Can serological methods help distinguish between prophylactic and alloimmune anti-D?

OBJECTIVES: Enzyme indirect antiglobulin test (EIAT) and polyethylene glycol IAT (PIAT) were evaluated for their potential use as tests to distinguish between prophylactic and alloimmune anti-D in plasma by comparing with a tube variation of the standard low ionic strength solution-IAT (LISS-IAT). BACKGROUND: Laboratories performing the screening of RhD-negative pregnant women are required to provide clinicians with guidance as to the source of detected RhD antibodies. Currently, this is derived from RhIg immunoprophylaxis history, agglutination scores and titration results, where performed. A serological test that can differentiate between prophylactic and alloimmune anti-D would be useful in the diagnosis of RhD alloimmunisation in pregnant women. MATERIALS AND METHODS: Plasma samples (n = 273) [fresh (collected from April 2014 to February 2015) and frozen (up to 2 years)] from antenatal females, preoperative males and females over child-bearing age were used in this study. Samples were identified as containing anti-D by routine column agglutination (CAT) and were tested by tube LISS-IAT, EIAT and PIAT, and a score difference was calculated. RESULTS: A total of 32% of alloimmune anti-D samples demonstrated an increase in agglutination score (+2 or +3) when tested by EIAT. A significant increase in agglutination score for alloimmune samples using EIAT compared with LISS-IAT was observed. EIAT had a sensitivity (Sn) of 59%, positive predictive value (PPV) of 100% and specificity (Sp) of 100% for alloimmune anti-D. CONCLUSION: EIAT is capable of confirming but not excluding the presence of alloimmune anti-D in samples where anti-D is detected in routine antibody screening.

Determining the stability of complete blood count parameters in stored blood samples using the SYSMEX XE-5000 automated haematology analyser.

INTRODUCTION: In this study, changes that occur in various blood parameters as determined by the Sysmex XE-5000 analyser upon storage of blood samples for 72 h were investigated. METHODS: Blood specimens (200) were processed through the SYSMEX XE-5000 haematology analyser within 4 h of collection. Each specimen was distributed into two aliquots and one stored at 4 °C and the other at room temperature (25 ± 1 °C). Both stored aliquots were retested after 24, 48 and 72 h. The mean, mean per cent change and mean absolute difference between the value at 4 h and each time point for all parameters were calculated. RESULTS: Among CBC parameters tested, the white cell count, red cell count and haemoglobin levels were found to be stable for up to 72 h. The mean cell volume and haematocrit changed significantly following 24-h storage at room temperature. Reticulocytes were stable for 72 h under both storage conditions. Among the differential parameters, results of the monocyte count displayed significant change after 24-h storage at room temperature. CONCLUSION: The data presented suggest that clinically reliable results for some CBC parameters can be obtained from specimens that are stored at 4 °C for up to 72 h.

Risk factors for alloimmunisation in the general patient population.

For hospitals providing services to regional populations, difficulties are associated with transferred patients with poorly communicated medical history and a risk of alloimmunisation. Identification of patients at risk would assist in treatment planning. A retrospective study of alloimmunised patients was undertaken, comparing the demographics and diagnoses of this population with a control patient population. A preponderance of diagnoses of Sepsis, Haematological Malignancy, GIT Bleeds and Renal Failure was demonstrated in the alloimmunised population. Consistent with prior studies, RhD negative patients and female patients were over-represented in the study group, which was also on average significantly older.

Rhodococcus equi (Prescottella equi) vaccines; the future of vaccine development.

For decades researchers have been targeting prevention of Rhodococcus equi (Rhodococcus hoagui/Prescottella equi) by vaccination and the horse breeding industry has supported the ongoing efforts by researchers to develop a safe and cost effective vaccine to prevent disease in foals. Traditional vaccines including live, killed and attenuated (physical and chemical) vaccines have proved to be ineffective and more modern molecular-based vaccines including the DNA plasmid, genetically attenuated and subunit vaccines have provided inadequate protection of foals. Newer, bacterial vector vaccines have recently shown promise for R. equi in the mouse model. This article describes the findings of key research in R. equi vaccine development and looks at alternative methods that may potentially be utilised.

Prevalence of equine adenovirus antibodies in horses in New South Wales, Australia.

There are currently two known serotypes of equine adenovirus (EAdV), equine adenovirus type 1 (EAdV1) and equine adenovirus type 2 (EAdV2); EAdV1 is predominantly associated with upper respiratory tract infections while EAdV2 appears to have a higher association with gastrointestinal infection, however, very little is known about the prevalence of these viruses in horse populations in Australia. In this study we tested 122 serum samples obtained from horses in New South Wales, Australia, using a standard serum neutralization (SN) assay and ELISA. Ninety-seven of the 122 sera displayed had moderate to high titers of antibodies to EAdV1 and/or EAdV2. Eighteen of the 122 sera were positive for both EAdV1 and EAdV2. In contrast, only thirty-seven positive samples were detected using the ELISA. These results suggest that EAdV1 and EAdV2 infections are widely prevalent in the horse population tested and that SN is currently the most suitable assay for the detection of EAdV1 and EAdV2 antibodies in equine serum.

