A framework for clinical evaluation of diagnostic technologies.

Most new diagnostic technologies have not been adequately assessed to determine whether their application improves health. Comprehensive evaluation of diagnostic technologies includes establishing technologic capability and determining the range of possible uses, diagnostic accuracy, impact on the health care provider, therapeutic impact and impact on patient outcome. Guidelines to determine whether each of these criteria have been met adequately are presented. Diagnostic technologies should be disseminated only if they are less expensive, produce fewer untoward effects and are at least as accurate as existing methods, if they eliminate the need for other investigations without loss of accuracy, or if they lead to institution of effective therapy. Establishing patient benefit often requires a randomized controlled trial in which patients receive the new test or an alternative diagnostic strategy. Other study designs are logistically less difficult but may not provide accurate assessment of benefit. Rigorous assessment of diagnostic technologies is needed for efficient use of health care resources.     

A sensitive proximity ligation assay for active PSA

Prostate-specific antigen (PSA) is a widely used marker for prostate cancer. The utility of PSA tests is limited by their inability to differentiate prostate cancer from non-malignant conditions such as benign prostatic hyperplasia and prostatitis. In circulation, PSA occurs in various complexed and free forms, and specific determination of some of these can be used to improve the diagnostic accuracy of PSA tests. We have previously identified peptides that specifically bind to enzymatically active PSA and using such a peptide we have developed an immunopeptidometric assay for this form of PSA. However, the sensitivity of that assay is too low to measure active PSA at clinically important levels. Recently a novel sensitive immunoassay for analysis of proteins, termed the proximity ligation assay, has been established. Here we describe a sensitive implementation of the proximity ligation assay, which utilizes a PSA-binding peptide and antibody as probes to detect active PSA. The assay has a sensitivity of 0.07 microg/l, which is approximately ten-fold lower than that of our previous assay. It does not cross-react with inactive proPSA or the highly similar kallikrein hK2. Our results show that a highly sensitive immunopeptidometric assay can be developed using proximity ligation. This principle should facilitate establishment of specific assays for active forms of other proteases.     

Implementation science in resource-poor countries and communities

Background

Implementation science in resource-poor countries and communities is arguably more important than implementation science in resource-rich settings, because resource poverty requires novel solutions to ensure that research results are translated into routine practice and benefit the largest possible number of people.

Methods

We reviewed the role of resources in the extant implementation science frameworks and literature. We analyzed opportunities for implementation science in resource-poor countries and communities, as well as threats to the realization of these opportunities.

Results

Many of the frameworks that provide theoretical guidance for implementation science view resources as contextual factors that are important to (i) predict the feasibility of implementation of research results in routine practice, (ii) explain implementation success and failure, (iii) adapt novel evidence-based practices to local constraints, and (iv) design the implementation process to account for local constraints. Implementation science for resource-poor settings shifts this view from “resources as context” to “resources as primary research object.” We find a growing body of implementation research aiming to discover and test novel approaches to generate resources for the delivery of evidence-based practice in routine care, including approaches to create higher-skilled health workers—through tele-education and telemedicine, freeing up higher-skilled health workers—through task-shifting and new technologies and models of care, and increasing laboratory capacity through new technologies and the availability of medicines through supply chain innovations. In contrast, only few studies have investigated approaches to change the behavior and utilization of healthcare resources in resource-poor settings. We identify three specific opportunities for implementation science in resource-poor settings. First, intervention and methods innovations thrive under constraints. Second, reverse innovation transferring novel approaches from resource-poor to research-rich settings will gain in importance. Third, policy makers in resource-poor countries tend to be open for close collaboration with scientists in implementation research projects aimed at informing national and local policy.

Conclusions

Implementation science in resource-poor countries and communities offers important opportunities for future discoveries and reverse innovation. To harness this potential, funders need to strongly support research projects in resource-poor settings, as well as the training of the next generation of implementation scientists working on new ways to create healthcare resources where they lack most and to ensure that those resources are utilized to deliver care that is based on the latest research results.

     

Accuracy and economics of Helicobacter pylori diagnosis.

Many diagnostic tests are available to establish Helicobacter pylori infection status. Most of the tests are accurate though none works perfectly, and no gold standard for diagnosis exists. Newly developed serum immunoassay kits can substitute for laboratory-based enzyme-linked immunosorbent assays, but whole blood immunoassays do not yet demonstrate adequate performance characteristics. Serologic diagnosis of H. pylori remains the most cost-effective option and should be utilized to establish initial infection in the majority of cases. If rapid urease testing is performed at endoscopy, negative results can be confirmed with a subsequent serologic test in those patients with a high probability of infection. Obtaining additional gastric tissue at endoscopy to evaluate for bacterial infection is reasonable if specimens are being taken for a mucosal defect. Confirmation of bacterial eradication cannot be justified for all post-treatment patients at present due to the expense. It is important to test for cure in those patients with complicated ulcer disease and those with recurrent symptoms after therapy.     

