Integration and validation of host transcript signatures, including a novel 3-transcript tuberculosis signature, to enable one-step multiclass diagnosis of childhood febrile disease.

BACKGROUND: Whole blood host transcript signatures show great potential for diagnosis of infectious and inflammatory illness, with most published signatures performing binary classification tasks. Barriers to clinical implementation include validation studies, and development of strategies that enable simultaneous, multiclass diagnosis of febrile illness based on gene expression. METHODS: We validated five distinct diagnostic signatures for paediatric infectious diseases in parallel using a single NanoString nCounter® experiment. We included a novel 3-transcript signature for childhood tuberculosis, and four published signatures which differentiate bacterial infection, viral infection, or Kawasaki disease from other febrile illnesses. Signature performance was assessed using receiver operating characteristic curve statistics. We also explored conceptual frameworks for multiclass diagnostic signatures, including additional transcripts found to be significantly differentially expressed in previous studies. Relaxed, regularised logistic regression models were used to derive two novel multiclass signatures: a mixed One-vs-All model (MOVA), running multiple binomial models in parallel, and a full-multiclass model. In-sample performance of these models was compared using radar-plots and confusion matrix statistics. RESULTS: Samples from 91 children were included in the study: 23 bacterial infections (DB), 20 viral infections (DV), 14 Kawasaki disease (KD), 18 tuberculosis disease (TB), and 16 healthy controls. The five signatures tested demonstrated cross-platform performance similar to their primary discovery-validation cohorts. The signatures could differentiate: KD from other diseases with area under ROC curve (AUC) of 0.897 [95% confidence interval: 0.822-0.972]; DB from DV with AUC of 0.825 [0.691-0.959] (signature-1) and 0.867 [0.753-0.982] (signature-2); TB from other diseases with AUC of 0.882 [0.787-0.977] (novel signature); TB from healthy children with AUC of 0.910 [0.808-1.000]. Application of signatures outside of their designed context reduced performance. In-sample error rates for the multiclass models were 13.3% for the MOVA model and 0.0% for the full-multiclass model. The MOVA model misclassified DB cases most frequently (18.7%) and TB cases least (2.7%). CONCLUSIONS: Our study demonstrates the feasibility of NanoString technology for cross-platform validation of multiple transcriptomic signatures in parallel. This external cohort validated performance of all five signatures, including a novel sparse TB signature. Two exploratory multi-class models showed high potential accuracy across four distinct diagnostic groups.

Correcting mortality estimates among children and youth on antiretroviral therapy in southern Africa: A comparative analysis between a multi-country tracing study and linkage to a health information exchange.

OBJECTIVES: The objective of this study is to assess the outcomes of children, adolescents and young adults with HIV reported as lost to follow-up, correct mortality estimates for children, adolescents and young adults with HIV for unascertained outcomes in those loss to follow-up (LTFU) based on tracing and linkage data separately using data from the International epidemiology Databases to Evaluate AIDS in Southern Africa. METHODS: We included data from two different populations of children, adolescents and young adults with HIV; (1) clinical data from children, adolescents and young adults with HIV aged ≤24 years from Lesotho, Malawi, Mozambique, Zambia and Zimbabwe; (2) clinical data from children, adolescents and young adults with HIV aged ≤14 years from the Western Cape (WC) in South Africa. Outcomes of patients lost to follow-up were available from (1) a tracing study and (2) linkage to a health information exchange. For both populations, we compared six methods for correcting mortality estimates for all children, adolescents and young adults with HIV. RESULTS: We found substantial variations of mortality estimates among children, adolescents and young adults with HIV reported as lost to follow-up versus those retained in care. Ascertained mortality was higher among lost and traceable children, adolescents and young adults with HIV and lower among lost and linkable than those retained in care (mortality: 13.4% [traced] vs. 12.6% [retained-other Southern Africa countries]; 3.4% [linked] vs. 9.4% [retained-WC]). A high proportion of lost to follow-up children, adolescents and young adults with HIV had self-transferred (21.0% and 47.0%) in the traced and linked samples, respectively. The uncorrected method of non-informative censoring yielded the lowest mortality estimates among all methods for both tracing (6.0%) and linkage (4.0%) approaches at 2 years from ART start. Among corrected methods using ascertained data, multiple imputation, incorporating ascertained data (MI(asc.)) and inverse probability weighting with logistic weights were most robust for the tracing approach. In contrast, for the linkage approach, MI(asc.) was the most robust. CONCLUSIONS: Our findings emphasise that lost to follow-up is non-ignorable and both tracing and linkage improved outcome ascertainment: tracing identified substantial mortality in those reported as lost to follow-up, whereas linkage did not identify out-of-facility deaths, but showed that a large proportion of those reported as lost to follow-up were self-transfers.

