Epidemiology of respiratory viruses in bronchoalveolar lavage samples in a tertiary hospital

BackgroundThe prevalence of respiratory viruses in adults is largely underexplored, as most studies focus on children. Additionally, in severely ill or immunocompromised adults, where respiratory infections are mostly attributed to bacteria and fungi; respiratory viruses can lead to severe complications.ObjectivesTo evaluate the epidemiology of respiratory viruses in bronchoalveolar lavage fluid (BAL) specimens from patients with lower respiratory tract disease. The study population consisted of different groups including immunocompetent patients (control patients), solid organ transplant recipients, patients with haematological malignancies and other immunocompromised adults.Study designA total of 134 BAL fluid specimens collected during 2009–2011 were retrospectively assessed with the new commercial multiplex real-time PCR FTD Respiratory 21 Plus^®, targeting 18 different viruses and 2 atypical bacterial pathogens.ResultsViral or atypical bacterial pathogens were detected in 29.1% of BAL fluid specimens. Coronaviruses were most prevalent (13.4%), followed by rhinoviruses (5.2%), RSV (4.5%) and bocaviruses (3.7%). Comparing the total number of viruses detected, a statistically significant difference was observed between the control group and patients with haematological malignancies (27.5% vs. 57.1%, p < 0.05).ConclusionIn conclusion, our study highlights the high prevalence of respiratory viruses in BAL fluid specimens from adult patients with lower respiratory tract disease. The methods to be used should be sensitive and cover a wide range of potential pathogens. The specific patient population can also influence the detection rates of respiratory viruses.     

Infectious diseases in paediatric pathology: experience from a developing country

Infectious and parasitic diseases have always challenged man. Although many of them are typically seen in some areas of the world and can be adequately managed by just improving socioeconomic status and sanitary conditions, they are still quite prevalent and may sometimes be seen outside their original geographical areas. Human migration due to different reasons, tourism, blood transfusion and solid organ transplantation has created new concerns for health professionals all over the world. If not for diagnostic purposes, at least these tropical and infectious diseases should be largely known because their epidemiology, pathogenesis, host/parasite interaction, inflammatory and reparative responses are quite interesting and teach us about human biology. Curiosity is inherent to pathology practice and so we are compelled to look for things other than tumours or degenerative diseases. This review focuses on infectious and parasitic diseases found in a developing country and brings up-to-date information on diseases caused by viruses (dengue, yellow fever), bacteria (typhoid fever, leprosy), parasites (Chagas' disease, cutaneous and visceral leishmaniasis, amoebiasis, Capillaria hepatica, schistosomiasis, cysticercosis) and caused by fungi (paracoccidioidomycosis, cryptococcosis, histoplasmosis) that may be useful for pathologists when facing somewhat strange cases from developing countries.     

TRARESA: a tissue microarray-based hospital system for biomarker validation and discovery

Formalin fixed and paraffin embedded tissue (FFPE) collections in pathology departments are the largest resource for retrospective biomedical research studies. Based on the literature analysis of FFPE related research, as well as our own technical validation, we present the Translational Research Arrays (TRARESA), a tissue microarray centred, hospital based, translational research conceptual framework for both validation and/or discovery of novel biomarkers. TRARESA incorporates the analysis of protein, DNA and RNA in the same samples, correlating with clinical and pathological parameters from each case, and allowing (a) the confirmation of new biomarkers, disease hypotheses and drug targets, and (b) the postulation of novel hypotheses on disease mechanisms and drug targets based on known biomarkers. While presenting TRARESA, we illustrate the use of such a comprehensive approach. The conceptualisation of the role of FFPE-based studies in translational research allows the utilisation of this commodity, and adds to the hypothesis-generating armamentarium of existing high-throughput technologies.     

Broadsheet. Number 50: Diagnosis of human cytomegalovirus infection and disease.

Human cytomegalovirus (CMV) remains an important cause of illness in immunocompromised individuals and the most common viral cause of congenital malformation. The tests available for diagnosis of CMV include serology, antigen detection, virus culture, tissue histopathology and nucleic acid detection. The diagnosis of CMV remains difficult because of the issues of virus latency, virus infection versus clinical disease and virus reactivation. The tests available and the use of these tests are undergoing significant changes. This Broadsheet presents a review of these tests, particularly in the diagnosis of congenital infection and infection in pregnant women and immunocompromised individuals.     

