Tools for diagnosis, monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels
SUMMARY The potential of various quantitative lateral flow (LF) based assays utilizing up-converting phosphor (UCP) reporters for the diagnosis of schistosomiasis is reviewed including recent developments. Active infections are demonstrated by screening for the presence of regurgitated worm antigens (genus specific polysaccharides), whereas anti-Schistosoma antibodies may indicate ongoing as well as past infections. The circulating anodic antigen (CAA) in serum or urine (and potentially also saliva) is identified as the marker that may allow detection of single-worm infections. Quantitation of antigen levels is a reliable method to study effects of drug administration, worm burden and anti-fecundity mechanisms. Moreover, the ratio of CAA and circulating cathodic antigen (CCA) is postulated to facilitate identification of either Schistosoma mansoni or Schistosoma haematobium infections. The UCP-LF assays allow simultaneous detection of multiple targets on a single strip, a valuable feature for antibody detection assays. Although antibody detection in endemic regions is not a useful tool to diagnose active infections, it gains potential when the ratio of different classes of antibody specific for the parasite/disease can be determined. The UCP-LF antibody assay format allows this type of multiplexing, including testing a linear array of up to 20 different targets. Multiple test spots would allow detection of specific antibodies, e.g. against different Schistosoma species or other pathogens as soil-transmitted helminths. Concluding, the different UCP-LF based assays for diagnosis of schistosomiasis provide a collection of tests with relatively low complexity and high sensitivity, covering the full range of diagnostics needed in control programmes for mapping, screening and monitoring.
Design Aspects of a Case-Control Clinical Investigation of the Effect of HIV on Oral and Gastrointestinal Soluble Innate Factors and Microbes
INTRODUCTION: The impaired host defense system in HIV infection impacts the oral and gastrointestinal microbiota and associated opportunistic infections. Antiretroviral treatment is predicted to partially restore host defenses and decrease the oral manifestation of HIV/AIDS. Well-designed longitudinal studies are needed to better understand the interactions of soluble host defense proteins with bacteria and virus in HIV/AIDS. "Crosstalk" was designed as a longitudinal study of host responses along the gastrointestinal (GI) tract and interactions between defense molecules and bacteria in HIV infection and subsequent therapy. PURPOSE: The clinical core formed the infrastructure for the study of the interactions between the proteome, microbiome and innate immune system. The core recruited and retained study subjects, scheduled visits, obtained demographic and medical data, assessed oral health status, collected samples, and guided analysis of the hypotheses. This manuscript presents a well-designed clinical core that may serve as a model for studies that combine clinical and laboratory data. METHODS: Crosstalk was a case-control longitudinal clinical study an initial planned enrollment of 170 subjects. HIV+ antiretroviral naive subjects were followed for 9 visits over 96 weeks and HIV uninfected subjects for 3 visits over 24 weeks. Clinical prevalence of oral mucosal lesions, dental caries and periodontal disease were assessed. RESULTS: During the study, 116 subjects (47 HIV+, 69 HIV-) were enrolled. Cohorts of HIV+ and HIV- were demographically similar except for a larger proportion of women in the HIV- group. The most prevalent oral mucosal lesions were oral candidiasis and hairy leukoplakia in the HIV+ group. DISCUSSION: The clinical core was essential to enable the links between clinical and laboratory data. The study aims to determine specific differences between oral and GI tissues that account for unique patterns of opportunistic infections and to delineate the differences in their susceptibility to infection by HIV and their responses post-HAART.
The use of upconverting phosphors in point-of-care (POC) testing [Meeting Abstract]
Point-of-care (POC) testing is increasingly applied as a cost effective alternative to many diagnostic tests. Key in POC testing is to create sufficient assay sensitivity with relatively low cost reagents and equipment. For this purpose we have employed a unique reporter, upconverting phosphor (UCP) particles, in combination with lateral flow (LF) assays. UCPs, submicron ceramic particles doped with rare earth ions (lanthanides), convert infrared to visible light and do not suffer from autofluorescence which limits conventional fluorescence based assays. Low cost handheld readers and microfluidics were evaluated in various applications. Designed assays are well suited for applications outside diagnostic laboratories, in resource poor settings, and can even be used by patients at home. Using two distinctly different UCP-LF assay formats, we focussed on assays for infectious diseases based on the detection of pathogen-specific antibodies and/or antigens including nucleic acids to demonstrate active infection with HIV. Only minor adaptation of the standard UCP-LF assay format is needed to render the format suitable for applications involving low affinity capture antibodies (e. g. in the detection of neurotoxin, botulism), capture of small molecules (e. g. detection of melatonin, a key hormone in chronopharmacology) or the use of dry UCP reagents (e. g. detection of protein based fruit-ripening markers, of economic interest in agriculture). Finally, we anticipate on developments in healthcare (personalized medicine) by discussing the potential of one of the UCP-LF assay formats to measure serum trough levels of immunodrugs (e. g. infliximab or adalimumab) in patients treated for inflammatory bowel disease and rheumatoid arthritis.
