Investigating the etiologies of non-malarial febrile illness in Senegal using metagenomic sequencing.

The worldwide decline in malaria incidence is revealing the extensive burden of non-malarial febrile illness (NMFI), which remains poorly understood and difficult to diagnose. To characterize NMFI in Senegal, we collected venous blood and clinical metadata in a cross-sectional study of febrile patients and healthy controls in a low malaria burden area. Using 16S and untargeted sequencing, we detected viral, bacterial, or eukaryotic pathogens in 23% (38/163) of NMFI cases. Bacteria were the most common, with relapsing fever Borrelia and spotted fever Rickettsia found in 15.5% and 3.8% of cases, respectively. Four viral pathogens were found in a total of 7 febrile cases (3.5%). Sequencing also detected undiagnosed Plasmodium, including one putative P. ovale infection. We developed a logistic regression model that can distinguish Borrelia from NMFIs with similar presentation based on symptoms and vital signs (F1 score: 0.823). These results highlight the challenge and importance of improved diagnostics, especially for Borrelia, to support diagnosis and surveillance.

Improving diagnosis of non-malarial fevers in Senegal: Borrelia and the contribution of tick-borne bacteria.

The worldwide decline in malaria incidence is revealing the extensive burden of non-malarial febrile illness (NMFI), which remains poorly understood and difficult to diagnose. To characterize NMFI in Senegal, we collected venous blood and clinical metadata from febrile patients and healthy controls in a low malaria burden area. Using 16S and unbiased sequencing, we detected viral, bacterial, or eukaryotic pathogens in 29% of NMFI cases. Bacteria were the most common, with relapsing fever Borrelia and spotted fever Rickettsia found in 15% and 3.7% of cases, respectively. Four viral pathogens were found in a total of 7 febrile cases (3.5%). Sequencing also detected undiagnosed Plasmodium, including one putative P. ovale infection. We developed a logistic regression model to distinguish Borrelia from NMFIs with similar presentation based on symptoms and vital signs. These results highlight the challenge and importance of improved diagnostics, especially for Borrelia, to support diagnosis and surveillance.

Modeling Optimal Laboratory Testing Strategies for Bacterial Meningitis Surveillance in Africa.

Since 2010, the introduction of an effective serogroup A meningococcal conjugate vaccine has led to the near-elimination of invasive Neisseria meningitidis serogroup A disease in Africa's meningitis belt. However, a significant burden of disease and epidemics due to other bacterial meningitis pathogens remain in the region. High-quality surveillance data with laboratory confirmation is important to monitor circulating bacterial meningitis pathogens and design appropriate interventions, but complete testing of all reported cases is often infeasible. Here, we use case-based surveillance data from 5 countries in the meningitis belt to determine how accurately estimates of the distribution of causative pathogens would represent the true distribution under different laboratory testing strategies. Detailed case-based surveillance data was collected by the MenAfriNet surveillance consortium in up to 3 seasons from participating districts in 5 countries. For each unique country-season pair, we simulated the accuracy of laboratory surveillance by repeatedly drawing subsets of tested cases and calculating the margin of error of the estimated proportion of cases caused by each pathogen (the greatest pathogen-specific absolute error in proportions between the subset and the full set of cases). Across the 12 country-season pairs analyzed, the 95% credible intervals around estimates of the proportion of cases caused by each pathogen had median widths of ±0.13, ±0.07, and ±0.05, respectively, when random samples of 25%, 50%, and 75% of cases were selected for testing. The level of geographic stratification in the sampling process did not meaningfully affect accuracy estimates. These findings can inform testing thresholds for laboratory surveillance programs in the meningitis belt.

Molecular insights into meningococcal carriage isolates from Burkina Faso 7 years after introduction of a serogroup A meningococcal conjugate vaccine.

