Using Multiplex Amplicon PCR Technology to Efficiently and Timely Generate Rift Valley Fever Virus Sequence Data for Genomic Surveillance.

Rift Valley fever (RVF) is a febrile vector-borne disease endemic in Africa and continues to spread in new territories. It is a climate-sensitive disease mostly triggered by abnormal rainfall patterns. The disease is associated with high mortality and morbidity in both humans and livestock. RVF is caused by the Rift Valley fever virus (RVFV) of the genus Phlebovirus in the family Phenuiviridae. It is a tripartite RNA virus with three genomic segments: small (S), medium (M) and large (L). Pathogen genomic sequencing is becoming a routine procedure and a powerful tool for understanding the evolutionary dynamics of infectious organisms, including viruses. Inspired by the utility of amplicon-based sequencing demonstrated in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Ebola, Zika and West Nile viruses, we report an RVFV sample preparation based on amplicon multiplex polymerase chain reaction (amPCR) for template enrichment and reduction of background host contamination. The technology can be implemented rapidly to characterize and genotype RVFV during outbreaks in a near-real-time manner. To achieve this, we designed 74 multiplex primer sets covering the entire RVFV genome to specifically amplify the nucleic acid of RVFV in clinical samples from an animal tissue. Using this approach, we demonstrate achieving complete RVFV genome coverage even from samples containing a relatively low viral load. We report the first primer scheme approach of generating multiplex primer sets for a tripartite virus which can be replicated for other segmented viruses.

Genomic surveillance of Rift Valley fever virus: from sequencing to lineage assignment.

Genetic evolution of Rift Valley fever virus (RVFV) in Africa has been shaped mainly by environmental changes such as abnormal rainfall patterns and climate change that has occurred over the last few decades. These gradual environmental changes are believed to have effected gene migration from macro (geographical) to micro (reassortment) levels. Presently, 15 lineages of RVFV have been identified to be circulating within the Sub-Saharan Africa. International trade in livestock and movement of mosquitoes are thought to be responsible for the outbreaks occurring outside endemic or enzootic regions. Virus spillover events contribute to outbreaks as was demonstrated by the largest epidemic of 1977 in Egypt. Genomic surveillance of the virus evolution is crucial in developing intervention strategies. Therefore, we have developed a computational tool for rapidly classifying and assigning lineages of the RVFV isolates. The computational method is presented both as a command line tool and a web application hosted at https://www.genomedetective.com/app/typingtool/rvfv/ . Validation of the tool has been performed on a large dataset using glycoprotein gene (Gn) and whole genome sequences of the Large (L), Medium (M) and Small (S) segments of the RVFV retrieved from the National Center for Biotechnology Information (NCBI) GenBank database. Using the Gn nucleotide sequences, the RVFV typing tool was able to correctly classify all 234 RVFV sequences at species level with 100% specificity, sensitivity and accuracy. All the sequences in lineages A (n = 10), B (n = 1), C (n = 88), D (n = 1), E (n = 3), F (n = 2), G (n = 2), H (n = 105), I (n = 2), J (n = 1), K (n = 4), L (n = 8), M (n = 1), N (n = 5) and O (n = 1) were also correctly classified at phylogenetic level. Lineage assignment using whole RVFV genome sequences (L, M and S-segments) did not achieve 100% specificity, sensitivity and accuracy for all the sequences analyzed. We further tested our tool using genomic data that we generated by sequencing 5 samples collected following a recent RVF outbreak in Kenya. All the 5 samples were assigned lineage C by both the partial (Gn) and whole genome sequence classifiers. The tool is useful in tracing the origin of outbreaks and supporting surveillance efforts.Availability: https://github.com/ajodeh-juma/rvfvtyping.

Estimating pediatric cases of COVID-19 over time in the United States: Filling in a gap in public use data.

