The implementation and utility of clinical exome sequencing in a South African infant cohort.

Genetic disorders are significant contributors to infant hospitalization and mortality globally. The early diagnosis of these conditions in infants remains a considerable challenge. Clinical exome sequencing (CES) has shown to be a successful tool for the early diagnosis of genetic conditions, however, its utility in African infant populations has not been investigated. The impact of the under-representation of African genomic data, the cost of testing, and genomic workforce shortages, need to be investigated and evidence-based implementation strategies accounting for locally available genetics expertise and diagnostic infrastructure need to be developed. We evaluated the diagnostic utility of singleton CES in a cohort of 32 ill, South African infants from two State hospitals in Johannesburg, South Africa. We analysed the data using a series of filtering approaches, including a curated virtual gene panel consisting of genes implicated in neonatal-and early childhood-onset conditions and genes with known founder and common variants in African populations. We reported a diagnostic yield of 22% and identified seven pathogenic variants in the NPHS1, COL2A1, OCRL, SHOC2, TPRV4, MTM1 and STAC3 genes. This study demonstrates the utility value of CES in the South African State healthcare setting, providing a diagnosis to patients who would otherwise not receive one and allowing for directed management. We anticipate an increase in the diagnostic yield of our workflow with further refinement of the study inclusion criteria. This study highlights important considerations for the implementation of genomic medicine in under-resourced settings and in under-represented African populations where variant interpretation remains a challenge.

HIV-associated neurodevelopmental delay: prevalence, predictors and persistence in relation to antiretroviral therapy initiation and viral suppression.

CONTEXT: HIV infection in infancy may influence the developing brain, leading to adverse neurodevelopmental consequences. OBJECTIVE: We aim to describe neurodevelopmental characteristics of a cohort of HIV-infected infants and young children prior to antiretroviral therapy (ART) initiation and after achieving viral suppression. METHODS: As part of the Neverest 2 trial, 195 HIV-infected children under 2 years of age were assessed using the Ages and Stages Questionnaire (ASQ) prior to ART initiation and at subsequent age-appropriate time points after ART had been started. The ASQ is a simple screening questionnaire used to identify children at risk of neurodevelopmental delays. Questionnaires completed by the parent/caregiver assess neurodevelopmental functioning in five domains: communication, gross motor, fine motor, problem solving and personal-social. RESULTS: Median age pre-ART was 8.8 months (range 2.2-24.9) and 53.9% were male. Mean time to viral suppression was 9.4 months (range 5.9-14.5). Compared with pre-ART better outcomes were reported at time of viral suppression with a lower proportion of children failing the gross motor (31.5% vs. 13%, p = 0.0002), fine motor (21.3% vs. 10.2%, p = 0.017), problem solving (26.9% vs. 9.3%, p = 0.0003) and personal-social (19.6% vs. 7.4%, p = 0.019) domains. However, there was no change in the communication domain (14.8% vs. 12.0%, p = 0.6072). CONCLUSION: Although achieving viral suppression on ART resulted in significant improvements in markers of neurodevelopmental function of young HIV-infected children, potential neurodevelopmental delays still persisted in a large proportion. Further interventions are needed to limit potential disabilities and maximize developmental outcomes.

Influence of intragenic CCL3 haplotypes and CCL3L copy number in HIV-1 infection in a sub-Saharan African population.

Two CCL3 haplotypes (HapA1 and Hap-A3) and two polymorphic positions shared by the haplotypes (Hap-2SNP (single nucleotide polymorphism)) were investigated together with CCL3L copy number (CN), for their role in HIV-1 disease. Hap-A1 was associated with protection from in utero HIV-1 infection: exposed uninfected (EU) infants had higher representation of wild type (WT)/Hap-A1 than infected infants (excluding intrapartum (IP)-infected infants), which maintained significance post maternal Nevirapine (mNVP) and viral load (MVL) correction (P=0.04; odds ratio (OR)=0.33). Mother-infant pair analyses showed the protective effect of Hap-A1 is dependent on its presence in the infant. Hap-A3 was associated with increased IP transmission: WT/Hap-A3 was increased in IP-transmitting vs non-transmitting (NT) mothers, and remained significant post mNVP and MVL correction (P=0.02; OR=3.50). This deleterious effect of Hap-A3 seemed dependent on its presence in the mother. Hap-2SNP was associated with lower CD4 count in the NT mothers (P=0.03). CCL3 Hap-A1 was associated with high CCL3L CN in total (P=0.001) and EU infants (P=0.006); the effect was not additive, however, having either Hap-A1 or high CCL3L CN was more significantly (P=0.0008) associated with protection from in utero infection than Hap-A1 (P=0.028) or high CCL3L CN (P=0.002) alone. Linkage disequilibrium between Hap-A1 and high CCL3L CN appears unlikely given that a Nigerian population showed an opposite relationship.

HAART and risk of tuberculosis in HIV-infected South African children: a multi-site retrospective cohort.

SETTING: Four human immunodeficiency virus (HIV) clinics located at South African tertiary hospitals. OBJECTIVE: To assess the effectiveness of highly active antiretroviral therapy (HAART) in reducing incident tuberculosis (TB) in HIV-infected children. DESIGN: Retrospective cohort. RESULTS: A total of 1132 children's records were included in the study. At entry to the cohort, the median (interquartile range [IQR]) age, CD4%, CD4 count and viral load of all children was respectively 6.3 years (4.1-8.8), 15% (9.0-22.2), 576 cells/mm(3) (287-960) and 160 000 copies/ml (54 941.5-449 683); 75.9% were started on HAART. The male:female ratio was 1:1, and median follow-up time was 1.7 years. In children whose follow-up included both pre-HAART and on-HAART periods, the incidence of clinically diagnosed TB was respectively 21.1 per 100 person-years (py; 95%CI 18.2-24.4) and 6.4/100 py (95%CI 4.8-8.1), and when restricted to confirmed cases, respectively 3.1/100 py (95%CI 2.2-4.2) and 0.8/100 py (95%CI 0.5-1.4). Only 23% of all cases of TB were microbiologically confirmed. Multivariate analyses showed that HAART reduced incident TB by approximately 70%, both for confirmed and all TB cases. CONCLUSIONS: In this high TB burden country, the incidence of diagnosis of TB in HIV-infected children is at least as high as that of adults. HAART reduces incident TB, but further prospective TB preventive and diagnostic studies are urgently needed in children.

PMTCT from research to reality--results from a routine service.

OBJECTIVES: Assessment of the efficacy of a prevention of mother-to-child transmission (PMTCT) programme in a routine service setting in comparison to a research environment. DESIGN: Descriptive study over a 13-month period utilising retrospective data obtained from hospital records complemented by prospective data on a sample of patients enrolled in a study to determine an affordable HIV diagnostic protocol for infants. SETTING: Routine PMTCT service at Coronation Women and Children's Hospital (CWCH) situated in Johannesburg and affiliated to the University of the Witwatersrand. SUBJECTS: Pregnant women known to be HIV infected who delivered at CWCH from 1 October 2001 to 31 October 2002. OUTCOME MEASURES: The HIV transmission rate to infants, which reflects nevirapine (NVP) delivery and infant feeding practices, and follow-up rates of perinatally exposed children. RESULTS: Of the 8,221 deliveries, 1,234 (15%) occurred in women known to be HIV infected. HIV transmission rates of 8.7% at 6 weeks and 8.9% at 3 months of age in the study population verifies the high rate of NVP administration and the ability of women to formula-feed their babies and abstain from breast-feeding. More than one-third of infants never return for follow-up and more than 70% are lost to follow-up by 4 months of age. CONCLUSIONS: The low HIV transmission rate confirms the efficacy of this routine service PMTCT programme. HIV-infected children are not being identified for medical management as part of PMTCT follow-up. It is imperative that record keeping is improved to facilitate ongoing monitoring.

Prolonged in vitro hemolysis of EDTA-anticoagulated blood after a delayed hemolytic transfusion reaction.

BACKGROUND: A novel case where in vitro hemolysis was observed in plasma, but not in serum samples, obtained after the onset of a severe delayed hemolytic transfusion reaction is presented. CASE REPORT: A 54-year-old woman received 2 units of blood during an orthopedic procedure. She had received transfusion 30 years earlier, and testing before transfusion revealed no alloantibodies. The patient returned 12 days after the transfusion with a Hb level of 54 g per L due to a severe delayed hemolytic reaction caused by anti-K. The plasma and serum samples were grossly hemolysed on Day 12. On Day 14, the serum samples showed no evidence of hemolysis; however, the EDTA sample remained grossly hemolysed. This discrepancy was not identified until Day 19. Due to concerns of ongoing apparent severe hemolysis, the patient was unnecessarily treated with IVIG and corticosteroids. The in vitro hemolysis was still present at 75 days, despite complete normalization of her Hb, bilirubin, and LDH levels. The phenomenon had resolved by 125 days. CONCLUSION: This in vitro artifact has not been previously reported and the mechanism remains unclear. Both plasma and serum samples should be observed for hemolysis when evaluating a patient with a severe delayed hemolytic transfusion reaction.

Reporting of near-miss events for transfusion medicine: improving transfusion safety.

BACKGROUND: Half of the reported serious adverse events from transfusion are a consequence of medical error. A no-fault medical-event reporting system for transfusion medicine (MERS-TM) was developed to capture and analyze both near-miss and actual transfusion-related errors. STUDY DESIGN AND METHODS: A prospective audit of transfusion-related errors was performed to determine the ability of MERS-TM to identify the frequency and patterns of errors. RESULTS: Events and near-miss events (total, 819) were recorded for a period of 19 months (median, 51/month). No serious adverse patient outcome occurred, despite these events, with the transfusion of 17,465 units of RBCs. Sixty-one events (7.4%) were potentially life-threatening or could have led to permanent injury (severity Level 1). Of most concern were 3 samples collected from the wrong patient, 13 mislabeled samples, and 22 requests for blood for the wrong patient. Near-miss events were five times more frequent than actual transfusion errors, and 68 percent of errors were detected before blood was issued. Sixty-one percent of events originated from patient areas, 35 percent from the blood bank, and 4 percent from the blood supplier or other hospitals. Repeat collection was required for 1 of every 94 samples, and 1 in 346 requests for blood components was incorrect. Education of nurses and alterations to blood bank forms were not by themselves effective in reducing severe errors. An artifactual 50-percent reduction in the number of errors reported was noted during a 6-month period when two chief members of the event-reporting team were on temporary leave. CONCLUSION: The MERS-TM allowed the recognition and analysis of errors, determination of patterns of errors, and monitoring for changes in frequency after corrective action was implemented. Although no permanent injury resulted from the 819 events, innovative mechanisms must be designed to prevent these errors, instead of relying on faulty informal checks to capture errors after they occur.

An evaluation of the process and costs associated with targeted lookbacks for HCV and general notification of transfusion recipients.

BACKGROUND: The Commission of Inquiry on the Blood System in Canada recommended that hospitals notify patients who received blood between 1978 and May 1990 of the risks of contracting HIV (up to the end of 1985 only) and HCV infection. The commission also recommended that patients should be informed of any transfusion received. STUDY DESIGN AND METHOD: General notifications for HIV and HCV for this period were begun in mid-1994. Notification after discharge of transfusions received after May 1990 was begun in 1997. Targeted HCV lookback was performed from 1995 to 1999. RESULTS: Of 21,016 transfusion recipients from January 1978 to May 1990 identified in the general look-back process and believed still alive, 13,549 (64%) were presumed contacted, by registered mail. The overall contact rate for the ongoing notifications (transfusions after May 1990) cannot be accurately determined, as registered mail was not used and a reply not requested. The total cost for these two processes was CAN$373,481, or $13 per patient believed contacted. Most (56%) of this cost was for the conversion to electronic form of paper transfusion records for the period 1978 through early 1984. In the targeted HCV lookback program 1995 through 1999, 94 percent of 256 recipients of specific components identified as likely to have transmitted HCV either were contacted or had died. Of 84 living recipients, 47 (56%) are HCV positive. The last documented potential seroconversion occurred after a transfusion in November 1991, during the period of first-generation EIA testing. If the targeted HCV lookback had been restricted to transfusions after 1987, as the FDA recommended, we would have failed to identify 39 living patients, of whom 21 are HCV positive. The cost per HCV-positive patient notified in the targeted HCV lookback was CAN $4,174. CONCLUSION: The cost of compliance with the com-mission's recommendations was CAN$569,636. Over 28,000 of 36,773 transfusion recipients were notified or presumed notified, and 272 targeted HCV lookbacks to 256 recipients were performed. Performance of this task required the existence of transfusion records back to 1978, conversion of paper records to electronic form, and adequate secretarial and financial support.

A gel technology system to determine postpartum RhIG dosage.

Failures of Rh immune globulin (RhIG) prophylaxis occur when the dose is too small. We report a test using a gel technology (GT) method to replace the Kleihauer-Betke (K-B) test to assess fetomaternal hemorrhage (FMH) and assist in determining the minimum necessary dose of RhIG. Cord blood (O, D+) was mixed with adult blood (O D-) to mimic an FMH of 10 mL, 20 mL, 28 mL, and 40 mL. Test samples were incubated with anti-D at known concentrations and centrifuged. The supernatant was titrated against D+ and D- red cells using GT and an interpretation of the required RhIG dose was made. Results were compared with the K-B test. Results were easily discernible and interpretations leading to determination of recommended RhIG dosage were reproducible. Correlation to standard K-B testing was confirmed. Elapsed time for result availability by GT testing was 60 minutes, with a direct technical time requirement of 30 minutes. The GT system is easier, objective, and quantitative, and compares well to the standard K-B test. A single procedure will allow assessment of the extent of FMH in the great majority of cases. This technique works well in determining the appropriate dose of anti-D required to treat D- patients with D+ newborns. There are potential cost savings in decreased use of RhIG, less direct technical time required, and more rapid availability of results.

Changes in red-cell transfusion practice in a tertiary care hospital during the 1990s--a 7-year study.

Use of red cells for transfusion in a tertiary care hospital has been studied over a 7-year period from 1990-1991 to 1996-1997. In this time, red-cell use has declined by 18% while new patients or admissions to programmes in oncology, trauma or cardiac bypass surgery have increased by 57%, 66% and 73%, respectively. This reduction in red-cell transfusion has been achieved by a combination of less patients (proportionately) receiving red cells and less red cells being transfused to individual recipients. When the trends are analysed for red-cell use in four elective surgical procedures there is a significant reduction in both the proportion of patients transfused and the mean number of units used per patient undergoing the procedure. Autologous presurgical blood deposit met about 45% of the blood requirement for those four procedures. A similar decreasing trend in units per patient and proportion of patients transfused red cells was seen for 'first-time' coronary artery bypass surgery. The question arises as to how far this trend may go before adverse effects of undertransfusion become apparent.

Assessment of an instrument for automated reagent and specimen dispensing in blood banking.

We have evaluated a semi-automated computer controlled dispensing device in conjunction with microtube technology for the performance of routine blood grouping and antibody screening procedures. A total of 787 specimens have been tested, 78 with unexpected antibodies previously identified by manual microtube methods. All were tested in duplicate in different order; there was complete agreement in blood group (ABO, RhD) determinations and the unexpected antibodies were found in the appropriate microtube location in all duplicate sets of results. We conclude that the device accurately and reproducibly dispenses cells, sera and reagents. The savings of technologists' time over traditional manual tube methods exceed 75%, excluding the time to perform elements common to both methods. The combination of automation and microtube technology offers the opportunity for substantial savings in technologist time with accuracy in reagent and specimen dispensing.

Transfusion practice in support of surgery during introduction of a hospital-based autologous presurgical blood donor program.

OBJECTIVES: To assess blood use in support of elective surgery during the introduction of a hospital-based presurgical autologous blood donor program and to identify changes in transfusion practice. DESIGN: Case series over a 3-year period. SETTING: A tertiary-care, university-affiliated hospital. PATIENTS: All patients (887) who underwent, electively, one of four major surgical procedures between Apr. 1, 1990, and Mar. 31, 1993, during introduction of the hospital's autologous blood transfusion program. The criteria for donation were wide. INTERVENTIONS: Hip and knee arthroplasty, radical hysterectomy and radical prostatectomy. MAIN OUTCOME MEASURES: Increase or decrease in the use of autologous or allogeneic blood for transfusion for the four surgical procedures over the study period. RESULTS: For hip arthroplasty, the use of blood decreased significantly overall. The use of blood in support of radical hysterectomy and prostatectomy decreased but not significantly. In knee arthroplasty, blood use increased for reasons still under investigation. The contribution of autologous blood for the four procedures increased over the 3 years of the study from 17% to 55%, and constituted 3.8% of red cell and whole blood transfusions. Avoidance of allogeneic transfusion in the 3rd year of the study was 64% for patients who underwent hip arthroplasty, 71% for those who underwent radical prostatectomy and 77% for those who underwent knee arthroplasty and radical hysterectomy. CONCLUSIONS: Hospital-based autologous blood collection with wide eligibility criteria can contribute significantly to the availability of blood for elective surgery and can prevent allogeneic blood exposure in about 75% of patients who undergo, electively, one of four common procedures. Compared with other centres, there is room for further reduction in allogeneic blood exposure.