Travellers' diarrhoea : Impact of TD definition and control group design on study results

AbstractBackground Travellers' diarrhoea (TD) is a common health problem among visitors to the (sub)tropics. Much research deals with aetiology, prevention, and post-infection sequalae, yet the data may not allow comparisons due to incompatible definitions of TD and No TD control groups. Method The impact of defining TD and No TD control groups was explored by revisiting our recent data. We set up two TD groups: classical TD i.e. ≥3 loose or liquid stools/day and WHO TD (diarrhoea as defined by the WHO) i.e. any diarrhoea, and four No TD groups by TD definition and timing (no classical/WHO TD during travel, no ongoing classical/WHO TD). Results TD was recorded for 37% versus 65% of subjects when using classical versus WHO definitions, respectively; the proportions of the various pathogens proved similar. The strictest criterion for the No TD control group (no WHO TD during travel) yielded pathogens among 61% and the least strict (no ongoing classical TD) among 73% of the travellers; the differences were greatest for enteroaggregative Escherichia coli and Campylobacter. Conclusions Definition of TD and control group design substantially impact on TD study results. The WHO definition yields more cases, but the pathogen selection is similar by both definitions. Design of the No TD control group was found critical: only those remaining asymptomatic throughout the journey should be included.Peer reviewe

Circulating pathogen-specific plasmablasts in female patients with upper genital tract infection

Mucosal antibodies constitute the first line of adaptive immune defence against invaders in the female genital tract (FGT), yet the sequence of events leading to their production is surprisingly poorly characterized. We explored the induction of pathogen-specific antibody-secreting cells (ASC) as a response to an acute infection in the upper FGT. We recruited 12 patients undergoing surgery due to an upper FGT infection (7/12 blood culture positive, 5/ 12 negative) and six healthy controls. Pathogens were sampled during surgery and PBMC collected in the acute phase of the disease (days 7-10). We searched by ELISPOT circulating pathogen-specific ASC and explored their frequency, immunoglobulin isotype distribution, and expressions of homing receptors (alpha(4)beta(7), L-selectin, and CLA). All patients had circulating ASC specific to the infective bacteria; the geometric mean was 434 (95%CI 155-1234) ASC (IgA + IgG + IgM)/10(6) PBMC. IgA ASC predominated in 7/12, IgG ASC in 3/12, and IgM ASC in 2/12 cases. Of all the pathogen-specific ASC, 60% expressed alpha(4)beta(7), 67% L-selectin, and 9% CLA. This study is the first to show induction of pathogen-specific ASC in the peripheral blood in bacterial infection in the human FGT. Our findings reveal that such FGT-originating pathogen-specific ASC are predominated by IgA ASC and exhibit a homing receptor profile resembling that of ASC in acute urinary tract infection. The data thus suggest a characteristic profile shared by the urogenital tract.Peer reviewe

Bacterial, viral and parasitic pathogens analysed by qPCR: Findings from a prospective study of travellers' diarrhoea

Background: The diagnostics of travellers' diarrhoea (TD) has been revolutionised by multiplex qPCR assays. While mostly of bacterial aetiology, viruses and parasites account for the disease among 10-20% of travellers. Despite this, prospective studies applying qPCR assays remain scarce that cover not only bacteria, such as the various diarrhoeagenic Escherichia coli (DEC), but also viral and parasitic pathogens. Method: We analysed by qPCR pre- and post-travel stool samples of 146 Finnish travellers for bacterial, viral and parasitic pathogens: enteropathogenic (EPEC), enteroaggregative (EAEC), enterotoxigenic (ETEC), enterohaemorrhagic (EHEC), and enteroinvasive (EIEC) E. coli; Shigella, Campylobacter, Salmonella, Yersinia and Vibrio cholerae; norovirus G1 and G2, rotavirus, enteroviruses, and sapovirus; and Giardia lamblia, Entamoeba histolytica, and Cryptosporidium. Symptoms and medication data during travel were collected by questionnaires. Results: We detected bacterial pathogens in 102/146 samples (69.9%; EAEC, EPEC, ETEC most common), viral ones in 13 (8.9%; norovirus most common), and parasitic ones in one (0.7%; Giardia). Noroviruses were associated with severe symptoms (23.5% versus non-severe 4.9%). In the TD group, 41.7% (5/12) of those with viral pathogens (vs. 13.3%; 11/83 without) took antibiotics. Conclusion: Viral pathogens, particularly noroviruses, prevail in severe TD. The symptoms of viral disease are often severe and lead to unwarranted use of antibiotics.Peer reviewe

