An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

1. Ethylenediaminetetraacetic Acid (EDTA) Whole Blood Processing

Note: Blood can be processed immediately after collection of can be stored at 4 °C for no more than 24 hr.

1. Working in a biosafety cabinet, allow the EDTA whole blood sample to reach room temperature.
2. For each sample, label enough cryovials with the sample identification (ID), storage material (plasma), and date.
3. Centrifuge the samples for 10 min at 1,000 x g. Do not use brakes to stop centrifuge. This will yield three layers (from top to bottom): plasma, leucocytes (buffy coat) - a very thin layer – and erythrocytes, including platelets.
4. Carefully aspirate the supernatant (plasma) and aliquot 500 ml into each cryovial. Take care not to disrupt the cell layer (buffy coat) or transfer any cells.
5. Store at -80 °C until needed for RNA extraction or proceed to RNA extraction immediately.

2. RNA Extraction

1. Prepare an extraction worksheet with the IDs of the samples to be extracted including positive and negative plasma controls.
2. For each sample to be extracted, label a 1.5 ml sterile microcentrifuge tube with the sample ID, extraction date and “RNA”. Also label an assembled column and collection tube as well as a 2 ml microcentrifuge tube containing working lysis solution with corresponding numbers from the extraction worksheet.
3. Working in the Bio-Safety Cabinet, add 200 µl sample to the corresponding 2 ml microcentrifuge tube of working lysis solution.
4. Vortex well and incubate for 10 min at room temperature.
5. After 10 min, centrifuge the tube briefly.
6. Add 800 ml of absolute ethanol to each of the tubes.
7. Mix by pulse vortexing and briefly centrifuge.
8. Transfer 600 µl of this solution to the corresponding column/collection tube assembly. Centrifuge at 6,000 x g for 1 min.
9. Transfer column to a new collection tube and discard the old collection tube containing the filtrate. Repeat the above step 2.8 (above) twice more.
10. Add 500 µl wash buffer AW1 to each column and centrifuge at 6,000 x g for 1 min.
11. Discard the filtrate and collection tube and transfer the column to a new collection tube.
12. Add 500 µl was buffer AW2 and centrifuge at 20,000 x g for 3 min. Repeat step 2.11.
13. Centrifuge in a new collection tube at 20,000 x g for an additional 2 min.
14. Discard filtrate and place column in 1.5 ml microcentrifuge tube.
15. Add 60 µl Buffer AVE (RNase free water) to the middle of the column ensuring that you do not dispense the liquid on the side of the column.
16. Incubate at room temperature for 1 min.
17. Centrifuge at 6,000 x g for 2 min.
18. Discard the column and cap the 1.5 ml microcentrifuge tubes.
19. The samples are now ready for reverse transcription.
20. If testing is to be performed immediately, store at 4 °C for up to 6 hr. However, if testing is to be delayed then place at -80 °C immediately. NB: do not freeze/thaw the samples more than 3x.

3. Reagent Preparation for Reverse Transcription

1. Before starting, calculate the volumes of each of the reagents required for the number of samples being processed including, the positive and negative plasma controls. Also add a reagent control.
2. Using the calculated volumes from step 3.1 (above), prepare the deoxyribonucleotide triphosphate (dNTP)-primer mix in a clean, sterile 200 µl PCR tube followed by briefly pulse vortexing. Each sample should have 0.5 µl of the reverse primer RT21 and 0.5 µl of the dNTP, see Table 2.
3. Aliquot 1.0 µl of the dNTP-primer mix to 200 µl PCR tubes.
4. Prepare reverse transcriptase (RT) enzyme mix by adding 1 µl of the 10x reverse transcription buffer, 1 µl of 0.1M DTT and 2 µl of 25mM MgCl₂ to a sterile tube followed by vortexing and briefly centrifuging, see Table 3.
5. Add 0.5 µl each of the enzymes RNAseOUT and Superscript III reverse transcriptase to the enzyme mix tube then tap the tube gently to mix.
6. Keep the tubes with the dNTP-primer mixes and enzyme mix on a cold block and move to the RNA station.

