.000 --> 00:07:13.733 like picture with fever, body aches, swollen lymph nodes 110 00:07:13.733 --> 00:07:16.733 and then the sores on the mucous membranes. 111 00:07:16.733 --> 00:07:20.533 And you can see in this picture the sores are very widespread. 112 00:07:20.533 --> 00:07:21.866 And that initial infection, 113 00:07:21.866 --> 00:07:25.000 they're going across the lips and even involving the tongue. 114 00:07:26.133 --> 00:07:28.300 After that initial infection 115 00:07:28.300 --> 00:07:31.500 the virus becomes dormant in the sensory nerve ganglion. 116 00:07:31.833 --> 00:07:35.000 So this is an infection that you never clear completely. 117 00:07:35.000 --> 00:07:40.300 It's always dormant in your body and can be reactivated when it is reactivated. 118 00:07:40.300 --> 00:07:42.300 You get these subsequent outbreaks. 119 00:07:42.300 --> 00:07:45.300 Usually there's heralding symptoms such as burning 120 00:07:45.300 --> 00:07:48.300 or tingling prior to the sores appearing. 121 00:07:48.833 --> 00:07:51.733 And this is a picture of a subsequent outbreak. 122 00:07:51.733 --> 00:07:54.200 Usually the sores are very local 123 00:07:54.200 --> 00:07:57.200 rather than widespread, like the initial infection, 124 00:07:58.300 --> 00:08:01.800 these sores are usually preceded by some sort of stressor, 125 00:08:01.800 --> 00:08:06.900 such as a cold or, just stress at work, things like that. And 126 00:08:06.900 --> 00:08:10.066 so that's probably where the colloquial, colloquial 127 00:08:10.066 --> 00:08:13.066 cold sores came from. 128 00:08:13.100 --> 00:08:17.066 It can also cause inflammation around the brain, or encephalitis, 129 00:08:17.333 --> 00:08:21.000 which would cause altered mental status, headache and seizures. 130 00:08:23.933 --> 00:08:26.833 The transmission is through contact with the virus. 131 00:08:26.833 --> 00:08:30.800 This could be through open sores, saliva, other body fluids 132 00:08:30.800 --> 00:08:33.033 such as semen or vaginal fluid. 133 00:08:33.033 --> 00:08:35.600 In contact with mucous membranes. 134 00:08:35.600 --> 00:08:40.866 The greatest risk of transmission is when coming in contact with active sores, 135 00:08:41.166 --> 00:08:45.100 but it can also be transmitted while the infection is dormant. 136 00:08:45.833 --> 00:08:49.133 Rarely, there's transmission from mother to baby. 137 00:08:49.500 --> 00:08:53.633 It happens in about ten out of 100,000 births worldwide. 138 00:08:54.000 --> 00:08:56.666 for the general population who are not pregnant. 139 00:08:56.666 --> 00:09:00.200 We only recommend testing for those that are symptomatic. 140 00:09:00.200 --> 00:09:03.833 So have lesions that are suspicious for an HSV infection. 141 00:09:04.333 --> 00:09:07.100 High risk populations, such as those that have ten 142 00:09:07.100 --> 00:09:11.600 or more sexual partners, are HIV positive or positive. 143 00:09:11.600 --> 00:09:14.600 For other sexually transmitted diseases, 144 00:09:15.100 --> 00:09:18.133 the symptoms are managed using antivirals. 145 00:09:18.433 --> 00:09:23.233 I have, bolded the acyclovir because that's the most common one. 146 00:09:23.833 --> 00:09:28.500 And again, I've emphasized that this is symptom management and not treatment, 147 00:09:28.733 --> 00:09:33.266 because the virus will always remain dormant in the person's system. 148 00:09:33.700 --> 00:09:35.633 It's never fully cleared. 149 00:09:35.633 --> 00:09:39.500 But by using these antivirals, we're able to reduce the viral shedding 150 00:09:39.500 --> 00:09:41.466 and allowed the source to heal quicker. 151 00:09:42.733 --> 00:09:44.500 Prevention is avoiding coming 152 00:09:44.500 --> 00:09:48.400 in contact with those, especially that have open sores. 153 00:09:48.766 --> 00:09:53.433 Condoms do offer some protection and for the oral lesions, 154 00:09:53.433 --> 00:09:56.833 not sharing food, beverages and cutlery is important. 