Transmitted HIV Type 1 Drug Resistance in Newly Diagnosed Cuban Patients

Abstract

Knowledge of the associated mutations to transmitted drug resistance (TDR) in strains of human immunodeficiency virus type 1 (HIV-1) constitutes a fundamental premise in epidemiological surveillance. In this present study, TDR from 200 Cuban patients who were diagnosed with HIV-1 between 2009 and 2011 was analyzed. By partial reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing of the HIV pol gene, an HIV subtype and transmitted resistance profile were determined. The prevalence of associated mutations to the TDR in the individuals studied was 21.5%. In the region of the reverse transcriptase, the most common mutations were K103N and M184V, while in the region of the protease they were L33F and M46L. The results of this study provide evidence of TDR in the Cuban seropositive population and suggest the necessity of making resistance assays before beginning antiretroviral therapy in HIV-1-infected patients in Cuba.

T he preexistence of antiretroviral (ARV) drug-resistant virus is strongly associated with treatment failure in naive treatment patients.¹ Because of the public health implications of this phenomenon, many studies on the prevalence of drug-resistant HIV-1 strains in treatment-naive individuals have been carried out in different countries.²

Cuba provides comprehensive care to all people living with AIDS and guarantees ARV treatment for these needing it. In 2003, Cuba achieved universal antiretroviral therapy with domestically manufactured ARVs for patients meeting international clinical criteria and in 2010, 5,692 people received ARV therapy.³ However, its massive use favored the appearance of antiretroviral resistance-associated mutations.

Although previous studies carried out in untreated Cuban patients have reported a low prevalence to ARV resistance,^4,5 the international guidelines recommend carrying out resistance tests before beginning antiretroviral therapy and the World Health Organization (WHO) has outlined the necessity of each country establishing programs of surveillance of transmitted resistance. Knowledge of transmitted resistance in untreated seropositive people will allow the Ministry of Public Health to optimize the distribution of the resources dedicated to combat the infection of HIV/AIDS, to design new therapeutic strategies, and to update the epidemiological and molecular situation of the HIV epidemic. The aim of the present study was to characterize drug resistance profiles in untreated Cuban patients.

For this purpose, 200 HIV-infected patients diagnosed between June 2009 and December 2011 were included in the study. Written informed consent from each subject was obtained. Patient age, sex, transmission route, and province were determined at the time of diagnosis. Patients were categorized as either recently or chronically infected, according to the presence or absence of documented seroconversion in the preceding 12 months or clinical evidence of acute HIV infection.⁶ Plasma HIV RNA was quantified using the COBAS Ampliprep/COBAS Taqman HIV-1 Test (Roche Diagnostics GmbH, Mannheim, Germany) with a limit of detection of 40 copies/ml.

For genotypic analyses, viral RNA was isolated from 200 μl using High Pure Viral Nucleic Acid kit (Roche Diagnostics, GmbH, Mannheim, Germany). The pol gene was amplified from positions 2143 to 3798 (HXB2 numbering) by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by nested PCR, and sequenced in an CEQ 8800 sequencer (Beckman Coulter, Fullerton, CA), as previously described.⁷ Phylogenetic analyses were performed with neighbor joining trees, based on Kimura's two parameter distances using the MEGA 3.0 program.⁸ The reliability of tree topologies was assessed by bootstrapping with 1,000 replicates, with a bootstrap support of ≥50%. Reference sequence viruses included in the phylogenetic analyses were downloaded from the Los Alamos database (www.hiv.lanl.gov/). The primary resistance mutations (PRMs) associated with reduced susceptibility to protease (PR) and RT inhibitors were detected by means of the Stanford University calibrated population resistance tool, using the 2009 surveillance drug resistance mutations list.⁹

Categorical data were analyzed using the X ² test. A comparison of two proportions with independent samples was carried out. For all statistical tests a p value≤0.05 was considered significant.

Two hundred samples of HIV-1-infected individuals were analyzed in the study. According to the presence or absence of documented seroconversion in the preceding 12 months or clinical evidence of acute HIV infection, 31 individuals were classified as recently infected and 169 as chronically infected. Table 1 shows the baseline characteristics of patients included in the study.

Table 1.