A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

Accurate and rapid diagnostic tools are needed for management of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Antibody tests enable detection of individuals past the initial phase of infection and help examine vaccine responses. The major targets of human antibody response in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the spike glycoprotein (SP) and nucleocapsid protein (NP). We have developed a rapid homogenous approach for antibody detection termed LFRET (protein L-based time-resolved Förster resonance energy transfer immunoassay). In LFRET, fluorophore-labeled protein L and antigen are brought to close proximity by antigen-specific patient immunoglobulins of any isotype, resulting in TR-FRET signal. We set up LFRET assays for antibodies against SP and NP and evaluated their diagnostic performance using a panel of 77 serum/plasma samples from 44 individuals with COVID-19 and 52 negative controls. Moreover, using a previously described SP and a novel NP construct, we set up enzyme linked immunosorbent assays (ELISAs) for antibodies against SARS-CoV-2 SP and NP. We then compared the LFRET assays with these ELISAs and with a SARS-CoV-2 microneutralization test (MNT). We found the LFRET assays to parallel ELISAs in sensitivity (90–95% vs. 90–100%) and specificity (100% vs. 94–100%). In identifying individuals with or without a detectable neutralizing antibody response, LFRET outperformed ELISA in specificity (91–96% vs. 82–87%), while demonstrating an equal sensitivity (98%). In conclusion, this study demonstrates the applicability of LFRET, a 10-min “mix and read” assay, to detection of SARS-CoV-2 antibodies

High number of diarrhoeal co-infections in travellers to Benin, West Africa

Peer reviewe

A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

Accurate and rapid diagnostic tools are needed for management of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Antibody tests enable detection of individuals past the initial phase of infection and help examine vaccine responses. The major targets of human antibody response in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the spike glycoprotein (SP) and nucleocapsid protein (NP). We have developed a rapid homogenous approach for antibody detection termed LFRET (protein L-based time-resolved Förster resonance energy transfer immunoassay). In LFRET, fluorophore-labeled protein L and antigen are brought to close proximity by antigen-specific patient immunoglobulins of any isotype, resulting in TR-FRET signal. We set up LFRET assays for antibodies against SP and NP and evaluated their diagnostic performance using a panel of 77 serum/plasma samples from 44 individuals with COVID-19 and 52 negative controls. Moreover, using a previously described SP and a novel NP construct, we set up enzyme linked immunosorbent assays (ELISAs) for antibodies against SARS-CoV-2 SP and NP. We then compared the LFRET assays with these ELISAs and with a SARS-CoV-2 microneutralization test (MNT). We found the LFRET assays to parallel ELISAs in sensitivity (90–95% vs. 90–100%) and specificity (100% vs. 94–100%). In identifying individuals with or without a detectable neutralizing antibody response, LFRET outperformed ELISA in specificity (91–96% vs. 82–87%), while demonstrating an equal sensitivity (98%). In conclusion, this study demonstrates the applicability of LFRET, a 10-min “mix and read” assay, to detection of SARS-CoV-2 antibodies

Scent dogs in detection of COVID-19 : triple-blinded randomised trial and operational real-life screening in airport setting

Objective To estimate scent dogs' diagnostic accuracy in identification of people infected with SARS-CoV-2 in comparison with reverse transcriptase polymerise chain reaction (RT-PCR). We conducted a randomised triple-blinded validation trial, and a real-life study at the Helsinki-Vantaa International Airport, Finland. Methods Four dogs were trained to detect COVID-19 using skin swabs from individuals tested for SARS-CoV-2 by RT-PCR. Our controlled triple-blinded validation study comprised four identical sets of 420 parallel samples (from 114 individuals tested positive and 306 negative by RT-PCR), randomly presented to each dog over seven trial sessions. In a real-life setting the dogs screened skin swabs from 303 incoming passengers all concomitantly examined by nasal swab SARS-CoV-2 RT-PCR. Our main outcomes were variables of diagnostic accuracy (sensitivity, specificity, positive predictive value, negative predictive value) for scent dog identification in comparison with RT-PCR. Results Our validation experiments had an overall accuracy of 92% (95% CI 90% to 93%), a sensitivity of 92% (95% CI 89% to 94%) and a specificity of 91% (95% CI 89% to 93%) compared with RT-PCR. For our dogs, trained using the wild-type virus, performance was less accurate for the alpha variant (89% for confirmed wild-type vs 36% for alpha variant, OR 14.0, 95% CI 4.5 to 43.4). In the real-life setting, scent detection and RT-PCR matched 98.7% of the negative swabs. Scant airport prevalence (0.47%) did not allow sensitivity testing; our only SARS-CoV-2 positive swab was not identified (alpha variant). However, ad hoc analysis including predefined positive spike samples showed a total accuracy of 98% (95% CI 97% to 99%). Conclusions This large randomised controlled triple-blinded validation study with a precalculated sample size conducted at an international airport showed that trained scent dogs screen airport passenger samples with high accuracy. One of our findings highlights the importance of continuous retraining as new variants emerge. Using scent dogs may present a valuable approach for high-throughput, rapid screening of large numbers of people.Peer reviewe

