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Periodontitis is a progressive disease of the periodontium with a complex, polymicrobial etiology. Recent Next-Generation Sequencing (NGS) studies of the microbial diversity associated with periodontitis have revealed strong, community-level differences in bacterial assemblages associated with healthy or diseased periodontal sites. In this study, we used NGS approaches to characterize changes in periodontal pocket bacterial diversity after standard periodontal treatment. Despite consistent changes in the abundance of certain taxa in individuals whose condition improved with treatment, post-treatment samples retained the highest similarity to pre-treatment samples from the same individual. Deeper phylogenetic analysis of periodontal pathogen-containing genera Prevotella and Fusobacterium found both unexpected diversity and differential treatment response among species. Our results highlight how understanding interpersonal variability among microbiomes is necessary for determining how polymicrobial diseases respond to treatment and disturbance.

Periodontal disease is a polymicrobial disease with a complex etiology. Many studies use the 16s rRNA gene as a molecular marker for bacterial identification associated with oral biofilm, known as dental plaque. However, the high level of conservation in the 16s rRNA gene does not provide enough resolution to discriminate between closely related bacterial species. Recent studies have successfully used the RNA polymerase β subunit (rpoB) gene as an alternative molecular marker to help resolve this issue. Although there are many studies that use the rpoB gene in bacterial identification, it is usually to describe specific groups, such as the family Pasteurellaceae or the genus Veillonella. In this study, we investigated the use of the rpoB gene as an alternative molecular marker to differentiate between closely related species in the microbial community of dental plaque. To analyze bacterial diversity, genomic DNA was extracted from 28 samples, and sequenced using 454 pyrosequencing. Eight out of a total of 17 identified genera were selected for phylogenetic analyses. The 16s rRNA gene sequences from these eight genera were used to compare to the rpoB gene sequences. Neisseria and Granulicatella showed greater diversity using the 16s rRNA gene. However, there was more diversity in the genera Pseudomonas, Lautropia, Eikenella, and Haemophilus using the rpoB gene. In Cardiobacterium and Aggregatibacter, there were no differences in diversity revealed with the rpoB or the 16s rRNA gene. The use of the rpoB gene to identify and differentiate between closely related bacterial species can provide insight into organisms that may be associated with health and disease states.

Periodontitis is a progressive disease of the periodontium with a complex, polymicrobial etiology. Recent Next-Generation Sequencing (NGS) studies of the microbial diversity associated with periodontitis have revealed strong, community-level differences in bacterial assemblages associated with healthy or diseased periodontal sites. In this study, we used NGS approaches to characterize changes in periodontal pocket bacterial diversity after standard periodontal treatment. Despite consistent changes in the abundance of certain taxa in individuals whose condition improved with treatment, post-treatment samples retained the highest similarity to pre-treatment samples from the same individual. Deeper phylogenetic analysis of periodontal pathogen-containing genera Prevotella and Fusobacterium found both unexpected diversity and differential treatment response among species. Our results highlight how understanding interpersonal variability among microbiomes is necessary for determining how polymicrobial diseases respond to treatment and disturbance.

The antibody response to vaccination and infection is a key component of the immune response to pathogens. Sequencing of peripheral B cells may not represent the complete B cell receptor repertoire. Here we present a method for sequencing human plasma-derived polyclonal IgG using a combination of mass spectrometry and B-cell sequencing. We investigate the IgG response to the Moderna Spikevax COVID-19 vaccine. From the sequencing data of the natural polyclonal response to vaccination, we generate 12 recombinant antibodies. Six derived recombinant antibodies, including four generated with de novo protein sequencing, exhibit similar or higher binding affinities than the original natural polyclonal antibody. Neutralization tests reveal that the six antibodies possess neutralizing capabilities against the target antigen. This research provides insights into sequencing polyclonal IgG antibodies and the potential of our approach in generating recombinant antibodies with robust binding affinity and neutralization capabilities. Directly examining the circulating IgG pool is crucial due to potential misrepresentations by B-cell analysis alone. The antibody response to infection and vaccination is an essential component of the anti-infective immune response. Here the authors present a de novo protein sequencing method for antibody discovery from polyclonal IgG from human plasma and characterise the antibody response to the Moderna Spikevax COVID-19 vaccine.

