Microbial populations of the upper respiratory tract in mid-aged adults and the elderly differ; it is possible that these differences contribute to the increased risk of respiratory infections experienced by the elderly.

BackgroundWe undertook a 2X2 factorial, randomized controlled trial (RCT) to assess whether vitamin D3 supplementation (10,000 international units per week) versus placebo and gargling versus no gargling could prevent viral, clinical upper respiratory tract infection (URTI) in university students.MethodsWe randomized 600 students into 4 treatment arms: 1) vitamin D3 and gargling, 2) placebo and gargling, 3) vitamin D3 and no gargling, and 4) placebo and no gargling. Students completed weekly electronic surveys and submitted self-collected mid-turbinate nasal flocked swabs during September and October in 2010 or 2011. Symptomatic students also completed an electronic symptom diary. The primary and secondary outcomes were the occurrence of symptomatic clinical URTI and laboratory confirmed URTI respectively.ResultsOf 600 participants, 471 (78.5%) completed all surveys while 43 (7.2%) completed none; 150 (25.0%) reported clinical URTI. Seventy participants (23.3%) randomized to vitamin D3 reported clinical URTI compared to 80 (26.7%) randomized to placebo (RR:0.79, CI95:0.61-1.03, p = 0.09). Eighty-five participants (28.3%) randomized to gargling reported clinical URTI compared to 65 participants (21.7%) randomized to the no gargling arm (RR:1.3, CI95:0.92-1.57, p = 0.19). Laboratory testing identified 70 infections (46.7 per 100 URTIs). Vitamin D3 treatment was associated with a significantly lower risk for laboratory confirmed URTI (RR: 0.54, CI95:0.34-0.84, p = 0.007) and with a significantly lower mean viral load measured as log10 viral copies/mL (mean difference: -0.89, CI95: -1.7, -0.06, p = 0.04). Fewer students assigned to gargling experienced laboratory confirmed URTI, however this was not statistically significant (RR:0.82, CI95:0.53-1.26, p = 0.36).ConclusionsThese results suggest that vitamin D3 is a promising intervention for the prevention of URTI. Vitamin D3 significantly reduced the risk of laboratory confirmed URTI and may reduce the risk of clinical infections.Trial registrationClinical Trials Registration: NCT01158560.

f Two-hundred eighty matched bulk stool and anatomically designed flocked rectal swab samples were collected from children admitted to the hospital with acute diarrhea in Botswana. Their parents were asked about the acceptability of the swab collection method compared with bulk stool sampling. All samples underwent identical testing with a validated 15-target (9 bacterial, 3 viral, and 3 parasite) commercial multiplex PCR assay. The flocked swabs had a 12% higher yield for bacterial pathogen targets (241 versus 212; P ‫؍‬ 0.003) compared with that of stool samples, as well as similar yields for viral targets (110 versus 113; P ‫؍‬ 0.701) and parasite targets (59 versus 65; P ‫؍‬ 0.345). One hundred sixty-four of the flocked swab-stool pairs were also tested with separate laboratory-developed bacterial and viral multiplex assays, and the flocked rectal swabs had a performance that was similar to that seen with commercial assay testing. Almost all parents/guardians found the swabs acceptable. Flocked rectal swabs significantly facilitate the molecular diagnosis of diarrheal disease in children. Diarrheal disease remains a leading cause of global childhood morbidity and mortality, yet access to diagnostic laboratory testing is rarely available in much of the world. One of the barriers to diagnosing diarrheal disease, either for clinical or surveillance purposes, is the difficulty and time delays in obtaining and transporting a bulk stool specimen. Several investigators have sought to overcome this barrier through the use of rectal swab specimens for culture, molecular, and antigen testing, with variable results (1-5). Flocked swabs designed for respiratory and genitourinary sampling have been shown to acquire better samples than those acquired with more traditional spun fiber swabs (6, 7). We used a specially designed flocked rectal swab (FLOQSwabs; Copan Italia, Brescia, Italy) developed specifically for the diagnosis of diarrheal disease in children (Fig. 1) and then compared matched flocked rectal swabs to bulk stool samples in a clinical setting. The samples were collected from children admitted to the hospital in Botswana with severe acute gastroenteritis and tested using a U.S. FDAcleared commercial multiplex PCR assay in order to assess performance across a broad number of bacterial, viral, and parasitic pathogens.(These data were presented in part at the 29th Annual Clinical Virology Symposium, Daytona Beach, FL, 28 April to 1 May 2013, and at the Annual Pediatric Academic Society Meeting, Vancouver, Canada, 5 May 2014.) MATERIALS AND METHODSChildren Ͻ13 years of age who were admitted to the hospital with a diagnosis of acute gastroenteritis were enrolled prospectively at the Princess Marina Hospital in Gaborone, Botswana. Princess Marina Hospital is the largest referral hospital in Botswana.Clinical data were collected, and both the pediatric flocked rectal swab and bulk stool samples were obtained from each child as soon as possible after enrollment. The swab and stool samples were collected simultaneousl...

