The ability of Interleukin-10 to negate haemozoin-related pro-inflammatory effects has the potential to restore impaired macrophage function associated with malaria infection.

BACKGROUND: Although pro-inflammatory cytokines are involved in the clearance of Plasmodium falciparum during the early stages of the infection, increased levels of these cytokines have been implicated in the pathogenesis of severe malaria. Amongst various parasite-derived inducers of inflammation, the malarial pigment haemozoin (Hz), which accumulates in monocytes, macrophages and other immune cells during infection, has been shown to significantly contribute to dysregulation of the normal inflammatory cascades. METHODS: The direct effect of Hz-loading on cytokine production by monocytes and the indirect effect of Hz on cytokine production by myeloid cells was investigated during acute malaria and convalescence using archived plasma samples from studies investigating P. falciparum malaria pathogenesis in Malawian subjects. Further, the possible inhibitory effect of IL-10 on Hz-loaded cells was examined, and the proportion of cytokine-producing T-cells and monocytes during acute malaria and in convalescence was characterized. RESULTS: Hz contributed towards an increase in the production of inflammatory cytokines, such as Interferon Gamma (IFN-γ), Tumor Necrosis Factor (TNF) and Interleukin 2 (IL-2) by various cells. In contrast, the cytokine IL-10 was observed to have a dose-dependent suppressive effect on the production of TNF among other cytokines. Cerebral malaria (CM) was characterized by impaired monocyte functions, which normalized in convalescence. CM was also characterized by reduced levels of IFN-γ-producing T cell subsets, and reduced expression of immune recognition receptors HLA-DR and CD 86, which also normalized in convalescence. However, CM and other clinical malaria groups were characterized by significantly higher plasma levels of pro-inflammatory cytokines than healthy controls, implicating anti-inflammatory cytokines in balancing the immune response. CONCLUSIONS: Acute CM was characterized by elevated plasma levels of pro-inflammatory cytokines and chemokines but lower proportions of cytokine-producing T-cells and monocytes that normalize during convalescence. IL-10 is also shown to have the potential to indirectly prevent excessive inflammation. Cytokine production dysregulated by the accumulation of Hz appears to impair the balance of the immune response to malaria and exacerbates pathology.

Evasion of MAIT cell recognition by the African Salmonella Typhimurium ST313 pathovar that causes invasive disease.

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal Salmonella enterica strains activate MAIT cells. However, S. Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of ribB This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium ST313 lineage 2.

A fast extraction-free isothermal LAMP assay for detection of SARS-CoV-2 with potential use in resource-limited settings.

BACKGROUND: To retain the spread of SARS-CoV-2, fast, sensitive and cost-effective testing is essential, particularly in resource limited settings (RLS). Current standard nucleic acid-based RT-PCR assays, although highly sensitive and specific, require transportation of samples to specialised laboratories, trained staff and expensive reagents. The latter are often not readily available in low- and middle-income countries and this may significantly impact on the successful disease management in these settings. Various studies have suggested a SARS-CoV-2 loop mediated isothermal amplification (LAMP) assay as an alternative method to RT-PCR. METHODS: Four previously published primer pairs were used for detection of SARS-CoV-2 in the LAMP assay. To determine optimal conditions, different temperatures, sample input and incubation times were tested. Ninety-three extracted RNA samples from St. George's Hospital, London, 10 non-extracted nasopharyngeal swab samples from Great Ormond Street Hospital for Children, London, and 92 non-extracted samples from Queen Elisabeth Central Hospital (QECH), Malawi, which have previously been tested for SARS-Cov-2 by quantitative reverse-transcription RealTime PCR (qRT-PCR), were analysed in the LAMP assay. RESULTS: In this study we report the optimisation of an extraction-free colourimetric SARS-CoV-2 LAMP assay and demonstrated that a lower limit of detection (LOD) between 10 and 100 copies/µL of SARS-CoV-2 could be readily detected by a colour change of the reaction within as little as 30 min. We further show that this assay could be quickly established in Malawi, as no expensive equipment is necessary. We tested 92 clinical samples from QECH and showed the sensitivity and specificity of the assay to be 86.7% and 98.4%, respectively. Some viral transport media, used routinely to stabilise RNA in clinical samples during transportation, caused a non-specific colour-change in the LAMP reaction and therefore we suggest collecting samples in phosphate buffered saline (which did not affect the colour) as the assay allows immediate sample analysis on-site. CONCLUSION: SARS-CoV-2 LAMP is a cheap and reliable assay that can be readily employed in RLS to improve disease monitoring and management.