BACKGROUND: COVID-19 continues to disturb nearly all aspects of life, leaving us striving to reach herd immunity. Currently, only weekly standardized incidence rate data per age group are publicly available, limiting assessment of herd immunity. Here, we estimate the time-series case counts of COVID-19 among age groups currently ineligible for vaccination in the USA. METHODS: This was a secondary analysis of publicly available data. COVID-19 case counts by age groups were computed using incidence rate data from the CDC and population estimates from the US Census Bureau. We also created a web-based application to allow on demand analysis. RESULTS: A total of 78 weeks of data were incorporated in the analysis, suggesting the highest peak in cases within the 5-11-year age group on week ending 2021-01-09 (n = 61,095) followed by the 12-15-year age group (n = 58,093). As of July 24, 2021, case counts in the 5-11-year age group have expanded beyond other groups rapidly. DISCUSSION: This study suggests it is possible to estimate pediatric case counts of COVID-19. National agencies should report COVID-19 time series case counts for pediatric age cohorts. These data will enhance our ability to estimate the population at risk and tailor interventions accordingly.

Near-Complete SARS-CoV-2 Seroprevalence among Rural and Urban Kenyans despite Significant Vaccine Hesitancy and Refusal.

Considering the early inequity in global COVID-19 vaccine distribution, we compared the level of population immunity to SARS-CoV-2 with vaccine uptake and refusal between rural and urban Kenya two years after the pandemic onset. A population-based seroprevalence study was conducted in the city of Nairobi (n = 781) and a rural western county (n = 810) between January and February 2022. The overall SARS-CoV-2 seroprevalence was 90.2% (95% CI, 88.6−91.2%), including 96.7% (95% CI, 95.2−97.9%) among urban and 83.6% (95% CI, 80.6−86.0%) among rural populations. A comparison of immunity profiles showed that >50% of the rural population were strongly immunoreactive compared to <20% of the urban population, suggesting more recent infections or vaccinations in the rural population. More than 45% of the vaccine-eligible (≥18 years old) persons had not taken a single dose of the vaccine (hesitancy), including 47.6% and 46.9% of urban and rural participants, respectively. Vaccine refusal was reported in 19.6% of urban and 15.6% of rural participants, attributed to concern about vaccine safety (>75%), inadequate information (26%), and concern about vaccine effectiveness (9%). Less than 2% of vaccine refusers cited religious or cultural beliefs. These findings indicate that despite vaccine inequity, hesitancy, and refusal, herd immunity had been achieved in Kenya and likely other African countries by early 2022, with natural infections likely contributing to most of this immunity. However, vaccine campaigns should be sustained due to the need for repeat boosters associated with waning of SARS-CoV-2 immunity and emergence of immune-evading virus variants.

Methodology minute: A statistical test primer for infection prevention and control.

Selecting the appropriate statistical tests for data analysis is a critical skill for the infection preventionist (IP), both for analyzing their own data as well as evaluating the scientific literature methodology. Obtaining results from data analyses has never been easier thanks to computational improvements, but the interpretation of results relies on a keen awareness that the approach was sound. The purpose of this primer is to introduce the infection preventionist to the ideas behind hypothesis testing with a focus on statistical test selection.

Endothelial activation is associated with intestinal epithelial injury, systemic inflammation and treatment regimen in children living with vertically acquired HIV-1 infection.

