Evaluation of the appropriate time period between sampling and analyzing for automated urinalysis

Introduction: Preanalytical specifications for urinalysis must be strictly adhered to avoid false interpretations. Aim of the present study is to examine whether the preanalytical factor ‘time point of analysis’ significantly influences stability of urine samples for urine particle and dipstick analysis. Materials and methods: In 321 pathological spontaneous urine samples, urine dipstick (Urisys™2400, Combur-10-Test™strips, Roche Diagnostics, Mannheim, Germany) and particle analysis (UF-1000 i™, Sysmex, Norderstedt, Germany) were performed within 90 min, 120 min and 240 min after urine collection. Results: For urine particle analysis, a significant increase in conductivity (120 vs. 90 min: P < 0.001, 240 vs. 90 min: P < 0.001) and a significant decrease in WBC (120 vs. 90 min P < 0.001, 240 vs. 90 min P < 0.001), RBC (120 vs. 90 min P < 0.001, 240 vs. 90 min P < 0.001), casts (120 vs. 90 min P < 0.001, 240 vs. 90 min P < 0.001) and epithelial cells (120 vs. 90 min P = 0.610, 240 vs. 90 min P = 0.041) were found. There were no significant changes for bacteria. Regarding urine dipstick analysis, misclassification rates between measurements were significant for pH (120 vs. 90 min P < 0.001, 240 vs. 90 min P < 0.001), leukocytes (120 vs. 90 min P < 0.001, 240 vs. 90 min P < 0.001), nitrite (120 vs. 90 min P < 0.001, 240 vs. 90 min P < 0.001), protein (120 vs. 90 min P < 0.001, 240 vs. 90 min P<0.001), ketone (120 vs. 90 min P < 0.001, 240 vs. 90 min P < 0.001), blood (120 vs. 90 min P < 0.001, 240 vs. 90 min P < 0.001), specific gravity (120 vs. 90 min P < 0.001, 240 vs. 90 min P < 0.001) and urobilinogen (120 vs. 90 min, P = 0.031). Misclassification rates were not significant for glucose and bilirubin. Conclusion: Most parameters critically depend on the time window between sampling and analysis. Our study stresses the importance of adherence to early time points in urinalysis (within 90 min)

Real-time PCR detection of the HhaI tandem DNA repeat in pre- and post-patent Brugia malayi infections: a study in Indonesian transmigrants

Background: Lymphatic filariasis caused by Wuchereria bancrofti or Brugia spp. is a public health problem in developing countries. To monitor bancroftian filariasis infections, Circulating Filarial Antigen (CFA) test is commonly used, but for brugian infections only microfilariae (Mf) microscopy and indirect IgG4 antibody analyses are available. Improved diagnostics for detecting latent infections are required. Methods: An optimized real-time PCR targeting the brugian HhaI repeat was validated with plasma from microfilariae negative Mongolian gerbils (jirds) infected with B. malayi. Plasma samples from microfilaremic patients infected with B. malayi or W. bancrofti were used as positive and negative controls, respectively. PCR results of plasma samples from a transmigrant population in a B. malayi endemic area were compared to those of life-long residents in the same endemic area; and to IgG4 serology results from the same population. To discriminate between active infections and larval exposure a threshold was determined by correlation and Receiver-Operating Characteristics (ROC) curve analyses. Results: The PCR detected HhaI in pre-patent (56 dpi) B. malayi infected jirds and B. malayi Mf-positive patients from Central Sulawesi, Indonesia. HhaI was also detected in 9/9 elephantiasis patients. In South Sulawesi 87.4% of the transmigrants and life-long residents (94% Mf-negative) were HhaI PCR positive. Based on ROC-curve analysis a threshold for active infections was set to &gt;53 HhaI copies/μl (AUC: 0.854). Conclusions: The results demonstrate that the HhaI PCR detects brugian infections with greater sensitivity than the IgG4 test, most notably in Mf-negative patients (i.e. pre-patent or latent infections)

Reductions in microfilaridermia by repeated ivermectin treatment are associated with lower Plasmodium-specific Th17 immune responses in Onchocerca volvulus-infected individuals