155 00:09:59.466 --> 00:10:00.733 There are multiple testing 156 00:10:00.733 --> 00:10:04.000 modalities available for herpes simplex virus, 157 00:10:04.266 --> 00:10:08.100 and we're going to go through each of them and their indications and how they work. 158 00:10:08.533 --> 00:10:11.533 The first, for those that have current lesions, 159 00:10:11.800 --> 00:10:15.833 we can either do a viral culture, which has been the traditional gold standard, 160 00:10:16.166 --> 00:10:19.633 and it's often paired with the direct fluorescent antibody stain. 161 00:10:20.000 --> 00:10:25.266 Or we can do PCR testing for those that have a history of lesions, 162 00:10:25.266 --> 00:10:30.133 but no current lesions, we'd recommend that antibody testing or serology is. 163 00:10:30.833 --> 00:10:34.766 And then for actual slides of tissue, 164 00:10:35.100 --> 00:10:38.100 we can recommend immunohistochemical staining. 165 00:10:40.866 --> 00:10:42.600 Starting off with the direct fluorescent 166 00:10:42.600 --> 00:10:47.033 antibody stain this is often paired with viral cultures. 167 00:10:47.400 --> 00:10:50.233 It is rapid results compared 168 00:10:50.233 --> 00:10:53.233 to viral cultures that take time to actually grow. 169 00:10:54.000 --> 00:10:57.000 But the problem is that it has a lower sensitivity, 170 00:10:57.333 --> 00:11:00.800 meaning that if you get a negative result, there's a good chance 171 00:11:00.800 --> 00:11:02.100 that it's a false negative. 172 00:11:02.100 --> 00:11:04.633 And so you have to confirm it with viral culture. 173 00:11:07.966 --> 00:11:09.033 So for these 174 00:11:09.033 --> 00:11:12.133 specimens, usually we get a swab 175 00:11:12.266 --> 00:11:17.233 that, has has gone over a lesion in some sort of matrix. 176 00:11:17.466 --> 00:11:21.000 And we just want to get all of those particles off of the swab 177 00:11:21.000 --> 00:11:22.433 and into the fluid. 178 00:11:22.433 --> 00:11:25.433 So usually we vortex it. 179 00:11:25.900 --> 00:11:27.433 And then we're going to take some of that 180 00:11:27.433 --> 00:11:30.433 fluid and pour it off into a tube. 181 00:11:31.633 --> 00:11:34.633 We then want to spin it down. 182 00:11:35.300 --> 00:11:37.633 And in this case we. 183 00:11:37.633 --> 00:11:40.633 So we get a supernatant and a pellet. 184 00:11:40.633 --> 00:11:42.800 And we want to remove the supernatant. 185 00:11:42.800 --> 00:11:45.533 That liquid on top and just leave the pellet. 186 00:11:45.533 --> 00:11:48.500 So we're concentrating all the cells, 187 00:11:48.500 --> 00:11:51.500 that might be infected by the herpes simplex virus. 188 00:11:53.266 --> 00:11:54.966 We're then going to re suspend 189 00:11:54.966 --> 00:11:57.966 that pellet into our own solution. 190 00:11:59.200 --> 00:12:00.700 By vortex again. 191 00:12:01.766 --> 00:12:04.233 And then we put a drop onto the plate. 192 00:12:04.233 --> 00:12:07.633 This is a method used for quite a few different viruses. 193 00:12:07.633 --> 00:12:10.700 But because we have herpes simplex one and two, 194 00:12:10.933 --> 00:12:14.000 we have two wells on our plate, one for each. 195 00:12:15.900 --> 00:12:17.833 We allow that to dry, 196 00:12:17.833 --> 00:12:22.933 and then we fix those cells to the plate using some sort of fixing solution 197 00:12:22.933 --> 00:12:25.933 that usually contains acetone on. 198 00:12:26.866 --> 00:12:31.066 We wait for that to dry and then we add our reagents. 199 00:12:31.066 --> 00:12:34.933 So there's different tests, but each one is going to have 200 00:12:34.933 --> 00:12:39.633 a reagent specific for HSV one and a reagent specific for HSV two. 201 00:12:40.233 --> 00:12:42.266 And we let that incubate. 202 00:12:42.266 --> 00:12:46.300 And what's happening is that in that reagent we have antibodies 203 00:12:46.533 --> 00:12:49.966 that are attached to a fluorescent molecule. 204 00:12:50.833 --> 00:12:53.033 And when they incubate, 205 00:12:53.033 --> 00:12:56.033 they can attach to the cells 206 00:12:56.066 --> 00:12:59.800 that are expressing antigens for HSV 1 or 2. 207 00:13:00.633 --> 00:13:03.633 And so they attach to the cells. 208 00:13:04.033 --> 00:13:08.400 We do a washing stage to wash off any antibody that's not found. 209 00:13:08.900 --> 00:13:12.133 And then we're able to look at them under a fluorescent microscope. 