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      Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children

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          Abstract

          Streptococcus pneumoniae ( S. pneumoniae) is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media. It is difficult to isolate and identify S. pneumoniae form clinical samples. To evaluate a novel, rapid, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay to detect S. pneumoniae pneumonia in children, we designed specific LAMP primers targeting lytA and psaA genes. We optimized the reaction time and reaction system, and evaluated its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation. We also analyzed the molecular characteristics of the isolates obtained from the positive samples. The primer sets LytA-1 and PsaA-2 amplified the genes in the shortest times, and 63°C was confirmed as the optimum reaction temperature. The detection sensitivity of each reaction was 10 and 100 copies/μL with primer sets LytA-1 and PsaA-2, respectively. This LAMP assay showed no cross-reactivity with other 27 pathogens. To describe the availability of this method, we collected 748 clinical samples from children with pneumonia. Among them, 135 were confirmed to be S. pneumoniae positive by LAMP. The sensitivity was 100% (95% CI 96.4–100%), specificity 99.0% (95% CI 97.8–99.6%). Including them, 50 were co-infected with Mycoplasma pneumoniae. This LAMP assay detected S. pneumoniae in 1 h and the results can be identified with visual naked eyes. Thus, it will be a powerful tool for S. pneumoniae early diagnosis and effective antibiotic therapy.

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          Most cited references30

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          Streptococcus pneumoniae: transmission, colonization and invasion

          Streptococcus pneumoniae as a complex relationship with its obligate human host. On the one hand, the pneumococci are highly adapted commensals, and their main reservoir on the mucosal surface of the upper airways of carriers enables transmission. On the other hand, they can cause severe disease when bacterial and host factors allow them to invade essentially sterile sites, such as the middle ear spaces, lungs, bloodstream and meninges. Transmission, colonization and invasion depend on the remarkable ability of S. pneumoniae to evade or take advantage of the host inflammatory and immune responses. The different stages of pneumococcal carriage and disease have been investigated in detail in animal models and, more recently, in experimental human infection. Furthermore, widespread vaccination and the resulting immune pressure have shed light on pneumococcal population dynamics and pathogenesis. Here, we review the mechanistic insights provided by these studies on the multiple and varied interactions of the pneumococcus and its host.
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            Global burden of childhood pneumonia and diarrhoea

            Summary Diarrhoea and pneumonia are the leading infectious causes of childhood morbidity and mortality. We comprehensively reviewed the epidemiology of childhood diarrhoea and pneumonia in 2010–11 to inform the planning of integrated control programmes for both illnesses. We estimated that, in 2010, there were 1·731 billion episodes of diarrhoea (36 million of which progressed to severe episodes) and 120 million episodes of pneumonia (14 million of which progressed to severe episodes) in children younger than 5 years. We estimated that, in 2011, 700 000 episodes of diarrhoea and 1·3 million of pneumonia led to death. A high proportion of deaths occurs in the first 2 years of life in both diseases—72% for diarrhoea and 81% for pneumonia. The epidemiology of childhood diarrhoea and that of pneumonia overlap, which might be partly because of shared risk factors, such as undernutrition, suboptimum breastfeeding, and zinc deficiency. Rotavirus is the most common cause of vaccine-preventable severe diarrhoea (associated with 28% of cases), and Streptococcus pneumoniae (18·3%) of vaccine-preventable severe pneumonia. Morbidity and mortality from childhood pneumonia and diarrhoea are falling, but action is needed globally and at country level to accelerate the reduction.
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              Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates.

              Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and sepsis in children worldwide. However, many countries lack national estimates of disease burden. Effective interventions are available, including pneumococcal conjugate vaccine and case management. To support local and global policy decisions on pneumococcal disease prevention and treatment, we estimated country-specific incidence of serious cases and deaths in children younger than 5 years. We measured the burden of pneumococcal pneumonia by applying the proportion of pneumonia cases caused by S pneumoniae derived from efficacy estimates from vaccine trials to WHO country-specific estimates of all-cause pneumonia cases and deaths. We also estimated burden of meningitis and non-pneumonia, non-meningitis invasive disease using disease incidence and case-fatality data from a systematic literature review. When high-quality data were available from a country, these were used for national estimates. Otherwise, estimates were based on data from neighbouring countries with similar child mortality. Estimates were adjusted for HIV prevalence and access to care and, when applicable, use of vaccine against Haemophilus influenzae type b. In 2000, about 14.5 million episodes of serious pneumococcal disease (uncertainty range 11.1-18.0 million) were estimated to occur. Pneumococcal disease caused about 826,000 deaths (582,000-926,000) in children aged 1-59 months, of which 91,000 (63,000-102,000) were in HIV-positive and 735,000 (519,000-825,000) in HIV-negative children. Of the deaths in HIV-negative children, over 61% (449,000 [316,000-501,000]) occurred in ten African and Asian countries. S pneumoniae causes around 11% (8-12%) of all deaths in children aged 1-59 months (excluding pneumococcal deaths in HIV-positive children). Achievement of the UN Millennium Development Goal 4 for child mortality reduction can be accelerated by prevention and treatment of pneumococcal disease, especially in regions of the world with the greatest burden. GAVI Alliance and the Vaccine Fund.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                17 January 2022
                2021
                : 12
                : 816997
                Affiliations
                [1] 1Department of Bacteriology, Capital Institute of Pediatrics , Beijing, China
                [2] 2Key Laboratory of Resources Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University , Xi’an, China
                [3] 3College of Biomedicine, Beijing City College , Beijing, China
                Author notes

                Edited by: Jens Andre Hammerl, Bundesinstitut für Risikobewertung, Germany

                Reviewed by: Yanhong Liu, United States Department of Agriculture (USDA), United States; Satoshi Hayakawa, Nihon University, Japan; Dongsheng Zhou, Beijing Institute of Microbiology and Epidemiology, China

                *Correspondence: Jing Yuan, yuanjing6216@ 123456163.com

                These authors have contributed equally to this work and share first authorship

                This article was submitted to Infectious Agents and Disease, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2021.816997
                8803124
                c853dab8-6d81-4a74-bd31-ba3876aa7757
                Copyright © 2022 Du, Yan, Du, Zhao, Xue, Zheng, Feng, Cui, Gan, Feng, Fan, Xu, Fu, Fu, Zhang, Li, Zhang, Liu, Li, Cui, Zhou, Zhang, Chen and Yuan.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 17 November 2021
                : 20 December 2021
                Page count
                Figures: 4, Tables: 3, Equations: 0, References: 30, Pages: 9, Words: 5971
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                streptococcus pneumoniae,loop-mediated isothermal amplification,detection,visual,children

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