Field study to determine the reliability of HIV viral load results shows minimal impact of delayed testing in South Africa.

Background: Understanding factors that impact HIV viral load (VL) accuracy in resource-limited settings is key to quality improvement. Objective: We evaluated whether testing delay and specimen storage between 25 °C and 30 °C before testing affected results. Methods: Between November 2019 and June 2023, 249 individuals on antiretroviral therapy, or with newly diagnosed HIV, were recruited from clinics in Cape Town and Gqeberha, South Africa, and three plasma preparation tubes were collected. One tube was tested within 24 h, while the others were stored uncentrifuged at ambient temperatures before testing. Centrifugation and testing of matched samples were performed on Day 4 and Day 7 after collection. Results: Time delay and ambient storage had minimal impact in specimens with a Day 1 VL of > 100 copies/mL. When grouped by Day 1 VL range, 96% - 100% of specimens at Day 4 and 93% - 100% at Day 7 had VLs within 0.5 log copies/mL of the first result. The greatest variability at Days 4 and 7 was observed when the Day 1 VL was < 100 copies/mL. However, there was no trend of increasing difference over time. Of Day 1 specimens with undetectable VL, or VL < 50 copies/mL, 80% had concordant results at Day 4 and 78% at Day 7. Conclusion: These results show that VL is stable in plasma preparation tubes for 7 days when stored at room temperature. There is significant variability in specimens with low VL, but variability is not affected by testing delay. What this study adds: Ideal HIV VL testing conditions are frequently unachievable in resource-limited settings. Data are needed on whether this impacts on the validity of test results. Our results provide reassurance that storage at ambient temperature for up to 7 days before testing does not substantially affect the VL result.

Effective radiation dose of skeletal surveys performed for suspected physical abuse.

BACKGROUND: A skeletal survey is an important diagnostic tool for patients presenting with suspected physical abuse. A relatively recent change in guidelines for skeletal surveys by the Royal College of Radiologists (RCR) in 2017 has led to more initial and follow-up images for these patients, which would be expected to have led to an increase in effective radiation dose. OBJECTIVE: To estimate the effective dose following the change in guidelines and to ascertain the difference between doses before and after the change in guidelines. MATERIALS AND METHODS: Data were collected retrospectively on children younger than 3 years old referred for skeletal surveys for suspected physical abuse at a tertiary paediatric centre. A Monte Carlo radiation patient dose simulation software, PCXMC v 2.0.1, was used to estimate the effective dose, expressed in millisieverts (mSv). RESULTS: Sixty-eight children underwent skeletal surveys for suspected physical abuse. The total estimated effective dose for skeletal surveys with the previous RCR guidelines was found to be 0.19 mSv. For initial skeletal surveys with the current RCR guidelines, the estimated effective radiation dose was 0.19 mSv. Eighteen children had both initial and follow-up skeletal surveys as indicated by the current RCR guidelines, with an estimated effective total radiation dose of 0.26 mSv. CONCLUSION: Skeletal surveys deliver a relatively low estimated effective radiation dose equivalent to 1 month of United Kingdom background radiation, with no significant change in dose following the change in guidelines. Therefore, the benefits of having a skeletal survey outweigh the main radiation risk. However, accurate data regarding the radiation dose are important for clinicians consenting parents/guardians for imaging in suspected physical abuse.

Molecular characterisation and epidemiology of enterovirus-associated aseptic meningitis in the Western and Eastern Cape Provinces, South Africa 2018-2019.