210 00:13:13.166 --> 00:13:15.300 And usually 211 00:13:15.300 --> 00:13:18.666 ones that are positive are going to have this green color. 212 00:13:18.900 --> 00:13:22.533 And those that are negative are going to have this red color in the background. 213 00:13:23.033 --> 00:13:26.033 And so this would be considered a positive test. 214 00:13:28.133 --> 00:13:29.866 But once again the viral 215 00:13:29.866 --> 00:13:33.866 culture is the the traditional gold standard. 216 00:13:33.866 --> 00:13:36.966 And for all of the previous testing that was negative, 217 00:13:36.966 --> 00:13:39.966 we'd want to confirm with viral cultures. 218 00:13:40.066 --> 00:13:43.066 And so again we have that specimen. 219 00:13:43.433 --> 00:13:46.033 And in this time we're going to centrifuge it down. 220 00:13:46.033 --> 00:13:47.500 And we don't want that pellet. 221 00:13:47.500 --> 00:13:50.500 We're getting rid of all of that cellular debris here. 222 00:13:50.833 --> 00:13:54.133 And just leaving any viral material in that supernatant. 223 00:13:54.766 --> 00:13:57.266 And we put some of that supernatant 224 00:13:57.266 --> 00:14:00.266 into these special viral vials 225 00:14:00.333 --> 00:14:03.333 that are coated with cells that, 226 00:14:04.266 --> 00:14:08.300 react in certain ways when infected with herpes simplex virus. 227 00:14:11.166 --> 00:14:14.733 We centrifuge again, we're getting all of that viral material 228 00:14:14.733 --> 00:14:18.966 from the supernatant down into the cells that are coating this vial. 229 00:14:20.466 --> 00:14:21.700 And then we're going to incubate. 230 00:14:21.700 --> 00:14:26.566 And again, this often is for quite a while because we need those cells to grow. 231 00:14:26.933 --> 00:14:29.866 So we're able to observe them. 232 00:14:29.866 --> 00:14:32.866 And then we examine them microscopically 233 00:14:33.133 --> 00:14:38.333 and depending on the test, sometimes we're looking for psychopathic effect. 234 00:14:38.333 --> 00:14:40.733 Like I talked about those three times. 235 00:14:40.733 --> 00:14:46.500 The molding margins and multi nucleation or some of the tests 236 00:14:46.500 --> 00:14:49.566 actually have changes that occur in the cell if they're infected. 237 00:14:49.866 --> 00:14:52.866 And we're able to see those visually as well. 238 00:14:55.166 --> 00:14:58.033 Next up PCR testing. 239 00:14:58.033 --> 00:15:03.133 So for PCR testing there's a lot of different things that go into each tube. 240 00:15:03.433 --> 00:15:05.800 The first is our DNA sample. 241 00:15:05.800 --> 00:15:09.566 So it has to be purified in some way and broken out of the cells. 242 00:15:09.933 --> 00:15:13.033 And in this case, again, this is a double stranded DNA. 243 00:15:13.766 --> 00:15:16.166 We have primers and probes, which I'll talk in 244 00:15:16.166 --> 00:15:19.166 about in a little bit of what they're used for. 245 00:15:19.533 --> 00:15:22.833 We have the nucleotides, which are the building blocks of DNA, 246 00:15:23.433 --> 00:15:27.833 the Tak polymerase, which is the enzyme that actually builds the DNA. 247 00:15:28.633 --> 00:15:33.300 There's some sort of buffer, and then either a PCR tube 248 00:15:33.566 --> 00:15:36.466 or for larger, runs 249 00:15:36.466 --> 00:15:39.900 with more samples, you can use a 96 well plate as well. 250 00:15:41.633 --> 00:15:44.666 And so the PCR goes through these cycles. 251 00:15:45.200 --> 00:15:49.200 And I will emphasize that these temperatures are not 252 00:15:49.866 --> 00:15:53.200 the like not the certain temperature that it has to be at. 253 00:15:53.200 --> 00:15:56.000 And it's more relative. 254 00:15:56.000 --> 00:15:58.933 And also I'll discuss that as I go. 255 00:15:58.933 --> 00:16:02.966 So first of all we have denaturing of the, the DNA. 256 00:16:03.366 --> 00:16:06.033 And so this is the separation of the strands. 257 00:16:06.033 --> 00:16:09.033 And for this it needs to be really, really hot. 258 00:16:10.533 --> 00:16:11.800 So the strands separate. 