Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

https://journals.asm.org/doi/pdf/10.1128/CMR.00022-06

Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

Background In 2007, a US Air Force training facility reported a cluster of severe respiratory illnesses associated with a rare human adenovirus (Ad) serotype, Ad14. We investigated this outbreak to better understand its epidemiology, clinical spectrum, and associated risk factors. Methods Data were collected from ongoing febrile respiratory illness (FRI) surveillance and from a retrospective cohort investigation. Because an Ad7 vaccine is in development, Ad7 antibody titers in pretraining serum samples from trainees with mild and those with severe Ad14 illness were compared. Results During 2007, an estimated 551 (48%) of 1147 trainees with FRI were infected with Ad14; 23 were hospitalized with pneumonia, 4 required admission to an intensive care unit, and 1 died. Among cohort members (n = 173), the Ad14 infection rate was high (50%). Of those infected, 40% experienced FRI. No cohort members were hospitalized. Male sex (risk ratio [RR], 4.7 [95% confidence interval {CI}, 2.2-10.1]) and an ill close contact (RR, 1.6 [95% CI, 1.2-2.2]) were associated with infection. Preexisting Ad7 neutralizing antibodies were found in 7 (37%) of 19 Ad14-positive trainees with mild illness but in 0 of 16 trainees with Ad14 pneumonia (P = .007). Conclusions Emergence of Ad14, a rare Ad serotype, caused a protracted outbreak of respiratory illness among military recruits. Most infected recruits experienced FRI or milder illnesses. Some required hospitalization, and 1 died. Natural Ad7 infection may protect against severe Ad14 illness.

https://www.jstor.org/stable/info/40254579

Outbreak of Severe Respiratory Disease Associated with Emergent Human Adenovirus Serotype 14 at a US Air Force Training Facility in 2007

Viral culture isolation has been widely accepted as the “gold standard” for laboratory confirmation of viral infection; however, it requires ultralow temperature specimen storage. Storage of specimens in ethanol at room temperature could expand our ability to conduct active surveillance and retrospective screenings of viruses with rapid and inexpensive real-time PCR tests, including isolates from remote regions where freezing specimens for culture is not feasible. Molecular methods allow for rapid identification of viral pathogens without the need to maintain viability. We hypothesized that ethanol, while inactivating viruses, can preserve DNA and RNA for PCR-based methods. To evaluate the use of ethanol-stored specimens for augmenting surveillance for detection of influenza viruses A and B and adenoviruses (AdV), paired nasal swab specimens were collected from 384 recruits with febrile respiratory illness at Fort Jackson, S.C., in a 2-year study. One swab was stored at ambient temperature in 100% ethanol for up to 6 months, and the other swab was stored at −70°C in viral medium. For viral detection, frozen specimens were cultured for a variety of respiratory viruses, and ethanol-fixed specimens were tested with TaqMan (TM) probe and LightCycler SYBR green (SG) melting curve assays with at least two different PCR targets for each virus. The sensitivities of the TM and SG assays on specimens stored in ethanol for 1 month were 75% and 58% for influenza A, 89% and 67% for influenza B, and 93 to 98% and 57% for AdV, respectively. Lower specificities of the real-time assays corresponded to the increased detection of PCR-positive but culture-negative specimens. Influenza virus RNA was detected as well or better after 6 months of storage in ethanol.

Evaluation of PCR Testing of Ethanol-Fixed Nasal Swab Specimens as an Augmented Surveillance Strategy for Influenza Virus and Adenovirus Identification

Objective To provide primary care physicians with clinical guidance for addressing parental vaccine hesitancy. Sources of information The PubMed database was searched for English-language articles published in the 10 years before January 1, 2018. Search terms included vaccine hesitancy or confidence or acceptance, parents or children, and communication, counseling, or clinical practice. References of identified articles were assessed for additional relevant articles. A separate gray literature search was conducted using Google to find best-practice guidelines from public health and health care organizations, knowledge translation materials for health care providers, and resources that could be used in discussions with parents about vaccines. Main message Practical tips for addressing parental vaccine hesitancy in primary care include starting early, presenting vaccination as the default approach, building trust, being honest about side effects, providing reassurance on a robust vaccine safety system, focusing on protection of the child and community, telling stories, and addressing pain. Also provided are statements that providers could use in vaccination-related conversations; answers to commonly asked questions on benefits, safety, and immunologic aspects of vaccines; and links to a number of online resources for physicians and parents. Conclusion Vaccine-hesitant parents who are on the fence far outnumber vaccine refusers; therefore, counseling this group might be more effective. Reasons behind vaccine hesitancy are complex and encompass more than just a knowledge deficit. As a trusted source of information on vaccines, family physicians play a key role in driving vaccine acceptance.

https://www.cfp.ca/content/cfp/65/3/175.full.pdf

Addressing vaccine hesitancy: Clinical guidance for primary care physicians working with parents.