Baseline Characteristics of Patients Included in the Study

	Recently infected	Chronically infected	Total
Participants, No. (%)	31 (16)	169 (84)	200 (100)
Sex, No. (%)
Male	28 (14)	142 (71)	170 (85)
Female	3 (1.5)	27 (13.5)	30 (15)
Age, mean (SD)	32.7 (8.1)	36.9 (11.2)	35.3 (11.3)
Transmission route, No. (%)
Homosexual contact	23 (11.5)	116 (58)	139 (69.5)
Heterosexual contact	8 (4.0)	53 (26.5)	61 (30.5)
HIV-RNA log₁₀ copies/ml, average (IQR)	4.0 (2.6–7.0)	4.7 (2.6–6.5)	4.4 (2.6–7.0)

No., number; SD, standard deviation; IQR, interquartile range.

The subtype analysis reported that 127 individuals (63.5%) were infected with non-B virus, where the recombinant forms CRF 19_cpx, CRF 20 BG, CRF 23 BG, CRF 18_cpx, URF, and CRF05_DF and subtypes G, H, and C are included; 73 individuals (36.5%) were infected with subtype B virus. Forty-three (21.5%) patients presented associated mutations to transmitted drug resistance (TDR); of these 36 (18%) were chronically infected patients and 7 (3.5%) were recently infected patients. Table 2 shows the behavior from resistance to the different ARVs in the studied groups. The mutation K103N, which confers resistance to all the nonnucleoside reverse transcriptase inhibitors (NNRTIs), was detected in 10 patients (5%), while mutation M184V, associated with high resistance to lamivudine (3TC) and emtricitabine (FTC), was detected in 14 individuals (7%). Table 3 shows the main mutations associated with TDR in the studied groups. Twenty-two (11%) and 33 (16.5%) strains classified as B and non-B subtypes, respectively, presented associated mutations to TDR. In Table 4, the number of resistant samples is shown according to viral subtype. In a patient infected with CRF19_cpx, seven mutations were associated with NRTIs and NNRTIs: D67N, K70R, M184V, L210W, T215Y, Y181C, and Q190A were detected.

Table 2.

Distribution of the Samples with Transmitted Resistance to Antiretrovirals in Recently Diagnosed Individuals

ARV families	Chronically infected n=169 (%)	Recently infected n=31 (%)	Total n=200 (%)
Protease inhibitors (PI)	2 (1.2)	1 (3.2)	3 (1.5)
Nucleoside reverse transcriptase inhibitors (NRTI)	12 (7.1)	2 (6.4)	14 (7)
Nonnucleoside reverse transcriptase inhibitors (NNRTI)	12 (7.1)	2 (6.4)	14 (7)
Reverse transcriptase inhibitors (includes the two families)	10 (5.9)	2 (6.4)	12 (6)
	36 (18)	7 (3.5)	43 (21.5)

ARV, antiretroviral.

Table 3.

Associated Mutations to Transmitted Resistance in Protease and Reverse Transcriptase Genes in Antiretroviral Treatment-Naíve Patients

ARV families	Mutations	Chronically infected (n=168)	Recently infected (n=32)
Protease inhibitors	L33F	1 (0.6)
	M46L		1 (3.2)
	F53L	1 (0.6)
Nucleoside reverse transcriptase inhibitors	M41L	3 (1.8)
	D67N	6 (3.6)
	T69D	1 (0.6)
	K70R	3 (1.8)
	L74V	2 (1.2)
	M184V	12 (7.1)	2 (6.4)
	L210W	3 (1.8)
	T215Y	1 (0.6)
	K219Q	4 (2.4)	2 (6.4)
Nonnucleoside reverse transcriptase inhibitors	K103N	9 (5.3)	1 (3.2)
	V106A	7 (4.1)
	V179F		1 (3.2)
	Y181C	7 (4.1)	1 (3.2)
	Q190A	5 (3.0)	1 (3.2)

The numbers inside the parentheses indicate the percent.

ARV, antiretroviral.

Table 4.

Distribution of Associated Mutations to Resistance Transmitted According to Viral Subtype

Subtype	NRTI n=26 ^a	NNRTI n=26 ^a	PI n=3	Total n=200
B	13 (50)	8 (30.7)	1 (33.3)	22 (11)
Non-B	15 (57.6)	16 (61.5)	2 (66.7)	33 (16.5)

Include the samples with mutations to the two families of RTI (NRTI and NNRTI).

The numbers inside the parentheses indicate the percent.

NRTI, nucleoside reverse transcriptase inhibitors; NNRTI, nonnucleoside reverse transcriptase inhibitors; PI, protease inhibitors.

Of the patients classified as recently infected, six presented associated mutations to transmitted resistance to reverse transcriptase inhibitors (RTI), while one presented mutation M46L, which decreases the susceptibility to protease inhibitors (PIs): atazanavir (ATV/r), indinavir (IDV), fosamprenavir (FPV), nelfinavir (NFV/r), and lopinavir (LPV/r) (Table 3).