259 00:16:11.800 --> 00:16:13.833 And then we lowered the temperature 260 00:16:13.833 --> 00:16:17.066 and we allowed the primers and the probes to bind. 261 00:16:17.633 --> 00:16:20.333 And so the probes or excuse me 262 00:16:20.333 --> 00:16:25.633 the primers are allow a starting point for the tack preliminary polymerase 263 00:16:25.633 --> 00:16:28.633 to work and replicate the DNA. 264 00:16:28.766 --> 00:16:32.066 And the probes allow us to monitor the reaction and tell 265 00:16:32.066 --> 00:16:34.400 how much DNA is there. 266 00:16:34.400 --> 00:16:36.966 And it can happen at two different phases. 267 00:16:36.966 --> 00:16:40.900 The first one, the annealing, where the probes and the primers attach is one spot. 268 00:16:40.900 --> 00:16:43.200 We can monitor it, which is why I have this. 269 00:16:43.200 --> 00:16:46.966 I hear it can also happen at the next stage 270 00:16:47.733 --> 00:16:50.833 where it gets a little bit warmer, and that allows the tack 271 00:16:50.900 --> 00:16:55.933 polymerase to work efficiently and this is when we're actually extending the DNA. 272 00:16:55.933 --> 00:16:59.766 And so when we end we have two new strands of DNA. 273 00:16:59.800 --> 00:17:03.866 So we've exponentially increased our DNA each cycle. 274 00:17:04.366 --> 00:17:07.333 And this is the other time where we're able to monitor 275 00:17:07.333 --> 00:17:10.333 the reaction and see how much DNA is present 276 00:17:10.733 --> 00:17:15.333 and where we analyze and see where the DNA 277 00:17:15.333 --> 00:17:19.466 or how much DNA we have depends on what probes we're using. 278 00:17:21.066 --> 00:17:23.966 The first is a hybridization probe. 279 00:17:23.966 --> 00:17:28.300 And so with this we have a fluorophore which would further us. 280 00:17:28.300 --> 00:17:32.700 And then we have a quencher that prevents the fluorophore from fluorescing. 281 00:17:33.433 --> 00:17:36.533 And when the probe is just floating in solution 282 00:17:36.533 --> 00:17:39.366 the quencher is close to the fluorophore. 283 00:17:39.366 --> 00:17:42.800 And so we don't see any light, we don't detect anything. 284 00:17:43.666 --> 00:17:46.466 But when there's DNA present and the probe 285 00:17:46.466 --> 00:17:49.700 binds, there's a confirmation change. 286 00:17:49.700 --> 00:17:52.900 And so the quencher is farther away from the fluorophore. 287 00:17:53.133 --> 00:17:56.833 And so we're able to see that fluorescence and actually measure it. 288 00:17:59.133 --> 00:18:00.833 So this one would be used 289 00:18:00.833 --> 00:18:05.233 for the annealing phase of the PCR cycle. 290 00:18:06.466 --> 00:18:10.733 The attack me on probe again is going to bind. 291 00:18:11.066 --> 00:18:16.233 But in this case when it's bound the quencher and the 4 or 4 are so close 292 00:18:16.233 --> 00:18:19.566 enough together that you don't get any fluorescence that you can see. 293 00:18:20.266 --> 00:18:22.833 But as the tag polymerase 294 00:18:22.833 --> 00:18:26.500 is going to replicate the DNA when it gets to the probe, it's 295 00:18:26.500 --> 00:18:31.566 going to cleave off the fluorophore and cleave up the probe. 296 00:18:31.900 --> 00:18:35.166 And so the quencher and the probe will be far away 297 00:18:35.166 --> 00:18:38.166 from each other, and you'll be able to see the fluorescence. 298 00:18:38.300 --> 00:18:41.600 And so this occurs in the extension portion of the PCR 299 00:18:41.600 --> 00:18:43.366 cycle because you actually need 300 00:18:43.366 --> 00:18:47.400 the tag polymerase making new DNA and cleaving the probe. 301 00:18:50.666 --> 00:18:54.033 Either way you get this data where you have the number of cycles, 302 00:18:54.033 --> 00:18:58.466 meaning how many, temperature changes we've gone through. 303 00:18:58.833 --> 00:19:03.966 And then with the fluorescence, we're able to correlate that to copy number, 304 00:19:04.566 --> 00:19:07.566 and plot on this y axis. 305 00:19:07.766 --> 00:19:10.666 And then each lab is going to set a threshold for 306 00:19:10.666 --> 00:19:13.933 how much DNA you need to call something positive. 307 00:19:14.500 --> 00:19:15.766 So everything under this 308 00:19:15.766 --> 00:19:19.233 threshold is going to be considered negative or background noise. 