Absence of <i>Pneumocystis jirovecii</i> colonization in human immunodeficiency virus-infected individuals with and without airway obstruction and with undetectable viral load

Pneumocystis jirovecii colonization has been associated with non-acquired immune deficiency syndrome (AIDS) pulmonary comorbidity. We used spirometry to measure pulmonary function and analyzed oral wash specimens by quantitative polymerase chain reaction (PCR), targeting the large mitochondrial ribosomal subunit. For sensitivity control, a blinded subsample was subjected to touch-down PCRs, targeting both large and small ribosomal subunits and the major surface glycoprotein. Pneumocystis jirovecii deoxyribonucleic acid (DNA) was detected in 1 of 156 (95% confidence interval, .1%–3.5%) virologically suppressed human immunodeficiency virus (HIV)-infected individuals confirmed by all PCR methods. Thus, prevalence of P jirovecii colonization was low and unlikely to be a major cause of pulmonary comorbidity in this group of well treated HIV-infected individuals

Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae

BACKGROUND: Chlamydia pneumoniae infection has been detected by serological methods, but PCR is gaining more interest. A number of different PCR assays have been developed and some are used in combination with serology for diagnosis. Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods. RESULTS: We compared the performance of a newly developed real-time PCR with a conventional PCR method for detection of C. pneumoniae. The PCR methods were tested on reference samples containing C. pneumoniae DNA and on 136 nasopharyngeal samples from patients with a chronic cough. We found the same detection limit for the two methods and that clinical performance was equal for the real-time PCR and for the conventional PCR method, although only three samples tested positive. To investigate whether the low prevalence of C. pneumoniae among patients with a chronic cough was caused by suboptimal PCR efficiency in the samples, PCR efficiency was determined based on the real-time PCR. Seventeen of twenty randomly selected clinical samples had a similar PCR efficiency to samples containing pure genomic C. pneumoniae DNA. CONCLUSIONS: These results indicate that the performance of real-time PCR is comparable to that of conventional PCR, but that needs to be confirmed further. Real-time PCR can be used to investigate the PCR efficiency which gives a rough estimate of how well the real-time PCR assay works in a specific sample type. Suboptimal PCR efficiency of PCR is not a likely explanation for the low positivity rate of C. pneumoniae in patients with a chronic cough

Effect of early measles vaccine on pneumococcal colonization: A randomized trial from Guinea-Bissau.

BACKGROUND: Measles vaccine (MV) may have non-specific beneficial effects for child health and particularly seems to prevent respiratory infections. Streptococcus pneumoniae is the leading cause of bacterial pneumonia among children worldwide, and nasopharyngeal colonization precedes infection. OBJECTIVE: We investigated whether providing early MV at 18 weeks of age reduced pneumococcal colonization and/or density up to 9 months of age. METHOD: The study was conducted in 2013-2014 in Guinea-Bissau. Pneumococcal vaccine was not part of the vaccination program. Infants aged 18 weeks were block-randomized 2:1 to early or no early MV; at age 9 months, all children were offered MV as per current policy. Nasopharyngeal swabs were taken at baseline, age 6.5 months, and age 9 months. Pneumococcal density was determined by q-PCR. Prevalence ratios of pneumococcal colonization and recent antibiotic treatment (yes/no) by age 6.5 months (PR6.5) and age 9 months (PR9) were estimated using Poisson regression with robust variance estimates while the pneumococcal geometric mean ratio (GMR6.5 and GMR9) was obtained using OLS regression. RESULTS: Analyses included 512 children; 346 early MV-children and 166 controls. At enrolment, the pneumococcal colonization prevalence was 80% (411/512). Comparing early MV-children with controls, the PR6.5 was 1.02 (95%CI = 0.94-1.10), and the PR9 was 1.04 (0.96-1.12). The GMR6.5 was 1.02 (0.55-1.89), and the GMR9 was 0.69 (0.39-1.21). Early MV-children tended to be less frequently treated with antibiotics prior to follow up (PR6.5 0.60 (0.34-1.05) and PR9 0.87 (0.50-1.53)). Antibiotic treatment was associated with considerably lower colonization rates, PR6.5 0.85 (0.71-1.01) and PR9 0.66 (0.52-0.84), as well as lower pneumococcal density, GMR6.5 0.32 (0.12-0.86) and GMR9 0.52 (0.18-1.52). CONCLUSION: Early MV at age 18 weeks had no measurable effect on pneumococcal colonization prevalence or density. Higher consumption of antibiotics among controls may have blurred an effect of early MV. TRIAL REGISTRATION: clinicaltrials.gov NCT01486355

Expanding the Diagnostic Use of PCR in Leptospirosis: Improved Method for DNA Extraction from Blood Cultures

Background: Leptospirosis is a neglected zoonosis of ubiquitous distribution. Symptoms are often non-specific and may range from flu-like symptoms to multi-organ failure. Diagnosis can only be made by specific diagnostic tests like serology and PCR. In non-endemic countries, leptospirosis is often not suspected before antibiotic treatment has been initiated and consequently, relevant samples for diagnostic PCR are difficult to obtain. Blood cultures are obtained from most hospitalized patients before antibiotic therapy and incubated for at least five days, thus providing an important source of blood for PCR diagnosis. However, blood cultures contain inhibitors of PCR that are not readily removed by most DNAextraction methods, primarily sodium polyanetholesulfonate (SPS). Methodology/Principal Findings: In this study, two improved DNA extraction methods for use with blood cultures are presented and found to be superior in recovering DNA of Leptospira interrogans when compared with three previously described methods. The improved methods were easy and robust in use with all tested brands of blood culture media. Applied to 96 blood cultures obtained from 36 patients suspected of leptospirosis, all seven patients with positive convalescence serology were found positive by PCR if at least one anaerobic and one aerobic blood culture, sampled before antibiotic therapy were tested. Conclusions/Significance: This study suggests that a specific and early diagnosis can be obtained in most cases of sever