Supplementary appendix

Transcription

1 Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Goedhals D, Rossouw I, Hallbauer U, Mamabolo M, de Oliveira T. The tainted milk of human kindness. Lancet 2012; 380: 702.

2 SUPPLEMENTARY INFORMATION: 1. Supplementary Notes: Background of this transmission case: The case infant was born by normal vaginal delivery at 37 weeks gestation at a midwife clinic on 29 June The 19 year old mother (Gravida 2 Para 2) had attended antenatal visits at the local clinic and her clinical chart showed her to be RPR and HIV negative. The 2.9kg baby was referred directly to a secondary hospital with transient tachypnea of the newborn. She was treated with supportive oxygen, fluids and antibiotics, and blood gases and infective markers were found to be normal. Breastfeeding was started during the hospital admission and the infant was discharged after 6 days in a stable condition. The child was then admitted to the Pediatric Intensive Care Unit at Pelonomi Hospital, Bloemfontein, South Africa on 11 September 2009 with a two day history of rapid breathing, coughing, weakness and lethargy. Clinical examination showed a respiratory rate of 75 breaths/minute, heart rate of 157/minute, and temperature of 35.8 C. Oxygen saturation was 34% on room air and 98% on supplemental oxygen administered via a head box. The infant appeared to be anemic, cyanosed and had palpable axillary lymphnodes. The respiratory examination showed nasal flaring, subcostal recession and bilateral coarse crepitations on auscultation. Laboratory testing confirmed a normochromic, normocytic anemia (Hb 9.8 g/dl) and neutrophil leukocytosis, with a C- reactive protein of 70.1 mg/l (reference range: 0-10 mg/l) and lactate dehydrogenase of 978 U/l (reference range: U/l) on the day of admission. Antibiotics including intravenous cotrimoxazole were administered, however, microbiological testing of cerebrospinal fluid and blood cultures

3 showed no growth, while a urine culture showed no significant organisms. Indirect immunofluorescent antibody testing for Pneumocystis jirovecii was negative, but cytomegalovirus (CMV) was isolated on shell vial culture of a respiratory sample and a CMV viral load of copies/ml was found in a paired blood sample. She was successfully weaned off oxygen therapy after eight days. An HIV ELISA taken on 11 September 2009, as well as a confirmatory sample on 16 September 2009, tested positive on the infant. An HIV DNA PCR was then submitted on 17 September 2009 which was also found to be positive. She had a CD4+ T cell count of 2189 x10 6 cells/l (40% of lymphocytes) and antiretroviral therapy was started. At the six month follow- up visit, the HIV viral load was undetectable and the CD4+ T cell count was 3378 x10 6 cells/l (29.4% of lymphocytes). An HIV ELISA test (combination antibody/antigen assay) was performed on the mother on 19 September 2009, which confirmed the history obtained from the antenatal clinic that she was HIV negative. The aunt and her own 117 day old child were subsequently found to be HIV positive by HIV ELISA (submitted 12 October 2009) and HIV DNA PCR (submitted 23 October 2009) respectively. Laboratory records subsequently confirmed that the aunt had tested HIV ELISA positive in February 2008 and a previous child of hers had tested HIV DNA PCR positive at that stage. It cannot be confirmed whether the aunt was counselled regarding this result, but she claimed that she was not aware of her HIV status. The characteristics of each individual involved are summarized in table 1. EDTA blood samples were obtained from the aunt, cousin and case infant on 27 November The samples were centrifuged at 4000 rpm for 10 min and the plasma was stored in cryotubes at - 80 C until testing. Informed consent was

