HIV transmitted drug resistance in adult and pediatric populations in Panama
Farmacorresistencia transmitida del VIH en poblaciones adultas y pediátricas en Panamá
Juan CastilloI; Griselda ArteagaI; Yaxelis MendozaI; Alexander A. MartínezI; Rigoberto SamaniegoII; Dora EstripeautIII; Kathleen R. PageIV; Rebecca E. SmithI; Nestor SosaI; Juan M. PascaleI
IInstituto Conmemorativo Gorgas de Estudios de la Salud, Panama City, Panama. Send correspondence to: Juan M. Pascale, firstname.lastname@example.org
IIHospital Santo Tomás, Departmento de Enfermedades Infecciosas, Panama City, Panama
IIIHospital del Niño, Departmento de Enfermedades Infecciosas, Panama City, Panama
IVJohns Hopkins University School of Medicine, Division of Infectious Diseases, Baltimore, Maryland, United States of America
OBJECTIVE: To investigate the prevalence of transmitted drug-resistant HIV among adults in Panama by using a modified World Health Organization Threshold Survey (WHO-TS) and to investigate rates of initial resistance among HIV-positive infants in Panama.
METHODS: At the Gorgas Memorial Institute, 47 HIV-positive adults were genotyped for mutations associated with transmitted drug resistance (TDR) in the reverse transcriptase and protease genes of HIV-1, according to WHO-TS guidelines, modified to include patients ≤ 26 years old. Prevalence rates for drug-resistance mutations against three classes of antiretroviral drugsnucleoside analog reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitorswere calculated as low (< 5.0%), moderate (5.0%15.0%), and high (> 15.0%). Twenty-five infant patients were also geno-typed and prevalence rates for drug-resistance mutations were calculated.
RESULTS: TDR among Panamanian adults was moderate: 6 of 47 HIV-positive adults showed one or more mutations associated with TDR. Horizontal TDR mutations were moderate for NRTIs and NNRTIs and low for protease inhibitors. Vertical transmission of HIV in Panama has decreased for 20022007, but vertical HIV TDR prevalence is moderate (12.0%) and is emerging as a problem due to incomplete antiretroviral coverage in pregnancy.
CONCLUSIONS: The prevalence of HIV TDR indicated by this study, combined with known rates of HIV infection in Panama, suggests more extensive surveys are needed to identify risk factors associated with transmission of HIV drug resistance. Specific WHO-TS guidelines for monitoring vertical transmission of drug-resistant HIV should be established.
Key words: HIV-1; drug resistance; infectious disease transmission, vertical; protease inhibitors; antiretroviral therapy, highly active; Panama.
OBJETIVO: Investigar la prevalencia de farmacorresistencia transmitida del VIH en adultos en Panamá mediante un estudio del umbral modificado de la Organización Mundial de la Salud (OMS) e investigar las tasas de resistencia inicial en lactantes seropositivos para el VIH en Panamá.
MÉTODOS: En el Instituto Conmemorativo Gorgas, en 47 adultos seropositivos al VIH se efectuó la genotipificación de las mutaciones asociadas con la farmacorresistencia transmitida en los genes de la transcriptasa inversa y la proteasa del VIH-1, según las directrices del estudio umbral de la OMS, modificadas para incluir a las personas ≤ 26 años de edad. Las tasas de prevalencia de las mutaciones farmacorresistentes contra tres clases de fármacos antirretroviral inhibidores de la transcriptasa inversa análogos de nucleósidos, inhibidores de la transcriptasa inversa no análogos de nucleósidos e inhibidores de la proteasa se clasificaron en bajas (< 5,0%), moderadas (5,0%15,0%) o altas (> 15,0%). También se llevó a cabo genotipificación y se calcularon las tasas de prevalencia de las mutaciones causantes de farmacorresistencia en 25 lactantes.
RESULTADOS: En los adultos de Panamá la farmacorresistencia transmitida fue moderada: 6 de 47 adultos seropositivos para el VIH presentaron una o más mutaciones asociadas con farmacorresistencia transmitida. Las mutaciones farmacorresitentes de transmisión horizontal fueron moderadas para los inhibidores de la transcriptasa inversa análogos de nucleósidos y los inhibidores de la transcriptasa inversa no análogos de nucleósidos, y bajas para los inhibidores de la proteasa. En Panamá la transmisión vertical del VIH ha disminuido en el período 20022007, pero la prevalencia de la farmacorresistencia del VIH transmitida por vía vertical es moderada (12,0%) y está surgiendo como un problema debido a la cobertura antirretroviral incompleta durante el embarazo.
