1. Module 1: Principles of Viral Load Monitoring

2. Learning Objectives
Understand the dynamics of viral load during the course of HIV infection
Understand how viral load affects risk for transmission and progression of HIV
Explain how viral load responds to antiretroviral therapy (ART)
Identify treatment failure using viral load
Describe schedule of viral load monitoring

3. Outline Understanding Viral Load Viral Load and HIV Infection
Disease Progression
Disease Transmission
Viral Load Measurement and Reporting
Viral Load Response to ART
Treatment Failure
Criteria
Routine and Targeted Viral Load Monitoring
Schedule of Viral Load Monitoring

4. Introduction: What is Viral Load? (1)
Viral load is the concentration of
HIV RNA copies in blood.
This is a reflection of ongoing virus
replication in a person’s body.

5. Introduction: What is Viral Load? (2)
Viral load is used as an indicator of:
Response to ART and risk for clinical disease progression
Risk of transmission of HIV between sex partners
Risk of transmission of HIV from mother to child
Since 2013, WHO guidelines recommend routine viral load testing for all HIV-infected children and adults on ART
Viral load is used as an indicator of:
A persons response to ART and risk for clinical disease progression
risk of transmission of HIV between sex partners
Risk of transmission of HIV from mother to child
Since 2013, WHO guidelines recommend routine viral load testing for all HIV-infected children and adults on ART
DHHS Guidelines, 2013; Murray et al, AIDS 1999; Marschner et al, JID 1998; Thiebaut et al, AIDS 2000

6. Viral Load during HIV Disease
To better understand how VL can be used for monitoring patients on ART is it helpful to briefly review the typical course of viral load trends in patients when no ART are provided. In this schematic we have CD4 on the left side IN BLUE and viral load on the right IN RED with time from HIV infection to death on horizontal axis. You can see that during acute infection there are typically dramatically elevated concentrations of viral load as measured in copies of RNA per ml of plasma during which time there is a sharp drop in CD4. Even without ART over the next 6 WEEKS there is a decline in VL – largely related to an immune response which allows for partial control (but as you can see not total suppression of VL). The figure shows that for many PLWHIV VL can stabilize for quite a long time (up to years for some). This level of stabilization is known as “viral set point”- which is highly predictive of rapidity of disease progression. With no treatment the higher the set point the more rapid progression to AIDS and death Without ART, VL increases over several years, gradually then more rapidly as symptoms develop.
Acute infection: Viral load rises rapidly and often to very high levels (>1 million c/ml)
6-12 weeks after infection: Immune response reduces viral load to steady level (“set point”)
Set point predicts disease progress, higher set point indicates a more rapid progression to AIDS
Without ART, viral load increases over several years, gradually and then more rapidly as symptoms develop

7. Progression to AIDS by Viral Load
These Kaplan Meier curves demonstrate just how well VL set point is as a prognostic measurement. We see that at 4 year approx. 45% of those with highest VL shown in lower blue curve have progressed to AIDS in contrast to virtually 0% IN THOSE WITH THE LOWEST CATEGORY OF VIRAL LOAD shown in green

8. Viral Load and HIV Transmission
Rate of transmission:
23 per 100 person-years
Rate of transmission:
2.2 per 100 person-years
These are results of an observational study conducted prior to availability of ART in Uganda whch assessed rates of HIV transmission in serodiscordant couples based on the HIV+ partners viral load. Among those with low viral load (<400) NO transmission was observed, and with increasing viral load the rate of transmission had a corresponding increase and those in the highest VL category (>50K) had transmission rate of 23 per 100 person years.
Zero transmissions
HIV viral load (copies/ml)
Quinn et al., NEJM 2000

9. Viral Load Measurement
Viral load can be measured using whole blood, plasma, or dried blood spots
Assays quantify HIV viral load as RNA copies/ml, also reported on a log10 scale
Lower limits of detection vary with assays (e.g.<20, 50, 400 copies/ml)
Optimal results include target not detected, or the lower limits of detection
In addition to content on the slide can say:
Assays quantify HIV VL by means of cycles of amplification then detection with indicator primers that target viral enzymes

10. Viral Load Log Scale Viral RNA copies/ml of plasma
Log-scale: a 1-log change = 10-fold change
Viral load changes less than 1 log are generally not of clinical significance
Log10 scale
Copies/ml
1.0 10
1.5 32
2.0
100
2.5
316
3.0
1,000
3.5
3,162
4.0
10,000
4.5
31,623
5.0
100,000
5.5
316,228
Different labs will report results differently. I’ve included this to familiarize you with log scale and how it relates to results reported as copies per ml. Each log represents an order of magnitude ( ie. 10 fold change) Like virtually any biologic measure performed in a lab there will be a certain amount of “noise” or variability. When interpreting changes this needs to be kept in mind so we are concerned about 1 log changes in VL before we consider it significant
So a change from 10,000 to 3,162 really is not a clinically meaningful change – as we can see here it is only a ½ log change.