Diagnostic validation of selected serological tests for detecting scrub typhus

Clinical diagnosis of scrub typhus is often difficult because the symptoms are very similar to those of other febrile illness such as dengue, leptospirosis, malaria and other viral hemorrhagic fevers. Though better diagnostic tests are available for rickettsial diseases and scrub typhus elsewhere, the Weil–Felix test is still commonly used in India, mainly because microimmunofluorescence assays (M‐IFA) were not available in India till recently and relevant staff had insufficient training. The present study was performed to investigate the performance of M‐IFA, IgM ELISA, and Weil–Felix test on 546 non‐repeated serum samples from subjects suspected of having scrub typhus. One hundred and forty‐three of these 546 samples were positive by M‐IFA; these cases were also confirmed clinically to have scrub typhus based on their dramatic responses to doxycycline therapy. IgM ELISA was positive in 122 of the 143 M‐IFA positive cases and the Weil–Felix test in 96. Though the Weil–Felix test is a heterophile agglutination test, it was found in this study to have good specificity but far too little sensitivity to use as a routine diagnostic test. IgM ELISA can be a good substitute for M‐IFA. Incorporation of multiple prototype antigens on M‐IFA slides is likely one of the reasons for its superior performance. As newer and better diagnostic assays become available for scrub typhus diagnosis in developed countries, it will be imperative to also use such tests in other endemic countries to prevent over‐ or under‐diagnosis of scrub typhus.     

Plasmonic-based platforms for diagnosis of infectious diseases at the point-of-care

Infectious diseases such as HIV-1/AIDS, tuberculosis (TB), hepatitis B (HBV), and malaria still exert a tremendous health burden on the developing world, requiring rapid, simple and inexpensive diagnostics for on-site diagnosis and treatment monitoring. However, traditional diagnostic methods such as nucleic acid tests (NATs) and enzyme linked immunosorbent assays (ELISA) cannot be readily implemented in point-of-care (POC) settings. Recently, plasmonic-based biosensors have emerged, offering an attractive solution to manage infectious diseases in the developing world since they can achieve rapid, real-time and label-free detection of various pathogenic biomarkers. Via the principle of plasmonic-based optical detection, a variety of biosensing technologies such as surface plasmon resonance (SPR), localized surface plasmon resonance (LSPR), colorimetric plasmonic assays, and surface enhanced Raman spectroscopy (SERS) have emerged for early diagnosis of HIV-1, TB, HBV and malaria. Similarly, plasmonic-based colorimetric assays have also been developed with the capability of multiplexing and cellphone integration, which is well suited for POC testing in the developing world. Herein, we present a comprehensive review on recent advances in surface chemistry, substrate fabrication, and microfluidic integration for the development of plasmonic-based biosensors, aiming at rapid management of infectious diseases at the POC, and thus improving global health.     

Clinical and diagnostic management of toxoplasmosis in the immunocompromised patient

With the advent of the highly active antiretroviral therapy (HAART), the natural course of HIV infection has markedly changed and opportunistic infections including toxoplasmosis have declined and modified in presentation, outcome and incidence. However, TE is a major cause of morbidity and mortality especially in resource-poor settings but also a common neurological complication in some countries despite the availability of HAART and effective prophylaxis. In most cases toxoplasmosis occurs in brain and toxoplasmic encephalitis (TE) is the most common presentation of toxoplasmosis in immunocompromised patients with or without AIDS. The need of a definitive diagnosis is substantial because other brain diseases could share similar findings. Rapid and specific diagnosis is thus crucial as early treatment may improve the clinical outcome. Classical serological diagnosis is often inconclusive as immunodeficient individuals fail to produce significant titres of specific antibodies. Polymerase chain reaction (PCR) has a high diagnostic value in the acute disease, but like many 'in-house' PCR assays, suffers from lack of standardization and variable performance according to the laboratory. Molecular diagnosis of toxoplasmosis can be improved by performing real-time PCR protocols. This article summarises the clinical manifestations, diagnostic procedures and management strategies for this condition.     

Real-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea.