4. Reverse Transcription

1. Add 6 µl of the RNA sample to the dNTP-primer mix tube followed by briefly vortexing to mix.
2. After the addition of the RNA, move to the PCR room with both dNTP/primer/RNA mix and RT Enzyme mix tubes on a cold block or ice.
3. Briefly centrifuge the dNTP/primer/RNA mix tubes (from step 4.2) and place them into a thermocycler.
4. Heat at 65 °C for 5 min to denature the RNA.
5. Rapidly cool to 4 °C, hold for 2 min.
6. Pause the thermocycler while still at 4 °C; take out the tubes.
7. Quickly add 5 µl of the enzyme mix while keeping the tubes on a cold block.
8. Mix gently by tapping the tube then briefly centrifuge the tubes and return to the thermocycler.
9. Hold the tubes at 50 °C for 60 min to reverse transcribe the RNA followed by enzyme denaturation at 85 °C for 5 min to stop the reverse transcription.
10. Cool to 37 °C. As soon the temperature gets to 37 °C, pause and take the tube out of the thermocycler.
11. Quickly add 0.5 µl of RNAse H to the tubes and return to the thermocycler.
12. Hold at 37 °C for 20 min and then cool to 4 °C.
13. The complementary DNA (cDNA) can be used immediately or can be stored at -20 °C or colder until needed. However, the long term storage of cDNA should be at -80 °C.

5. Reagent Preparation for PCR

1. Before starting, calculate the volumes of each of the reagents required for the number of samples being processed and the controls. In addition to the three controls (Positive, Negative, and Reagent), you can also add a PCR control (HIV DNA). The first and second round PCR mixes can be prepared simultaneously and the second master mix stored at -20 °C until needed. Mixes can be stored for approximately 8 hr.
2. Add 18.4 µl water, 2.5 µl 10x buffer, 1.0 µl MgCl₂, 0.5 µl dNTPs, and 0.25 µl of each of the primers as shown on Table 4 and vortex.
3. Add 0.1 µl of Platinum Taq polymerase (5U/µl) and gently mix the tube by tapping it.
4. Aliquot 23 μl of the master mix to 200 μl PCR tubes.
5. With the master mix tubes on a cold block or ice move to the PCR room.

6. Nested PCR

1. Add 2 µl of the cDNA to 23 µl of the 1st round PCR master mix.
2. Close the tubes, put the samples in the thermocycler and use the following PCR cycling conditions: 94 °C for 2 min, 30 cycles of 95 °C for 30 sec, 58 °C for 20 sec, and 72 °C for 2 min, followed by a final extention at 72 °C for 10 min as shown on Figure 1.

Figure 1. Nested PCR cycling conditions. Click here to view larger image.

3. Continue to the 2nd round PCR stage or store the 1st round PCR products at -20 °C or colder until required at a later stage.
4. For 2nd round PCR, add 2 µl of the 1st round PCR product to 23 µl of the 2nd round PCR master mix and use the same PCR program on Figure 1.

7. Gel Electrophoresis

1. Gel preparation
   1. Add a 0.5 g of agarose tablet to a 250 ml glass flask and add 50 ml of 1x TBE buffer to the flask.
   2. Heat in microwave to boiling; swirl frequently (approximately every 30 sec) until completely solubilized. Use a silicone grip or silicone oven glove to grasp the hot flask. The agarose solution can boil out of the flask very easily so closely monitor this process.
   3. Cool at room temperature for approximately 10 min.
   4. Pour agarose into a gel casting tray containing appropriate size comb; gel is ready to use in approximately 20-30 min.
   5. Place gel in electrophoresis chamber and run as recommended by the manufacturer.
2. Gel electrophoresis and visualization.
   1. Vortex Novel Juice for 10 sec prior to use.
   2. Dilute 1 µl of Novel Juice with 5 µl of DNA sample and mix.
   3. Dilute 3 µl of Novel Juice with 3 µl of molecular weight marker and mix.
   4. Load the mixes from sections 7.2.2 and 7.2.3 (above) and run the gel at 100 V and 400 mA for 40 min to evaluate the PCR amplification.
   5. Positive amplification can be visualized under UV light as 1,315 bp fragment, Figure 2.
      
      Figure 2. Gel confirmation of PCR amplification using 1% agarose gel electrophoresis and a 200 bp ladder. Click here to view larger image.
   6. There should be no amplification in the Negative and reagent controls, thus indicating absence of contamination.

