Considerable rewards could be obtained from early identification of Type 2 diabetes mellitus (T2DM). One of the most obvious, as suggested in a recent report on diabetes’ global burden, would be better disease management. The report, by the University of East Anglia in the UK, concludes that “early investments into prevention and disease management may therefore be particularly worthwhile.”
Risk factors
Such perspectives are strengthened by evidence that the onset of T2DM can be delayed by behaviour modification. A study in the ‘British Medical Journal’ in 2007 noted that lifestyle changes could be “at least as effective as drug treatment” in slowing the onset of diabetes. It concluded that the only barrier to the effectiveness of such a strategy was to identify diabetes quickly enough.
Much is now known about the risk factors associated with T2DM such as parental history, age, body mass index and elevated blood glucose levels. Combining these with measurable indicators of metabolic syndrome – high blood pressure, LDL and HDL cholesterol and excess triglyceride – can result in a credible degree of prediction. However, there are several barriers to the process.
Fasting glucose and oral glucose tolerance
The typical method for assessing T2DM risk is to measure fasting plasma glucose (FPG). However, the test’s specificity is poor. Two decades ago, the so-called Hoorn study at Amsterdam warned about significant levels of variation in blood glucose levels. Although many individuals are identified as having impaired fasting glucose (IFG), their absolute risk of conversion to diabetes is a mere 5 to 10% per year.
Over this period, differences have also emerged about how best to measure glucose. In the year 2000, while some experts (including the American Diabetes Association) recommended the use of fasting plasma glucose (FPG) alone, others noted that many diabetic subjects would have been classified as non-diabetic on the FPG test. As a result, they recommended use of the two-hour oral glucose tolerance test (OGTT). Nevertheless, in spite of its greater accuracy, OGTT is rarely used since it requires two hours to perform and is an unpleasant experience for the patient.
Glucose tolerance only one risk indicator
The above factors have provoked a search for new approaches to predict T2DM. Some beliefs about OGTT have been brought into question, too. In 2002, clinical epidemiologists at the University of Texas Health Center in San Antonio published the results of a prospective cohort study to identify people at high risk of T2DM.
The results were unequivocal. Impaired glucose tolerance was only one indicator of risk. Persons at high risk for T2DM, the study concluded, were “better identified by using a simple prediction model than by relying exclusively on the results of a 2-hour oral glucose tolerance test.”
Predictive models
Subsequent years have been witness to significant efforts to develop and refine predictive models for T2DM. However, five years after the San Antonio study, the choices are still less than wholly clear.
In 2007, the Framingham Offspring study in the US estimated seven-year T2DM risk based on a pyramid of metrics consisting – at the base – of age, sex, parental history and body mass index. This was followed by the inclusion of simple clinical measurements on metabolic syndrome traits, and thereafter, the 2-hour post-oral glucose tolerance test, fasting insulin and C-reactive protein levels. At its most complex, the model used the Gutt insulin sensitivity index or a homoeostasis model of insulin resistance.
For proponents of new alternatives to impaired glucose tolerance, the conclusions of the Framingham study were stark. Complex clinical models, it stated, were not superior to the simple one, and in spite of the definite existence of T2DM prediction rules, “we lack consensus for the most effective approach.”
The limitations of biotech
More recently, investigations at the frontiers of biotech have also faced challenges to clear-cut answers. Although it is clear that multiple genetic loci are associated with the risk of T2DM, researchers have not managed to connect the genetics underlying a family history of diabetes with predictability.
In 2008, researchers at Harvard/Massachusetts General and Emory University published results of a study on 18 single-nucleotide polymorphisms (SNPs) known to have associations with the risk of T2DM, to predict new cases in a large, prospectively examined, community-based cohort. However, the outcome, in terms of risk prediction, was less than encouraging. In reality, it proved to be only slightly better at making a prediction than did traditional risk factors on their own. The authors concluded: “Our findings underscore the view that identification of adverse phenotypic characteristics remains the cornerstone of approaches to predicting the risk of type 2 diabetes.”
Adiponectin and ferritin
Meanwhile, the effort to identify and validate alternate biomarkers for prediction and screening continue. Two especially promising ones appear to be adiponectin, an adipocyte-derived, insulin-sensitizing peptide, and ferritin, a protein that binds to iron and accounts for most of the iron stored in the body.
Studies in the early 2000s in the US and Germany confirmed that adiponectin was independently associated with a reduced risk of type 2 diabetes.
Interest in this area goes back a long time, to a cross-sectional and longitudinal study of Arizona’s Pima Indians, who have the world’s highest reported prevalence and incidence of non-insulin-dependent diabetes mellitus (NIDDM). The study dates to the early 1980s when it sought to document the sequence of metabolic events occurring with “the transition from normal to impaired glucose tolerance and then to diabetes.”
In 2004, a prospective study within the US Nurses’ Health Study investigated iron storage, given a belief that T2DM was a manifestation of hemochromatosis, due to iron overload. Researchers have established that higher iron store (reflected by an elevated ferritin concentration and a lower ratio of transferrin receptors to ferritin) is associated with increased T2DM risk in healthy women, independent of known diabetes risk factors.
However, there still are reasons for caution. In July 2014, or more than a decade after the US Nurses’ Health Study, a meta-analysis of T2DM risk and ferritin in the journal ‘Diabetes/Metabolism Research and Reviews’ warned that though evidence suggested a causal link, “publication bias and unmeasured confounding cannot be excluded.”
