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Development of multiplex PCR for neglected infectious diseases.

ABSTRACT: Scrub typhus, murine typhus, and leptospirosis are widely neglected infectious diseases caused by Orientia tsutsugamushi, Rickettsia typhi, and pathogenic Leptospira spp., respectively. Patients usually present with non-specific symptoms and therefore are commonly diagnosed with acute undifferentiated febrile illness. Consequently, patients face delayed treatment and increased mortality. Antibody-based serological test currently used as gold standard has limitations due to insufficient antibody titers, especially in the early phase of infection. In this study, we aimed to develop multiplex PCR to combine 3 primer pairs that target specific genes encoding 56-kDa TSA of O. tsutsugamushi, 17-kDa antigen of R. typhi, and LipL32 of L. Interrogans and evaluate its performance in comparison to the standard serological tests. Using EDTA blood samples of known patients, the sensitivity and specificity of our multiplex PCR was 100% and 70%, respectively. In addition, the assay was able to diagnose the co-infection of scrub typhus and leptospirosis. The assay may be useful in identifying causative agents during the early phase of these diseases, enabling prompt and appropriate treatment.

SUBMITTER: Sea-Liang N

PROVIDER: S-EPMC6613674 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Development of multiplex PCR for neglected infectious diseases.

Sea-Liang Nutchanart N Sereemaspun Amornpun A Patarakul Kanitha K Gaywee Jariyanart J Rodkvamtook Wuttikon W Srisawat Nattachai N Wacharaplusadee Supaporn S Hemachudha Thiravat T

PLoS neglected tropical diseases 20190708 7

Scrub typhus, murine typhus, and leptospirosis are widely neglected infectious diseases caused by Orientia tsutsugamushi, Rickettsia typhi, and pathogenic Leptospira spp., respectively. Patients usually present with non-specific symptoms and therefore are commonly diagnosed with acute undifferentiated febrile illness. Consequently, patients face delayed treatment and increased mortality. Antibody-based serological test currently used as gold standard has limitations due to insufficient antibody ...[more]

PMID: 31283768

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Project description:BackgroundA strategy to combat infectious diseases, including neglected tropical diseases (NTDs), will depend on the development of reliable epidemiological surveillance methods. To establish a simple and practical seroprevalence detection system, we developed a microsphere-based multiplex immunoassay system and evaluated utility using samples obtained in Kenya.MethodsWe developed a microsphere-based immuno-assay system to simultaneously measure the individual levels of plasma antibody (IgG) against 8 antigens derived from 6 pathogens: Entamoeba histolytica (C-IgL), Leishmania donovani (KRP42), Toxoplasma gondii (SAG1), Wuchereria bancrofti (SXP1), HIV (gag, gp120 and gp41), and Vibrio cholerae (cholera toxin). The assay system was validated using appropriate control samples. The assay system was applied for 3411 blood samples collected from the general population randomly selected from two health and demographic surveillance system (HDSS) cohorts in the coastal and western regions of Kenya. The immunoassay values distribution for each antigen was mathematically defined by a finite mixture model, and cut-off values were optimized.FindingsSensitivities and specificities for each antigen ranged between 71 and 100%. Seroprevalences for each pathogen from the Kwale and Mbita HDSS sites (respectively) were as follows: HIV, 3.0% and 20.1%; L. donovani, 12.6% and 17.3%; E. histolytica, 12.8% and 16.6%; and T. gondii, 30.9% and 28.2%. Seroprevalences of W. bancrofti and V. cholerae showed relatively high figures, especially among children. The results might be affected by immunological cross reactions between W. bancrofti-SXP1 and other parasitic infections; and cholera toxin and the enterotoxigenic E. coli (ETEC), respectively.InterpretationA microsphere-based multi-serological assay system can provide an opportunity to comprehensively grasp epidemiological features for NTDs. By adding pathogens and antigens of interest, optimized made-to-order high-quality programs can be established to utilize limited resources to effectively control NTDs in Africa.

Project description:IntroductionPrior research on neglected disease drug development suggested inadequate funding was responsible for relatively few new approvals. In response, significantly more resources have been allocated towards development of drugs targeting neglected diseases. Our objective was to reassess drug development between 1975 and 1999, evaluate progress in neglected disease drug development since 2000, and explain how increased numbers of approvals are a necessary but insufficient condition to improving access.MethodsTo assess numbers of approvals targeting neglected diseases, we employed two distinct methodologies: First, to revisit numbers published in Trouiller et al. (2002) we used their method to count marketed new chemical entities (NCEs) between 1975 and 1999. Second, using the G-Finder report as a benchmark, we identified which diseases are currently considered "neglected" to tally approvals in the 1975-1999 and 2000-2009 periods. Searching PharmaProjects and IMS R&D Focus databases as well as websites from numerous drug regulatory agencies, we identified new drug approvals and indications. Also, we examined the World Health Organization's (WHO) Essential Drug List (EDL) to see which drugs and indications were on the list.FindingsUpon recount, using Trouiller et al. methodology, we found that between 1975 and 1999 more NCEs (n = 32) targeting tropical diseases and tuberculosis were approved than reported in Trouiller et al. (n = 16). Using the G-Finder method of defining neglected diseases, we found 46 new drug approvals between 1975 and 1999. WHO included 85% of these drugs on the EDL. In the period 2000 to May 2009, despite much greater funding, only 26 new drugs and vaccines for neglected diseases were marketed. Of these, WHO placed 50% on the EDL.ConclusionsProduct approvals for neglected diseases have increased, though progress has been uneven, with malaria appearing to benefit most in the short run from increased funding, while less success has been booked in other disease categories. Uneven progress suggests funding could be better targeted, particularly with regard to neglected diseases that have hitherto received scant attention. In addition, policymakers should focus on other aspects related to access. Besides drug development, there are the issues of EDL listing, architecture, availability, affordability, and adoption.

