Development and Evaluation of an Affordable Real-Time Qualitative Assay for Determining HIV-1 Virological Failure in Plasma and Dried Blood Spots

Virological failure (VF) has been identified as the earliest, most predictive determinant of HIV-1 antiretroviral treatment (ART) failure. Due to the high cost and complexity of virological monitoring, VF assays are rarely performed in resource-limited settings (RLS). Rather, ART failure is determined by clinical monitoring and to a large extent immunological monitoring. This paper describes the development and evaluation of a low-cost, dried blood spot (DBS)-compatible qualitative assay to determine VF, in accordance with current WHO guideline recommendations for therapy switching in RLS. The assay described here is an internally controlled qualitative real-time PCR targeting the conserved long terminal repeat domain of HIV-1. This assay was applied to HIV-1 subtypes A to H and further evaluated on HIV-1 clinical plasma samples from South Africa (n = 191) and Tanzania (n = 42). Field evaluation was performed in Uganda using local clinical plasma samples (n = 176). Furthermore, assay performance was evaluated for DBS. This assay is able to identify VF for all major HIV-1 group M subtypes with equal specificity and has a lower detection limit of 1.00E+03 copies/ml for plasma samples and 5.00E+03 copies/ml for DBS. Comparative testing yielded accurate VF determination for therapy switching in 89% to 96% of samples compared to gold standards. The assay is robust and flexible, allowing for “open platform” applications and producing results comparable to those of commercial assays. Assay design enables application in laboratories that can accommodate real-time PCR equipment, allowing decentralization of testing to some extent. Compatibility with DBS extends access of sampling and thus access to this test to remote settings.     

