Community-based health workers implementing universal access to HIV testing and treatment: lessons from South Africa and Zambia-HPTN 071 (PopART).

The global expansion of HIV testing, prevention and treatment services is necessary to achieve HIV epidemic control and promote individual and population health benefits for people living with HIV (PLHIV) in sub-Saharan Africa. Community-based health workers (CHWs) could play a key role in supporting implementation at scale. In the HPTN 071 (PopART) trial in Zambia and South Africa, a cadre of 737 study-specific CHWs, working closely with government-employed CHW, were deployed to deliver a 'universal' door-to-door HIV prevention package, including an annual offer of HIV testing and referral services for all households in 14 study communities. We conducted a process evaluation using qualitative and quantitative data collected during the trial (2013-2018) to document the implementation of the CHW intervention in practice. We focused on the recruitment, retention, training and support of CHWs, as they delivered study-specific services. We then used these descriptions to: (i) analyse the fidelity to design of the delivery of the intervention package, and (ii) suggest key insights for the transferability of the intervention to other settings. The data included baseline quantitative data collected with the study-specific CHWs (2014-2018); and qualitative data from key informant interviews with study management (n = 91), observations of CHW training events (n = 12) and annual observations of and group discussions (GD) with intervention staff (n = 68). We show that it was feasible for newly recruited CHWs to implement the PopART intervention with good fidelity, supporting the interpretation of the trial outcome findings. This was despite some challenges in managing service quality and CHW retention in the early years of the programme. We suggest that by prioritizing the adoption of key elements of the in-home HIV services delivery intervention model-including training, emotional support to workers, monitoring and appropriate remuneration for CHWs-these services could be successfully transferred to new settings.

Identification of the K103N resistance mutation in Ugandan women receiving nevirapine to prevent HIV-1 vertical transmission.

OBJECTIVE: A recent trial in Uganda demonstrated that a simple, inexpensive regimen of nevirapine (NVP) prophylaxis can dramatically reduce HIV-1 vertical transmission risk. In this regimen, women receive a single dose of NVP at the onset of labor and infants receive a single dose of NVP within 72 h of birth. The objective of this study was to determine whether HIV-1 variants with NVP resistance mutations were selected in Ugandan women who received this regimen in the Phase I/II trial HIVNET 006. METHODS: Reverse transcriptase (RT) sequences from plasma HIV-1 were analyzed from 15 women 6 weeks after NVP dosing. RT sequences from plasma collected prior to NVP dosing were also analyzed. RESULTS: The K103N NVP resistance mutation was detected 6 weeks after NVP administration in three (20%) out of 15 women (95% confidence interval, 0-40%). Pre-dose samples were available from two of the three women; both pre-dose samples lacked the mutation. Other NVP resistance mutations were absent from all 15 women. Women with the K103N mutation had a longer median NVP elimination half-life, decreased median oral clearance, and increased median area under the concentration time curve than those without the mutation. An evaluable sample was obtained from one of these three women 33 months after delivery; the K103N mutation was not detected in that sample. CONCLUSIONS: This preliminary study demonstrates that HIV-1 with the RT K103N mutation can be detected in some Ugandan women following a single dose of NVP. This suggests that non-nucleoside RT inhibitor resistance may be selected in some people by single dose NVP prophylaxis. Pharmacokinetic data suggested that a more prolonged exposure to NVP after dosing may favor selection of NVP-resistant HIV-1.

Selection and fading of resistance mutations in women and infants receiving nevirapine to prevent HIV-1 vertical transmission (HIVNET 012).

OBJECTIVE: To examine the emergence and fading of NVP resistance (NVP(R)) mutations in HIV-1-infected Ugandan women and infants who received single dose NVP to prevent HIV-1 vertical transmission. DESIGN: We examined NVP(R) in women and infants who received NVP in the HIVNET 012 clinical trial, including 41 out of 48 women with infected infants, 70 randomly-selected women with uninfected infants, and 33 out of 49 infected infants. METHODS: Plasma HIV-1 was analyzed using the Applied Biosystems ViroSeq HIV-1 Genotyping System. RESULTS: NVP(R) mutations were detected in 21 out of 111 (19%) women tested 6-8 weeks after delivery. The rate of NVP(R) was similar among women whose infants were or were not HIV-1 infected. K103N was the most common mutation detected. NVP(R) mutations faded from detection within 12-24 months in all 11 evaluable women. High baseline viral load and low baseline CD4 cell count were associated with development of NVP(R). NVP(R) mutations were detected in 11 out of 24 (46%) evaluable infants who were infected by 6-8 weeks of age. The most common NVP(R) mutation detected in infants was Y181C. Those mutations faded from detection by 12 months of age in all seven evaluable infants. Of nine evaluable infants with late HIV-1 infection, only one had evidence of NVP(R). CONCLUSIONS: NVP(R) was detected more frequently in infants than women following NVP prophylaxis, and different patterns of NVP(R) mutations were detected in women versus infants. NVP(R) was detected infrequently in infants with late HIV-1 infection. NVP-resistant HIV-1 faded from detection in women and infants over time.