4 signed by the mother and aunt to publish details of the case and laboratory testing pertaining to themselves and their respective children. Ethics approval was obtained from the University of the Free State Ethics Committee (ETOVS 220/09). 2. Supplementary methods: RNA Extraction, PCR Amplification and Sequencing. RNA was extracted from plasma using the QIAmp Viral RNA kit (Qiagen, Heiden, Germany). Briefly 140µl of plasma was added to lysis buffer containing carrier RNA and loaded onto a column. The spin protocol was followed using a microcentrifuge and after washing the sample was eluted in 40µl of nuclease free water. Sequencing was performed using the TRUGENE HIV- 1 Genotyping kit according to the manufacturer s instructions (Visible Genetics, Inc). From the extracted RNA, 17µl was mixed with RT- PCR reagents including buffer, primers, dntps, dithiothreitol, RNAse inhibitor, reverse transcriptase enzyme, and Taq DNA polymerase. The RT- PCR primers used in the kit are designed to amplify the entire protease (codons 1-99) and codons of reverse transcriptase. The reverse transcriptase reaction involves an incubation at 90 C for 2 min, followed by 50 C for 5 min. Following the addition of reverse transcriptase enzyme, the reaction continued for a further 55 min before ending at 94 C for 2 min. DNA amplification followed with the following cycling conditions: 20 cycles of 94 C for 30 s, 57 C for 30 s, and 68 C for 2 min; then 17 cycles of 94 C for 30 s, 60 C for 30 s, and 68 C for 2.5 min; lastly 1 incubation of 68 C for 7 min a 4 C hold. 5µl of the RT- PCR product from each specimen was used in each of 16 sequencing reactions. This is based on the CLIP principle 1 which uses four pairs of primers, namely two pairs that sequence protease and one pair each for the beginning

5 and middle of reverse transcriptase. Four sequencing reactions were run for each segment, using one of four dideoxy terminators (ddatp, ddctp, ddgtp, and ddttp) and at least two primers. The forward and reverse primers were labeled with fluorescent dyes (Cy5 and Cy5.5 respectively). The cycling conditions were as follows: 30 cycles of 94 C for 20 s, 56 C for 20 s, and 70 C for 1.5 min; followed by 70 C for 5 min. The CLIP sequencing products were analyzed using Surefill 6% sequencing polyacrylamide gel cartridges and Microcel cassettes, which underwent gel electrophoresis at 2000 V for 50 min on the Long Read Tower Sequencers. 2 Sequence data analysis Sequence data was analysed using the OpenGene system software. The forward and reverse sequences are combined and compared to the sequence of HIV- 1 LAI and a mutated reference sequence in order to determine the presence of all mutations, including drug resistant mutations. Manual editing was limited to bases in positions known to be associated with resistance mutations and bases that were discordant between the two sequencing reactions. The edited sequences were exported from the software in fasta format for analysis. 2 Reference Sequences and Alignment The first reference dataset contained HIV- 1 subtype C sequences isolated in the Free State Province of South Africa. This dataset was obtained from the paper by Huang and colleagues. 3 In this paper, the authors have sequenced 425 highly active antiretroviral therapy (HAART)- naïve HIV- 1 positive patients from the Free State province in South Africa. In total, 108 reference strains with highest BLAST search similarity score to the case were chosen for phylogenetic

6 construction. The alignment was 880 nucleotide base pairs in length and spanned the protease amino- acid positions 4 to 99 and the reverse transcriptase positions 38 to 235. The second dataset for HIV- 1 subtype C was obtained from the HIV Sequence Database at Los Alamos ( and from GenBank ( In total 1,379 reference strains were chosen for the analysis. All of the sequences selected spanned the 880bp region used in the first analysis. These include HIV- 1 subtype C strains from Argentina (4), BE (9), Brazil (24), Botswana (137), CD (10), CH (1), CM (1), CN (2), CZ (11), DE (1), DJ (1), DK (18), ER (2), ES (1), ET (11), France (7), GA (1), GE (1), Israel (6), India (20), Italy (14), Kenya (3), LC (1), MM (1), Malawi (146), Nigeria (1), Portugal (1), SD (11), SE (40), SN (7), Somalia (1), Tanzania (22), UA (3), Uganda (10), United States of America (6), Uruguay (1), Yemen (1) and South Africa (797). All of the reference sequences from the first and second dataset and the three case sequences were subtyped using the Rega HIV- 1 Subtyping Tool version ,5 All sequences were confirmed as HIV- 1 subtype C. The case sequences were then aligned with the two reference datasets using ClustalW 6 and subsequently edited by hand using Se- Al The consensus HIV- 1 subtype B sequence from the Los Alamos Database was added to both datasets and was used as the outgroup in the phylogenetic analysis. Phylogenetic Analysis First dataset (111 sequences: 3 cases, 107 Free State and 1 reference subtype B): The best fitting nucleotide substitution model was evaluated for the first dataset using hierarchical likelihood ratio tests the methodology implemented in ModelTest. 8 The model chosen for the HIV- 1 alignment was GTR+G (general time