Reliable methods for disease severity assessment are of crucial importance in the study of plant pathogen interactions, either for disease diagnostic on the field or to assess phenotypical differences in plants or pathogen strains. Currently, most of the assays used in fungal disease diagnostic rely on visual assessment of the symptoms, lesion diameter measurement or spore counting. However, these tests are tedious and often cannot discriminate between slightly different levels of resistance. Besides, they are not well suited to assess fungal development in the early phases of the infection, before macroscopical symptoms are visible or before sporulation. We describe here a pathogenicity assay based on the relative quantification of fungal and plant DNA in infected Arabidopsis thaliana leaves by means of real-time quantitative PCR. We show that it allows to monitor quantitatively the growth of the fungi Alternaria brassicicola and Botrytis cinerea in a sensitive and reliable way. Although highly sensitive, this test also exhibits a high robustness, which is crucial to significantly discriminate between lines displaying slightly different levels of resistance. Therefore, it allows to assess fungal development from the very first stages of infection and provides a fast and very practical alternative to currently described assays for phenotyping either plant mutant lines or fungal strains.     

Identifying insecticide-resistant greenbugs (Homoptera: Aphididae) with diagnostic assay tests

Laboratory bioassays were used to develop a diagnostic assay test for identifying greenburg, Schizaphis graminum (Rondani), populations that are insecticide-resistant. Petri dish assays with chlorpyrifos showed greenbug mortality should be monitored after 2 h of exposure. One-hour exposure did not kill a high percentage of susceptible greenbugs, and a 3-h exposure killed too many resistant greenbugs. Ethanol and methanol were both good solvents for mixing with chlorpyrifos in the petri dish assay. From the laboratory bioassays, four diagnostic concentrations of chlorpyrifos (3, 10, 30, and 100 ppm) were evaluated in the field by Texas A&M University agricultural research and extension entomologists across the Texas High Plains. Results from the diagnostic assay tests were compared with gel-electrophoresis resistance tests to validate resistance detection. The diagnostic assay tests gave the same greenbug resistance identification as the gel-electrophoresis analysis in 21 of 22 field bioassays in 1994 and 35 of 39 field bioassays in 1995. Diagnostic concentrations of 30 and 100 ppm chlorpyrifos killed > or = 85 and > or = 90%, respectively, of greenbugs identified by gel-electrophoresis as susceptible and < 40% and < 55%, respectively, of resistant greenbugs. The diagnostic assay technique is a quick, reliable, and inexpensive method for detecting insecticide resistance in greenbug populations.     

Focus: Zoonotic Disease: Ending the Neglect of Treatable Bacterial Zoonoses Responsible for Non-Malaria Fevers

Bacterial zoonotic diseases such as leptospirosis, Q fever, melioidosis, spotted fever group rickettsioses, and brucellosis are increasingly recognized causes of non-malaria acute fevers. However, though readily treatable with antibiotics, these diseases are commonly misdiagnosed resulting in poor outcomes in patients. There is a considerable deficit in the understanding of basic aspects of the epidemiology of these neglected diseases and diagnostic tests for these zoonotic bacterial pathogens are not always available in resource-poor settings. Raising awareness about these emerging bacterial zoonoses is directly beneficial to the patients by allowing a test-and-treat approach and is essential to control these life-threatening diseases.     

Measuring and Understanding Depression in Women in Kisoro,Uganda

Depression is highly prevalent and the cause of considerable suffering for peoples across the globe. Case finding for depression is challenging because individuals often do not recognize the symptoms in themselves or may resist the diagnosis as a result of cultural stigma. Screening instruments, to be accurate, must be valid in the particular setting in which they are being applied, and diagnosis in primary care settings, is further made challenging because patients often present with a wide variety of somatic symptoms that could be medical. 115 women were screened for depression in this study in one community in Uganda, and 87 were found to be depressed using the SRQ-20. The cognitive impairment and decreased energy sub-scales of the SRQ-20 seemed to best differentiate for depression. We then interviewed the 87 women and found that, overwhelmingly, their complaints were somatic, and that their expectation for treatment was to receive medical tests and medications. Caregivers in primary care clinics in Uganda should know that in the reporting of their somatic symptoms patients may be trying to communicate more about themselves than just the state of their physical health; and that feelings of uselessness or of hopelessness when expressed by a patient should lead them to suspect severe mental illness since these symptoms were not found to be characteristic of the milder depression that is highly prevalent in Ugandan women.

     

On a chip

The future of clinical and POC BioMEMS is very bright. With an increasing emphasis on the personalization of medicine and the rising costs of health care, early detection and diagnostics at the POC will be even more important. Early detection implies early intervention, resulting in the saving of lives and reducing overall spending. The potential impact of these technologies on the early diagnosis and management of both communicable and noncommunicable diseases is very high. Many grand challenges applications are possible, e.g., routine tests such as complete blood cell count on a chip that an individual can perform at home; detection of cardiac markers from blood after a perceived heart attack; detection of cancer markers such as exosomes, CTCs from blood, or protein biomarkers in serum; and detection of infectious agents such as virus and bacteria for public health. These applications are expected to result in new diagnostic assays for home, doctor's office, clinical laboratories, and various POC settings.     

