Retrospective Investigations Research Articles

Molecular Characterization of Leishmania Parasites in Giemsa-Stained Slides from Cases of Human Cutaneous and Visceral Leishmaniasis, Eastern Algeria.

In Algeria, visceral leishmaniasis (VL) is due to Leishmania (L.) infantum, while three cutaneous forms (CL) are caused by Leishmania major, Leishmania tropica and Leishmania infantum. In this study, the use of Giemsa-stained slides was evaluated with two PCR techniques, in Eastern Algeria. A total of 136 samples corresponding to 100 CL smears (skin scrapings) and 36 VL slides (bone marrow aspirates) collected from 2008 to 2014 were tested. Upon DNA extraction, two PCRs were used to amplify the ribosomal Internal Transcribed Spacer 1 (ITS1) and mini-exon genes. Amplified products were digested (PCR-RFLP) and profiles analyzed for Leishmania species identification. A statistical analysis was also performed. ITS1-PCR was found significantly more sensitive than mini-exon-PCR (77.95% positives vs. 67.65%; p = 0.001). Comparison of PCR positivity showed statistically significant differences between old and recently prepared slides suggesting a better use of recent slides in PCR analyses. For species identification, PCR-restriction fragment length polymorphism (RFLP) results of ITS1 and mini-exon were concordant. L. infantum was identified from VL cases and L. infantum, L. major, and L. tropica from CL ones. According to geographical origin, L. infantum was found in North-Eastern provinces, while L. major was distributed from the North to the Center-East of Algeria. Interestingly, two L. tropica samples were identified in Annaba, located far North-East Algeria. Distribution of leishmaniasis in Eastern parts of Algeria, besides finding of L. tropica in the far North, is in this study described for the first time using molecular tools, thus confirming the usefulness of slides for PCR identification of Leishmania parasites in retrospective epidemiological investigations.

The presence of the gadolinium-based contrast agent depositions in the brain and symptoms of gadolinium neurotoxicity - A systematic review.

Background and purposeGadolinium based contrast agents (GBCAs) are widely used in magnetic resonance imaging, but recently, high signal intensity in the cerebellum structures was reported after repeated administrations of contrast- enhanced magnetic resonance images. The aim of this systematic review was to investigate the association between increased signal intensity in the dentate nucleus and globus pallidus in the brain and repeated administrations of GBCAs. Additionally, we focused on possible short- and long-term consequences of gadolinium use in those patients.MethodsSystematic review of retrospective investigations in PubMed and Medline was performed in July 2016. Primary outcomes included the presence of increased signal intensity within the dentate nucleus and globus pallidus on unenhanced T1-weighted MR images in patients following administrations of GBCAs. Two independent reviewers were responsible for search and data extraction.Results25 publications satisfied inclusion criteria (19 magnetic resonance images analyses, 3 case reports; 3 autopsy studies). Magnetic resonance images of 1247 patients with increased signal intensity on unenhanced T1-weighted MR images were analyzed as well as tissue specimens from 27 patients. Signal intensity correlated positively with the exposure to GBCAs and was greater after serial administrations of linear nonionic than cyclic contrast agents. Gadolinium was detected in all tissue examinations.ConclusionsHigh signal intensity in the dentate nucleus and globus pallidus on unenhanced T1-weighted magnetic resonance images were associated with previous administration of GBCAs. Signal intensity correlated negatively with stability of contrast agents. Clinical significance of gadolinium deposition in the brain remains unclear. There is a strong need for further research to identify type of gadolinium deposited in the brain as well as to gather knowledge about long-term consequences.

Systematic review of β blocker, aspirin, and statin in critically ill patients: importance of severity of illness and cardiac troponin

Non-cardiac critically ill patients with type II myocardial infarction (MI) have a high risk of mortality. There are no evidence-based interventions to mitigate this risk. We systematically reviewed the literature...

Detective effort: what contributes to arrests during retrospective criminal investigations?