Nevertheless, ferritin and adiponectin do appear to play a key role in predicting T2DM when combined with other selected biomarkers.
The Danish model
One predictive model that has emerged in Denmark selected a panel of six biomarkers out of a total of 64, to assess T2DM risk. The selected biomarkers include adiponectin and ferritin, as well as four of their more common counterparts: glucose and insulin, as well as the inflammation markers C-reactive protein (CRP) and interleukin-2 receptor A (IL2RA).
The model was developed by a research team from Copenhagen’s Glostrup Hospital and Steno Diabetes Centre, along with the Copenhagen and Aarhus universities, and Tethys Bioscience of the US.
The researchers used the so-called Inter99 cohort, a study of about 6,600 Danes with the primary outcome of 5-year conversion to T2DM, to select 160 individuals who developed T2DM and 472 who did not. They carefully measured several clinical variables and candidate biomarkers from a multitude of diabetes-associated pathways, using an ultrasensitive immunoassay microsample molecular counting technology.
Their effort ultimately led to six biomarkers that gave a Diabetes Risk Score. This, they concluded in a July 2009 issue of ‘Diabetes Care’, provided “an objective and quantitative estimate of the 5-year risk of developing type 2 diabetes, performs better than single risk indicators and a noninvasive clinical model, and provides better stratification than fasting plasma glucose alone.”
Expert acclaim
The researchers who developed the Danish Diabetes Risk Score are modest in their claims. In an appendix to their report in ‘Diabetes Care’, they point out that their selection process for biomarkers may not have identified the best possible model, but do state that they identified a ‘good’ model.
Some outside observers are however less circumspect, given what many acknowledge to be one of the most exhaustive and profound selection efforts to date. James Meigs of Harvard Medical School calls the Danish Diabetes Risk Score “the most robust multimarker prediction model possible.”
Beyond Europeans to Chinese
One of the only major caveats in the Danish effort consisted of demographics. The report on the Danish model in ‘Diabetes Care’ noted that it “may only apply to white Northern Europeans enrolled in a lifestyle intervention trial” and that it was an open question whether the model “would produce the same biomarkers or discriminate well in race/ethnicity populations that are differentially affected by diabetes.”
Answers to these are still emerging. In 2013, a study on 2,198 community-living Chinese by the Shanghai Institutes for Biological Sciences endorsed the use of ferritin as a biomarker. Though the focus of the research was on iron storage, two of three other biomarkers used in the effort were the same as those in the Danish study, namely adiponectin and CRP (the fourth was γ-glutamyltransferase).
Biomarker search continues
Meanwhile, the search for TD2M biomarkers continues.
Two endothelial dysfunction biomarkers being investigated for T2DM risks consist of E-selectin and ICAM-1. The US Nurses Health Study mentioned above also found that significantly elevated levels of the latter predicted incident diabetes in women independent of traditional risk factors such as BMI, family history, diet and activity. In addition, adjustment for baseline levels of C-reactive protein, fasting insulin, and hemoglobin A (1c) did not alter these associations.
Incretins and melatonin
Incretins, metabolic hormones which lower blood glucose by causing an increase in insulin after eating, are another potentially significant biomarker. An ‘incretin effect’ is associated with the fact that oral glucose elicits a higher insulin response than does intravenous glucose. There are two hormones responsible for the incretin effect: glucose-dependent insulinotropic hormone (GIP) and glucagon-like peptide-1 (GLP-1).
In patients with type 2 diabetes, the incretin effect is reduced. In addition, about half first-degree relatives of patients with T2DM show reduced responses toward GIP, without any significant change in GIP or GLP-1 secretion after oral glucose. To some researchers, this opens the possibility that a reduced responsiveness to GIP is an early step in the pathogenesis of type 2 diabetes.
Variation in the Circadian system has also drawn a great deal of attention.
Reverse transcription polymerase chain reaction (RT-PCR) analyses, led by a team at the University of Lille in France, investigated melatonin receptor 2 (MT2 transcripts) in neural tissues and MT2 expression in human pancreatic islets and beta cells. Their findings suggest a link between circadian rhythm regulation and glucose homoeostasis through the melatonin signalling pathway.
Point-of-care testing (POCT) enables quick test results with minimal manual interference nearer to the site of patient care, which leads to better health outcomes via rapid diagnosis, quick clinical decisions and the early start of treatment. The emerging technologies would further improve POCT by low-cost analysis with increased performance characteristics.
by Dr Sandeep Kumar Vashist and Prof. John H.T. Luong
Existing point-of-care testing (POCT) technologies
A variety of POCT technologies are being used such as POCT analysers, biosensor devices, lab-on-chips (LOC), test strips, and lateral flow assay (LFA) cartridges. Such cost-effective technologies offer rapid analysis in just a few minutes using minimal sample volumes. As well as at a patient’s bedside, POCT have been employed in the operating theatre, emergency department and critical care/maternity unit. Other deployments include nursing homes, physician’s office, prison, emergency vehicles, etc. Local pharmacies have adopted the POCT technology to provide a one-stop service for glucose, cholesterol, pregnancy, etc.
The POCT analysers are standard bench-top devices that can determine a broad range of analytes, based on spectrophotometry, reflectometry, immunoassay, turbidimetry, potentiometry/amperometry, oximetry and hematological particle counting. The target analytes are small metabolites, enzymes, drugs-of-abuse, inflammation biomarkers, heart and kidney injury biomarkers, infectious agents, humoral and cellular coagulation markers, hematological parameters, etc.
The biosensor-based devices, notably the blood glucose meters, are the most common POCT technology, which have been widely used for the detection of glucose. Different LOC platforms are being used in various POCT technologies. They are fully automated platforms that integrate all microfluidics-based bioanalytical steps such as sample treatment, separation, biomolecular detection, washing, signal detection, and data processing, storage, and transmission. The signal detection in most LOC platforms employs optical readout. The technologies and materials used for the production of LOC platforms have been fully characterized and standardized.
Test strips are another prominent POCT technology for detecting different analytes in a patient’s blood or urine sample. They are easy to use and easy to read, leading to immediate on-the-spot analysis. The test strip comprises a solid support onto which porous matrices with dried assay reagents are integrated. The reaction starts as the biorecognition element present on the test strip detects the analyte, which leads to a visual change in colour on the test strip. The signal is read by inserting the test strip into a reader device.
LFA, one of the most widely used POCT technologies, is based on an immunochromatography format where the assay reagents are stored in dried form on various porous materials. Once the sample (urine or diluted blood) is dispensed at the designated area of the LFA cartridge, the sample flows in the lateral direction by capillary forces. It first interacts with the capture antibodies spotted at the reaction area leading to the formation of the immune complex, which is followed by binding to the detection antibodies immobilized at another area of the cartridge, thereby resulting in the formation of the sandwich immune complex. This leads to a visible colour change in the test and control lines, which facilitates rapid qualitative or semi-quantitative analysis. The POC pregnancy testing is done exclusively by LFA.
The multiplex analysis using DNA and protein microarrays is also being intensively investigated although the technology has not yet been commercialized for clinical POCT. It is envisaged that this upcoming technology would be fully automated, which would involve advanced microfluidics and the signal readout by electrochemical, chemiluminescence, fluorescence or evanescent wave techniques.
Emerging POCT technologies
Various POCT technologies have emerged during the last decade, which have tremendous potential for next-generation healthcare monitoring and management [1]. In 2011, the estimated total POCT market was US$15 billion and projected to reach US$18 billion by 2016. Of the total POCT market in 2011, 55% of it was in the US market, followed by 30% in Europe and 12% in Asia [2].
Cellphone (CP)-based devices
The most prospective emerging technology is CP-based devices. CPs have become ubiquitous with more than 7 billion global users that account for more than 95% of the world’s population. Moreover, about 70% of the CP users reside in the developing countries, where there is an imminent need for mobile healthcare (mH). The current generation of CPs are cost-effective and equipped with all the desired advanced features that facilitate personalized mH monitoring and management. The spatiotemporal tagging of the data by CP enables the real-time active response to epidemics and emergency situations. Various FDA approved and CE certified CP-based personalized healthcare devices have already been commercialized for the monitoring of basic physiological parameters such as blood glucose, blood pressure, pulse rate, blood oxygen saturation, body weight, body analysis parameters, electrocardiogram, physical activity, sleep and cardiac parameters (Fig. 1) [3]. Most of such commercial CP-based devices are developed by iHealth Labs, France. Similarly, CP-based technologies have been prepared for many POCT applications [4]. Cellmic, USA has developed a compact CP-based rapid-diagnostic-test reader for the readout of colorimetric and fluorometric LFA (Fig. 2). Of notice is the conversion of the CP into a compact and lightweight computational microscope for bright field, fluorescence, darkfield, transmission and polarized microscopy modes. Moreover, a CP-based flow cytometer based on optofluidic fluorescent imaging enabled the screening of pathogens in whole blood or water samples [5]. Another similar endeavour is the development of smartphone-based spectrophotometers for the detection of absorbance, fluorescent or chemiluminescent signals [6]. Various CP-based colorimetric readers [7], electrochemical sensing platform [8], angle-resolved surface plasmon resonance (SPR) system [9], and multiplex assays have also been developed [10].
Paper-based diagnostics
Considering simplicity and cost-effectiveness, paper-based diagnostics (PBD) are available in various formats: LFA, dipstick and microfluidic paper-based analytical devices (µPADs) [11, 12]. LFA are widely used in home pregnancy test strips to detect human chorionic gonadotropin in urine. It employs the dispensing of the sample onto the sample pad of the LFA test strip (fabricated from a nitrocellulose membrane). The sample flows laterally over a conjugate pad due to the capillary action provided by the absorbent pad, which leads to the binding of the analyte to conjugate particles (gold nanoparticles and upconversion nanoparticles). The signal detection can be visual, colorimetric, electrochemical, photoelectrochemical, chemiluminescent and electrochemiluminescent. The colorimetric PBD provide qualitative analysis by comparing the colour against a predetermined score chart. But the colour intensity can also be quantified using cameras, scanners, commercial test strip readers and hand-held colorimeters. A CP-based rapid-diagnostic-test reader is the most recent development that enables precise determination of colour intensity [13].
Paper can be patterned to fabricate two-dimensional (2D) or three-dimensional (3D) µPADs. The 3D µPADs, formed by stacking layers of the 2D paper, are ideal for multiplexing. The microfluidic paper-based electrochemical devices (µPEDs) can be fabricated by printing electrodes on paper.
The sensitivity of PBDs can be increased by employing enzymes and nanomaterials based signal enhancement strategies, which increases the costs and assay duration, and decreases the shelf-life. However, PBDs suffer from poor reproducibility, non-uniformity and variable accuracy on µPADs due to the passive capillary transport in paper substrates.
Lab-on-a-chip platforms
Various LOC platforms, such as the most widely used blood glucose testing strips, have been made for POCT. These platforms enable fully automated analysis by integrating all process steps in the operational procedure. The Piccolo Xpress™ whole blood chemistry analyser, developed by Abaxis Inc, is a prospective LOC-based POCT device that performs 14 tests on a single reagent LabDisk (8 cm diameter, barcoded).
Rapid assay formats
A prospective assay format is Optimiser™ ELISA [14] by Siloam Biosciences Inc, which employs a novel microfluidic microtiter plate. It detects an analyte in just a few minutes using minimal reagent volumes and least number of steps. Other prospective formats are the wash-free AlphaLISA® by Perkin Elmer, wash-free electrochemiluminescent ELISA by Meso Scale Diagnostics LLC, rapid one-step kinetics-based formats [15], CP-based easy immunoassay platforms [16], and COBAS® Lab-in-a-Tube (LIAT) system by Roche Diagnostics.
Prolonged reagent storage strategies
The most prospective prolonged reagent storage strategies are the use of polysaccharides and saccharides (such as pullulan and trehalose), sugar alcohols, stabilizers, freeze drying, lyophilization, and reagent pouches. Use of POCT in remote areas, particularly in developing countries might be problematic due to inconsistent electrical power, lighting, and refrigeration.
Conclusions
The next decade will witness the revolutionary breakthroughs in POCT that will drastically cut down the costs and lead to considerably improved analysis with increased bioanalytical performance and capabilities. Multi-channel, high-throughput instruments are expected to expand the new concept of POCT, and there is an obvious need for the combination of POCT technology and communication technology. Non-invasive blood glucose monitoring remains the most important market for POCT, followed by coagulation, blood gas, chemistry, hematology, urinalysis, and cardiac. Also, molecular POCT technology has matured and began to move toward commercialization.
Future trends for POCT will rise due to new emerging technologies to make them well suited for low-resource or remote areas. POCT vendors increasingly offer more available types of tests with improved accuracy and minimal turnaround times. Thus, POCT will be more widely accepted as an addition to the current method of managing patients. Considering its limited test menus, clinicians still have to wait for other sophisticated laboratory tests before the action can be taken for treatment or further testing [17]. POCT evolves continuously and becomes an ‘extension’ of laboratory services, not a replacement of routine core laboratory testing.
References
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The authors
Sandeep Kumar Vashist*1 PhD, John H.T. Luong2 PhD
1Vallo Med Health Care GmbH, Castrop-Rauxel, Germany
2Innovative Chromatography Group, Irish Separation Science Cluster (ISSC), Department of Chemistry and Analytical, Biological Chemistry Research Facility (ABCRF), University College Cork, Cork, Ireland
*Corresponding author
E-mail: sandeep.vashist@vallomed.com
Diagnosis of Pneumocystis jirovecii pneumonia (PCP) is conventionally based on direct staining and visualization. Challenges in obtaining alveolar samples have stimulated interest in techniques for detection of Pneumocystis DNA in non-invasive samples, which can give good sensitivity and specificity. Robust diagnosis is key to ensuring appropriate therapy.
by Dr Farnaz Dave, Dr Ashley Horsley, Dr Thomas Whitfield
and Dr Clare van Halsema
Introduction
Pneumocystis jirovecii (previously Pneumocystis carinii) is a pathogen capable of causing life threatening Pneumocystis pneumonia (PCP) in the immunocompromised with case fatality rates among those hospitalized of around 10% [1]. PCP typically occurs in individuals with hematological malignancies on chemotherapy or with other causes of acquired cellular immunodeficiency or, most frequently, in human immunodeficiency virus (HIV)-positive individuals with CD4 T-cell counts <200 cells/µL or <14% of total white cell count [2, 3]. First-line treatment is co-trimoxazole, a combination of the antibiotics sulfamethoxazole and trimethoprim, at high dose for 3 weeks, which has the clinically significant potential side effects of bone marrow suppression, rash and bronchial hypersensitivity. Unfortunately the classical clinical presentation of PCP of progressive dry cough, dyspnoea and malaise is non-specific and chest examination and radiographs are often normal or near normal [4]. Oxygen desaturation on exercise is a helpful clinical sign in the right patient population [5]. Furthermore, many individuals with PCP do not produce sputum, so laboratory confirmation can be challenging. Given the serious nature of the illness and the possible side effects of treatment, accurate diagnosis is key to making an informed treatment decision.
Incidence of PCP among HIV-positive individuals has declined since the widespread availability of antiretroviral therapy (ART) and has also declined as a cause of hospitalization of HIV-positive individuals [6]. In a study in a large HIV centre in London, mortality for all hospitalizations was around 10% during the first 10 years of availability of effective ART [1]. In our unit a decrease in mortality among those admitted to intensive care with PCP was seen between the periods of 1986–1995 and 1996–2004. This improvement is thought to be due to advances in critical care rather than treatment of PCP itself [7].
Reaching a diagnosis
Traditional methods
P. jirovecii is a fungus that cannot be cultured in vitro and so the organism is identified using histochemical staining techniques of fluid samples. Grocott-Gomori methenamine silver nitrate or direct immunofluorescence monoclonal antibody (IFA) stains on deep respiratory samples are generally regarded as the gold standard in diagnosis [8]. The life cycle of P. jirovecii is demonstrated by trophic, pre-cystic and cystic forms by morphological criteria. Diagnosis through microscopy of the cystic stage requires significant technical expertise and can still lead to false-negative results. In florid disease, P. jirovecii is present throughout the bronchial tree, from the upper respiratory tract down to the alveolar surface. Induced sputum samples are recommended by most guidelines, if routinely available, as spontaneously expectorated sputum is not considered an adequate alveolar sample and microscopy could be falsely negative. If the results of testing on induced sputum are not conclusive then a bronchoalveolar lavage (BAL) is recommended (sensitivity 86–98%) [9, 10]. In clinical practice, induced sputum is often not readily available, and BAL may not be possible due to hypoxia or may lead to a delay in sampling of several days; therefore, in clinical practice spontaneously expectorated samples are often processed. The disadvantages of this are twofold: a lower yield of cystic forms for visualization and potential for false-positive results due to colonization. Only in rare cases would a lung biopsy be appropriate (sensitivity 95–98%) in circumstances of poor response to empirical treatment and negative initial testing [9].
Molecular techniques
Molecular testing of lower respiratory tract secretions and blood is an alternative and operator-independent method for confirming the presence of P. jirovecii. Nucleic acid amplification techniques (NAAT) can be used with a number of primers targeting different substrates – most commonly the major surface glycoprotein (MSG), mitochondrial large subunit (MTLSU) rRNA and internal transcribed spacer (ITS) region genes [11]. One potential pitfall with these techniques is that the detection of specific nucleic acid sequence does not distinguish between colonization and disease or between viable and non-viable organisms [12]. P. jirovecii RNA is less stable and rapidly degraded after cell death so is a more reliable marker of viable organisms. Modification of standard PCR protocols with quantitative methods (e.g. quantitative touch-down PCR) may help to differentiate between colonization and infection through the selection of thresholds to maximize sensitivity [13]. An added advantage of these molecular techniques is they may provide information on molecular epidemiology and resistance-associated mutations in the gene encoding dihydropteroate synthase (DHPS), the target of sulfamethoxazole, though the benefit of this is controversial [14]. One caveat to this is the potential for point mutations in DNA paired with primer sequences and risk of false negatives as a result.
These molecular tests are said to have increased sensitivity compared with cyst staining techniques but variable specificity depending on the specimens used, the primer chosen and whether treatment has been started [15]. The three most commonly assessed specimen groups are sputa (ideally induced), oropharyngeal washes (OPW) and blood. The clinical relevance of the known detectability of P. jirovecii DNA in whole blood has not been fully established but could represent colonization as well as disease [16]. The use of cycle threshold values has been proposed as a method to distinguish colonization from disease using BAL samples, although further studies are needed to validate cut offs on different samples [17].
Although there has been a widespread adoption of NAATs the current British HIV Association guidance, and that of similar professional bodies, still suggests combining them where available with a traditional visualization technique as described previously and performing them on alveolar specimens where possible to increase sensitivity and specificity [10, 18].
PCP diagnosis by detection of DNA in non-respiratory samples
Due to the variability in sampling methods, the challenges in obtaining ideal samples and the need for prompt diagnosis research has been conducted on the use of NAATs on OPW and blood. Samples are relatively non-invasive, collection is straight-forward and no special equipment or preparation is required. A study in our unit compared NAATs on OPW and blood with sputum, spontaneously expectorated or induced, using primers for the P. jirovecii MTLSU rRNA gene [19].
All patients were consenting adults presenting to a regional infectious disease unit who were being investigated for PCP as part of routine care. A spectrum of patients was included of different pre-test probabilities to allow estimates of sensitivity and specificity. Each participant was asked to provide sputum (spontaneous or induced), OPW and blood for analysis. OPW was obtained by gargling of normal saline for 10 to 30 seconds without any additional preparation.
Forty-five participants were included, 41 male (91%), 38 Caucasian (84%) with a median age of 39 years. One participant was an HIV-negative renal transplant recipient. Forty-four were HIV-positive with a median CD4 count of 64 cells/mL. Thirty-five of the 44 were not on ART with a median HIV RNA of 164 550 copies/mL. Thirty-nine of the 45 started empirical treatment for PCP a median of 2 days before sampling. We compared the sensitivity and specificity of tests on blood and OPW compared with sputum. Sputum PCR was positive in 60% of participants and in this group 47% of OPW and 50% of blood PCRs were positive. None with negative sputum PCR had positive OPW or blood PCR. A diagnosis of PCP could be reached in 14 of 16 patients with positive NAAT on sputum using these non-respiratory specimens.
Among those with P. jirovecii DNA detected in sputum a sensitivity of 47% for OPW was increased to 80% when considering only OPW samples taken within 48 hours of starting treatment. When this was combined with blood sample testing in the same time frame the sensitivity increased to 88%, which is comparable to that quoted in previous similar studies [12, 13, 15]. There were no false positives based on no OPW or blood PCR positives in those with negative PCR on sputum. As the laboratory techniques used were routine, few additional skills or resources were required.
Overall, using molecular tests on non-respiratory samples was of diagnostic benefit and show potential for savings in time and resources. The molecular tests provide excellent specificity and good sensitivity comparable with sputum without proceeding to time-consuming and invasive tests [20]. However, in view of uncertainty regarding the specificity of testing these non-invasive samples at all, results must be interpreted with care and in the right clinical context.
Conclusions
PCP diagnosis remains a combination of clinical suspicion and physical examination, supported by radiological and microbiological investigations. Using a combination of traditional microscopy with staining and NAAT on appropriate specimens, plus interpretation of results in the clinical context a clear diagnosis can be reached in most cases and this may prevent unnecessary treatment. Using non-respiratory specimens taken early to maximize sensitivity could reduce the requirement for invasive testing or diagnostic uncertainty.
Future developments
We expect the use of NAATs to become even more widely available and useful diagnostic aids alongside traditional techniques. With a plethora of protocols the sensitivity, specificity and utility of these will improve further over time. Combination with other laboratory techniques such as β-D-glucan may be similarly useful. Given the inability to culture the organism and so look for in vitro susceptibility to sulfamethoxazole-based treatment, molecular methods for detecting mutations and potential resistance may develop as a routinely used test.
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9. Broaddus C, Dake MD, Stulbarg MS, Blumenfeld W, Hadley WK, Golden JA, Hopewell PC. Bronchoalveolar lavage and transbronchial biopsy for the diagnosis of pulmonary infections in the acquired immunodeficiency syndrome. Ann Intern Med. 1985; 102: 747–752.
10. Nelson M, Dockrell D, Edwards S; BHIVA Guidelines Subcommittee, Angus B, Barton S, Beeching N, Bergin C, Boffito M, et al. British HIV Association and British Infection Association guidelines for the treatment of opportunistic infection in HIV-seropositive Individuals 2011. HIV Med. 2011; 12(Suppl 2): 1–140.
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13. Larsen H, Huang L, Kovacs J, Crothers K, Silcott V, Morris A, Turner J, Beard C, Masur H, Fischer S. A prospective, blinded study of quantitative touch-down polymerase chain reaction using oral-wash samples for diagnosis of Pneumocystis pneumonia in HIV-infected patients. J Infect Dis. 2004; 189: 1679–1683.
14. Durand-Joly I, Chabé M, Fabienne Soula F, Delhaes L, Camus D, Dei-Cas E. Molecular diagnosis of Pneumocystis pneumonia. FEMS Immunol Med Microbiol. 2005; 45: 405–410.
15. Olsson M, K. Strålin K, Holmberg H. Clinical significance of nested polymerase chain reaction and immunofluorescence for detection of Pneumocystis carinii pneumonia. Clin Microbiol Infect. 2001; 7: 492–497.
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19. van Halsema C, Johnson L, Baxter J, Douthwaite S, Clowes Y, Guiver M, Ustianowski A. Diagnosis of Pneumocystis jirovecii pneumonia by detection of DNA in blood and oropharyngeal wash, compared with sputum. AIDS Res Hum Retroviruses 2016; 32: 463–466.
20. de Oliveira A, Unnasch T, Crothers K, Eiser S, Zucchi P, Moir J, Beard C, Lawrence G, Huang L. Performance of a molecular viability assay for the diagnosis of Pneumocystis pneumonia in HIV-infected patients. Diagn Microbiol Infect Dis. 2007; 57: 169–176.
The authors
Farnaz Dave MBChB, MRCP; Ashley Horsley MBChB, MRCP; Thomas Whitfield MBChB, MSc, MRCP; Clare van Halsema* MBChB, MRCP, MD, DipHIVMed
North West Infectious Diseases Unit, North Manchester
General Hospital, Manchester M8 5RB, UK
*Corresponding author
E-mail: clare.vanhalsema@pat.nhs.uk
When Dr Margaret Chan, Director-General of WHO, stated that the health risks for both participants and spectators at the Rio Olympic games were “low and manageable” she was referring to possible exposure to Zika virus. But the other major health concern raised was the quality of the water, particularly for the one to two thousand athletes who competed in aquatic events.
Both the CDC and WHO provided advice on managing the risk of Zika virus infection whilst in Brazil, including use of insect repellent and wearing light clothing covering most of the body. Visitors were also urged to stay in air-conditioned accommodation so that open windows would not admit mosquitoes, and to avoid impoverished areas where lack of suitable sanitation encourages Zika vectors to breed. Abstention or the correct and consistent use of condoms was also advocated whilst in Brazil and for at least eight weeks after returning. Unfortunately we now know that the virus can persist in semen for much longer than eight weeks; two recent cases in men who contacted symptomatic Zika infection (and around 80% of cases are estimated to be asymptomatic) still had virus in their semen after 181 and 188 days respectively.