8. PCR Product Cleanup

1. In preparation for the sequencing reaction, the positive second round PCR products are cleaned up using the PureLink PCR purification kit.
2. Add 80 μl of working Binding buffer High-Cutoff (B3) to 20 µl of PCR product and pipette mix.
3. Add the sample mixed with the binding buffer to a spin column in a collection tube.
4. Centrifuge the column at 10,000 x g for 1 min. Transfer the column into a new collection tube.
5. Wash the column with 650 µl of Wash Buffer with ethanol.
6. Centrifuge the column at 10,000 x g for 1 min. Transfer the column into a new collection tube.
7. Centrifuge the column at maximum speed for 2-3 min to remove any residual wash buffer.
8. Place the spin column in a clean 1.7 ml elution tube supplied with the kit.
9. Add 40 µl of elution buffer to the center of the column and incubate the column at room temperature for 1 min.
10. Centrifuge the column at maximum speed for 2 min (>10,000 x g).
11. The elution tube contains your purified PCR product ready for sequencing. Discard the column.
12. Determine the concentration and quality of the DNA using a Nanodrop.
13. If no in-house sequencing facilities are available, the purified PCR products can be sent to a commercial sequencing lab at this stage.

9. Sequencing Reactions

1. The PCR products are sequenced using the big dye terminator kit version 3.1 and 4 primers for each sample (two forward and two reverse). The primer sequences are shown in Table 2. Therefore, after the sequencing run, each sample will have four sequences to be assembled into a contig.
2. Set up the sequencing reactions as indicated in Table 5 for each of the four primers.
3. Mix the sequencing buffer and the primers by vortexing before use.
4. Mix the water, buffer and primer before the addition of the big dye sequencing. Mix by vortexing.
5. Gently mix the master mix after adding the big dye sequencing mix by inverting the tube or tapping it gently.
6. Aliquot 9 µl of the master mix into a 96-well optical plate.
7. In order to run 24 samples/plate, set up the plate as indicated below Figure 3.

Figure 3. Scheme representation of a 96-well plate with 12 patient samples being sequenced with 4 primers each (RTC1F, RTC2R, RTC3F, and RTC4R). Click here to view larger image.

8. Add 1.0 µl of the DNA sample (~20-40 ng), cover the plate with an adhesive aluminum cover and then gently mix it.
9. Centrifuge at 3,000 x g for 1 min. Remove the aluminum cover and add a rubber sealing mat.
10. Place the plate on the thermocycler and run the following cycling program shown in Figure 4.

Figure 4. PCR cycling conditions for sequencing. Click here to view larger image.

11. When the PCR finishes, clean up the sequencing product immediately.

10. Sequencing Cleanup

1. For each sequencing reaction, mix 50 μl absolute ethanol and 5 μl 3 M sodium acetate.
2. Using a multichannel pipette, add 55 μl of the sodium acetate/EtOH solution to each well.
3. Seal wells with adhesive aluminum cover, ensuring that each well is sealed properly.
4. Centrifuge at 3,000 x g for 20 min.
5. After 20 min, remove cover and invert the plate, in one smooth motion, onto a folded lab tissue (DO NOT bang to get rid of supernatant as this will dislodge the pellet!).
6. Centrifuge the inverted plate on the same tissue at 150 x g for 2 min.
7. Immediately add 150 μl cold 70% EtOH. DO NOT delay addition of ethanol at this step.
8. Seal with the same adhesive aluminum cover and vortex.
9. Centrifuge at 3,000 x g for 5 min.
10. Invert plate onto a new folded tissue and centrifuge inverted at 150 x g for 1 min.
11. After the centrifugation, place uncovered in thermocycler and dry it at 50 °C for 2 min.
12. Once the plates is dry, seal it with adhesive foil covers, wrap in foil and store at -20 ˚C until ready to proceed with the sequencing electrophoresis.
13. When ready to sequence, dissolve cleaned sequencing products in 10 ml Hi-Di formamide, denature and load for electrophoresis.

