Thick blood film examination for Plasmodium falciparum malaria has reduced sensitivity and underestimates parasite density.

BACKGROUND: Thick blood films are routinely used to diagnose Plasmodium falciparum malaria. Here, they were used to diagnose volunteers exposed to experimental malaria challenge. METHODS: The frequency with which blood films were positive at given parasite densities measured by PCR were analysed. The poisson distribution was used to calculate the theoretical likelihood of diagnosis. Further in vitro studies used serial dilutions to prepare thick films from malaria cultures at known parasitaemia. RESULTS: Even in expert hands, thick blood films were considerably less sensitive than might have been expected from the parasite numbers measured by quantitative PCR. In vitro work showed that thick films prepared from malaria cultures at known parasitaemia consistently underestimated parasite densities. CONCLUSION: It appears large numbers of parasites are lost during staining. This limits their sensitivity, and leads to erroneous estimates of parasite density.

Quantitative real-time polymerase chain reaction for malaria diagnosis and its use in malaria vaccine clinical trials.

The demand for an effective malaria vaccine is high, with millions of people being affected by the disease every year. A large variety of potential vaccines are under investigation worldwide, and when tested in clinical trials, researchers need to extract as much data as possible from every vaccinated and control volunteer. The use of quantitative real-time polymerase chain reaction (PCR), carried out in real-time during the clinical trials of vaccines designed to act against the liver stage of the parasite's life cycle, provides more information than the gold standard method of microscopy alone and increases both safety and accuracy. PCR can detect malaria parasites in the blood up to 5 days before experienced microscopists see parasites on blood films, with a sensitivity of 20 parasites/mL blood. This PCR method has so far been used to follow 137 vaccinee and control volunteers in Phase IIa trials in Oxford and on 220 volunteer samples during a Phase IIb field trial in The Gambia.

Impact on malaria parasite multiplication rates in infected volunteers of the protein-in-adjuvant vaccine AMA1-C1/Alhydrogel+CPG 7909.

BACKGROUND: Inhibition of parasite growth is a major objective of blood-stage malaria vaccines. The in vitro assay of parasite growth inhibitory activity (GIA) is widely used as a surrogate marker for malaria vaccine efficacy in the down-selection of candidate blood-stage vaccines. Here we report the first study to examine the relationship between in vivo Plasmodium falciparum growth rates and in vitro GIA in humans experimentally infected with blood-stage malaria. METHODS: In this phase I/IIa open-label clinical trial five healthy malaria-naive volunteers were immunised with AMA1/C1-Alhydrogel+CPG 7909, and together with three unvaccinated controls were challenged by intravenous inoculation of P. falciparum infected erythrocytes. RESULTS: A significant correlation was observed between parasite multiplication rate in 48 hours (PMR) and both vaccine-induced growth-inhibitory activity (Pearson r = -0.93 [95% CI: -1.0, -0.27] P = 0.02) and AMA1 antibody titres in the vaccine group (Pearson r = -0.93 [95% CI: -0.99, -0.25] P = 0.02). However immunisation failed to reduce overall mean PMR in the vaccine group in comparison to the controls (vaccinee 16 fold [95% CI: 12, 22], control 17 fold [CI: 0, 65] P = 0.70). Therefore no impact on pre-patent period was observed (vaccine group median 8.5 days [range 7.5-9], control group median 9 days [range 7-9]). CONCLUSIONS: Despite the first observation in human experimental malaria infection of a significant association between vaccine-induced in vitro growth inhibitory activity and in vivo parasite multiplication rate, this did not translate into any observable clinically relevant vaccine effect in this small group of volunteers. TRIAL REGISTRATION: ClinicalTrials.gov [NCT00984763].

Blood-stage challenge for malaria vaccine efficacy trials: a pilot study with discussion of safety and potential value.

There is increasing interest in malaria vaccines targeting the asexual blood stage of Plasmodium falciparum. Without accepted immunologic correlates of clinical protection, challenge studies are useful for assessing the efficacy of candidate vaccines in vivo in healthy volunteers. We report a pilot study of a safe and robust challenge protocol using a blood-stage inoculum. We have applied well-validated trial endpoints and twice daily real-time quantitative polymerase chain reaction monitoring of parasitemia to blood-stage challenge, which enabled direct comparison with sporozoite challenge. We found that greater accuracy in quantification of blood-stage growth rates can be achieved with blood-stage challenge. This finding may provide greater power to detect partial efficacy of many blood-stage candidate vaccines. We discuss the potential utility of blood-stage challenge studies in accelerating malaria vaccine development.

Immunohistochemical analysis of negative inguinal lymph nodes in men with squamous cell carcinoma of the penis: are we missing micrometastases which could predict recurrence?

OBJECTIVE: To determine the presence or absence of micrometastases in lymph node dissection specimens of men with squamous cell carcinoma (SCC) of the penis using immunohistochemistry, and to correlate the results with clinical outcome. PATIENTS AND METHODS: In all, 13 men (mean age 55.8 years) with penile SCC, treated at St George's hospital between 2001 and 2005, were selected. All had prophylactic lymph node dissections; (12 bilateral and one unilateral). All lymph nodes were negative on standard haematoxylin and eosin (H&E) staining. Each block was stained for cytokeratins MNF 116 and LP34. In all, 217 lymph nodes were examined (mean 16.7 per patient). The median (range) follow-up was 23 (10-52) months. RESULTS: The mean tumour size and depth of invasion were 30.2 mm and 15.9 mm, respectively. There were eight T2 and four T3 tumours. Four tumours showed lymphovascular invasion. Information on tumour size and pathological grade was not available in one case. None of the patients have to date developed a local recurrence. A single lymph node was found to contain a micrometastasis, which was identified retrospectively on the original H&E slide. This measured 0.24 mm in maximum dimension. This patient had a T2G2 tumour with no vascular invasion; he has not progressed clinically in 10 months of follow-up. CONCLUSIONS: Anticytokeratin immunohistochemistry on H&E-negative inguinal dissection specimens in men with SCC of the penis can detect micrometastases that might be overlooked on routine stains. Their prognostic and therapeutic significance needs further study.