Drug-resistant HIV-1 in Cuban Children and their Seropositive Mothers

INTRODUCTION The use of highly active antiretroviral therapy has reduced progression to AIDS and increased survival among seropositive persons; yet, appearance of resistant viruses may jeopardize these benefits. In Cuba, HIV mainly affects adults; at the end of 2009 of the 41 children infected, 25 were still alive; of these, 22 were under antiretroviral treatment. Until now, nothing was known about HIV-1 antiviral resistance and viral subtypes in the pediatric population in Cuba. OBJECTIVE This study aims to identify presence of antiretroviral-resistant HIV-1 strains in Cuban children and their mothers, and to provide a phylogenetic characterization and comparison of pol gene sequences in the same. METHODS Plasma samples were collected from 22 children and their mothers, all HIV-1-infected, from 2004 through 2009. Reverse transcription polymerase chain reaction was used to amplify the pol gene fragment coding for HIV protease and reverse transcriptase enzymes; this was then sequenced and subjected to phylogenetic analysis of HIV subtypes and recombinant forms to compare sequences between mothers and children. HIV mutations conferring antiretroviral resistance were determined. RESULTS Viral amplification was achieved in samples from 11 children and 8 mothers. Subtypes detected were: CRF19_cpx in five children, subtype B in three, CRF18_cpx in two, and subtype C in one child. In all mother-child pairs, samples were grouped within the same viral subtype in the phylogenetic tree. One mother was under treatment and five children had been treated before the sample was collected. In viruses amplified from samples of children under treatment, resistance was most frequently found to lamivudine (3 cases) and nevirapine (4 cases). Two untreated children carried resistant viruses possibly acquired from their mothers. CONCLUSIONS This is the first study to describe HIV-1 antiviral resistance in the pediatric population in Cuba; it also identified viral subtypes infecting the mother-child pairs studied. We recommend antiretroviral resistance assays before initiating treatment in pregnant seropositive women and their newborns.