CONCLUSIONES: La prevalencia de farmacorresistencia transmitida del VIH observada en este estudio, junto con las tasas de infección por el VIH registradas en Panamá, indican que se necesitan estudios más amplios para determinar los factores de riesgo asociados con la transmisión de la farmacorresistencia del VIH. Deben establecerse directrices específicas del estudio del umbral de la OMS a fin de vigilar la transmisión vertical del VIH farmacorresistente.
Palabras clave: VIH-1; resistencia a medicamentos; transmisión vertical de enfermedad infecciosa; inhibidores de proteasas; terapia antirretroviral altamente activa; Panamá.
Throughout the developing world, access to antiretroviral therapy (ART) for treatment of HIV infections is increasing. As a consequence, drug-resistant HIV (HIVDR) is emerging and diminishing treatment options. In developing countries, first-line options for treatment are limited, second-line treatment regimens are much more expensive than first-line drugs, and the opportunity to perform drug-resistance genotyping is restricted (1). In Latin America, there are a number of studies of secondary drug resistance, initial drug resistance (IDR), and primary or transmitted drug resistance (TDR) (215), but discerning continental trends for antiretroviral (ARV) resistance in this region is complex.
Prolonging the lifetimes of ARVs is vital to the sustainability of HIV treatment programs in developing nations (1). Recommendations for limiting HIVDR in resource-limited countries are defined in the World Health Organization (WHO) global strategy for prevention and assessment of HIVDR (16). Surveillance of HIV TDR in recently infected individuals is key to the WHO strategy (16).
HIV/AIDS was first observed in Panama in 1984. There are approximately 20 000 HIV-positive persons in a concen-trated epidemic (17) and there have been a total of 10 381 AIDS cases since 1984.1 In 2006, 0.5% of pregnant women were HIV positive, and the share dropped to 0.3% for 20072009.1
Panama has provided free diagnosis, monitoring, and ART to 70.0% of all eligible patients since 1999 and to all patients since 2001 (18). Currently, 4 463 (19) to 8 700 (Panama Ministry of Health, personal communication, 30 March 2011) adult and minor patients receive ART. In the absence of a national database of patients receiving ARV, 70.0%80.0% are estimated to receive first-line therapy, 15.0%20.0% receive second-line therapy, and 5.0%10.0% receive salvage therapy (Panama Ministry of Health, personal communication, 30 March 2011).
First-line ART for adults in Panama follows WHO guidelines (20): one non-nucleoside reverse transcriptase inhibitor (NNRTI), efavirenz, combined with two nucleoside analog reverse transcrip-tase inhibitors (NRTIs), for which recommendations have recently changed (21, 22). Since 2007, NRTIs were lamivudine (3TC) and zidovudine (AZT) (21) and, since July 2011, tenofovir with 3TC or emtricitabine. Current recommendations for second-line therapy are two NRTIs with a ritonavir-boosted protease inhibitor, usually lopinavir (22). Since 2007, Panama's ART to prevent mother-to-child-transmission has involved AZT and 3TC, with lopinavir (21, 22). From 2007 to July 2011, ART for infants born to HIV-positive mothers was AZT until 6 weeks of age (21). Current guidelines recommend this regimen only for babies born to mothers receiving ART prenatally and perinatally (22). Babies born to mothers not taking ART prenatally and perinatally should receive AZT, 3TC, and nevirapine (22).
Despite broad and prolonged ART coverage in Panama, few studies have examined HIVDR prevalence (2) and there are no reports of HIV TDR from horizontal or vertical transmission. HIVDR studies in this country are important, but financial, human, and laboratory capacities to manage HIVDR are limited in developing countries. In recognition of this problem and as a pillar of the WHO global strategy against HIVDR (16), the WHO Threshold Survey (WHO-TS) surveillance and classification strategy was developed (23, 24). WHO-TS allows for low-cost classification of the prevalence of HIV TDR in adults to individual drugs or drug classes as low (< 5.0%), moderate (5.0%15.0%), or high (> 15.0%) (24).
