Øyvind Kommedal

Direct 16S rRNA Gene Sequencing from Clinical Specimens, with Special Focus on Polybacterial Samples and Interpretation of Mixed DNA Chromatograms

ABSTRACT RipSeq (iSentio, Bergen, Norway) is a web-based application for the analysis of mixed DNA chromatograms. It opens the possibility to analyze chromatograms obtained by direct 16S rRNA gene sequencing from polybacterial human clinical samples. In this study, we used direct 16S rRNA gene sequencing to investigate 264 samples from a wide range of suspected human bacterial infections. The sequence-based identification was compared with the results from routine culture-based identification. A total of 151 samples were positive by the first PCR, producing 85 pure and 66 mixed DNA chromatograms. All mixed chromatograms were analyzed by RipSeq, although seven were so complex that only the dominant bacterial sequences could be identified. In general, sequence-based identification detected a larger number of species than did culture for samples from patients who had received antibiotics prior to sample collection and for samples containing anaerobic bacteria. RipSeq made it possible to apply this supplementary diagnostic tool to typical polybacterial specimens, such as internal abscesses, pleural fluids, and bile.

Analysis of Mixed Sequencing Chromatograms and Its Application in Direct 16S rRNA Gene Sequencing of Polymicrobial Samples

ABSTRACT Investigation of clinical samples by direct 16S rRNA gene sequencing provides the possibility to detect nonviable bacteria and bacteria with special growth requirements. This approach has been particularly valuable for the diagnosis of patients who have received antibiotics prior to sample collection. In specimens containing more than one bacterium, direct sequencing gives mixed chromatograms that complicate further interpretation. We designed an algorithm able to analyze these ambiguous chromatograms and implemented it as a Web-based service. The algorithm contains both a new base-calling procedure and a new database search procedure. 16S rRNA gene sequencing was performed on polybacterial suspensions prepared in the laboratory. The computer program identified all bacteria correctly to the species level in 23 out of 23 samples containing two different bacteria. For samples containing three different bacteria, correct identification to the species level was achieved for three out of five and to the genus level for five out of five.

Dual-priming oligonucleotides for broad-range amplification of the bacterial 16S rRNA gene directly from human clinical specimens

ABSTRACT Broad-range amplification and sequencing of the bacterial 16S rRNA gene directly from clinical specimens are offered as a diagnostic service in many laboratories. One major pitfall is primer cross-reactivity with human DNA which will result in mixed chromatograms. Mixed chromatograms will complicate subsequent sequence analysis and impede identification. In SYBR green real-time PCR assays, it can also affect crossing threshold values and consequently the status of a specimen as positive or negative. We evaluated two conventional primer pairs in common use and a new primer pair based on the dual priming oligonucleotide (DPO) principle. Cross-reactivity was observed when both conventional primer pairs were used, resulting in interpretation difficulties. No cross-reactivity was observed using the DPOs even in specimens with a high ratio of human to bacterial DNA. In addition to reducing cross-reactivity, the DPO principle also offers a high degree of flexibility in the design of primers and should be considered for any PCR assay intended for detection and identification of pathogens directly from human clinical specimens.

Characterization of polybacterial clinical samples using a set of group-specific broad-range primers targeting the 16S rRNA gene followed by DNA sequencing and RipSeq analysis

The standard use of a single universal broad-range PCR in direct 16S rDNA sequencing from polybacterial samples leaves the minor constituents at risk of remaining undetected because all bacterial DNA will be competing for the same reagents. In this article we introduce a set of three broad-range group-specific 16S rDNA PCRs that together cover the clinically relevant bacteria and apply them in the investigation of 25 polybacterial clinical samples. Mixed DNA chromatograms from samples containing more than one species per primer group were analysed using RipSeq Mixed (iSentio, Norway), a web-based application for the interpretation of chromatograms containing up to three different species. The group-specific PCRs reduced complexity in the resulting DNA chromatograms and made the assay more sensitive in situations with unequal species concentrations. Together this allowed for identification of a significantly higher number of bacterial species than did standard direct sequencing with a single universal primer pair and RipSeq analysis (95 vs 51). The method could improve microbiological diagnostics for important groups of patients and can be established in any laboratory with experience in direct 16S rDNA sequencing.