In 2010, Burkina Faso completed the first nationwide mass-vaccination campaign of a meningococcal A conjugate vaccine, drastically reducing the incidence of disease caused by serogroup A meningococci. Since then, other strains, such as those belonging to serogroups W, X and C, have continued to cause outbreaks within the region. A carriage study was conducted in 2016 and 2017 in the country to characterize the meningococcal strains circulating among healthy individuals following the mass-vaccination campaign. Four cross-sectional carriage evaluation rounds were conducted in two districts of Burkina Faso, Kaya and Ouahigouya. Oropharyngeal swabs were collected for the detection of Neisseria meningitidis by culture. Confirmed N. meningitidis isolates underwent whole-genome sequencing for molecular characterization. Among 13 758 participants, 1035 (7.5 %) N. meningitidis isolates were recovered. Most isolates (934/1035; 90.2 %) were non-groupable and primarily belonged to clonal complex (CC) 192 (822/934; 88 %). Groupable isolates (101/1035; 9.8 %) primarily belonged to CCs associated with recent outbreaks in the region, such as CC11 (serogroup W) and CC10217 (serogroup C); carried serogroup A isolates were not detected. Phylogenetic analysis revealed several CC11 strains circulating within the country, several of which were closely related to invasive isolates. Three sequence types (STs) were identified among eleven CC10217 carriage isolates, two of which have caused recent outbreaks in the region (ST-10217 and ST-12446). Our results show the importance of carriage studies to track the outbreak-associated strains circulating within the population in order to inform future vaccination strategies and molecular surveillance programmes.

The Strengthening of Laboratory Systems in the Meningitis Belt to Improve Meningitis Surveillance, 2008-2018: A Partners' Perspective.

Laboratories play critical roles in bacterial meningitis disease surveillance in the African meningitis belt, where the highest global burden of meningitis exists. Reinforcement of laboratory capacity ensures rapid detection of meningitis cases and outbreaks and a public health response that is timely, specific, and appropriate. Since 2008, joint efforts to strengthen laboratory capacity by multiple partners, including MenAfriNet, beginning in 2014, have been made in countries within and beyond the meningitis belt. Over the course of 10 years, national reference laboratories were supported in 5 strategically targeted areas: specimen transport systems, laboratory procurement systems, laboratory diagnosis, quality management, and laboratory workforce with substantial gains made in each of these areas. To support the initiative to eliminate meningitis by 2030, continued efforts are needed to strengthen laboratory systems.

Epidemiology of Bacterial Meningitis in the Nine Years Since Meningococcal Serogroup A Conjugate Vaccine Introduction, Niger, 2010-2018.

BACKGROUND: In 2010, Niger and other meningitis belt countries introduced a meningococcal serogroup A conjugate vaccine (MACV). We describe the epidemiology of bacterial meningitis in Niger from 2010 to 2018. METHODS: Suspected and confirmed meningitis cases from January 1, 2010 to July 15, 2018 were obtained from national aggregate and laboratory surveillance. Cerebrospinal fluid specimens were analyzed by culture and/or polymerase chain reaction. Annual incidence was calculated as cases per 100 000 population. Selected isolates obtained during 2016-2017 were characterized by whole-genome sequencing. RESULTS: Of the 21 142 suspected cases of meningitis, 5590 were confirmed: Neisseria meningitidis ([Nm] 85%), Streptococcus pneumoniae ([Sp] 13%), and Haemophilus influenzae ([Hi] 2%). No NmA cases occurred after 2011. Annual incidence per 100 000 population was more dynamic for Nm (0.06-7.71) than for Sp (0.18-0.70) and Hi (0.01-0.23). The predominant Nm serogroups varied over time (NmW in 2010-2011, NmC in 2015-2018, and both NmC and NmX in 2017-2018). Meningococcal meningitis incidence was highest in the regions of Niamey, Tillabery, Dosso, Tahoua, and Maradi. The NmW isolates were clonal complex (CC)11, NmX were CC181, and NmC were CC10217. CONCLUSIONS: After MACV introduction, we observed an absence of NmA, the emergence and continuing burden of NmC, and an increase in NmX. Niger's dynamic Nm serogroup distribution highlights the need for strong surveillance programs to inform vaccine policy.

