Essay: Window period of HIV

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  • Window period of HIV
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Abstract
Window period of HIV is from the first day of infection which is the presence of the HIV in blood to the development of the specific antibody against it. Most of the tests detect the viral immune response. But some test can detect the viral antigen or viral nucleic acid in this period. However these test some can be more accurate and faster than the others, because early detection of HIV can help in increasing the ability of controlling the virus. So, as a result of the comparison between some tests like ELISA, combo test and PCR. And, found that the PCR is the fastest and more accurate test for detection of HIV in window period, also it is the mostly used test in hospitals as a confirmatory test or for some hospitals as an initial test.
Acknowledgment
First of all, this project would have been impossible without the support of my mother and my father. And, I would like to thank Ms. Basmah Al-maarik for her extraordinary help and for her advices and encouragement in writing this project. As well as, Dr. Maha Al-mozaini for giving me good sources and letting me understand some of the tests in the research.
Contents
Introduction: 1
Window period: 4
Incidence: 7
Lab tests to identify HIV: 8
Human immunodeficiency virus (HIV) immunoassays
Polymerase chain reaction (PCR)
Combination test
Oral test
Treatment: 13
Conclusion 14
References 16
List of figures:
Figure 1: Human immunodeficiency virus structure, and the viral proteins. 1
Figure 2: HIV mechanism to inter CD4 cells and its replication (AIDSinfo, 2016). 3
Figure 3: show the viral load and the CD4 cell count in the three phases of HIV infection. 5
Figure 4: number of deaths from AIDS worldwide, a significant increase in the recent years (Upload.wikimedia.org, 2016). 6
Figure 5: the three steps of PCR (Ncbi.nlm.nih.gov, 2016). 11
Figure 6: OraQuick HIV test 12
Figure 7: effect of HAART on the virus 13
Abbreviation:
HIV: Human immunodeficiency virus
RNA: Ribonucleic Acid
USA: United States of America
SIV: Simian Immunodeficiency Virus
DNA: Deoxyribonucleic Acid
CRF: Circulating Recombinant Form
AIDS: Acquired Immune Deficiency Syndrome
WHO: world health organization
PCP: Pneumocystis Carinii PneumoniaG
GCC: Gulf Cooperation Council
FDA: Food and Drug Administration
HAART: Highly Active Antiretroviral Therapy
CCR5: Chemokine Receptor 5 gene
Introduction:
Human immunodeficiency virus (HIV), a rotavirus which alters the immune system. In which, the virus attack different cells like B lymphocytes, monocytes, macrophages, dendritic cells, and natural killer cells (Lederman, Rodriguez and Sieg, 2006), but mainly it infect the CD4 T lymphocytes, and any damage to any these cells will lead to decrease of the immune system and make the infected person much more susceptible for infections and diseases (Cdc.gov, 2016).
HIV virus is RNA virus, icosaheadral (20 sided), enveloped. It transcribe RNA to DNA. There are two viral strands of RNA found in core surrounded by protein capsid that made viral protein p24 core antigen (Gelderblom et al., 1987). The outer envelope contains a lipid matrix which have a specific viral glycoprotein gp120, and a stem gp14 (Gelderblom et al., 1987). Within the viral envelop there is a HIV protein called p17 which is located at the inner part of the lipid membrane (figure.1) (Gelderblom et al., 1987).
Figure 1: Human immunodeficiency virus structure, and the viral proteins.
The first case of HIV in humans was 1920 in the Democratic Republic of Congo, in which the virus spread was due to a lot of sexual trade and migrants around Kinshasa city (Avert.org, 2015). by 1980, half of the cases were outside Kinshasa, which reflect the growing of the epidemics. For example, in 1960’s some scientist from Haiti were working in the Democratic Republic of Congo. Then they get back to Haiti so, they were the cause of HIV epidemic (Avert.org, 2015). Moreover, in 1981 HIV identified as a new health condition for the first time among homosexual individuals in United States of America (USA), in which they were having rare malignancies like Kaposi sarcoma, and other rare infections (Sharp and Hahn, 2011).
