Kathy Luinstra

The Loss of Topography in the Microbial Communities of the Upper Respiratory Tract in the Elderly

RATIONALE The microbial communities inhabiting the upper respiratory tract protect from respiratory infection. The maturity of the immune system is a major influence on the composition of the microbiome and, in youth, the microbiota and immune system are believed to mature in tandem. With age, immune function declines and susceptibility to respiratory infection increases. Whether these changes contribute to the microbial composition of the respiratory tract is unknown. OBJECTIVES Our goal was to determine whether the microbes of the upper respiratory tract differ between mid-aged adults (18-40 yr) and the elderly (>65 yr). METHODS Microbiomes of the anterior nares and oropharynx of elderly individuals were evaluated by 16S rRNA gene sequencing. These communities were compared with data on mid-aged adults obtained from the Human Microbiome Project. MEASUREMENTS AND MAIN RESULTS The microbiota of the elderly showed no associations with sex, comorbidities, residence, or vaccinations. Comparisons of mid-aged adults and the elderly demonstrated significant differences in the composition of the anterior nares and oropharynx, including a population in the anterior nares of the elderly that more closely resembled the oropharynx than the anterior nares of adults. The elderly oropharyngeal microbiota were characterized by increased abundance of streptococci, specifically, Streptococcus salivarius group species, but not Streptococcus pneumoniae, carriage of which was low (<3% of participants), as demonstrated by PCR (n = 4/123). CONCLUSIONS 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.

High Analytical Sensitivity and Low Rates of Inhibition May Contribute to Detection of Chlamydia trachomatis in Significantly More Women by the APTIMA Combo 2 Assay

ABSTRACT 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.

Multiplex PCR tests sentinel the appearance of pandemic influenza viruses including H1N1 swine influenza.

Abstract Background Since the turn of the century seven new respiratory viruses have infected man and two of these have resulted in worldwide epidemics. Both SARS Coronavirus which quickly spread to 29 countries in February 2003 and H1N1 swine influenza that recently spread from Mexico to 30 countries in three weeks represent major pandemic threats for mankind. Diagnostic assays are required to detect novel influenza strains with pandemic potential. Objective In this report we evaluate the ability of a multiplex PCR test (xTAG™ RVP) to detect new, “non-seasonal” influenza viruses including the H1N1 swine influenza A/swine/California/04/2009. Study design Laboratory based study using retrospective and prospective specimens. Results This multiplex PCR test detected the present of non-seasonal (non-H1, non-H3) influenza in 20 of 20 patients infected with H1N1 swine flu virus. In addition to detecting the current swine flu the xTAG™ RVP test detected the H5N1 A/Vietnam/1203/2004 high pathogenicity avian influenza virus that circulated in South East Asia in 2003 as well as 17 out of 17 influenza A viruses representing 11 HA subtypes isolated from birds, swine and horses not yet seen in the human population. Conclusion Based on these results we believe that this molecular test can perform an important role as a sentinel test to detect novel non-seasonal influenza A viruses in patients presenting with influenza-like illness (ILI) and therefore act as an early warning system for the detection of future pandemic influenza threats.

Screening urine with a leukocyte esterase strip and subsequent Chlamydial testing of asymptomatic men attending primary care practitioners