Bacterial Meningitis Epidemiology in Five Countries in the Meningitis Belt of Sub-Saharan Africa, 2015-2017.

BACKGROUND: The MenAfriNet Consortium supports strategic implementation of case-based meningitis surveillance in key high-risk countries of the African meningitis belt: Burkina Faso, Chad, Mali, Niger, and Togo. We describe bacterial meningitis epidemiology in these 5 countries in 2015-2017. METHODS: Case-based meningitis surveillance collects case-level demographic and clinical information and cerebrospinal fluid (CSF) laboratory results. Neisseria meningitidis, Streptococcus pneumoniae, or Haemophilus influenzae cases were confirmed and N. meningitidis/H. influenzae were serogrouped/serotyped by real-time polymerase chain reaction, culture, or latex agglutination. We calculated annual incidence in participating districts in each country in cases/100 000 population. RESULTS: From 2015-2017, 18 262 suspected meningitis cases were reported; 92% had a CSF specimen available, of which 26% were confirmed as N. meningitidis (n = 2433; 56%), S. pneumoniae (n = 1758; 40%), or H. influenzae (n = 180; 4%). Average annual incidences for N. meningitidis, S. pneumoniae, and H. influenzae, respectively, were 7.5, 2.5, and 0.3. N. meningitidis incidence was 1.5 in Burkina Faso, 2.7 in Chad, 0.4 in Mali, 14.7 in Niger, and 12.5 in Togo. Several outbreaks occurred: NmC in Niger in 2015-2017, NmC in Mali in 2016, and NmW in Togo in 2016-2017. Of N. meningitidis cases, 53% were NmC, 30% NmW, and 13% NmX. Five NmA cases were reported (Burkina Faso, 2015). NmX increased from 0.6% of N. meningitidis cases in 2015 to 27% in 2017. CONCLUSIONS: Although bacterial meningitis epidemiology varied widely by country, NmC and NmW caused several outbreaks, NmX increased although was not associated with outbreaks, and overall NmA incidence remained low. An effective low-cost multivalent meningococcal conjugate vaccine could help further control meningococcal meningitis in the region.

Implementation of Case-Based Surveillance and Real-time Polymerase Chain Reaction to Monitor Bacterial Meningitis Pathogens in Chad.

BACKGROUND: Meningococcal serogroup A conjugate vaccine (MACV) was introduced in Chad during 2011-2012. Meningitis surveillance has been conducted nationwide since 2003, with case-based surveillance (CBS) in select districts from 2012. In 2016, the MenAfriNet consortium supported Chad to implement CBS in 4 additional districts and real-time polymerase chain reaction (rt-PCR) at the national reference laboratory (NRL) to improve pathogen detection. We describe analysis of bacterial meningitis cases during 3 periods: pre-MACV (2010-2012), pre-MenAfriNet (2013-2015), and post-MenAfriNet (2016-2018). METHODS: National surveillance targeted meningitis cases caused by Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae. Cerebrospinal fluid specimens, inoculated trans-isolate media, and/or isolates from suspected meningitis cases were tested via culture, latex, and/or rt-PCR; confirmed bacterial meningitis was defined by a positive result on any test. We calculated proportion of suspected cases with a specimen received by period, and proportion of specimens with a bacterial meningitis pathogen identified, by period, pathogen, and test. RESULTS: The NRL received specimens for 6.8% (876/12813), 46.4% (316/681), and 79.1% (787/995) of suspected meningitis cases in 2010-2012, 2013-2015, and 2016-2018, respectively, with a bacterial meningitis pathogen detected in 33.6% (294/876), 27.8% (88/316), and 33.2% (261/787) of tested specimens. The number of N. meningitidis serogroup A (NmA) among confirmed bacterial meningitis cases decreased from 254 (86.4%) during 2010-2012 to 2 (2.3%) during 2013-2015, with zero NmA cases detected after 2014. In contrast, proportional and absolute increases were seen between 2010-2012, 2013-2015, and 2016-2018 in cases caused by S. pneumoniae (5.1% [15/294], 65.9% [58/88], and 52.1% [136/261]), NmX (0.7% [2/294], 1.1% [1/88], and 22.2% [58/261]), and Hib (0.3% [1/294], 11.4% [10/88], and 14.9% [39/261]). Of specimens received at the NRL, proportions tested during the 3 periods were 47.7% (418), 53.2% (168), and 9.0% (71) by latex; 81.4% (713), 98.4% (311), and 93.9% (739) by culture; and 0.0% (0), 0.0% (0), and 90.5% (712) by rt-PCR, respectively. During the post-MenAfriNet period (2016-2018), 86.1% (678) of confirmed cases were tested by both culture and rt-PCR, with 12.5% (85) and 32.4% (220) positive by culture and rt-PCR, respectively. CONCLUSIONS: CBS implementation was associated with increased specimen referral. Increased detection of non-NmA cases could reflect changes in incidence or increased sensitivity of case detection with rt-PCR. Continued surveillance with the use of rt-PCR to monitor changing epidemiology could inform the development of effective vaccination strategies.

