Oropharyngeal Samples Research Articles

The dynamics of circulating SARS-CoV-2 lineages in Bogor and surrounding areas reflect variant shifting during the first and second waves of COVID-19 in Indonesia.

BackgroundIndonesia is one of the Southeast Asian countries with high case numbers of COVID-19 with up to 4.2 million confirmed cases by 29 October 2021. Understanding the genome of SARS-CoV-2 is crucial for delivering public health intervention as certain variants may have different attributes that can potentially affect their transmissibility, as well as the performance of diagnostics, vaccines, and therapeutics.ObjectivesWe aimed to investigate the dynamics of circulating SARS-CoV-2 variants over a 15-month period in Bogor and its surrounding areas in correlation with the first and second wave of COVID-19 in Indonesia.MethodsNasopharyngeal and oropharyngeal swab samples collected from suspected patients from Bogor, Jakarta and Tangerang were confirmed for SARS-CoV-2 infection with RT-PCR. RNA samples of those confirmed patients were subjected to whole genome sequencing using the ARTIC Network protocol and sequencer platform from Oxford Nanopore Technologies (ONT).ResultsWe successfully identified 16 lineages and six clades out of 202 samples (male n = 116, female n = 86). Genome analysis revealed that Indonesian lineage B.1.466.2 dominated during the first wave (n = 48, 23.8%) while Delta variants (AY.23, AY.24, AY.39, AY.42, AY.43 dan AY.79) were dominant during the second wave (n = 53, 26.2%) following the highest number of confirmed cases in Indonesia. In the spike protein gene, S_D614G and S_P681R changes were dominant in both B.1.466.2 and Delta variants, while N439K was only observed in B.1.466.2 (n = 44) and B.1.470 (n = 1). Additionally, the S_T19R, S_E156G, S_F157del, S_R158del, S_L452R, S_T478K, S_D950N and S_V1264L changes were only detected in Delta variants, consistent with those changes being characteristic of Delta variants in general.ConclusionsWe demonstrated a shift in SARS-CoV-2 variants from the first wave of COVID-19 to Delta variants in the second wave, during which the number of confirmed cases surpassed those in the first wave of COVID-19 pandemic. Higher proportion of unique mutations detected in Delta variants compared to the first wave variants indicated potential mutational effects on viral transmissibility that correlated with a higher incidence of confirmed cases. Genomic surveillance of circulating variants, especially those with higher transmissibility, should be continuously conducted to rapidly inform decision making and support outbreak preparedness, prevention, and public health response.

A High-Risk Profile for Invasive Fungal Infections Is Associated with Altered Nasal Microbiota and Niche Determinants.

It is becoming increasingly clear that the communities of microorganisms that populate the surfaces exposed to the external environment, termed microbiota, are key players in the regulation of pathogen-host cross talk affecting the onset as well as the outcome of infectious diseases. We have performed a multicenter, prospective, observational study in which nasal and oropharyngeal swabs were collected for microbiota predicting the risk of invasive fungal infections (IFIs) in patients with hematological malignancies. Here, we demonstrate that the nasal and oropharyngeal microbiota are different, although similar characteristics differentiate high-risk from low-risk samples at both sites. Indeed, similar to previously published results on the oropharyngeal microbiota, high-risk samples in the nose were characterized by low diversity, a loss of beneficial bacteria, and an expansion of potentially pathogenic taxa, in the presence of reduced levels of tryptophan (Trp). At variance with oropharyngeal samples, however, low Trp levels were associated with defective host-derived kynurenine production, suggesting reduced tolerance mechanisms at the nasal mucosal surface. This was accompanied by reduced levels of the chemokine interleukin-8 (IL-8), likely associated with a reduced recruitment of neutrophils and impaired fungal clearance. Thus, the nasal and pharyngeal microbiomes of hematological patients provide complementary information that could improve predictive tools for the risk of IFI in hematological patients.

Comparison of reverse-transcription qPCR and droplet digital PCR for the detection of SARS-CoV-2 in clinical specimens of hospitalized patients

Accurate detection of severe acute respiratory syndrome coronavirus 2 is not only necessary for viral load monitoring to optimize treatment in hospitalized coronavirus disease 2019 patients, but also critical for deciding whether the patient could be discharged without any risk of viral shedding. Digital droplet PCR (ddPCR) is more sensitive than reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and is usually considered the superior choice. In the current study, we compared the clinical performance of RT-qPCR and ddPCR using oropharyngeal swab samples from patients hospitalized in the temporary Huoshenshan Hospital, Wuhan, Hubei, China. Results demonstrated that ddPCR was indeed more sensitive than RT-qPCR. Negative results might be caused by poor sampling technique or recovered patients, as the range of viral load in these patients varied significantly. In addition, both methods were highly correlated in terms of their ability to detect all three target genes as well as the ratio of copies of viral genes to that of the IC gene. Furthermore, our results evidenced that both methods detected the N gene more easily than the ORF gene. Taken together, these findings imply that the use of ddPCR, as an alternative to RT-qPCR, is necessary for the accurate diagnosis of hospitalized coronavirus disease 2019 patients.

Vertical Virus Transmission from SARS-CoV-2-positive Mothers to Neonates: A Tertiary Care Hospital Experience

Objectives: The coronavirus disease 2019 (COVID-19) outbreak is evolving rapidly worldwide. However, little is known about the association between pregnant women with COVID-19 and its transmission to neonates. This investigation aimed to see if COVID-19 infection could be transmitted vertically into the uterus. Methods: We conducted a prospective observational study. 48 COVID-19 infected mothers were enrolled during their third trimester. A qRT-PCR assay of the nasal and oropharyngeal swab samples was performed to confirm positive for COVID-19 infection as per WHO protocol. In addition, characteristics of pregnant women with confirmed SARS -CoV-2 infection and newborns were documented. Results: Forty-eight expectant mothers, 10 (20.8%) were found symptomatic, and 38 (79.2%) were asymptomatic, with COVID-19 infection were delivered (33 cesarean section & 15 vaginal deliveries). One female child (4.1%) out of 48 newborns was initially diagnosed with COVID-19 infection based on a nucleic acid qRT-PCR. The female child showed no or negligible signs and recovered completely, whereas 47 neonates (95.9%) confirmed negative. None of the mothers or neonates died from COVID-19 related pulmonary problems. Conclusion: There is insufficient evidence on vertical virologic transmission of COVID-19 infection during the third trimester of pregnancy. Additionally, research and surveillance involving adequate testing of samples of placental tissue, breast milk, vaginal swab, amniotic fluid, and cord blood will be needed to establish the possibility of vertical transmission of infection. J Microbiol Infect Dis 2022; 12(1):1-5.

Oropharyngeal Pathogenic Bacteria: Carriage, Antimicrobial Susceptibility Pattern and Associated Risk Factors among Febrile Patients