11. Viral Load Result Categories
Once VL lab report is received classify results as follows:
<1,000 copies/ml
>1,000 copies/ml
<1,000 copies/ml
“Suppressed”
Results below lower limit of detection
Results up through 999 (e.g. not detected, < 20, 367 copies/ml, 934 copies/ml)
>1,000 copies/ml
Includes all results greater than or equal to 1,000 copies/ml (e.g. 2,000 copies/ml, 30,000 copies/ml, 100,00 copies/ml)
Viral load result reports may vary in appearance, here we discuss the different categories relevant to patient management
We use the term “suppressed” for VL <1000 because WHO is currently using that cut off. Traditionally this term has been used for when the VL is below the lower limit of detection. Ongoing studies are examining this cut off and we will have an increased understanding of the risk of regimen failure with VL > 50 < 1000 cp/mL, there may be changes in definition of VF, but at this time current WHO guidelines still use > 1000 copies

12. Virologic Response to ART
ART prevents HIV replication by inhibiting viral enzymes causing the viral load to decline
The goal of treatment is to achieve a suppressed viral load:
Associated with better clinical outcomes
Lower risk of HIV transmission
Persistent viral replication while taking ART can lead to resistance to one or more antiretrovirals (ARVs)
Now that we have reviewed how VL is measured and what the typical natural history of VL is over the course of infection without treatment I want to describe briefly what the desired and expected VL response is when ART are given.
ART prevents HIV replication by inhibiting viral enzymes causing the viral load to decline. The goal of treatment is to achieve an undetectable viral load:
associated with better clinical outcomes
lower risk of HIV transmission.

13. Virologic Response to ART
Viral load <1000 copies/ml is considered acceptable
Risk of transmission and disease progression <1,000 copies/ml is low
In most patients taking ART, the viral load should be <1000 copies/ml after 6 months of treatment
Those with high baseline viral load, such as infants, may take longer to achieve suppression
VL target: <1000 copies/ml
Better outcome
Lower risk of transmission
While the goal of treatment is an undetectable viral load, VL <1000 copies/ml is considered acceptable given that risk of transmission and disease progress are low at this level.
You can see from the curve here that in most patients on ART, VL will decline to below 1000 copies/ml after 6 months of treatment.
Those with high baseline VL such as infants may take longer to reach viral load <1000 copies/ml.
DHHS Guidelines, 2013; Tsibris and Hirsch, PPID 2010; Kaufmann et al., Arch Intern Med 2003

14. Immunologic and Clinical Responses to ART with Viral Suppression
CD4 count increases
50 to 150 cells/µl expected in the 1st year
50 to 100 cells/µl expected in the 2nd year
Improved clinical status (weight gain, resolution of diarrhea, no new opportunistic infections)

15. Viral Load Suppression Allows for CD4 Recovery
While trajectories vary, suppression of VL with ART allow for immunologic recovery and improvements even normalization of CD4 count. Those who start at very low CD4 counts however may not achieve robust CD4 recovery. Here is plot of a single individual patient in which you can see over the course of 2 years following initiation of ART and persistently suppressed vl, an improved CD4 from low 400’s to sustained above 500.

16. Monitoring for Treatment Failure
Treatment failure can be defined using the following criteria:
Resource-limited settings have relied on suboptimal clinical and immunologic methods
May not detect early failure
Some with clinical or immunologic failure may not actually have virologic failure
Clinical
Immunologic
Virologic

17. Increased Sensitivity Higher Positive Predictive Value
How Does Virologic Monitoring Compare to Clinical or Immunologic Monitoring?
How Does Virologic Monitoring Compare to Clinical or Immunologic Monitoring?
Virologic monitoring has greater sensitivity and positive predictive value for detecting treatment failure than clinical or CD4 criteria.
Increased Sensitivity
Earlier detection
Timely regimen changes
Decreased time with ongoing viral replication
May prevent drug resistance and decrease transmission risk
Higher Positive Predictive Value
Less misclassification
Helps prevent unnecessary switches to second-line ART in those who are actually suppressed
Kantor et al., CID 2009; Rawizza et al., CID 2011; Mermin et al., BMJ 2011

18. Viral Load, CD4 and Clinical Criteria for Treatment Failure
Viral load (virologic monitoring) is more sensitive and reliable for determining treatment failure for those on ART compared to clinical monitoring or CD4 criteria (immunologic monitoring).
Availability of viral load allows clinicians to detect treatment failure more reliably AND EARLIER than currently available methods such as worsening of clinical status or decline in patient CD4 count. This schematic compares time that the various methods are able to first detect treatment failure. As you can see virologic detection by viral load shown here in red, precedes immunologic detection by CD4 count as well as clinical detection by clinical status (e.g. WHO stage.)