There was a rather bizarre occurrence during the games, when water in certain dedicated swimming pools turned from blue to green overnight. However the Fédération Internationale de Natation (FINA) Sports Medicine Committee confirmed that this resulted from “some of the chemicals used in the water treatment process running out”, causing the pH to be outside the normal range. FINA’s assurance that there was no risk to the health and safety of athletes was trusted; hopefully there was sufficient supporting evidence. However the potentially most serious health threat was the quality of the natural recreational water used for many aquatic events, water that is still significantly contaminated with untreated sewage. Athletes and visitors were urged to have vaccinations such as typhoid and Hepatitis A before travelling to Brazil, to avoid swallowing recreational water and to shower after being exposed to it.
But the risks to visiting athletes and participants were hopefully only transient compared with the enduring health hazards faced daily by the poorer citizens of Brazil. The best possible legacy of the Rio Olympic games would not be an increased interest in athletics but rather continued intensive Aedes mosquito control, widely disseminated information on sexual and vertical transmission of Zika virus, the sustained treatment of raw sewage and the provision of safe drinking water.
Pre-eclampsia is a major cause of maternal and perinatal mortality. Scientific advances in recent decades have meant new possibilities for enhancing the prediction and diagnosis of pre-eclampsia. Here we detail current biomarker-based approaches under development or validated for clinical translation that will revolutionize obstetric practice in the years ahead.
by Dr Kirsten Palmer1, Associate Professor Fabricio da Silva Costa1
Pre-eclampsia
Pre-eclampsia affects 3–8% of all pregnancies and is a major cause of maternal and perinatal morbidity and mortality. Globally, pre-eclampsia is responsible for over 60 000 maternal deaths and greater than 500 000 neonatal deaths every year. It is a heterogeneous condition, likely having multiple underlying etiologies, producing a clinical syndrome typically characterized by maternal hypertension and multi-organ dysfunction, including fetoplacental, renal, hepatic, hematological and/or neurological dysfunction. Currently, the mainstay of treatment is delivery, with delivery of the placenta curing the condition. Although this is a reasonable option when the disease presents at term (37–42 weeks gestation), when pre-eclampsia arises prematurely delivery places the neonate at the significant risks of prematurity. As such, current medical approaches for preterm pre-eclampsia, especially if occurring <34 weeks gestation, are centred around close observation of both maternal and fetal well-being, aiming to prolong gestation towards term, but timing delivery to minimize risks for mother and child due to evolving pre-eclampsia. On average this approach will only see a gestational advancement of 7–14 days [1]. Therefore, these women often require care in highly specialized obstetric units with access to a neonatal intensive care unit. The ability to predict those women most at risk of developing pre-eclampsia, may afford the opportunity to commence preventative therapies, such as aspirin, but may also enable better allocation of healthcare models.
Pre-eclampsia had long been considered to solely be of concern during the time of pregnancy; however, it is now appreciated that it is also associated with negative long-term health implications for the mother and child. Women who had pre-eclampsia are at increased risk of cardiovascular disease and death in the decades that follow [2]. Similarly, children born from pregnancies affected by pre-eclampsia are also at greater risk of hypertension later in life [3]. As such, it is becoming increasingly important to accurately diagnose pre-eclampsia and provide ongoing long-term healthcare following birth to minimize the morbidity.
The pathophysiology of pre-eclampsia
Multiple phenotypes of pre-eclampsia exist. Currently, these are understood to be early-onset and late-onset forms of pre-eclampsia. Underlying abnormal placentation is a key component of early-onset disease, where the shallowly implanted placenta leads to ischemia-reperfusion injuries within the placenta. This impacts on placental gene expression leading to an upregulation of hypoxia-regulated genes including anti-angiogenic proteins, such as soluble fms-like tyrosine kinase 1 (sFLT-1) and soluble endoglin. sFLT-1, a soluble version of the vascular endothelial growth factor receptor 1, is able to bind and antagonize the actions of the angiogenic proteins, vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). These proteins are essential in maintaining endothelial homeostasis and a reduction in their bioavailability produces the widespread end-organ endothelial dysfunction that yields the clinical pre-eclamptic phenotype.
In comparison, late-onset pre-eclampsia is thought to result from pre-existing endothelial dysfunction, such as exists in women with chronic hypertension, obesity and diabetes. In normal pregnancy, sFLT-1 within the maternal circulation gradually rises across gestation. In women with pre-existing endothelial dysfunction, this normal rise in sFLT-1 will lead to a worsening of endothelial function, which may tip the balance towards the clinical development of term pre-eclampsia.
With the discovery of sFLT-1 in 2003 [4], pre-eclampsia research has been firmly focused both on better prediction and diagnosis of the disease, but also the development of therapeutics. The use of anti-angiogenic and angiogenic biomarkers for pre-eclampsia is now entering into clinical use or in the process of translation to improve prediction and diagnosis across pregnancy.
Predicting pre-eclampsia during the first trimester
First-trimester screening for fetal aneuploidy is the most commonly employed test in early gestation for the prediction of later pregnancy complications, namely, delivery of an infant with a chromosomal anomaly. The principles underpinning these multiparametric tests have informed the development of screening strategies using multiple biomarkers in early gestation for the prediction of other complications, such as pre-eclampsia [5].
In 2009, researchers from the Fetal Medicine Foundation (FMF; UK) evaluated a multiparametric test incorporating maternal factors, mean arterial pressure, uterine artery Doppler pulsatility index, circulating PlGF, and PAPP-A. It detected 93% of early-onset pre-eclampsia with a false positive rate (FPR) of 5% [6]. This approach has subsequently been externally validated producing similar rates of detection for early-onset pre-eclampsia (80.8–91.7%), but with a 10% FPR [7, 8].