ABSTRACTClearance rates among retrospective criminal investigations have been a cause for concern in the last 20 years. Unfortunately, little is known about how detectives contribute to case outcomes. To explore this topic, 184 detectives during a 60 day period completed time-task logs that were paired with incident report data. Through a unique temporal sampling procedure, 564 index offenses were disaggregated into subsamples based on the case’s suspect status at the time the case was assigned. The curvilinearity of these estimates was tested and recalculated into binary terms when quadratic relationships were observed. Continuous and re-estimated quadratic variables were then evaluated for multivariate suitability and regressed to predict a cases likelihood of arrest. The results of these analyses found that (a) detectives continue to exercise great investigative discretion once cases are assigned; (b) suspect status influences arrests; (c) detective time spent interviewing the suspect was the most consistent predictor of arrests; (d) the influence of individual investigative activities on arrests tended to vary by the suspect status; (e) detective effort matters most among cases without a known suspect; and (f) more time performing investigative activities is not always positively associated with arrests. These findings are discussed as they relate to organisation issues, such as supervision, case management, and resource allocation within the context of criminal investigations. In doing so, this quantification of detective effort contributes to policy and practice debates on detective caseloads and seeks to improve the efficacy of criminal investigations.

Literature Review: Herbal Medicine Treatment after Large-Scale Disasters.

Large-scale natural disasters, such as earthquakes, tsunamis, volcanic eruptions, and typhoons, occur worldwide. After the Great East Japan earthquake and tsunami, our medical support operation's experiences suggested that traditional medicine might be useful for treating the various symptoms of the survivors. However, little information is available regarding herbal medicine treatment in such situations. Considering that further disasters will occur, we performed a literature review and summarized the traditional medicine approaches for treatment after large-scale disasters. We searched PubMed and Cochrane Library for articles written in English, and Ichushi for those written in Japanese. Articles published before 31 March 2016 were included. Keywords "disaster" and "herbal medicine" were used in our search. Among studies involving herbal medicine after a disaster, we found two randomized controlled trials investigating post-traumatic stress disorder (PTSD), three retrospective investigations of trauma or common diseases, and seven case series or case reports of dizziness, pain, and psychosomatic symptoms. In conclusion, herbal medicine has been used to treat trauma, PTSD, and other symptoms after disasters. However, few articles have been published, likely due to the difficulty in designing high quality studies in such situations. Further study will be needed to clarify the usefulness of herbal medicine after disasters.

Natural history of graves' orbitopathy after treatment.

Intravenous glucocorticoids are used for Graves' orbitopathy, alone or associated with/followed by additional treatments (orbital radiotherapy, orbital decompression, palpebral or eye surgery). However, the relation between associated/additional treatments and other variables with Graves' orbitopathy outcome following intravenous glucocorticoids is not clear. Thus, the present study was conducted to investigate retrospectively the impact of associated/additional treatments and other variables on Graves' orbitopathy outcome after intravenous glucocorticoids. We evaluated 226 untreated Graves' orbitopathy patients. Following first observation, patients were given intravenous glucocorticoids and re-examined after a median of 46.5 months. The end-points were the relation between Graves' orbitopathy outcome, outcome of NOSPECS score and of the single Graves' orbitopathy features with several variables, including associated/additional treatments. All Graves' orbitopathy features improved significantly after treatment. Overall, Graves' orbitopathy improved in ~60 % of patients (responders), whereas it was stable or worsened in ~40 % of patients (non-responders). Time between first and last observation and clinical activity score at first observation correlated significantly with Graves' orbitopathy outcome. The outcomes of NOSPECS, eyelid aperture, clinical activity score and diplopia correlated with time between the first and last observation. The NOSPECS outcome correlated with gender. The outcomes of proptosis, eyelid aperture and visual acuity correlated with orbital decompression. The outcome of diplopia correlated with orbital radiotherapy. Taking into account the limitations of retrospective investigations, our findings confirm that time (i.e. the natural history of Graves' orbitopathy) is a key factor in determining the long-term outcome of Graves' orbitopathy, radiotherapy is effective for diplopia, and orbital decompression is followed by an amelioration of several Graves' orbitopathy features.