In 1999, researchers found that HIV was related to another virus that attack the immune system of monkeys which is called simian immunodeficiency virus (SIV), specially a strain found in chimpanzee called (SIVcpz) which is almost identical to HIV (Avert.org, 2015). The most acceptable theory of how the virus pass’s from chimpanzee to humans, is the hunter theory. In which, the SIVcpz was transmitted to the hunter by eating a small chimpanzee or their blood getting into the hunter’s wounds (Avert.org, 2015). In addition, what support the theory is, every time the SIV pass from chimpanzee to human, there will be slight different genetic make-up, in which that what make the different strains of HIV (Avert.org, 2015).
When the virus infect the CD4 cells, the virus will go through seven steps (figure.2) (Waghmare, 2010). First step is binding, in which the virus binds to specific receptor on CD4 cells with the help of CCR5 co-receptor or CXCR4 co-receptor. Second step is fusion, it is when viral envelop fuses with the membrane of CD4 cells, and once the virus entre the cells it will release its RNA and enzymes like reverse transcriptase into the cell (AIDSinfo, 2016). Third step is reverse transcription, it is when the virus release reverse transcriptase enzyme that will transform viral RNA genome to DNA which will fuse with the host cell DNA (Johnson, 2001). Fourth step is integration, in which the virus release integrase enzyme that will integrate virus DNA with the DNA of host CD4 cells, and this integrated DNA can be in active for several years (Aids.gov, 2016). Fifth step is replication, it is when the virus uses the machinery of CD4 cells to produce long chains of HIV proteins, in which these proteins are the building blocks for more HIV (AIDSinfo, 2016). The sixth step is assembly, where the new HIV protein and RNA that made by host CD4 cells are move to the surface of cells and produce immature non-infectious HIV. The last step is budding, once the new immature HIV is outside CD4 cells, it will release protease enzyme that will break long protein chains into smaller proteins to be combined together and form mature infectious HIV (AIDSinfo, 2016).
Some mutations may occur each time HIV replicate (Smyth, Davenport and Mak, 2012). This means there will be so many different types or forms for HIV within the body of infected person (Avert.org, 2016). There are two types for HIV, HIV type1 and HIV type2. The predominant worldwide type is HIV-1 (Avert.org, 2016). The HIV-2 is relatively uncommon, but mostly found in West Africa, also, it is progress slower and less infectious than HIV-1 (Campbell-Yesufu and Gandhi, 2011). Also, in HIV-1 there are four groups, and the most important group is group M, in which the most of HIV epidemics are caused by this group, and the other three groups are group O, group N and group P, in which they are not really common (Avert.org, 2016) (Hemelaar, 2013). And, within HIV-1 group M there are at least nine subtypes, which are known as subtypes A, B, C, D, F, G, H, J and K (Avert.org, 2016). In addition, there is another subtype known as circulating recombinant form (CRFs), in which it is a different subtypes that form a combined genetic material to form hybrid virus (Hemelaar, 2013).
The Human immunodeficiency virus found in body fluids such as blood, saliva, and breast milk (Nordqvist, 2016). The virus can be transmitted from blood to blood, sexual contact, from mother to baby through breast feeding, during delivery, or pregnancy. Also, the virus could cause fatal syndrome called Acquired immune deficiency syndrome (AIDS) to some infected individuals with HIV (Nordqvist, 2016). According to world health organization (WHO) statistics that 34 million people died from AIDS, and the latest estimation is 1.2 million people died in 2014 (Aids.gov, 2016) (Labtestsonline.org, 2016).
Window period:
Window period of HIV is from the first day of infection which is the presence of the HIV RNA in blood to the development of the specific antibody, which then can be detected in plasma, which is known as the seroconversion (Lee, Park and Kang, 2013). There are three phases of infection (figure.3) (Nordqvist, 2016).
Acute phase and chronic phase and the third is when chronic phase develops into AIDS (Nordqvist, 2016). Window period in the acute phase of the infection with HIV is from 10 to 27 days (Lee, Park and Kang, 2013). And, in the chronic phase it could last for so many years, during these years the virus will develop and damage the immune system (Nordqvist, 2016). The symptoms of acute phase are fever, lymph node swelling, headache, weight loss, and night sweating (Watanabe et al., 2015). Some patients get positive test result without having any symptoms and appear healthy, this patient is in chronic phase and him or her in greater risk to be an AIDS patient (Tallmer, 1991). In which the AIDS considered as the third phase which is known as the last stage that lead to death if not treated early (Cdc.gov, 2016) (figure.4).