Forty-one WHO-TS studies have been conducted in Africa, Asia, and Mexico (1, 23, 2532).2 An adaptation of the WHO-TS strategy was used in Brazil (33). Combined analysis of WHO-TS from 20 countries showed a low overall level of HIV TDR (3.7%), although 17.0% of surveys showed moderate levels of HIV TDR (1). In Central America, the WHO-TS has not been applied.
Our first objective is to apply WHO-TS, with modifications, to investigate HIV TDR among Panamanian adults. For the second objective, we investigate IDR in Panama, for the first time at the molecular level (34). While WHO clearly describes its goal in surveying IDR (35), it does not have a formal IDR surveil-lance strategy.
MATERIALS AND METHODS
WHO-TS methodology and its adaptations
The WHO-TS strategy (23, 24) focuses on regions where ART has been available to ≥ 20.0% of eligible individuals for ≥ 3 years; this study focuses on Panama City. WHO-TS methodology requires collection and analysis of 47 eligible specimens, preferably within 12 months.
Samples were collected March 2008 to October 2010 at the clinic of the Gorgas Memorial Institute for Health Studies (ICGES), where all HIV patients in Panama City were referred for baseline viral load measurement after their initial HIV diagnoses elsewhere.
In this descriptive study, plasma samples for 47 HIV-positive, ART-naive adults from the general population were collected and genotyped for drug resistance at ICGES and their resistance levels were evaluated (24). A median of 3 months had elapsed between initial HIV diagnosis and genotyping. Mean CD4+ count was 400 cells per μL. At ICGES, two of the WHO-TS mandatory eligibility criteria for patient inclusion were met: laboratory confirmation of HIV-positive status and, if female, no previous pregnancies (23). The third criterion, patient age < 25 years, was extended to ≤ 26 years: patients ≥ 25 years had a confirmed HIV-negative serology in the previous 3 years. The sample collection period was extended from the recommended 12 months.
All infant patients from the general population with an HIV-positive diagnosis in the Panama City region attend ICGES for molecular confirmation of HIV status and measurement of viral load. Twenty-five infant patients were confirmed as HIV positive at ICGES and their samples, collected February 2007 to October 2009, were genotyped. Some data were available on the ART regimens of the children and their mothers.
An in-house method was used to genotype and analyze reverse transcriptase and protease genes for specific mutations associated with drug resistance in HIV-1. HIV-1 RNA was extracted with the QIAamp viral RNA mini kit (Qiagen) and reverse-transcribed, amplified, and sequenced with the primers indicated in Table 1 (36).
Sequences were edited and analyzed with Sequencher software, version 4.5. Consensus sequences were analyzed with the Stanford University Calibrated Population Resistance tool to identify mutations in HIV-1 reverse transcriptase and protease genes (3740). The 47 geno-types were classified with the WHO-TS binomial sampling and classification scheme, which classifies HIV TDR prevalence to individual drugs or drug classes but is not powered for punctual estimates of prevalence (24).
Ethics approval and patient consent
Panama's National Institutional Review Board approved this study as part of the ICGES HIV Epidemiology Study (RV165) and informed consent regarding sample use was obtained.
Forty-seven HIV-positive men (n = 30) and women (n = 17), aged 1626 years (mean 21.6 years), all residing in Panama City, were included (Table 2). Six patients showed one or more mutations associated with TDR: one patient showed multiple mutations associated with NNRTI resistance (patient 3) and another showed multiple resistance-associated mutations against NRTIs and NNRTIs (patient 15). The prevalence of TDR mutations overall and against NRTIs and NNRTIs was moderate (5.0%15.0%). The prevalence of TDR mutations against protease inhibitors was low (< 5.0%) (Table 2). Polymor-phisms not associated with drug resistance were also observed (Table 2).
Neonates and infants
Twenty-five blood samples from male (n = 12) and female (n = 13) patients aged between 9 days and 1 year were collected (Table 3). HIV IDR was observed in 1 of 6 HIV-positive babies tested in 2007, in 0 of 11 in 2008, and in 2 of 8 in 2009. Two patients had mutations associated with NRTI resistance (patients 3 and 5) and one patient had an NNRTI-resistance mutation (patient 13). On the basis of genotyping results and information about ART given to the infant patients and their mothers, the overall prevalence of vertically transmitted HIVDR was 12.0%.
