Minimum Standards for Laboratories in SADC Regions

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1 Minimum Standards for Laboratories in SADC Regions Facilitator Guide Day 2 Minimum Laboratory Testing Standards: Diagnosis of HIV Infection at the NRL

2 Table of Contents 1. Learner Information About this Course Learning Styles Keys to Icons Course Agenda Minimum Laboratory Testing Standards: Diagnosis of HIV Infection at the NRL Module Module Objectives Activity Diagnostic Tests for HIV Infection HIV Serological Diagnosis: Requirement at the NRL Window Period Following HIV Infection HIV Infection and Laboratory Markers WHAT IS ELISA? False Negatives and Positives St and 2nd Generation EIA rd Generation Sandwich EIA Fourth Generation ELISA Principle Fourth Generation ELISA Fourth Generation ELISA (continued ) Western Blot Assay Western Blot Sequence of Test Positivity Relative to WB LAg (Limiting Antigen) Avidity Assay LAg (Limiting Antigen) Avidity Assay (continued ) Vaccine Induced Sero-Positivity/Reactivity (VISP) Vaccine Induced Sero-Positivity/Reactivity (continued ) Examples of VISP HIV Testing Algorithms and Reporting Strategies What is a strategy? HIV Testing Strategies HIV Testing Strategies (continued )

3 HIV Testing Strategies (continued ) What is an Algorithm? HIV Testing Algorithms Testing Algorithm (continued ) Testing Algorithm (continued ) Testing Strategies and Algorithm Test Methods Used in Serology Test Methods Used in Serology (continued ) Verification of test methods Verification of test methods (continued ) Verification of test methods (continued ) Verification of test methods (continued ) Sensitivity and Specificity (continued ) Sample types used in Serology Tests Result Reporting for HIV Serology tests LAg Testing Algorithm New HIV Diagnostic Algorithm Activity Activity Summary References and Links Daily Wrap Up

4 Learner Information Details Please Complete details Name of Learner Participant s Country Job Title Facilitators: Diagnosis of HIV Infection at the NRL Mini-Pooling of Patient Samples for Acute HIV Diagnosis HIV Drug Resistance TB diagnostics - State of Art Testing in Reference lab Malaria Diagnostics -Functions of the malaria ref lab in SADC Beverley Singh Ewaldé Cutler Gillian Hunt Nazir Ismail John Frean 4

5 About this Course The National Health Laboratory Services welcomes you to the Minimum Standards for National Reference Laboratories in SADC Regions course. In this course the participant will learn about the different Learning Styles, understand the Key Icons used in the manual and will be taken through the training material developed for Day 2 of this course on Minimum Standards required to run an effective National Reference Laboratory. The Course includes discussions, activities and workshop sessions. The Modules covered in Day 2 are as follows: 1. Diagnosis of HIV Infection at the NRL 2. Mini-Pooling of Patient Samples for Acute HIV Diagnosis 3. HIV Drug Resistance 4. TB diagnostics - N(TB)RLs in SADC Regions 5. Malaria Diagnostics -Functions of the malaria ref lab in SADC This guide covers Minimum Laboratory Testing Standards: Diagnosis of HIV Infection at the NRL 5

6 Learning Styles What are learning styles? Learning styles are simply different approaches or ways of learning. The different types of learning styles are as follows: Visual Learners: learn through seeing... These learners need to see the teacher's body language and facial expression to fully understand the content of a lesson. They tend to prefer sitting at the front of the classroom to avoid visual obstructions (e.g. people's heads). They may think in pictures and learn best from visual displays including: diagrams, illustrated text books, overhead transparencies, videos, flipcharts and hand-outs. During a lecture or classroom discussion, visual learners often prefer to take detailed notes to absorb the information. Auditory Learners: learn through listening... They learn best through verbal lectures, discussions, talking things through and listening to what others have to say. Auditory learners interpret the underlying meanings of speech through listening to tone of voice, pitch, speed and other nuances. Written information may have little meaning until it is heard. These learners often benefit from reading text aloud and using a tape recorder. 6

7 Tactile/Kinesthetic Learners: learn through moving, doing and touching... Tactile/Kinesthetic persons learn best through a hands-on approach, actively exploring the physical world around them. They may find it hard to sit still for long periods and may become distracted by their need for activity and exploration. Read / Write Learners: learn through reading and writing... Read / Write persons prefer to learn and memorize through reading and writing things down. 7

8 Keys to Icons The following icons are used throughout the study guide to indicate specific functions: ACTIVITY Written activities to be completed DISCUSSION Take part in the discussion IMPORTANT / TAKE NOTE This icon indicates information of particular importance Notes The participant should write notes, should he/she wish to do so 8

