Probe-based qPCR as an alternative to modified Knott's test when screening dogs for heartworm (Dirofilaria immitis) infection in combination with antigen detection tests.

BACKGROUND: Current recommendations for diagnosis of Dirofilaria immitis infection in dogs rely on the detection of antigen produced largely by adult females coupled with the visualization of microfilariae (mf) in the circulation via a microfilaria detection test (MFDT). It is hypothesized that qPCR assays used in parallel with antigen detection tests will perform better in detecting mf than modified Knott's test (MK), when combined with antigen detection. This study compares probe-based qPCR and MK techniques for mf detection used in parallel with the DiroCHEK® antigen test to screen for heartworm infection in shelter dogs. METHODS: Matching blood and serum samples were collected from 300 shelter dogs in Brazos and Harris counties, Texas, USA. Blood was assessed for the presence of mf via MK and the presence of D. immitis DNA by a species-specific probe-based qPCR assay. Serum samples were tested for the presence of heartworm antigen using DiroCHEK® before and after immune complex dissociation (ICD) via heat treatment. In addition, the performance of each diagnostic test was evaluated via Chi-square test, Cochran's Q test, and post hoc analysis. RESULTS: Qualitatively, MK detected mf in 22.0% (66/300) of samples, 55 of which were morphologically identified as D. immitis and 11 as Acanthocheilonema reconditum. The range of heartworm mf was 28 to 88,803 mf/ml (median: 6627.5). Real-time PCR detected D. immitis DNA in 20.7% (62/300) of samples. Heartworm antigen was detected in 24.7% (74/300) of samples pre-ICD, and in 29.3% (88/300) post-ICD. When comparing tests, the Chi-square and McNemar's tests showed that the difference between positive and negative proportions was statistically significant. The Cochran test showed the difference in the distributions of cases and non-cases was significant when individual tests were combined (χ2 = 62.3, df = 3, P < 0.0001) and when parallel methods were combined (χ2 = 43.1, df = 4, P < 0.0001). CONCLUSION: Considering individual and combined test performances, practicality, and efficient use of bench time, this heartworm-specific probe-based qPCR method is a viable option as a mf detection test to be used in parallel with antigen tests for canine heartworm infection in diagnostic and research settings.

Detection of Dirofilaria immitis via integrated serological and molecular analyses in coyotes from Texas, United States.

Wild canids serve as reservoir for various vector-borne pathogens of veterinary and medical importance, including the canine heartworm, Dirofilaria immitis. In North and Central America, coyotes (Canis latrans) may be a relevant reservoir host for heartworm transmission. The objective of this study was to determine the occurrence of D. immitis in coyotes across Texas using integrated antigen detection test and molecular assays. Matching whole blood and serum samples were collected from 122 coyotes from different locations across the state of Texas, United States, encompassing nine counties. Collections occurred from February to April 2016, and December 2016. Samples were assessed serologically using a commercial microtiter plate ELISA (DiroCHEK®), and molecularly by conventional PCR targeting the cytochrome oxidase c subunit 1 (cox1) and NADH dehydrogenase subunit 5 (nad5) of the mitochondrial DNA, and via a TaqMan© probe-based real-time PCR protocol, also targeting a fragment of the cox1 gene. Overall, 12 (9.83%) samples tested positive when serological and molecular results were combined. Seven of 122 samples (5.73%) were antigen-positive, 8 (6.55%) were qPCR-positive, and 4 (3.27%) were positive using conventional PCR. Of 12 positive samples, 4 tested antigen-positive by DiroCHEK® but were negative in all molecular tests, another 4 tested positive by at least one of the molecular assays but tested negative by DiroCHEK®, and 3 samples tested positive by both antigen test and at least one of the molecular assays. Two samples (16.67%) tested positive on both the antigen test and both conventional PCR and qPCR. Our study confirmed the presence of D. immitis infection in coyotes from southern and northern Texas. The combination of serologic and molecular diagnostic tests was proven synergistic for the identification of D. immitis infections, including occult dirofilariosis, and revealed a more accurate picture of heartworm occurrence in the sampled coyotes.

Association between vitamin D levels and blood pressure in a group of Puerto Ricans.

OBJECTIVE: Hypertension is a global public health problem. The apparent association between vitamin D (VitD) and blood pressure (BP) has been studied in several populations but not in a Puerto Rican population. Therefore, we determined the association between serum 25(OH)D levels and BP in a convenience sample in Puerto Rico. METHODS: A cross-sectional study of patients attending a local laboratory with an order for serum 25(OH)D levels was performed. Participants completed a questionnaire that solicited the following information: age, sex, weight, height, VitD intake, and history of both sun exposure and hypertension. BP was measured by a phlebotomist on-site and classified according to the Joint National Committee; serum 25(OH)D levels were taken from the record, and VitD status was classified as either optimal (25(OH)D > or = 30 ng/dl) or non-optimal (< 30 ng/dl). Mean, standard deviation, and percentiles were used for descriptive analysis. VitD status and BP were compared by gender, age, and body mass index (BMI) using ANOVA or chi-square. The association between VitD status and blood pressure was assessed by polytomous logistic regression adjusting for several variables. RESULTS: Two hundred nineteen individuals were included in the analysis; most were females aged 21 to 50 years who were overweight/obese; 53% of the total population was pre-hypertensive or hypertensive; 60% had non-optimal VitD status; and the sun exposure index was 19.6. No significant associations were found between VitD status and blood pressure after adjusting for age, sex, BMI, total VitD intake, and solar exposure index. CONCLUSION: VitD status was not found to be associated with blood pressure in this sample population; more studies with a larger sample population are needed to determine whether such an association does in fact exist.