BACKGROUND: Enteroviruses are amongst the most common causes of aseptic meningitis. Between November 2018 and May 2019, an outbreak of enterovirus-associated aseptic meningitis cases was noted in the Western and Eastern Cape Provinces, South Africa. OBJECTIVES: To describe the epidemiology and phylogeography of enterovirus infections during an aseptic meningitis outbreak in the Western and Eastern Cape Provinces of South Africa. METHODS: Cerebrospinal fluid samples from suspected cases were screened using a polymerase chain reaction targeting the 5'UTR. Confirmed enterovirus-associated meningitis samples underwent molecular typing through species-specific VP1/VP2 primers and pan-species VP1 primers. RESULTS: Between November 2018 and May 2019, 3497 suspected cases of aseptic meningitis were documented in the Western and Eastern Cape Provinces. Median age was 8 years (range 0-61), interquartile range (IQR=4-13 years), 405/735 (55%) male. 742/3497 (21%) cases were laboratory - confirmed enterovirus positive by routine diagnostic PCR targeting the 5'UTR. 128/742 (17%) underwent molecular typing by VP1 gene sequencing. Echovirus 4 (E4) was detected in 102/128 (80%) cases. Echovirus 9 was found in 7%, Coxsackievirus A13 in 3%. 10 genotypes contributed to the remaining 10% of cases. Synonymous mutations were found in most cases, with sporadic amino acid changes in 13 (12.7%) cases. CONCLUSION: The aseptic meningitis outbreak was associated with echovirus 4. Stool samples are valuable for molecular typing in CSF confirmed EV-associated aseptic meningitis.

HIV-1 viral load testing in resource-limited settings: Challenges and solutions for specimen integrity.

HIV-1 viral load (VL) testing is a crucial element in providing an antiretroviral treatment monitoring program. The success of these programs depends on the availability and quality of the VL testing services. There are several pre-analytic factors which can affect the quality of VL testing. Many of the challenges faced by resource-limited countries result in a compromise of specimen integrity, thus limiting widespread access to VL monitoring. The various logistic and financial challenges that exist are not insurmountable and several innovative solutions currently exist to overcome these barriers to providing widespread VL testing. This review summarizes the VL testing challenges in resource-limited settings and provides an overview of potential solutions including testing dried blood spots, dried plasma spots, plasma separation cards and the use of point of care tests.

Rapid testing for respiratory syncytial virus in a resource-limited paediatric intensive care setting.

We analysed the performance characteristics of the respiratory syncytial virus lateral flow rapid antigen assay in use when compared to a multiplex polymerase chain reaction for detection of respiratory viruses. The study was conducted at a tertiary paediatric hospital in Port Elizabeth, South Africa, from 01 January 2017 to 31 December 2018. We found the clinical sensitivity (36.8%) of the rapid test to be too low for routine diagnostic use. Knowledge of assay performance characteristics of rapid tests are important for appropriate interpretation of rapid test results.

Lessons in diagnostic virology: expected and unexpected sources of error.

Viral diagnostics have shown continued innovation, with serological and molecular diagnostic assays pushing the limits of sensitivity. Technology has provided new automated shared diagnostic platforms that reduce hands-on time, while with globalisation of the diagnostic market, commercial assays are applied across epidemiologically diverse settings on different patient and viral populations. However, with these novel developments, new and often unexpected sources of diagnostic error emerge. In this review we will reflect on case studies that highlight these often underappreciated or unexpected diagnostic errors spanning pre-analytical, analytical, and post-analytic processes. We will also suggest approaches that could help identify error and reduce the impact on patient management.

Simplified approach to uncertainty of measurement in the clinical virology laboratory.

Medical laboratories are required to ensure the quality of their diagnostic results. Quality assurance procedures include quality assessments (internal and external), quality controls (negative, positive, or internal controls), equipment monitoring, and audits. Quality control data may be used to evaluate the uncertainty of measurement. All clinical virology laboratories require a standard operating procedure detailing their consideration of uncertainty of measurement, as this parameter may impact on the overall quality of diagnostic results as well as the clinical interpretation thereof. This review aims to provide a simplified approach to the concept of uncertainty of measurement, specific for clinical virology laboratories.

Basic overview of method validation in the clinical virology laboratory.