19. Criteria for Treatment Failure Determined by Viral Load
Virologic treatment failure:
Persistent plasma viral load >1000 copies/ml when measured at least 6 months after starting ART
While the most desirable viral load result for any patient on ART is undetectable, bear in mind that virologic failure is NEVER determined with single test alone but requires repeated testing. Virologic treatment FAILURE is defined as follows:
Persistent plasma VL >1000 copies/ml when measured after at least 6 months of starting ART
“Persistent” ≥2 or more consecutive VL 3 months apart with adherence support between measurements.
“Persistent” defined as >2 consecutive viral load results 3 months apart with adherence support between measurements

20. Routine and Targeted Viral Load Monitoring
Routine Viral Load Monitoring
Ideally, viral load should be measured at regular intervals for all those on ART to monitor response to treatment and for early detection of treatment failure
Targeted Viral Load Monitoring
In settings where routine viral load is not feasible or available, viral load testing should be used to confirm treatment failure if suspected by CD4 or clinical criteria

21. WHO 2016 Guidelines: Viral Load Schedule
Check viral load 6 months after ART initiation
If ≤1000 copies/ml , then repeat testing 6 months later, then repeat at least every 12 months if remains ≤1000 copies/ml
If viral load is >1000 copies/ml, provide adherence support and re-check in 3 months
Virologic failure only if repeat viral load result is still >1000 copies/ml after adherence support
Those with high baseline viral load, such as infants, young children and some adults, may take longer to achieve suppression
While the WHO 2016 guidelines define virologic failure as two VL results >1000 within 3 months with adherence support in between, if there has been no meaningful change in adherence 3 months after the first VL, then it is too soon to recheck it

22. Routine and Targeted Viral Load Monitoring Adapted from WHO Guidelines

23. Adherence Interventions Improve Suppression of Viral Load
53% of those with initial VL >1,000 copies/ml achieved virologic suppression after early viral load testing and a targeted intensive adherence intervention
Why do enhanced adherence counseling?
This study from S. Africa looked at the effect of early viral load testing and adherence interventions on viral load suppression and preservation of first line regimens. VL was checked every 4 months while on ART and if the result was above 1000 copies/ml, participants received intensive adherence interventions including home visits and then the VL was repeated 6-8 weeks after the adherence interventions
From the graph here it can be seen that the early VL result and intensive adherence interventions (pill box, dosing diary, home visits, education sessions) resulted in half of those with initial VL >1000 shown with the dashed line, subsequently achieving full suppression. The solid line shows those whose second VL remained above 1000 copies/ml
Orrell et al., Antivir Ther 2007

24. Enhanced Adherence Counseling
Enhanced Adherence Counseling is a continual and repeated process that involves:
Repeat Steps 1-4 during follow-up sessions.
Step 1: A structured assessment of current level of adherence
Step 2: Exploration of the specific barriers the patient must overcome
Step 3: Motivating and assisting patients to identify solutions and address barriers
Step 4: Develop and individualized adherence intervention plan

25. Achieving Good Adherence
Adherence sessions should be repeated until good adherence is achieved
Once good adherence is achieved, set a date for repeat viral load measurement after 3 months of good adherence and provide to patient
Once good adherence is achieved based on calculating adherence using the table, a date for follow up viral load testing is set and given to the patient. Adherence support may need to continue on a monthly basis to maintain good adherence until the viral load drops to 1000 copies/ml or less. Remember it will takes some months for vl to decline once good adherence achieved so starting counting the 3-6 m interval from time good adherence has begun

26. Viral Load Monitoring: Not Too Much, Not Too Little
More evidence is needed to determine the optimal schedule for viral load monitoring.
Monitoring too often:
Resource intensive, costly and may not contribute to improved patient outcomes
If improperly implemented may lead to premature regimen change (e.g. viral load testing prior to expected time of suppression, or repeat viral load testing without improvement in adherence)
Infrequent monitoring may lead to late detection of treatment failure and progression of disease
In partially adherent patients delayed viral load testing may allow for emergence of drug resistance
There is little evidence for any cost benefit for a specific VL monitoring scheduled. When considering a monitoring schedule a number of there are a number of important considerations to take into account including feasibility, cost, practicalities, biology of HIV replication and ART resistance.

