HIV Testing Blog

Introduction

Polymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in molecular biology and for good reason: it is a rapid, inexpensive and simple means of producing relatively large numbers of copies of DNA molecules from minute quantities of source DNA material–even when the source DNA is of relatively poor quality.

PCR involves preparation of the sample, the master mix and the primers, followed by detection and analysis of the reaction products. These steps are discussed below.

Sample Preparation

PCR is very versatile. Many types of samples can be analyzed for nucleic acids. Most PCR uses DNA as a target, rather than RNA, because of the stability of the DNA molecule and the ease with which DNA can be isolated. By following a few basic rules, problems can be avoided in the preparation of DNA for the PCR. The essential criteria for any DNA sample are that it contain at least one intact DNA strand encompassing the region to be amplified and that any impurities are sufficiently diluted so as not to inhibit the polymerization step of the PCR reaction.

Although any protocol is acceptable for PCR purposes, it is often best to use the fewest steps possible in DNA preparation in order to prevent accidental contamination with unwanted DNA. Usually a 1:5 dilution of the sample with water is sufficient to dilute out any impurities which may result from the purifying protocol.

The simplest method of isolating DNA from cells is as follows:

1. Cells can be obtained by using a toothpick to scrape under the fingernails, swabbing the inside of the mouth or from the roots of plucked hairs. Regardless of source, cells are resuspended in 20 ul of water. Skip to step four.
2. If you are using cells suspended in media, centrifuge at 1200- 1500Xg for 5 minutes. Resuspend the cell pellet in 1 ml of phosphate buffered saline (PBS) and repellet by spinning at 1200- 1500Xg for 5 minutes. Repeat. These PBS washes remove medium, and its inhibitory factors, from the surface of the cells. After the last wash resuspend the cell pellet in 20 ul of distilled water. Be aware that too much cell debris can inhibit the PCR reaction. If this happens, it may be necessary to further dilute the DNA sample. Go to step four.
3. For bacterial samples take a toothpick and scrape the teeth, or swab the throat, ears or between the toes. Resuspend material in 500ul of water. Freeze and thaw sample three times with vigorous shaking or vortexing between repetitions to break the bacterial cell wall. Although not all DNA will be released from the cells, there will be a sufficient quantity for PCR. Go to step four.
4. Place the sample in a 95oC heating block, or in boiling water, for 5 minutes. This step inactivates the DNase molecules that are found in the sample preparation. If left intact, DNase could clip the desired DNA template molecule into fragments which would be unsuitable for PCR. If there is very little DNA in the sample preparation, the DNA can be concentrated by ethanol precipitation. The sample is now ready for PCR.

DNA samples for PCR–regardless of preparation method–are generally run in duplicate in order to provide a control for the relative quality and purity of the original sample. Adding a small amount of DNA to the control just after the master mix step allows the detection of anything in the completed sample prep which would inhibit the PCR reaction.

Preparation of Master Mix

The Master Mix contains all of the components necessary to make new strands of DNA in the PCR process. The Master Mix reagents include:

http://www.accessexcellence.org/LC/SS/PS/PCR/PCR_technology.php

Notes on the Master Mix

The Master mix buffer is often stored as a 10X stock solution (100 mM Tris-HCL, pH 8.3, 500 mM KCL, 1.5 mM MgCl2) which is diluted to 1X for use. Both the Master mix buffer and the purified water can be stored at room temperature. Store deoxynucleotides, primers and Taq DNA polymerase enzyme at -20oC.

Although 100ul of master mix per reaction is generally used, it is possible to use as little as 25 or 50ul to save on cost of reagents. Regardless of the total volume, be certain to keep the final concentrations of reagents constant.

Master mix reagents can be optained from a variety of companies. Often the initial concentration of the reagent will differ depending on which company produced it. It is easy to figure out how much stock reagent to use by following a simple formula:

(initial concentration) X ( volume needed ) =

                                    (final concentration) X (volume of sample)

For example: I have 10X buffer, 10 mM of each nucleotide, 0.5 mM primers and Taq DNA polymerase at 5 Units/ul. I want to make one 50 ul reaction. Calculations are as follows:

10 X buffer: (10X) X (5 ul) = (1X) X (50 ul) Nucleotides: (10,000 uM) X (1 ul) = (200 uM) X (50 ul) (10mM=10,000uM) primers (500uM) X (O.1ul)= (1.0uM) X (50 ul) Since it is impossible to pipet 0.1ul accurately, a dilution needs to be made first. Add 10 ul of stock primer solution to 990 ul of water to get 5uM concentration of primers. This new primer dilution can be stored at 4oC. Calculation for 5uM stock: (5uM) X (10 ul) = (1.0 uM ) X (50 ul) Taq DNA polymerase (5Units/ul) X ( 0.25 ul) = (.025 Units/ul) X (50 ul) 2.5 Units/100ul= Since it is impossible to pipet 0.25ul accurately, a .025 Units/ul dilution needs to be made first. Add 1.25 ul stock to 3.75 ul water to get a 1.25 Units/ul concentration. Discard and make fresh with each use. Calculation for 1.25 Units/ul stock: (1.25 Units/ul) X (1 ul) = (.025 Units/ul) X (50 ul) To make the master mix for one reaction add:

• 5 ul 10X buffer
• 4ul Each nucleotide (1ul each of dATP, dCTP, dGTP, dTTP))
• 20 ul Each primer (10ul of each)
• 1 ul Taq DNA polymerase (Total volume = 30ul)
• add 15 ul of water
• 5 ul of template (Total volume = 50 ul)

If want to make 3 reactions, 3 X 50ul = 150ul. Use this number in the formula for “volume of sample.”

Primers

A primer is a short segment of nucleotides which is complementary to a section of the DNA which is to be amplified in the PCR reaction. Primers are annealed to the denatured DNA template to provide an initiation site for the elongation of the new DNA molecule. Primers can either be specific to a particular DNA nucleotide sequence or they can be “universal.” Universal primers are complementary to nucleotide sequences which are very common in a particular set of DNA molecules. Thus, they are able to bind to a wide variety of DNA templates.

Bacterial ribosomal DNA genes contain nucleotide sequences that are common to all bacteria. Thus, bacterial universal primers can be made by creating primers which are complementary to these sequences.
Examples of bacteria universal primer sequences are:
Forward 5′ GAT CCT GGC TCA GGA TGA AC 3′ (20 mer)
Reverse 5′ GGA CTA CCA GGG TAT CTA ATC 3′ (21 mer)

Animal cell lines contain a particular sequence known as the “alu gene”. There are approximately 900,000 copies of the alu gene distributed throughout the human genome, and multiple copies distributed through the genome of other animal cells, as well. Thus, the alu gene provides the sequence for a universal primer for animal cell lines. The alu primer is especially useful in that it binds in both forward and reverse directions.
The alu universal primer seqeunce is as follows:
5′ GTG GAT CAC CTG AGG TCA GGA GTT TC 3′ (26mer)

When using universal primers the annealing temperature on the thermal cycler is lowered to 40-55 degrees C.

Sometimes primer units are listed in optical density reading (OD). If this is a problem you will need to convert to molarity using the following equations: Change optical density reading of primer to molarity (uM units)-

1. N = # of primer bases
2. SIGMA 260 =~ 10,000 X N/ m X cm
3. Molecular weight =~ 330 X N
4. OD₂₆₀ / SIGMA 260 X 10⁶ = Concentration (uM)

For example- primer is 20 bases long/ OD₂₆₀ = 10.

1. N = 20
2. SIGMA 260 =~ 10,000 X 20/m X cm = 20,000/m X cm
3. molecular weight =~ 330 X 20 = 6,600
4. 10 OD₂₆₀/20,000 m^-1cm^-1 X 10⁶ = 50uM

Detection and analysis of the reaction product

The PCR product should be a fragment or fragments of DNA of defined length. The simplest way to check for the presence of these fragments is to load a sample taken from the reaction product, along with appropriate molecular-weight markers, onto an agarose gel which contains 0.8-4.0% ethidium bromide. DNA bands on the gel can then be visualized under ultraviolet trans-illumination. By comparing product bands with bands from the known molecular-weight markers, you should be able to identify any product fragments which are of the appropriate molecular weight.

MAJOR RECOMMENDATIONS

Responsibilities of Child Healthcare Providers

As part of the initial newborn evaluation, the pediatric clinician should determine whether human immunodeficiency virus (HIV) testing of the mother has been completed properly and should follow up on any outstanding laboratory values.

Pediatric clinicians should obtain testing for HIV beyond the neonatal period if the child presents with signs and symptoms of HIV disease. Testing should be performed in children who have not yet been tested when risk factors for HIV infection exist in the child or one of his/her parents.

Laboratory Tests for HIV in Newborns, Children, and Adolescents

Because positive antibody results alone do not establish infection in children younger than 18 months of age, assays to detect virus (HIV deoxyribonucleic acid [DNA] polymerase chain reaction [PCR] or viral culture) should be used for diagnosis (see Figure 1 in the original guideline document).

In children older than 18 months of age, HIV infection may be diagnosed on the basis of a positive HIV antibody test (enzyme-linked immunosorbent assay [ELISA]) and a confirmatory test, such as Western blot.

Because of the time period between infection and the development of detectable antibodies, children/adolescents exposed via sexual activity, sexual abuse/assault, or infected blood who have an initial negative test result should be retested at 1 month, 3 months, and 6 months after exposure.