A PhD completed 5. Restoration survival in the composite resin age

The article describes studies aimed at investigating factors that can influence composite restoration survival in posterior teeth, focusing on the main reasons for failure of direct restorations - fracture and secondary-caries. Since direct composite resin restaurations seem to be more prone to secondary caries when compared to amalgam, one study was performed to evaluate the influence of materials on secondary caries lesion formation in situ. Also, in most clinical studies evaluating restoration survival, there is a tendency to focus on materials factors. Two retrospective clinical investigations, a meta-analysis with primary data of clinical studies and a systematic review were performed to assess the influence of patient-, materials-, and tooth-related variables in posterior composite restoration survival. Composite resins were not related with higher mineral loss when compared to amalgam. Tooth type, number of restored surfaces and patient-related factors like a high caries risk and occlusal wear were consistently found to influence composite resin restoration survival in posterior teeth, whereas materials factors were not consistently implicated with the survivalrate.

Rationale for local treatment in the management of metastatic prostate cancer.

To evaluate the rationale supporting the role of local treatment in the management of patients with metastatic prostate cancer (PCa). Not all patients with metastatic PCa share the same prognosis, in which selected individuals with oligometastatic PCa might benefit from local therapies. These men would harbor a biologically different disease as compared with their counterparts with widespread metastases. Local treatment would eliminate the source of tumor-promoting factors, destroy the origin of metastatic cells, and stop the self-seeding process. Moreover, decreasing tumor burden would eventually allow for an improved response to systemic therapies. Recent clinical studies support an oncologic role of surgery or radiotherapy in metastatic PCa. However, their retrospective nature limits the relevance of these findings. Results of ongoing trials assessing the impact of local treatment in metastatic patients are needed to comprehensively address its role. Preclinical observations provide a rationale for treatment of the primary tumor in selected patients with metastatic PCa. However, available clinical evidence comes from retrospective investigations, and only results of ongoing randomized trials would clarify the role of local treatment in the metastatic setting.

Issues Surrounding Testosterone Replacement Therapy.

Testosterone is derived from Leydig cells and exerts its effects on androgen receptors to influence growth, mood, voice, and several other bodily functions. As men age, their testosterone levels decline. Human immunodeficiency virus (HIV) infection has also been associated with lowered serum testosterone levels. Subtherapeutic levels of testosterone may lead to fatigue, loss of libido, and dysphoria. Exogenous replacement of testosterone can be accomplished by several modalities (oral, topical, injection), with each having distinct advantages and disadvantages. Even though testosterone replacement has become a popular medical intervention, recent reports have made the practice increasingly controversial. Several small retrospective investigations have recently associated testosterone replacement with an increased risk of cardiovascular complications. Replacement should be used sparingly until further conclusive data regarding the efficacy and safety of testosterone become available.

Predicting water-to-cyclohexane partitioning of the SAMPL5 molecules using dielectric balancing of force fields.

Alchemical transformation of solutes using classical fixed-charge force fields is a popular strategy for assessing the free energy of transfer in different environments. Accurate estimations of transfer between phases with significantly different polarities can be difficult because of the static nature of the force fields. Here, we report on an application of such calculations in the SAMPL5 experiment that also involves an effort in balancing solute and solvent interactions via their expected static dielectric constants. This strategy performs well with respect to predictive accuracy and correlation with unknown experimental values. We follow this by performing a series of retrospective investigations which highlight the potential importance of proper balancing in these systems, and we use a null hypothesis analysis to explore potential biases in the comparisons with experiment. The collective findings indicate that considerations of force field compatibility through dielectric behavior is a potential strategy for future improvements in transfer processes between disparate environments.Electronic supplementary materialThe online version of this article (doi:10.1007/s10822-016-9950-z) contains supplementary material, which is available to authorized users.

Navigating Microbiological Food Safety in the Era of Whole-Genome Sequencing.

