Recent advances in the understanding of endothelial barrier function and fluid therapy.

Elucidation of the structural basis of endothelial barrier function and the study of transcapillary fluid exchange dynamics are areas of active research. There has been significant enhancement in our understanding of the ultrastructural basis of endothelial barrier function. The role of glycocalyx has received special attention. Experimental evidence has called for a revision in the classic Starling principle of transcapillary exchange. The glycocalyx model provides a potential structural mechanism for the revised Starling principle. This knowledge can provide the framework for understanding the volume expansion effect of fluid therapy and the physiological basis of fluid therapy.

Circulating antibodies against Plasmodium falciparum histidine-rich proteins 2 interfere with antigen detection by rapid diagnostic tests.

BACKGROUND: Rapid diagnostic tests (RDTs) for detection of Plasmodium falciparum infection that target P. falciparum histidine-rich protein 2 (PfHRP2), a protein that circulates in the blood of patients infected with this species of malaria, are widely used to guide case management. Understanding determinants of PfHRP2 availability in circulation is therefore essential to understanding the performance of PfHRP2-detecting RDTs. METHODS: The possibility that pre-formed host anti-PfHRP2 antibodies may block target antigen detection, thereby causing false negative test results was investigated in this study. RESULTS: Anti-PfHRP2 antibodies were detected in 19/75 (25%) of plasma samples collected from patients with acute malaria from Cambodia, Nigeria and the Philippines, as well as in 3/28 (10.7%) asymptomatic Solomon Islands residents. Pre-incubation of plasma samples from subjects with high-titre anti-PfHRP2 antibodies with soluble PfHRP2 blocked the detection of the target antigen on two of the three brands of RDTs tested, leading to false negative results. Pre-incubation of the plasma with intact parasitized erythrocytes resulted in a reduction of band intensity at the highest parasite density, and a reduction of lower detection threshold by ten-fold on all three brands of RDTs tested. CONCLUSIONS: These observations indicate possible reduced sensitivity for diagnosis of P. falciparum malaria using PfHRP2-detecting RDTs among people with high levels of specific antibodies and low density infection, as well as possible interference with tests configured to detect soluble PfHRP2 in saliva or urine samples. Further investigations are required to assess the impact of pre-formed anti-PfHRP2 antibodies on RDT performance in different transmission settings.

Bullous pemphigoid associated with dipeptidyl peptidase IV inhibitors. A case report and review of literature.

BACKGROUND: Bullous pemphigoid is a cutaneous autoimmune blistering disorder. The etiology for what precipitates this disease is not entirely clear at this point, although it has been associated with certain medications. MAIN OBSERVATION: We describe the case of a 70-year-old male with a past medical history of diabetes type 2 who developed a diffuse eruption of bullae with skin biopsy positive for bullous pemphigoid. He had previously been prescribed sitagliptin 50 mg daily for at least one year prior to onset of his disease. The medication was discontinued and the patient was treated with first IV and then oral steroids with good clinical outcome. There have been a few reports that have explored the relationship between DPP-IV inhibitors (gliptins) and bullous pemphigoid, including three case series and a report on sitagliptin associated allergic skin reactions submitted to the Adverse Event Reports System database of the FDA. According to the Naranjo ADR probability score there is a "possible" cause and effect relationship for this case. CONCLUSION: The enzyme DPP-IV is ubiquitously expressed in almost every organ system, including the skin. The exact mechanism at this time is unknown but is believed to be multifactorial involving many aspects of the immune system. Our case and the findings from our literature review further demonstrate a link between dipeptidyl peptidase-IV inhibitors and the development of bullous pemphigoid.

Patient satisfaction with clinical nurse specialists' practice.

A co-ordinated approach was adopted to monitor practice standards among clinical nurse specialists (CNSs) in West Midlands breast screening assessment nursing teams. A regional working party was assembled and a patient satisfaction survey was produced. Results of the survey show that for women attending for breast screening assessment, interaction with a CNS is perceived to be highly beneficial. Contact points vary with local practice, but the majority of women had contact with a CNS at some point during the assessment process for support and information. The results of the survey are used as an integral part of the quality assurance process in relation to CNS provision in the West Midlands. Services should aim to ensure that sufficient CNSs are available for women attending breast screening assessment centres.