Detection of Entebbe Bat Virus after 54 Years

Entebbe bat virus (ENTV; Flaviviridae: Flavivirus), closely related to yellow fever virus, was first isolated from a little free-tailed bat (Chaerephon pumilus) in Uganda in 1957, but was not detected after that initial isolation. In 2011, we isolated ENTV from a little free-tailed bat captured from the attic of a house near where it had originally been found. Infectious virus was recovered from the spleen and lung, and the viral RNA was sequenced and compared with that of the original isolate. Across the polypeptide sequence, there were 76 amino acid substitutions, resulting in 97.8% identity at the amino acid level between the 1957 and 2011 isolates. Further study of this virus would provide valuable insights into the ecological and genetic factors governing the evolution and transmission of bat- and mosquito-borne flaviviruses.Bat virus investigations in Uganda were initially inspired in the mid-1950s by the isolations of rabies virus and a novel flavivirus, later to become known as Rio Bravo virus (Flaviviridae: Flavivirus), from the salivary glands of insectivorous bats in the United States.^1–3 Subsequently, investigators at the Uganda Virus Research Institute (UVRI) collected bats from the attic of UVRI, dissected salivary glands, and isolated viruses by intracerebral inoculation of triturated salivary gland extracts into neonatal mice.^4,5 This first effort resulted in the isolation of Entebbe bat salivary gland virus, strain IL-30 (Flaviviridae: Flavivirus) from a little free-tailed bat (Chaerephon pumilus) (Cretzchmar).^4,5 The taxon was then recognized by one of its synonyms: Tadarida (Chaerephon) limbata (Peters). Lumsden and others^4,5 determined that this isolate was a novel flavivirus through a series of neutralization tests using immune sera against 20 arboviruses as well as against Rio Bravo virus. By 1964, bat virus research had expanded at UVRI, and sampling of 1,022 additional bats yielded 14 strains of viruses including Dakar bat virus (Flaviviridae: Flavivirus) also from little free-tailed bats, and Bukalasa bat virus (Flaviviridae: Flavivirus) from Angolan free-tailed bats (Mops condylurus) (A. Smith).⁶ Isolation of Mount Elgon bat virus (Rhabdoviridae: Ledantevirus) followed shortly thereafter from an eloquent horseshoe bat (Rhinolophus hildebrandtii eloquens) (K. Anderson) captured in Kenya, with the virus being isolated and characterized at UVRI.^7,8 Additional bat virus discoveries in Uganda later included Kasokero virus (Bunyaviridae, unassigned) from an Egyptian fruit bat (Rousettus aegyptiacus) (E. Geoffroy).⁹ Entebbe bat virus (ENTV, renamed), Bukalasa bat virus, and Dakar bat virus were all isolated from insectivorous bats captured in the attic of UVRI.¹⁰ Serological surveys suggested that the infection prevalence of ENTV in wild populations of little free-tailed bats was high, but ENTV was never isolated again after the original discovery.^10,11To follow up on these early studies, arbovirus surveillance of bats was conducted in Uganda from 2011 to 2013. Bats were captured from attics in several locations around Entebbe and Kampala as part of a larger, country-wide sampling effort to be reported elsewhere. All bat captures were conducted under the approval of CDC/DVBD IACUC protocol 010-015. Bats were captured in attics using mist nets, taking appropriate biosafety precautions. On capture, bats were placed individually in cloth holding bags. Bats were anesthetized with halothane, exsanguinated by cardiac puncture, and euthanized by halothane overdose and cervical dislocation. Blood from bats was collected directly into serum separator tubes, centrifuged in the field, and placed immediately in a nitrogen dry vapor shipper. Liver, spleen, heart, lung, and kidney were all collected directly into cryovials and stored immediately on dry ice. In total, 95 little free-tailed bats and 34 Angolan free-tailed bats were captured in 2011, 15 little free-tailed bats in 2012, and 70 little free-tailed bats in 2013.Virus isolation was attempted first from spleens, and on a positive result, virus isolation was also performed from the remaining tissues harvested from the infected bat. A single virus was isolated from 1 of 180 (0.5%) little free-tailed bats. The infected bat was an adult male, captured in Banga, Nakiwogo (0°04.884′ N, 32°27.030′ E) on June 23, 2011, near UVRI. Infectious virus was isolated from the spleen and lung; no infectious virus was recovered from the heart, liver, or kidney. No other tissues, oral or fecal swabs were collected from the insectivorous bats captured in 2011. The isolate was initially identified as ENTV by sequencing of the flavivirus NS5 amplicon using primers FU2/CFD3,¹² followed by next-generation sequencing (NGS). Virus isolations, nucleic acid preparations, and NGS were performed as described previously.¹³ Novel primers were designed from contigs generated from the NGS data using Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA)¹⁴ (Supplemental Table 1), and the sequence for the entire genome was confirmed by direct sequencing. End terminal sequences were obtained using 5′/3′ RACE (FirstChoice^® RLM-RACE, Life Technologies, Carlsbad, CA) according to the manufacturer''s instructions. Virus-specific RACE primers are provided in Supplemental Table 1. The 5′ outer reaction used primer 514R in combination with the 5′ RACE outer primer. The 5′ RACE inner reactions used primer 388R together with the 5′ RACE inner primer; amplification was also achieved with primers 15F + 388R. The 3′ outer reaction used either primer 9882F or 10328F in combination with the 3′ RACE outer primer. The 3′ inner RACE was not successful. A maximum likelihood phylogenetic tree was generated in MEGA version 6.0¹⁵ (Center for Evolutionary Medicine and Informatics, Biodesign Institute, Tempe, AZ) using 10,493 nucleotides spanning the open reading frame sequence of select flavivirus genomes available in GenBank (Figure 1 ). The analysis was performed using a complete deletion substitution model and 1,000 bootstrap replicates.Open in a separate windowFigure 1.Maximum likelihood tree based on open reading frame sequence (complete gap deletion). Scale bar indicates nucleotide substitutions per site. Bootstrap values > 50% are shown (1,000 replicates). CFAV = cell fusing agent virus (NC001564); DENV1 = dengue type 1 ({"type":"entrez-nucleotide","attrs":{"text":"M87512","term_id":"323660","term_text":"M87512"}}M87512); DENV2 = dengue type 2 ({"type":"entrez-nucleotide","attrs":{"text":"M20558","term_id":"323449","term_text":"M20558"}}M20558); DENV3 = dengue type 3 ({"type":"entrez-nucleotide","attrs":{"text":"M93130","term_id":"323468","term_text":"M93130"}}M93130); DENV4 = dengue type 4 ({"type":"entrez-nucleotide","attrs":{"text":"AF326573","term_id":"12659201","term_text":"AF326573"}}AF326573); EHV = Edge Hill virus ({"type":"entrez-nucleotide","attrs":{"text":"DQ859060","term_id":"146411782","term_text":"DQ859060"}}DQ859060); ENTV = Entebbe bat virus ({"type":"entrez-nucleotide","attrs":{"text":"DQ837641","term_id":"112818954","term_text":"DQ837641"}}DQ837641); RBV = Rio Bravo virus ({"type":"entrez-nucleotide","attrs":{"text":"JQ582840","term_id":"383215103","term_text":"JQ582840"}}JQ582840); SEPV = Sepik virus (NC008719); SLEV = St. Louis encephalitis virus (NC007580); SOKV = Sokoluk virus ({"type":"entrez-nucleotide","attrs":{"text":"NC_026624","term_id":"765702601","term_text":"NC_026624"}}NC_026624); TBEV = tick-borne encephalitis virus (NC001672); WNV = West Nile virus ({"type":"entrez-nucleotide","attrs":{"text":"DQ211652","term_id":"77166600","term_text":"DQ211652"}}DQ211652); WSLV = Wesselsbron virus ({"type":"entrez-nucleotide","attrs":{"text":"DQ859058","term_id":"146411778","term_text":"DQ859058"}}DQ859058); YFV = yellow fever virus ({"type":"entrez-nucleotide","attrs":{"text":"K02749","term_id":"336192","term_text":"K02749"}}K02749); YOKV = Yokose virus (NC005039); ZIKV = Zika virus ({"type":"entrez-nucleotide","attrs":{"text":"AF013415","term_id":"2731554","term_text":"AF013415"}}AF013415).In total, 10,663 nucleotides of the ENTV genome were obtained (GenBank Accession number {"type":"entrez-nucleotide","attrs":{"text":"KP233893","term_id":"873167403","term_text":"KP233893"}}KP233893), of which the polypeptide is encoded between nucleotides 120 and 10,355. Kuno and Chang¹⁶ published the only other existing ENTV genome ({"type":"entrez-nucleotide","attrs":{"text":"DQ837641","term_id":"112818954","term_text":"DQ837641"}}DQ837641) sequenced from the original IL-30 strain. Compared with that original sequence, we report an additional 153 nucleotides in the 3′ noncoding region, but also experienced difficulty in obtaining the complete 3′ terminal sequence.¹⁶ Across the polypeptide sequence, there were 76 amino acid substitutions, resulting in 97.8% identity at the amino acid level between the 1957 and 2011 isolates. Before sequencing, the 1957 isolate had been passaged in suckling mouse brain, but the earlier passage history is unclear; the 2011 isolate was passaged twice in Vero cells. This 2.2% amino acid divergence is within the range that has been observed for other flaviviruses with isolations ranging between 1 and 49 years apart,^17–19 indicating relatively little change in the ENTV genome in 54 years since the first isolation.Every gene had at least one amino acid change except for PrM and M (20 It has also been shown to associate with host cellular vimentin during dengue virus replication to stabilize the replication complex.²¹ The functional significance of this high percentage of amino acid divergence has yet to be determined, but Hahn and others²² surmised that a large number of amino acid substitutions could be accommodated among the small, hydrophobic polypeptides NS2A, NS2B, NS4A, and NS4B so long as the hydrophobicity profile remained unchanged. In this case, changes to the NS4A hydrophobicity profile between these two isolates were inconsequential. Two of the 76 amino acid changes occurred at the junctions of NS2A/NS2B, and at NS4A/2K. The lengths of all of the genes were identical to those previously reported.¹⁶