A real-time PCR assay for the detection of four Leishmania complexes (L. Viannia, L. mexicana, L. donovani/infantum, and L. major) was developed and evaluated. The assay was developed to detect the glucosephosphate isomerase gene and capitalizes on DNA sequence variability within that gene for Leishmania complex identification. Primer/probe sets were created and tested against a panel of 21 known negative controls and on DNA extracted from cultured promastigotes or from tissue biopsies from patients with cutaneous leishmaniasis. The assay was highly specific, as no amplification products were detected in the negative control samples while simultaneously retaining a high degree of complex-specific diagnostic accuracy for cultured organisms and patient clinical samples. Real-time PCR offers rapid (within hours) identification of Leishmania to the complex level and provides a useful molecular tool to assist both epidemiologists and clinicians.

Rapid identification of Leishmania complexes by a real-time PCR assay.

Provider-parent Communication When Discussing Vaccines: A Systematic Review

Acute respiratory disease (ARD) due to adenoviruses is a reemerging disease in military recruits. It is a challenge for clinicians to accurately diagnose this disease and to appropriately treat affected individuals. This study investigated the utility of a quantitative, rapid-cycle, real-time fluorogenic polymerase chain reaction (PCR) technique for detecting adenovirus type 4 (Ad4) in a clinical setting. Throat swab specimens and clinical data were collected from US Army basic trainees hospitalized with ARD at Fort Jackson, South Carolina. A total of 140 throat swab specimens were collected from 83 subjects. Rapid PCR results (obtained in <2 h) had a sensitivity of 100% and a specificity of 100%, compared with viral culture. There was no difference, qualitative or quantitative, between frozen and fresh samples for PCR detection of Ad4. Individuals with test results positive for Ad4 were hospitalized longer than were individuals with negative test results. Higher virus loads at hospital admission corresponded to longer lengths of stay for Ad4-positive subjects.

Evaluation of a rapid quantitative diagnostic test for adenovirus type 4.

Purpose: To perform a literature review of barriers to and facilitators of parents’ decisions to have their children vaccinated. Both differences and similarities between the civilian sector and the military health system (MHS) were explored.



Data sources: Articles and documents were identified from the following databases: CINAHL, PubMed, Defense Technical Information Center (DTIC), ISI, and Google Scholar. Reference lists from articles were reviewed.



Conclusions: Common themes identified in the civilian sector and the MHS included an electronic registry, care access issues, provider characteristics, and child illness. Several themes served as a barrier in one system while being a facilitator in the other, such as provider characteristics. Literature addressing the MHS and factors affecting vaccine coverage is scant; however, a large disparity in vaccination coverage exists between the MHS and the civilian sector.



Implications: The theme of provider characteristics was seen as a barrier in both systems; a better understanding of this theme (i.e., provider effectiveness at interacting with parents) would benefit primary care practice. An electronic vaccine registry and targeted education and media campaign used to facilitate vaccinations in the MHS should be piloted in the civilian sector. Additionally, future studies should be performed on identified themes in the MHS.

A description of barriers and facilitators to childhood vaccinations in the military health system.

Background and purpose Little research has been performed investigating the effect of using a vaccine hesitancy (VH) screening tool to address specific vaccination concerns. The purpose of this study was to determine whether using a VH screening tool in conjunction with provider discussions addressing parental concerns affected the parental intent to vaccinate (ITV). Methods This study used a pretest/posttest design to measure and categorize VH and ITV. Conclusions The total sample size was 89. Differences between previsit and postvisit questionnaires showed mean responses for questions that measured specific VH categories had slight increases in the level of VH. A regression analysis was conducted to predict the postvisit ITV. Questions measuring parental trust in the provider, beliefs on vaccine efficacy, and beliefs about the prevalence of vaccine preventable disease (VPD) in the community predicted 72% of the postvisit ITV responses. Implications for practice The utilization of a VH screening tool used in conjunction with provider education in an educated and mildly VH population did not positively affect the level of VH or the parental ITV. Additional research should investigate the role of parental trust in the provider, beliefs on vaccine efficacy, and the prevalence of VPD in the community affect parental ITV.

https://cdr.lib.unc.edu/downloads/x633f156c

John T. Connors

Papers

Provider-parent Communication When Discussing Vaccines: A Systematic Review

Evaluation of a rapid quantitative diagnostic test for adenovirus type 4.

A description of barriers and facilitators to childhood vaccinations in the military health system.

Implementing vaccine hesitancy screening for targeted education.