BACKGROUND: Premature development of cardiovascular disease in children living with HIV-1 (CLWH) may be associated with compromised gut barrier function, microbial translocation, immune activation, systemic inflammation and endothelial activation. Biomarkers of these pathways may provide insights into pathogenesis of atherosclerotic disease in CLWH. METHODS: This was a cross-sectional study of CLWH enrolled in the multicentre Early Pediatric Initiation-Canadian Child Cure Cohort (EPIC4 ) who were on antiretroviral therapy (ART) with undetectable viral load. Plasma biomarkers of intestinal epithelial injury [intestinal fatty acid binding protein-1 (IFABP)], systemic inflammation [tumour necrosis factor (TNF) and interleukin-6 (IL-6)] and endothelial activation [angiopoietin-2 (Ang2), soluble vascular endothelial growth factor-1 (sVEGFR1) and soluble endoglin (sEng)] were quantified by enzyme-linked immunosorbent assay. Correlation and factor analysis of biomarkers were used to examine associations between innate immune pathways. RESULTS: Among 90 CLWH, 16% of Ang2, 15% of sVEGFR1 and 23% of sEng levels were elevated relative to healthy historic controls. Pairwise rank correlations between the three markers of endothelial activation were statistically significant (ρ = 0.69, ρ = 0.61 and ρ = 0.65, P < 0.001 for all correlations). An endothelial activation index, derived by factor analysis of the three endothelial biomarkers, was correlated with TNF (ρ = 0.47, P < 0.001), IL-6 (ρ = 0.60, P < 0.001) and intestinal fatty acid binding protein-1 (ρ = 0.67, P < 0.001). Current or past treatment with ritonavir-boosted lopinavir (LPV/r) was associated with endothelial activation (odds ratio = 5.0, 95% CI: 1.7-17, P = 0.0020). CONCLUSIONS: Endothelial activation is prevalent in CLWH despite viral suppression with combination ART and is associated with intestinal epithelial injury, systemic inflammation and treatment with LPV/r.

HIV-associated neurocognitive impairment in stable people living with HIV on ART in rural Tanzania.

OBJECTIVES: Few studies have assessed cognitive impairment among healthy people living with HIV (PLWH) who are stable on antiretroviral treatment (ART) in sub-Saharan Africa. METHODS: We conducted a cross-sectional study among a random sample of stable adult PLWH from rural Tanzania on ART for more than 1 year and without immunological failure or pre-existing neurological disease. We evaluated the prevalence and risk factors for neurocognitive impairment (NCI), assessed through neuropsychological tests, functional and depression questionnaires and defined as a mean Z-score ≤ -1 in two or more cognitive domains. RESULTS: Among 243 participants [median age = 44.3 years (interquartile range: 36-52] and 71% female] we found a rate of NCI of 19.3% (95% confidence interval: 14.8-24.8%). Memory and psychomotor domains demonstrated the highest impairment. Independent predictors of NCI were age and self-reported alcohol use. Other classical risk factors were not associated with HIV-associated NCI. CONCLUSION: Despite effective ART roll-out, NCI remained a prevalent condition in this healthy rural Tanzanian population of PLWH on ART. Age and alcohol use were key risk factors.

Thresholds versus Anomaly Detection for Surveillance of Pneumonia and Influenza Mortality.

Computational surveillance of pneumonia and influenza mortality in the United States using FluView uses epidemic thresholds to identify high mortality rates but is limited by statistical issues such as seasonality and autocorrelation. We used time series anomaly detection to improve recognition of high mortality rates. Results suggest that anomaly detection can complement mortality reporting.

Effects of infection history on dengue virus infection and pathogenicity.

The understanding of immunological interactions among the four dengue virus (DENV) serotypes and their epidemiological implications is often hampered by the lack of individual-level infection history. Using a statistical framework that infers full infection history, we analyze a prospective pediatric cohort in Nicaragua to characterize how infection history modulates the risks of DENV infection and subsequent clinical disease. After controlling for age, one prior infection is associated with 54% lower, while two or more are associated with 91% higher, risk of a new infection, compared to DENV-naive children. Children >8 years old have 55% and 120% higher risks of infection and subsequent disease, respectively, than their younger peers. Among children with ≥1 prior infection, intermediate antibody titers increase, whereas high titers lower, the risk of subsequent infection, compared with undetectable titers. Such complex dependency needs to be considered in the design of dengue vaccines and vaccination strategies.

Movement amplitude on the Functional Re-adaptive Exercise Device: deep spinal muscle activity and movement control.