Analysis of HIV type 1 protease and reverse transcriptase in antiretroviral drug-naive Ugandan adults.

We analyzed plasma HIV-1 from 27 antiretroviral drug-naive Ugandan adults. Previous subtype analysis of env and gag sequences from these samples identified subtypes A, C, D, and recombinant HIV-1. Sequences of HIV-1 protease and reverse transcriptase (RT) were obtained with a commercial HIV-1 genotyping system. Subtypes based on protease sequences differed from gag subtypes for 5 of 27 samples, demonstrating a high rate of recombination between the gag and pol regions. Protease and RT sequences were analyzed for the presence of amino acid polymorphisms at positions that are sites of previously characterized drug resistance mutations. At those sites, frequent polymorphisms were detected at positions 36 and 69 in protease and positions 179, 211, and 214 in RT. Subtype-specific amino acid motifs were identified in protease. Most of the subtype A sequences had the amino acids DKKM at positions 35, 57, 69, and 89, whereas most subtype D sequences had the amino acids ERHL at those positions. Detection of those polymorphisms may provide a useful approach for rapid identification of subtype A and D isolates in Uganda. This analysis significantly increases the number of Ugandan protease and RT sequences characterized to date and demonstrates successful use of a commercial HIV-1 genotyping system for analysis of diverse non-B HIV-1 subtypes.

Comparison of nevirapine (NVP) resistance in Ugandan women 7 days vs. 6-8 weeks after single-dose nvp prophylaxis: HIVNET 012.

We compared nevirapine (NVP) resistance (NVPR) mutations in maternal plasma 7 days vs. 6-8 weeks after single-dose NVP prophylaxis. In the HIVNET 012 trial, Ugandan women received a single dose of NVP in labor for prevention of HIV-1 mother-to-child transmission. NVPR mutations were detected in 70 (25%) of 279 women 6-8 weeks after NVP. Samples collected 7 days after NVP were analyzed from a subset of those 279 women. Genotyping was performed with the ViroSeq HIV-1 Genotyping System. NVPR was analyzed using paired samples from 7 days and 6-8 weeks after NVP. Sixty-five women had genotyping results obtained for samples collected at both 7 days and 6-8 weeks post-NVP. Twenty-one (32%) of those women had NVPR mutations detected in one or both samples. This included three women with NVPR at 7 days only, seven with NVPR at 6-8 weeks only, and 11 with NVPR at both time points. Eight women had >1 NVPR mutation detected 7 days after NVP. Y181C was the most common NVPR mutation detected at 7 days, whereas K103N was the most common NVPR mutation detected at 6-8 weeks. We conclude that NVPR may be detected in women as early as 7 days after single-dose NVP. Complex patterns of NVPR are detected in some women. The Y181C NVPR mutation often fades from detection by 6-8 weeks. In contrast, the K103N mutation emerges more slowly, but often remains detectable 6-8 weeks after NVP.

Identification of diverse HIV type 1 subtypes and dual HIV type 1 infection in pregnant Ugandan women.

Vertical (mother-to-child) transmission accounts for the majority of pediatric HIV-1 infections. Many factors are involved in vertical transmission, however it is not clear which factors are most important for determining whether a mother will transmit HIV-1 to her infant. It has been suggested that HIV-1 subtype may influence vertical transmission and that subtype D viruses may be less likely to be transmitted in this setting. We analyzed HIV-1 gp120 V3 region sequences from the plasma of 20 pregnant Ugandan women of known transmission status who did not receive antiretroviral prophylaxis. V3 regions were cloned, sequenced, and subtyped by phylogenetic analysis. Among 11 women who transmitted HIV-1 to their infants, we detected subtypes A, C, D, and G. Two of the transmitters had dual infection with subtypes A and D. In addition, a third was infected with two distinct strains of subtype G viruses. HIV-1 subtype A and D viruses were found in 9 women who did not transmit the virus to their infants. This study reveals that pregnant Ugandan women harbor diverse HIV-1 subtypes, including women who transmit HIV-1 to their infants. Transmission of HIV-1 with subtype D V3 regions was confirmed in 4 of the 11 transmitters, including 2 who had dual infection with subtype A and D HIV-1.