INTRODUCTION
The use of highly active antiretroviral treatment (HAART) has reduced progression to AIDS and remarkably increased survival in HIV-positive patients.These benefi ts are threatened by emergence of antiviral resistance-due to mutations in HIV genes coding for viral proteins targeted by antiretroviral (ARV) drugsresulting in partial to total loss of susceptibility to inhibitors, impeding their therapeutic effects.[1] Mother-to-child transmission (MTCT) of HIV-1 is the main cause of pediatric HIV-1 infection and may occur in utero, during delivery or with breastfeeding.[2] Worldwide, about 1600 children are infected daily, 90% of them in Sub-Saharan Africa, where HIV-1 MTCT is the main contributor to infant mortality.[3] Earlier, only sporadic reports appeared in the literature describing MTCT by drug-resistant HIV-1 strains.[4] However, more recent studies have found that 9% to 30% of HIV-1-infected children were born to mothers who received prophylactic zidovudine (AZT), demonstrating resistance to the drug among these newborns.[5][6][7][8][9][10] Thus, while antiretroviral therapy (ART) has led to important advances in the last decade in preventing MTCT HIV-1 transmission, there remain some situations that are particularly risky: unknown maternal HIV status, primary HIV infection during pregnancy, and suboptimal maternal ART prophylaxis, among others.[10,11] Since the effi cacy of AZT was fi rst demonstrated in 1994, a gradual decrease of vertical HIV transmission has been observed in Europe and the United States, reducing rates from 25% to below 2%.[10,12,13] Based on such evidence, all newborns of HIV-1 seropositive mothers in Cuba are treated with AZT in the fi rst six weeks after birth.If HIV-1 infection is confi rmed, they are started on ARV treatment.Domestically-manufactured ARVs have been used in Cuba since 2001.At fi rst, limited supplies meant only some patients could be treated; children and pregnant women were among those given priority.[14] ARV drugs most frequently used are: nucleoside reverse transcriptase inhibitors (NRTI)-lamivudine (3TC), stavudine (D4T) and zidovudine (AZT); non-nucleoside reverse transcriptase inhibitors (NNRTI)-nevirapine (NVP); and protease inhibitors (PI)-indinavir (IDV).The most frequently used regimen includes 3TC, AZT and NVP.The most widely used antiretroviral in combined treatments is 3TC.[14] Until 2008, all HIV-positive pregnant women in Cuba were treated with AZT beginning at 14 weeks of gestation; and depending on immune status, additionally with 3TC and NVP.At the end of 2008, the therapeutic protocol was changed, introducing the triple therapy used today (3TC, AZT and NVP).When HIV infection is diagnosed late (during third trimester of pregnancy), AZT, 3TC and a lopinavir/ritonavir (Kaletra) booster are used.[15] By the end of 2009, 12,217 persons had been diagnosed with HIV-1 infection in Cuba.Of 402 children born to HIV-1-positive mothers, 36 were infected by vertical transmission (9%) and fi ve (1.2%) through blood.At the time of this study, 25 children were alive and 22 were receiving ART (National HIV Registry 2009 Annual Report, Ministry of Public Health, Havana).
Previous molecular epidemiology reports on HIV-1 describing most frequent subtypes and ARV resistance have been limited to adult populations.[9][10][11][12][13][14][15] Reverse transcription and amplifi cation with polymerase chain reaction (PCR) Reverse transcription (conversion of the HIV RNA molecule into a DNA copy) followed by PCR amplifi cation of 2060 bp of the HIV pol gene was carried out using the commercial kit SuperScript One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen, USA) under the following conditions: 15 μL of 2X reaction buffer (0.4 mM of each of the deoxinucleotides, dNTP, and 2.4 mM MgSO 4 ), 0.5 μL of each primer (av159 and av192) at 10 uM and 8 μL of 40 mM MgSO 4 , 1 μL of 50 U SuperScript III RT with Platinum Taq High Fidelity enzyme supplemented with 0.25 μL of 10 U RNAse protector (Roche, Germany) and the 40 μL volume was completed with water.Finally, 10 μL of extracted RNA were added to this mixture, for a fi nal reaction volume of 50 μL.
Reverse transcription took place at 55 o C for 30 minutes, followed by an initial denaturation at 94 o C for 2 minutes and 40 cycles of: denaturation at 94 o C for 15 seconds, hybridization at 61 o C for 30 seconds, and elongation at 68 o C for 3 minutes.A last elongation step took place at 68 o C for 5 minutes.
A second polymerase chain reaction (nested PCR) was performed to obtain a 1745 bp internal fragment of the pol gene using the Expand High Fidelity PCR System commercial kit (Roche, Germany).Two tubes were prepared for each sample to carry out this reaction.The fi rst tube contained a mixture of 0.4 μL of 10mM dNTPs, 1.25 μL of each primer (av190 and av191) at 2.5 mM concentration, and water to complete a volume of 23 μL.The second tube contained a mixture prepared with 5 μL 10X Buffer Expand, 6 μL of 150 mM MgCl 2, 0.75 μL of 2.625 U Expand HF Enzyme (Roche, Germany), and water to complete a volume of 25 μL.Two milliliters of amplifi ed cDNA (complementary DNA, synthesized by reverse transcriptase from a Messenger RNA template) were then added to the fi rst tube, for a total volume of 25 μL.The second mixture was added to the fi rst for a fi nal reaction volume of 50 μL.This reaction was subjected to 94 °C for 2 minutes and then cycles of: 94 °C for 15 seconds, 59 °C for 30 seconds, and 68 °C for 3 minutes.Finally there was an elongation at 72 °C for minutes.
Purifi cation and sequencing Ten microliters of the nested PCR product were used, plus 2 μL of run indicator (Blue/Orange 6 X loading Dye, Promega, USA) and applied to a 1% agarose gel stained with ethidium bromide (BDH, 0.1 μg/mL) to perform horizontal electrophoresis using a DNA standard of 1 kb molecular weight (Step Ladder, Promega, USA).Presence of amplifi ed virus DNA was detected by observing the electrophoresis-separated samples through a UV transilluminator.A visible band should show of the size expected (1745 bp).The electrophoresis-positive PCR products were subjected to column purifi cation using the QIAquickPCR Purifi cation (QIAGEN, Germany) kit following manufacturer's instructions.
To obtain both sense nucleotide sequences of the pol gene bp fragment-which codes for the HIV-1 99 protease amino acids and 335 reverse transcriptase amino acids-6 sequence reactions were performed with different primers for each purifi ed DNA (KVL162, KVL163, KVL164, KVL165, AV5, KVL176).For the sequencing reactions, mixtures formed by 1 μL of each sequencing primer, 8 μL of sequence reaction mixture DTCS Quick Star Master Mix (supplied with the Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit, from Beckman Coulter, USA), 5 μL of purifi ed DNA (approximately 100 ng) and 6 μL of water to complete 20 μL of reaction mixture.The sequencing reaction consisted of two minutes denaturation at 96 ºC, followed by 50 cycles of: denaturing at 96 ºC for 20 seconds, 20 seconds of hybridization at 50 ºC and 4 minutes of extension at 60 ºC.Once the sequence reaction was concluded, it was purifi ed, following the protocol described in the DTCS Quick Star Master Mix commercial kit (Beckman Coulter, USA).The sequencing reaction run was done in a Beckman Coulter model CEQ8800 (USA automatic sequencer), using the crude data analysis procedure for PCR products.
The primers to amplify and sequence the corresponding HIV-1 pol gene region were designed by Leathem and Vandamme at the Rega Medical Research Institute in Belgium, using OLIGO 5.0, 2007 (Personal Communication: Kristel Van Leathem and Anne Mieke Vandamme, Rega Institute for Medical Research, Louvain Catholic University, Belgium).