AIDS occur when the immune system is badly damage and number of CD4 cells falls below 200 cells/mm3 and having a high viral load, in which, in the normal immune system, CD4 cells count is between 500 and 1,600 cells/mm3. And, the symptoms of last stage are blurred vision, diarrhea, dry cough, and fever last for weeks, night sweat, shortness of breath, weight loss, and white spots on the tongue or mouth (Nordqvist, 2016).
In addition, viral load of HIV is the amount of the virus in patient blood sample, so when the viral load is high that mean the immune system is not fighting the virus (Aids.gov, 2016). However, even when the viral load is low or undetectable it can be transmitted through body fluids other than blood. Because the viral load test only measure the viral load in blood (Aids.gov, 2016), in which this test measures the genetic material copies of HIV in a milliliter of blood, and it is usually tested when the patient is on treatment.
Any delay in diagnosing an infected person with HIV or having AIDS will make him susceptible to opportunistic infections because of his damaged immune system (The 3rd epidemic, 1990). The most opportunistic infection is Pneumocystis Carinii Pneumonia (PCP) which is infection of the lung and it is the most cause of death of AIDS patients. Also, become more susceptible to rare skin cancer called Kaposi sarcoma (Tallmer, 1991). And so many other infections like tuberculosis that it is caused by Mycobacterium tuberculosis, herpes simplex virus and Epstein-Barr virus.
Incidence:
Number of currently infected people with HIV worldwide are 33 million people according to (WHO) (WHO, 2016). However, there are limited information about the cases of HIV in the Middle East – North Africa region (MENA) and the Gulf Cooperation Council (GCC) states (A. Al Mozaini, 2014). Moreover, the estimated numbers of infected people with HIV in MENA region in decade ending in 2009 is 460,000 people and 24,000 of them died from AIDS, and the numbers are increasing (Shawky et al., 2009). Now, HIV considered to be a major Health problem in GCC states and Kingdom of Saudi Arabia as well (Obermeyer, 2006). As in Saudi Arabia the majority of infected people were 70% of male and 30% of female which is considered to be high rate (A. Al Mozaini, 2014).
The first reported case in Kingdom of Saudi Arabia was in 1984 of a blood transfusion recipient, and between 1984 and 2001 there was a 6,046 HIV infected people in Kingdom of Saudi Arabia, and 1,285 people of them were Saudi citizens (Madani et al., 2004). Because of this epidemic the Saudi Ministry Of Health (MOH) established a national AIDS program to prevent, diagnose and to treat HIV patient (A. Al Mozaini, 2014). Most of these patient between 1986 and 1990 were infected by HIV because of blood transfusion and organs transplantations, otherwise between 1999 and 2003, moste of the infection was related to heterosexual transmission (A. Al Mozaini, 2014). By 2004, the cases of infected people with HIV has been decreased in Saudi Arabia (A. Al Mozaini, 2014).
In 1990’s, 184 opportunistic infections related to AIDS were recorded in Saudi Arabia. As the most diagnosed infection was Pneumocystis Carinii Pneumonia (PCP) in percentage of 27 (A. Al Mozaini, 2014). Also, Mycobacterium tuberculosis and cytomegalovirus were recorded in 29 patient each which represent 16% for each. In the other hand, Kaposi sarcoma was less recorded in percentage of 3 (A. Al Mozaini, 2014).
Any delay in detection and treating of HIV infection will lead to increase in the mortality and to get opportunistic infections (A. Al Mozaini, 2014). As the mortality rates were high between 1989 and 1993, especially in 1992 the death rates reach to 90 deaths per 1000 person. Then, between 1993 and 2000 the mortality rates decreased to 20 deaths per 1000 person (A. Al Mozaini, 2014).
Lab tests to identify HIV:
There are so many tests that can detect the presence of HIV in the blood. Some of these tests can detect the immune response to the virus, and some tests that can detect the virus antigen, in which it is the earliest detection tests can detect the viral RNA or p24 antigen of the HIV, and some test that is combination of both that detect the HIV p24 antigen and HIV antibodies (Masciotra et al., 2013).
HIV immunoassays:
The main HIV specific antibodies found in plasma are IgM and IgG. Within the third week of infection, the first antibodies that appear are IgM, and peak between the fourth and fifth week (Buttò et al., 2010). However, IgM cannot be detected early in all infected individual because the detection of the antibodies it depend on the immunity of the individual and the assay that been used, in which there are different immunoassays with different designed principles to detect HIV infected person (Buttò et al., 2010). In addition, after the fourth week of infection, IgG antibodies will appear. However, some individuals may require up to 6 months for antibodies to appear (Buttò et al., 2010).