PURPOSE: Lumbar multifidus (LM) and transversus abdominis (TrA) show altered motor control, and LM is atrophied, in people with low-back pain (LBP). The Functional Re-adaptive Exercise Device (FRED) involves cyclical lower-limb movement against minimal resistance in an upright posture. It has been shown to recruit LM and TrA automatically, and may have potential as an intervention for non-specific LBP. However, no studies have yet investigated the effects of changes in FRED movement amplitude on the activity of these muscles. This study aimed to assess the effects of different FRED movement amplitudes on LM and TrA muscle thickness and movement variability, to inform an evidence-based exercise prescription. METHODS: Lumbar multifidus and TrA thickness of eight healthy male volunteers were examined using ultrasound imaging during FRED exercise, normalised to rest at four different movement amplitudes. Movement variability was also measured. Magnitude-based inferences were used to compare each amplitude. RESULTS: Exercise at all amplitudes recruited LM and TrA more than rest, with thickness increases of approximately 5 and 1 mm, respectively. Larger amplitudes also caused increased TrA thickness, LM and TrA muscle thickness variability and movement variability. The data suggests that all amplitudes are useful for recruiting LM and TrA. CONCLUSIONS: A progressive training protocol should start in the smallest amplitude, increasing the setting once participants can maintain a consistent movement speed, to continue to challenge the motor control system.

Randomized clinical trial of dexketoprofen/tramadol 25 mg/75 mg in moderate-to-severe pain after total hip arthroplasty.

BACKGROUND: The aim was to evaluate the analgesic efficacy and safety of the dexketoprofen/tramadol 25 mg/75 mg fixed-dose combination vs dexketoprofen (25 mg) and tramadol (100 mg) in moderate-to-severe acute pain after total hip arthroplasty. METHODS: This was a randomized, double-blind, parallel-group study in patients experiencing pain of at least moderate intensity on the day after surgery, compared with placebo at first administration to validate the pain model. The study drug was administered orally every 8 h throughout a 5 day period. Rescue medication, metamizole 500 mg, was available during the treatment period. The evaluation of efficacy was based on patient assessments of pain intensity and pain relief. The primary end point was the mean sum of the pain intensity difference values throughout the first 8 h (SPID8). RESULTS: Overall, 641 patients, mean age 62 (range 29-80) yr, were analysed; mean (sd) values of SPID8 were 247 (157) for dexketoprofen/tramadol, 209 (155) for dexketoprofen, 205 (146) for tramadol, and 151 (159) for placebo. The primary analysis confirmed the superiority of the combination over dexketoprofen 25 mg (P=0.019; 95% confidence interval 6.4-73) and tramadol 100 mg (P=0.012; 95% confidence interval 9.5-76). The single components were superior to placebo (P<0.05), confirming model sensitivity. Most secondary analyses supported the superiority of the combination. The incidence of adverse drug reactions was low and similar among active treatment groups. CONCLUSION: The efficacy results confirmed the superiority of dexketoprofen/tramadol over its single components, even at higher doses (tramadol), with a safety profile fully in line with that previously known for these agents in monotherapy. CLINICAL TRIAL REGISTRATION: EudraCT 2012-004548-31 (https://www.clinicaltrialsregister.eu/ctr-search/search?query=eudract_number:2012-004548-31);ClinicalTrials.gov NCT01902134 (https://www.clinicaltrials.gov/ct2/show/NCT01902134?term=NCT01902134&rank=1).

Addition of chlorine during water purification reduces iodine content of drinking water and contributes to iodine deficiency.

Drinking water is the major natural source of iodine in many European countries. In the present study, we examined possible sites of iodine loss during the usual water purification process.Water samples from 6 sites during the technological process were taken and analyzed for iodine content. Under laboratory circumstances, prepared iodine in water solution has been used as a model to test the effect of the presence of chlorine. Samples from the purification sites revealed that in the presence of chlorine there is a progressive loss of iodine from the water. In the chlorine concentrations employed in the purification process, 24-h chlorine exposure eliminated more than 50% of iodine when the initial iodine concentration was 250 µg/l or less. Iodine was completely eliminated if the starting concentration was 16 µg/l.We conclude that chlorine used during water purification may be a major contributor to iodine deficiency in European communities.