Sequence edition
The six sequence reactions for the pol gene fragment obtained from each purifi ed HIV-1 DNA, were assembled and edited with the Sequencher Version 4.9 (Gene Codes Corporation, USA) platform, using as a reference the nucleotide sequence of HIV-1 strain B.FR.83.HXB2_LAI_IIIB_BRU.K03455, to obtain a single HIV-1 consensus sequence for every patient's virus.

Original Research
Peer Reviewed Phylogenetic analysis To defi ne HIV-1 subtypes or genetic variants in samples studied and similarity or divergence between pairs, phylogenetic analysis constructing trees by neighbor joining was performed based on Kimura's method for distance between two parameters [16] using the MEGA program version 4. [17] The analysis of recombinant genetic forms was done by bootscanning [18], using the Simplot V, version 3.5.1.[19] Tree topology reproducibility was evaluated by bootstrapping using 1000 replicas.A phylogenetic group was defi ned as having a bootstrap value of ≥70%.[20] Evolutionary distances were estimated with the DNA-Dist program using Kimura's two parameter method.[16] Mutation analysis and resistance inference The complete 99 amino-acid protease (PR) sequence and 335 amino-acid sequence of reverse transcriptase (RT) of each previously sequenced and edited virus was sent to the Stanford University database to determine mutations conferring resistance to transaminase and protease inhibitors, as well as resistance levels (high, intermediate, low or susceptible) to each specifi c drug.[21]