Candida bloodstream infection among children hospitalised in three public-sector hospitals in the Metro West region of Cape Town, South Africa.

BACKGROUND: Candida bloodstream infection (BSI) causes appreciable mortality in neonates and children. There are few studies describing the epidemiology of Candida BSI in children living in sub-Saharan Africa. METHODS: A retrospective descriptive study was conducted at three public sector hospitals in Cape Town, South Africa. Demographic and clinical details, antifungal management and patient outcome data were obtained by medical record review. Candida species distribution and antifungal susceptibility testing results were obtained from the National Health Laboratory Service database. RESULTS: Of the 97 Candida BSI episodes identified during a five-year period, 48/97 (49%) were Candida albicans (C. albicans), and 49/97 (51%) were non-C. albicans species. The overall incidence risk was 0.8 Candida BSI episodes per 1000 admissions at Red Cross War Memorial Children's Hospital. Of the 77/97 (79%) Candida BSI episodes with available clinical information, the median age (interquartile range) at the time of BSI was 7 (1-25) months, 36/77 (47%) were associated with moderate or severe underweight-for-age and vasopressor therapy was administered to 22/77 (29%) study participants. Most of the Candida BSI episodes were healthcare-associated infections, 63/77 (82%). Fluconazole resistance was documented among 17%, 0% and 0% of C. parapsilosis, C. tropicalis and C. albicans isolates, respectively. All Candida isolates tested were susceptible to amphotericin B and the echinocandins. The mortality rate within 30 days of Candida BSI diagnosis was 13/75 (17%). On multivariable analysis, factors associated with mortality within 30 days of Candida BSI diagnosis included vasopressor therapy requirement during Candida BSI, adjusted Odds ratio (aOR) 53 (95% confidence interval 2-1029); hepatic dysfunction, aOR 13 (95% CI 1-146); and concomitant bacterial BSI, aOR 10 (95% CI 2-60). CONCLUSION: The study adds to the limited number of studies describing paediatric Candida BSI in sub-Saharan Africa. Non-C. Albicans BSI episodes occurred more frequently than C. albicans episodes, and vasopressor therapy requirement, hepatic dysfunction and concomitant bacterial BSI were associated with an increase in 30-day mortality.

A rare mutation causing autosomal dominant STAT1 deficiency in a South African multiplex kindred with disseminated BCG infection.

BACKGROUND: Autosomal dominant signal transducer and activator of transcription 1 (STAT1) deficiency, part of the Mendelian susceptibility to mycobacterial disease (MSMD) group, frequently causes disseminated Bacillus Calmette-Guérin (BCG) infections, but has not been reported from Sub-Saharan Africa (SSA) where routine birth BCG vaccination is practiced. CASE PRESENTATION: Two half-siblings presented five years apart, with multifocal osteomyelitis as the dominant feature of disseminated BCG, which was successfully treated with antimycobacterial therapy. Whole exome sequencing demonstrated a novel heterozygous substitution in the splice site between intron 13 and exon 14 of the STAT1 gene, NM_007315: c.1128-1G>A, in the proband and his mother and was later confirmed in his half-brother. CONCLUSIONS: Children with BCG vaccine complications in SSA should be referred for further investigation and particular consideration of MSMD.

Viral load testing among pregnant women living with HIV in Mutare district of Manicaland province, Zimbabwe.