Mobile phone-based infectious disease surveillance system, Sri Lanka.

Because many infectious diseases are emerging in animals in low-income and middle-income countries, surveillance of animal health in these areas may be needed for forecasting disease risks to humans. We present an overview of a mobile phone-based frontline surveillance system developed and implemented in Sri Lanka. Field veterinarians reported animal health information by using mobile phones. Submissions increased steadily over 9 months, with ≈4,000 interactions between field veterinarians and reports on the animal population received by the system. Development of human resources and increased communication between local stakeholders (groups and persons whose actions are affected by emerging infectious diseases and animal health) were instrumental for successful implementation. The primary lesson learned was that mobile phone-based surveillance of animal populations is acceptable and feasible in lower-resource settings. However, any system implementation plan must consider the time needed to garner support for novel surveillance methods among users and stakeholders.

Evaluation of the thyroid function of healthy pregnant women by five different hormone assays.

A normal function of the thyroid gland during pregnancy is essential. Any change can affect both the pregnant woman and the fetus. Thyroid hormones play a crucial role in the brain development of the fetus, thus proper maternal free thyroid hormone levels are important especially during the first trimester. We compared the free thyroid hormone levels FT3 and FT4 in forty pregnant women with no thyroidal disease by five different assays available on the market. The blood samples were collected between the 8th and 22nd weeks of pregnancy. The correlation coefficient "r" between different assays was 0.908-0.975 for TSH, 0.676-0.892 for FT4 and 0.480-0.789 for FT3. These data show that the inter-assay results varied widely in the studied population. One reasonable explanation may be that during pregnancy the serum levels of the thyroid hormone binding proteins are altered and "free" hormone measurements by immunoassays are influenced by these alterations. Thus, the results may show higher or lower thyroid hormone values depending upon the assay used. Therefore, it is strongly suggested that every laboratory should establish its own pregnant reference ranges for the tests used for the evaluation of thyroid function, based on values of the population served.

DNA repair modulates the vulnerability of the developing brain to alkylating agents.

Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag(-/-)) or O(6)-methylguanine methyltransferase (Mgmt(-/-)), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt(-/-) neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag(-/-) neurons were for the most part significantly less sensitive than wild type or Mgmt(-/-) neurons to MAM and HN2. Aag(-/-) neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt(-/-) mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM-treated Aag(-/-) or MGMT-overexpressing (Mgmt(Tg+)) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in Mgmt(Tg+) mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant.

Alkylation-induced colon tumorigenesis in mice deficient in the Mgmt and Msh6 proteins.

O(6)-methylguanine DNA methyltransferase (MGMT) suppresses mutations and cell death that result from alkylation damage. MGMT expression is lost by epigenetic silencing in a variety of human cancers including nearly half of sporadic colorectal cancers, suggesting that this loss maybe causal. Using mice with a targeted disruption of the Mgmt gene, we tested whether Mgmt protects against azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF), against AOM and dextran sulfate sodium (DSS)-induced colorectal adenomas and against spontaneous intestinal adenomas in Apc(Min) mice. We also examined the genetic interaction of the Mgmt null gene with a DNA mismatch repair null gene, namely Msh6. Both Mgmt and Msh6 independently suppress AOM-induced ACF, and combination of the two mutant alleles had a multiplicative effect. This synergism can be explained entirely by the suppression of alkylation-induced apoptosis when Msh6 is absent. In addition, following AOM+DSS treatment Mgmt protected against adenoma formation to the same degree as it protected against AOM-induced ACF formation. Finally, Mgmt deficiency did not affect spontaneous intestinal adenoma development in Apc(Min/+) mice, suggesting that Mgmt suppresses intestinal cancer associated with exogenous alkylating agents, and that endogenous alkylation does not contribute to the rapid tumor development seen in Apc(Min/+) mice.