The 1st generation can detect viral antibodies which means detect infection after the window period. It uses purified and lysed HIV particles to capture virus IgG in plasma (Louie et al., 2006). This test was used in clinical and public health laboratories due to their reliability and low coast. But, it has some limitations such as it is not capable of detecting antibodies generated from HIV type 2 and non-group M HIV (Louie et al., 2006).
The 2nd generation it is also detect viral antibodies, which also means it detect the infection after the window period. It uses synthetic protein antigens alone like protein A of staphylococcus auruse, which have great affinity with IgG antibodies, or combined with lysed HIV particle to capture virus IgG in the plasma (FDA, 2006). In addition, it is more sensitive and specific test to detect antibodies for HIV-1 group O and HIV-2 (FDA, 2006).
The 3rd generation antibody assay which is known as enzyme linked immunosorbent assays (ELISA), that can detect the antibodies of HIV-1 group M and O, after 21 – 24 days of infection (Mahajan, Pace and Jarolim, 2010) (Lee, Park and Kang, 2013). To be more specific the HIV antibody which is IgG and IgM antibodies will attach to HIV antigen in the assay that congregated to indicator molecule, and sensitivity will increase during the seroconversion “after the window period” (FDA, 2006). Because of its low coast this immunoassay was used to reduce the coast of using PCR, in which if ELISA give a nonreactive results we will do western plot test if it is also give negative results, in which it is a confirmatory test for ELIZA that allow visualization of HIV antibodies (Fearon, 2005), we will do the PCR and if it is give positive results that means the patient having acute HIV infection (Branson et al., 2014). Now, ELISA alone is used as an initial test for HIV, not confirmatory, because it can’t detect the HIV within the acute phase of the window period (Branson et al., 2014).
Combination test:
The combo test which is Known as the 4th generation test, that is frequently used because it can detect the antigen (p-24 antigen) within 4 to 10 days of infection, which is within the window period (Masciotra et al., 2013) (Gürtler et al., 1998), besides detection of IgM and IgG antibodies like the 3rd generation test (FDA, 2006). However, detection of p-24 antigen is transient because when antibodies develops they will attach to the p-24 antigen and forming an antigen-antibody complex that will interfere the results (Branson et al., 2014). Furthermore, the specificity of 4th generation test is lower than 3rd generation when used for screening for blood donors, and have high rates of false-positive results (Lee, Park and Kang, 2013). Also, 4th generation immunoassay used as initial test for the acute phase (Branson et al., 2014).
Polymerase Chain Reaction (PCR)
PCR it is used to confirm the diagnosis of the test because it can detect the nucleic acid of the antigen in the host cell after 10 days of infection or less “within window period” (Vermeulen et al., 2013). It’s a molecular testing that amplifies viral nucleic acid by using a set of primers from envelop, polymerase, reverse transcriptase, and core protein (A. Al Mozaini, 2014) (Fearon, 2005). The primers are a short sequence of nucleotides (Fearon, 2005). The method of PCR is in three steps, first by annealing which is heating of the DNA template and then cool it again to have separated strands that is complementary to each other (Fearon, 2005). Second step, is adding the primers that is designed to be complementary to a part of the separated strands (Fearon, 2005). Third step, is the amplification, that the DNA will be copied by the polymerase enzyme starting from the primers (Fearon, 2005) (figure.5). And this process will be repeated for 30 to 40 times and during each cycle of the process will doubles the target DNA which is the DNA of HIV in the host cell, and by the end of the process will have a million of copies of the original DNA strand, so it is a rapid, sensitive and specific test that can detect a very small numbers of viral particle (Fearon, 2005). In addition, PCR is mostly used in testing infants of HIV positive mothers (Fearon, 2005).
Oral test:
Oral test is a home test for HIV, which is immune-chromatography test that look like the home pregnancy test. This home test will help in decreasing the epidemic of HIV by improving detection and to let the patient follow-up his condition (Schnall, Carballo-Diéguez and Larson, 2014). In 2012, the Food and Drug Administration (FDA) approve the sale of this rapid HIV home test for people above 17 years, like OraQuick, and OraSure Technologies for example (Schnall, Carballo-Diéguez and Larson, 2014) (figure.6) .
Also, this test have high sensitivity (92%) that mean only one false negative result will be found every 12 tests that shows positive results, and high specificity (99.8%) that mean only one false positive will be found every 5,000 tests that shows negative results (Fda.gov, 2016). This test uses oral fluid to detect HIV antibodies, which means after the window period, by swabbing the upper and lower gums with the test devise, then put the collected sample in the kit’s vial that contains a developer solution, then wait for 20 to 40 minutes and read the results (Fda.gov, 2016). If an individual get a positive result that does not mean definitely infected with HIV and need medical tests to confirm the results (Fda.gov, 2016). Even if, the test give negative results that does not mean not infected with HIV, because the individual might be infected but antibodies are not yet produced (Fda.gov, 2016). In addition, almost 60% of infected people in United States they didn’t know their HIV status, but this test give much more help in early identification of HIV (Schnall, Carballo-Diéguez and Larson, 2014).
Treatment:
Until now, there is no treatment that will cure infected people with HIV, there is only a combination of three or four antiretroviral drugs; two or one nucleoside reverse transcriptase inhibitors (NRTIs) which inhibit the viral reverse transcriptase enzyme (Sarafianos et al., 2009), non-nucleoside reverse transcriptase inhibitors (NNRTIs), which also inhibit the reverse transcriptase enzyme, and protease inhibitors (PIs) that act on HIV protease enzyme (Danner et al., 1995). This combination known as Highly Active Antiretroviral Therapy (HAART) (A. Al Mozaini, 2014), which decrease the severity and slows the progression of the acute phase of infection. Furthermore, decrease the viral mutations and suppress viral replication (Branson et al., 2014). However, when the patient stop taking the therapy, or after a period of time, some of these viruses will mutate to be resistant to the drugs, which will lead to rebounding of the virus to CD4 cells (Johnson, 2001) (Figure.7) (Biotechnologyforums.com, 2016).
The recent trials is focused on the treatment to cure HIV infected people. The latest discovery is stem cells generating Chemokine Receptor 5 gene (CCR5). In which, the CCR5 act as a co-receptor for HIV to enter the CD4 T lymphocytes (Dean et al., 1996) (Liu et al., 1996). The homozygous deletion of CCR5 gene is the cause of the resistance of the HIV, so heterozygous deletion will delay the HIV progression to the CD4 cells (Manotham, Chattong and Setpakdee, 2015).
There is only one patient with HIV positive was completely cured from HIV in 2008. The patient was a 40 years old male that was diagnosed with HIV in 1995 and was treated with HAART for 4 years, then after 10 years were diagnosed with acute myeloid leukemia. After 7 months, the patient needed to stem cell transplantation CD34+ cells, from a Human Leukocyte Antigen (HLA) identical donor. Because he took two courses of chemotherapy to treat acute myeloid leukemia, but this resulted in sever hepatic toxic effect and renal failure, and the HAART needed to be stopped so the viral count will increase again. In 2007, the patient received the first stem cell transplantation from a donor that was homozygous for the CCR5 delta32 allele deletion mutation, so he was resistant to HIV. Then in 2008, the leukemia have been relapsed so, he received a second stem cell transplantation from the same donor, which led to complete remission of acute myeloid leukemia and decrease in HIV count after 20 months of not taking HAART and increase in CD4 cells count (Hütter et al., 2009).
Conclusion:
In conclusion, the window period can be different from one individual to another, but most of infected people develop HIV antibodies within 3 months of infection (hivguidelines.org, 2016). It should be noted that an HIV infected person can transmit the virus during window period, if a patient get tested for HIV and get negative results, by tests that can detect the antibodies not the antigen itself, he need to repeat the test after 3 months to detect the antibodies because he might be in the window period. PCR is the test of choice to be used during window period because it is more sensitive than immunoassays in detection viral antigens. However, follow up is required if an individual get negative results but still having repeated potential exposures like, if an individual was a health care worker, and get an infected blood into the body through needle prick (hivguidelines.org, 2016) (Singh et al., 2012). However, the transmitted infection through needle prick from an infected individual to health care workers is approximately 1 in 300 exposures (Singh et al., 2012).
Moreover, according to CDC that recommends all individual at high risk and between 13 and 64 years must be tested for HIV at least once, in which it is a rational step to improve early detection and treatment of infected individuals (A. Al Mozaini, 2014).

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