Because a child with end-stage HIV disease may become HIV-antibody seronegative as a result of severe humoral immunodeficiency, children who are clinically suspected to be HIV-infected yet test HIV antibody negative should be tested by DNA PCR (or HIV culture).

Children older than 18 months of age with an indeterminate Western blot result should be retested as soon as possible. If the Western blot result remains indeterminate, the patient should be tested for HIV-2 or specific viral tests (e.g., DNA PCR) for HIV-1 should be performed.

Rapid testing and expedited preliminary test results prior to Western blot confirmation should generally be used only when immediate information is needed to determine the need for post-exposure prophylaxis in the labor/delivery, newborn, or other acute exposure settings, or when the person who is being tested is unlikely to return for a follow-up visit.

When preliminary diagnostic tests are used for expedited HIV testing, a preliminary positive test result must be confirmed with a Western blot as soon as possible.

Testing for HIV Antibody

See the original guideline document for discussions of screening tests, confirmatory testing of positive results, and rapid test assays.

Testing for HIV or Viral Components

Clinicians should test children younger than 18 months of age who are born to an HIV-infected mother for HIV using one of the following methods:

* HIV DNA PCR (preferred method)

* HIV culture (acceptable method)

Because infection can only be confirmed with two positive test results performed on samples collected at different times, a repeat sample should be obtained promptly for any child with a single positive test result.

In an infant younger than 18 months of age, HIV can be reasonably excluded with two negative HIV viral tests, one at 1 month of age or older, and the other at age 4 months or older.

Ideally, a DNA PCR should be obtained for HIV-exposed infants at each of the following time points:

* at birth

* at 2 weeks of age

* at 4 to 6 weeks of age

* at 6 to 12 weeks of age

* at 4 to 6 months of age

See the original guideline for discussions of HIV DNA PCR, HIV culture, plasma HIV RNA, and HIV antigen detection.

HIV Counseling and Testing

In New York State, written informed consent from the child’s biological parent or legal guardian must be obtained before HIV testing can be performed in children except in certain specific circumstances, such as expedited testing, newborn screening, and follow-up PCR testing, and when testing is urgently necessary to provide medical care for a life-threatening condition.

When HIV testing of a child is performed, the parents should be considered for testing as well.

If a child is found to be perinatally HIV infected, his/her siblings also should be tested.

If HIV infection is newly diagnosed in a woman, all of her children should be strongly considered for testing, even if they are asymptomatic.

Pre-Test Counseling

The clinician should counsel the child’s parent or guardian or the child/adolescent with capacity to consent prior to HIV testing (see Table 2 in the original guideline document).

In New York State, a minor’s right to consent for or refuse HIV testing is based on his/her capacity to understand, without regard to chronological age, what an HIV antibody test actually tests for, the implications/consequences of being HIV infected, and why he/she is at risk for HIV.

The clinician should arrange for follow-up visits at the time of testing and should note in the patient’s medical record that counseling was provided and written consent was obtained when required.

When rapid testing is obtained and will yield a preliminary result during the visit, the clinician should first ensure that the patient/parent is emotionally able to receive a positive result and that mental health services are available for patients receiving a positive result.

Obtaining Consent

See the original guideline document for a discussion New York State laws on obtaining consent for HIV testing in children and adolescents for HIV testing.

Post-test Counseling

Counseling after a Patient Receives a Positive Test Result

Positive HIV test results should be presented in person to the appropriate individual (patient, parent, or guardian). A clinician should not communicate results to a patient or family member by telephone or mail.

Clinicians must respect an adolescent’s right to confidentiality concerning HIV status.

The clinician should explain the test results and should provide general information about available treatment.

The clinician should discuss the implications of the HIV Reporting/Partner Notification law (refer to the section “HIV Reporting and Partner Notification” below).

The clinician should provide or arrange for necessary referrals for treatment and supportive services.

The clinician should discuss methods of risk reduction and advise the family to inform medical personnel of the child’s HIV status during any medical care visit.

Counseling After the Patient Receives a Negative Test Result

When telling a patient that his/her test result is negative, the clinician should educate the patient on how to reduce the risk of transmission in the future.

HIV Reporting and Partner Notification

Since June 2000, New York State has required HIV reporting and partner notification for all confirmed positive HIV tests (unless testing occurred at an anonymous site) and HIV-related tests.

During pre-test counseling, parents/children should be informed that if their HIV test result is positive, their names will be reported to the New York State Department of Health.

Parents/children should be informed during pre-test counseling that if they provide the names of sexual or needle-sharing partners, the provider is required to report these names to the State Health Department. They should also be informed that if the test results are positive, their partners will be notified that they have been exposed to HIV.

All sexually active HIV-infected adolescents should be informed about the importance and benefits of notifying partners of their possible exposure to HIV.

Adolescents who are undergoing HIV testing should be questioned regarding the potential for domestic violence if their partners were notified. If domestic violence is a concern, partner notification should be deferred until the risk of harm to the patient (or one close to the patient, e.g., child) is eliminated.