Project description:IntroductionResearch quality can be improved with reliable and reproducible experimental results when animal experiments are conducted using laboratory animals with guaranteed microbiological and genetic quality through health monitoring. Therefore, health monitoring requires the rapid and accurate diagnosis of infectious diseases in laboratory animals.MethodsThis study presents a performance evaluation of a commercially available multiplex real-time PCR (mRT-PCR) assay for the rapid detection of 12 infectious pathogens (Set 1: Sendai virus [SeV, formally murine respirovirus], Mycoplasma spp., Rodentibacter pneumotropicus, and Rodentibacter heylii; Set 2: Helicobacter spp., Murine norovirus [MNV], Murine hepatitis virus [MHV], and Salmonella spp.; Set 3: Staphylococcus aureus, Streptobacillus moniliformis, Corynebacterium kutscheri, and Pseudomonas aeruginosa). To evaluate the efficacy of the mRT-PCR assay, 102 clinical samples encompassing fecal and cecal specimens were analyzed. The resulting data were then compared with the findings from sequence analysis for validation.ResultsThe assay's detection limit ranged from 1 to 100 copies per reaction. Specificity testing involving various viruses and bacteria indicated no cross-reactivity between strains. Additionally, the assay exhibited good reproducibility, with mean coefficients of variation for inter- and intra assay variation below 3%. The overall positive rate was 52.9% (n = 54), with the mRT-PCR assay findings matching sequence analysis results (κ = 1). MHV (n = 29, 28.4%) was the most prevalent pathogen, followed by Helicobacter spp. (n = 28, 27.5%), R. heylii (n = 18, 17.6%), Mycoplasma spp. (n = 14, 13.7%), MNV (n = 12, 11.8%), S. aureus (n = 9, 8.8%), P. aeruginosa (n = 4, 3.9%), and R. pneumotropicus (n = 1, 0.9%).DiscussionThis assay offers a rapid turnaround time of 100 min, including 30 min for DNA preparation and 70 min for target DNA/RNA amplification. It ensures accuracy, minimizing false positives or negatives, making it a convenient tool for the simultaneous detection of infectious diseases in many samples. Overall, the propose‑d assay holds promise for the effective detection of the most important pathogens in laboratory animal health monitoring.

Project description:BackgroundTropical infectious diseases are called neglected, because they are, inter alia, characterized by an R&D deficit. A similar deficit exists for rare (orphan) diseases which neither promise a sufficient return on R&D investment. To encourage the development of treatments for rare diseases, orphan drug acts were created which contain financial and non-financial incentives for the pharmaceutical industry. Similar instruments aimed exclusively at neglected diseases do not yet exist. Proposals for a regulatory approach to promote R&D for neglected diseases include the application of selected orphan drug incentives, or the implementation of a Medical Research and Development Treaty (MRDT) with national funding obligations for medical R&D. We compiled and analyzed experts' opinions on causes for the treatment deficit for neglected diseases and on desirable and feasible measures to promote neglected disease R&D. Hereby, the focus was on mechanisms contained in orphan drug regulations and in the Medical Research and Development Treaty draft (Discussion draft 4, 2005). Lastly, we solicited experts' opinions on the desirability and feasibility of a regulatory instrument to foster R&D for neglected diseases.MethodsAn international online-Delphi survey was conducted with 117 (first round) and 56 (second round) experts of different professional backgrounds and professional affiliations who formulated and ranked causes and solutions related to the treatment deficit for neglected diseases.ResultsIn both rounds of survey, the majority of the participating experts (88.4% first round, 86.8% second round) advocated the development of a regulatory instrument to promote R&D for neglected diseases. Most experts (77.9% first round, 79.3% second round) also considered this to be a feasible option. With the exception of market exclusivity, which was viewed critically, key provisions contained in orphan drug regulations were judged favorably also for neglected diseases. A majority (87.1% first round, 77.2% second round) supported national funding obligations for neglected diseases which are proposed by the Medical Research and Development Treaty draft.ConclusionsWhile not all features of orphan drug regulations and of the MRDT draft received equal support, the view was expressed that a regulatory instrument would be a desirable and feasible measure to promote R&D for neglected diseases.

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Development of multiplex PCR for neglected infectious diseases.

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Development of multiplex PCR for neglected infectious diseases.

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