An Electrochemiluminescent Assay for gp340 (DMBT1)
gp340 is a member of the scavenger receptor cysteine-rich family of innate immune molecules and also functions as a tumor suppressor. This study describes a picogram level assay using electrochemiluminescent technology on the MesoScale Discovery (MSD) platform. Antibodies were evaluated and the best pair used to assay whole mouth stimulated saliva (WMSS) and cervical vaginal lavage (CVL). The assay was tested using specimens obtained from healthy volunteers in order to determine if gp340 concentration in saliva correlates with levels in vaginal lavage fluid. Interestingly, no correlation was determined between gp340 content in these two fluids.
Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis
BACKGROUND: A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas. METHODS: A semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene. RESULTS: This study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix. CONCLUSIONS: The development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to < two hours. Analysis of the PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples.
Development of a generic microfluidic device for simultaneous detection of antibodies and nucleic acids in oral fluids
A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive "sample-to-result" diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30â€‰min, and the confirmation by RNA amplification takes another 60â€‰min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test.
Detecting viruses by using salivary diagnostics
BACKGROUND: Diagnostics that involve the use of oral fluids have become increasingly available commercially in recent years and are of particular interest because of their relative ease of use, low cost and noninvasive collection of oral fluid for testing. TYPES OF STUDIES REVIEWED: The authors discuss the use of salivary diagnostics for virus detection with an emphasis on rapid detection of infection by using point-of-care devices. In particular, they review salivary diagnostics for human immunodeficiency virus, hepatitis C virus and human papillomavirus. Oral mucosal transudate contains secretory immunoglobulin (Ig) A, as well as IgM and IgG, which makes it a good source for immunodiagnostic-based devices. CLINICAL IMPLICATIONS: Because patients often visit a dentist more regularly than they do a physician, there is increased discussion in the dental community regarding the need for practitioners to be aware of salivary diagnostics and to be willing and able to administer these tests to their patients.
HIV Infection Affects Streptococcus mutans Levels, but Not Genotypes
We report a clinical study that examines whether HIV infection affects Streptococcus mutans colonization in the oral cavity. Whole stimulated saliva samples were collected from 46 HIV-seropositive individuals and 69 HIV-seronegative control individuals. The level of S. mutans colonization was determined by conventional culture methods. The genotype of S. mutans was compared between 10 HIV-positive individuals before and after highly active antiretroviral therapy (HAART) and 10 non-HIV-infected control individuals. The results were analyzed against viral load, CD4+ and CD8+ T-cell counts, salivary flow rate, and caries status. We observed that S. mutans levels were higher in HIV-infected individuals than in the non-HIV-infected control individuals (p = 0.013). No significant differences in S. mutans genotypes were found between the two groups over the six-month study period, even after HAART. There was a bivariate linear relationship between S. mutans levels and CD8+ counts (r = 0.412; p = 0.007), but not between S. mutans levels and either CD4+ counts or viral load. Furthermore, compared with non-HIV-infected control individuals, HIV-infected individuals experienced lower salivary secretion (p = 0.009) and a positive trend toward more decayed tooth surfaces (p = 0.027). These findings suggest that HIV infection can have a significant effect on the level of S. mutans, but not genotypes.
Patient and Provider Acceptance of Oral HIV Screening in a Dental School Setting
In 2006, the Centers for Disease Control and Prevention (CDC) recommended routine HIV screening in health care settings regardless of the patient's level of risk. This pilot study was developed in response to the suggestion by some health care professionals that dental settings would be appropriate for expansion of HIV testing. This project consisted of two parts: oral fluid HIV testing of patients in the clinic of a dental school and a survey of the clinical dental faculty members' attitudes about acceptability of routine HIV testing in the dental clinic. When patients' agreement to participate in oral fluid HIV testing was examined, 8.2 percent of the patients contacted by the clinic administration staff completed testing. When approached by a faculty member or student during the dental visit admission and tested during the dental visit, however, 88.2 percent completed testing. Of the faculty members who took the survey, 27.4 percent were neutral, 26.4 percent were somewhat in agreement, and 32.1 percent were willing to incorporate HIV testing into routine dental care. In this pilot study, HIV testing of dental patients was most successful when a dental care provider approached patients about testing. If consent was given, the testing was performed during the visit. For the faculty members, the major barrier to testing was a lack of protocol familiarity.
Human Microbiome and HIV/AIDS
Understanding of the human microbiome continues to grow rapidly; however, reports on changes in the microbiome after HIV infection are still limited. This review surveys the progress made in methodology associated with microbiome studies and highlights the remaining challenges to this field. Studies have shown that commensal oral, gut, vaginal, and penile bacteria are vital to the health of the human immune system. Our studies on crosstalk among oral and gastrointestinal soluble innate factors, HIV, and microbes indicated that the oral and gut microbiome was altered in the HIV-positive samples compared to the negative controls. The importance of understanding the bacterial component of HIV/AIDS, and likelihood of "crosstalk" between viral and bacterial pathogens, will help in understanding the role of the microbiome in HIV-infected individuals and facilitate identification of novel antiretroviral factors for use as novel diagnostics, microbicides, or therapeutics against HIV infection.