One of the main causes for the failure of anti-HIV therapy involves the appearance of viral variants with mutations that confer resistance to ARV. Due to HIV-1 population dynamics, these resistant variants have been detected in individuals who have not undergone antiretroviral treatment.⁹

In countries in which there has been widespread use of antiretroviral drugs for many years, it has been shown that the frequency of drug-resistant variants in drug-naive populations ranges from 1% to 14%.¹⁰ The results of the present study suggest a high frequency of mutations associated with resistance in the group of studied patients (21.5%). Similar results were obtained in the southeast of Switzerland, where 16.6% of the studied patients presented mutations associated with TDR.¹¹ A study carried out in Cuba in 2003 reported a prevalence of 5.2% in 250 untreated patients.⁵ The significant increase of TDR in untreated individuals may be due to the larger number of patients who have access to highly active antiretroviral therapy (HAART), to nonadherence to the treatment, and to the persistence of viral-resistant variants to drugs in an individual, which facilitate a prolonged “window of opportunity” for secondary transmission of drug-resistant variants at a time when plasma viral load measures are routinely very high (from 5.5 to 6.3 log₁₀ copies/ml).¹²

There are no significant differences in the prevalence of TDR among recently infected and chronically infected patients. Although the HIV-1-resistant variants are detected with less frequency in chronically infected individuals,¹⁰ in this study, 18% of the naive drug individuals with chronic infection presented mutations associated with TDR. It has been demonstrated that the resistant variants can persist for up to 3 years in antiretroviral-naive subjects and that they can be detected in patients with chronic infection and in patients with infection dates less than 10 years.¹² In the studied population, the most frequent mutations were K103N and M184V. In previous studies carried out in Cuba, the most frequent substitutions were M41L, T215E/D/N/S, and V179D.⁵ The greatest access to HAART and the most frequently used regimen that includes lamivudine (3TC), zidovudine (AZT), and nevirapine (NVP) would explain the frequent detection of the mutations and their later transmission. Bennett et al. have reported a low prevalence of the mutation M184V in untreated patients and an association of these results with the possible elimination by the cytotoxic T lymphocyte (CTL) of the viral strains that present this polymorphism, which is situated at terminal anchoring positions of known CTL epitopes.⁹

Twenty-two infected people with subtype B (11%) presented associated mutations to TDR. Pérez et al. reported that 6.4% of the infected individuals with subtype B presented substitutions of this type.⁵ In an international study carried out in the years 1996–2002 in 19 European countries that included 2,208 patients with recent and chronic infection, the frequency of mutations associated with resistance to virus subtype B was 12.9%, while in non-B subtype viruses it was 4.8%. This result was sustained throughout the exhibition of the B viruses to ARV in the European countries studied.¹³ In Belgium in the years 2003–2006 it was reported that subtype B was significantly associated with transmission of resistance to ARV.¹⁴

The presence of associated mutations to TDR in samples corresponding to non-B subtypes reflects the versatility of the genetic variants in the Cuban epidemic. The detection of multiple associated mutations to resistance to RTI in a sample classified as CRF19_cpx illustrates that this phenomenon is not exclusive to subtype B virus. The prevalence of CRF19_cpx has increased in the recently diagnosed population with regard to previous studies,¹⁵ a fact to keep in mind in the surveillance of transmitted resistance. In a previous study carried out in Cuba, only the presence of the mutation E169D was detected in an untreated patient infected with this recombinant.⁵ The number of patients with non-B virus subtypes and associated mutations to transmitted resistance endorses the importance of maintaining epidemiological surveillance, due to the increase of these genetic variants in the Cuban epidemic.^5,15

Indinavir (IDV) is one of the PIs manufactured in Cuban laboratories and it is used, in combination with two NRTIs, in the treatment of seropositive Cuban patients; the presence of mutation M46L constitutes an indication of the systematic employment of this drug.

In conclusion, this study reported an increment of mutations associated with TDR in untreated patients with regard to previous studies,^4,5 and a high prevalence of transmitted resistance to NRTIs and NNRTIs in the evaluated period, according to the approaches of WHO, which endorses the necessity of maintaining surveillance of TDR in HIV-1 recently diagnosed Cuban patients.

Sequence Data

GenBank accession numbers for the sequences reported here are HQ655334–HQ655409, JN000005–JN000070, and JX194180–JX194237.

Footnotes

Acknowledgments

The authors thank Otto Cruz, Ph.D., for useful comments and manuscript revision.

Author Disclosure Statement

No competing financial interests exist.

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