RESULTS
Of all samples processed, HIV amplifi cation was achieved in only 15 of the children's samples (11 children) and in 8 maternal samples (Table 1).This was probably because the viral load in negative cases was under 1000 copies/mL-the sensitivity limit for the PCR used to amplify the HIV-1 pol gene fragment in patients with ARV therapy failure.
Five children and one mother with viral amplifi cation had received ARV; the mother had received AZT monotherapy.One child was sexually infected and the rest, vertically (transplacentally, perinatally or from breastfeeding) according to clinical records.Serial HIV specimens from three children were amplifi ed and sequenced; three in Patient 4 (sequences NCB4, NCB4a and NCB4b), and two samples from the other two, Patient 1 (sequences NCB1 and NCB1a) and Patient 2 (sequences NCB2 and NCB2a) (Table 1).
The sample was too small to permit statistical inferences for proportions, subtypes, or mutation development.
Phylogeny Subtype analysis showed recombinant form CRF19_cpx present in four children; subtype B in three; CRF18_cpx, in two and subtype C in one.All viruses obtained in children of mother−child pairs were coincident for one subtype, results supported by high bootstrapping values observed in trees (≥75%) (Figure 1; Table 2).Estimated evolutionary divergence between sequences in mother-child dyads MCB1-NCB1, MCB4-NCB4, MCB5-NCB5, and MCB9-NCB9 is almost zero, with values of 0.008, 0.002, 0.003, and 0.002, respectively.This is because the samples were obtained within two years of diagnosis; moreover, in all four pairs the dates of diagnosis were within fi ve weeks of one another.The greatest divergence values were found between MCB7-NCB7 (0.010) and MCB11-NCB11 (0.082).Samples from these patients were collected several years after HIV infection diagnosis, which could affect the values obtained (Table 1).Estimated evolutionary divergence between sequences of HIV-1 serial specimens of the same patient was zero (0.000) for NCB1 and NCB1a and for NCB2 and NCB2a; that is, serial sequences of these two children were almost identical.However, between the fi rst NCB4 virus and the third NCB4b, this value was 0.012; while between the second (NCB4a) and third (NCB4b) the value was 0.008, demonstrating that this child's virus changed over time.Of the serial specimens from Patient 4 (NCB4, NCB4a y NCB4b), the second one was obtained three months later, when the child was already in ARV therapy, and two mutations were detected in the virus.The third sample was collected 33 months after the fi rst; by that time, a larger number of mutations had accumulated, presenting even greater divergence between the fi rst and last samples.
HIV-1 mutations associated with ARV resistance Treated children Mutations conferring resistance to ARV were detected in viruses of all fi ve children under treatment at time of sample collection (NCB1, NCB2, NCB4, NCB8 and NCB11).In patients with serial plasma samples (seven samples), mutations were detected in more than one sample (Table 2).
Four children were being treated with 3TC, NVP, D4T and one with 3TC, NVP, AZT, corresponding to viral mutations and resistances found (Table 2).
The most frequent mutations were M184V (conferring high resistance to 3TC and FTC medications), found in fi ve samples from three patients and K103N (conferring resistance to NNRTIs), detected in six samples from four patients.Mutations K70KN, F116Y, A98AG, V108IV, L210F, H221HY, F227FL and G190AG, which affect sensitivity to NRTI and NNRTI drugs, were observed less frequently.Mutation Q151M, found in amplifi ed virus of the third sample of Patient NCB4b is noteworthy, since it is accompanied by sequential accumulation of other mutations increasing loss of sensitivity to all NNRTI (Table 2).
In repeat samples from three patients (NCB1, NCB2, NCB4), viruses were detected that maintained resistance mutations from the fi rst sample.This is because treatments were not changed, either because patients did not fulfi ll clinical, immunologic or virologic criteria for therapeutic failure; or because the country lacked genotype assays for assessing how best to manage these patients.
Untreated children Mutations conferring ARV resistance were detected in two of six viruses sequenced from children who had not been treated at the time of sampling (NCB3, NCB10).Mutations L90LM and V106IV were found in Patient NCB10's virus.The former is associated with reduced PI sensitivity.The exact time this child was infected is unknown, but his mother was not infected at time of delivery, since she was tested twice during her pregnancy, both were diagnosed more than fi ve years later, and he had been breastfed for three years.We therefore infer that this patient acquired HIV from breastfeeding and that his mother transmitted the resistant strain amplifi ed in his sample, although it was not found in hers.The virus population detected in the child could have gone undetected in the mother if it was not the majority virus at the time of sample collection; minority virus populations (less than 20%) may not be detected by the usual techniques used for resistance studies.[22] Patient NCB3's virus had multiple mutations (M184V, T215Y, A98G, K103S and G190A) conferring resistance to NRTIs and NNRTIs (Table 2).This child was born to an HIV-infected mother under ARV therapy, so measures had been taken to avoid vertical transmission (birth was by caesarean section and the baby was

Original Research
Peer Reviewed not breastfed).Even so, the child was infected, probably because of the mother's poor compliance with therapy (data from clinical records).There was no virus amplifi cation in the mother's sample, so we could not demonstrate presence of these mutations, but we infer that she transmitted this multi-resistant strain to her child, since she was treated in 2004 with 3TC, NVP and D4T, and in 2005 with 3TC, NVP y AZT.
Child NCB7 did not show the V118I mutation detected in the mother (MCB7), possibly because four years had passed between the child's birth and the time of sample collection.

Untreated mothers
In the analysis of viruses obtained from untreated mothers, mutations were detected in two of eight patients (MCB7 and MCB12); one had the V118I mutation and the other, K219Q/E, both of which affect sensitivity to NRTIs in the presence of other mutations, but not on their own.