309 00:19:19.533 --> 00:19:22.333 And everything above this threshold is going to be 310 00:19:22.333 --> 00:19:25.333 considered positive. 311 00:19:26.166 --> 00:19:29.433 In a case like this, we're not able to tell 312 00:19:29.466 --> 00:19:33.366 between, say, this line and this line, which one is HSV one? 313 00:19:33.366 --> 00:19:35.500 Which one is HSV two? 314 00:19:35.500 --> 00:19:40.200 But with the annealing, phase 315 00:19:40.333 --> 00:19:44.833 for those that use that probe, a melting curve analysis 316 00:19:44.833 --> 00:19:48.866 can be performed to help distinguish between one probe and another. 317 00:19:50.000 --> 00:19:50.366 And so 318 00:19:50.366 --> 00:19:53.533 with this you have your probes attach. 319 00:19:54.366 --> 00:19:58.900 And then instead of heating it to where the polymerase can work, 320 00:19:59.233 --> 00:20:02.200 we're going to slowly heat it and wait for that probe 321 00:20:02.200 --> 00:20:05.200 to fall off or denature from the DNA. 322 00:20:05.733 --> 00:20:07.633 And so we'll see the fluorescence. 323 00:20:07.633 --> 00:20:10.633 And then when it falls off, we'll lose that fluorescence. 324 00:20:11.233 --> 00:20:15.433 And how we're able to tell between one virus and the other is based 325 00:20:15.433 --> 00:20:20.900 on the energy between those bonds, between the DNA strand and the probe. 326 00:20:20.900 --> 00:20:25.100 So those are hydrogen bonds that require energy to break. 327 00:20:25.466 --> 00:20:30.400 And the better match that you have, the more energy is required 328 00:20:30.400 --> 00:20:32.233 for that break. 329 00:20:32.233 --> 00:20:34.300 And so for example, 330 00:20:34.300 --> 00:20:39.733 if you have a probe that's a perfect match for HSV two, it's going to have a higher 331 00:20:39.733 --> 00:20:42.800 melting temperature than if it is not 332 00:20:42.800 --> 00:20:45.800 quite a perfect match for HSV one. 333 00:20:46.033 --> 00:20:47.400 So in that example, 334 00:20:48.433 --> 00:20:49.633 HSV one 335 00:20:49.633 --> 00:20:54.166 would denature or melt sooner and at a lower temperature, 336 00:20:54.466 --> 00:20:57.466 and HSV two would melt at a higher temperature. 337 00:20:57.600 --> 00:21:01.633 And because of that difference, we're able to tell apart, 338 00:21:01.666 --> 00:21:04.666 if the person's been infected by 1 or 2. 339 00:21:07.400 --> 00:21:08.633 Most of the serologic 340 00:21:08.633 --> 00:21:11.833 testing is going to be done by it can be luminescent assay. 341 00:21:12.400 --> 00:21:16.533 It's important to note that these tests should not be performed in neonates, 342 00:21:16.833 --> 00:21:21.933 because mom's antibodies can cross the placenta and enter baby. 343 00:21:21.933 --> 00:21:25.600 And so you may be testing moms antibodies and not babies. 344 00:21:25.866 --> 00:21:29.766 And you would not have, an accurate result 345 00:21:29.766 --> 00:21:32.833 for if baby has been infected and formed antibodies. 346 00:21:34.133 --> 00:21:36.633 There are also options of different testing 347 00:21:36.633 --> 00:21:41.800 where they look for combined, meaning both HSV one and two and one test 348 00:21:41.800 --> 00:21:45.900 that are unable to determine between them versus type specific 349 00:21:45.900 --> 00:21:51.300 that looks for specific antibodies against HSV one or HSV two. 350 00:21:52.466 --> 00:21:53.266 For any of these 351 00:21:53.266 --> 00:21:59.533 testing, you're going to have some sort of either beads or the bottom 352 00:21:59.533 --> 00:22:02.533 of the well coated with antigen, 353 00:22:02.700 --> 00:22:05.700 which you add patient serum to. 354 00:22:06.133 --> 00:22:09.133 There's then washing and incubation 355 00:22:09.866 --> 00:22:12.100 and then cami luminescent 356 00:22:12.100 --> 00:22:15.100 antibody is, is added. 357 00:22:15.833 --> 00:22:18.833 And then again you incubate, incubate and wash 358 00:22:19.600 --> 00:22:22.066 and then you add a starter reagent. 359 00:22:22.066 --> 00:22:25.566 And so what's happening inside of the the test tube 360 00:22:25.900 --> 00:22:30.433 is that we have either the bottom of the plate or the, the beads 361 00:22:30.433 --> 00:22:33.466 that are labeled with these antigens 362 00:22:33.466 --> 00:22:36.733 specific for HSV one or HSV two or both. 363 00:22:37.500 --> 00:22:40.600 This green antibody is from the patient serum. 