11. Bioinformatics

1. Sequence Assembly
   1. Launch the program Geneious.
   2. Create a working folder to store the sequences.
   3. Import the ABI files generated by the sequencing machine to the working folder using the import tool. Geneious will allocate percentage quality score for each sequence imported.
   4. Open sequences with quality scores >70% by double clicking on them.
   5. Each file should open in a new window. The software will indicate the quality at each nucleotide position of the chromatogram of the sequence quality using light blue bars. The higher the bar, the better the quality of the base call.
   6. Using the cursor, select the mid section of the sequence leaving out the ends, which are usually of poor quality.
   7. Click on the extract button to extract the region with good quality sequence.
   8. Select all four extracted sequences for each sample and assemble them against a reference sequence.
   9. Inspect the assembled sequence to ensure that you are in the correct reading frame. If you are in the correct reading frame, the beginning of Protease should start with the following amino acids: PQITLW. The beginning of RT will start with PISPIE.
   10. Extract the contig region covering the beginning of PR to the 300th RT codon. During this process, also check for insertions or deletions.
   11. Go through the consensus sequence of the extracted contig, identifying any ambiguities and verify positions with mixed bases by inspecting quality (symmetry, height, background and shoulders of flanking regions) of the base calls.
   12. Select the consensus sequence and click the extract button to create a separate file of the consensus sequence from the four primers and label it appropriately.
   13. Export the sequence to a backup storage folder on the computer or a network folder.
2. Sequence Quality Assessment (HIVDB)
   1. Analyze the sequence using the HIVDB program at http://hivdb.stanford.edu.
   2. Check for deletions and insertions in the summary data and ensure that the sequence covers all the 99 protease (PR) codons and the 1st 300 RT codons.
   3. Check for any highlighted quality assurance (QA) issues in both the PR and RT regions, such as stop codons, frame shifts, ambiguous positions and unusual residues.
3. Sequencing Quality Control
   1. Blast the new sequence against a local sequence database from previous run.
   2. If the new sequence is >97% similar to any sequence in the database, all the stages of the protocol should be reviewed, starting with the sequence analysis and going back to the RNA extraction to ensure that there were no mix ups (sample switching, mislabeling) or contamination.
   3. If no problems are identified, repeat the analysis of both the old and new samples from the RNA extraction stage.
   4. If the sequences are still >97% similar, review the patient history to assess for any epidemiologic linkage between the individuals.
4. Phylogenetic Analysis
   1. Align all the sequences from the database using ClustalW program in Geneious.
   2. Manually check the alignment for misaligned sequences, deletions and insertions and edit accordingly.
   3. Construct a phylogenetic tree using PHYML, Geneious tree builder or other tree builders in Geneious.
   4. Examine the tree for samples with short branch lengths.
   5. Review the samples with short branch lengths for possible contamination.

12. REGA DB Informatics

1. Sequence Upload
   1. Log into the RegaDB using a unique username and password.
   2. On the drop down menu, under Patient ID, select “Begins with”.
   3. Add the patient ID and select the individual whose genotype is to be uploaded.
   4. On the menu to the left, select “viral isolate”.
   5. From the options under viral isolate select “add”.
   6. Enter the Sample date, Sample I.D, Sequence I.D and Sequence date.
   7. Select “choose file” and then navigate to the fasta file of the sequence to be uploaded.
   8. After selecting the fasta file to be uploaded, click on upload.
   9. Once the uploaded sequence appears in the nucleotide box under the sequence identifies and dates, click the ok button at the bottom right of the window.
   10. Check for PR and RT protein alignment by clicking the button protein and selecting either PR or RT.
   11. Check for the drug resistance mutation by clicking on the resistance button. This gives you the resistance profiles from three algorithms: ANRS, Stanford HIVDB and RegaDB.
2. Report generation using REGA
   1. Log into the RegaDB using your unique username and password.
   2. On the drop down menu, under Patient ID, select “Begins with”.
   3. Add the patient ID and select the individual whose report is to be generated.
   4. On the menu to the right, select viral isolate.
   5. From the options under viral isolate click on “view”.
   6. Double click on the viral isolate for which you want to create a report.
   7. On the viral isolate window, click on the viral isolate report tab.
   8. Select the algorithms for the interpretation of the genotype from the drop down menu and then select report template to use.
   9. Once the algorithm and the template are selected, click on the button “generate”.
   10. Download the rtf document generated.
   11. Open the rtf document as a word document.
   12. Resize the treatment history chart.
   13. After the chart, add the section “Clinical chart and resistance interpretation”.
   14. Using the data on the resistance table and the clinical chart, add a description of the patient’s resistance profile starting with the patient’s treatment history, and the drugs to which the viral isolate is resistant. Also add a description of the patient’s viral load and CD4+ cell count profiles from the chart.
   15. Send the report to the Infectious Diseases (ID) specialist for review and recommendations on future patient management. This process is also a very important quality assurance stage. Any errors in the genotype or inconsistences in the treatment history, virological and immunological profiles can be identified and reviewed before a final report is sent, with all the recommendations, to the clinician managing the patient.