Background: Pharyngeal and respiratory infections due to bacteria are global concerns especially because of the emergence of multi-drug resistant strains. The Oropharynx is one of the regions of the human body that is heavily colonized by microbial flora. So, the Oropharyngeal carriage is a major risk factor for an invasion and developing the disease. Therefore, this study was aimed at determining the carriage rate of potential pathogenic Oropharyngeal bacteria, associated risk factors, and antimicrobial susceptibility patterns among febrile patients at AHMC. Methods: A Cross-sectional study was conducted from November 2018 to April 2019. A systematic random technique was implemented to collect Oropharyngeal samples from 253 selected febrile patients. Specimen collection and processing were done following standard bacteriological procedures in Adama Public Health, Research, and Referral Laboratory. Antimicrobial susceptibility testing was done using the disk diffusion method in accordance with the Clinical and Laboratory Standard Institutes guideline. Data were analyzed using SPSS version 20. Results: Overall oropharyngeal bacteria carriage rate was 75.1%. A total of 190 bacterial organisms were isolated with 96 Coagulase-Negative Staphylococci (CNS), 55 Streptococcus pyogenes, and 19 Staphylococcus aureus were dominant isolates among gram-positive. Klebsiella pneumoniae, Neisseria meningitides, Moraxella catarrhalis, and Escherichia coli were among gram-negative bacteria isolates. Living in urban, cigarette smoking, and poor oral hygiene was significantly associated with the Oropharyngeal bacterial carriage rate. The majority of CNS, S.aureus, and S. pyogenes were resistant to different classes of antibiotics. Conclusion: Potentially pathogenic Oropharyngeal bacterial colonization rate was high. Place of living and behavioral factors are risk factors that were associated with bacterial colonization of the oropharynx. Several bacterial isolates were resistant to frequently prescribed antibiotic classes. Therefore, determining the antimicrobial susceptibility pattern of the isolates is important to intervene and prevent the risk of infection and also the emergence of multiple drug-resistant strains and disease management.

Development of Duplex and Multiplex Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) Assays for Clinical Diagnosis of SARS-COV-2 in Sri Lanka

PurposeDespite the rollout of several vaccines targeting SARS-CoV-2, attainment of near-universal vaccination is a challenging task, particularly for low- and middle-income nations such as Sri Lanka. Rapid, reliable diagnostics for the detection of the virus is of vital importance for the predominantly export- and tourism-based economy of the country. Herein, we report the development of a RT-LAMP assay as an alternative to the gold-standard RT-qPCR method for diagnostic laboratories in Sri Lanka in a cost-effective and highly reliable manner.Methods & MaterialsAbout 313 nasopharyngeal and oropharyngeal samples from the community were collected and subjected to RNA purification and subjected to simultaneous RT-qPCR and RT-LAMP experiments by using previously published primers in a thermocycler. Duplex (containing N and E gene primers) and multiplex (containing N, E and ORF1ab gene primers) RT-LAMP assay results were compared with standard RT-qPCR results using an agreement attribute statistical test. The effect of guanidine hydrochloride was also analyzed.ResultsThe limit of detection for the duplex assay was found to be 10 copies µL-1 at a constant temperature of 63°C, and 5 copies µL-1 for multiplex assays at 66.4°C. Both types of RT-LAMP assay were specific only for the SARS-COV-2 virus, successfully distinguishing it from multiple other human viruses. Attribute agreement analysis between duplex- and multiplex RT-LAMP vs RT-qPCR yielded 93% and 96.5% scores, respectively. Moreover, both RT-LAMP assays showed 100% agreement with RT-qPCR when Ct was <25 in positive samples and showed 100% (duplex) or 97.22% (multiplex) at 35≥ Ct ≥25. The discrepancy between agreements at higher Ct values was attributed to the higher sensitivity of the multiplex RT-LAMP assay. The addition of guanidine hydrochloride increased the sensitivity and decreased detection time significantly for both the duplex and multiplex assays.ConclusionOverall, we have demonstrated a potentially rapidly deployable diagnostic test kit not only for widespread community use but particularly for high-risk locations such as ports of entry or manufacturing facilities to mitigate the effects of the SARS-CoV-2 virus in Sri Lanka.

Development of an immunofluorescence assay for detection of SARS-CoV-2.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis in 2019. Currently, the main method for identification of SARS-CoV-2 is a reverse transcription (RT)-PCR assay designed to detect viral RNA in oropharyngeal (OP) or nasopharyngeal (NP) samples. While the PCR assay is considered highly specific and sensitive, this method cannot determine the infectivity of the sample, which may assist in evaluation of virus transmissibility from patients and breaking transmission chains. Thus, cell-culture-based approaches such as cytopathic effect (CPE) assays are routinely employed for the identification of infectious viruses in NP/OP samples. Despite their high sensitivity, CPE assays take several days and require additional diagnostic tests in order to verify the identity of the pathogen. We have therefore developed a rapid immunofluorescence assay (IFA) for the specific detection of SARS-CoV-2 in NP/OP samples following cell culture infection. Initially, IFA was carried out on Vero E6 cultures infected with SARS-CoV-2 at defined concentrations, and infection was monitored at different time points. This test was able to yield positive signals in cultures infected with 10 pfu/ml at 12 hours postinfection (PI). Increasing the incubation time to 24 hours reduced the detectable infective dose to 1 pfu/ml. These IFA signals occur before the development of CPE. When compared to the CPE test, IFA has the advantages of specificity, rapid detection, and sensitivity, as demonstrated in this work.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-022-05392-z.