Immune Defense in Upper Airways : A Single-Cell Study of Pathogen-Specific Plasmablasts and Their Migratory Potentials in Acute Sinusitis and Tonsillitis

Background Despite the high frequency of upper respiratory tract (URT) infections and use of the nasal mucosa as route for vaccination, the local immune mechanism and dissemination of effector lymphocytes from the URT have been insufficiently characterized. To devise a single-cell approach for studying the mucosal immune response in the URT, we explored URT-originating B effector lymphocytes in the circulation of patients with one of two common respiratory infections, acute sinusitis or tonsillitis. Methods Patients with acute sinusitis (n = 13) or tonsillitis (n = 11) were investigated by ELISPOT for circulating pathogen-specific antibody-secreting cells (ASCs) of IgA, IgG and IgM isotypes approximately one week after the onset of symptoms. These cells' potential to home into tissues was explored by assessing their expression of tissue-specific homing receptors alpha(4)beta(7), L-selectin, and cutaneous lymphocyte antigen (CLA). Results Pathogen-specific ASCs were detected in the circulation of all patients, with a geometric mean of 115 (95% CI 46-282) /10(6) PBMC in sinusitis, and 48 (27-88) in tonsillitis. These responses were mainly dominated by IgG. In sinusitis alpha(4)beta(7) integrin was expressed by 24% of the ASCs, L-selectin by 82%, and CLA by 21%. The proportions for tonsillitis were 15%, 80%, and 23%, respectively. Healthy individuals had no ASCs. Conclusions URT infections-acute sinusitis and tonsillitis-both elicited a response of circulating pathogen-specific plasmablasts. The magnitude of the response was greater in sinusitis than tonsillitis, but the homing receptor profiles were similar. Human nasopharynx-associated lymphoid structures were found to disseminate immune effector cells with a distinct homing profile.Peer reviewe

Specific and cross-reactive immune response to oral Salmonella Typhi Ty21a and parenteral Vi capsular polysaccharide typhoid vaccines administered concomitantly

Peer reviewe

Characterization of low-density granulocytes in COVID-19

Author summary The emergence of SARS-COV-2 and the ensuing COVID-19 disease has revealed an unprecedented need to understand the pathological mechanisms of acute respiratory infections in more detail. Granulocytes are highly abundant cells of the innate immunity, and thus first responders towards acute infections. However, their excessive activation can cause unwanted tissue damage and detrimental effects in humans. This study identifies a population of low-density granulocytes (LDGs) in COVID-19 patient samples, which has been poorly described in the context of acute infections so far. These cells were subclassified and found to be mainly of immature phenotypes. Further characterization revealed COVID-19 LDGs as a phenotypically diverse population with immunosuppressive characteristics, which seemed to be in line with an elevated recruitment and activation of granulocytes. Altogether, these findings suggest LDG may play a role in COVID-19 disease progression. Severe COVID-19 is characterized by extensive pulmonary complications, to which host immune responses are believed to play a role. As the major arm of innate immunity, neutrophils are one of the first cells recruited to the site of infection where their excessive activation can contribute to lung pathology. Low-density granulocytes (LDGs) are circulating neutrophils, whose numbers increase in some autoimmune diseases and cancer, but are poorly characterized in acute viral infections. Using flow cytometry, we detected a significant increase of LDGs in the blood of acute COVID-19 patients, compared to healthy controls. Based on their surface marker expression, COVID-19-related LDGs exhibit four different populations, which display distinctive stages of granulocytic development and most likely reflect emergency myelopoiesis. Moreover, COVID-19 LDGs show a link with an elevated recruitment and activation of neutrophils. Functional assays demonstrated the immunosuppressive capacities of these cells, which might contribute to impaired lymphocyte responses during acute disease. Taken together, our data confirms a significant granulocyte activation during COVID-19 and suggests that granulocytes of lower density play a role in disease progression.Peer reviewe