BACKGROUND AND OBJECTIVES The detection of asymptomatic urethritis using a leukocyte esterase (LE) strip may have a role in primary care screening to select men who need diagnostic testing for Chlamydia trachomatis and Neisseria gonorrhoeae. STUDY DESIGN Eight-hundred and eighty-two men, 16 to 35 years of age were studied when they attended their family physician or university health clinic for nongenitourinary complaints. First void urine (FVU) was tested by an LE strip (Chemstrip 2 LN, Boehringer Mannheim Corp., Indianapolis, IN), Chlamydiazyme (Abbott Laboratories, N. Chicago, IL) enzyme immunoassay with confirmatory blocking and polymerase chain reaction (PCR) with chlamydial plasmid primers. RESULTS Forty-five men (5.1%) were positive (> trace) by LE strip. Of the LE-positive urines, 9 (20.0%) were positive by EIA or PCR, and none of the LE-negatives were positive by EIA or PCR. Twenty-three LE positives (5 EIA/PCR-positive; 1 PCR-positive; 17 EIA/PCR-negative) were able to be followed with a second urine and 2 urethral swabs. All of the 6 chlamydia-positives who had follow-up tests were positive by both immunoassay and PCR on urine. Based on the FVU results, the prevalence of asymptomatic chlamydial infection was 1.0% (9/88) (95% CL, 0.5 to 1.9) for which the LE urine strip was 100% (9/9) sensitive and 95.9% (837/873) specific. Analyses based on screening 1,000 men, 16 to 25 years of age, showed that the cost per case detected was

Development and Evaluation of a Flocked Nasal Midturbinate Swab for Self-Collection in Respiratory Virus Infection Diagnostic Testing

192.00 using the LE strip (> 1+) to select urine specimens for EIA testing, compared to

Accuracy of Results Obtained by Performing a Second Ligase Chain Reaction Assay and PCR Analysis on Urine Samples with Positive or Near-Cutoff Results in the LCx Test for Chlamydia trachomatis

1,326.00 using the EIA to test all urine specimens. CONCLUSION In this low prevalence, primary care setting, the LE urine strip was an accurate screening test, which if used to preselect urine specimens for subsequent chlamydial testing, would be less costly per case detected than assaying each specimen for chlamydia.

Detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, and Mycoplasma genitalium in first-void urine specimens by multiplex polymerase chain reaction

ABSTRACT 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.

Can serology diagnose upper genital tract Chlamydia trachomatis infection ? Studies on women with pelvic pain, with or without chlamydial plasmid DNA in endometrial biopsy tissue

ABSTRACT Nucleic acid amplification assays such as the ligase chain reaction and PCR have encountered reproducibility problems. The initial extract and a newly extracted aliquot of urine specimens (n = 120) which had signal-to-cutoff (S/CO) ratios above 0.80 by the LCx Chlamydia assay were retested. Nucleic acid was extracted from an additional urine sample for testing by the AMPLICOR PCR Chlamydia assay. Fifteen percent (18 of 120) of the urine specimens were negative by all repeat tests (initial mean S/CO ratio by the LCx Chlamydia assay, 0.93; S/CO ratio range, 0.80 to 3.30). Repeat testing of the 102 specimens with possible positive results by the LCx Chlamydia assay by use of the initially extracted aliquot confirmed the results for 95 (93.1%) of the specimens; repeat testing of a newly extracted aliquot confirmed the results for 87 (85.3%) of the specimens. Twenty specimens had discordant results by the two repeat LCx Chlamydia assays. A total of 78 of 102 (76.5%) of the specimens were positive by the AMPLICOR PCR, and the AMPLICOR PCR confirmed the results for 82.1% (78 of 95) and 89.6% (78 of 87) of the specimens positive by the two repeat LCx Chlamydia assays, respectively. Some of the discrepancies observed by multiple repeat tests may have been due to specimen mislabeling or contamination during performance of the procedure rather than to the LCx Chlamydia assay. Both assays suffered from a lack of reproducibility on repeat testing with a small proportion of specimens, probably due to the presence of low levels of DNA, the presence of variable amounts of amplification inhibitors, and the loss of DNA during extraction.