Nuclear organization of some immunohistochemically identifiable neural systems in two species of the Euarchontoglires: A Lagomorph,Lepus capensis,and a Scandentia,Tupaia belangeri

The present study describes the organization of the nuclei of the cholinergic, catecholaminergic, serotonergic and orexinergic systems in the brains of two members of Euarchontoglires, Lepus capensis and Tupaia belangeri. The aim of the present study was to investigate the nuclear complement of these neural systems in comparison to previous studies on Euarchontoglires and generally with other mammalian species. Brains were coronally sectioned and immunohistochemically stained with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. The majority of nuclei revealed in the current study were similar between the species investigated and to mammals generally, but certain differences in the nuclear complement highlight potential phylogenetic interrelationships within the Euarchontoglires and across mammals. In the northern tree shrew the nucleus of the trapezoid body contained neurons immunoreactive to the choline acetyltransferase antibody with some of these neurons extending into the lamellae within the superior olivary nuclear complex (SON). The cholinergic nature of the neurons of this nucleus, and the extension of cholinergic neurons into the SON, has not been noted in any mammal studied to date. In addition, cholinergic neurons forming the medullary tegmental field were also present in the northern tree shrew. Regarding the catecholaminergic system, the cape hare presented with the rodent specific rostral dorsal midline medullary nucleus (C3), and the northern tree shrew lacked both the ventral and dorsal divisions of the anterior hypothalamic group (A15v and A15d). Both species were lacking the primate/megachiropteran specific compact portion of the locus coeruleus complex (A6c). The nuclei of the serotonergic and orexinergic systems of both species were similar to those seen across most Eutherian mammals. Our results lend support to the monophyly of the Glires, and more broadly suggest that the megachiropterans are more closely related to the primates than are any other members of Euarchontoglires studied to date.     

Sensitive and high sensitivity next generation cardiac troponin assays: more than just a name

In the 20 years that cardiac troponin testing has been available in clinical laboratories, the biomarker has revolutionised testing of patients with acute coronary syndromes. Cardiac troponin I and T testing has become the cornerstone for diagnosis of myocardial infarction and is useful for risk assessment and management of suspected acute coronary syndrome patients. As evidence and knowledge have evolved, it has become clear that even small troponin elevations are associated with adverse health outcomes. As a result there have been several generations of troponin assays, all toward tests that reliably detect lower concentrations of this critical analyte. Guidance for cardiac troponin interpretation has been in the form of myocardial infarction redefinition and evidence-based clinical and analytical guidelines. Although terminology naming generations for cardiac troponin assays has been inconsistent, state-of-the-art cardiac troponin assays are generally referred to as 'sensitive' assays and are in general compliance with analytical guidelines. Evidence shows that use of a sensitive troponin assay can result in diagnosis of myocardial infarction earlier. Next generation cardiac troponin I and T assays will likely be termed 'high sensitivity'; these assays should have the ability to measure troponin with a CV of total error of <10% at concentrations significantly lower than the 99 percentile of the normal reference population. As such, these assays should reliably measure troponin in most normal individuals and detect troponin changes (delta values) below the 99 percentile. This property may result in earlier ACS diagnosis and better management. Utilisation of high sensitivity troponin measurements may be useful for applications other than acute coronary syndromes including risk stratifying patients with renal insufficiency, heart failure, cardiac amyloid and screening elderly patients.     

Mutations in rpoB gene and rifabutin susceptibility of multidrug-resistant Mycobacterium tuberculosis strains isolated in Australia.

Control of tuberculosis, the single largest killer among the infectious diseases, has been threatened by the emergence of multidrug-resistant Mycobacterium tuberculosis (MDRTB) infection due to the limited treatment options. Rifampicin (RIF) resistance is considered as a marker for MDRTB. The aim of this study was the detection of rpoB gene mutations and rifabutin resistance in MDRTB strains recently isolated in Australia by a line probe assay (INNO-LiPA Rif. TB, Innogenetics). Rifabutin and RIF susceptibility of 20 MDRTB and 16 RIF-sensitive M. tuberculosis complex clinical isolates were studied. The overall concordance of the line probe assay (LiPA) with phenotypic RIF susceptibility test was 96%. Seven distinct nucleotide substitutions were identified in 21 of 22 RIF-resistant isolates of diverse geographical origins, but in none of the RIF-sensitive strains. The majority (71%) of mutations occurred in the 526-533 codons and were associated with resistance to rifabutin and RIF. Of the RIF-resistant MDRTB strains, 18% appeared to be rifabutin-sensitive and produced delta S2 and delta S3 INNO-LiPA patterns. We conclude that amino acid substitutions at Asp516 and Ser522 in the rpoB gene in RIF-resistant M. tuberculosis predict rifabutin susceptibility for MDRTB. Use of the LiPA for RIF and rifabutin resistance may facilitate the rapid response required to limit the extent and severity of MDRTB transmission and infection.     