Prevalence and molecular characterisation of human adenovirus in diarrhoeic children in Tanzania; a case control study

BackgroundHuman adenovirus (HAdV) causes acute diarrhoea sporadically, as well as in outbreaks. Understanding the prevalence and types of HAdV in diarrhoea is important for control and preventive measures, especially in the African region where there is a high burden of diarrhoeal disease. The present study assessed the prevalence, molecular characteristics, seasonality and associated clinical features of HAdV infection Tanzanian children below two years of age with and without diarrhoea between 2010–2011.MethodsStool specimens, demographic and clinical information were collected in 690 cases and 545 controls. All stool samples were screened for HAdV-antigen using ELISA. Positive samples subsequently underwent real-time PCR and sequencing for molecular typing.ResultsHAdV was detected in 37 children, corresponding to a prevalence of 3.5% (24/690) in diarrhoeic and 2.4% (13/545) in non-diarrhoeic children (P > 0.05). Among HAdV-infected children, the median age was significantly lower in diarrhoeic than in non-diarrhoeic children (10 vs. 14 months, P˂0.001). More than half of HAdV infected (54.2%) were dehydrated as compared to diarrhoeic children without HAdV (45.8%, P = 0.01). The proportion of the enteric HAdV type 40/41 in diarrhoeic and non-diarrhoeic children was (50.0%, 12/24) and (46.2%, 6/13) respectively. Other HAdV types detected were; 1, 2, 7, 18, 19 and 31. The prevalence of adenovirus was not significantly different between rainy and dry seasons. HAdV was not detected in the 33 known HIV positive children. There was no significant association between HAdV infection and gender, nutritional status of the child and parent educational level.ConclusionThe present study provides further evidence of the contribution of adenovirus in causing gastroenteritis in young children, with symptomatic infection being significantly more prevalent in children below one year. We found similar prevalence of adenovirus in non-diarrhoeic children and in diarrhoeic children. This first report on molecular epidemiology of human adenovirus in Tanzania observed diversity of HAdV types that circulate the study setting. The study findings suggest that HAdV is not an important cause of diarrhoea in young HIV-positive children.

Massive Parallel Sequencing Provides New Perspectives on Bacterial Brain Abscesses

ABSTRACT Rapid development within the field of massive parallel sequencing (MPS) is about to bring this technology within reach for diagnostic microbiology laboratories. We wanted to explore its potential for improving diagnosis and understanding of polymicrobial infections, using bacterial brain abscesses as an example. We conducted a prospective nationwide study on bacterial brain abscesses. Fifty-two surgical samples were included over a 2-year period. The samples were categorized as either spontaneous intracerebral, spontaneous subdural, or postoperative. Bacterial 16S rRNA genes were amplified directly from the specimens and sequenced using Ion Torrent technology, with an average of 500,000 reads per sample. The results were compared to those from culture- and Sanger sequencing-based diagnostics. Compared to culture, MPS allowed for triple the number of bacterial identifications. Aggregatibacter aphrophilus, Fusobacterium nucleatum, and Streptococcus intermedius or combinations of them were found in all spontaneous polymicrobial abscesses. F. nucleatum was systematically detected in samples with anaerobic flora. The increased detection rate for Actinomyces spp. and facultative Gram-negative rods further revealed several species associations. We suggest that A. aphrophilus, F. nucleatum, and S. intermedius are key pathogens for the establishment of spontaneous polymicrobial brain abscesses. In addition, F. nucleatum seems to be important for the development of anaerobic flora. MPS can accurately describe polymicrobial specimens when a sufficient number of reads is used to compensate for unequal species concentrations and principles are defined to discard contaminant bacterial DNA in the subsequent data analysis. This will contribute to our understanding of how different types of polymicrobial infections develop.

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.

Genetic diversity of norovirus in hospitalised diarrhoeic children and asymptomatic controls in Dar es Salaam, Tanzania