Prevention of respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract infection in young children, with significant numbers of premature infants and those with other risk factors requiring hospitalization in Canada each year. Palivizumab, an RSV-specific monoclonal antibody, can reduce the hospitalization rate and severity of illness for a small group of high-risk or premature infants during their first RSV season. The present statement reviews the published literature and provides recommendations regarding its use in premature and other at-risk infants, for Canadian physicians.

Comparison of the new InoDiag automated fluorescence multiplexed antigen microarray to the reference technique in the serodiagnosis of atypical bacterial pneumonia.

The aetiological diagnosis of pneumonia depends largely on culture-, antigen- or PCR-based tests. Atypical agents of pneumonia include Coxiella burnetii, Chlamydophila pneumoniae, Chlamydia psittaci, Legionella pneumophila, Francisella tularensis and Mycoplasma pneumoniae. In these cases, serological tests are commonly used for diagnosis. All of the above species were comparatively screened for by using the InoDiag multiplexed automatic immunofluorescence assay and established reference techniques. The InoDiag assay required 5 microL of serum, took 76 min per serum sample, and required an incubator, a fluorescence reader and interpretation software. In total, 248 single sera from patients were tested, for the diagnosis of pneumonia, and the results obtained with selected serum samples were compared with results obtained with the reference method. It was shown that, for the detection of Coxiella burnetii IgM, the automated assay had a sensitivity and specificity of 100%. For the detection of M. pneumoniae IgM, sensitivity was 100% and specificity was 98%. For the detection of Chlamydophila pneumoniae and Chlamydia psittaci IgG, sensitivity was 81% and specificity was 94%. For the detection of L. pneumoniae IgG, sensitivity was 63% and specificity was 98%. For the detection of F. tularensis IgG and IgM, sensitivity was 100% for both, and specificity was 95% and 100%, respectively. The performance of this serological assay was comparable to that of other assays reported in the literature. This preliminary study shows that the automatic InoDiag assay opens the way to immunofluorescence assay standardization.

Multiplexed whole bacterial antigen microarray, a new format for the automation of serodiagnosis: the culture-negative endocarditis paradigm.

The serological diagnosis of blood culture-negative endocarditis due to Coxiella burnetii, Bartonella spp., Brucella melitensis and Legionella pneumophila is based on a manual immunofluorescence assay (IFA), which is taken to be the reference method. The automated IFA InoDiag multiplexed antigenic microarray, which includes a slide with all the above bacteria and four internal controls, an incubator, a fluorescent reader and software with an algorithm of interpretation for infectious endocarditis (IE) was evaluated. A single serum dilution at 1/128 was used. Eleven patients with Bartonella spp. IE and ten with C. burnetii IE, diagnosed using the modified Duke criteria, as well as one patient with B. melitensis infection and three patients with L. pneumophila IE were tested. In total, 236 sera were used as negative controls, with the reference method. The results of IgG detection were: C. burnetii phase I, 'sensitivity (Se) = 88% and specificity (Sp) = 99%', and C. burnetii phase II, Se = 88% and Sp = 99%; for Bartonella henselae, Se = 100% and Sp = 100%; for Bartonella quintana, Se = 78% and Sp = 96%; for B. melitensis, Se = 100% and Sp = 99%; and for L. pneumophila, Se = 100% and Sp = 99%. With the algorithm interpretation, the negative and positive predictive values of the test 'were 100% for the diagnosis of IE caused by the four bacteria tested. These results were confirmed by two other assays, one using triplicate testing and one blind testing performed by another centre. This multiplexed test is therefore a valuable tool for the rapid diagnosis of blood-culture negative IE.