Parasite vaccines: the new generation.

Parasites cause some of the most devastating and prevalent diseases in humans and animals. Moreover, parasitic infections increase mortality rates of other serious non-parasitic infections caused by pathogens such as HIV-1. The impact of parasitic diseases in both industrialised and developing countries is further exacerbated by the resistance of some parasites to anti-parasitic drugs and the absence of efficacious parasite vaccines. Despite years of research, much remains to be done to develop effective vaccines against parasites. This review focuses on the more recent vaccine strategies such as DNA and viral vector-based vaccines that are currently being used to develop vaccines against parasites. Obstacles yet to be overcome and possible advantages and disadvantages of these vaccine modalities are also discussed.

DNA immunization using a non-viral promoter.

Most DNA vaccines rely on strong viral promoters to optimize levels of transgene expression. Some studies have demonstrated that the potency of viral promoters does not necessarily correlate with DNA vaccine efficacy in vivo. This has partly been attributed to downregulation of these promoters by cytokines such as interferon gamma induced by the CpG motives of these vaccines. In an attempt to avoid downregulation of viral promoters by IFN-gamma, we tested vaccine vectors driven by the MHC class II promoter. To enhance the activity of this promoter, another plasmid expressing the human MHC class II transactivator driven by a viral promoter, the native IFN-gamma inducible CIITA type IV promoter (PIV) or a synthetic promoter containing IFN-gamma inducible elements was co-inoculated. Our data show that a non-viral promoter such as the MHC class II promoter tested in this study can indeed be used in DNA vaccines.

Adenoviral gene delivery for HIV-1 vaccination.

The AIDS epidemic continues to spread throughout nations of Africa and Asia and is by now threatening to undermine the already frail infrastructure of developing countries in Sub-Saharan Africa that are hit the hardest. The only option to stem this epidemic is through inexpensive and efficacious vaccines that prevent or at least blunt HIV-1 infections. Despite decades of pre-clinical and clinical research such vaccines remain elusive. Most anti-viral vaccines act by inducing protective levels of virus-neutralizing antibodies. The envelope protein of HIV-1, the sole target of neutralizing antibodies, is constantly changing due to mutations, B cell epitopes are masked by heavy glycosylation and the protein's structural unfolding upon binding to its CD4 receptor and chemokine co-receptors. Efforts to induce broadly cross-reactive virus-neutralizing antibodies able to induce sterilizing or near sterilizing immunity to HIV-1 have thus failed. Studies have indicated that cell-mediated immune responses and in particular CD8+ T cell responses to internal viral proteins may control HIV-1 infections without necessarily preventing them. Adenoviral vectors expressing antigens of HIV-1 are eminently suited to stimulate potent CD8+ T cell responses against transgene products, such as antigens of HIV-1. They performed well in pre-clinical studies in rodents and nonhuman primates and are currently in human clinical trials. This review summarizes the published literature on adenoviral vectors as vaccine carriers for HIV-1 and discusses advantages and disadvantages of this vaccine modality.

B-Cell epitope mapping of the VapA protein of Rhodococcus equi: implications for early detection of R. equi disease in foals.

Linear B-cell epitopes of the Rhodococcus equi virulence-associated protein (VapA) were mapped using a synthetic peptide bank in this study. The peptides were screened in an enzyme-linked immunosorbent assay (ELISA) with a total of 70 sera from foals with current R. equi disease (51 sera), as well as from foals that had either recovered from R. equi infection 10 months previously (3 sera) or that had no known history of R. equi disease (16 sera). An epitope with the sequence NLQKDEPNGRA was identified and was universally recognized by all 51 sera from foals with R. equi disease and was not recognized by any of the other sera. There was poor reactivity between all sera and peptides relating to other areas of the VapA protein. It is proposed that an ELISA based upon a defined peptide epitope may be used in an improved serological diagnostic test for R. equi infection in foals.

PCR for the detection of Campylobacter spp. in clinical specimens.

The suitability of PCR (based on the amplification of the 16S rRNA gene) for use as a diagnostic test for the detection of Campylobacter spp. in human faecal specimens was assessed. A total of 493 faecal specimens from patients with symptoms of enteritis were tested for the presence of campylobacters using PCR. Results were compared with those obtained from the analyses of the same specimens by culture techniques, using chi 2 square with Fisher's exact test. PCR was found to detect significantly more positive specimens than culture (chi 2 = 200.086; P < 0.0001). The sensitivity and specificity of PCR when compared with the culture technique were found to be 91 and 97%, respectively. It is proposed that the PCR is a reliable and sensitive method which may be used as a routine diagnostic technique for the detection of campylobacters in clinical specimens.