Managing Contamination and Diverse Bacterial Loads in 16S rRNA Deep Sequencing of Clinical Samples : Implications of the Law of Small Numbers

In this article, we investigate patterns of microbial DNA contamination in targeted 16S rRNA amplicon sequencing (16S deep sequencing) and demonstrate how this can be used to filter background bacterial DNA in diagnostic microbiology. We also investigate the importance of sequencing depth. We first determined the patterns of contamination by performing repeat 16S deep sequencing of negative and positive extraction controls. This process identified a few bacterial species dominating across all replicates but also a high intersample variability among low abundance contaminant species in replicates split before PCR amplification. Replicates split after PCR amplification yielded almost identical sequencing results. On the basis of these observations, we suggest using the abundance of the most dominant contaminant species to define a threshold in each clinical sample from where identifications with lower abundances possibly represent contamination. We evaluated this approach by sequencing of a diluted, staggered mock community and of bile samples from 41 patients with acute cholangitis and noninfectious bile duct stenosis. All clinical samples were sequenced twice using different sequencing depths. We were able to demonstrate the following: (i) The high intersample variability between sequencing replicates is caused by events occurring before or during the PCR amplification step. (ii) Knowledge about the most dominant contaminant species can be used to establish sample-specific cutoffs for reliable identifications. (iii) Below the level of the most abundant contaminant, it rapidly becomes very demanding to reliably discriminate between background and true findings. (iv) Adequate sequencing depth can be claimed only when the analysis also picks up background contamination. IMPORTANCE There has been a gradual increase in 16S deep sequencing studies on infectious disease materials. Management of bacterial DNA contamination is a major challenge in such diagnostics, particularly in low biomass samples. Reporting a contaminant species as a relevant pathogen may cause unnecessary antibiotic treatment or even falsely classify a noninfectious condition as a bacterial infection. Yet, there are few studies on how to filter contamination in clinical microbiology. Here, we demonstrate that sequencing of extraction controls will not reveal the full spectrum of contaminants that could occur in the associated clinical samples. Only the most abundant contaminant species were consistently detected, and we present how this can be used to set sample specific thresholds for reliable identifications. We believe this work can facilitate the implementation of 16S deep sequencing in diagnostic laboratories. The new data we provide on the patterns of microbial DNA contamination is also important for microbiome research

Genetic diversity of circulating rotavirus strains in Tanzania prior to the introduction of vaccination.

BACKGROUND: Tanzania currently rolls out vaccination against rotavirus-diarrhea, a major cause of child illness and death. As the vaccine covers a limited number of rotavirus variants, this study describes the molecular epidemiology of rotavirus among children under two years in Dar es Salaam, Tanzania, prior to implementation of vaccination. METHODS: Stool specimens, demographic and clinical information, were collected from 690 children admitted to hospital due to diarrhea (cases) and 545 children without diarrhea (controls) during one year. Controls were inpatient or children attending child health clinics. Rotavirus antigen was detected using ELISA and positive samples were typed by multiplex semi-nested PCR and sequencing. RESULTS: The prevalence of rotavirus was higher in cases (32.5%) than in controls (7.7%, P<0.001). The most common G genotypes were G1 followed by G8, G12, and G4 in cases and G1, G12 and G8 in controls. The Tanzanian G1 variants displayed 94% similarity with the Rotarix vaccine G1 variant. The commonest P genotypes were P[8], P[4] and P[6], and the commonest G/P combination G1 P[8] (n = 123), G8 P[4] and G12 P[6]. Overall, rotavirus prevalence was higher in cool (23.9%) than hot months (17.1%) of the year (P = 0.012). We also observed significant seasonal variation of G genotypes. Rotavirus was most frequently found in the age group of four to six months. The prevalence of rotavirus in cases was lower in stunted children (28.9%) than in non-stunted children (40.1%, P = 0.003) and lower in HIV-infected (15.4%, 4/26) than in HIV-uninfected children (55.3%, 42/76, P<0.001). CONCLUSION: This pre-vaccination study shows predominance of genotype G1 in Tanzania, which is phylogenetically distantly related to the vaccine strains. We confirm the emergence of genotype G8 and G12. Rotavirus infection and circulating genotypes showed seasonal variation. This study also suggests that rotavirus may not be an opportunistic pathogen in children infected with HIV

Validation and Retrospective Clinical Evaluation of a Quantitative 16S rRNA Gene Metagenomic Sequencing Assay for Bacterial Pathogen Detection in Body Fluids.