Background: 37 million individuals are currently infected with Onchocerca volvulus (O. volvulus), a parasitic nematode that elicits various dermal manifestations and eye damage in man. Disease control is primarily based on distributing ivermectin in mass drug administration (MDA) programmes which aim at breaking transmission by eliminating microfilariae (MF), the worm's offspring. The majority of infected individuals present generalized onchocerciasis, which is characterized by hyporesponsive immune responses and high parasite burden including MF. Recently, in areas that have been part of MDA programmes, individuals have been identified that present nodules but are amicrofilaridermic (a-MF) and our previous study showed that this group has a distinct immune profile. Expanding on those findings we determined the immune responses of O. volvulus-infected individuals to a Plasmodium-derived antigen MSP-1 (merozoite surface protein-1), which is required by the parasite to enter erythrocytes. Methods: Isolated PBMCs from O. volvulus-infected individuals (164 MF+ and 46 a-MF) and non-infected volunteers from the same region (NEN), were stimulated with MSP-1 and the resulting supernatant screened for the presence of IL-5, IL-13, IFN-γ, TNF-α, IL-6, IL-17A and IL-10. These findings were then further analyzed following regression analysis using the covariates MF, ivermectin (IVM) and region. The latter referred to the Central or Ashanti regions of Ghana, which, at the time sampling, had received 8 or 1 round of MDA respectively. Results: IL-5, IL-13 and IFN-γ responses to MSP-1 were not altered between NEN and O. volvulus-infected individuals nor were any associations revealed in the regression analysis. IL-10, IL-6 and TNF-α MSP-1 responses were, however, significantly elevated in cultures from infected individuals. Interestingly, when compared to a-MF individuals, MSP-induced IL-17A responses were significantly higher in MF+ patients. Following multivariable regression analysis these IL-10, IL-6, TNF-α and IL-17A responses were all dominantly associated with the regional covariate. Conclusions: Consequently, areas with a lowered infection pressure due to IVM MDA appear to influence bystander responses to Plasmodium-derived antigens in community members even if they have not regularly participated in the therapy

Histopathological evaluation of Onchocerca volvulus nodules by microscopy and by digital image analysis for the study of macrofilaricidal drug efficacy

BACKGROUND: Novel drugs or drug combinations that kill or permanently sterilize adult MATERIAL AND METHODS: Two expert microscopists independently assessed 605 nodules by direct microscopy. At least two sections with two different stains hematoxylin & eosin (H&E, APR immunostain) of paraffin-embedded, ethanol-fixed whole-nodule cross-sections were analyzed. After variables were identified prone to observer discrepancies, we performed a second study to compare consolidated results for 100 nodules obtained by the two readers by microscopy and by analysis of scanned, high resolution digital images (20x magnification). The last data set analyzed was a quality panel of 100 nodules that has been previously examined by microscopy, and included additional immunostains for RESULTS: The degree of agreement between assessors varied for different parameters. Agreement for female worm counts in nodules was approximately 80%, while agreement regarding female worm viability was 98%. There were no major differences observed between results obtained by microscopy or digital images. Good agreement for important parameters was also observed for the nodules of the quality panel. CONCLUSION: Nodule analysis by experienced microscopists was reproducible with regard to important parameters such as identification of living female worms or detection of normal embryogenesis. Assessments varied more for other parameters, and we recommend continued use of two independent readers for detailed analyzes. Analysis of scanned images provided similar results to direct microscopy. This facilitates training and comparison of nodule findings by readers in different locations. Analysis of high quality digital images that can be viewed remotely should improve the quality and availability of nodule assessments that are primary endpoints for onchocerciasis clinical trials

Doxycycline Leads to Sterility and Enhanced Killing of Female Onchocerca volvulus Worms in an Area With Persistent Microfilaridermia After Repeated Ivermectin Treatment: A Randomized, Placebo-Controlled, Double-Blind Trial