7 reversible model with gamma distributed among- site rate heterogeneity). Maximum likelihood (ML) phylogeny was estimated for the first dataset (smaller dataset, which contained 111 sequences) under the abovementioned models, starting with a NJ starting tree and using the TBR (tree bisection and reconnection) and SPR (subtree pruning and regrafting) heuristic search algorithms. The final ML phylogeny is shown in Supplementary Figure 1. Calculations were performed with PAUP* 4.0b10. 9 Statistical support for ML phylogeny structures was evaluated by bootstrapping analysis of the original sequence alignments (1000 NJ replicates). The tree was rooted using the HIV- 1 subtype B consensus reference dataset and presented using the program FigTree. 10 The model parameter estimations and ML assessment took 123 hours and 16 minutes to be computed (5,14 days), using 2 x 2.66 GHz 6- Core Intel Xeon MacPro computer. Maximum likelihood parameters estimates are seen on supplementary table 2. Bayesian estimates of phylogeny were obtained using MrBayes 11, under the GTR+G model for the first HIV- 1 subtype C alignment. Four Markov Chain Monte Carlo (MCMC) runs were calculated independently. Each MCMC run was 10,000,000 steps long and was sampled every 1000 steps, with 0.2 as the temperature parameter. The average standard deviation of split frequencies was calculated for the four chains to check for convergence. Subsequently, the Effective Sampling Size (ESS) was calculated by combining the output of the four runs using Tracer, excluding an initial burn- in of 10% for each chain. 12 The ESS values were >100, indicating a sufficient level of sampling. The four runs were combined using LogCombiner. 13 A final Bayesian majority- rule consensus tree was obtained with the combined runs using TreeAnnotator. 14 The tree was rooted using the HIV- 1 subtype B consensus reference dataset and presented

8 using the program FigTree. 10 The model parameter estimations and four Bayesian chains assessment took 72 hours and 32 minutes to be computed (3,02 days), Bayesian parameters estimates are seen on supplementary table Z. Second dataset (1,379 sequences; 3 cases, 1375 Subtype C worldwide reference sequences and 1 reference subtype B): Given the number of sequences on this alignment, phylogenetic reconstruction was done with the use of neighbor- joining (NJ) and minimum evolution (ME) methods. These are the two most appropriate methods for large phylogenies construction and assessment. The assessment of the phylogenies was performed with 1,000 bootstrap resampling. Neighbor- joining (NJ) phylogeny was estimated under the GTR+G evolutionary model. Model parameters were estimated using a maximum likelihood framework and a NJ starting tree. Model parameters were estimated using three rounds, with start values from the previous round. The final model estimates were RMatrix (1.37, 9.12, 0.80, 0.75, 9.12, 1.0), Gamma rate variation across sites alpha shape value (0.5). These were used for the construction of the NJ phylogeny. Calculations were performed with PAUP* 4.0b10. 9 Statistical support for NJ phylogeny structures was evaluated by bootstrapping analysis of the original sequence alignments (1000 NJ replicates). The model parameter estimations and NJ assessment took 3,5 days, using 2 x 2.66 GHz 6- Core Intel Xeon MacPro computer. The final NJ phylogeny is shown in Supplementary Figure 3. Minimum evolution (ME) phylogeny was estimated under the F84 model of evolution with Gamma rate variation across sites The reason to not use the GTR was due to the absence of this model in FastMe 15, the software used for the

9 ME phylogeny construction. The ME phylogeny was constructed with Nearest Neighbor Interchange (NNI) algorithm. NNI is a heuristic algorithm for searching treespace. During each round, the best tree is retained, in total 718 rounds were necessary for the construction of the final tree, which is shown in Supplementary Figure 4. The model parameter estimations and ME assessment took 21 hours and 21 minutes. The tree was assessed with 1,000 bootstrap replicates. It is important to note that the phylogenetic methods used in this analysis cannot be used to determine the direction of transmission, and cannot exclude horizontal transmission between the two children, or transmission from the infant- case to the aunt. However, this may be an unlikely event and the fact that the aunt breastfed her niece suggests that transmission is more likely to have happened via breastfeeding. The possibility that the aunt gave birth to both children and that one of them was given to her sister is excluded due to the dates of birth of the children, which are separated by 44 days. Supplementary Material References: 1. Yager, T. D., Baron L., Batra R., et al. High performance DNA sequencing, and the detection of mutations and polymorphisms, on the Clipper sequencer. Electrophoresis 20: (1999). 2. Grant RM, Kuritzkes DR, Johnson VA, et al. Accuracy of the TRUGENE HIV- 1 genotyping kit. J Clin Microbiol 41: (2003). 3. Huang K.H., Goedhals D., Fryer H., et al. Prevalence of HIV type- 1 drug- associated mutations in pre- therapy patients in the Free State, South Africa. Antivir Ther.,x14(7): (2009).