27. Schedule for Routine Viral Load Monitoring
Adults (Non-Pregnant or Breastfeeding )
Fill in schedule according to national guidelines

28. Schedule for Routine Viral Load Monitoring
Children and Adolescents on ART
Fill in schedule according to national guidelines
Each country has different guidelines for how frequently to test VL in specific populations. (Fill in your country’s guidelines on this and the next few slides.)

29. Schedule for Routine Viral Load Monitoring
Pregnant or Breastfeeding Women on ART
Fill in schedule according to national guidelines

30. Role of CD4 Count Tests
For those not on ART: CD4 test at baseline and every 6 months to prioritize for ART and determine opportunistic infection (OI) prophylaxis
For targeted viral load monitoring: CD4 test every 6 months for those on ART to assess treatment response
If routine viral load testing is available:
CD4 may no longer be necessary for those on ART with confirmed viral load <1000 copies/ml
CD4 may still be useful if ill and OI suspected to assist with diagnosis
A question that comes up for which there is no clear answer is whether CD4 count testing should be continued once vl is established. The utility and cost effectiveness of CD4 testing has been further called in to question with move to treat all PLWHIV. Here we have potential continued use of CD4 testing once VL monitoring services have been established. But again this is an area where each country will have different guidelines therefore on the next slide country specific guidelines are given

31. CD4 Count Testing Fill in national guidelines on CD4 count testing
A question that comes up for which there is no clear answer is whether CD4 count testing should be continued once vl is established. The utility and cost effectiveness of CD4 testing has been further called in to question with move to treat all PLWHIV. Here we have potential continued use of CD4 testing once VL monitoring services have been established. But again this is an area where each country will have different guidelines therefore on the next slide country specific guidelines are given

32. Knowledge Check
As a group, I’m going to conduct a quick knowledge check.

33. Question 1
Which of the following patients has the highest risk of transmitting HIV during an unprotected sexual act?
On ARVs with VL = 10,000 copies/ml
Not on ARVs with VL = 100,000 copies/ml
On ARVs with VL < 20 copies/ml
On ARVs with VL = 2,000 copies/ml
As a group, I’m going to conduct a quick three-question knowledge assessment on viral load. Please raise your hand when I call out the answer you think is correct.

34. Answer
Which of the following patients has the highest risk of transmitting HIV during an unprotected sexual act?
On ARVs with VL = 10,000
Not on ARVs with VL = 100,000
On ARVs with VL < 20
On ARVs with VL = 2,000
The correct answer is B = Not on ARVs with VL = 100,000. Can you tell me why this is the correct answer?
This patient has the highest viral load and thus has the highest risk of transmitting HIV. Notice that this patient also is not on ART. Remember that ART prevents HIV replication by inhibiting viral enzymes causing the viral load to decline.

35. Question 2
Patient A has a VL = 3,500 after 6 weeks on ART. This represents treatment failure.
True
False
Please raise your hand when I call out the answer you think is correct.

36. Answer
Patient A has a VL = 3,500 after 6 weeks on ART. This represents treatment failure.
True
False
The correct answer is B = False. Can you tell me why this is the correct answer?
This is false – the patient has not been on ART long enough to determine whether or not he is experiencing treatment failure. Patients should undergo follow up viral load testing six months after starting ART. If viral load is >1000 copies/ml, provide adherence support and re-check in 3 months. Treatment failure is only considered if repeat viral load result is still >1000 copies/ml after adherence support.

37. Question 3
Patient B has an undetectable VL after 6 months on ART. When is the next VL due?
3 months
6 months
1 year
Not needed
Please raise your hand when I call out the answer you think is correct.

38. Answer
Patient B has an undetectable VL after 6 months on ART. When is the next VL due?
Not needed
1 year
6 months
3 months
The correct answer is C = 6 months. Can you tell me why this is the correct answer?
After 6 months on ART, if VL is undetectable (<1,000 copies/ml), then repeat testing 6 months later, followed by repeat testing at least every 12 months if VL remains <1,000 copies/ml.

39. Summary
Viral load predicts progression of disease in an individual, and onward transmission of HIV to sex partners or from mother to baby
In most individuals, viral load will drop to below levels detectable by viral load blood tests after 6 months of ART
Viral load testing is the preferred method for detecting treatment failure for ART patients and should be checked after 6 months on ART
At this time, viral load <1000 copies/ml indicates acceptable response to ART (at this time, research is ongoing)
Virologic treatment failure: persistent (>2 viral load tests 3 months apart with adherence support in between ) plasma viral load ≥1000 copies/ml when measured after at least 6 months of ART

40. Questions?