Duodenal and colonic mucosal S100A8/A9 (calprotectin) expression is increased and correlates with the severity of select histologic lesions in dogs with chronic inflammatory enteropathy.

BACKGROUND: Calprotectin, a damage-associated molecular pattern protein of the S100/calgranulin family, is a potential marker of gastrointestinal inflammation in dogs and mainly originates from activated macrophages and granulocytes. Increased calprotectin concentrations are reported in feces and serum samples from dogs with chronic inflammatory enteropathy (CIE), but mucosal calprotectin expression has not been extensively investigated in canine CIE. Thus, we aimed to evaluate gastrointestinal mucosal concentrations of calprotectin in 62 dogs (44 dogs with CIE compared to 18 healthy Beagles) using a particle-enhanced turbidimetric immunoassay method. Additionally, we assessed the relationship of gastric, duodenal, jejunal, ileal, and colonic mucosal calprotectin levels with the clinical disease severity (canine clinical inflammatory bowel disease activity index, CIBDAI), histopathologic findings, clinical outcome, and serum albumin concentrations to further evaluate the potential of calprotectin as a biomarker for CIE. RESULTS: Mucosal calprotectin concentrations in dogs with CIE were significantly higher in the duodenum (median: 276.2 µg/g) and colon (median: 298.2 µg/g) compared to healthy controls (median: 94.3 µg/g, P = 0.0039; and median: 112.0 µg/g, P = 0.0061). Similar numerical differences in the ileum and cecum were not statistically significant, and mucosal calprotectin concentrations correlated significantly among the different gastrointestinal segments. Histologic lesion severity was linked to mucosal calprotectin concentrations for inflammatory and structural histology criteria in the duodenum and colon (all P < 0.05). Higher mucosal calprotectin levels in the duodenum and across all segments correlated with lower serum albumin concentrations (both P < 0.05); duodenal mucosal calprotectin concentrations were more than sixfold higher in hypoalbuminemic dogs (median: 1441 µg/g, n = 4) than normoalbuminemic dogs (median: 227 µg/g, n = 40). There was no significant association of mucosal calprotectin levels with CIBDAI scores or individual clinical outcomes. CONCLUSIONS: These results show that duodenal and colonic mucosal calprotectin concentrations are increased in dogs with CIE, providing further supporting evidence for the diagnostic potential of fecal calprotectin (presumably reflecting mucosal) concentrations and in dogs with CIE. Further longitudinal research is needed to assess changes in mucosal calprotectin concentrations with clinical response to treatment vs. mucosal disease remission and to determine the clinical utility of fecal calprotectin concentrations to diagnose and monitor dogs with CIE in clinical practice.

Utility of ultra-sensitive qPCR to detect Plasmodium falciparum and Plasmodium vivax infections under different transmission intensities.

BACKGROUND: The use of molecular diagnostics has revealed an unexpectedly large number of asymptomatic low-density malaria infections in many malaria endemic areas. This study compared the gains in parasite prevalence obtained by the use of ultra-sensitive (us)-qPCR as compared to standard qPCR in cross-sectional surveys conducted in Thailand, Brazil and Papua New Guinea (PNG). The compared assays differed in the copy number of qPCR targets in the parasite genome. METHODS: Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) parasites were quantified by qPCR amplifying the low-copy Pf_ and Pv_18S rRNA genes or the multi-copy targets Pf_varATS and Pv_mtCOX1. Cross-sectional surveys at the three study sites included 2252 participants of all ages and represented different transmission intensities. RESULTS: In the two low-transmission areas, P. falciparum positivity was 1.3% (10/773) (Thailand) and 0.8% (5/651) (Brazil) using standard Pf_18S rRNA qPCR. In these two countries, P. falciparum positivity by Pf_varATS us-qPCR increased to 1.9% (15/773) and 1.7% (11/651). In PNG, an area with moderate transmission intensity, P. falciparum positivity significantly increased from 8.6% (71/828) by standard qPCR to 12.2% (101/828) by us-qPCR. The proportions of P. falciparum infections not detected by standard qPCR were 33%, 55% and 30% in Thailand, Brazil and PNG. Plasmodium vivax was the predominating species in Thailand and Brazil, with 3.9% (30/773) and 4.9% (32/651) positivity by Pv_18S rRNA qPCR. In PNG, P. vivax positivity was similar to P. falciparum, at 8.0% (66/828). Use of Pv_mtCOX1 us-qPCR led to a significant increase in positivity to 5.1% (39/773), 6.4% (42/651) and 11.5% (95/828) in Thailand, Brazil, and PNG. The proportions of P. vivax infections missed by standard qPCR were similar at all three sites, with 23%, 24% and 31% in Thailand, Brazil and PNG. CONCLUSION: The proportional gains in the detection of P. falciparum and P. vivax infections by ultra-sensitive diagnostic assays were substantial at all three study sites. Thus, us-qPCR yields more precise prevalence estimates for both P. falciparum and P. vivax at all studied levels of endemicity and represents a significant diagnostic improvement. Improving sensitivity in P. vivax surveillance by us-qPCR is of particular benefit, because the additionally detected P. vivax infections signal the potential presence of hypnozoites and subsequent risk of relapse and further transmission.

Plasmodium vivax molecular diagnostics in community surveys: pitfalls and solutions.

A distinctive feature of Plasmodium vivax infections is the overall low parasite density in peripheral blood. Thus, identifying asymptomatic infected individuals in endemic communities requires diagnostic tests with high sensitivity. The detection limits of molecular diagnostic tests are primarily defined by the volume of blood analysed and by the copy number of the amplified molecular marker serving as the template for amplification. By using mitochondrial DNA as the multi-copy template, the detection limit can be improved more than tenfold, compared to standard 18S rRNA targets, thereby allowing detection of lower parasite densities. In a very low transmission area in Brazil, application of a mitochondrial DNA-based assay increased prevalence from 4.9 to 6.5%. The usefulness of molecular tests in malaria epidemiological studies is widely recognized, especially when precise prevalence rates are desired. Of concern, however, is the challenge of demonstrating test accuracy and quality control for samples with very low parasite densities. In this case, chance effects in template distribution around the detection limit constrain reproducibility. Rigorous assessment of false positive and false negative test results is, therefore, required to prevent over- or under-estimation of parasite prevalence in epidemiological studies or when monitoring interventions.