We developed and evaluated flocked nasal midturbinate swabs obtained from 55 asymptomatic and 108 symptomatic volunteers. Self-collected swabs obtained from asymptomatic volunteers yielded numbers of respiratory epithelial cells comparable to those of staff-collected nasal (n ‫؍‬ 55) or nasopharyngeal (n ‫؍‬ 20) swabs. Specific viruses were detected in swabs self-collected by 42/108 (38.9%) symptomatic volunteers by multiplex PCR.We report herein the design and evaluation of a novel selfcollected nasal midturbinate swab for respiratory virus diagnosis. We previously demonstrated that Copan flocked nasopharyngeal swabs (NPS) collected significantly more respiratory epithelial cells than conventional rayon swabs and improved sample collection for direct fluorescent antibody (DFA) testing of respiratory viruses (2). We observed that the cell yield obtained from sampling the nose using a flocked swab designed for nasopharyngeal sampling was equivalent to that obtained from nasopharyngeal sampling using rayon NPS. This led us to hypothesize that a flocked nasal swab designed to contact a larger nasal surface area would further improve cell sampling and enable self-collection.We measured the nasal passages of adult white cadavers at the Michael G. DeGroote School of Medicine anatomy laboratory, McMaster University, and designed a tapered coneshaped swab with a greater length and diameter of flocked nylon ( Fig. 1). A collar was added at 5.5 cm as a guide to maximum insertion depth for adults. The nasal swab samples a large surface area of respiratory mucosa, covering the inferior and middle turbinate bones, and is now commercially available in pediatric and adult sizes (FLOQSwabs; Copan Italia S.p.A., Brescia, Italy).Our primary study objectives were to determine the feasibility, acceptability, and performance characteristics of selfsampled nasal midturbinate swabs. We tested the adequacy of self-collected flocked nasal swabs, the equivalence of nasal and nasopharyngeal sampling, and the diagnostic yield for specific respiratory viruses by multiplex PCR. The study protocol was approved by the Research Ethics Board at St. Joseph's Healthcare, Hamilton, Ontario, Canada.To examine the adequacy of respiratory specimen self-collection, 55 healthy asymptomatic adult volunteers were recruited from hospital staff and visitors. After providing signed informed consent, volunteers self-collected two flocked nasal swabs by following the printed instructions with illustrations. Each swab was inserted up to 5.5 cm, as tolerated, into the same nostril of their choice. An experienced staff member then collected the following two additional nasal swabs, using the opposite nostril in randomized order: a flocked midturbinate nasal swab and a rayon pernasal swab. A self-administered questionnaire assessed the ease of self-collection, discomfort, and swab preferences.All four nasal swabs were placed into universal transport medium (UTM; Copan Italia S.p.A.) and coded to maintain blinding. Samples were processed identically, according to c...

