Abstract
The Kento-Mwana project was carried out in Pointe Noire, Republic of the Congo, to prevent mother-to-child HIV-1 transmission. To determine the prevalence of different subtypes and transmitted drug resistance-associated mutations, 95 plasma samples were collected at baseline from HIV-1-positive naive pregnant women enrolled in the project during the years 2005–2008. Full protease and partial reverse transcriptase sequencing was performed and 68/95 (71.6%) samples were successfully sequenced. Major mutations to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 4/68 (5.9%), 3/68 (4.4%), and 2/68 (2.9%) samples, respectively. Phylogenetic analysis of HIV-1 isolates showed a high prevalence of unique recombinant forms (24/68, 35%), followed by CRF45_cpx (7/68, 10.3%) and subsubtype A3 and subtype G (6/68 each, 8.8%). Although the prevalence of transmitted drug resistance mutations appears to be currently limited, baseline HIV-1 genotyping is highly advisable in conjunction with antiretroviral therapy scale-up in resource-limited settings to optimize treatment and prevent perinatal transmission.
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HIV-1 genetic variability has a relevant impact in different settings such as vaccine design and the accuracy of laboratory assays used in clinical practice for the diagnosing and monitoring of HIV-1 infection. In addition, clinical evidence suggests that certain HIV-1 non-B subtypes are associated with faster disease progression and have different pathways to drug resistance. For example, mutations at codon 17, 36, 64, and 89 of HIV-1 protease and 65, 138, 181, and 348 of HIV-1 reverse transcriptase have been found to be favored in specific non-B subtypes, suggesting that subtype determination may have a role in treatment decisions. 9
The global scale-up of antiretroviral treatments in low to middle income countries resulted in a significant decrease of HIV-related deaths, the reduction of new infections, and an increased efficacy in the prevention of mother-to-child transmission (PMTCT). However, the supply of antivirals in countries with a high prevalence of non-B subtypes has raised concern about the clinical management, treatment efficacy, and development of drug resistance in uncommon HIV strains.
The Kento-Mwana project was a PMTCT program conducted in the region around Pointe Noire, Republic of the Congo from September 2005 to December 2008. This project provided counseling, serological and molecular tests, prophylaxis, and therapy to all pregnant women and their children attending four antenatal care units located at the Army Hospital of Pointe Noire and in the districts of Ndaka Susu, Mbota, and Ngoyo. The project was conducted in accordance with the Declaration of Helsinki and was approved by the Congolese Ministry of Health and the Congolese Council against AIDS. All participants provided written informed consent or an equivalent. 10
The aim of this work was to characterize the genetic variability of protease and reverse transcriptase sequences obtained from samples collected during the project as well as to determine the prevalence of resistance-associated mutations (RAMs). Briefly, 12.830/13.164 (97.5%) contacted pregnant women agreed to be screened for HIV and 615/12.830 (4.8%) were found to be seropositive; 464 of them (75.4%) agreed to be enrolled in the prophylactic and therapeutic program. Due to different reasons, such as stillbirth, only 415 (89.4%) started the therapeutic protocol, most of them (368, 88.6%) being naive to antiretroviral therapy. Antiviral drugs available in this region before and at the start of the project were zidovudine, stavudine, lamivudine, nevirapine, and efavirenz. 10
Ninety-five samples from naive HIV-1-positive pregnant women were used for subtype determination and analysis of RAMs. EDTA blood samples were collected at the peripheral health care unit, centralized daily in the project's dedicated laboratory where plasma was separated and stored at −20°C within 18 h. Frozen plasma samples were maintained in dry ice during the transportation to Italy. Sequencing was performed at the laboratory of Hygiene Unit, IRCCS AOU San Martino-IST, Genoa, Italy using the Trugene HIV-1 Genotyping Kit (Siemens HealthCare Diagnostics), according to the manufacturer's recommendations after automated extraction through the NucliSens EasyMAG instrument (BioMérieux). RAMs selected by nucleoside reverse transcriptase inhibitors (NRTIs), nonnucleoside analogue reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) were evaluated according to the surveillance drug resistance mutations (SDRM) list. 11 Phylogenetic analysis was performed as previously described. 5
Viral sequences were successfully obtained for 74/95 (77.9%) samples. In the remaining 21 samples, either amplification was unsuccessful (11/21) or too short sequence fragments were obtained (10/21). Finally, 6/74 sequences were not included in the analysis because no anamnestic data of the patients were available.