The epidemiological investigation of a foodborne outbreak, including identification of related cases, source attribution, and development of intervention strategies, relies heavily on the ability to subtype the etiological agent at a high enough resolution to differentiate related from nonrelated cases. Historically, several different molecular subtyping methods have been used for this purpose; however, emerging techniques, such as single nucleotide polymorphism (SNP)-based techniques, that use whole-genome sequencing (WGS) offer a resolution that was previously not possible. With WGS, unlike traditional subtyping methods that lack complete information, data can be used to elucidate phylogenetic relationships and disease-causing lineages can be tracked and monitored over time. The subtyping resolution and evolutionary context provided by WGS data allow investigators to connect related illnesses that would be missed by traditional techniques. The added advantage of data generated by WGS is that these data can also be used for secondary analyses, such as virulence gene detection, antibiotic resistance gene profiling, synteny comparisons, mobile genetic element identification, and geographic attribution. In addition, several software packages are now available to generate in silico results for traditional molecular subtyping methods from the whole-genome sequence, allowing for efficient comparison with historical databases. Metagenomic approaches using next-generation sequencing have also been successful in the detection of nonculturable foodborne pathogens. This review addresses state-of-the-art techniques in microbial WGS and analysis and then discusses how this technology can be used to help support food safety investigations. Retrospective outbreak investigations using WGS are presented to provide organism-specific examples of the benefits, and challenges, associated with WGS in comparison to traditional molecular subtyping techniques.

Ischemic pituitary adenoma apoplexy—Clinical appearance and prognosis after surgical intervention

BackgroundSeveral retrospective investigations have recommended more passive surgical indications for intratumoral hemorrhage of pituitary adenomas due to probable spontaneous resolution. However, no definitive analyses have compared pituitary adenomas with hemorrhagic apoplexy and intratumoral hemorrhage without evident apoplectic symptoms or pituitary adenoma infarction. MethodsThis study retrospectively identified 43 patients with symptomatic pituitary apoplexy among 1067 patients with pituitary adenomas initially treated by surgery at a single institute between April 2005 and May 2015, with 27 cases of hemorrhagic (2.53%) and 16 cases of ischemic apoplexy (1.50%). The inclusion criteria involved evident and sudden onset of symptoms and simultaneous histological confirmation as hemorrhagic or ischemic pituitary apoplexy. Diagnostic differentiation with magnetic resonance (MR) imaging was performed to examine the agreement between MR imaging and histological findings, and the clinical appearance and mid-term prognosis were compared for ischemic pituitary apoplexy and hemorrhagic apoplexy. ResultsDiagnostic matching with MR imaging could be performed in 41 of 43 patients (25 with hemorrhagic and 16 with ischemic apoplexy). Agreement with the histological finding was found in 32 of 41 patients overall (78%), 23 of 25 patients with hemorrhagic apoplexy (92%), and 9 of 16 patients with ischemic apoplexy (56%). The main reason for diagnostic discrepancy was thought to be the difficulty in identifying ischemic lesion. All patients in the ischemic group suffered progression of symptoms from initial onset including various cranial nerve palsies, aseptic meningitis, and decreased level of consciousness, whereas the hemorrhagic group suffered progression in 4 of 27 patients. Ischemic group showed a statistically stronger tendency to disease progression than the hemorrhagic group (P<0.001). Endocrinological examinations showed 4 patients required no hormone supplement therapies but the other 11 patients had persistent hypopituitarism and required hormone supplementation in the ischemic group, whereas 2 of 25 patients required hormone supplementation in the hemorrhagic group. Endocrinological recovery showed a significant difference between the ischemic group and hemorrhagic group (P<0.01). ConclusionsIschemic pituitary adenoma apoplexy has a more severe clinical course than hemorrhagic apoplexy. Development of preoperative diagnostic technology to differentiate ischemic from hemorrhagic apoplexy is required to improve the low rate of agreement between the histological and MR imaging findings in patients with ischemic apoplexy.

SU‐F‐T‐111: Investigation of the Attila Deterministic Solver as a Supplement to Monte Carlo for Calculating Out‐Of‐Field Radiotherapy Dose