NOTCH1 Intracellular Domain and the Tumor Microenvironment as Prognostic Markers in HNSCC.

Simple SummaryIn the head and neck, a large proportion of squamous cell carcinoma demonstrate a mutation of the NOTCH1 gene. The aim of this project was to investigate the role of NOTCH1 and immunological characteristics and highlight a potential rationale for therapy. We found that a high expression of NOTCH1 intracellular domain in these patients is associated with reduced overall survival. In vitro experiments additionally showed a reduction of migration and proliferation of cancer cells when NOTCH1 was knocked down. NOTCH1 is, therefore, most likely involved in migration and proliferation of head and neck squamous cell carcinoma and is a prognostic marker in these patients.(1) Background: NOTCH1 is the second most common mutated gene in whole-exome sequencing of HNSCC. The aim of this project was to gain further insight into the relevance of NOTCH1 in HNSCC, potentially establishing NOTCH1 as a prognostic marker or therapeutic target; (2) Methods: NOTCH1 was silenced via RNA interference in six HNSCC cell lines and the impact was evaluated in migration and proliferation assays. Subsequently, the protein expression of NOTCH1 intracellular domain (NICD) and NOTCH1 mRNA expression were examined in 70 oropharyngeal squamous cell cancer tissue samples. Lastly, the NICD expression was compared with the local infiltration of lymphocytes, measured with the immunoscore; (3) Results: Knockdown of NOTCH1 decreased migration and proliferation. A high NICD expression was associated with lower OS. A high immunoscore resulted in significantly better OS. NICD expression was independent of the immunoscore and as a whole differentiated three distinct prognostic groups; (4) Conclusions: These data suggest that NOTCH1 is involved in migration and proliferation of HNSCC cell lines. In vivo, NICD expression was associated with overall survival and could, therefore, be used as a prognostic marker. NICD expression differs from NOTCH1 mRNA levels, potentially explaining the previously suggested bimodal role as an oncogene and tumor suppressor in HNSCC.

Early life inter-kingdom interactions shape the immunological environment of the airways

BackgroundThere is increasing evidence that the airway microbiome plays a key role in the establishment of respiratory health by interacting with the developing immune system early in life. While it has become clear that bacteria are involved in this process, there is a knowledge gap concerning the role of fungi. Moreover, the inter-kingdom interactions that influence immune development remain unknown. In this prospective exploratory human study, we aimed to determine early post-natal microbial and immunological features of the upper airways in 121 healthy newborns.ResultsWe found that the oropharynx and nasal cavity represent distinct ecological niches for bacteria and fungi. Breastfeeding correlated with changes in microbiota composition of oropharyngeal samples with the greatest impact upon the relative abundance of Streptococcus species and Candida. Host transcriptome profiling revealed that genes with the highest expression variation were immunological in nature. Multi-omics factor analysis of host and microbial data revealed unique co-variation patterns.ConclusionThese data provide evidence of a diverse multi-kingdom microbiota linked with local immunological characteristics in the first week of life that could represent distinct trajectories for future respiratory health.Trial registrationNHS Health Research Authority, IRAS ID 199053. Registered 5 Oct 2016. https://www.hra.nhs.uk/planning-and-improving-research/application-summaries/research-summaries/breathing-together/-dqCA8KxpBwUxTCR4xx6DaVideo abstract

Screening of Anorectal and Oropharyngeal Samples Fails to Detect Bacteriophages Infecting Neisseria gonorrhoeae.

