Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay

AbstractLegionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non-pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5′-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non-pneumophila Legionella species in human disease

Composition for personal growth : program design and evaluation.

<p>Two categories were used for cross-validation of the model, either type 1 or type 2. Clinical isolates were treated as an unknown class and cross-validated sensitivity, specificity, and class error were based on their classification prediction score with their respective reference strain control class. CV, cross-validated.</p><p>Cross-validated PLS-DA modeling statistics for the prediction performance for NA-SERS typing of individual type 1 and 2 <i>M</i>. <i>pneumoniae</i> clinical isolates.</p

Outbreak of Pneumonia in the Setting of Fatal Pneumococcal Meningitis among US Army Trainees: Potential Role of Chlamydia pneumoniae Infection

<p>Abstract</p> <p>Background</p> <p>Compared to the civilian population, military trainees are often at increased risk for respiratory infections. We investigated an outbreak of radiologically-confirmed pneumonia that was recognized after 2 fatal cases of serotype 7F pneumococcal meningitis were reported in a 303-person military trainee company (Alpha Company).</p> <p>Methods</p> <p>We reviewed surveillance data on pneumonia and febrile respiratory illness at the training facility; conducted chart reviews for cases of radiologically-confirmed pneumonia; and administered surveys and collected nasopharyngeal swabs from trainees in the outbreak battalion (Alpha and Hotel Companies), associated training staff, and trainees newly joining the battalion.</p> <p>Results</p> <p>Among Alpha and Hotel Company trainees, the average weekly attack rates of radiologically-confirmed pneumonia were 1.4% and 1.2% (most other companies at FLW: 0-0.4%). The pneumococcal carriage rate among all Alpha Company trainees was 15% with a predominance of serotypes 7F and 3. <it>Chlamydia pneumoniae </it>was identified from 31% of specimens collected from Alpha Company trainees with respiratory symptoms.</p> <p>Conclusion</p> <p>Although the etiology of the outbreak remains unclear, the identification of both <it>S. pneumoniae </it>and <it>C. pneumoniae </it>among trainees suggests that both pathogens may have contributed either independently or as cofactors to the observed increased incidence of pneumonia in the outbreak battalion and should be considered as possible etiologies in outbreaks of pneumonia in the military population.</p

Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for SARS-associated Coronavirus

A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection

Epidemiology and molecular identification and characterization of mycoplasma pneumoniae, South Africa, 2012-2015.

CAPRISA, 2018.Abstract available in pdf

Survey of Legionella

Members of the Gram-negative genus Legionella are typically found in freshwater environments, with the exception of L. longbeachae, which is present in composts and potting mixes. When contaminated aerosols are inhaled, legionellosis may result, typically as either the more serious pneumonia Legionnaires’ disease or the less severe flu-like illness Pontiac fever. It is presumed that all species of the genus Legionella are capable of causing disease in humans. As a followup to a prior clinical study of legionellosis in rural Thailand, indigenous soil samples were collected proximal to cases’ homes and workplaces and tested for the presence of legionellae by culture. We obtained 115 isolates from 22/39 soil samples and used sequence-based methods to identify 12 known species of Legionella represented by 87 isolates

Causes and incidence of community-acquired serious infections among young children in south Asia (ANISA): an observational cohort study.

BACKGROUND: More than 500 000 neonatal deaths per year result from possible serious bacterial infections (pSBIs), but the causes are largely unknown. We investigated the incidence of community-acquired infections caused by specific organisms among neonates in south Asia. METHODS: From 2011 to 2014, we identified babies through population-based pregnancy surveillance at five sites in Bangladesh, India, and Pakistan. Babies were visited at home by community health workers up to ten times from age 0 to 59 days. Illness meeting the WHO definition of pSBI and randomly selected healthy babies were referred to study physicians. The primary objective was to estimate proportions of specific infectious causes by blood culture and Custom TaqMan Array Cards molecular assay (Thermo Fisher, Bartlesville, OK, USA) of blood and respiratory samples. FINDINGS: 6022 pSBI episodes were identified among 63 114 babies (95·4 per 1000 livebirths). Causes were attributed in 28% of episodes (16% bacterial and 12% viral). Mean incidence of bacterial infections was 13·2 (95% credible interval [CrI] 11·2-15·6) per 1000 livebirths and of viral infections was 10·1 (9·4-11·6) per 1000 livebirths. The leading pathogen was respiratory syncytial virus (5·4, 95% CrI 4·8-6·3 episodes per 1000 livebirths), followed by Ureaplasma spp (2·4, 1·6-3·2 episodes per 1000 livebirths). Among babies who died, causes were attributed to 46% of pSBI episodes, among which 92% were bacterial. 85 (83%) of 102 blood culture isolates were susceptible to penicillin, ampicillin, gentamicin, or a combination of these drugs. INTERPRETATION: Non-attribution of a cause in a high proportion of patients suggests that a substantial proportion of pSBI episodes might not have been due to infection. The predominance of bacterial causes among babies who died, however, indicates that appropriate prevention measures and management could substantially affect neonatal mortality. Susceptibility of bacterial isolates to first-line antibiotics emphasises the need for prudent and limited use of newer-generation antibiotics. Furthermore, the predominance of atypical bacteria we found and high incidence of respiratory syncytial virus indicated that changes in management strategies for treatment and prevention are needed. Given the burden of disease, prevention of respiratory syncytial virus would have a notable effect on the overall health system and achievement of Sustainable Development Goal. FUNDING: Bill & Melinda Gates Foundation