The clinical sensitivity of nucleic acid amplification tests may be determined by analytical sensitivity and inhibitors in patient samples. We established endpoints for detection of propagated Chlamydia trachomatis L2 434, diluted according to swab and urine protocols for APTIMA Combo 2 (AC2), ProbeTec ET (PT), and Amplicor (AMP) assays. AC2 was 1,000-fold more sensitive than PT and 10-fold more sensitive than AMP on mock swab specimens. For urine, AC2 analytical sensitivity was 100-fold greater than those of the other assays. Spiking an aliquot of each clinical-trial sample from 298 women demonstrated inhibition rates in first-void urine (FVU), cervical swabs (CS), and vaginal swabs (VS) of 12.1%, 12.8%, and 10.4% for AMP; 27.2%, 2%, and 2%, for PT; and 0.3%, 1.7%, and 1.3% for AC2. Inhibition of our C. trachomatis spike and the PT or AMP amplification controls from the manufacturers showed less than 50% correlation. Using an infected-patient reference standard (a specimen positive in at least two tests or a single test positive in two of three samples) in AC2, the VS identified 68/69 (98.6%) infected women compared to CS (89.9%) or FVU (81.2%). Significantly fewer women were identified by PT (65.2%, 63.8%, and 66.7%) or AMP (65.2%, 59.4%, and 56.5%) with the three specimens. By individual specimen type, AC2 confirmed virtually all PT-and AMP-positive specimens, but rates of AC2 confirmation by AMP or PT ranged from 62.9 to 80.3%. The AC2 test identified significantly more women infected with C. trachomatis (P ‫؍‬ 0.001). Vaginal swabs appear to be the specimen of choice for screening.Chlamydia trachomatis is one of the most common sexually transmitted infections in the United States and worldwide (17), largely due to high rates of asymptomatic infection in the lower genital tracts of women and men (24). Early diagnosis followed by treatment of this infection can prevent upper genital tract infection, such as pelvic inflammatory disease (21).For the past 10 years, a great deal of research has led to the development and evaluation of sensitive and specific diagnostic tests for C. trachomatis. These newer nucleic acid amplification tests (NAATs) have been commercialized and are now in routine use in many parts of the world. The important aspect of these assays is their capacity to be used on less invasive specimens, such as vaginal swabs (VS) and first-void urine (FVU), which can be self-collected. Studies have shown that NAATs performed on these less invasive samples are able to detect as many or more infected patients than traditional swabs from the urethra or cervix (2-4, 6-10, 19-22, 25-27, 31).The analytical sensitivity of each test and its susceptibility to inhibitors in different specimen types may determine the clinical sensitivity of each test. We compared detection thresholds and determined the inhibitor and infection rates in three different specimens from 298 women by using three commercial assays for C. trachomatis. MATERIALS AND METHODSSpecimens. Three cervical swabs (CS), three VS, and the first 3...

Several laboratories have demonstrated that the polymerase chain reaction (PCR) is more sensitive than culture or enzyme immunoassay (EIA) for detecting Chlamydia trachomatis in genitourinary tract specimens when various DNA targets are used for amplification, including the cryptic plasmid, major outer membrane protein (MOMP), or rRNA genes. We compared the performances of five different PCR assays, including assays with two plasmid, two MOMP, and one rRNA targets, by amplifying serial dilutions of C. trachomatis DNA and testing genitourinary tract specimens. By using published procedures, two different plasmid primers had sensitivities of 0.1 fg for C. trachomatis plasmid DNA and 10 fg for total cellular DNA. The sensitivities of the assays with the two MOMP primers were 0.1 and 10 pg, and the sensitivity for the assay with the rRNA primers was 1 pg for cellular DNA. Both plasmid-based assays detected 38 of 38 confirmed Chlamydiazyme-positive specimens, whereas the assays with the MOMP and rRNA primers detected 36 of 38 and 29 of 38 confirmed Chlamydiazyme-positive specimens, respectively. Six of 18 Chlamydiazyme-negative specimens collected from individuals whose specimens were positive by culture or immunofluorescence were positive by both plasmid-based PCRs; 4 of these were positive by PCR with the MOMP primers and 3 were positive by PCR with the rRNA primers. The results obtained with both purified DNA and genitourinary tract specimens indicated that the plasmid-based PCRs are more sensitive than bacterial chromosome-based PCRs for detecting C. trachomatis.