Epidemiological evidence about the etiology and antimicrobial resistance of neonatal infections remains limited in low-resource settings. We aimed to describe the etiology of neonatal infections in a prospective observational cohort study conducted at two hospital sites in Kampala, Uganda. Babies admitted to either unit with risk factors or signs of sepsis, pneumonia, or meningitis had a blood culture, nasopharyngeal swab, and lumbar puncture (if indicated) collected. Basic demographics were collected, and babies were followed up until discharge or death to determine admission outcome. Blood cultures were processed using the BACTEC system and identification confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Cerebrospinal fluid was processed using standard microbiological testing and swabs were processed using the multiplex real-time polymerase chain reaction assay. Antimicrobial susceptibilities of bacterial isolates to World Health Organization-recommended first-line antibiotics (ampicillin or benzylpenicillin and gentamicin) were assessed using e-tests. A total of 7323 infants with signs or risk factors for sepsis had blood cultures, 2563 had nasopharyngeal swabs, and 23 had lumbar punctures collected. Eleven percent of blood cultures and 8.6% of swabs were positive. Inpatient mortality was 12.1%, with 27.7% case fatality observed among infants with Gram-negative bloodstream infections. (14.8%), spp. (10.3%), and spp. (7.6%), were notable contributors to Gram-negative sepsis, whereas Group B was the predominant Gram-positive pathogen identified (13.5%). Almost 60% of Gram-negative pathogens were ampicillin- and gentamicin-resistant. Our study demonstrates high levels of antimicrobial resistance and inpatient mortality from neonatal sepsis in the first months of life in Uganda. This underscores the pressing need for revised, context-specific antimicrobial treatment guidelines that account for the evolving landscape of antimicrobial resistance in neonatal sepsis.

Low- and middle-income countries lack data on culture-confirmed sepsis in HIV-exposed infants, despite the reported heightened risk of infectious morbidity. This study describes culture-confirmed sepsis and antibiotic resistance patterns among HIV-exposed children in a large etiological cohort study in Kampala, Uganda. This was a prospective birth cohort study based at 2 Ugandan sites, as part of the Progressing Group B Streptococcal Vaccines (PROGRESS) study. Any infant with risk factors, signs, or symptoms of infection presenting before 3 months of age had a blood culture and nasopharyngeal swab taken to determine the etiology of neonatal and young infant sepsis. Among 4492 blood cultures, 460 were obtained from HIV-exposed infants. Nine infants (1.9%) had positive blood cultures. The most frequently isolated organisms were , group B , and , and these organisms demonstrated resistance to the common antibiotics (aminoglycosides, penicillins, and cephalosporins) used for management of suspected sepsis. A higher proportion of the exposed babies died vs HIV-unexposed (15.8 vs 11.2; = .005). Nasopharyngeal swabs were collected from 114 infants, with 7.9% positive for at least one virus or bacterium. Future work is needed to investigate why mortality among HIV-exposed infants persists despite maternal antiretroviral treatment. Antimicrobial resistance is an increasing concern in this setting.

We investigated awareness of neonatal infections among a population of pregnant women and other community members in Kampala, Uganda. We explored perceived causes of neonatal infections and perceptions of appropriate treatments. We conducted focus group discussions (FGDs) and in-depth interviews (IDIs) with 97 participants: 25 community leaders who took part in 3 FGDs, 12 pregnant women who took part in IDIs, and 60 pregnant women who took part in 8 FGDs, between November 2019 and October 2020. Data were analyzed thematically. This work formed part of the PROGRESS study, an observational cohort study undertaken in Kampala, Uganda, between November 2018 and April 2021. Beliefs about causes, signs, symptoms, and treatment of infants with suspected infections impacted health-seeking behavior. Some illnesses were perceived to be caused by environmental factors while others were believed to have social or behavioral causes, such as the promiscuity of the male partner causing infections or the mother being bewitched. Local herbs and traditional remedies were the most preferred method of treatment and were commonly relied on to address various health issues rather than conventional medicines. Notably, no participant mentioned vaccines as a way of preventing infections. Pregnant women and community members' understanding of the causes and treatment of neonatal illnesses were diverse, including environmental, social-behavioral, and supernatural causes, while both conventional and traditional remedies were perceived as appropriate treatments and sought accordingly. Understanding community perceptions and practices around neonatal infections is key to improving neonatal health interventions and outcomes.