Structural pituitary abnormalities associated with CHARGE syndrome.

INTRODUCTION: CHARGE syndrome is a multisystem disorder that, in addition to Kallmann syndrome/isolated hypogonadotrophic hypogonadism, has been associated with anterior pituitary hypoplasia (APH). However, structural abnormalities such as an ectopic posterior pituitary (EPP) have not yet been described in such patients. OBJECTIVE: The aims of the study were: 1) to describe the association between CHARGE syndrome and a structurally abnormal pituitary gland; and 2) to investigate whether CHD7 variants, which are identified in 65% of CHARGE patients, are common in septo-optic dysplasia /hypopituitarism. METHODS: We describe 2 patients with features of CHARGE and EPP. CHD7 was sequenced in these and other patients with septo-optic dysplasia/hypopituitarism. RESULTS: EPP, APH, and GH, TSH, and probable LH/FSH deficiency were present in 1 patient, and EPP and APH with GH, TSH, LH/FSH, and ACTH deficiency were present in another patient, both of whom had features of CHARGE syndrome. Both had variations in CHD7 that were novel and undetected in control cohorts or in the international database of CHARGE patients, but were also present in their unaffected mothers. No CHD7 variants were detected in the patients with septo-optic dysplasia/hypopituitarism without additional CHARGE features. CONCLUSION: We report a novel association between CHARGE syndrome and structural abnormalities of the pituitary gland in 2 patients with variations in CHD7 that are of unknown significance. However, CHD7 mutations are an uncommon cause of septo-optic dysplasia or hypopituitarism. Our data suggest the need for evaluation of pituitary function/anatomy in patients with CHARGE syndrome.

Perceptions of barriers to discussing and testing for sexually transmitted infections in a convenience sample of general practice patients.

We aimed to identify patient perceptions of barriers to discussing sexually transmitted infections (STIs) at the primary care level. An anonymous questionnaire was available to patients (16-70 years) in the waiting room of four metropolitan Perth general practices. Results are based on 370 participant views (9.5% of the potential target population). Patients felt comfortable discussing STIs with their general practitioner (GP) and their level of comfort would be enhanced if they knew their GP had a special interest or qualification in sexual health. Willingness to discuss issues increased or remained unchanged if the GP took time to explain it to them or was a good listener. Patients were willing to discuss STIs if they were a new patient and irrespective of the GP's gender and age. Fewer patients were willing to discuss STIs if they knew the GP socially. Patients who had sex with a new partner were willing to request a STI test from their GP. Patients were not embarrassed if discussion was initiated in a consultation unrelated to sexual health and did not mind discussing the topic in the presence of a partner or parent, though this depended on circumstances. Waiting room STI test advertising did not affect patient comfort level. Patients would involve their GP when seeking information about STIs. Patients have fewer barriers to discussing sexual health matters than perceived by GPs.

Laboratory demonstration of a prozone-like effect in HRP2-detecting malaria rapid diagnostic tests: implications for clinical management.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) are now widely used for prompt on-site diagnosis in remote endemic areas where reliable microscopy is absent. Aberrant results, whereby negative test results occur at high parasite densities, have been variously reported for over a decade and have led to questions regarding the reliability of the tests in clinical use. METHODS: In the first trial, serial dilutions of recombinant HRP2 antigen were tested on an HRP2-detectiing RDT. In a second trial, serial dilutions of culture-derived Plasmodium falciparum parasites were tested against three HRP2-detecting RDTs. RESULTS: A prozone-like effect occurred in RDTs at a high concentration of the target antigen, histidine-rich protein-2 (above 15,000 ng/ml), a level that corresponds to more than 312000 parasites per µL. Similar results were noted on three RDT products using dilutions of cultured parasites up to a parasite density of 25%. While reduced line intensity was observed, no false negative results occurred. CONCLUSIONS: These results suggest that false-negative malaria RDT results will rarely occur due to a prozone-like effect in high-density infections, and other causes are more likely. However, RDT line intensity is poorly indicative of parasite density in high-density infections and RDTs should, therefore, not be considered quantitative. Immediate management of suspected severe malaria should rely on clinical assessment or microscopy. Evaluation against high concentrations of antigen should be considered in malaria RDT product development and lot-release testing, to ensure that very weak or false negative results will not occur at antigen concentrations that might be seen clinically.