The emergence of a novel coronavirus (CoV) as the cause of severe acute respiratory syndrome (SARS) catalyzed the development of rapid diagnostic tests. Stool samples have been shown to be appropriate for diagnostic testing for SARS CoV, although it has been recognized to be a heterogeneous and difficult sample that contains amplification inhibitors. Limited information on the efficiency of extraction methods for the purification and concentration of SARS CoV RNA from stool samples is available. Our study objectives were to determine the optimal extraction method for SARS CoV RNA detection and to examine the effect of increased specimen volume for the detection of SARS CoV RNA in stool specimens. We conducted a multicenter evaluation of four automated and four manual extraction methods using dilutions of viral lysate in replicate mock stool samples, followed by quantitation of SARS CoV RNA using real-time reverse transcriptase PCR. The sensitivities of the manual methods ranged from 50% to 100%, with the Cortex Biochem Magazorb method, a magnetic bead isolation method, allowing detection of all 12 positive samples. The sensitivities of the automated methods ranged from 75% to 100%. The bioMérieux NucliSens automated extractor and miniMag extraction methods each had a sensitivity of 100%. Examination of the copy numbers detected and the generation of 10-fold dilutions of the extracted material indicated that a number of extraction methods retained inhibitory substances that prevented optimal amplification. Increasing the volume of sample input did improve detection. This information could be useful for the extraction of other RNA viruses from stool samples and demonstrates the need to evaluate extraction methods for different specimen types.The emergence of a novel coronavirus (CoV) as the cause of severe acute respiratory syndrome (SARS) and its spread throughout the world catalyzed the development of rapid diagnostic tests. SARS CoV has been shown to replicate in the gastrointestinal tract (4), and consistent with this, stool samples were shown to be appropriate for diagnostic testing for SARS CoV. Peiris et al. (7) found a positivity rate of 97% (65/67 samples) for detection of SARS CoV nucleic acid in stool samples at 14 days after the onset of symptoms. By contrast, Chan et al. (1) found a lower overall stool positivity rate of 26.2% (70/267), with a 42.9% (9/21) positivity rate within 1 week of the onset of symptoms, a 68.0% (17/25) positivity rate between 1 and 2 weeks of onset, and a 70.8% (34/48) positivity rate between 2 and 4 weeks of onset. Preliminary studies performed in our laboratories indicated that variations in RNA extraction methods could explain the differences seen in these studies. We anticipated that the optimization of extraction methods for stool samples could potentially increase the sensitivity of amplification detection of SARS CoV, especially early in infection. Stool samples have been recognized to be heterogeneous and difficult samples for use for molecular analysis. Bile salts, he...

The presence of endogenous amplification inhibitors in urine may produce false-negative results for the detection of Chlamydia trachomatis nucleic acids by tests such as PCR, ligase chain reaction (LCR), and transcription-mediated amplification (TMA). Consecutive urine specimens from 101 pregnant women and 287 nonpregnant women submitted for urinalysis were processed for C. trachomatis detection. Aliquots were spiked with the equivalent of one C. trachomatis elementary body and were tested by three commercial assays: AMPLICOR CT/NG, Chlamydia LCX, and Chlamydia TMA. The prevalence of inhibitors resulting in complete inhibition of amplification was 4.9% for PCR, 2.6% for LCR, and 7.5% for TMA. In addition, all three assays were partially inhibited by additional urine specimens. Only PCR was more often inhibited by urine from pregnant women than by urine from nonpregnant women (9.9 versus 3.1%;P = 0.011). A complete urinalysis including dipstick and a microscopic examination was performed. Logistic regression analysis revealed that the following substances were associated with amplification inhibition: beta-human chorionic gonadotropin (odds ratio [OR], 3.3) and crystals (OR, 3.3) for PCR, nitrites for LCR (OR, 14.4), and hemoglobin (OR, 3.3), nitrites (OR, 3.3), and crystals (OR, 3.3) for TMA. Aliquots of each inhibitory urine specimen were stored at 4 and −70°C overnight or were extracted with phenol-chloroform and then retested at dilutions of 1:1, 1:4, and 1:10. Most inhibition was removed by storage overnight at 4 or −70°C and a dilution of 1:10 (84% for PCR, 100% for LCR, and 92% for TMA). Five urine specimens (three for PCR and two for TMA) required phenol-chloroform extraction to remove inhibitors. The results indicate that the prevalence of nucleic acid amplification inhibitors in female urine is different for each technology, that this prevalence may be predicted by the presence of urinary factors, and that storage and dilution remove most of the inhibitors.