There are real concerns that Neisseria gonorrhoeae may become untreatable in the near future due to the rapid emergence of antimicrobial resistance. Alternative therapies are thus urgently required. Bacteriophages active against N. gonorrhoeae could play an important role as an antibiotic-sparing therapy. To the best of our knowledge, no bacteriophages active against N. gonorrhoeae have ever been found. The aim of this study was to screen for bacteriophages able to lyse N. gonorrhoeae in oropharyngeal and anorectal swabs of 74 men who have sex with men attending a sexual health clinic in Antwerp, Belgium. We screened 210 swabs but were unable to identify an anti-gonococcal bacteriophage. This is the first report of a pilot screening that systematically searched for anti-gonococcal phages directly from clinical swabs. Further studies may consider screening for phages at other anatomical sites (e.g., stool samples, urine) or in environmental settings (e.g., toilet sewage water of sex clubs or sexually transmitted infection clinics) where N. gonorrhoeae can be found.

Infectivity Period After Symptom Relief in SARS-CoV-2 Patients: An Experimental Trial

Background: This study aimed to understand the infectivity period after symptom relief in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients using real-time reverse transcription polymerase chain reaction (RT-PCR). This would probably help prevent the disease spread by partially recovered patients to some extent. Methods: A total of 10 coronavirus disease 2019 (COVID-19) infected patients with mild to moderate illness, approved by RT-PCR, were followed daily. After 2 days of cessation of clinical symptoms, a nasopharyngeal and oropharyngeal sample RT-PCR was taken. If the patients’ test had a negative result, a repeated RT-PCR was performed 24 hours later. Moreover, if two consecutive tests had negative results, the patient was considered definitively treated, and the follow-up was terminated. However, if RT-PCR was positive, the test was performed 3 days later. The infectivity period after symptom relief and total infectivity period were calculated. The SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies were measured 1 month after symptom relief. The SPSS Software (version 20) was used for data analysis. Results: A total of 10 patients met the inclusion criteria to enter the study. There were 6 (60%) male and 4 (40%) female patients. The mean age of patients was 37.40 ± 7.975 years (minimum: 28 years, maximum: 51 years). The mean days passed from the onset of symptoms at the time of referring to Kowsar Hospital in Semnan, Iran, was 3.2 ± 0.919 days (minimum: 2 days, maximum: 5 days). The mean infectivity period after symptom relief was 6.9 ± 5.152 days indicating that it took about 7 days after symptom relief that the patients were considered completely treated according to RT-PCR results. However, the mean period of total infectivity was 16.6 ± 5.73 days. Moreover, 70% of patients had an IgM negative/IgG positive result 1 month after symptom relief; nevertheless, 30% of patients showed a delayed antibody response. Conclusions: The virus could be detected in nasopharyngeal and oropharyngeal samples in the studied patients up to 7 days after symptom relief. This finding highlights the importance of isolation and distancing in patients after recovery and the adoption of hygiene measures.

Whole-Genome Sequencing of SARS-CoV-2 Strains from Asymptomatic Individuals in India.

ABSTRACTWe performed targeted genome sequencing of 11 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples collected from asymptomatic individuals. These nasopharyngeal and oropharyngeal samples were collected during the first wave of coronavirus disease 2019 (COVID-19) in Karnataka, India. Nine strains were found to be Nextstrain clade 20B (PANGO lineage B.1.1.519, GISAID clade GR), and two were identified as clade 20A (PANGO lineage B.1.619, GISAID clade G). The spike protein mutation D614G was observed across all the sequenced strains.

SARS-CoV-2 Direct Real Time PCR without RNA Extraction: Appropriate Method or Not?