DNA typing of the human MN and Ss blood group antigens in amniotic fluid and following massive transfusion.

Although red blood cell (RBC) antigen typing by agglutination is generally useful, several situations exist where this approach is difficult or impossible. For example, following a massive transfusion, a patient's residual RBCs are mixed with transfused normal donor RBCs. In this case, typing by hemagglutination primarily detects the antigens on the heterogeneous population of transfused RBCs. Agglutination testing is also of limited use for determining the phenotype of a fetus at risk for hemolytic disease of the newborn because fetal RBCs must be obtained by periumbilical blood sampling. Determining the genotype of an individual by analyzing genomic DNA isolated from peripheral blood nucleated cells or amniocytes is an alternative approach for determining the RBC antigen type. In this report, the allele specific polymerase chain reaction (AS-PCR) was used to identify the alleles at the MN and Ss loci that encode the corresponding antigens on glycophorin A (GPA) and glycophorin B (GPB), respectively. This method was used to type these alleles in peripheral blood samples obtained from normal individuals and from patients following massive transfusion. Of 23 peripheral blood specimens analyzed, all were correctly typed by this method. The allele specific polymerase chain reaction was also used to determine these alleles using amniotic fluid samples. Of 11 amniotic fluid specimens analyzed, 8 were correctly typed at both loci. Mistyping of three amniotic fluid specimens was explained by possible maternal blood contamination.

Improved detection of human immunodeficiency virus type 1 variants by analysis of replicate amplification reactions: relevance to studies of human immunodeficiency virus type 1 vertical transmission.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1)-infected individuals typically harbor mixtures of HIV-1 variants. For HIV-1 transmission studies, methods used for genotypic analysis should reliably detect variant mixtures. Such studies typically analyze complementary DNAs (cDNAs) from a single polymerase chain reaction (PCR) amplification. This approach may fail to detect variant mixtures in some samples because of analytic bias. METHODS: To evaluate the impact of analytic bias on the detection of HIV-1 variants, we analyzed samples from a mother and infant known to contain both subtypes A and D HIV-1. The env third variable region of HIV-1 gp120 (V3 region) was amplified and cloned in five replicate experiments using a single plasma sample from each individual. Ten cDNAs from each experiment were analyzed. RESULTS: The subtype mixture was detected in only four of 10 amplification experiments (three of five for the mother and one of five for the infant). Sequencing of uncloned PCR products showed that a single subtype, either A or D, was preferentially amplified in each experiment. However, the subtype mixture was detected for each sample by analyzing the five replicate experiments as a group. CONCLUSIONS: This shows that mixtures of HIV-1 variants may be more readily detected when replicate amplification reactions are analyzed. This approach may be useful for characterizing HIV-1 variants for studies of HIV-1 transmission.

Massive theophylline overdose with atypical metabolic abnormalities.

We report a case of fatal theophylline overdose in a 16-year-old asthmatic boy who presented with seizures, respiratory arrest, and a theophylline concentration of 117 mg/L in serum. His hospital course was complicated by refractory hypotension and severe ischemic necrosis of skeletal muscle, bowel, and liver. The metabolic abnormalities observed early in his hospital course included severe hyperkalemia, hyperphosphatemia, hypermagnesemia, hypocalcemia, and profound metabolic acidosis. These metabolic abnormalities differ from those previously reported in cases of massive theophylline overdose. The metabolic abnormalities observed in this patient probably reflected his extensive ischemic tissue damage with release of intracellular ions and associated acidemia. Markedly increased catalytic activities of creatine kinase, aspartate aminotransferase, and alanine aminotransferase in serum were also noted.

Automated RBC exchange transfusion:treatment for cerebral malaria.

