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.

Subacute sclerosing panencephalitis in South African children following the measles outbreak between 2009 and 2011.

Between 2009 and 2011, there was an outbreak of measles throughout South Africa (SA). The largest age category infected was children<5 years of age. In 2014, four patients, with a median age of 4 years and 5 months (range 4 years 3 months-4.5 years), three males and one female, presented with subacute sclerosing panencephalitis (SSPE). All were infected with measles during the period of the 2009-2011 outbreak in early infancy, at a time when their immune systems were immature and before they were vaccinated against the measles virus. One patient was immunocompromised, with vertically acquired HIV infection. All the children presented with cognitive and behavioural decline, abnormal movements and medically intractable myoclonic and atonic seizures. Outcome was poor in all and no reversibility was evident with standard therapeutic interventions. Optimal seizure control with carbamazepine is reported in patients with SSPE. Three of our patients who received carbamazepine experienced improved seizure control, but their neuroregression continued. Since submission of this case series, patient 1 (see Table 1) has died, and a further child has presented with the same clinical phenotype as described. On the basis of this clustering of patients in the Western Cape Province, SA, it is important to screen children admitted with acute cognitive decline and intractable seizures for SSPE, especially those who were infants during the measles outbreak.

Molecular characterisation of virus in the brains of patients with measles inclusion body encephalitis (MIBE).

BACKGROUND: During 2009/10 a major measles epidemic caused by genotype B3 occurred in South Africa. Measles inclusion body encephalitis (MIBE) was diagnosed in a number of highly immuno-compromised HIV patients. The diagnosis was based on typical clinical and MRI findings and positive measles virus PCR in brain or CSF.To characterize the brain virus, nucleoprotein, matrix, fusion and haemagglutinin genes from 4 cases was compared with virus from acutely infected patients. METHODS: cDNA was synthesized using random primers and viral genes were amplified by nested RT-PCR. PCR products were sequenced in the forward and reverse direction and a contig of each gene was created. Sequences were aligned with reference sequences from GenBank and other local sequences. RESULTS: Brain virus was very similar to the South African epidemic virus. Features characteristic of persistent measles virus in the brain were absent. Mutation frequency in brain virus was similar to epidemic virus and had the same substitution preference (U to C and C to U). The virus of 2 patients had the same L454W mutation in the fusion protein. CONCLUSION: The brain virus was very similar to the epidemic strain. The relatively few mutations probably reflect the short time from infection to brain disease in these highly immuno-compromised patients.

Cytomegalovirus load in whole blood is more reliable for predicting and assessing CMV disease than pp65 antigenaemia.

CMV is a common cause of disease in immunocompromised patients. Because sampling of the diseased organ can be invasive, markers of systemic CMV reactivation such as pp65 and CMV viral load are commonly used to monitor patients at risk of CMV disease. In this retrospective analysis, the performance of these markers was compared in solid organ transplant recipients, patients with haematological malignancies and HIV infection. Both assays were sensitive markers of reactivation, however, the predictive value for disease of a positive result for both was low. Compared to viral load, the pp65 assay was a less sensitive marker of CMV reactivation. It was only positive when the viral load was greater than 3 log (10) copies/ml whole blood and was negative in 10 instances when the viral load was between 3 and 5 logs. In concordantly positive samples, the number of pp65 positive cells varied widely relative to the viral load and the number of positive cells counted could not be used to predict disease likelihood with any certainty. To conclude, CMV viral load provides a more consistent guide to determine likelihood of disease than pp65 count and is a more sensitive marker of CMV reactivation.

An investigation into the prevalence and outcome of patients admitted to a pediatric intensive care unit with viral respiratory tract infections in Cape Town, South Africa.

OBJECTIVES: To describe the prevalence and outcome of patients admitted to a pediatric intensive care unit with viral respiratory tract infections. DESIGN: Retrospective descriptive study. SETTING: Pediatric intensive care unit in a tertiary pediatric hospital situated in Cape Town, South Africa. PATIENTS: All children (n = 195; 20% pediatric intensive care unit admissions) with positive respiratory viral isolates between April 1 and December 31, 2009. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Demographic, clinical, laboratory, and outcome data were recorded from medical folders. Complete data were available for 175 patients (median age [interquartile range] 4.7 months [2.3-12.9 months]; 49% male). One hundred four (59.4%) patients had comorbid conditions; 30 (17%) were HIV-infected. Rhinovirus (n = 76 [39%]), respiratory syncytial virus (n = 54 [27.7%]), adenovirus (n = 30 [15.4%]), influenza A (n = 26 [13.3%]), parainfluenza (n = 23 [11.8%]), and human metapneumovirus (n = 12 [6.2%]) were most commonly isolated. Ninety-five infections (51.4%) were isolated >48 hrs after admission. Seasonal patterns were identified for respiratory syncytial virus, human metapneumovirus, and influenza A, whereas others occurred throughout the year. Twenty-five patients (14.3%) had more than one viral isolate. Presumed bacterial coinfection, which occurred in 68 (39%) patients (18 [26.5%] HIV-infected), was associated with significantly longer pediatric intensive care unit and hospital stays but not with mortality. Twenty patients died (11%, standardized mortality ratio 0.64). High Pediatric Index of Mortality scores, HIV exposure and infection, nosocomial infection, and influenza A infection were associated with mortality. CONCLUSIONS: Viral respiratory tract infection is common in this pediatric intensive care unit associated with significant morbidity and mortality, which may relate to the high burden of comorbidity and HIV.