364 00:22:40.600 --> 00:22:45.666 And so if they have antibodies against HSV, they'll bind to the antigen. 365 00:22:46.333 --> 00:22:49.333 Anything not bound we wash away. 366 00:22:49.466 --> 00:22:51.333 And then we add this reagent. 367 00:22:51.333 --> 00:22:57.100 And so again it's an antibody against the FC region of the patient's antibody. 368 00:22:57.433 --> 00:23:01.333 And it's attached to a Cami luminescent molecule. 369 00:23:02.233 --> 00:23:05.233 We again wash away anything that's not bound. 370 00:23:05.233 --> 00:23:08.633 And so then when we add the starter reagent anything that's left 371 00:23:08.800 --> 00:23:10.700 will come cami Luminesce. 372 00:23:10.700 --> 00:23:14.566 So the more antibody of the patient's that's present 373 00:23:14.566 --> 00:23:18.566 and reactive to HSV, the more cami luminescence we'll see. 374 00:23:21.933 --> 00:23:22.733 It's important 375 00:23:22.733 --> 00:23:26.366 to note though with antibodies they can be a little bit fickle. 376 00:23:27.433 --> 00:23:32.600 The first for the combined IgG it can take days to weeks to form. 377 00:23:32.600 --> 00:23:35.600 And so you do have this time where you might be negative. 378 00:23:36.333 --> 00:23:40.966 Likewise for the specific type antibodies, 379 00:23:40.966 --> 00:23:45.566 it can take as long as six months to form for visual learners. 380 00:23:45.566 --> 00:23:48.066 I put it into a little graph. 381 00:23:48.066 --> 00:23:50.900 So the first at time zero 382 00:23:50.900 --> 00:23:53.900 weeks zero is the initial infection. 383 00:23:54.200 --> 00:23:58.200 And then this blue line is going to be that combined antibody. 384 00:23:58.700 --> 00:24:01.700 And it takes between ten days 385 00:24:01.833 --> 00:24:04.833 and three weeks to form. 386 00:24:04.933 --> 00:24:08.066 And so during this time I have a dotted line because it may or may 387 00:24:08.066 --> 00:24:09.400 not be present. 388 00:24:09.400 --> 00:24:13.300 But after that three weeks usually everybody has antibodies. 389 00:24:13.600 --> 00:24:15.266 And so this line becomes solid. 390 00:24:16.800 --> 00:24:17.433 The type 391 00:24:17.433 --> 00:24:21.066 specific antibody on the other hand takes longer to form. 392 00:24:21.466 --> 00:24:23.966 On average it takes between 2 to 3 weeks. 393 00:24:23.966 --> 00:24:26.633 But it can take as long as six months. 394 00:24:26.633 --> 00:24:31.166 And some individuals never form the the type specific antibodies. 395 00:24:31.800 --> 00:24:35.400 And so you can imagine if you're testing either in this window 396 00:24:35.400 --> 00:24:40.200 period right after infection, you may see no antibodies formed 397 00:24:40.566 --> 00:24:46.133 or you may see a variety of antibodies during this period where you may have type 398 00:24:46.566 --> 00:24:50.533 combined specific antibodies, but not the type 399 00:24:50.533 --> 00:24:53.533 specific antibodies. 400 00:24:55.100 --> 00:24:57.066 And emphasizing why? 401 00:24:57.066 --> 00:24:58.300 Why do we even care? 402 00:24:58.300 --> 00:25:01.300 Why do we even know need to know which type it is. 403 00:25:02.433 --> 00:25:06.533 So for HSV two again there's more subclinical shedding, 404 00:25:06.533 --> 00:25:08.233 more frequent recurrences. 405 00:25:08.233 --> 00:25:12.033 And so it's important for clinicians to be able to counsel the patients 406 00:25:12.033 --> 00:25:15.033 appropriately based on which infection they have. 407 00:25:15.333 --> 00:25:20.466 And then also just tracking these viruses over time for epidemiologic information. 408 00:25:21.566 --> 00:25:22.100 And so we would 409 00:25:22.100 --> 00:25:25.100 recommend that testing be repeated. 410 00:25:25.400 --> 00:25:29.300 And that time period should be at least a month later, 411 00:25:29.300 --> 00:25:32.333 if not longer, to just give the patient time 412 00:25:32.333 --> 00:25:35.333 to develop these antibodies. 413 00:25:36.433 --> 00:25:39.433 Lastly, we have the immunohistochemical staining. 