BACKGROUND: Cerebral malaria is a life-threatening complication of Plasmodium falciparum infection. RBC exchange transfusion can reduce the level of parasitemia in this setting. Experience with automated RBC exchange for cerebral malaria may be limited, as most cases occur when the necessary equipment and blood components are not readily available. CASE REPORTS: Three patients were admitted with cerebral malaria. Parasites were found in more than 30 percent of RBCs in two cases and in more than 60 percent of RBCs in the third case. Many RBCs contained multiple organisms. In each case, antimalarial therapy was begun, and an automated RBC exchange was performed emergently with a cell separator. Exchange transfusion was repeated within 24 hours for two patients. Parasitemia levels were less than 1 percent in all patients 24 hours after the last exchange. The neurologic status of these patients returned to baseline, and they were discharged 7 to 18 days after admission. CONCLUSION: Automated RBC exchange transfusion can rapidly reduce the level of parasitemia and restore neurologic functioning in patients with cerebral malaria.

Influence of duplexes 3' to the mRNA initiation codon on the efficiency of monosome formation.

The structural features of mRNA molecules that determine their relative translational rates are at present poorly defined. An early and potentially rate-limiting step in this process is the assembly of an intact 80S ribosome at the translational initiation codon. It is generally assumed that the efficiency of this reaction is controlled by structures in the 5' nontranslated region and in the immediate proximity of the AUG initiation codon. In this paper, we present an assay of initial monosome formation and measure the effects of hybridizing mRNA to complementary DNA fragments on the efficiency of this reaction. This hybridization serves to block specific regions of the mRNA from sequence-specific and intramolecular (secondary structure) interactions. We find that cDNAs that block the 5' nontranslated region, the initiation codon, or regions immediately 3' to the initiation codon markedly inhibit 80S ribosome attachment. These results are consistent with previous studies by ourselves and others which suggest that the introduction of secondary structures into this region can result in decreased translational efficiency. In addition, however, we note that cDNAs that hybridize to segments of the coding region significant distances (as many as several hundred bases) 3' to the initiation codon can also inhibit initial ribosome binding. This effect appears to be limited to duplexes within the mRNA coding region since a cDNA hybridizing exclusively within the 3' nontranslated region does not inhibit, and may actually stimulate, monosome formation. The results of this monosome formation assay therefore suggest that mRNA structures remote from the 5' terminus and initiation codon may also be important in determining the efficiency of translational initiation.

N-linked glycosylation of beta-amyloid precursor protein.

The beta-amyloid peptide that accumulates in the brain of patients with Alzheimer's disease is derived by proteolytic processing of a family of membrane bound beta-amyloid precursor proteins (beta APPs). The three major isoforms of beta APP, derived by alternative splicing, contain 695, 751, and 770 amino acids. They are heavily O-glycosylated and contain two N-linked glycosylation sites. The pathways leading to beta-amyloid deposition in brain are not clear. It is possible that defects in metabolic and processing pathways of beta APP lead to the increased production and deposition of beta-amyloid. In many cases post-translational modifications, such as glycosylation, are important in regulating such pathways. We studied N-linked glycosylation of the 695 amino acid form of beta APP in detail by deleting the two potential glycosylation sites at Asn467 and Asn496. The mutants were examined both in a cell-free transcription/translation/glycosylation system and in transfected Chinese hamster ovary (CHO) cells. In both systems, only Asn467 was glycosylated. In CHO cells the N-linked oligosaccharide on beta APP was completely resistant to Endoglycosidase H, suggesting that it is of complex type. These mutants will be useful for studying the role of glycosylation in the metabolism of beta APP.

Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors.

N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr sequons are efficiently glycosylated, even when Xaa=Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr sequons in other glycoproteins confirms that sequons with Xaa=Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.

The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency.

N-Linked glycosylation is a common form of protein processing that can profoundly affect protein expression, structure, and function. N-Linked glycosylation generally occurs at the sequon Asn-X-Ser/Thr, where X is any amino acid except Pro. To assess the impact of the X amino acid on core glycosylation, rabies virus glycoprotein variants were generated by site-directed mutagenesis with each of the 20 common amino acids substituted at the X position of an Asn-X-Ser sequon. The efficiency of core glycosylation at the sequon in each variant was quantified in a rabbit reticulocyte lysate cell-free translation system supplemented with canine pancreas microsomes. The presence of Pro at the X position completely blocked core glycosylation, whereas Trp, Asp, Chi, and Leu were associated with inefficient core glycosylation. The other variants were more efficiently glycosylated, and several were fully glycosylated. These findings demonstrate that the X amino acid is an important determinant of N-linked core-glycosylation efficiency.