Real time PCR (RT-PCR) detection method is the widely used for COVID-19 virus detection. This includes sample collection in viral transport medium (VTM), viral RNA extraction followed by detection of virus using fluorescence dye-based system using RT-PCR machine. Several studies have demonstrated a new method which replaces the extraction step by a simple method involving DTT and Proteinase-K and heat treatment. ICMR and few other governing bodies have approved such protocols but are they appropriate in clinical context? In present study, we tried to evaluate one such protocol by using ICMR and WHO approved COVID-19 detection protocol (of CoviPath™ COVID-19 RT-PCR Kit) by replacing RNA extraction step. We used 228 clinical COVID-19 samples for studying method which includes 176 positive (CT values from 14 to 23; 24 to 31 and 32 to 37 were considered as high, moderate and low positive respectively) and 52 negative nasopharyngeal and oropharyngeal swabs samples. We got 100% concordant results with negative samples and 92% concordant and 8% non-concordant results for positive samples. Non-concordant results are with low positive samples. Low level of positivity in the samples could indicate the initial/end stage of COVID-19 disease. If they are at the initial stage, they can be the potential carrier and spread the disease. Authors believe that direct methods can be used for screening bit not for diagnosis of COVID-19 disease.

Clinical performance of the Xpert® Xpress Flu/RSV assay for the detection of influenza A, B, and respiratory syncytial virus on ESwab™ medium

BackgroundPrior to the SARS-CoV-2 pandemic, the Xpert® Xpress Flu/RSV assay (GXA) was regularly used, while during the pandemic the Xpert® Xpress SARS-CoV-2/Flu/RSV (plus) assay was developed by Cepheid. In contrast to this new assay, the use of oropharyngeal ESwabs in combination with the Flu/RSV assay is designated as off CE-IVD label use, while these were used frequently in The Netherlands. AimTo investigate the clinical performance of the GXA using 1,289 ESwab™ oropharyngeal samples for the detection of Influenza A virus, Influenza B virus and respiratory syncytial virus (RSV). MethodsThe clinical performance of the GXA was prospectively investigated during the influenza seasons of 2017 and 2018 by testing fresh oropharyngeal samples, collected with ESwab™, contemporaneously with both the GXA and a laboratory developed Flu/RSV real time RT-PCR assay (LDA) (reference method). Results1,289 Samples from 1,213 patients (46% men, median age 73 (IQR 62–82)) were tested with both tests. Positive percent agreement (95% CI) was 98% (95%-99%) for Influenza A virus, 92% (88%-95%) for influenza B virus, and 88% (73%-96%) for RSV. Negative percent agreement was ≥ 99% for all three viruses. Ct-values of the GXA were on average higher than the LDA. ConclusionThis study showed a good clinical performance of the GXA in oropharyngeal samples for the detection of Influenza A and B virus. The positive percent agreement of the GXA for the detection of RSV in oropharyngeal samples was somewhat lower and in particular for the detection of RSV-A.

Oral Abundance of Actinomyces spp. in Breast Cancer Patients

Objectives: Pathophysiology of medication-related osteonecrosis of the jaw (MRONJ) is still unclear, and disease development is associated with adverse reaction of bisphosphonates and denosumab, and Actinomyces spp. as well. In this study, we evaluated the abundance of Actinomyces spp. in breast cancer patients undergoing chemotherapy compared to healthy controls. Methods: Oropharyngeal samples were collected from treatment-naive early-stage breast cancer patients, who were scheduled for standard of care therapy (eight samples throughout chemotherapy, one prior to radiotherapy and one after a year of start), as well as from healthy controls at matched timepoints. We quantified Actinomyces spp. in the samples with a highly sensitive and specific quantitative polymerase chain reaction. Results: Twenty-one patients and 16 healthy subjects were enrolled. Forty-eight percent of patients suffered from estrogen receptor-positive/progesterone receptor-positive or -negative/human epidermal growth factor receptor 2 (HER2)-negative disease, 38% were HER2-positive, and 14% were triple-negative. Comparison of Actinomyces spp. loads in cancer patients and healthy controls did not reveal significant difference. Fluctuations on bacterial quantity were observed in both groups over time. Tumor receptor status or different chemotherapy schemes of patients were not correlated with a particular pattern on abundance of Actinomyces spp. Conclusions: We suggest that Actinomyces spp. are not the initiative factors in MRONJ development. These bacteria are not altered in abundance during chemotherapy, but they behave opportunistic when there is a bone disruption in the oropharynx in the first place caused by antiresorptive drugs or dental trauma and proliferate in their new niche. Thus, Actinomyces spp. plays a latter role in MRONJ development, rather than a primary causative one.