p24 antigen rapid test for diagnosis of acute pediatric HIV infection.

Currently, the majority of HIV-infected infants are found within limited-resource settings, where inadequate screening for HIV due to the lack of access to simple and affordable point-of-care tests impedes implementation of antiretroviral therapy. Here we report development of a low-cost dipstick p24 antigen assay using a visual readout format that can facilitate the diagnosis of HIV for infants in resource-poor conditions. A heat shock methodology was developed to optimize disruption of immune complexes present in the plasma of infected infants. The analytical sensitivity of the assay using recombinant p24 antigen is 50 pg/mL (2 pM) with whole virus detection as low as 42.5k RNA copies per milliliter plasma. In a blinded study comprising 51 archived infant samples from the Women and Infants Transmission Study, our assay demonstrated an overall sensitivity and specificity of 90% and 100%, respectively. In field evaluations of 389 fresh samples from South African infants, a sensitivity of 95% and specificity of 99% was achieved. The assay is simple to perform, requires minimal plasma volume (25 µL), and yields a result in less than 40 minutes making it ideal for implementation in resource-limited settings.

The predictive value of cerebrospinal fluid Epstein-Barr viral load as a marker of primary central nervous system lymphoma in HIV-infected persons.

BACKGROUND: The presence of Epstein-Barr virus (EBV) DNA in cerebrospinal fluid (CSF) is used as a marker of HIV-associated primary central nervous system lymphoma (PCNSL). In our setting, EBV DNA is frequently detected in the CSF of HIV-infected patients with miscellaneous neurological diseases and thus its presence is a poor predictor of PCNSL. OBJECTIVES: To determine whether quantification of EBV DNA in CSF improves its diagnostic specificity for PCNSL. STUDY DESIGN: EBV viral loads were determined on CSF samples from 55 HIV-infected patients with CNS disease. RESULTS: Twenty of the 55 patients had detectable EBV DNA in their CSF (median viral load 6120copies/ml, range 336-1,034,000copies/ml). PCNSL was confirmed in 2 patients. Their CSF EBV loads were 1,034,000 and 15,460copies/ml, respectively. Using a cut-off of 10,000copies/ml improved the specificity and positive predictive value (PPV) compared to a qualitative result for the diagnosis of PCNSL (96% vs. 66% and 50% vs. 10%, respectively). CONCLUSION: EBV DNA is commonly detected in CSF of HIV-infected patients. Quantitative PCR improves the diagnostic specificity, however, the PPV remains too low for it to be used as an isolated marker for PCNSL.

Seroprevalence of rubella antibodies among antenatal patients in the Western Cape.

OBJECTIVES: To determine the seroprevalence of rubella virus infection among antenatal patients aged between 15 and 45 years in the Western Cape province of South Africa, in order to provide data to determine the need for vaccination to protect women of childbearing age. DESIGN: A cross-sectional study. Setting. Virology laboratory, Groote Schuur Hospital, National Health Laboratory Service (NHLS), South Africa. SUBJECTS AND METHODS: One thousand two hundred provincial serum specimens from participants in the 2003 Department of Health antenatal HIV/syphilis serosurvey were selected from the 4 districts of the Western Cape. The specimens were age-stratified and screened qualitatively for rubella immunoglobulin G (IgG) antibodies by means of a commercial immunoassay during October 2004. RESULTS: Within the Western Cape a total of 95.3% of women in the 15-24-year age group, 97.5% in the 25-34-year group and 98% in the 35-45-year age group were immune to rubella. There was no statistically significant difference in the rate of rubella susceptibility between the 4 districts tested. CONCLUSIONS: The study is an important step in addressing the seroprevalence of rubella infection in women of childbearing age in South Africa. Further information is needed on rubella seroprevalence from the other provinces in South Africa as well as formal implementation of rubella and congenital rubella syndrome surveillance to determine the feasibility of routine rubella immunisation.