Cytotoxicity evaluation and antioxidant enzyme expression related to heavy metals found in tuna by-products meal: An in vitro study in human and rat liver cell lines

Heavy metals can accumulate in organisms via various pathways, including respiration, adsorption and ingestion. They are known to generate free radicals and induce oxidative and/or nitrosative stress with depletion of anti-oxidants. Tuna by-product meal (TBM) is rich in proteins and can, therefore, offer an attractive protein source for animals. This study was undertaken to assess the effects of metals present in TBM, namely cadmium (Cd), lead (Pb), and mercury (Hg), separately or in combination with oxidative stress, on cell viability. Three cell models: rat liver FTO2B, human hepatoma HepG2, and human hepatic WRL-68, were used. Cell viability was determined following exposure to various concentrations of the metals. Two antioxidant genes, catalase (CAT) and superoxide dismutase (SOD), were measured to obtain a better understanding of oxidative stress-associated gene expression. Among the metals present in TBM, only Cd at a concentration of 30 μM was noted to exhibit cytotoxic effects. This cytotoxicity was even more pronounced after co-stimulation with H₂O₂, used to mimic systemic oxidative stress. At non-toxic concentrations, Hg and Pb were noted to aggravate oxidative stress toxicity. The results further revealed that exposure to Cd, Pb, and a co-stimulation of H₂O₂ with Hg resulted in the increased expression of antioxidant gene SOD. A risk assessment of toxic contaminants in TBM indicated that food safety objectives should consider the human health impacts of foods derived from animals fed on contaminated meal and that much care should be taken when TBM is used in animal diet.     

Cellular therapy to treat haematological and other malignancies: progress and pitfalls

The recent Food and Drug Administration (FDA) approval of a cellular therapy to treat castration resistant prostate cancer has reinforced the potential of cellular therapy to consolidate current pharmacological approaches to treating cancer. The emergence of the cell manufacturing facility to facilitate clinical translation of these new methodologies allows greater access to these novel therapies. Here we review different strategies currently being explored to treat haematological malignancies with a focus on adoptive allogeneic or autologous transfer of antigen specific T cells, NK cells or dendritic cells. These approaches all aim to generate immunological responses against overexpressed tissue antigens, mismatched minor histocompatability antigens or tumour associated antigens. Current successes and limitations of these different approaches will be discussed with an emphasis on challenges encountered in generating long term engraftment, antigen selection and implementation as well as therapeutic immune monitoring of clinical responses, with examples from recent clinical trials.     

Complex phenotypes in the haemoglobinopathies: recommendations on screening and DNA testing

This document considers a number of scenarios involving complex haemoglobinopathies and provides 28 recommendations at both the clinical and laboratory levels on how these should be managed.     

The epigenomics revolution in myelodysplasia: a clinico-pathological perspective

Rapid advances in molecular technologies are continually re-shaping the way we view and understand the mechanisms driving oncogenesis. The last decade has witnessed unparalleled change in the biology and therapy of the myelodysplastic syndromes (MDS), a heterogeneous collection of clonal myeloid disorders characterised by ineffective haematopoiesis and susceptibility to acute leukaemia transformation. Pivotal studies demonstrating the positive effects of hypomethylating agents on clinical outcome have brought an 'epigenomics revolution' to this disease, emphasising the importance of epigenetic mechanisms to the underlying pathogenesis of MDS. One of the most important future challenges in the MDS field will be to determine whether epigenetic therapies can be made more 'targeted' through identification of biomarkers which define subsets of patients most likely to benefit from treatment. A wave of novel mutations have recently been reported in MDS and other myeloid disorders, several of which regulate endogenous methylation networks within cells (including TET2, DNMT3A, IDH and EZH2). The relevance of these lesions in being able to predict response to epigenetic modulators and their correlation with epigenetic signatures in MDS are beginning to emerge.     