Seroprevalence of anti-SARS-CoV-2 antibodies in a population living in Bomassa village, Republic of Congo

ObjectivesWith limited data available from Central Africa, the aim of our study was to evaluate the anti-SARS-CoV-2 Ab prevalence in indigenous residents of Bomassa, a village located in the Sangha region in the Republic of Congo. MethodsPlasma and oropharyngeal swab samples were collected from 304 healthy adult individuals, randomly recruited in May 2021 before vaccine introduction in the area. In addition, 82 plasma samples from the same area in 2019 were included as controls for the investigation of cross-reactivity against other coronaviruses. The SARS-CoV-2 virus was detected by qRT-PCR and sequenced using next-generation sequencing. ELISA was used for detecting IgG, IgM, and neutralizing Ab against SARS-CoV-2 antigens. ResultsAround 4.9% (15/304) of the participants were SARS-CoV-2 positive, with B.1.631 being the only variant identified. Of 109 individuals harboring anti-SARS-CoV-2 IgG and/or IgM Ab, 45.9% (50/109) had anti-SARS-CoV-2 neutralizing Ab. Of the control samples collected before the pandemic, 3.7% (3/82) were positive for IgG, but negative for neutralizing Ab. ConclusionsSeroprevalence against SARS-CoV-2 occurred in 25% of the indigenous population sample, with almost 50% of these seropositive participants possessing neutralizing antibodies. These findings suggest that the spread of SARS-CoV-2 has been underestimated in the Republic of Congo.

Viral co-pathogens in COVID-19 acute respiratory syndrome – what did we learn from the first year of pandemic?

Objective: This study aimed to describe the distribution of respiratory pathogens and the occurrence of co-pathogens during the first year of the COVID-19 pandemic. MethodsWe used a multiplex polymerase chain reaction (PCR) panel targeting 23 microorganisms to analyze the oro-pharyngeal samples of patients admitted to our hospital with acute respiratory infection (ARI) between March 1, 2020, and February 28, 2021. We matched 40 to 50 patients who were SARS-CoV-2 positive and SARS-CoV-2 negative per month for age and sex. ResultsA total of 939 patients with multiplex PCR test results were included in the study. Respiratory pathogens where detected in only 8/476 (1.6%) patients with COVID-19 versus 87/463 (18.7%) patients with non–COVID-19 ARI patients. Diversity and rates of pathogens vastly differed from previous years but showed seasonal variance. ConclusionPatients with SARS-CoV-2 infection presenting with ARI during the first year of the COVID-19 pandemic demonstrated paucity of respiratory co-pathogens.

Prevalence and risk factors of Avian Influenza Viruses among household ducks in Chattogram, Bangladesh.