Purpose:To use the Attila deterministic solver as a supplement to Monte Carlo for calculating out‐of‐field organ dose in support of epidemiological studies looking at the risks of second cancers. Supplemental dosimetry tools are needed to speed up dose calculations for studies involving large‐scale patient cohorts.Methods:Attila is a multi‐group discrete ordinates code which can solve the 3D photon‐electron coupled linear Boltzmann radiation transport equation on a finite‐element mesh. Dose is computed by multiplying the calculated particle flux in each mesh element by a medium‐specific energy deposition cross‐section. The out‐of‐field dosimetry capability of Attila is investigated by comparing average organ dose to that which is calculated by Monte Carlo simulation. The test scenario consists of a 6 MV external beam treatment of a female patient with a tumor in the left breast. The patient is simulated by a whole‐body adult reference female computational phantom. Monte Carlo simulations were performed using MCNP6 and XVMC. Attila can export a tetrahedral mesh for MCNP6, allowing for a direct comparison between the two codes. The Attila and Monte Carlo methods were also compared in terms of calculation speed and complexity of simulation setup. A key perquisite for this work was the modeling of a Varian Clinac 2100 linear accelerator.Results:The solid mesh of the torso part of the adult female phantom for the Attila calculation was prepared using the CAD software SpaceClaim. Preliminary calculations suggest that Attila is a user‐friendly software which shows great promise for our intended application. Computational performance is related to the number of tetrahedral elements included in the Attila calculation.Conclusion:Attila is being explored as a supplement to the conventional Monte Carlo radiation transport approach for performing retrospective patient dosimetry. The goal is for the dosimetry to be sufficiently accurate for use in retrospective epidemiological investigations.

Establishment of health management package for native sheep of Bangladesh

The present study was designed to explore the disease status of sheep in Bangladesh with the aim of developing health management package for sheep for better and efficient sheep production in Bangladesh. Both retrospective and prospective investigations on the incidence and prevalence of different diseases of sheep have been conducted in the study areas. Information on the disease related factors like health status, sex and age, vaccination, deworming, etc. were collected. Samples (faeces, blood, etc.) were collected from the diseased/dead animals and preserved following the standard procedure. Faecal samples were examined to determine the parasitic loads and faecal as well as blood samples were examined to determine any protozoan infection present in the study animals. Samples from diarrhoeic animals were studied to isolate and identify causal organisms. Antibiotic resistance and sensitivity studies of the aetiological agents responsible for common infectious diseases in sheep were also conducted in order to suggest the most suitable antibiotic to treat the concerned diseases in the field. Since helminthic infections, diarrhea, pneumonia and foot rot were found to be the mostly occurring health hazards in sheep of all ages, especial attempts were made to develop herbal based novel approaches to treat and control major intestinal helminthic infestations of sheep. However, gentamicin and ciprofloxacin were found most effective antibiotics and recommended to use in diarrhoeal cases in the field. On the other hand, methanol extract of mahogany seeds (100 mg), betel leaf (100 mg) and dodder (100 mg) were found significantly effective against 100% worms in 2 hours in vitro and thus recommended to be used in the field against helminthic infection in sheep. Therefore, a health management package for native sheep of Bangladesh can be designed using the present findings with some fluctuations for different sites after doing the in vivo evaluation of the medicinal plants used in this experiment.Res. Agric., Livest. Fish.3(1): 151-155, April 2016

Validation of the Mass-Extraction-Window for Quantitative Methods Using Liquid Chromatography High Resolution Mass Spectrometry.

A paradigm shift is underway in the field of quantitative liquid chromatography-mass spectrometry (LC-MS) analysis thanks to the arrival of recent high-resolution mass spectrometers (HRMS). The capability of HRMS to perform sensitive and reliable quantifications of a large variety of analytes in HR-full scan mode is showing that it is now realistic to perform quantitative and qualitative analysis with the same instrument. Moreover, HR-full scan acquisition offers a global view of sample extracts and allows retrospective investigations as virtually all ionized compounds are detected with a high sensitivity. In time, the versatility of HRMS together with the increasing need for relative quantification of hundreds of endogenous metabolites should promote a shift from triple-quadrupole MS to HRMS. However, a current "pitfall" in quantitative LC-HRMS analysis is the lack of HRMS-specific guidance for validated quantitative analyses. Indeed, false positive and false negative HRMS detections are rare, albeit possible, if inadequate parameters are used. Here, we investigated two key parameters for the validation of LC-HRMS quantitative analyses: the mass accuracy (MA) and the mass-extraction-window (MEW) that is used to construct the extracted-ion-chromatograms. We propose MA-parameters, graphs, and equations to calculate rational MEW width for the validation of quantitative LC-HRMS methods. MA measurements were performed on four different LC-HRMS platforms. Experimentally determined MEW values ranged between 5.6 and 16.5 ppm and depended on the HRMS platform, its working environment, the calibration procedure, and the analyte considered. The proposed procedure provides a fit-for-purpose MEW determination and prevents false detections.