Diagnostic virology laboratories are an essential part of the health system and are often relied upon to provide information to clinicians that will inform clinical decision making. It is therefore imperative that diagnostic results produced in the laboratory are reliable. One way of ensuring quality results is by ensuring that all tests are either validated (for tests developed in-house) or verified (for commercial assays that are FDA-approved or CE-labeled). In the diagnostic virology laboratory, these processes can be complex as both qualitative and quantitative measurements for serological and molecular tests are routinely offered. While there are numerous guidelines governing quality assurance in the virology laboratory, all accrediting agencies would insist on tests being validated or verified prior to implementation without providing explicit guidance to the process. As there is no universal guideline on the optimal way to perform validation/verification experiments, this review will provide a basic overview of method validation/verification, specific for clinical virology laboratories, and includes explanation of statistical analysis and acceptance/rejection criteria.

A qualitative PCR minipool strategy to screen for virologic failure and antiretroviral drug resistance in South African patients on first-line antiretroviral therapy.

BACKGROUND: The high cost of commercial HIV-1 viral load tests for monitoring of patients on antiretroviral treatment limits their use in resource-constrained settings. Commercial genotypic antiretroviral resistance testing is even more costly, yet it provides important benefits. OBJECTIVES: We sought to determine the sensitivity and negative predictive value of a qualitative PCR targeting partial reverse transcriptase for detection of virologic failure when 5 patient specimens are pooled. STUDY DESIGN: A total of 300 South African routine patient samples were included and tested in 60 pools of 5 samples each. A qualitative nested PCR was optimised for testing pools and individual samples from positive pools. All positive samples were sequenced to detect drug resistance-associated mutations. Results were compared to those of conventional viral load monitoring. RESULTS: Twenty-two of 60 pools tested positive. Individual testing yielded 29 positive individual samples. Twenty-six patients had viral loads of above 1000 copies/ml. The pooling algorithm detected 24 of those 26 patients, resulting in a negative predictive value of 99.3%, and a positive predictive value of 89.7%. The sensitivity for detecting patients failing therapy was 92%, with a specificity of 98.9%. Of the patients failing first-line ART, 83.3% had NRTI and 91.7% NNRTI resistance mutations. CONCLUSIONS: The pooled testing algorithm presented here required 43% fewer assays than conventional viral load testing. In addition to offering a potential cost saving over individual viral load testing, it also provided drug resistance information which is not available routinely in resourced-limited settings.

Viral ocular manifestations: a broad overview.

The viruses able to affect the eye are taxonomically diverse, ranging from double-stranded DNA viruses, to single stranded RNA viruses, to retroviruses. Any part of the eye may be affected, frequently producing blepharitis, conjunctivitis, keratitis, uveitis, cataract and retinitis. The more common ocular viral infections include the Herpesviruses such as HSV-1, VZV and CMV. The HIV pandemic is placing a serious burden on ophthalmology clinics, particularly in sub-Saharan Africa as the number of viral ocular diseases is increasing. In particular, CMV retinitis is becoming more prevalent where antiretroviral therapy is not available and is replaced by immune-recovery uveitis where antiretrovirals are given. This review aims to improve knowledge of the common viral ocular diseases, their diagnosis and management, as well as the fairly uncommon viral ocular diseases that may also cause considerable morbidity.

Pyrimidine containing RSV fusion inhibitors.

The knowledge of SAR in a series of biphenyl anionic RSV inhibitors has been broadened by synthesis and testing of analogs with pyrimidine linkers. Generally, pyrimidine compounds were much harder to synthesize, and their anti-RSV activity was lower in comparison with triazine analogs.

Synthesis and activity of novel benzoxazole derivatives of mannopeptimycin glycopeptide antibiotics.

A series of benzoxazole derivatives of the mannopeptimycin glycopeptide antibiotics was synthesized via a novel benzoxazole formation reaction by treating aminophenol of mannopeptimycin-beta with an aldehyde and DDQ in DMF. Some of these derivatives (e.g., 5b, 5d, 5m, and 7b) showed good activity against Gram-(+) bacteria when compared to the parent compound mannopeptimycin-beta.