Sequential acquisition of T cells and antibodies to nontyphoidal Salmonella in Malawian children.

BACKGROUND: Salmonella Typhimurium (STm) remain a prominent cause of bacteremia in sub-Saharan Africa. Complement-fixing antibodies to STm develop by 2 years of age. We hypothesized that STm-specific CD4⁺ T cells develop alongside this process. METHODS: Eighty healthy Malawian children aged 0-60 months were recruited. STm-specific CD4⁺ T cells producing interferon γ, tumor necrosis factor α, and interleukin 2 were quantified using intracellular cytokine staining. Antibodies to STm were measured by serum bactericidal activity (SBA) assay, and anti-STm immunoglobulin G antibodies by enzyme-linked immunosorbent assay. RESULTS: Between 2006 and 2011, STm bacteremias were detected in 449 children <5 years old. STm-specific CD4⁺ T cells were acquired in infancy, peaked at 14 months, and then declined. STm-specific SBA was detectable in newborns, declined in the first 8 months, and then increased to a peak at age 35 months. Acquisition of SBA correlated with acquisition of anti-STm-lipopolysaccharide (LPS) immunoglobulin G (r = 0.329 [95% confidence interval, .552-.062]; P = .01) but not anti-STm-outer membrane protein or anti-STm-flagellar protein (FliC). CONCLUSIONS: Acquisition of STm-specific CD4⁺ T cells in early childhood is consistent with early exposure to STm or cross-reactive protein antigens priming this T-cell development. STm-specific CD4⁺ T cells seem insufficient to protect against invasive nontyphoidal Salmonella disease, but sequential acquisition of SBA to STm LPS is associated with a decline in its incidence.

Suppression of host gene expression is associated with latent TB infection: a possible diagnostic biomarker.

The World Health Organization End TB strategy aims for a 90% reduction of tuberculosis (TB) incidence by 2035. Systematic testing and treatment of latent TB infection (LTBI) among contacts of active TB patients is recommended as one of the ways to curtail TB incidence. However, there is a shortage of tools to accurately diagnose LTBI. We assessed the appropriateness of whole blood host transcriptomic markers (TM) to diagnose LTBI among household contacts of bacteriologically confirmed index cases compared to HIV negative healthy controls (HC). QuantiFERON-TB Gold Plus Interferon gamma release assay (IGRA) and reverse-transcriptase quantitative PCR were used to determine LTBI and quantify TM expression respectively. Association between TM expression and LTBI was evaluated by logistic regression modelling. A total of 100 participants, 49 TB exposed (TBEx) household contacts and 51 HC, were enrolled. Twenty-five (51%) TBEx individuals tested positive by IGRA, and were denoted as LTBI individuals, and 37 (72.5%) HC were IGRA-negative. Expression of 11 evaluated TM was significantly suppressed among LTBI compared to HC. Out of the 11 TM, ZNF296 and KLF2 expression were strongly associated with LTBI and successfully differentiated LTBI from HC. Paradoxically, 21 (49%) TBEx participants who tested IGRA negative exhibited the same pattern of suppressed TM expression as IGRA positive (LTBI-confirmed individuals). Results suggest that suppression of gene expression underlies LTBI and may be a more sensitive diagnostic biomarker than standard-of-care IGRA.

Vaccine value profile for invasive non-typhoidal Salmonella disease.

Invasive non-typhoidal Salmonella (iNTS) disease is an under-recognized high-burden disease causing major health and socioeconomic issues in sub-Saharan Africa (sSA), predominantly among immune-naïve infants and young children, including those with recognized comorbidities such as HIV infection. iNTS disease is primarily caused by Salmonella enterica serovar Typhimurium sequence type (ST) 313 and 'African-restricted clades' of Salmonella Enteritidis ST11 that have emerged across the African continent as a series of epidemics associated with acquisition of new antimicrobial resistance. Due to genotypes with a high prevalence of antimicrobial resistance and scarcity of therapeutic options, these NTS serovars are designated by the World Health Organization as a priority pathogen for research and development of interventions, including vaccines, to address and reduce NTS associated bacteremia and meningitis in sSA. Novel and traditional vaccine technologies are being applied to develop vaccines against iNTS disease, and the results of the first clinical trials in the infant target population should become available in the near future. The "Vaccine Value Profile" (VVP) addresses information related predominantly to invasive disease caused by Salmonella Enteritidis and Salmonella Typhimurium prevalent in sSA. Information is included on stand-alone iNTS disease candidate vaccines and candidate vaccines targeting iNTS disease combined with another invasive serotype, Salmonella Typhi, that is also common across sSA. Out of scope for the first version of this VVP is a wider discussion on either diarrheagenic NTS disease (dNTS) also associated with Salmonella Enteritidis and Salmonella Typhimurium or the development of a multivalent Salmonella vaccines targeting key serovars for use globally. This VVP for vaccines to prevent iNTS disease is intended to provide a high-level, holistic assessment of the information and data that are currently available to inform the potential public health, economic, and societal value of pipeline vaccines and vaccine-like products. Future versions of this VVP will be updated to reflect ongoing activities such as vaccine development strategies and a "Full Vaccine Value Assessment" that will inform the value proposition of an iNTS disease vaccine. This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, public private partnerships, and multi-lateral organizations, and in collaboration with stakeholders from the World Health Organization African Region. All contributors have extensive expertise on various elements of the iNTS disease VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using only existing and publicly available information.

Environmental surveillance for Salmonella Typhi in rivers and wastewater from an informal sewage network in Blantyre, Malawi.

Environmental surveillance for Salmonella Typhi may provide information on the community-level dynamics of typhoid fever in resource poor regions experiencing high disease burden. Many knowledge gaps concerning the feasibility of ES remain, especially in areas lacking formal sewage systems. We implemented protocols for S. Typhi ES, including site selection and catchment population estimation, sample concentration and testing using qPCR for S. Typhi specific gene targets. Between May 2021 and May 2022, we collected grab samples and Moore swabs from 43 sites in Blantyre, Malawi. Catchment characteristics, water quality, and human faecal contamination (qPCR for Bacteroides HF183) were also recorded. Their association with S. Typhi detection was investigated using a logistic mixed-effects regression analysis. Prevalence of S. Typhi in ES samples was 2.1% (1.1-4.0%) and 3.9% (1.9-7.9%) for grab and Moore swab samples, respectively. HF183 was associated S. Typhi positivity, with a unit increase in log genome copies/microlitre increasing the odds of detection of S. Typhi by 1.56 (95% CI: 1.29-1.89) and 1.33 (1.10-1.61) in Moore swabs and grab samples, respectively. The location and timing of S. Typhi detection through ES was not associated with the incidence of typhoid fever reported in associated catchment populations. During this period of relatively low typhoid fever incidence, wastewater surveillance continued to detect S. Typhi in human sewage and wastewater suggesting that ES using natural river systems can be a sensitive indicator of transmission.

Household costs and care seeking patterns associated with COVID-19 in Blantyre, Malawi.

Economic consequences of COVID-19 illness and healthcare use for households in low income countries are not well known. We estimated costs associated with COVID-19 care-seeking and treatment from a household perspective and assessed determinants of treatment costs. A cross-sectional household survey was conducted between December 2020 and November 2021 in urban and peri-urban areas of Blantyre district. Adults (age ≥18 years) with confirmed COVID-19 were asked to report the symptoms they experienced or prompted them to seek COVID-19 tests as well as healthcare seeking behaviors preceding and following COVID-19 diagnosis. For individuals who sought healthcare, information on out-of-pocket expenditures incurred while seeking and receiving care including on transport, food etc. by both the patients and their guardians was collected. Finally, data on time use seeking, receiving care and during convalesces was recorded. Multivariate Generalized Linear Models were used to evaluate association between household COVID-19 costs and their determinants. Of 171 individuals who took part in the study, the average age was 40.7 years, standard deviation (SD) 15.0, and 50.8% were females. Most participants (85.3%) were symptomatic. Of these, 67.8% sought care at health facilities and the majority (91.7%) were treated as outpatients. The average total household cost associated with COVID-19 seeking, receiving care and convalescence was $62.81 (SD $126.02). Average costs for outpatient and inpatient cases were $52.96 (SD $54.35) and $172.39 (SD $407.08), respectively. Average out-of-pocket household expenditures were $42.62 (SD $123.10), accounting for 62% of total household costs. Being a male COVID-19 patient and engagement in formal employment were significantly associated with high COVID-19 household costs. Households face high economic burden related to COVID-19 sickness and healthcare use. Social policies that support households cope with both the direct and indirect COVID-19 cost are needed to ensure access to healthcare and protect households from COVID-19 related shocks.

Nontyphoidal Salmonella Invasive Disease: Challenges and Solutions.

Nontyphoidal Salmonella are a leading cause of community-onset bacteremia and other serious infections in sub-Saharan African countries where large studies indicate that they are an uncommon cause of moderate-to-severe diarrhea. Approximately 535 000 nontyphoidal Salmonella invasive disease illnesses and 77 500 deaths were estimated to occur in 2017; 422 000 (78.9%) illnesses and 66 500 (85.9%) deaths in countries in sub-Saharan Africa. Lineages of Salmonella enterica serovar Typhimurium sequence type (ST) 313 and lineages of Salmonella enterica serovar Enteritidis ST11 dominate as causes of invasive disease. A major reservoir for these specific strains outside of humans has not been identified to date. Human fecal shedding of such strains is common in areas where nontyphoidal Salmonella invasive disease incidence is high. The case-fatality ratio of nontyphoidal Salmonella invasive disease is approximately 15%. Early diagnosis and treatment are needed to avert fatal outcomes. Antimicrobial resistance, including multiple drug resistance, decreased fluoroquinolone susceptibility, and resistance to third-generation cephalosporins, is increasing in prevalence and is likely to further compromise patient outcomes. Naturally acquired immunity against invasive disease develops in children aged >3 years in endemic areas, likely mediated in part by the sequential acquisition of T-cell immunity, followed by antigen-specific immunoglobulin G antibodies. Vaccines in preclinical or clinical development include live-attenuated S. enterica serovar Typhimurium, nontyphoidal S. enterica core and O-polysaccharide glycoconjugates, multiple antigen-presenting system complexes, and generalized modules for membrane antigens vaccines. The latter are in phase I trials in Europe and Africa. Both vaccine use, and other effective, evidence-based nonvaccine interventions, are needed to prevent and control nontyphoidal Salmonella invasive disease.

Characterization of Enzyme-Linked Immunosorbent Assay (ELISA) for Quantification of Antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-Antigens in Human Sera.

Nontyphoidal Salmonella (NTS) is a leading cause of morbidity and mortality caused by enteric pathogens worldwide in both children and adults, and vaccines are not yet available. The measurement of antigen-specific antibodies in the sera of vaccinated or convalescent individuals is crucial to understand the incidence of disease and the immunogenicity of vaccine candidates. A solid and standardized assay used to determine the level of specific anti-antigens IgG is therefore of paramount importance. In this work, we presented the characterization of a customized enzyme-linked immunosorbent assay (ELISA) with continuous readouts and a standardized definition of EU/mL. We assessed various performance parameters: standard curve accuracy, dilutional linearity, intermediate precision, specificity, limits of blanks, and quantification. The simplicity of the assay, its high sensitivity and specificity coupled with its low cost and the use of basic consumables and instruments without the need of high automation makes it suitable for transfer and application to different laboratories, including resource-limiting settings where the disease is endemic. This ELISA is, therefore, fit for purpose to be used for quantification of antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-antigens in human samples, both for vaccine clinical trials and large sero-epidemiological studies.

Characterizing the evolving SARS-CoV-2 seroprevalence in urban and rural Malawi between February 2021 and April 2022: A population-based cohort study.

OBJECTIVES: This study aimed to investigate the changing SARS-CoV-2 seroprevalence and associated health and sociodemographic factors in Malawi between February 2021 and April 2022. METHODS: In total, four 3-monthly serosurveys were conducted within a longitudinal population-based cohort in rural Karonga District and urban Lilongwe, testing for SARS-CoV-2 S1 immunoglobulin (Ig)G antibodies using an enzyme-linked immunosorbent assay. Population seroprevalence was estimated in all and unvaccinated participants. Bayesian mixed-effects logistic models estimated the odds of seropositivity in the first survey, and of seroconversion between surveys, adjusting for age, sex, occupation, location, and assay sensitivity/specificity. RESULTS: Of the 2005 participants (Karonga, n = 1005; Lilongwe, n = 1000), 55.8% were female and median age was 22.7 years. Between Surveys (SVY) 1 and 4, population-weighted SARS-CoV-2 seroprevalence increased from 26.3% to 89.2% and 46.4% to 93.9% in Karonga and Lilongwe, respectively. At SVY4, seroprevalence did not differ by COVID-19 vaccination status in adults, except for those aged 30+ years in Karonga (unvaccinated: 87.4%, 95% credible interval 79.3-93.0%; two doses: 98.1%, 94.8-99.5%). Location and age were associated with seroconversion risk. Individuals with hybrid immunity had higher SARS-CoV-2 seropositivity and antibody titers, than those infected. CONCLUSION: High SARS-CoV-2 seroprevalence combined with low morbidity and mortality indicate that universal vaccination is unnecessary at this stage of the pandemic, supporting change in national policy to target at-risk groups.

Utilizing river and wastewater as a SARS-CoV-2 surveillance tool in settings with limited formal sewage systems.

The COVID-19 pandemic has profoundly impacted health systems globally and robust surveillance has been critical for pandemic control, however not all countries can currently sustain community pathogen surveillance programs. Wastewater surveillance has proven valuable in high-income settings, but less is known about the utility of water surveillance of pathogens in low-income countries. Here we show how wastewater surveillance of SAR-CoV-2 can be used to identify temporal changes and help determine circulating variants quickly. In Malawi, a country with limited community-based COVID-19 testing capacity, we explore the utility of rivers and wastewater for SARS-CoV-2 surveillance. From May 2020-May 2022, we collect water from up to 112 river or defunct wastewater treatment plant sites, detecting SARS-CoV-2 in 8.3% of samples. Peak SARS-CoV-2 detection in water samples predate peaks in clinical cases. Sequencing of water samples identified the Beta, Delta, and Omicron variants, with Delta and Omicron detected well in advance of detection in patients. Our work highlights how wastewater can be used to detect emerging waves, identify variants of concern, and provide an early warning system in settings with no formal sewage systems.

Utilizing river and wastewater as a SARS-CoV-2 surveillance tool to predict trends and identify variants of concern in settings with limited formal sewage systems.