Very recently, a prospective multicentre study of first-trimester screening for pre-eclampsia in singleton pregnancies was published [9]. The study population had 239 (2.7%) cases that developed pre-eclampsia, including 17 (0.2%), 59 (0.7%) and 180 (2.0%) at <32, <37 and >37 weeks, respectively. Using combined screening by maternal factors, mean arterial pressure, uterine artery pulsatility index and serum PlGF the detection rate was 100% (95% CI 80–100) for pre-eclampsia at <32 weeks, 75% (95% CI 62–85) at <37 weeks and 43% (95% CI 35–50) at >37 weeks, with a 10% FPR. As such, The FMF model is the most accurate and thoroughly validated algorithm available at 11–13 weeks to predict pre-eclampsia in a low-risk population.
A clear benefit to predicting women at high risk of pre-eclampsia in the first trimester is the opportunity it affords to institute preventative therapies. Currently, low dose aspirin is the only medication that appears to reduce the rate of pre-eclampsia in high-risk women, with the results of the ASPRE study (Aspirin for evidence-based PRE-eclampsia prevention) [10], a European multicentre randomized controlled trial, eagerly awaited to define how effective preventative aspirin truly is. This trial applied the FMF screening model in 30 000 women at 11–13 weeks gestation, with those at increased risk of pre-eclampsia randomly assigned to aspirin (n=798) or placebo (n=822). However, the FMF algorithm cannot detect all cases of preterm pre-eclampsia and detection of term disease was poor, furthermore, aspirin cannot prevent all cases. Thus, further studies with novel biomarkers to improve screening (especially for term pre-eclampsia) and new treatments are needed to reduce the global burden of this disease.
Predicting pre-eclampsia later in pregnancy
Circulating anti-angiogenic factor levels are often below the detection limit of available assays during the first trimester; however, both sFLT-1 and soluble endoglin levels rise as gestation advances and are significantly raised in the maternal circulation, while PlGF levels are significantly reduced, weeks before the clinical development of pre-eclampsia [11, 12]. These findings have prompted widespread study into the use of these biomarkers in predictive algorithms for pre-eclampsia. PlGF appears the most useful biomarker in first-trimester screening, as well as in the second trimester either on its own or in a ratio with sFLT-1. Automated systems able to rapidly process these biomarkers are already available and the utility of PlGF and sFLT-1 has now been assessed in multiple trials. PlGF appears the most accurate biomarker, capable of detecting approximately 75% of women who will develop early-onset pre-eclampsia [13], whereas the sFLT-1:PlGF ratio appears to perform well as a negative predictor of early-onset pre-eclampsia [14].
Recently, an assay able to detect a placental-specific variant of sFLT-1, known as sFLT-1 e15a, has been developed [15]. This may provide improved positive predictive performance in predicting who will develop pre-eclampsia. As anticipated, total sFLT-1 and sFLT-1 e15a, are most useful in predicting early-onset disease [16], in which abnormal placental pathology is central to disease development. However, measurement in the third trimester and assessing the longitudinal change in expression may be useful for the prediction of late-onset pre-eclampsia [17]. Certainly, total sFLT-1, as well as PlGF and maternal factors currently appear the most promising for predicting term disease; however, none have been validated or perform favourably enough for translation to clinical
practice [18].
Improving the diagnosis of pre-eclampsia
The use of angiogenic and anti-angiogenic biomarkers may provide significant improvement for the accurate diagnosis of pre-eclampsia itself. Current markers used to diagnose pre-eclampsia consist of a history of pre-eclamptic symptoms (such as headache, visual disturbance or epigastric pain), physical signs (such as high blood pressure, hyper-reflexia or tender liver edge) and biochemical markers (such as proteinuria; elevated liver transaminases, uric acid or creatinine; or thrombocytopenia). However, these can also be present in other pre-existing medical conditions, such as renal disease, which can make the accurate diagnosis of pre-eclampsia a challenge.
PlGF currently appears the most promising biomarker for improving the accurate diagnosis of pre-eclampsia, even in the setting of pre-existing renal disease or chronic hypertension [19]. PlGF outperforms our current diagnostic approach for pre-eclampsia, particularly for disease occurring <35 weeks gestation. However, the challenge remains to find an accurate diagnostic test to identify late-onset pre-eclampsia and improve test performance to minimize the FPR.
Conclusion
The last 15 years has seen a rapid increase in our knowledge of the pre-eclamptic process enabling development of promising new approaches to disease prediction and diagnosis. However, the pathway to translation of these tests into widespread clinical use has been slowed by the heterogeneity of pre-eclampsia itself, as it is likely that no one test or approach will work for all forms of pre-eclampsia. New approaches and ongoing progression in our understanding of the disease process provide hope that our clinical approach to pre-eclampsia will change significantly in the years ahead, hopefully to the betterment of the women and infants we care for.
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The authors
Kirsten Palmer1 MBBS, PhD; Fabricio da Silva Costa1 MD, PhD
1Department of Obstetrics and Gynaecology, Monash University,
Monash Medical Centre, Clayton 3168,
Victoria, Australia
*Corresponding author
E-mail: kirsten.palmer@monash.edu
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