The WHO-TS guidelines were adapted to investigate HIV TDR in Panamanian adults. The overall prevalence was moderate and justifies more advanced surveillance for drug-resistant HIV among newly infected patients, especially from groups at high risk of HIV infection.
The prevalence of TDR mutations against protease inhibitors (< 5.0%) and NRTIs and NNRTIs (5.0%15.0%) in Panama is similar to findings from a Brazilian adaptation of the WHO-TS, reporting a moderate level of HIV TDR (33), and to other regional HIV TDR studies: low to moderate HIV TDR has been identified in Argentina (2.4%8.8%), Brazil(4.2% 11.0%), Chile (1.7%12.0%), Colombia (5.8%), the Dominican Republic (7.8%) (7), Honduras (7.0%) (5), Peru (3.0% 3.4%), and Venezuela (3.2%11.0%) (41). In the region, therefore, Panama's prevalence classification is not unusual, but Latin America's overall rates of HIV TDR appear to be higher than in Africa and Asia, according to the combined analysis of WHO-TS from 20 African and Asian countries (1). This general conclusion justifies closer surveillance of HIV TDR in the region and adoption of methodologic adaptations, which will increase Latin American participation in the WHO-TS.
Panama's moderate levels of HIV TDR reflect 10 years of using ART and the types of ART available. K103N, P225H, and Y181C mutations alter the effectiveness of efavirenz, which is used as a first-line drug for nonpregnant Panamanians; I85V is associated, in ART-naive patients, with resistance to atazanavir (42), a second-line ART in Panama (21); and M41L and T215F reduce the susceptibility of HIV-1 to stavudine and AZT, first-line drugs in Panama for pregnant women and newborns and formerly for adults and young people. M41L, one of the few transmitted mutations not observed to revert over time (43), was seen in four patients. Its high prevalence is particularly important in Panama: until July 2011, first-line therapy was efavirenz with AZT and 3TC, a regimen whose efficacy would have been compromised by a high prevalence of the M41L mutation, but the therapy has since changed to efavirenz, tenofovir with 3TC, or emtricitabine, a regimen compromised only by the M41L mutation combined with other thymidine analog mutations.3 The prevalence of TDR mutations found here agrees with our work showing that mutations associated with resistance to NRTIs and NNRTIs, singularly or combined, are the most prevalent causes of HIV secondary drug resistance in Panama.4
The 47 samples in this study were collected from Panama City, where ART has been available since 2001 (Panama Ministry of Health, personal communication, 30 March 2011). Samples were collected at ICGES, which at the time of this study received 100.0% of newly diagnosed HIV-positive patients from Panama City's wider population. Reference laboratories having the opportunity to consecutively enroll eligible HIV-positive patients from the general population may be considered for WHO-TS studies.
Ages within this study's patient population ranged from 16 to 26 years: 96.0% of the cohort was ≤ 25 years old and all patients ≥ 25 years old (25, n = 2; 26, n = 2) had an HIV-negative test in the 3 years before diagnosis. However, the age range of patients was at the limit of WHO-TS feasibility: the mandatory age-eligibility criterion stipulates < 25 years. The period of sample collection was 2.5 years: WHO recommends only 12 months.
While ICGES received 100.0% of newly diagnosed HIV-positive patients from Panama City, identifying 47 age-eligible individuals in 12 months was difficult and was the reason for adapting the WHO-TS methodology to older patients and longer sampling. Panama has the second highest HIV prevalence rate in Central America (17), but HIV infections are concentrated in specific groups (44) and absolute numbers of new HIV-positive patients from the general population attending ICGES were low.
Identifying sufficient numbers of eligible HIV-positive patients will be a problem faced by similar countries in Latin America, with small populations, low numbers of HIV infections, low prevalence, and concentrated epidemics. The WHO publication on WHO-TS (23) states: "generally no more than 50 individuals < 25 years of age, without previous pregnancies and ineligible for ART, were likely to be diagnosed with HIV... within 36 months, even in areas of high HIV prevalence." For these reasons, either WHO-TS HIV TDR studies are best applied only in populations or subpopulations with a high prevalence of HIV, or WHO could consider adapting survey guidelines to increase participation by small populations with a low prevalence of HIV and generalized epidemics.