Background Ivermectin (IVM) has been the drug of choice for the treatment of onchocerciasis. However, there have been reports of persistent microfilaridermia in individuals from an endemic area in Ghana after many rounds of IVM, raising concerns of suboptimal response or even the emergence of drug resistance. Because it is considered risky to continue relying only on IVM to combat this phenomenon, we assessed the effect of targeting the Onchocerca volvulus Wolbachia endosymbionts with doxycycline for these individuals with suboptimal response. Methods One hundred sixty-seven patients, most of them with multiple rounds of IVM, were recruited in areas with IVM suboptimal response and treated with 100 mg/day doxycycline for 6 weeks. Three and 12 months after doxycycline treatment, patients took part in standard IVM treatment. Results At 20 months after treatment, 80% of living female worms from the placebo group were Wolbachia positive, whereas only 5.1% in the doxycycline-treated group contained bacteria. Consistent with interruption of embryogenesis, none of the nodules removed from doxycycline-treated patients contained microfilariae, and 97% of those patients were without microfilaridermia, in contrast to placebo patients who remained at pretreatment levels (P < .001). Moreover, a significantly enhanced number of dead worms were observed after doxycycline. Conclusions Targeting the Wolbachia in O. volvulus is effective in clearing microfilariae in the skin of onchocerciasis patients with persistent microfilaridermia and in enhanced killing of adult worms after repeated standard IVM treatment. Strategies can now be developed that include doxycycline to control onchocerciasis in areas where infections persist despite the frequent use of IVM

Clinical, haematological and biochemical profiling of podoconiosis lymphoedema patients prior to their involvement in a clinical trial in the Northwest Region of Cameroon

Background Prior to carrying out clinical trials, it is important to assess the health status of the study participants to be able to interpret subsequent changes that may be related to the effects of the treatments during the follow-up of patients. This study presents the clinical, haematological and biochemical profiles of podoconiosis patients prior to their involvement in the PodoLEDoxy clinical trial. Methods All lower limb lymphoedema patients visiting the centre were screened and a podoconiosis diagnosis was based on clinical manifestation and detailed medical history. Patients who satisfied the eligibility criteria were enrolled in the study and their demographic data, vital signs and medical history were collected followed by biochemical and haematological examinations. Results Of the 222 participants enrolled in the study, 55.4% and 41.4% had either stage 3 or 2 podoconiosis as their highest stages, respectively. On physical examination, gastritis (46%) and poor vision (2.7%) were the most prevalent health issues identified. The majority of haematological and biochemical values were within the normal range except for mean platelet volume (47.7%), plateletcrit (58.1%), platelet distribution width (66.2%), mean corpuscular volume (67.6%) and red cell distribution width-standard deviation (79.3%), where >40% of the study participants had values out of the normal. Conclusion The clinical, haematological and biochemical profiles of the study participants were largely within the normal range except for certain haematological parameters that might be worth investigating

MR

Mass drug administration (MDA) programmes against Onchocerca volvulus use ivermectin (IVM) which targets microfilariae (MF), the worm's offspring. Most infected individuals are hyporesponsive and present regulated immune responses despite high parasite burden. Recently, with MDA programmes, the existence of amicrofilaridermic (a-MF) individuals has become apparent but little is known about their immune responses. Within this immunoepidemiological study, we compared parasitology, pathology and immune profiles in infection-free volunteers and infected individuals that were MF+ or a-MF. The latter stemmed from villages in either Central or Ashanti regions of Ghana which, at the time of the study, had received up to eight or only one round of MDA respectively. Interestingly, a-MF patients had fewer nodules and decreased IL-10 responses to all tested stimuli. On the other hand, this patient group displayed contrary IL-5 profiles following in vitro stimulation or in plasma and the dampened response in the latter correlated to reduced eosinophils and associated factors but elevated neutrophils. Furthermore, multivariable regression analysis with covariates MF, IVM or the region (Central vs. Ashanti) revealed that immune responses were associated with different covariates: whereas O. volvulus-specific IL-5 responses were primarily associated with MF, IL-10 secretion had a negative correlation with times of individual IVM therapy (IIT). All plasma parameters (eosinophil cationic protein, IL-5, eosinophils and neutrophils) were highly associated with MF. With regards to IL-17 secretion, although no differences were observed between the groups to filarial-specific or bystander stimuli, these responses were highly associated with the region. These data indicate that immune responses are affected by both, IIT and the rounds of IVM MDA within the community. Consequently, it appears that a lowered infection pressure due to IVM MDA may affect the immune profile of community members even if they have not regularly participated in the programmes

Combinations of registered drugs reduce treatment times required to deplete Wolbachia in the Litomosoides sigmodontis mouse model.

