Research Article: Early infant diagnosis of HIV-1 infection in Luanda, Angola, using a new DNA PCR assay and dried blood spots

Date Published: July 17, 2017

Publisher: Public Library of Science

Author(s): Francisco Martin, Claudia Palladino, Rita Mateus, Anna Bolzan, Perpétua Gomes, José Brito, Ana Patrícia Carvalho, Yolanda Cardoso, Cristovão Domingos, Vanda Sofia Lôa Clemente, Nuno Taveira, Chiyu Zhang.


Early diagnosis and treatment reduces HIV-1-related mortality, morbidity and size of viral reservoirs in infants infected perinatally. Commercial molecular tests enable the early diagnosis of infection in infants but the high cost and low sensitivity with dried blood spots (DBS) limit their use in sub-Saharan Africa.

To develop and validate a sensitive and cheap qualitative proviral DNA PCR-based assay for early infant diagnosis (EID) in HIV-1-exposed infants using DBS samples.

Chelex-based method was used to extract DNA from DBS samples followed by a nested PCR assay using primers for the HIV-1 integrase gene. Limit of detection (LoD) was determined by Probit regression using limiting dilutions of newly produced recombinant plasmids with the integrase gene of all HIV-1 subtypes and ACH-2 cells. Clinical sensitivity and specificity were evaluated on 100 HIV-1 infected adults; 5 infected infants; 50 healthy volunteers; 139 HIV-1-exposed infants of the Angolan Pediatric HIV Cohort (APEHC) with serology at 18 months of life.

All subtypes and CRF02_AG were amplified with a LoD of 14 copies. HIV-1 infection in infants was detected at month 1 of life. Sensitivity rate in adults varied with viral load, while diagnostic specificity was 100%. The percentage of HIV-1 MTCT cases between January 2012 and October 2014 was 2.2%. The cost per test was 8-10 USD which is 2- to 4-fold lower in comparison to commercial assays.

The new PCR assay enables early and accurate EID. The simplicity and low-cost of the assay make it suitable for generalized implementation in Angola and other resource-constrained countries.

Partial Text

HIV-1 mother-to-child-transmission (MTCT) is the main mode of infection among the pediatric population and is disproportionately affecting children in impoverished countries. Despite the decline in MTCT rate in recent years in most of the sub-Saharan Africa, it is estimated that 150,000 children became newly infected with HIV in 2015 [1]. Children infected perinatally are at high risk of rapid disease progression and death during the first year of life without antiretroviral therapy (ART) [2]. Given the reported benefits of early ART initiation in reducing HIV-1-related mortality and long-term morbidity [3] and reducing the size of the HIV-1 reservoirs [4], early HIV-1 diagnosis in newborns represents the critical gateway to timely initiation of life-saving ART. Serological assays do not permit the early diagnosis of HIV-1 infection because of the persistence of maternal HIV-1 antibodies in infants during the first 12-18 months of life. The WHO recommends the use of molecular-based virological testing to determine the infection status for HIV-1-exposed infants during the first 4-6 weeks of life or at the earliest opportunity thereafter [5]. Despite the high accuracy of tests which detect HIV RNA or p24, their sensitivity could potentially be affected in settings of expanded ART for prevention of MTCT (option B and B+), which reduce circulating HIV-1 RNA and viral particles [6]. Qualitative DNA PCR test which detect proviral DNA in peripheral blood mononuclear cells (PBMCs) is recommended for early infant diagnosis (EID) of HIV-1 and is the most widely implemented test in resource-limited settings [7–8]. The considerable uptake of HIV-1 DNA molecular tests is driven by the lower costs compared with quantitative assays along with their good sensitivity when performed on blood microsamples or dried blood spots (DBS) [7]. The use of DBS presents several advantages such as reduced costs for collection, storage and shipping. Thus, DBS samples are convenient for increasing access to testing in settings with poor healthcare provision and referral laboratories [9]. Currently, two HIV-1 DNA assays are commercially available for EID using DBS: the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Qualitative Test (Roche Molecular System Inc., Branchburg, NJ), which recently replaced the Roche Amplicor® DNA test v1.5, and the RealTime HIV-1 Qualitative Test (Abbott Molecular, Des Plaines, IL) [8]. These assays are highly sensitive but require sophisticated instrument platforms whose cost with related equipment (e.g. centrifuge; biosafety cabinet; freezer) can range from about US$ 100,000 to more than US$ 200,000. Recently, innovative technologies designed for use at or near the point-of-care (such as the Cepheid Xpert® HIV-1 Qual assay and the Alere™ q HIV-1/2 Detect) have been developed and the Xpert® HIV-1 Qual assay has been validated for both whole blood and DBS specimens [8].

Children are among the most vulnerable to be at risk for HIV infection but in spite of this the AIDS response in sub-Saharan Africa has largely left them behind [1]. In this regard, Angola has only registered a 25% reduction of new infections among infants since 2009 [1] which led the Assistant Secretary-General of the United Nations to declare in 2015 that the epidemic in Angola might worsen if an effective AIDS response is not reinvigorated. EID of HIV-1 infection in infants at risk enables early treatment and care of the infected infants. Significant progress has been made in many sub-Saharan countries in implementing EID services following the introduction of HIV DNA testing on DBS [27]. However, the high cost of commercially available HIV-1 DNA tests and the perceived sensitivity problems related to the very diverse and complex viral strains circulating in Angola have prevented their implementation in this country. At the HDP where our cohort is based and in most other hospitals in Angola, pediatric diagnosis of HIV-1 infection is still done by serology at month 12 of life which significantly delays the initiation of treatment [28]. To support EID service expansion in Luanda we developed and validated a new HIV-1 DNA assay to be used on DBS samples. To account for the very diverse HIV-1 strains present in Luanda, we had to produce a new set of control plasmids containing the IN gene, our target for amplification, from local HIV-1 subtypes. Phylogenetic analysis showed that most of the new IN sequences fall at basal positions on the phylogenetic trees (pre-subtype branches) which is consistent with the complexity of the HIV-1 strains circulating in Luanda and with Angola being one of the epicenters of the HIV-1 epidemic [10–12, 16–18].

The high analytical and clinical sensitivity of our EID assay have enabled accurate, early and low cost diagnosis of HIV-1 infection in exposed infants in Angola. The low percentage of HIV-1 MTCT case observed within the APEHC cohort is consistent with the current high standard of pediatric care provided at HDP. The simplicity and low-cost of the assay make it suitable for generalized implementation in Angola and other resource-constrained countries.




0 0 vote
Article Rating
Notify of
Inline Feedbacks
View all comments