Evaluation of the safety and tolerability of a short higher-dose primaquine regimen for presumptive anti-relapse therapy in healthy subjects.

The safety and tolerability of primaquine (PQ) administered as a short higher-dose (30mg twice daily for 7 days) regimen in 203 Australian Defence Force personnel was evaluated in an open-label presumptive anti-relapse therapy study. No clinically significant differences were measured in the subjects' haematological and biochemical indices before and after PQ treatment. The most common adverse events were nausea, abdominal pain, headache and insomnia, many of which were mild in severity (30%; 60/203) and transient; 19% of subjects (39/203) experienced moderate (with some interference with daily duties requiring no or minimal medical therapy) adverse events. Two subjects (1%) had severe gastrointestinal adverse events requiring cessation of medication, but neither was seriously ill. Ten subjects (5%) had peripheral cyanosis (blueness of the lips), but none reported any respiratory compromise. These findings suggest that the short higher-dose PQ regimen is safe and well tolerated, which could improve PQ compliance and effectiveness.

Transcription and expression of Plasmodium falciparum histidine-rich proteins in different stages and strains: implications for rapid diagnostic tests.

BACKGROUND: Although rapid diagnostic tests (RDTs) for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates. METHODS: Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle. RESULTS: Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed. CONCLUSIONS: The levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs.

Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests.

BACKGROUND: Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. METHODS: The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. RESULTS: Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. CONCLUSIONS: The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation.

Assessing the genetic diversity of the aldolase genes of Plasmodium falciparum and Plasmodium vivax and its potential effect on performance of aldolase-detecting rapid diagnostic tests.

Malaria-specific rapid diagnostic tests (RDTs) targeting aldolase show highly variable sensitivities. We assessed diversity in Plasmodium falciparum and P. vivax aldolases by sequencing the coding genes from parasites of various origins. The results show that aldolases are highly conserved, indicating that antigenic diversity is not a cause of variable RDT sensitivity.

Effect of sequence variation in Plasmodium falciparum histidine- rich protein 2 on binding of specific monoclonal antibodies: Implications for rapid diagnostic tests for malaria.

The ability to accurately diagnose malaria infections, particularly in settings where laboratory facilities are not well developed, is of key importance in the control of this disease. Rapid diagnostic tests (RDTs) offer great potential to address this need. Reports of significant variation in the field performance of RDTs based on the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) (PfHRP2) and of significant sequence polymorphism in PfHRP2 led us to evaluate the binding of four HRP2-specific monoclonal antibodies (MABs) to parasite proteins from geographically distinct P. falciparum isolates, define the epitopes recognized by these MABs, and relate the copy number of the epitopes to MAB reactivity. We observed a significant difference in the reactivity of the same MAB to different isolates and between different MABs tested with single isolates. When the target epitopes of three of the MABs were determined and mapped onto the peptide sequences of the field isolates, significant variability in the frequency of these epitopes was observed. These findings support the role of sequence variation as an explanation for variations in the performance of HRP2-based RDTs and point toward possible approaches to improve their diagnostic sensitivities.

Efficacy of sulfadoxine-pyrimethamine in the treatment of uncomplicated Plasmodium falciparum malaria in East Timor.

The efficacy of sulfadoxine-pyrimethamine (SP) in East Timor is unknown. We treated 38 individuals with uncomplicated Plasmodium falciparum malaria with SP and monitored the outcome for 28 days. Recrudescent parasitemia, confirmed by genotyping, were detected in three individuals resulting in a late treatment failure rate of 7.9% (95% confidence interval = 1.7-21.4%). The results suggest that SP is still efficacious in treating uncomplicated P. falciparum malaria in East Timor. However, the useful life of SP in East Timor may be limited because 80% of the parasites in our samples were found to already carry double mutations in P. falciparum dihydrofolate reductase (S108N/C59R). The data from this study also highlights that the presence of gametocytes may significantly influence the estimate of SP efficacy determined by genotyping.