9 Course Agenda Day 2 Diagnosis of HIV Infection at the NRL Mini-Pooling of Patient Samples for Acute HIV Diagnosis HIV Drug Resistance TB diagnostics - N(TB)RLs in SADC Regions Malaria Diagnostics -Functions of the malaria ref lab in SADC Times Topic Duration Presenter 08:00 Registration 5 minutes 08:05 Day 1 Questions 10 minutes 08:15 Diagnosis of HIV Infection at the NRL 1 hour Beverley Singh 09:15 Mini-Pooling of Patient Samples for Acute 1 hour Ewaldé Cutler HIV Diagnosis 10:15 Tea 15 minutes 10:30 HIV Drug Resistance 1 hour Gillian Hunt 11:30 TB diagnostics - State of Art Testing in 1 hours Nazir Ismail Reference lab 12:30 Lunch 45 minutes 13:15 TB diagnostics - State of Art Testing in Reference lab 1 hr 15 minutes Nazir Ismail 14:30 Malaria Diagnostics -Functions of the malaria ref lab in SADC 1 hr John Frean 15:30 Tea 15 minutes 15:45 Malaria Diagnostics -Functions of the 1 hr 15 minutes John Frean malaria ref lab in SADC 17:00 Day 2 Summary wrap up 5 minutes 9

10 SPECIALIST TALK 10

11 Minimum Laboratory Testing Standards: Diagnosis of HIV Infection at the NRL Module Module Objectives Instructions for Facilitator Total Module Time: 1 hour The Facilitator must go through the Module Objectives. At the end of this module, participants will be able to: At the end of this module, participants will be able to: Describe the role of NRL in HIV laboratory testing; Discuss diagnosis of HIV infection using serological methods (ELISA testing) as the standard; Explain what advances have been done in diagnostic ELISA testing; Discuss Performance in detection for HIV infection; Describe the use of HIV Algorithms; Discuss Reporting Strategies; Discuss Test Method Verification; Describe new Algorithms/VISP. 11

12 Activity Facilitator to ask participants to work individually and explain the activity Instructions: Participants to work individually for 2 minutes. Read the brief scenario and jot down the answer. Suggested Time: 5 minutes discussion The facilitator can ask the participants what indicated the right answer to them. Scenario: Enzyme-Linked Immunosorbent Assay! What assay does the above terminology relate to? ANSWER: ELISA/Serology assay that detects antibody/ antigen in serum or plasma specimens. 12

13 Diagnostic Tests for HIV Infection Instructions for Facilitator The Facilitator must go through all the slides and expand on the points in the slides. Facilitator to inform participants that they should make notes while the facilitator talks. 13

14 HIV Serological Diagnosis: Requirement at the NRL 14

15 Window Period Following HIV Infection 15

16 HIV Infection and Laboratory Markers 16

17 WHAT IS ELISA? Elisa- is enzyme linked immunosorbent assay sometimes called EIA enzyme immunoassay a technique used mainly in serology to detect the presence of an antibody or an antigen in a sample 17

18 False Negatives and Positives 18

19 1St and 2nd Generation EIA 19

20 3 rd Generation Sandwich EIA 20

21 Fourth Generation ELISA Principle 21

22 Fourth Generation ELISA 22

23 Fourth Generation ELISA (continued ) 23

24 Western Blot Assay 24

25 Western Blot 25

26 Sequence of Test Positivity Relative to WB 26

27 LAg (Limiting Antigen) Avidity Assay 27

28 LAg (Limiting Antigen) Avidity Assay (continued ) 28

29 Vaccine Induced Sero-Positivity/Reactivity (VISP) 29

30 Vaccine Induced Sero-Positivity/Reactivity (continued ) 30

31 Examples of VISP 31

32 HIV Testing Algorithms and Reporting Strategies 32

33 What is a strategy? 33

34 HIV Testing Strategies 34

35 HIV Testing Strategies (continued ) 35

36 HIV Testing Strategies (continued ) 36

37 What is an Algorithm? 37

38 HIV Testing Algorithms 38

39 Testing Algorithm (continued ) 39

40 Testing Algorithm (continued ) 40

41 Testing Strategies and Algorithm 41

42 Test Methods Used in Serology 42

43 Test Methods Used in Serology (continued ) ANSWER: Qualitative: ELISA, Rapid, PCR, Western Blot Quantitative: Incidence EIA, Viral Load 43

44 Verification of test methods 44

45 Verification of test methods (continued ) 45

46 Verification of test methods (continued ) 46

47 Verification of test methods (continued ) 47

48 Sensitivity and Specificity 48

49 Sensitivity and Specificity (continued ) 49

50 Sample types used in Serology Tests 50

51 Result Reporting for HIV Serology tests The WB assay can be performed to determine final HIV results where results are discordant 51