A high-throughput pH-based colorimetric assay: application focus on alpha/beta hydrolases.

Research involving α/ß hydrolases, including α-amino acid ester hydrolase and cocaine esterase, has been limited by the lack of an online high throughput screening assay. The development of a high throughput screening assay capable of detecting α/ß hydrolase activity toward specific substrates and/or chemical reactions (e.g., hydrolysis in lieu of amidase activity and/or synthesis instead of thioesterase activity) is of interest in a broad set of scientific questions and applications. Here we present a general framework for pH-based colorimetric assays, as well as the mathematical considerations necessary to estimate de novo the experimental response required to assign a 'hit' or a 'miss,' in the absence of experimental standard curves. This combination is valuable for screening the hydrolysis and synthesis activity of α/ß hydrolases on a variety of substrates, and produces data comparable to the current standard technique involving High Performance Liquid Chromatography (HPLC). In contrast to HPLC, this assay enables screening experiments to be performed with greater efficiency.

Stabilizing biocatalysts.

The area of biocatalysis itself is in rapid development, fueled by both an enhanced repertoire of protein engineering tools and an increasing list of solved problems. Biocatalysts, however, are delicate materials that hover close to the thermodynamic limit of stability. In many cases, they need to be stabilized to survive a range of challenges regarding temperature, pH value, salt type and concentration, co-solvents, as well as shear and surface forces. Biocatalysts may be delicate proteins, however, once stabilized, they are efficiently active enzymes. Kinetic stability must be achieved to a level satisfactory for large-scale process application. Kinetic stability evokes resistance to degradation and maintained or increased catalytic efficiency of the enzyme in which the desired reaction is accomplished at an increased rate. However, beyond these limitations, stable biocatalysts can be operated at higher temperatures or co-solvent concentrations, with ensuing reduction in microbial contamination, better solubility, as well as in many cases more favorable equilibrium, and can serve as more effective templates for combinatorial and data-driven protein engineering. To increase thermodynamic and kinetic stability, immobilization, protein engineering, and medium engineering of biocatalysts are available, the main focus of this work. In the case of protein engineering, there are three main approaches to enhancing the stability of protein biocatalysts: (i) rational design, based on knowledge of the 3D-structure and the catalytic mechanism, (ii) combinatorial design, requiring a protocol to generate diversity at the genetic level, a large, often high throughput, screening capacity to distinguish 'hits' from 'misses', and (iii) data-driven design, fueled by the increased availability of nucleotide and amino acid sequences of equivalent functionality.