Next-generation sequencing-based 16S rRNA gene metagenomic sequencing (16S MG) technology has tremendous potential for improving diagnosis of bacterial infections given its quantitative capability and culture-independent approach. We validated and used a quantitative 16S MG assay to identify and quantify bacterial species in clinical samples from a wide spectrum of infections, including meningitis, septic arthritis, brain abscess, intra-abdominal abscess, soft tissue abscess, and pneumonia. Twenty clinical samples were tested, and 16S MG identified a total of 34 species, compared with 22 species and three descriptive findings identified by culture. 16S MG results matched culture results in 75% (15/20) of the samples but detected at least one more species in five samples, including one culture-negative cerebrospinal fluid sample that was found to contain Streptococcus intermedius. Shotgun metagenomic sequencing verified the presence of all additional species. The 16S MG assay is highly sensitive, with a&nbsp;limit of detection of 10 to 100 colony-forming units/mL. Other performance characteristics, including linearity, precision, and specificity, all met the requirements for a clinical test. This assay showed the&nbsp;advantages of accurate identification and quantification of bacteria in culture-negative and polymicrobial infections for which conventional microbiology methods are limited. It also showed promises to serve unmet clinical needs for solving difficult infectious diseases cases

Seasonal variation of rotavirus infection and G genotypes among children admitted with diarrhea.

<p>The graph shows the total number of rotavirus infected children admitted due to diarrhea (cases) per month with G genotypes.</p

A and B. Phylogenetic trees of the rotavirus nucleotide sequence of the partial VP7 and VP4 genes.

<p>The phylogenetic tree: Phylogenetic trees based on the nucleotide sequence of the partial VP7 gene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097562#pone-0097562-g001" target="_blank">figure 1A</a>) and VP4 gene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097562#pone-0097562-g001" target="_blank">figure 1B</a>) of rotaviruses from Tanzania with known rotavirus reference strains from GenBank database and rotavirus vaccine strains i.e Rotateq and Rotarix. Reference strains, vaccine strains and strains from this study are indicated by accession numbers. The Genius software package was used to build the tree with the UPGMA method and bootstrapped with 1,000 repetitions; The Kimura-2 substitution model was used. The bar indicates nucleotide substitutions per site.</p

Sequencing results of rotavirus G and P genotypes circulating in children with diarrhea (cases) and children without diarrhea (controls) in Dar es Salaam, Tanzania (n = 211; 190 cases and 21 controls).

<p>Sequencing results of rotavirus G and P genotypes circulating in children with diarrhea (cases) and children without diarrhea (controls) in Dar es Salaam, Tanzania (n = 211; 190 cases and 21 controls).</p

Prevalence of rotavirus infection in different age groups.

<p>The graph shows the prevalence of rotavirus from ELISA results per age group in cases and controls.</p

Comparison of rapid molecular testing methods for detecting respiratory viruses in emergency care: a prospective study

Background: Respiratory tract infections (RTIs) caused by contagious viruses are common among patients presenting to the emergency department (ED). Early detection of these viruses can help prevent nosocomial transmission.Aim: To investigate the efficacy of three rapid molecular methods, namely FilmArray® Pneumonia Panel plus (FAP plus), ID NOW™ Influenza A and B 2 (ID NOW2) point-of-care test, and an in-house real-time polymerase chain reaction (RT-PCR) test, to identify patients with viral RTIs requiring isolation in an emergency setting.Methods: We included a FilmArray® Pneumonia Panel plus in the initial workup of patients with suspected RTIs during a flu season. The RT-PCR and the influenza point-of-care test were performed as part of routine diagnostics, on demand from the treating physicians. We compared viral detections and compared time to positive test results for each method.Findings: The FAP plus significantly reduced the turnaround time and was able to identify 95% patients with potential contagious viral RTI. Routine diagnostics ordered by the treating physician had a turnaround time of a median 22 h and detected 87% of patients with potential contagious viral RTI. In patients that had all three tests, the ID NOW2 detected 62% of patients with influenza.Conclusions: The FAP plus was able to rapidly and reliably identify patients with potential contagious viral RTIs; its use was feasible in the ED setting. Failing to test patients with viral RTI and using tests with long turnaround time may lead to nosocomial transmission of viral infections and adverse patient outcomes