Phylogenetic analysis showed that HIV-1 viral strains circulating in the Pointe Noire area were characterized by a high genetic variability. In particular, 24/68 (35%) of the sequences were classified as URF, 7/68 (10.3%) as CRF45_cpx, 6/68 (8.8%) as subtype G, 6/68 (8.8%) as subsubtype A3, 5/68 (7.4%) as CRF37_cpx, 4/68 (5.9%) as CRF18_cpx, 3/68 (4.4%) as subtype D, 2/68 (2.9%) as subtype B, 2/68 (2.9%) as subtype H, CRF02_AG, and 2/68 (2.9%) as subsubtype A1, while subtypes C and J, subsubtypes F1 and F2, and CRF25_cpx were found in only one sample each (Fig. 1).
Phylogenetic tree of the HIV-1 pol sequences created with MEGA 6.
According to the SDRM list, major mutations to NRTIs, NNRTIs, and PIs were detected in six out of 68 (8.8%) sequenced viruses. NRTI mutations were identified in four viruses (5.9%), two with M184V alone, one with L210W plus T215S, and one with M41L, L210W, T215Y, plus M184V. NNRTI mutations were found in three patients (4.4%) harboring K103N, G190A, or K101E, respectively, while two patients (2.9%) carried PI mutations, one D30N plus F53Y and the other V32I, I54M, and I84V, respectively. Other mutations detected are not included in the SDRM list but are included in the IAS-USA drug mutations list 12 (Table 1). The reverse transcriptase substitutions V90I, E138A/G, V179D, and H221Y were considered because of their impact in the development of resistance to NNRTIs, even though they occur as natural polymorphisms in one or more subtypes. The rare mutation K65E was detected in two sequences and is potentially involved in resistance to NRTI, although this variant has been shown to confer a reduced replication capacity as compared to wild-type reference virus in vitro. 13
SDRMs, surveillance drug resistance mutations; PIs, protease inhibitors; NRTIs, nucleoside reverse transcriptase inhibitors; NNRTIs, nonnucleoside reverse transcriptase inhibitors.
All the SDRMs detected in reverse transcriptase were consistent with the drugs in use in the country before our intervention (zidovudine, stavudine, lamivudine, nevirapine, and efavirenz). However, since PIs had not been introduced in the area of Pointe Noire before sample collection, we can only presume that the two viruses carrying PI resistance mutations were transmitted from a donor treated with PIs elsewhere.
Although these data suggest that the Pointe Noire area has a relatively low rate of transmitted drug resistance, it must be noted that the size of the population analyzed was small and samples were obtained in years 2005–2008. However, this estimate is in agreement with other studies from different African countries. 14 Furthermore, the global effort to increase access to antiretroviral therapy in resource-limited settings is expected to result in a significant reduction in HIV-related morbidity as well as to favor the development as well as transmission of RAMs, particularly in the context of irregular drug supplies and/or poor adherence. The extremely high genetic variability in this area poses an additional challenge, potentially affecting the efficacy of antiviral therapy with highly divergent strains and warranting systematic monitoring of the response to the commonly used treatment strategies.
Sequence Data
GenBank accession numbers of the sequences are KP889062–KP889098 and EU914159–EU914187. 5
Footnotes
Acknowledgments
We thank all the women who agreed to participate in the study and all the doctors and biologists who through the years contributed to its fulfillment. The Kento-Mwana project was financially supported mainly by Eni E&P and Eni Congo SA; support for the project was also provided by Esther Italia (Istituto Superiore di Sanità): convenzione no. 3M41 and no. 521E/2-II.
Author Disclosure Statement
No competing financial interests exist.