BACKGROUND: Viral load (VL) monitoring of pregnant women living with HIV (PWLHIV) and antiretroviral therapy (ART) may contribute to lowering the risk of vertical transmission of HIV. The aims of this study were to assess the uptake of HIV VL testing among PWLHIV at entry to the prevention-of-mother-to-child transmission (PMTCT) services and identify facilitatory factors and barriers to HIV VL access. METHODS: A retrospective, cross-sectional study was conducted at 15 health facilities in Mutare district, Manicaland Province, Zimbabwe from January to December 2018. This analysis was complemented by prospective interviews with PWLHIV and health care providers between October 2019 and March 2020. Quantitative data were analysed using descriptive and inferential statistical methods. Risk factors were evaluated using multivariate logistic regression. Open-ended questions were analysed and recurring and shared experiences and perceptions of PWLHIV and health care providers identified. RESULTS: Among 383 PWLHIV, enrolled in antenatal care (ANC) and receiving ART, only 121 (31.6%) had a VL sample collected and 106 (88%) received their results. Among these 106 women, 93 (87.7%) had a VL < 1000 copies/mL and 77 (73%) a VL < 50 copies/mL. The overall median duration from ANC booking to VL sample collection was 87 (IQR, 7-215) days. The median time interval for the return of VL results from date of sample collection was 14 days (IQR, 7-30). There was no significant difference when this variable was stratified by time of ART initiation. VL samples were significantly less likely to be collected at local authority compared to government facilities (aOR = 0.28; 95% CI 0.16-0.48). Barriers to VL testing included staff shortages, non-availability of consumables and sub-optimal sample transportation. Turnaround time was prolonged by the manual results feedback system. CONCLUSIONS AND RECOMMENDATION: The low rate of HIV VL testing among PWLHIV in Mutare district is a cause for concern. To reverse this situation, the Ministry of Health should consider interventions such as disseminating antiretroviral guidelines and policies electronically, conducting regular PMTCT mentorship for clinical staff members, and utilising point of care testing and telecommunication devices like mHealth to increase uptake of VL testing and improve results turnaround time.

Delivering an integrated sexual reproductive health and rights and HIV programme to high-school adolescents in a resource-constrained setting.

Southern Africa remains the epicentre of the human immunodeficiency virus (HIV) epidemic with AIDS the leading cause of death amongst adolescents. Poor policy translation, inadequate programme implementation and fragmentation of services contribute to adolescents' poor access to sexual and reproductive health and rights (SRHR) services. This study assessed an integrated, school-based SRHR and HIV programme, modelled on the South African Integrated School Health Policy in a rural, high HIV-prevalence district. A retrospective cohort study of 1260 high-school learners was undertaken to assess programme uptake, change in HIV knowledge and behaviour and the determinants of barrier-methods use at last sexual intercourse. Programme uptake increased (2%-89%; P�<�0.001) over a 16-month period, teenage-pregnancy rates declined (14%-3%; P�<�0.050) and accurate knowledge about HIV transmission through infected blood improved (78.3%-93.8%; P�<�0.050), a year later. Post-intervention, attending a clinic perceived as adolescent-friendly increased the odds of barrier-methods use during the last sexual encounter (aOR=1.85; 95% CI: 1.31-2.60), whilst being female (aOR=0.69; 95% CI: 0.48-0.99), <15 years (aOR=0.44; 95% CI: 0.24-0.80), or having >5 sexual partners in the last year (aOR=0.59; 95% CI: 0.38-0.91) reduced the odds. This study shows that the unmet SRHR needs of under-served adolescents can be addressed through integrated, school-based SRHR programmes.

Accelerated Immunodeficiency-associated Vaccine-derived Poliovirus Serotype 3 Sequence Evolution Rate in an 11-week-old Boy With X-linked Agammaglobulinemia and Perinatal Human Immunodeficiency Virus Exposure.

Primary B-cell immunodeficiencies are risk factors for the generation of vaccine-derived polioviruses. We report immunodeficiency-associated vaccine-derived poliovirus serotype 3 in an 11-week-old boy with X-linked agammaglobulinemia. Unique characteristics of this case include early age of presentation, high viral evolutionary rate, and the child's perinatal exposure to human immunodeficiency virus.

Analysis of a Subacute Sclerosing Panencephalitis Genotype B3 Virus from the 2009-2010 South African Measles Epidemic Shows That Hyperfusogenic F Proteins Contribute to Measles Virus Infection in the Brain.