Mucosal immune responses to HIV-1 C4/V3 epitopes following genetic or peptide immunizations of rabbits

The studies reported herein were designed to elicit secretory IgA (S-IgA) responses to specific epitopes of the HIV-1 envelope protein gp120. Since HIV is most often acquired after sexual contact at mucosal surfaces, a S-IgA response may reduce transmission rates. The synthetic peptide T1- SP10MN(A) is derived from the V3 loop and C4 domains of gp120 and contains T-helper, B-cell, and CTL epitopes. This 40 amino acid peptide was first tested for its ability to induce immunity after systemic immunizations and generated high titered antibody responses. The mucosal immunogenicity of this peptide was subsequently examined using the Thiry-Vella loop model in rabbits. Groups of five rabbits were intestinally immunized with either T1-SP10MN(A) alone, T1-SP10MN(A) plus the mucosal adjuvant cholera toxin (CT), or T1-SP10MN(A) linked to the mucosal carrier protein LT-B and admixed with CT. Intestinal secretions and sera demonstrated specific S-IgA and serum IgG responses, respectively. These results indicated that T1-SP10MN(A) was immunogenic upon mucosal administration and allowed for additional studies intended to induce specific S-IgA antibody responses in vaginal and fecal secretions.^ The first vaccine strategy consisted of intranasal peptide immunizations where five rabbits received T1-SP10MN(A) co-administered with CT. Three animals generated specific S-IgA in nasal and vaginal secretions, while all five contained serum anti-T1-SP10MN(A) IgG. This study further establishes the correlation between nasal immunizations and vaginal immunity.^ The second study focused on a novel technique known as DNA vaccination. A mammalian-based plasmid containing the sequence encoding T1-SP10MN(A) was delivered to the Peyer\u27s patches (PP) of five rabbits using the helium-driven gene gun. Two of five animals received intradermal boosts of plasmid at week 6. The PP was chosen as a target because it is the principal inductive site found in the intestine. Following immunization, high titered S-IgA responses were observed in specific vaginal and fecal samples and serum IgG was detected in two animals. The two boosted animals displayed an augmentation of S-IgA responses in both vaginal and fecal samples. This study establishes the PP as a promising mucosal target tissue for DNA immunization.^ These studies may assist in future HIV vaccine research, especially those focusing on eliciting mucosal immunity.

The evolution of advanced molecular diagnostics for the detection and characterization of Mycoplasma pneumoniae

Over the past several years there have been significant advancements in the methods used for detecting and characterizing Mycoplasma pneumoniae, a common cause of respiratory illness and community-acquired pneumonia worldwide. The repertoire of available molecular diagnostics has greatly expanded from nucleic acid amplification techniques (NAATs) that encompass a variety of chemistries used for detection, to more sophisticated characterizing methods such as multi-locus variable-number tandem-repeat analysis and sequencing typing (MLVA and MLST, respectively), matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS), single nucleotide polymorphism (SNP) typing, and numerous macrolide susceptibility profiling methods, among others. These many molecular-based approaches have been developed and employed to continually increase the level of discrimination and characterization in order to better understand the epidemiology and biology of M. pneumoniae. This review will summarize recent molecular techniques and procedures and lend perspective to how each has enhanced the current understanding of this organism and will emphasize how Next Generation Sequencing may serve as a resource for researchers to gain a more comprehensive understanding of the genomic complexities of this insidious pathogen