DISCUSSION
HIV evolutionary studies and detection of virus mutations conferring ARV resistance in the pediatric population require serial specimens from children from birth, and from mothers before starting treatment, at time of delivery and thereafter.Thus identifying resistant strains transmitted provides a timely and effective aid in clinical management.[23] Among the limitations of this study are the absence of viral amplifi cation and lack of serial specimens from the women in pregnancy and from some of the children.Ob-taining serial specimens from pregnant women is diffi cult, since most HIV-positive women decide to become pregnant when they are clinically, immunologically and virologically compensated and so have undetectable viral loads.
In the present study, most mothers in whose samples virus amplifi cation was achieved were diagnosed at the end of pregnancy or after delivery-in some cases, more than a year later-infecting their children during breastfeeding.So in some cases we are missing the children's samples at birth because they were not diagnosed until later; in some for whom we did have a sample taken at birth, the viral load was undetectable.Note that the PCR technique used in ARV resistance studies is not the same that is used for diagnosis and has different sensitivity limits.[24−27] The technique used in this study can detect viral loads between 500 and 1000 copies/mL.
All analyzed sequences of the children's pol gene fragments coincided with those of their respective mothers and were consistent with subtypes previously seen in Cuba.[28][29][30] Evolutionary divergences between viruses obtained from mothers and their children were very small (in some cases zero), consistent with the short time elapsed between collection of samples from mother and child at the time of diagnosis.This was also true for children's serial specimens.The greatest divergences occurred when mothers had been infected more than fi ve years previously.[31,32] Original Research Peer Reviewed  Most pediatric patients studied were infected by vertical transmission as a result of delayed diagnosis of HIV infection in the mother.Cuba has reported reduced rates of vertical transmission with correct ARV compliance in the infected mother.[33,34] However, possibly due to inadequate ARV compliance, we have detected ARV resistance mutations which may then be transmitted from mother to child.[35−37] Both timely diagnosis and more powerful therapy are increasingly important, [38] which is why Cuba changed its treatment protocol for HIV-1-infected pregnant women in 2008.[15] It is critical that resistance assays be done before starting ARV.Therapeutic options for children are already limited because not all drugs are available in pediatric formulations.[39][40][41] Verticallytransmitted resistances further limit therapeutic options.[39,42] Delaugerre and colleagues describe two mechanisms involved in MTCT of resistant strains.The fi rst involves a majority population in the mother transmitted to the child during delivery; the second, a minority-resistant population that becomes majority in the child.[10] As is seen in this study, in some cases MTCT of resistant viruses can be inferred, if not proven, since some mutations are observed in children that for various reasons are not found in their mothers.
Since commencing domestic ARV manufacture, Cuba's health system has given priority to mothers and children.
[14] However, mutations have been found in all children treated, sometimes after as little as 4 months' therapy.This underscores the need for resistance assays in pregnant and puerperal women and in the infected child to search for transmitted resistant strains.Early appearance of mutations may result from the use of low genetic barrier medications, for which just one mutation can cause loss of sensitivity, or may occur when an undetected minorityviral population rapidly emerges in the presence of an antiviral to which it is resistant.[43] Rapid emergence of resistance to 3TC and NVP with the appearance of M184V and K103N mutations, respectively, has been reported and worldwide.[40,[44][45][46][47] In our pediatric patients under treatment, we also found the NRTI-resistant HIV- Pediatric treatment of HIV infection has the same goals regardless of context: to limit vertical transmission; [38,39] to minimize emergence of resistant viruses in mother and child if vertical transmission does occur; and in such cases, to characterize the viral ARV resistance profi le in the child beginning treatment.[23] Without resistance tests, effective fi rst-line therapy is essential.

CONCLUSIONS
All sequences of the HIV-1 pol gene obtained from children in this study were the same as those of their respective mothers and are among the subtypes previously seen in Cuba.We detected mutations conferring resistance to NRTI in all amplifi ed viruses from treated children, consistent with the drugs used in that population.In samples taken from mothers and children at the time of diagnosis, we detected possible resistant-strain transmission.The results of this study highlight the importance of resistance assays in pediatric HIV patients at the time of diagnosis prior to starting therapy, and, if treatment failure is suspected, during therapy.Strict adherence to ARV protocols is critical in both pregnant women and children, since early infection with resistant strains can have dire long-term consequences for both mother and child, undermining treatment effectiveness.
This study is the fi rst in Cuba to determine levels of ARV-resistant viruses in HIV-1-infected mothers and children, describe related mutations, and correlate them with therapy used.The results of this study make an important contribution to patient management and selection of appropriate therapy and follow-up, helping improve their prognosis and quality of life.

Figure 1 :
Figure 1: Phylogenetic trees (by neighbor-joining) of 23 hiv-1 pol gene sequences from mothers and children

Table 1 : Information on patients whose samples achieved viral amplifi cation
BF: Breastfeeding; TP: transplacental; NCB: Cuban child; MCB: Cuban mother; NT: not treated at time of sample Source: Clinical records, IPK