HIV Testing of Older Children and Adolescents With the Capacity to Consent

Clinicians should be knowledgeable about New York State laws pertaining to adolescent consent and confidentiality and should educate their patients about these laws (see the National Guideline Clearinghouse (NGC) summary of the New State Department of Health guideline Identification and Ambulatory Care of HIV-exposed and -infected Adolescents).

In New York State, older children and adolescents who are judged capable of understanding the informed consent process may give written informed consent for HIV testing.

Parents cannot be informed of their child’s HIV test results without the explicit consent of the child or adolescent who is deemed capable of providing consent.

Ideally, HIV testing of older children and adolescents should occur in a comprehensive care setting that provides social support, ancillary services, and ongoing health care.

HIV Testing in Children in Foster Care

Within 5 days of entering the foster care system, all children must be assessed for capacity to consent for HIV testing. If a child is determined not to have capacity to consent, an HIV risk assessment must also be completed within the first 5 days of entering foster care. Children already in foster care must be assessed for HIV risk factors at least 60 days prior to their next scheduled periodic medical examination. If it is determined that a child may have the capacity to consent, an assessment of capacity to consent must be made and documented by authorized foster care agency staff within 30 days of the child’s entry into foster care. An HIV risk assessment must also be completed within this timeframe.

If one or more risk factors are present, a child in foster care should be offered HIV testing, or if the child lacks capacity to consent, he/she should be tested for HIV infection.

Adolescents and older children in foster care with the capacity to consent for HIV testing have the right to either consent for their own test or refuse testing.

For the commplete article, please refer to http://www.guidelines.gov/summary/summary.aspx?doc_id=6834

Emergency room patients who are most at risk for HIV are opting out of HIV testing at a huge rate amid a hospital atmosphere cold to such testing. Hospital personnel view it as too time-consuming, and insurers are reluctant to reimburse hospitals for their test-related expenses. In 2006, the Centers for Disease Control and Prevention recommended that everyone visiting a hospital for a major disease condition be tested for the virus that causes AIDS, with the opportunity for them to opt out of the testing, if they so chose.

Since then, only about 5 percent of such patients have been tested, according to Veronica Miller, director of the Forum for Collaborative HIV Research, an independent public-private partnership operating at the George Washington University School of Public Health and Health Services.

“HIV is a life-threatening disease that is so grossly underdiagnosed and undertreated in this country,” Miller said in a briefing on the two-day Summit on HIV Testing. It’s been found that infection rates in urban emergency rooms are from 0.5 percent to 1 percent of those tested – though many refuse testing, which involves a simple saliva test followed, if necessary, by a confirmatory blood test, all of which cost $80 to $120.

In Washington, D.C., where it’s estimated that 5 percent of people are infected with HIV, the George Washington University Medical Center emergency department found that only 0.8 percent of people tested were HIV positive. But half of those in the city’s wealthiest ward chose not to be tested, as did a third of people in the poorest ward. So it’s probably the case that the HIV rate is sharply higher among those who refuse the test.

A study done at Hahnemann University Hospital in Philadelphia found that patients’ acceptance of testing was boosted to 83 percent when trained counselors spent just five minutes pitching each emergency room patient. Such an increase could greatly benefit those who are HIV positive by catching the infection at an early stage, when it’s more treatable.

All too often, teenagers are going to their doctor’s office with flu-like symptoms, aches and pains, etc.- having some routine blood work done – and when everything comes back negative they are sent home with no-questions-asked. A few weeks later (symptoms still persistent) the teens return to have more blood work. This time the HIV test comes back positive. “What caused this,” or “how did this happen” are often the response that doctors get.

Most often the tests used to detect HIV are antibody tests. The antibodies they are trying to detect usually do not fully develop for several weeks in most people, so if someone were to try to take this test only a week or two after infection chances are they would receive a false negative.

Allison Agwu, M.D., a pediatric infectious disease specialist at John Hopkins Children’s Center, explains that these false negatives usually occur during the most contagious stage of HIV infection – the earliest one. If teenagers are engaging in risky sexual behavior, their need for more extensive testing is increased. Often doctors will ignore these possibilities because of the age of the patients.

If the teen is at high risk they should consider the use of a polymerase chain reaction (PCR) test, which detects genetic markers instead of antibodies. These tests have significantly smaller window periods than antibody tests. While they are more expensive than the standard antibody test, PCR tests allow us to get accurate results at around two or three weeks.

Doctors should consider using a PCR if the patient has used injectible drugs or has two or more of the following symptoms: enlarged lymph nodes, night sweats, malaise/fatigue/headaches or rash, fever/chills, or a persistent sore throat or cough.

* For the complete article, please visit http://hivtestingblog.com/original-articles/