Maternal Group B (GBS) rectovaginal colonization is an important risk factor for invasive disease in neonates, yet availability of culture-based methods for detection is limited in low-resource settings. We evaluated the diagnostic performance of the HiberGene (HG) GBS loop-mediated isothermal amplification (LAMP) assay for the rapid detection of GBS in rectal/vaginal swabs collected from women in Uganda. This work forms a part of the PROGRESS GBS study. In phase 1, 1294 rectal and vaginal swabs were collected from pregnant women and inoculated in enrichment (Lim) broth, which was then tested using the HG GBS LAMP assay ( gene target) and culture on chromogenic agar. In phase 2, 166 swabs from nonpregnant women were tested directly (without the enrichment step). For samples with discordant results, an additional method of testing against multiplex real-time polymerase chain reaction assay was used. Overall, the HG GBS LAMP assay detected more GBS-positive samples (31.3%; 452/1445) than culture-based methods (13.3%; 192/1445). Multiplex polymerase chain reaction-tested results were concordant with LAMP results in 96.3% of cases. The sensitivity and specificity of the LAMP assay, after adjusting for the tiebreaker results of discordant samples, were 94.4% (95% confidence interval, 86.2-99.4) and 99.0% (95% confidence interval, 94.3-100), respectively. The results of this study demonstrate high sensitivity and specificity of the HG GBS LAMP assay for the detection of GBS rectovaginal colonization in our setting. Given its rapid turnaround time, the HG GBS LAMP assay could appropriately be used to screen women for GBS rectovaginal colonization during labor to enable provision of intrapartum antibiotic prophylaxis.

Maternal primary cytomegalovirus (CMV) infection is associated with abortion and congenital anomalies. In Uganda, the burden of maternal CMV infection is not well studied. This study thus assessed the seroprevalence and factors associated with CMV infection among pregnant women at Kawempe National Referral Hospital in Kampala. This work forms a part of the PROGRESS study, an observational cohort study undertaken in Kampala, Uganda, between November 2018 and April 2021. We conducted a cross-sectional study between September 2020 and January 2021 among the 639 pregnant women admitted to the labor ward at a government hospital. Sociodemographic, medical, obstetric, and socioeconomic data were collected. Blood samples from study participants were drawn and analyzed for the presence of CMV immunoglobulin G (IgG) and IgM using enzyme-linked immunosorbent assay-based quantitative assays. Further analysis of all IgM-positive samples was conducted using CMV IgG avidity assays. All infants had a nasal polymerase chain reaction (PCR) on the first day of life to investigate CMV positivity. Logistic regression was performed to determine the factors associated with CMV infection. Seroprevalence of CMV IgG among the 637 women was universal (100%), and that of CMV (IgM) was 5.8% (37/637). CMV (IgM) was associated with being low socioeconomic status (odds ratio, 3.44; 95% CI, 1.05-11.32; = .04). Transmission risk was low, and no infant had a positive PCR for CMV at birth. Universally, by the time women in Kampala conceive, they will have been exposed to CMV. Women of lower socioeconomic status were more likely to have recent CMV infection than their more affluent counterparts, highlighting the need for screening guidelines in this setting.

Hepatitis B virus (HBV) infection is a significant cause of morbidity and mortality globally. The World Health Organization estimates that just 10.5% of individuals living with HBV globally are aware of their status. Antenatal care provides an opportunity to screen pregnant women for HBV and to treat those who are eligible to reduce the risk of vertical transmission. We conducted an observational study to determine the proportion of pregnant women with active HBV infection delivering at a government-funded hospital in Kampala, Uganda, to estimate the number of missed opportunities to prevent vertical transmission. Eligible participants were enrolled via the PROGRESS study, an observational cohort study undertaken in Kampala, Uganda, between November 2018 and April 2021. Results presented here describe data from April 2019 to November 2020. Five milliliters of venous blood was drawn shortly after delivery. Serum aliquots were analyzed for hepatitis B surface antigen (HBsAg). HBsAg-positive participants were informed of their result by telephone and referred to the gastroenterology service for specialist management. In total, 6062 women were enrolled between April 2019 and November 2020. Results were available for 6012 (99.6%) participants, among whom 131 (2.2%) were HBsAg positive. Only 10 of 131 (7.6%) HBsAg-positive participants were successfully referred to the gastroenterology service at Mulago Hospital for treatment of their infection. Our study identified a number of missed opportunities to identify active HBV infection among our pregnant cohort. Additional resources are urgently required to increase the coverage of antenatal HBV screening while also improving treatment pathways for pregnant women with HBV infection in this region.