Implementing root cause analysis in Iranian hospitals: challenges and benefits.

Root cause analysis (RCA) has been widely used for retrospective investigations of patient safety incidents. To increase patient safety competencies, RCA has recently been introduced in Iranian hospitals. The aims of the current study were to explore team members' experiences and perceptions of RCA and to identify the challenges and benefits of using it in Iranian hospitals from their perspective. A qualitative study was conducted consisting of 32 semi-structured interviews with health professionals who participated in the national training programme and were involved in RCA investigations. Data were analysed using the thematic analysis method. The participants encountered a range of obstacles while conducting RCA, including time constraints, a lack of resources, the blame culture and unsupportive colleagues. They stressed the need for further leadership support and cultural change within the Iranian healthcare system to facilitate the application of RCA. RCA was perceived as a beneficial analytical tool that improved patient care, fostered teamwork and communication among staff and promoted safety culture. This study concluded that applying RCA in the Iranian healthcare setting has had a significant impact on improving commitment to safety. However, the general adoption of this method is hindered by the lack of workplace and system supports. To maximize profits from RCA, clinical leaders must assign a high priority to RCA investigations and support RCA team efforts. Copyright © 2016 John Wiley & Sons, Ltd.

Translamina Cribrosa Pressure Difference as Potential Element in the Pathogenesis of Glaucomatous Optic Neuropathy.

The main proven risk factor for glaucomatous optic neuropathy (GON) is an intraocular pressure (IOP) higher than the pressure sensibility of the optic nerve head allows. Fulfilling Koch postulates, numerous studies have shown that the presence of high IOP leads to GON, that lowering IOP stops the progression of GON, and that a re-increase in IOP again causes the progression of GON. There are, however, many patients with glaucoma who have statistically normal or low IOP, and despite low IOP values, they develop progressing GON. These observations led to findings that IOP is only 1 of 2 determinants of the translamina cribrosa pressure difference (TLCPD), which is the main pressure-related parameter for the physiology and pathophysiology of the optic nerve head. The second parameter influencing TLCPD is orbital cerebrospinal fluid pressure (CSFP) as the counter pressure against IOP across the lamina cribrosa. Recent experimental and clinical studies have suggested that a low CSFP could be associated with GON in normal-pressure glaucoma. These investigations included studies with an experimental long-term reduction in CSFP in monkeys, population-based studies, and clinical retrospective and prospective investigations on patients with normal-pressure glaucoma. Besides TLCPD, other ocular parameters influenced by CSFP may be choroidal thickness, retinal vein pressure and diameter, occurrence of retinal vein occlusions, and occurrence and severity of diabetic retinopathy.

Reliability (Revalidation) of MSU MRI Classification of Lumbar Disc Herniation in 100 Patients Series: Severity and Objective Surgical Criteria

Background Data: Success rate of discectomy is greatly variable between surgeons due to lack of clear categorization of lumbar disc patients. Valid radiological objective criteria are required especially for patients with uncertain surgical indication to help in pre-operative patient assessment, surgical selection and research of post-operative outcomes of comparable level of pathology. MSU Classification reports size and location in three precise increments, described simply 1-2-3 and A-B-C providing clear objective classification of disc herniation resulting in excellent interexaminarreliability. Purpose To test reliability of MSU MRI classification of lumbar disc herniation in helping surgical selection and correlation with preoperative clinical presentation and postoperative clinical and functional outcome. Study Design Observational analytic retrospective study. Patients and Methods Retrospective investigations of one hundred patientswho underwent lumbar discectomy at Ain Shams University between January 2014 and June 2015 in term of MSU classification. Results Only 2 patients (2%) in our series had grade 1-A. All other 98 patients had MSU grades with size 2 or 3 and/or B or C location or 2 or 3 combination of horizontal disc locations (A, B or C). Two cases of unintended durotomy occurred in MSU grade 3-AB (40%) and the other 3 cases occurred in grade 2-AB (60%). One case of CSF leak occurred in 3-AB herniated disc lesion (50%) and another one in 2-AB lesion (50%). Twelve patients (12%) developed recurrent sciatica with mean interval of 47.92 weeks after first disc surgery. Only 5 patients (5%) underwent second disc surgery with mean interval of 6.2 months. 95 patients (95%) had good to excellent result. Conclusion: MSU classification provides solid, universal and objective radiological description of lumbar disc herniation helping patient selection for surgery. (2016ESJ097)