414 00:25:39.833 --> 00:25:43.100 So this is a stain that goes on a very thin section 415 00:25:43.100 --> 00:25:46.100 of tissue on a glass microscope slide. 416 00:25:46.433 --> 00:25:50.733 We have specific antigens that are targeted by antibodies. 417 00:25:51.133 --> 00:25:51.933 And then it's either 418 00:25:51.933 --> 00:25:55.600 coupled with a fluorophore where we use a fluorescent microscope 419 00:25:55.866 --> 00:25:59.300 or a stain that we're able to visualize with a light microscope. 420 00:26:00.033 --> 00:26:02.900 For the case of the herpes simplex viruses, it's 421 00:26:02.900 --> 00:26:07.066 going to be a dark brown nuclear staining. 422 00:26:08.066 --> 00:26:10.100 And so you can see some of those here. 423 00:26:10.100 --> 00:26:13.666 This one is a very vibrantly staining one. 424 00:26:13.966 --> 00:26:16.966 Often you may just have one cell positive. 425 00:26:17.033 --> 00:26:19.300 And then there's always a background stain. 426 00:26:19.300 --> 00:26:22.966 And so in this case it's a blue where these cells are all negative. 427 00:26:24.133 --> 00:26:25.166 And so again this is going to 428 00:26:25.166 --> 00:26:28.166 be used in actual tissue sections. 429 00:26:30.166 --> 00:26:35.733 I know this is a busy busy slide, but I'm going to walk through line by line. 430 00:26:35.733 --> 00:26:39.333 And just re-emphasize when we use each of these tests, 431 00:26:40.000 --> 00:26:43.266 the first one the herpes virus culture, 432 00:26:43.600 --> 00:26:47.000 it's going to be used for acute infections with active lesions. 433 00:26:47.500 --> 00:26:50.233 This is especially used in neonates. 434 00:26:50.233 --> 00:26:52.333 It's going to be highly specific. 435 00:26:52.333 --> 00:26:54.233 The problem is going to be time. 436 00:26:54.233 --> 00:26:58.333 It takes time for us to grow and incubate these cells. 437 00:26:58.633 --> 00:27:01.633 So it's not a quick test. 438 00:27:01.633 --> 00:27:05.466 Depending on the assay it may or may not differentiate the type. 439 00:27:05.866 --> 00:27:08.900 And there can be false negatives late in the disease. 440 00:27:09.433 --> 00:27:12.766 And this is kind of a mid level range for cost. 441 00:27:13.733 --> 00:27:16.433 The DFA testing again is going to be used 442 00:27:16.433 --> 00:27:19.433 with acute infections with active lesions. 443 00:27:20.100 --> 00:27:24.433 Generally it's not used alone again because it has that lower sensitivity. 444 00:27:24.733 --> 00:27:29.000 And so there can be false negatives that must be confirmed by culture. 445 00:27:29.633 --> 00:27:33.100 But it is very quick compared to the viral cultures. 446 00:27:33.666 --> 00:27:37.200 And this too is about a mid level test for cost. 447 00:27:38.566 --> 00:27:42.133 PCR testing can be performed on blood or CSF. 448 00:27:42.133 --> 00:27:45.233 So this is a great, test for those that 449 00:27:45.500 --> 00:27:48.500 one suspects encephalitis in. 450 00:27:48.700 --> 00:27:49.833 It's going to be quick. 451 00:27:49.833 --> 00:27:51.966 Again very sensitive and specific. 452 00:27:51.966 --> 00:27:56.033 You're able to determine type if you're using the annealing probes 453 00:27:56.300 --> 00:27:58.866 that can do a melting point curve. 454 00:27:58.866 --> 00:28:02.066 There can be some false negatives early in the disease. 455 00:28:02.300 --> 00:28:05.166 And there's little data about its use in neonates. 456 00:28:05.166 --> 00:28:08.533 And so it's not recommended just because we don't have the data. 457 00:28:08.833 --> 00:28:12.166 And so for neonates you'd want to do the viral cultures. 458 00:28:12.833 --> 00:28:15.833 This is going to be one of the more expensive testing. 459 00:28:17.800 --> 00:28:19.133 For the antibody 460 00:28:19.133 --> 00:28:22.566 testing again you don't have to have active lesions. 461 00:28:22.600 --> 00:28:25.833 Usually this is performed in some sort of blood products. 462 00:28:25.833 --> 00:28:29.766 So blood serum plasma, no active lesions. 463 00:28:30.100 --> 00:28:34.933 It should be noted that some of these tests will give you an exam result. 