Laboratory testing of anticoagulants: the present and the future

This review provides an update on laboratory testing and monitoring for existing and emerging anticoagulants, starting with an overview of haemostasis and the routine coagulation tests currently employed within most haemostasis laboratories, including the prothrombin time (PT)/international normalised ratio (INR) and the activated partial thromboplastin time (APTT). Current anticoagulant therapy and laboratory monitoring is then discussed in terms of benefits and limitations, followed by a similar brief discussion of the new and emerging anticoagulants. The main focus, however, is laboratory testing related to vitamin K antagonists, heparin, lepirudin and the new agents dabigatran etexilate and rivaroxaban. Although the newer agents do not require laboratory monitoring, laboratory testing will occasionally be required, and pathology laboratories should become proactive in developing appropriate strategies. The tests most likely to fulfill this role are the ecarin clotting time (or chromogenic alternatives), and the chromogenic anti-Xa assay. Nevertheless, the dilute Russell viper venom time (dRVVT) assay may provide another alternative, and existing routine tests are also likely to be utilised for the foreseeable future, potentially also for laboratory testing of the new anticoagulants, albeit perhaps in modified form.     

Serum lipoprotein(A) profiles in a Singaporean population.

Lipoprotein(a) [Lp(a)] is formed when apolipoprotein(a) is linked to low density lipoprotein (LDL)-cholesterol via a single disulfide bond. It is an independent risk factor for myocardial infarction and raised concentrations are associated with an increased risk of developing coronary artery disease. Singapore has a multi-racial population of 77% Chinese, 14% Malays and 7% Indians. Studies have shown that the Indians have significantly higher standardised mortality ratios (SMR) compared to the Chinese and the Malays. We measured serum Lp(a) concentrations in 803 healthy individuals recruited from the Multiphasic Health Screening Programme, using the Macra Lp(a) sandwich enzyme immunoassay kit (Strategics Diagnostics, Delaware, USA). Lp(a) concentrations were skewed in all three groups. Our population mean was 9.0 mg/dl, with 50th, 75th and 95th percentile values of 10.2, 19.8 and 43.1 mg/dl, respectively, which are lower than values reported from Caucasian populations (15.0, 29.0 and 60.0 mg/dl, respectively). Males had lower Lp(a) concentrations than females (P < 0.05). The Indian group had significantly higher concentrations (median 12.3 mg/dl) compared to their Chinese (median 9.6 mg/dl) and Malay (median 8.4 mg/dl) counterparts (P < 0.05). This could partly account for the higher SMR seen in the Indian population in Singapore. As serum Lp(a) concentrations are method- and population-dependent, we recommend that laboratories determine their own reference ranges by their method to avoid misclassification of the coronary heart disease (CHD) risk of patients.     

Viral regulators of complement activation: Structure,function and evolution

The complement system surveillance in the host is effective in controlling viral propagation. Consequently, to subvert this effector mechanism, viruses have developed a series of adaptations. One among these is encoding mimics of host regulators of complement activation (RCA) which help viruses to avoid being labeled as ‘foreign’ and protect them from complement-mediated neutralization and complement-enhanced antiviral adaptive immunity. In this review, we provide an overview on the structure, function and evolution of viral RCA proteins.     

Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium

We investigated the relationship between mouse taste bud development and innervation of the soft palate. We employed scanning electron microscopy and immunohistochemistry using antibodies against protein gene product 9.5 and peripherin to detect sensory nerves, and cytokeratin 8 and α-gustducin to stain palatal taste buds. At E14, nerve fibers were observed along the medial border of the palatal shelves that tracked toward the epithelium. At E15.5, primordial stages of taste buds in the basal lamina of the soft palate first appeared. At E16, the taste buds became large spherical masses of columnar cells scattered in the soft palate basal lamina. At E17, the morphology and also the location of taste buds changed. At E18–19, some taste buds acquired a more elongated shape with a short neck, extending a variable distance from the soft palate basal lamina toward the surface epithelium. At E18, mature taste buds with taste pores and perigemmal nerve fibers were observed on the surface epithelium of the soft palate. The expression of α-gustducin was demonstrated at postnatal day 1 and the number of pored taste buds increased with age and they became pear-shaped at 8 weeks. The percent of pored fungiform-like papillae at birth was 58.3% of the whole palate; this increased to 83.8% at postnatal day 8 and reached a maximum of 95.7% at 12 weeks. The innervation of the soft palate was classified into three types of plexuses in relation to taste buds: basal nerve plexus, intragemmal and perigemmal nerve fibers. This study reveals that the nerve fibers preceded the development of taste buds in the palate of mice, and therefore the nerve fibers have roles in the initial induction of taste buds in the soft palate.