Avian influenza viruses (AIV) increase commercial and backyard poultry mortality and morbidity, reduces egg production, and elevates public health risk. Household ducks propagate and transmit HPAI and LPAI viruses between domesticated and wild birds in Southeast Asian countries, including Bangladesh. This study was conducted to identify epidemiological factors associated with AIV infection among household ducks at Chattogram, Bangladesh. We randomly selected and collected blood and oropharyngeal swab samples from 281 households ducks. We evaluated the serum for AIV antibody using cELISA and tested for H5 and H9 subtypes using the HI test. We tested the swabs with real-time reverse transcriptase PCR (rRT-PCR) for M gene, and H5, H9 subtypes. In the duck populations, the household level AIV sero-prevalence was 57.7% (95% CI: 51.6-63.3) and RNA prevalence was 2.4% (95% CI: 1.0-5.0). H5 and H9 subtype sero-prevalence was 31.5% (95% CI: 22.2-42.0) and 23.9% (95% CI: 15.6-33.9). H5 and H9 subtype RNA prevalence were 0% (95% CI: 0.0-1.3) and 2.4% (95% CI: 1.0-5.0). We determined household-level OR (Odds Ratios) for the "combined (mixed materials-mud and concrete or metallic)" category was 2.2 (95% CI: 1.1-4.2) compared with "wooden/bamboo" category (p = 0.02); 2.8 (95% CI: 1.2-6.6) in households with duck plague vaccine coverage compared with no coverage (p = 0.01); and 2.4 (95% CI: 0.6-9.7) in households that threw dead birds in bushes and the roadside compared with households that buried or threw dead birds in garbage pits (p = 0.21). M gene phylogenetic analysis compared M gene sequences to previously reported Bangladeshi H9N2 isolates. The evidence presented here shows AIV circulation in the Chattogram, Bangladesh study areas. AIV reduction can be achieved through farmer education of proper farm management practices.

Meningococcal Carriage in 'Men Having Sex With Men' With Pharyngeal Gonorrhoea.

We assessed the characteristics of Neisseria meningitidis pharyngeal carriage in a cohort of ‘men having sex with men’, including patients with pharyngeal Neisseria gonorrhoeae infection. In the period 2017-2019, among all the oropharyngeal samples tested for gonorrhoea from MSM attending a STI Clinic in Bologna (Italy), we randomly selected 244 N. gonorrhoeae-positive samples and 403 negatives (n=647). Pharyngeal specimens were tested for N. meningitidis presence, by the detection of sodC gene. N. meningitidis-positive samples were further grouped by PCR tests for the major invasive genogroups (i.e., A, B, C, W, and Y). A molecular assay, targeting capsule transporter gene, was used to determine meningococcal capsular status. Overall, 75.8% (491/647) of samples tested positive for sodC gene, indicating a pharyngeal meningococcal carriage. Meningococcal colonisation was significantly more frequent in younger subjects (P=0.009), with no association with HIV infection. Non-groupable meningococci represented most of pharyngeal carriages (about 71%). The commonest N. meningitidis serogroup was B (23.6%), followed by C (2.1%), Y (1.8%) and W (1.1%). Meningococci were often characterized by the genetic potential of capsule production. Interestingly, a negative association between N. meningitidis and N. gonorrhoeae was found: pharyngeal gonorrhoea was significantly more present in patients without meningococcal carriage (P=0.03). Although preliminary, our data added knowledge on the epidemiology of meningococcal carriage in MSM communities at high risk of gonococcal infections, gaining new insights into the interactions/dynamics between N. meningitidis and N. gonorrhoeae.

Direct RNA Nanopore Sequencing of SARS-CoV-2 Extracted from Critical Material from Swabs

In consideration of the increasing prevalence of COVID-19 cases in several countries and the resulting demand for unbiased sequencing approaches, we performed a direct RNA sequencing (direct RNA seq.) experiment using critical oropharyngeal swab samples collected from Italian patients infected with SARS-CoV-2 from the Palermo region in Sicily. Here, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retrotranscription. Using an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapsid (N) gene, which have been reported previously in studies conducted in other countries. In conclusion, to the best of our knowledge, the technique used in this study has not been used for SARS-CoV-2 detection previously owing to the difficulties in the extraction of RNA of sufficient quantity and quality from routine oropharyngeal swabs. Despite these limitations, this approach provides the advantages of true native RNA sequencing and does not include amplification steps that could introduce systematic errors. This study can provide novel information relevant to the current strategies adopted in SARS-CoV-2 next-generation sequencing.