Differential effects of frozen storage on the molecular detection of bacterial taxa that inhabit the nasopharynx

<p>Abstract</p> <p>Background</p> <p>Frozen storage often precedes metagenomic analysis of biological samples; however, the freezing process can have adverse effects on microbial composition. The effect of freezing on the detection of bacteria inhabiting the infant nasopharynx, a major reservoir of bacterial pathogens, was investigated.</p> <p>Methods</p> <p>16S ribosomal RNA (rRNA) gene-based terminal restriction fragment length polymorphism (T-RFLP) analysis of nasopharyngeal (NP) swabs from twelve Gambian infants was employed. NP swabs were analysed within hours of collection and then after 30 days of storage at -70°C.</p> <p>Results</p> <p>There was substantial heterogeneity among subjects with respect to the effect of freezing on the number of operational taxonomic units (OTUs) detected. Nevertheless, the mean number of OTUs decreased after frozen storage and the relative abundance for 72% of the OTUs changed by less than 0.5% after deep frozen storage. There were differences in the odds of detection and relative abundance of OTUs matched with <it>Moraxella sp</it>., <it>Haemophilus sp</it>./<it>Burkholderia sp.</it>, and <it>Pseudomonas sp</it>. A strong interaction between sex and the effect of freezing was found, whereby there was no significant change observed for males while the mean number of OTUs significantly declined among female infants following frozen storage.</p> <p>Conclusions</p> <p>Although frozen storage of biological samples is often necessary for archiving and logistic purposes, the potential effects on the number of taxa (composition) detected in microbial community studies are significant and should not be overlooked. Moreover, genetic factors such as sex may influence the integrity of nucleic acids during the freezing process.</p

Differential effects of frozen storage on the molecular detection of bacterial taxa that inhabit the nasopharynx

<p>Abstract</p> <p>Background</p> <p>Frozen storage often precedes metagenomic analysis of biological samples; however, the freezing process can have adverse effects on microbial composition. The effect of freezing on the detection of bacteria inhabiting the infant nasopharynx, a major reservoir of bacterial pathogens, was investigated.</p> <p>Methods</p> <p>16S ribosomal RNA (rRNA) gene-based terminal restriction fragment length polymorphism (T-RFLP) analysis of nasopharyngeal (NP) swabs from twelve Gambian infants was employed. NP swabs were analysed within hours of collection and then after 30 days of storage at -70°C.</p> <p>Results</p> <p>There was substantial heterogeneity among subjects with respect to the effect of freezing on the number of operational taxonomic units (OTUs) detected. Nevertheless, the mean number of OTUs decreased after frozen storage and the relative abundance for 72% of the OTUs changed by less than 0.5% after deep frozen storage. There were differences in the odds of detection and relative abundance of OTUs matched with <it>Moraxella sp</it>., <it>Haemophilus sp</it>./<it>Burkholderia sp.</it>, and <it>Pseudomonas sp</it>. A strong interaction between sex and the effect of freezing was found, whereby there was no significant change observed for males while the mean number of OTUs significantly declined among female infants following frozen storage.</p> <p>Conclusions</p> <p>Although frozen storage of biological samples is often necessary for archiving and logistic purposes, the potential effects on the number of taxa (composition) detected in microbial community studies are significant and should not be overlooked. Moreover, genetic factors such as sex may influence the integrity of nucleic acids during the freezing process.</p

Pharmacodynamic modeling of bacillary elimination rates and detection of bacterial lipid bodies in sputum to predict and understand outcomes in treatment of pulmonary tuberculosis

This work was supported by a Wellcome Trust Clinical PhD Fellowship (086757/Z/08/A to D. J. S.), the Malawi Liverpool Wellcome Trust Core grant, and Medical Research Council (grant number G0300403 to M. R. B.).Background. Antibiotic-tolerant bacterial persistence prevents treatment shortening in drug-susceptible tuberculosis, and accumulation of intracellular lipid bodies has been proposed to identify a persister phenotype of Mycobacterium tuberculosis cells. In Malawi, we modeled bacillary elimination rates (BERs) from sputum cultures and calculated the percentage of lipid body-positive acid-fast bacilli (%LB + AFB) on sputum smears. We assessed whether these putative measurements of persistence predict unfavorable outcomes (treatment failure/relapse). Methods. Adults with pulmonary tuberculosis received standard 6-month therapy. Sputum samples were collected during the first 8 weeks for serial sputum colony counting (SSCC) on agar and time-to positivity (TTP) measurement in mycobacterial growth indicator tubes. BERs were extracted from nonlinear and linear mixed-effects models, respectively, fitted to these datasets. The %LB + AFB counts were assessed by fluorescence microscopy. Patients were followed until 1 year posttreatment. Individual BERs and %LB + AFB counts were related to final outcomes. Results. One hundred and thirty-three patients (56% HIV coinfected) participated, and 15 unfavorable outcomes were reported. These were inversely associated with faster sterilization phase bacillary elimination from the SSCC model (odds ratio [OR], 0.39; 95% confidence interval [CI], .22-.70) and a faster BER from the TTP model (OR, 0.71; 95% CI, .55-.94). Higher %LB + AFB counts on day 21-28 were recorded in patients who suffered unfavorable final outcomes compared with those who achieved stable cure (P = .008). Conclusions. Modeling BERs predicts final outcome, and high %LB + AFB counts 3-4 weeks into therapy may identify a persister bacterial phenotype. These methods deserve further evaluation as surrogate endpoints for clinical trials.Publisher PDFPeer reviewe

Predictors of poor treatment outcomes of MDR-TB patients: univariate analysis (N = 43).

<p>*Missing information.</p><p>**After exclusion of 4 cases with mixed or double infection.</p><p>Km: kanamycin; Ofx: ofloxacin.</p

Baseline characteristics of patients, N = 326.

a<p>1 missing information.</p>b<p>2 missing informations.</p>c<p>One case without DST result for all 2^nd line drugs.</p>d<p>Exclusion of 15 patients with mixed or double infection.</p><p>E: ethambutol; S: streptomycin; H: isoniazid; MDR: multidrug resistance; XDR: extensively drug-resistance.</p

Evolution of drug resistance during treatment: amplification and re-infection.

<p>A. Drug resistance increase in fully susceptible and PDR-TB patients during treatment B. Drug resistance increase to ofloxacin, kanamycin, capreomycin and XDR during MDR-TB treatment.</p