Performance of Applied Biosystems ViroSeq HIV-1 Genotyping System for sequence-based analysis of non-subtype B human immunodeficiency virus type 1 from Uganda.

The Applied Biosystems ViroSeq HIV-1 Genotyping System is a commercially available, integrated system for sequence-based analysis of drug resistance mutations in human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT). We evaluated the performance of this system for analysis of non-subtype B HIV-1 by analyzing plasma samples from Ugandan women and infants. Plasma samples were obtained from 105 women and 25 infants enrolled in a Ugandan clinical trial. HIV-1 analysis was performed with the ViroSeq system according to the manufacturer's instructions, except that the volume of plasma used for analysis was less than the recommended 0.5 ml for some samples. Viral loads ranged from 2,313 to 2,336,400 copies/ml. PCR products suitable for sequencing were amplified from all samples tested. Complete sequences for protease (amino acids 1 to 99) and RT (amino acids 1 to 320) were obtained for 102 of 105 (97%) of the maternal samples tested and all 25 of the infant samples tested. Complete double-stranded sequences were obtained for 90 of 105 (86%) of the maternal samples tested and 22 of 25 (88%) of the infant samples tested. The sequences obtained with this system were used for HIV-1 subtyping. The subtypes identified were A, C, D, and A/D recombinant HIV-1. The performances of the seven sequencing primers were similar for the subtypes examined. The ViroSeq system performs well for analysis of Ugandan plasma samples with subtypes A, C, D, and A/D recombinant HIV-1. The availability of this genotyping system should facilitate studies of HIV-1 drug resistance in countries where these subtypes are prevalent.

Impact of human immunodeficiency virus type 1 (hiv-1) subtype on women receiving single-dose nevirapine prophylaxis to prevent hiv-1 vertical transmission (hiv network for prevention trials 012 study).

In Uganda, the HIV Network for Prevention Trials (HIVNET) 012 study recently demonstrated that single-dose nevirapine (Nvp) prophylaxis is effective for preventing mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1). This exploratory study examines the relationship between HIV-1 subtype, MTCT, and the development of Nvp resistance (Nvp(R)) in women enrolled in HIVNET 012. For 102 women (32 whose infants were HIV-1 infected by age 6-8 weeks and 70 whose infants were uninfected), HIV-1 subtypes included 50 (49%) subtype A, 35 (34%) subtype D, 4 (4%) subtype C, 12 (12%) recombinant subtype, and 1 unclassified. There was no apparent difference in the rate of MTCT among women with subtype A versus D (adjusted odds ratio [OR], 1.24; 95% confidence interval [CI], 0.45-3.43). Nvp(R) mutations were detected more frequently at 6-8 weeks postpartum in women with subtype D than in women with subtype A (adjusted OR, 4.94; 95% CI, 1.21-20.22). Additional studies are needed to further define the relationship between HIV-1 subtype and Nvp(R) among women receiving Nvp prophylaxis.

Performance of the applied biosystems ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system for sequence-based analysis of HIV-1 in pediatric plasma samples.

The ViroSeq HIV-1 Genotyping System is a commercially available, integrated sequence-based system for analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance. We evaluated the performance of this system by analyzing HIV-1 in pediatric plasma samples. Plasma samples from children 4 months to 17 years of age were obtained from a clinical trial protocol (PACTG 377). Children in PACTG 377 were randomized to four treatment arms, including different combinations of antiretroviral drugs. HIV-1 genotyping was performed using samples collected prior to antiretroviral therapy (baseline) and at the time of virologic failure. Performance of the genotyping system was compared in three university laboratories. A total of 196 samples were analyzed, including 135 baseline and 61 failure samples. Plasma volumes ranged from 0.05 to 0.5 ml, and viral loads ranged from 1,084 to 3,484,991 copies/ml. PCR products suitable for sequencing were obtained for 192 of the 196 samples. Complete sequences for protease and reverse transcriptase were obtained for all of these 192 samples. For 180 samples, data were obtained from both DNA strands for the entire region analyzed. There was no evidence of sample cross-contamination based on phylogenetic analysis of HIV-1 sequences. Performance of the genotyping system was similar in three laboratories. This genotyping system performs well for analysis of HIV-1 in pediatric plasma samples, including those with low volume and low viral load. The availability of this system should facilitate studies of HIV-1 drug resistance.