Plasmodium falciparum dihydroartemisinin-piperaquine failures in Cambodia are associated with mutant K13 parasites presenting high survival rates in novel piperaquine in vitro assays: retrospective and prospective investigations.

BackgroundThe declining efficacy of dihydroartemisinin-piperaquine against Plasmodium falciparum in Cambodia, along with increasing numbers of recrudescent cases, suggests resistance to both artemisinin and piperaquine. Available in vitro piperaquine susceptibility assays do not correlate with treatment outcome. A novel assay using a pharmacologically relevant piperaquine dose/time exposure was designed and its relevance explored in retrospective and prospective studies.MethodsThe piperaquine survival assay (PSA) exposed parasites to 200 nM piperaquine for 48 hours and monitored survival 24 hours later. The retrospective study tested 32 culture-adapted, C580Y-K13 mutant parasites collected at enrolment from patients treated with a 3-day course of dihydroartemisinin-piperaquine and having presented or not with a recrudescence at day 42 (registered ACTRN12615000793516). The prospective study assessed ex vivo PSA survival rate alongside K13 polymorphism of isolates collected from patients enrolled in an open-label study with dihydroartemisinin-piperaquine for uncomplicated P. falciparum malaria in Cambodia (registered ACTRN12615000696594).ResultsAll parasites from recrudescent cases had in vitro or ex vivo PSA survival rates ≥10 %, a relevant cut-off value for piperaquine-resistance. Ex vivo PSA survival rates were higher for recrudescent than non-recrudescent cases (39.2 % vs. 0.17 %, P <1 × 10−7). Artemisinin-resistant K13 mutants with ex vivo PSA survival rates ≥10 % were associated with 32-fold higher risk of recrudescence (95 % CI, 4.5–224; P = 0.0005).ConclusionPSA adequately captures the piperaquine resistance/recrudescence phenotype, a mainstay to identify molecular marker(s) and evaluate efficacy of alternative drugs. Combined ex vivo PSA and K13 genotyping provides a convenient monitor for both artemisinin and piperaquine resistance where dihydroartemisinin-piperaquine is used.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-015-0539-5) contains supplementary material, which is available to authorized users.

Molecular and Immunohistochemical Diagnosis of Photobacterium damselae Subspecies piscicida During Naturally Occurring Disease in Egypt

AbstractIn the marine aquaculture industry, photobacteriosis caused by the Photobacterium damselae subspecies piscicida, Phdp is a globally significant disease. A number of clinical photobacteriosis outbreaks among yearling cultured and broodstocks of gilthead sea bream were sampled and submitted to our laboratory during the summer and autumn of 2013. The tissues of infected fish were subjected to an ordinary bacteriological identification and were analyzed using the polymerase chain reaction (PCR) and immunohistochemistry techniques. The results indicated that the selective primers that have been designed for detecting the gene encoding the apoptotic‐induced protein, AIP56, represent a powerful tool for sensitive and specific detection of virulent strains of Phdp. AIP56 toxin triggers apoptosis of host macrophages and neutrophils, contributing to the lesions observed during the pathological investigation. Immunohistochemistry allows bacterial identification and antigen expression to be directly correlated to the disease; the immune‐positive bacteria were detected in gills, liver, kidneys, spleen, and brain tissues in acute photobacteriosis. These also appeared in the necrotic areas of the granulomas of chronically infected fish. Molecular and immunohistochemical methods were useful as research and diagnostic tools in different stages of the disease; moreover, they appear to have enormous potential in retrospective epidemiological investigations.