This study investigated and reports norovirus diarrhoea, genetic diversity and associated clinical symptoms, HIV status and seasonality in a paediatric population of Tanzania. Stool specimens and demographic/clinical information, were prospectively collected from 705 hospitalised children with diarrhoea (cases) and 561 children without diarrhoea (controls) between 2010 and 2011. Norovirus detection was done by real-time RT-PCR. Genotype was determined using Gel-based and real time RT-PCR methods and sequencing targeting the polymerase and the capsid region respectively. Norovirus was detected in 14.3%, 181/1266 children. The prevalence of norovirus was significantly higher in cases (18.3%, 129/705) than in controls, (9.2%, 52/561), P<0.05. Except for one child who had double infection with GI and GII all 129 cases had GII. Among controls, 23.1% had GI and 76.9% had GII. Norovirus GII.4 was significantly more prevalent in cases 87.9% than in controls 56.5%. Other genotypes detected in both cases and controls were GII.21, GII.16 and GII.g. The highest numbers of norovirus were detected in April 2011. The number of norovirus detected was significantly higher during the first than second year of life (109/540, 20.2% vs. 20/165, 12.1%). The prevalence of norovirus in HIV-positive and negative children was (21.2%, 7/33) and (10.3%, 40/390, P=0.05) respectively, regardless of diarrhoea symptoms. No significant difference in gender, parents level of education or nutritional status with norovirus infection was observed within cases or controls. This study confirms the significant role of norovirus infection, especially GII.4 in diarrhoeic children who need hospitalisation and adds knowledge on norovirus epidemiology in the African region.

High prevalence of faecal carriage of ESBL-producing enterobacteriaceae among children in Dar es Salaam, Tanzania

Background Faecal carriage of ESBL-producing bacteria is a potential risk for transmission and infection. Little is known about faecal carriage of antibiotic resistance in Tanzania. This study aimed to investigate the prevalence of faecal carriage of ESBL-producing Enterobacteriaceae and to identify risk factors for carriage among young children in Tanzania. Methodology/Principal Findings From August 2010 to July 2011, children below 2 years of age were recruited in Dar es Salaam, including healthy community children (n = 250) and children hospitalized due to diarrhoea (n = 250) or other diseases (n = 103). ChromID ESBL agar and ChromID CARBA SMART agar were used for screening. Antimicrobial susceptibility testing was performed by the disk diffusion method. ESBL genotypes were identified by Real-Time PCR and sequencing. The overall prevalence of ESBL carriage was 34.3% (207/ 603). The prevalence of ESBL carriage was significantly higher among hospitalized children (50.4%), compared to community children (11.6%; P < 0.001; OR = 7.75; 95% CI: 4.99–12.03). We found high prevalence of Multidrug-resistance (94%) among Escherichia coli and Klebsiella pneumoniae isolates. No resistance to carbapenems was detected. For the majority of isolates (94.7%) we detected a blaCTX-M-15-like gene. In addition, the plasmid mediated AmpC beta-lactamase CMY-2 was detected for the first time in Tanzania. ESBL prevalence was significantly higher among HIV positive (89.7%) than HIV negative (16.9%) children (P = 0.001; OR = 9.99; 95% CI: 2.52–39.57). Use of antibiotics during the past 14 days and age below 1 year was also associated with ESBL carriage. Conclusions/Significance We report a high rate of faecal carriage of ESBL-producing Enterobacteriaceae among children below 2 years of age in Tanzania, particularly those with HIV-infection. Resistance to a majority of the available antimicrobials commonly used for children in Tanzania leaves few treatment options for infections when caused by these bacteria.

Simultaneous Sequence Analysis of the 16S rRNA and rpoB Genes by Use of RipSeq Software To Identify Mycobacterium Species

ABSTRACT The 16S rRNA gene is commonly used to identify Mycobacterium spp., but alternative DNA targets can provide better resolution to the species level. We evaluated a novel system that enables simultaneous amplification, sequencing, and analysis of two different DNA targets in a single tube to identify clinical isolates of Mycobacterium spp. For 139 clinical isolates, we found that the 16S rRNA gene alone identified 67 (48%) isolates as single species, 68 (49%) isolates to the complex or group level, and 4 (3%) isolates to the genus level only. The rpoB gene alone identified 117 (84%) isolates as single species, 10 (7%) isolates to the complex or group level, and 12 (8%) isolates to the genus level only. Combining the separate analyses for sequencing of two gene targets, 119 (86%) isolates were identified as single species and 10 (7%) isolates were identified to the complex or group level. Seven (5%) isolates were identified as novel species within established groups, and 3 (2%) were identified to the genus level only. Dual-locus identification identified 110 (79%) isolates as single species and 22 (16%) isolates to the complex or group level. Six (4%) were identified as novel species within established groups, and 1 (1%) was identified to the genus level only. Identifications were more accurate when both the 16S rRNA and rpoB genes were screened, and reliance on a single gene target was suboptimal. RipSeq dual-locus software provides an accurate alternative method for laboratories using two different gene targets for microorganism identification.