Filarial parasites can be targeted by antibiotic treatment due to their unique endosymbiotic relationship with Wolbachia bacteria. This finding has led to successful treatment strategies in both, human onchocerciasis and lymphatic filariasis. A 4-6 week treatment course using doxycycline results in long-term sterility and safe macrofilaricidal activity in humans. However, current treatment times and doxycycline contraindications in children and pregnant women preclude widespread administration of doxycycline in public health control programs; therefore, the search for shorter anti-wolbachial regimens is a focus of ongoing research. We have established an in vivo model for compound screening, using mice infected with Litomosoides sigmodontis. We could show that gold standard doxycycline treatment did not only deplete Wolbachia, it also resulted in a larval arrest. In this model, combinations of registered antibiotics were tested for their anti-wolbachial activity. Administration of rifamycins in combination with doxycycline for 7 days successfully depleted Wolbachia by > 2 log (>99% reduction) and thus resulted in a significant reduction of the treatment duration. Using a triple combination of a tetracycline (doxycycline or minocycline), a rifamycin and a fluoroquinolone (moxifloxacin) led to an even greater shortening of the treatment time. Testing all double combinations that could be derived from the triple combinations revealed that the combination of rifapentine (15mg/kg) and moxifloxacin (2 x 200mg/kg) showed the strongest reduction of treatment time in intraperitoneal and also oral administration routes. The rifapentine plus moxifloxacin combination was equivalent to the triple combination with additional doxycycline (>99% Wolbachia reduction). These investigations suggest that it is possible to shorten anti-wolbachial treatment times with combination treatments in order to achieve the target product profile (TPP) requirements for macrofilaricidal drugs of no more than 7-10 days of treatment

Expression of the lymphatic marker podoplanin (D2-40) in human fetal eyes

During human ocular development, expression of proteins varies in different maturation stages. This study aims to characterize structures in human fetal eyes stained by the lymphatic marker podoplanin (D2-40) with emphasis on the stage of maturation and the presence of intraocular lymphatic structures. Formalin-fixed paraffin-embedded eyes from 40 human fetuses between 10 and 38 weeks of gestation (WoG) were investigated. Immunohistochemical stains were performed for D2-40, LYVE-1 as a secondary lymphatic marker, and CD34 as a control for endothelial reactivity. A semiquantitative analysis of antigen expression in different segments of the eye was performed by light microscopy. The intensity of antigen expression was graded with a score ranging from 0 to 3. Podoplanin expression was found with a variable intensity in 97.5% of the eyes, in particular in lymphatic vessels of the conjunctiva (n = 26), conjunctival and corneal epithelium (n = 33), corneal endothelium (n = 4), trabecular meshwork (n = 28), and optic nerve sheaths (n = 23). A slight, equivocal staining reaction was noted in the choroid (n = 14). There was a correlation of antigen reactivity and the gestational age for corneal endothelial reactivity in earlier gestational stages (p = 0.003) and trabecular meshwork in older eyes (p = 0.031). D2-40 positive Muller cells were detected in two eyes >= 32 WoG. Thus, aside from conjunctival lymphatic vessels, podoplanin was expressed in several structures of the human fetal eye and the ocular adnexae at different gestational stages. Podoplanin positive structures were also found in the choroid and the chamber angle. However, lymphatic vessels or its progenitors could not be unequivocally identified in intraocular structures during 10-38 weeks of gestation. There is no evidence from our data that transient intraocular lymphactics develop in the fetal eye between 10 and 38 weeks of gestation. (C) 2014 Elsevier Ltd. All rights reserved