These alterations may involve relaxing the < 25-year age criterion. In WHO-TS, patient age is an indicator of recent infection, and if some other marker of recent infection can be consistently used, it should be satisfactory. The BED test has been rejected for this function (30). Alternative criteria, such as a negative HIV test or a well-defined risk event in the previous 12 months may be considered.
A Tanzanian study on ART-naive patients failing WHO-TS eligibility criteria due to age (45) reported a statistically significant difference in the prevalence of HIVDR between patients < 25 years old (0.0%) and those aged 2563 years (19.1%). Failing to involve patients > 25 years old in threshold studies may exclude some recently infected HIV-positive patients who are at increased risk of acquiring drug-resistant HIV strains and disregards underlying social factors leading to increased risk of infection, such as older-age repartnering.
WHO-TS methodology was designed to help developing countries assess HIV TDR economically. We suggest it is important that WHO consider alternative criteria for site selection and patient eligibility to maximize WHO-TS participation, especially in Latin American countries underrepresented in this program. There is some urgency in increasing participation, as future opportunities for WHO-TS may be limited: ART for preventing transmission is increasingly discussed (46) and, if adopted, wider up-take throughout the developing world will make it more difficult to identify ARV-naive patients.
This study also represents Panama's first molecular examination of IDR in 25 HIV-positive infants. Between 2007 and 2009, the overall prevalence of IDR was 12.0% (n = 3), the prevalence of IDR mutations against NRTIs was moderate (8.0%), and the prevalence of IDR was < 5.0% for NNRTIs and protease inhibitors.
Data from Panama's Ministry of Health for the number of HIV-positive pregnancies (Panama Ministry of Health, personal communication, 30 March 2011) can be matched to reports from the Joint United Nations Programme on HIV/AIDS on ART cover-age among pregnant Panamanians: in 2006, 100.0% of 153 pregnant women received Panama's recommended ART (AZT, 3TC, lopinavir) (47), resulting in no cases of vertical transmission (21); in 2007, 71.0% of 100 mothers received ART (47), resulting in one infant with NRTI resistance (K219Q); for 2008, no data on ART coverage were available and no cases of IDR were detected; and in 2009, 73.0% of 162 mothers received ART (17) and our data show two cases of HIV IDR, with one case of NRTI resistance (T215Y) and one case of NNRTI resistance (K103N). The mother of the K103N infant was a known HIV-positive patient of poor compliance who did not take ART prenatally.
The overall prevalence of HIV TDR was moderate. This fact, combined with known rates of HIV infection among adult Panamanians, suggests that it is important that more extensive surveys better identify factors associated with horizontal HIV TDR and assist Panama's Ministry of Health in choosing first-and second-line regimens. Applying WHO-TS to high-risk subpopulations in Panama may reveal a broader range of resistance mutations, while larger sur-veillance studies may more accurately indicate the prevalence of HIV TDR in the general population.
Some HIV-positive mothers in Panama transmit HIV and HIVDR to their infants. To better understand vertical infection and IDR, we also need new surveys among HIV-positive pregnant women and their newborns. This will require collection of sociodemographic data providing early warnings about mothers at increased risk of transmitting HIV, information about ART use by infants and their mothers before and during pregnancy, and introduction of genotyping tests at defined times during pregnancy and neo-natal life. Establishing public health protocols in Panama whereby HIV-positive mothers and their newborns would be automatically subject to HIVDR analysis would be a useful collaboration between Panama's Ministry of Health, ICGES, and antenatal clinics. Finally, we recommend establishing WHO-TS guidelines specifically for assessing IDR.
The authors thank Aurelio Nuñez Maitin from Panama's Ministry of Health for providing important statistical information on the incidence of HIV/AIDS in Panama and Alma Ortiz and Dayana Best from the Gorgas Memorial Institute for their technical assistance. This study was partially supported by grant 5-N-2008 from the Network for Research and Training in Tropical Diseases in Central America; by grant Col 059 from the National Council for Science and Technology, Panama; and by grant PAN-6011from the International Atomic Energy Agency.
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Manuscript received on 9 April 2011. Revised version accepted for publication on 23 September 2011.
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