Diana Hardie

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 failure of routine rapid HIV testing: a case study of improving low sensitivity in the field.

BackgroundThe rapid HIV antibody test is the diagnostic tool of choice in low and middle-income countries. Previous evidence suggests that rapid HIV diagnostic tests may underperform in the field, failing to detect a substantial number of infections. A research study inadvertently discovered that a clinic rapid HIV testing process was failing to detect cases of established (high antibody titer) infection, exhibiting an estimated 68.7% sensitivity (95% CI [41.3%-89.0%]) over the course of the first three weeks of observation. The setting is a public service clinic that provides STI diagnosis and treatment in an impoverished, peri-urban community outside of Cape Town, South Africa.MethodsThe researchers and local health administrators collaborated to investigate the cause of the poor test performance and make necessary corrections. The clinic changed the brand of rapid test being used and later introduced quality improvement measures. Observations were made of the clinic staff as they administered rapid HIV tests to real patients. Estimated testing sensitivity was calculated as the number of rapid HIV test positive individuals detected by the clinic divided by this number plus the number of PCR positive, highly reactive 3rd generation ELISA patients identified among those who were rapid test negative at the clinic.ResultsIn the period of five months after the clinic made the switch of rapid HIV tests, estimated sensitivity improved to 93.5% (95% CI [86.5%-97.6%]), during which time observations of counselors administering tests at the clinic found poor adherence to the recommended testing protocol. Quality improvement measures were implemented and estimated sensitivity rose to 95.1% (95% CI [83.5%-99.4%]) during the final two months of full observation.ConclusionsPoor testing procedure in the field can lead to exceedingly low levels of rapid HIV test sensitivity, making it imperative that stringent quality control measures are implemented where they do not already exist. Certain brands of rapid-testing kits may perform better than others when faced with sub-optimal use.

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.

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.

Genetic Variants of Human Parvovirus B19 in South Africa: Cocirculation of Three Genotypes and Identification of a Novel Subtype of Genotype 1

ABSTRACT Parvovirus B19 comprises three distinct genotypes (1, 2, and 3). The distribution of B19 genotypes has not before been examined in South Africa. Two hundred thirty-nine laboratory samples submitted to a diagnostic virology laboratory for parvovirus DNA detection were analyzed retrospectively. Of the 53 PCR-positive samples investigated, 40 (75.4%) were identified as genotype 1 by genotype-specific PCR or consensus NS1 PCR and sequencing and 3 (5.7%) as genotype 2 and 10 (18.9%) as genotype 3 by analysis of NS1 sequences. Furthermore, phylogenetic analysis identified two genotype 1 sequences which were distinct from the previously described genotypes 1A and 1B. Interestingly, a genotype 2 virus was detected in the serum of an 11-year-old child, providing evidence for its recent circulation. This is the first study to demonstrate the concurrent circulation of all three genotypes of B19 in South Africa and the provisional identification of a novel subtype of genotype 1. The implications of parvovirus B19 variation are discussed.

Cytomegalovirus viraemia in HIV exposed and infected infants: prevalence and clinical utility for diagnosing CMV pneumonia.

BACKGROUND Human cytomegalovirus (HCMV) is an important pathogen in HIV exposed infants with pneumonia. However, the diagnosis of HCMV pneumonia in this setting is challenging due to limited access to bronchoscopy, lung biopsy and direct sampling of the lower respiratory tract. HCMV viraemia is more accessible, but their diagnostic performance in this context has not been studied. OBJECTIVE To describe the prevalence of HCMV viraemia and evaluate its clinical utility in HIV exposed infants. STUDY DESIGN In this cross-sectional study, we performed qualitative and quantitative PCR to detect HCMV viraemia in HIV exposed asymptomatic infants and in infants with severe pneumonia in the Western Cape province of South Africa. RESULTS 283 asymptomatic HIV exposed infants and 142 HIV exposed infants with severe pneumonia were studied. Infants with pneumonia had a higher prevalence of HCMV viraemia compared to asymptomatic infants (68% vs 24% OR 6.7, 95% CI 4.2-10.8). This increased prevalence remained significant (OR 4.3 95% CI 2.6-7.0) after adjusting for HIV infection. Of the infants with pneumonia, the level of HCMV viraemia was significantly higher in a subset of infants diagnosed with HCMV pneumonia (median HCMV viral load 4.6 vs 2.5 log copies/ml p<0.001). Receiver operator characteristic (ROC) analysis showed the area under the curve was 0.78 (95% CI 0.71-0.86) and a threshold of 4.1 log copies/ml was able to correctly identify 70% of HCMV pneumonia cases. CONCLUSION Prevalence and level of HCMV viraemia in sub-Saharan HIV-exposed and infected infants peaks at 3-4 months of age. Quantitative HCMV PCR may be useful in diagnosing HCMV pneumonia.

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.

Silent casualties from the measles outbreak in South Africa

South Africa, home to the worlds largest population of people living with HIV (5.7 million), experienced a measles outbreak that started in late 2009. There was a stepped increase in cases of measles, with the highest incidence reported in March 2010. By September 2010, more than 17 000 new measles cases had been reported to the National Institute of Communicable Diseases since January 2009. A mass vaccination campaign from mid-April to early May 2010 resulted in a significant decline in new measles cases.

A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR.

Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2 min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100 μl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants.

DNA single strand conformation polymorphism identifies five defined strains of hepatitis B virus (HBV) during an outbreak of HBV infection in an oncology unit

An outbreak of hepatitis B virus (HBV) infection in a childrens oncology unit was identified in which 61 children were shown to have been infected, 59 of them asyptomatically. In order to establish whether intra‐unit cross infection had occurred, we used the single strand conformational polymorphism (SSCP) technique to analyse viral isolates from 57 of the infected children and 40 unrelated controls. HBV‐specific primers were designed to amplify a 189 bp fragment of DNA encompassing part of the hypervariable pre‐S1 region of the HBV genome. Denatured PCR products were compared after electrophoresis through polyacrylamide gels and staining with silver. By SSCP analysis, the unrelated infections each yielded a unique electrophoretic banding pattern, indicative of a variety of distinct virus strains. In contrast, most of the oncology patients had been infected with one of only five different strains. Three major groups comprising 19, 16, and 9 patients, respectively, and two minor groups of 5 and 3 patients were identified. Results indicate the occurrence of multiple episodes of cross infection, and demonstrate the sensitivity and value of SSCP as a technique to establish common sources of infection.