During a measles virus (MeV) epidemic in 2009 in South Africa, measles inclusion body encephalitis (MIBE) was identified in several HIV-infected patients. Years later, children are presenting with subacute sclerosing panencephalitis (SSPE). To investigate the features of established MeV neuronal infections, viral sequences were analyzed from brain tissue samples of a single SSPE case and compared with MIBE sequences previously obtained from patients infected during the same epidemic. Both the SSPE and the MIBE viruses had amino acid substitutions in the ectodomain of the F protein that confer enhanced fusion properties. Functional analysis of the fusion complexes confirmed that both MIBE and SSPE F protein mutations promoted fusion with less dependence on interaction by the viral receptor-binding protein with known MeV receptors. While the SSPE F required the presence of a homotypic attachment protein, MeV H, in order to fuse, MIBE F did not. Both F proteins had decreased thermal stability compared to that of the corresponding wild-type F protein. Finally, recombinant viruses expressing MIBE or SSPE fusion complexes spread in the absence of known MeV receptors, with MIBE F-bearing viruses causing large syncytia in these cells. Our results suggest that alterations to the MeV fusion complex that promote fusion and cell-to-cell spread in the absence of known MeV receptors is a key property for infection of the brain.IMPORTANCE Measles virus can invade the central nervous system (CNS) and cause severe neurological complications, such as MIBE and SSPE. However, mechanisms by which MeV enters the CNS and triggers the disease remain unclear. We analyzed viruses from brain tissue of individuals with MIBE or SSPE, infected during the same epidemic, after the onset of neurological disease. Our findings indicate that the emergence of hyperfusogenic MeV F proteins is associated with infection of the brain. We also demonstrate that hyperfusogenic F proteins permit MeV to enter cells and spread without the need to engage nectin-4 or CD150, known receptors for MeV that are not present on neural cells.

Pseudomonas aeruginosa bloodstream infection at a tertiary referral hospital for children.

BACKGROUND: This study describes the disease burden, clinical characteristics, antibiotic management, impact of multidrug resistance and outcome of Pseudomonas aeruginosa bloodstream infection (PABSI) among children admitted to a tertiary referral hospital for children in Cape Town, South Africa. METHODS: A retrospective descriptive study was conducted at a paediatric referral hospital in Cape Town, South Africa. Demographic and clinical details, antibiotic management and patient outcome information were extracted from medical and laboratory records. Antibiotic susceptibility results of identified organisms were obtained from the National Health Laboratory Service database. RESULTS: The incidence risk of PABSI was 5.4 (95% CI: 4.34-6.54) PABSI episodes / 10,000 hospital admissions and the most common presenting feature was respiratory distress, 34/91 (37.4%). Overall, 69/91 (75.8%) of the PA isolates were susceptible to all antipseudomonal antibiotic classes evaluated. Fifty (54.9%) of the PABSI episodes were treated with appropriate empiric antibiotic therapy. The mortality rate was 24.2% and in multivariable analysis, empiric antibiotic therapy to which PA isolates were not susceptible, infections present on admission, and not being in the intensive care unit at the time that PABSI was diagnosed were significantly associated with 14-day mortality. CONCLUSIONS: PABSI caused appreciable mortality, however, appropriate empiric antibiotic therapy was associated with reduced 14-day mortality.

Stroke transcranial Doppler in children with human immunodeficiency virus.

AIMS: To describe stroke syndromes and transcranial Doppler (TCD) findings in children with human immunodeficiency virus (HIV) and examine the associations between TCD and clinical and laboratory data. METHOD: We enrolled 42 children (24 males, 18 females) with HIV (median age=7y 6mo; 2y 7mo-15y 6mo), with and without stroke who underwent a TCD examination of the anterior and posterior circulations to derive time-averaged maximum mean velocity (TAMMV) measurements for comparison with previous studies. Clinical and laboratory variables were extracted from the medical records. RESULTS: Of the 42 children with HIV, five had right-sided hemiparesis, three had chronic lung disease, two occurred post-varicella infection, one after herpetic oral ulceration, and one had a poorly functioning left ventricle. Neuroimaging showed middle cerebral artery (MCA) TAMMV greater than 200cm/s, moyamoya-like arteriopathy, left basal ganglia infarction with ipsilateral stenosis, hygroma consistent with venous thrombosis, and a hyperdense left MCA. Eight neurologically asymptomatic children had atypical TCD. The CD4 cell count was non-significantly lower in 6 out of 30 children with atypical TCD (median=21.5; interquartile range=16.1-26.5) compared with the remainder (median=29; interquartile range=21.3-35.0; p=0.09). INTERPRETATION: A variety of stroke syndromes occur in children with HIV. TCD suggests atypical intracranial vessels and/or haemodynamics in some children with HIV infection, consistent with vasculopathy, possibly related directly to immunodeficiency and/or infection. WHAT THIS PAPER ADDS: A range of stroke syndromes are found in children with human immunodeficiency virus (HIV). Transcranial Doppler (TCD) velocities in HIV are commonly outside the range for typically developing children. TCD and neuroimaging data in children with HIV suggest intracranial vasculopathy as one mechanism for stroke. CD4 cell count is non-significantly lower in children with HIV and atypical TCD.