Table 1

Length of each gene region in the Entebbe bat virus genome and the number (%) of amino acid (aa) substitutions in the 2011 isolate ({"type":"entrez-nucleotide","attrs":{"text":"KP233893","term_id":"873167403","term_text":"KP233893"}}KP233893) relative to the 1957 isolate ({"type":"entrez-nucleotide","attrs":{"text":"DQ837641","term_id":"112818954","term_text":"DQ837641"}}DQ837641)

Building capacity for the assessment of HIV drug resistance: experiences from the PharmAccess African Studies to Evaluate Resistance network

The PharmAccess African Studies to Evaluate Resistance (PASER) network was established as a collaborative partnership of clinical sites, laboratories, and research groups in 6 African countries; its purpose is to build research and laboratory capacity in support of a coordinated effort to assess population-level acquired and transmitted human immunodeficiency virus type-1 drug resistance (HIVDR), thus contributing to the goals of the World Health Organization Global HIV Drug Resistance Network. PASER disseminates information to medical professionals and policy makers and conducts observational research related to HIVDR. The sustainability of the network is challenged by funding limitations, constraints in human resources, a vulnerable general health infrastructure, and high cost and complexity of molecular diagnostic testing. This report highlights experiences and challenges in the PASER network from 2006 to 2010.     

Direct fluorochrome labeling of phage display library clones for studying binding specificities: applications in flow cytometry and fluorescence microscopy

Phage display technology is increasingly employed to identify high-affinity peptides and single-chain antibodies with binding specificities for a diversity of target types. The analysis of phage-binding sensitivity and specificity typically employs directly labeled secondary antiphage antibodies and potentially tertiary labels, such as fluorochromes and enzymes, when biotinylated antibodies are used. However, secondary or tertiary reagents may not be feasible or desirable for some target types and applications. Here, we present a simple approach for directly labeling phage clones with two common amine-reactive fluorochromes. We show that these fluorochromes label the pVIII major coat protein and that the binding selectivity of peptides displayed on the pIII protein of several well-characterized phage clones is maintained in flow cytometric analysis and immunofluorescence microscopy. Uniquely, such labeled phage, in part, represent self-propagating reagents because conjugation does not impair the ability to efficiently reproduce in bacteria, although relabeling with fluorochrome would be necessary. Our data suggest that primary labeled phage clones may be used similarly to primary antibody conjugates.     