The COVID-19 pandemic continues to impact health systems globally and robust surveillance is critical for pandemic control, however not all countries can sustain community surveillance programs. Wastewater surveillance has proven valuable in high-income settings, but little is known about how river and informal sewage in low-income countries can be used for environmental surveillance of SARS-CoV-2. In Malawi, a country with limited community-based COVID-19 testing capacity, we explored the utility of rivers and wastewater for SARS-CoV-2 surveillance. From May 2020 - January 2022, we collected water from up to 112 river or informal sewage sites/month, detecting SARS-CoV-2 in 8.3% of samples. Peak SARS-CoV-2 detection in water samples predated peaks in clinical cases. Sequencing of water samples identified the Beta, Delta, and Omicron variants, with Delta and Omicron detected well in advance of detection in patients. Our work highlights wastewater can be used for detecting emerging waves, identifying variants of concern and function as an early warning system in settings with no formal sewage systems.

Intracellular survival of Streptococcus pneumoniae in human alveolar macrophages is augmented with HIV infection.

People Living with HIV (PLHIV) are at an increased risk of pneumococcal pneumonia than HIV-uninfected adults, but the reasons for this are still not well understood. We investigated whether alveolar macrophages (AM) mediated control of pneumococcal infection is impaired in PLHIV compared to HIV-uninfected adults. We assessed anti-bactericidal activity against Streptococcus pneumoniae of primary human AM obtained from PLHIV and HIV-uninfected adults. We found that pneumococcus survived intracellularly in AMs at least 24 hours post ex vivo infection, and this was more frequent in PLHIV than HIV-uninfected adults. Corroborating these findings, in vivo evidence showed that PLHIV had a higher propensity for harboring S. pneumoniae within their AMs than HIV-uninfected adults. Moreover, bacterial intracellular survival in AMs was associated with extracellular propagation of pneumococcal infection. Our data suggest that failure of AMs to eliminate S. pneumoniae intracellularly could contribute to the increased risk of pneumococcal pneumonia in PLHIV.

Development and Characterization of a Luminescence-Based High-Throughput Serum Bactericidal Assay (L-SBA) to Assess Bactericidal Activity of Human Sera against Nontyphoidal Salmonella.

Salmonella Typhimurium and Salmonella Enteritidis are leading causative agents of invasive nontyphoidal Salmonella (iNTS) disease, which represents one of the major causes of death and morbidity in sub-Saharan Africa, still partially underestimated. Large sero-epidemiological studies are necessary to unravel the burden of disease and guide the introduction of vaccines that are not yet available. Even if no correlate of protection has been determined so far for iNTS, the evaluation of complement-mediated functionality of antibodies generated towards natural infection or elicited upon vaccination may represent a big step towards this achievement. Here we present the setup and the intra-laboratory characterization in terms of repeatability, intermediate precision, linearity, and specificity of a high-throughput luminescence-based serum bactericidal assay (L-SBA). This method could be useful to perform sero-epidemiological studies across iNTS endemic countries and for evaluation of antibodies raised against iNTS vaccine candidates in upcoming clinical trials.

Case-control investigation of invasive Salmonella disease in Malawi reveals no evidence of environmental or animal transmission of invasive strains, and supports human to human transmission.