10 4. De Oliveira, T., Deforche, K., Cassol, S., et al. An automated genotyping system for analysis of HIV- 1 and other microbial sequences. Bioinformatics 21: (2005). 5. Alcantara L.C.J., Cassol S., Libin P., et al. A Standardized Framework for Accurate, High- throughput Genotyping of Recombinant and Non- recombinant Viral Sequences. Nucleic Acids Research, 1-9, doi: /nar/gkp455 (2009). 6. Thompson, J.D., Higgins, D.G., Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position- specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, (1994). 7. Rambaut, A Se- Al: Sequence Alignment Editor. Available at (1996) 8. Posada D., Crandall K.A. MODELTEST: testing the model of DNA substitution. Bioinformatics. 14, (1998). 9. Swofford, D.L. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts (2003). 10. Rambaut, A. FigTree. Available from (2011). 11. Ronquist, F., Huelsenbeck, J.P. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, (2003) 12. Rambaut, A., Drummond, A.J. Tracer. Available from (2011)

11 13. Rambaut, A., Drummond, A.J. & Marc Suchard. LogCombiner. Available from (2011) 14. Rambaut, A., Drummond, A.J. & Marc Suchard. TreeAnnotator. Available from (2011) 15. Desper R., Gascuel O Fast and accurate phylogeny reconstruction algorithms based on the minimum- evolution principle. FastME algorithms (balanced and OLS GME, balanced and OLS NNI, balanced and OLS branch length optimization): Journal of Computational Biology. 9(5):

12 Supplementary Table 1: Individual characteristics Infant-Case Mother Aunt Infant- Cousin DOB 29/06/ /11/ /09/ /05/2009 Age (when 74 days 19 years 25 years 117 days seen by clinicians in Sept 2009) HIV ELISA Positive Negative Positive Not done test HIV PCR test Positive Not done Not done Positive HIV Yes No Yes Yes genotyping On ART No Not applicable No No when first seen

13 Supplementary Table 2 - Maximum Likelihood (ML) and Bayesian parameters for the First dataset (111 sequences: 3 cases, 107 Free State and 1 reference subtype B). Method Model LnL R- Matrix Alpha PInv Maximum GTR+G+I , 10.16, likelihood 0.86, 0.84, Bayesian run1 Bayesian run2 Bayesian run3 Bayesian run4 Bayesian combined GTR+G+I , 0.38, 0.04, 0.02, 0.41, 0.04 GTR+G+I , 0.38, 0.04, 0.02, 0.44, 0.04 GTR+G+I , 0.38, 0.04, 0.02, 0.44, 0.04 GTR+G+I , 0.38, 0.04, 0.02, 0.44, 0.04 GTR+G+I , 0.38, 0.04, 0.02, 0.44,

14 Supplementary Figure 1 Maximum likelihood phylogenetic tree showing the relationship among the three (n=3) transmission case s sequences and other HIV- 1 subtype C sequences from the Free State (n=107). The tree is rooted with a HIV- 1 Subtype B reference sequence (BRef). The red branches and red text represent sequences from the transmission cases and their bootstrap support values, respectively.

15 Supplementary Figure 2 HIV- 1 Consensus Majority Rule Bayesian Tree showing posterior probabilities for the internal branch of the tree that connect the three case sequences. Sequences from the transmission case are shown in red; the tree is rooted with the HIV- 1 subtype B reference sequence; the remaining 107 sequences are the most similar HIV- 1 subtype C sequences in the Free State Province, South Africa.

16 Supplementary Figure 3 Neighbor- Joining (NJ) phylogenetic tree showing the relationship among the three (n=3) transmission case s sequences and other HIV- 1 subtype C sequences (n=1375). The tree is rooted with a HIV- 1 Subtype B reference sequence (BRef). The red branches and red text represent sequences from the transmission cases and their bootstrap support v alues, respectively.

17 Supplementary Figure 4 Minimum evolution phylogenetic tree showing the relationship among the three (n=3) transmission case s sequences and other HIV- 1 subtype C sequences (n=1375). The tree is rooted with a HIV- 1 Subtype B reference sequence (BRef). The red branches and red text represent sequences from the transmission cases and their bootstrap support v alues, respectively.