Low-Frequency Drug Resistance in HIV-Infected Ugandans on Antiretroviral Treatment Is Associated with Regimen Failure

Most patients failing antiretroviral treatment in Uganda continue to fail their treatment regimen even if a dominant drug-resistant HIV-1 genotype is not detected. In a recent retrospective study, we observed that approximately 30% of HIV-infected individuals in the Joint Clinical Research Centre (Kampala, Uganda) experienced virologic failure with a susceptible HIV-1 genotype based on standard Sanger sequencing. Selection of minority drug-resistant HIV-1 variants (not detectable by Sanger sequencing) under antiretroviral therapy pressure can lead to a shift in the viral quasispecies distribution, becoming dominant members of the virus population and eventually causing treatment failure. Here, we used a novel HIV-1 genotyping assay based on deep sequencing (DeepGen) to quantify low-level drug-resistant HIV-1 variants in 33 patients failing a first-line antiretroviral treatment regimen in the absence of drug-resistant mutations, as screened by standard population-based Sanger sequencing. Using this sensitive assay, we observed that 64% (21/33) of these individuals had low-frequency (or minority) drug-resistant variants in the intrapatient HIV-1 population, which correlated with treatment failure. Moreover, the presence of these minority HIV-1 variants was associated with higher intrapatient HIV-1 diversity, suggesting a dynamic selection or fading of drug-resistant HIV-1 variants from the viral quasispecies in the presence or absence of drug pressure, respectively. This study identified low-frequency HIV drug resistance mutations by deep sequencing in Ugandan patients failing antiretroviral treatment but lacking dominant drug resistance mutations as determined by Sanger sequencing methods. We showed that these low-abundance drug-resistant viruses could have significant consequences for clinical outcomes, especially if treatment is not modified based on a susceptible HIV-1 genotype by Sanger sequencing. Therefore, we propose to make clinical decisions using more sensitive methods to detect minority HIV-1 variants.     

Characterization of penicillin intermediate serotypes of Streptococcus pneumoniae carried by human immunodeficiency virus-infected adults and healthy children in Uganda

There are little data on the genetic relatedness between antibiotic-resistant pneumococcal isolates colonizing the Ugandan population. Penicillin-intermediate pneumococci of serogroups or serotypes rarely or not previously reported as being penicillin nonsusceptible were selected out of 166 isolates representing 26 capsular serogroups or serotypes isolated from Ugandan children in 1995 and human immunodeficiency virus (HIV) infected Ugandan adults in 2004-2005. Pairs of penicillin-intermediate pneumococci of the same serogroup or serotype present in both patient populations were characterized further by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Seven such pairs of isolates were found and included serogroups 7, 11, 15B/C, and 16 as well as serotypes 13, 21, and 35B. PFGE of these seven pairs showed no clonality between serogroups or serotypes, and clonality only within serogroup 11 and serotype 13. MLST of the 14 individual isolates revealed 13 different sequence types (STs), 11 of which had not previously been recorded. Comparisons with all known STs revealed that most of these strains were related only to strains of the same serotype in other countries, with these related strains frequently also being penicillin intermediate. These findings suggest that penicillin nonsusceptibility in Uganda is likely due to the introduction of antibiotic-resistant pneumococcal clones into Uganda rather than development of resistance within the country.     

T cell activation in HIV-seropositive Ugandans: differential associations with viral load, CD4+ T cell depletion, and coinfection

Immune activation is thought to play a major role in the pathogenesis of human immunodeficiency virus (HIV). This effect may be particularly relevant in Africa, where endemic coinfections may contribute to disease progression, perhaps as a consequence of enhanced immune activation. We investigated the expression of CD38 and human leukocyte antigen (HLA)-DR on T cells in 168 HIV-seropositive volunteers in Uganda. We observed higher levels of CD4(+) and CD8(+) T cell activation in Uganda, compared with those reported in previous studies from Western countries. Coexpression of CD38 and HLA-DR on both CD4(+) and CD8(+) T cell subsets was directly correlated with viral load and inversely correlated with CD4(+) T cell counts. In antiretroviral therapy (ART)-naive volunteers, viral load and CD4(+) T cell count had stronger associations with CD8(+) and CD4(+) T cell activation, respectively. Virus suppression by ART was associated with a reduction in T cell activation, with a stronger observed effect on reducing CD8(+) compared with CD4(+) T cell activation. The presence of coinfection was associated with increased CD4(+) T cell activation but, interestingly, not with increased CD8(+) T cell activation. Our results suggest that distinct mechanisms differentially drive activation in CD4(+) and CD8(+) T cell subsets, which may impact the clinical prognostic values of T cell activation in HIV infection.