BACKGROUND: Invasive Salmonella infections cause significant morbidity and mortality in Sub-Saharan Africa. However, the routes of transmission are uncertain. We conducted a case-control study of index-case and geographically-matched control households in Blantyre, Malawi, sampling Salmonella isolates from index cases, healthy people, animals, and the household environment. METHODOLOGY: Sixty index cases of human invasive Salmonella infection were recruited (March 2015-Oct 2016). Twenty-eight invasive Non-Typhoidal Salmonella (iNTS) disease and 32 typhoid patients consented to household sampling. Each index-case household was geographically matched to a control household. Extensive microbiological sampling included stool sampling from healthy household members, stool or rectal swabs from household-associated animals and boot-sock sampling of the household environment. FINDINGS: 1203 samples from 120 households, yielded 43 non-Typhoidal Salmonella (NTS) isolates from 25 households (overall sample positivity 3.6%). In the 28 iNTS patients, disease was caused by 3 STs of Salmonella Typhimurium, mainly ST313. In contrast, the isolates from households spanned 15 sequence types (STs). Two S. Typhimurium isolates from index cases closely matched isolates from their respective asymptomatic household members (2 and 3 SNP differences respectively). Despite the recovery of a diverse range of NTS, there was no overlap between the STs causing iNTS disease with any environmental or animal isolates. CONCLUSIONS: The finding of NTS strains from index cases that matched household members, coupled with lack of related animal or environmental isolates, supports a hypothesis of human to human transmission of iNTS infections in the household. The breadth of NTS strains found in animals and the household environment demonstrated the robustness of NTS sampling and culture methodology, and suggests a diverse ecology of Salmonella in this setting. Healthy typhoid (S. Typhi) carrier state was not detected. The lack of S. Typhi isolates from the household environment suggests that further methodological development is needed to culture S. Typhi from the environment.

Differential localization and limited cytotoxic potential of duodenal CD8+ T cells.

The duodenum is a major site of HIV persistence during suppressive antiretroviral therapy despite harboring abundant tissue-resident memory (Trm) CD8+ T cells. The role of duodenal Trm CD8+ T cells in viral control is still not well defined. We examined the spatial localization, phenotype, and function of CD8+ T cells in the human duodenal tissue from people living with HIV (PLHIV) and healthy controls. We found that Trm (CD69+CD103hi) cells were the predominant CD8+ T cell population in the duodenum. Immunofluorescence imaging of the duodenal tissue revealed that CD103+CD8+ T cells were localized in the intraepithelial region, while CD103-CD8+ T cells and CD4+ T cells were mostly localized in the lamina propria (LP). Furthermore, HIV-specific CD8+ T cells were enriched in the CD69+CD103-/lo population. However, the duodenal HIV-specific CD8+ Trm cells rarely expressed canonical molecules for potent cytolytic function (perforin and granzyme B) but were more polyfunctional than those from peripheral blood. Taken together, our results show that duodenal CD8+ Trm cells possess limited perforin-mediated cytolytic potential and are spatially separated from HIV-susceptible LP CD4+ T cells. This could contribute to HIV persistence in the duodenum and provides critical information for the design of cure therapies.

An international, interlaboratory ring trial confirms the feasibility of an extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

Antimicrobial susceptibility testing profiles of ESBL-producing Enterobacterales isolated from hospital and community adult patients in Blantyre, Malawi.

Objective: There is a paucity of data on antimicrobial resistance (AMR) in Malawi. Here we present a study of AMR of extended-spectrum ß-lactamases-producing Enterobacterales (ESBL-E) isolated from hospital and community settings in Blantyre, Malawi. Design and Methods: A cross-sectional study was conducted between March and November 2020, involving 403 adult participants aged ≥18 years. Screening for ESBL-E was performed using CHROMagar ESBL medium. Production of ESBLs was confirmed by a combination disk test method. Antimicrobial susceptibility was tested using the agar disk diffusion method in accordance with the Clinical Laboratory Standards Institute's 2019 guidelines. Results: The mean resistance rate of ESBL-E to antimicrobial agents tested was 49.2% (range from 1.4%-92%). The highest resistance rates were observed for trimethoprim-sulfamethoxazole (92%), amoxicillin and ceftriaxone (79%), doxycycline (75%) and gentamicin (72%). Carbapenems (meropenem and imipenem) were highly active against isolates. The overall rate of multi-drug resistant (MDR) ESBL-E was 47%. The highest MDR was found in Yersinia enterocolitica (51%) and the least in Serratia spp. (40%). Conclusions: We found a high resistance rate of ESBL-E isolates to antimicrobial agents; the majority were MDR. Surveillance systems are recommended to monitor AMR in Malawi.