464 00:28:35.266 --> 00:28:39.666 And for other infections it is used to show chronicity. 465 00:28:39.733 --> 00:28:44.000 It's often seen in the kind of acute infection phase of the disease. 466 00:28:44.233 --> 00:28:47.266 But that's not the case for herpes simplex virus. 467 00:28:48.066 --> 00:28:51.066 You can see elevated exam 468 00:28:51.133 --> 00:28:55.166 throughout the course of someone's life with HSV. 469 00:28:55.533 --> 00:29:00.700 You can also see it re elevate when subsequent infections occur. 470 00:29:01.500 --> 00:29:04.066 And it can also have cross-reactivity 471 00:29:04.066 --> 00:29:07.133 with other viruses in the herpes verde family. 472 00:29:07.700 --> 00:29:10.666 And so it really should not be used clinically. 473 00:29:12.366 --> 00:29:13.933 The IgG 474 00:29:13.933 --> 00:29:16.966 for the combined does not differentiate the type. 475 00:29:17.300 --> 00:29:21.733 There may be some false negatives early on before those antibodies are formed. 476 00:29:22.300 --> 00:29:25.300 This is a mid-level test for cost as well. 477 00:29:26.100 --> 00:29:30.766 For the type specific this can help to aid in the treatment 478 00:29:30.766 --> 00:29:34.433 and counseling of patients usually performed in blood. 479 00:29:34.433 --> 00:29:36.900 No active lesions necessary. 480 00:29:36.900 --> 00:29:39.900 Again, there can be false negatives early on. 481 00:29:39.900 --> 00:29:43.500 And there are some patients that never develop these antibodies. 482 00:29:43.733 --> 00:29:46.733 And this is going to be the cheapest of these tests. 483 00:29:47.866 --> 00:29:51.433 Lastly the immunohistochemical this is going to be performed 484 00:29:51.433 --> 00:29:55.266 on paraffin embedded tissue and potentially body fluids. 485 00:29:56.533 --> 00:30:00.566 This is if it's going to be like a tissue collection is going to be a little bit 486 00:30:00.566 --> 00:30:05.500 more invasive than a blood draw or just a swab of a lesion, 487 00:30:05.733 --> 00:30:09.833 that you'd actually have to say, like take a punch biopsy of a lesion, 488 00:30:10.033 --> 00:30:11.533 things like that. 489 00:30:11.533 --> 00:30:14.633 And you're not able to distinguish between HSV 490 00:30:14.633 --> 00:30:17.633 one in speech, you they're all going to stay in the same. 491 00:30:17.933 --> 00:30:21.200 And this like PCR is a expensive test. 492 00:30:23.833 --> 00:30:25.533 In summary, the herpes Verde 493 00:30:25.533 --> 00:30:30.733 family, is a family with an envelope and double stranded linear DNA virus. 494 00:30:31.366 --> 00:30:34.833 These symptoms of infection are initially flu like 495 00:30:34.833 --> 00:30:37.833 with fever, swollen lymph nodes, 496 00:30:38.000 --> 00:30:41.000 and afterwards, 497 00:30:41.333 --> 00:30:43.000 subsequent infections 498 00:30:43.000 --> 00:30:46.066 are held by a burning or tingling sensation. 499 00:30:46.433 --> 00:30:51.366 And then with sores of the mucosa of the mouth, nose, eyes or genitals. 500 00:30:52.100 --> 00:30:55.333 Transmission occurs when you come in contact with the virus 501 00:30:55.333 --> 00:30:57.200 from an infected person. 502 00:30:57.200 --> 00:31:00.700 The risk is highest if that person is symptomatic, 503 00:31:00.733 --> 00:31:03.733 but can occur when they're asymptomatic as well. 504 00:31:04.333 --> 00:31:07.333 Symptoms are managed using antivirals. 505 00:31:07.533 --> 00:31:11.033 Usually a cycle ver and testing 506 00:31:11.033 --> 00:31:14.800 includes DFA viral culture, PCR, serology 507 00:31:14.966 --> 00:31:17.966 in immunohistochemical staining. 508 00:31:18.666 --> 00:31:21.666 These are my references. 509 00:31:22.766 --> 00:31:25.966 I wanted to give a special thanks to the virology lab 510 00:31:26.033 --> 00:31:29.166 and those in it that helped me understand these tests better. 511 00:31:30.100 --> 00:31:32.366 Sterling in the Molecular Infectious Disease 512 00:31:32.366 --> 00:31:36.366 Rapid Lab that helped provide data on the PCR testing 513 00:31:36.766 --> 00:31:40.933 and my attendings that helped to make this what it is today. 514 00:31:41.233 --> 00:31:42.600 Thank you so much for listening.