52 LAg Testing Algorithm 52

53 New HIV Diagnostic Algorithm 53

54 Activity Facilitator to explain how the activity works. Ask the participants to write down the answers to the questions below. Duration: 5 minutes (3 minutes [preparation time] and 2 minutes [discussion]) 1. What type of testing algorithm was displayed on slide 43? 2. What testing strategy was used to report results? ANSWER: Serial Algorithm Strategy 3: similar to 2, except a third test is performed. All samples that are reactive on the first ELISA (Screen) is tested on the second (confirmatory 1) ELISA, samples reactive on the second ELISA is tested on the third ELISA (confirmatory 2). Samples that are positive on all 3 ELISA s is considered Infected. Samples that are non-reactive on the first ELISA are considered uninfected and not tested further _ 54

55 Activity Facilitator to initiate and lead the group discussion Duration: 20 minutes [discussion]) Discussions: Algorithms and Strategies used in NRL labs currently The place for HIV RTD in diagnostics at the NRL Acute HIV Infections Incidence Testing - why Incidence? Discuss Incidence Testing Algorithm ANSWER: Discuss various algorithms used at NRL as well as strategies used. Reason for selection of algorithm and strategy. NICD uses strategy 3 and serial algorithm, wider selection of ELISA tests( include 3 rd and 4 th generation) as well as Western Blot for discordant results or to confirm positive results. Serial algorithm used high sample volume At NICD RTD not used for diagnostic purposes: evaluate new kits and perform Post Marketing Surveillance. Groups to discuss the role of RTD at the NRL Acute HIV Infections: 4 th generation assays detect early sero-conversion. Also p24 antigen assays available, but limited available. HIV RNA assays also to determine acute infection: on plasma samples only Why Incidence: Determine number of recent infections in a defined cohort. HIV positive samples only; screening test done on Incidence assay; samples that are >2.0 OD-N are then confirmed in triplicate on the assay; samples with <1.5 OD-N are reported as Recent Infections and samples >1.5OD-n are reported as Long Term Infections. Samples with OD-n <0.4 are repeated on the serology /RNA assays to confirm reactivity. If samples are still positive, then report as Recent and if samples are negative, then exclude from the number of Positive samples and add to the negative count. 55

56 56

57 Summary Facilitator to go through the points on the summary slides indicated below. Let us sum up what has been covered in the module MINIMUM LABORATORY TESTING STANDARDS: DIAGNOSIS OF HIV INFECTION AT THE NRL Serological methods for HIV Diagnosis: ELISA s, Western Blot and HIV Rapid tests Principles of ELISA s assays: 1 st, 2 nd, 3 rd and 4 th generation ELISA s 1 st, 2 nd and 3 rd based on antibody detection 4 th generation based on antibody/antigen detection early diagnosis of infection Western Blot used to confirm positive/discordant results LAg assay determines number of recent and long term infections in cohorts Qualitative assays: ELISA, WB and HIV Rapid. Either Negative or Positive result. Quantitative assays: Viral Load and LAg. Results are quantified. VISP: Vaccine induced seropositivity whereby a person is reactive after being administered a vaccine against disease even though they did not have the disease Choose testing strategy suitable for your laboratory Select an algorithm based on assays available Test methods/assay must be verified prior to use. Determine Sensitivity, Specificity, Precision and Accuracy of all test methods selected for use Use most sensitive assay as screening and the confirmatory assays must be greater in specificity than sensitivity. 57

58 References and Links REFERENCES Ingrid V. Bassett, Senica Chetty, Janet Giddy, Shabashini Reddy, Karen Bishop, Zhigang Lu, Elena Losina, Kenneth A. Freedberg and Rochelle P. Walensky. Screening for acute HIV infection in South Africa: finding acute and chronic disease. HIV Med January ; 12(1): Celia Serna-Boleaa, Jose Muñoza,b, Jose M. Almeidab, Ariel Nhacolob, Emilio Letanga, Tacilta Nhampossab,c, Eliana Ferreirac,d, Pedro Alonsoa,b and Denise Nanichea High prevalence of symptomatic acute HIV infection in an outpatient ward in southern Mozambique: identification and follow-up AIDS 2010, 24: Evaluation of World Health Organisation Antibody Testing Strategy for individual patient diagnosis of HIV Infection (Strategy ΙΙΙ) written by: DJ. Martin, NK Blackburn, KF. O Connell, ET. Brant, EA. Goetsch Branson BM. The Future of HIV Testing. J Acquir Immune Defic Syndr 2010, 55:S102-S105 World Health Organization. Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy

59 Daily Wrap Up The Facilitator must go through the objectives and confirm that the participants have understood everything and are able to do the objectives below. This is the end of module MINIMUM LABORATORY TESTING STANDARDS: DIAGNOSIS OF HIV INFECTION AT THE NRL Participants are now able to: Describe the role of NRL in HIV laboratory testing; Discuss diagnosis of HIV infection using serological methods (ELISA testing) as the standard; Explain what advances have been done in diagnostic ELISA testing; Discuss Performance in detection for HIV infection; Describe the use of HIV Algorithms; Discuss Reporting Strategies; Discuss Test Method Verification; Describe new Algorithms/VISP. Facilitator to ask if there are any additional questions Notes 59

60 60

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