Evaluation of Anatomically Designed Flocked Rectal Swabs for Molecular Detection of Enteric Pathogens in Children Admitted to Hospital with Severe Gastroenteritis in Botswana

Background: Sexually transmitted diseases are often caused by one or more microorganisms, and asymptomatic carriage and transmission may be of significance. Testing for more than one organism in a single assay could be a useful approach to laboratory diagnosis. Methods and Results: A multiplex polymerase chain reaction (PCR) assay was developed that employed specific primers targeted to the 7.5-kb cryptic plasmid of Chlamydia trachomatis, the cppB gene of the 4.2-kb cryptic plasmid of Neisseria gonorrhoeae, the 140-kd major adhesion protein gene of Mycoplasma genitlium, and the urease gene of Ureaplasma urealyticum. All four polymerase chain reaction products were detectable by agarose gel electorphoresis and were confirmed by Southern hybridization using fluorescein isothiocyanate-labeled oligonucleotide probes and enhanced chemiluminescent detection. Using purified DNA preparations, multiplex PCR had a reproducible detection limit of 1 fg of C. trachomatis DNA, 100 fg of N. gonorrhoeae DNA, and 10 fg U. urealyticum DNA and M. genitalium DNA, which converts to 1-2 genomic equivalents (ge) of C. trachomatis and N. gonorrhoeae, 4 ge of M. genitalium, and 10 ge U. urealyticum. Multiplex PCR was compared with individual uniplex polymerase chian reaction PCR assays by testing 117 first-void urine samples (91 men, 26 women) from Canadian or Kenyan patients. Multiplex PCR detected 45 of 46 (97.8%) urines with C. trachomatis DNA, 42 of 42 (100%) urines with N. gonorrhoeae DNA, 17 of 17 (100%) urines with U. urealyticum DNA, 4 of 4 (100%) urines with M. genitalium DNA, 12 of 12 urines that had DNA from two bacteria, and 2 of 2 urines with DNA from three bacteria. Multiplex PCR correctly identified bacteria in 92 of 93 urines for an overall sensitivity of 98.9%. Specificity calculations were 100% for C. trachomatis (71/71), N. gonorhoeae (75/75), U. urealyticum (100/100), and M. genitalium (113/113). Conclusions: Multiplex PCR provided a single sensitive and specific test for the detection of four bacteria in first-void urine samples. Testing of first-void urine samples by multiplex PCR could facilitate studies aimed at improving our understanding of the epidemiology of these important sexually transmitted diseases.

Evaluation and Clinical Validation of an Alcohol-Based Transport Medium for Preservation and Inactivation of Respiratory Viruses

Background: Upper genital tract chlamydial infections in women are on the increase, and serology might be a convenient tool for diagnosis. Evaluations of this approach are needed in women with or without microbiologic evidence of organisms in the upper genital tract. Goals: To compare the results of antibody assays with cervical culture and upper genital tract histopathology in women with pelvic pain and chlamydial plasmid DNA in endometrial biopsies. Study Design: Chlamydia trachomatis plasmid DNA was detected by polymerase chain reaction (PCR) on extracted deparaffinized endometrial biopsy tissue. Five antichlamydial antibody assays were performed measuring total antibodies or immunoglobulin G (IgG), IgM, and IgA classes on sera from 14 women with plasmid DNA as well as 31 without plasmid DNA. Results: Accepting the presence of plasmid DNA as the gold standard, no single test had total diagnostic accuracy. The best sensitivity and specificity occurred with the following assays: whole inclusion fluorescence (WIF) (100% and 80.6%); microimmunofluorescence IgM (MIF IgM) (78.6% and 93.6%); and heatshock protein‐60 enzyme immunoassay (42.9% and 100%). Although recombinant anti‐lipopolysaccharide enzyme‐linked immunosorbent assays measured antichlamydial antibodies in a large proportion of these women, specificity was low. The sensitivity and specificity of cervical culture was 28.6% and 100% and of endometrial histopathology was 71.4% and 48.4%. Analysis of patient serological profiles suggested that 6 women without plasmid DNA may have been cases that were missed by PCR. Conclusions: Evaluations of assays to diagnosis Chlamydia trachomatis upper genital tract infections could use the presence of organisms or their markers in the upper genital tract as a standard of comparison. Some of these serological assays, such as WIF or MIF IgM, may be helpful in diagnosis, but more studies are needed.