The study of Alzheimer’s disease biomarkers

Alzheimer’s disease (AD) is by far the most common dementing illness of late life and is increasing with the ever-growing number of older adults, in particular, in developed countries. The disease is often referred to as the “Long-Goodbye” because the person with the illness slowly becomes lost to everyone a long time before the body finally gives out. Being able to detect AD earlier on during the course of the disease offers better prospects for the future, for AD individuals, their families and friends as well as on the economy, as a whole. Unfortunately, such a detection technique is not yet, available. However, there are a number of promising biological markers (biomarkers) that correlate well with clinical cognitive tests of individuals and/or postmortem histopathological manifestations of the disease, especially when at least two markers are used for the diagnosis. Biosensors are tools that combine a biochemical binding element to a signal conversion unit and are already being used in the study of some AD biomarkers. However, their use in clinical diagnosis remains a challenge. Introduction of nanotechnology leading to nanobiosensors has several potential advantages over other clinical and/or existing analytical tools, including increased assay speed, flexibility, reduced cost of diagnostic testing, potential to deliver molecular diagnostic tools to family general practitioners, and other health care systems. Even more important, nano-based assays have the potential to detect target proteins at attomolar concentration level. They are, therefore, being increasingly exploited for the detection of early metabolic changes associated with diseases. Because brain damage is irreversible, the use of nanotechnology is particularly important in AD and other neurodegenerative disorders. Nanosensors can also facilitate and enable pointofcare-testing (POCT). This article reviews the basic biochemical processes that lead to AD pathology, current biomarkers for AD, and the current role of nanosensor technology for the study of AD biomarkers. Furthermore, it discusses the huge potential of nanosensing to deliver new molecular diagnostic strategies to AD research.     

The role of laboratory diagnostics in emerging viral infections: the example of the Middle East respiratory syndrome epidemic

Rapidly emerging infectious disease outbreaks place a great strain on laboratories to develop and implement sensitive and specific diagnostic tests for patient management and infection control in a timely manner. Furthermore, laboratories also play a role in real-time zoonotic, environmental, and epidemiological investigations to identify the ultimate source of the epidemic, facilitating measures to eventually control the outbreak. Each assay modality has unique pros and cons; therefore, incorporation of a battery of tests using traditional culture-based, molecular and serological diagnostics into diagnostic algorithms is often required. As such, laboratories face challenges in assay development, test evaluation, and subsequent quality assurance. In this review, we describe the different testing modalities available for the ongoing Middle East respiratory syndrome (MERS) epidemic including cell culture, nucleic acid amplification, antigen detection, and antibody detection assays. Applications of such tests in both acute clinical and epidemiological investigation settings are highlighted. Using the MERS epidemic as an example, we illustrate the various challenges faced by laboratories in test development and implementation in the setting of a rapidly emerging infectious disease. Future directions in the diagnosis of MERS and other emerging infectious disease investigations are also highlighted.     

HIV and tuberculosis in India

The global impact of the converging dual epidemics of tuberculosis (TB) and human immunodeficiency virus (HIV) is one of the major public health challenges of our time. The World Health Organization (WHO) reports 9.2 million new cases of TB in 2006 of whom 7.7% were HIV-infected. Tuberculosis is the most common opportunistic infection in HIV-infected patients as well as the leading cause of death. Further, there has been an increase in rates of drug resistant tuberculosis, including multi-drug (MDRTB) and extensively drug resistant TB (XDRTB), which are difficult to treat and contribute to increased mortality. The diagnosis of TB is based on sputum smear microscopy, a 100-year old technique and chest radiography, which has problems of specificity. Extra-pulmonary, disseminated and sputum smear negative manifestations are more common in patients with advanced immunosuppression. Newer diagnostic tests are urgently required that are not only sensitive and specific but easy to use in remote and resource-poor settings. Treatment of HIV-TB co-infection is complex and associated with high pill burden, overlapping drug toxicities, risk of immune reconstitution inflammatory syndrome (IRIS) and challenges related to adherence. From a programmatic point of view, screening of all HIV-infected persons for tuberculosis and vice-versa will help identify co-infected patients who require treatment for both infections. This requires good coordination and communication between the TB and AIDS control programs, in India.     

Non-Instrumented Incubation of a Recombinase Polymerase Amplification Assay for the Rapid and Sensitive Detection of Proviral HIV-1 DNA

Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25–43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS.