The role of new hepatitis B vaccines in South Africa.

Vaccination is key to eliminating hepatitis B virus infection in South Africa (SA). Despite introducing immunisation in 1995, as part of the expanded programme of immunisation (EPI), hepatitis B virus infection remains endemic, and EPI vaccine coverage is incomplete. In addition to infants, non-immune adults at risk of infection through their occupation or with behavioural risk factors should receive vaccination. SA has many individuals with diabetes mellitus (a prevalence of almost 13%), obesity, HIV (8.45 million) or older age (5 million >60 years old), associated with a poorer vaccine response. Recently two new hepatitis B vaccines have been licensed: HEPLISAV-B includes an adjuvant that improves immunogenicity and has shown improved vaccine response in individuals with HIV, old age or diabetes mellitus. PreHevbrio, which includes three hepatitis B surface protein domains, instead of one, may also be more immunogenic, although clinical study data are still limited. These two novel vaccines have not yet been investigated in children and licensed in SA. Should HEPLISAV-B become available in SA, it may be particularly valuable to target high-risk groups in the country, such as people living with HIV, who show a poor response to the currently licensed vaccine.

High burden of viral respiratory co-infections in a cohort of children with suspected pulmonary tuberculosis.

BACKGROUND: The presentation of pulmonary tuberculosis (PTB) in young children is often clinically indistinguishable from other common respiratory illnesses, which are frequently infections of viral aetiology. As little is known about the role of viruses in children with PTB, we investigated the prevalence of respiratory viruses in children with suspected PTB at presentation and follow-up. METHODS: In an observational cohort study, children < 13 years were routinely investigated for suspected PTB in Cape Town, South Africa between December 2015 and September 2017 and followed up for 24 weeks. Nasopharyngeal aspirates (NPAs) were tested for respiratory viruses using multiplex PCR at enrolment, week 4 and 8. RESULTS: Seventy-three children were enrolled [median age 22.0 months; (interquartile range 10.0-48.0); 56.2% male and 17.8% HIV-infected. Anti-tuberculosis treatment was initiated in 54.8%; of these 50.0% had bacteriologically confirmed TB. At enrolment, ≥1 virus were detected in 95.9% (70/73) children; most commonly human rhinovirus (HRV) (74.0%). HRV was more frequently detected in TB cases (85%) compared to ill controls (60.6%) (p = 0.02). Multiple viruses were detected in 71.2% of all children; 80% of TB cases and 60.6% of ill controls (p = 0.07). At follow-up, ≥1 respiratory virus was detected in 92.2% (47/51) at week 4, and 94.2% (49/52) at week 8. CONCLUSIONS: We found a high prevalence of viral respiratory co-infections in children investigated for PTB, irrespective of final PTB diagnosis, which remained high during follow up. Future work should include investigating the whole respiratory ecosystem in combination with pathogen- specific immune responses.

Pooling strategies to reduce the cost of HIV-1 RNA load monitoring in a resource-limited setting.

BACKGROUND: Quantitative human immunodeficiency virus (HIV) RNA load testing surpasses CD4 cell count and clinical monitoring in detecting antiretroviral therapy (ART) failure; however, its cost can be prohibitive. Recently, the use of pooling strategies with a clinically appropriate viral load threshold was shown to be accurate and efficient for monitoring when the prevalence of virologic failure is low. METHODS: We used laboratory request form information to identify specimens with a low pretest probability of virologic failure. Patients aged ≥15 years who were receiving first-line ART had individual viral load results available were eligible. Blood plasma, dried blood spots, and dried plasma spots were evaluated. Two pooling strategies were compared: minipools of 5 samples and a 10 ×10 matrix platform (liquid plasma specimens only). A deconvolution algorithm was used to identify specimens(s) with detectable viral loads. RESULTS: The virologic failure rate in the study sample was <10%. Specimens included were liquid plasma specimens tested in minipools(n = 400), of which 300 were available for testing by matrix, and specimens tested with minipools only: dried blood spots (n = 100) and dried plasma spots (n = 185). Pooling methods resulted in 30.5%-60% fewer HIV RNA tests required to screen the study sample. For plasma pooling, the matrix strategy had the better efficiency, but minipools of 5 dried blood spots had the best efficiency overall and were accurate at a >95% negative predictive value with minimal technical requirements. CONCLUSIONS: In resource-constrained settings, a combination of preselection of patients with low pretest probability of virologic failure and pooled testing can reduce the cost of virologic monitoring without compromising accuracy.

NucliSens EasyQ HIV-1 V1.2 system: Detection of human plasma-derived background signal.

Until recently the NucliSens EasyQ HIV-1 V1.2 system has been used throughout South Africa as part of the national antiretroviral roll-out programme for the monitoring of HIV-1 RNA load in patients on antiretroviral treatment. Shortly after changing to a new assay lot number an increased proportion of patient specimens, showing detectable but low viral loads, was observed (<200 IU/ml). The test runs remained valid as the lysis buffer-only no-template controls (NTCs) remained negative. Contamination with amplification product was excluded. Subsequently the same phenomenon was observed in at least three other South African laboratories across different assay lot numbers. When testing aliquots of plasma, freshly obtained from HIV-negative donors, at two of these laboratories, 33/134 aliquots showed detectable values (range 26-370, median: 64 IU/ml), while all NTCs remained negative. These findings emphasize the importance of appropriate specimen controls in all diagnostic assays. In this case HIV-negative human plasma should be included routinely in addition to NTCs, which would allow rapid detection of a background signal.

Extraction buffer contaminated bacterially as a cause of invalid HIV-1 viral load results on the NucliSens EasyQ system.

Shortly after starting to use the NucliSens EasyQ HIV-1 V1.1 system for HIV-1 RNA load testing, the number of invalid tests per assay run gradually increased. Within five days, approximately 50% of tests showed a total lack of amplification of the calibrator and in most cases also of the HIV-1 template. According to the manufacturer's specifications, the lysis buffer and three extraction buffers remain on the automated NucliSens easyMAG extraction system between assay runs. Therefore possible microbial contamination of these buffers was investigated, after they had been on the automated system for approximately one week. The NucliSens easyMAG extraction buffer 2 yielded bacterial growth identified as Acinetobacter baumannii. After regular decontamination of the machine's tubing system with 70% alcohol and storage of the buffers at 4 degrees C between assay runs were commenced, invalid results due to failed internal calibrator signal occurred no longer. It is likely that bacterial contamination of the buffer was the cause of assay failure, probably due to ribonuclease (RNase) activity. Bacterial contamination of PCR systems should be added to the list of potential hazards in diagnostic virology. This experience underlines the necessity of state-of-the-art assay design incorporating adequate internal controls and calibrators.

Zidovudine with nevirapine for the prevention of HIV mother-to-child transmission reduces nevirapine resistance in mothers from the Western Cape, South Africa.

In the Western Cape province of South Africa, an intensified regimen for the prevention-of-mother-to-child-transmission-of-HIV consisting of zidovudine (AZT) from 34 weeks of pregnancy plus single dose (sd) nevirapine (NVP) during labor was instituted in 2004. The newborn baby receives a single dose of NVP and AZT for 7 days. Similar strategies in Thailand and Africa have been shown to be more effective in reducing transmission than NVP alone. The use of sd NVP only for the prevention-of-mother-to-child-transmission-of-HIV has a high risk of inducing resistance (25-69%) with an average of 35.7% by a recent meta-analysis and has been shown to adversely affect non-nucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy when initiated within 6 months. In this study the prevalence of resistance to NVP and AZT in mothers who had received the intensified regimen was measured. Specimens collected from mothers were genotyped by in-house PCR and sequencing. In specimens obtained within 60 days of delivery, acquired NVP resistance mutations were detected in 13 of 76 patients (17.1%, 95% confidence interval: 8.7-25.6%), which appears to be lower than in studies with sd NVP alone (37.5%, 95% confidence interval: 23.0-50.6%).

Pitfalls with rapid HIV antibody testing in HIV-infected children in the Western Cape, South Africa.

BACKGROUND: Rapid HIV antibody tests are commonly used for HIV diagnosis in the developing world. These tests are generally reported as sensitive, despite paucity of evaluations in paediatric populations. OBJECTIVES: We tested specimens of paediatric patients, known to be HIV-infected, to detect any false negative tests and determine associations with such an outcome. STUDY DESIGN: One hundred and fifty-three specimens, from 109 patients, recorded to be HIV-infected by standard testing, were tested on the Capillustrade mark HIV-1/HIV-2 test (Trinity Biotech, Ireland); 150 specimens also had sufficient volume to be tested on Abbott Determinetrade mark HIV1/2 assay (Abbott GmbH, Wiesbaden, Germany). Treatment information, CD4 counts and HIV-1 viral load measurements were obtained from patient files and laboratory databases. RESULTS: Twenty-one of 153 specimens tested negative on the Capillus (sensitivity 86.3%). False negative results by Capillus were associated with antiretroviral treatment (ART) (p=0.0018) and lower HIV-1 viral load (p=0.013). Serial dilutions of some of the specimens indicated that both rapid tests, and the Capillus in particular, became negative at lower dilutions than an HIV enzyme immunoassay (EIA). CONCLUSIONS: The Capillus test had an unexpectedly low sensitivity in a South African population of HIV-infected children that had access to antiretroviral treatment, posing a risk of false negative HIV testing.