Hepatitis C virus antigen detection is an appropriate test for screening and early diagnosis of hepatitis C virus infection in at-risk populations and immunocompromised hosts.

Early diagnosis remains key for effective prevention and treatment. Unfortunately, current screening with anti-hepatitis C virus antibody (anti-HCV Ab) test may have limited utility in the diagnosis of HCV infection and reinfection. This is of special concern to at-risk population, such as immunocompromised hosts and end-stage renal failure patients on hemodialysis. HCV antigen (Ag) could be useful in identifying the ongoing infection in such clinical scenarios. Hence, we aimed to study the utility of HCV Ag testing for the diagnosis of acute and chronic hepatitis C. Of 89 samples studied, 19 were from acute hepatitis C patients who were immunocompromised or were on hemodialysis, 43 were from active chronic hepatitis C patients and 27 were from patients treated for chronic hepatitis C. All samples were tested for HCV Ag using the Abbott ARCHITECT HCV Ag assay. HCV Ag was reactive in 19/19 samples from acute hepatitis C patients and 42/43 samples from active chronic hepatitis C patients. It was nonreactive in all samples from treated patients. The test showed a sensitivity and specificity of 98.4% and 100.0%, respectively. The positive and negative predictive values were 100.0% and 96.4%, respectively. The HCV antigen test has high clinical sensitivity and specificity and is useful for the diagnosis of acute and chronic hepatitis C infection in at-risk and immunocompromised patients. Its short turnaround time and relatively low cost are advantageous for use in patients on hemodialysis and other at-risk patients who require monitoring of HCV infection and reinfection.

Epidemiologic Features and Clinical Course of Patients Infected With SARS-CoV-2 in Singapore.

Importance: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2019 and has spread globally with sustained human-to-human transmission outside China. Objective: To report the initial experience in Singapore with the epidemiologic investigation of this outbreak, clinical features, and management. Design, Setting, and Participants: Descriptive case series of the first 18 patients diagnosed with polymerase chain reaction (PCR)-confirmed SARS-CoV-2 infection at 4 hospitals in Singapore from January 23 to February 3, 2020; final follow-up date was February 25, 2020. Exposures: Confirmed SARS-CoV-2 infection. Main Outcomes and Measures: Clinical, laboratory, and radiologic data were collected, including PCR cycle threshold values from nasopharyngeal swabs and viral shedding in blood, urine, and stool. Clinical course was summarized, including requirement for supplemental oxygen and intensive care and use of empirical treatment with lopinavir-ritonavir. Results: Among the 18 hospitalized patients with PCR-confirmed SARS-CoV-2 infection (median age, 47 years; 9 [50%] women), clinical presentation was an upper respiratory tract infection in 12 (67%), and viral shedding from the nasopharynx was prolonged for 7 days or longer among 15 (83%). Six individuals (33%) required supplemental oxygen; of these, 2 required intensive care. There were no deaths. Virus was detectable in the stool (4/8 [50%]) and blood (1/12 [8%]) by PCR but not in urine. Five individuals requiring supplemental oxygen were treated with lopinavir-ritonavir. For 3 of the 5 patients, fever resolved and supplemental oxygen requirement was reduced within 3 days, whereas 2 deteriorated with progressive respiratory failure. Four of the 5 patients treated with lopinavir-ritonavir developed nausea, vomiting, and/or diarrhea, and 3 developed abnormal liver function test results. Conclusions and Relevance: Among the first 18 patients diagnosed with SARS-CoV-2 infection in Singapore, clinical presentation was frequently a mild respiratory tract infection. Some patients required supplemental oxygen and had variable clinical outcomes following treatment with an antiretroviral agent.

Microsporidial keratoconjunctivitis after rugby tournament, Singapore.

We investigated an outbreak of 47 probable and 6 confirmed cases of microsporidial keratoconjunctivitis involving participants of an international rugby tournament in Singapore in April 2012.The mode of transmission was eye contact with soil. Vittaforma corneae was identified in 4 of 6 corneal scrapings and in 1 of 12 soil water samples.

Successful treatment of NDM-1 Klebsiella pneumoniae bacteraemia in a neutropenic patient.

The rapid emergence of novel multidrug-resistant organisms coupled with the slow development of new antibiotics is of great concern. With the discovery of New Delhi metallo-beta-lactamase 1 (NDM-1)-producing Klebsiella pneumoniae, clinicians are faced with problems in the treatment of infections caused by this multidrug-resistant organism. Therapeutic experiences are limited. We share our experience of the successful treatment of a febrile neutropenic patient with NDM-1-producing K. pneumoniae bacteraemia.

Corrigendum: Immune Response in Myocardial Injury: In Situ Hybridization and Immunohistochemistry Techniques for SARS-CoV-2 Detection in COVID-19 Autopsies.

[This corrects the article DOI: 10.3389/fmolb.2021.658932.].

Emergence of SARS-CoV-2 Spike Mutations during Prolonged Infection in Immunocompromised Hosts.

Immunocompromised hosts with prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been implicated in the emergence of highly mutated SARS-CoV-2 variants. Spike mutations are of particular concern because the spike protein is a key target for vaccines and therapeutics for SARS-CoV-2. Here, we report the emergence of spike mutations in two immunocompromised patients with persistent SARS-CoV-2 reverse transcription (RT)-PCR positivity (>90 days). Whole-genome sequence analysis of samples obtained before and after coronavirus disease 2019 (COVID-19) treatment demonstrated the development of partial therapeutic escape mutations and increased intrahost SARS-CoV-2 genome diversity over time. This case series thus adds to the accumulating evidence that immunocompromised hosts with persistent infections are important sources of SARS-CoV-2 genome diversity and, in particular, clinically important spike protein diversity. IMPORTANCE The emergence of clinically important mutations described in this report highlights the need for sustained vigilance and containment measures when managing immunocompromised patients with persistent COVID-19. Even as jurisdictions across the globe start lifting pandemic control measures, immunocompromised patients with persistent COVID-19 constitute a unique group that requires close genomic monitoring and enhanced infection control measures, to ensure early detection and containment of mutations and variants of therapeutic and public health importance.

SARS-CoV-2 N Gene G29195T Point Mutation May Affect Diagnostic Reverse Transcription-PCR Detection.

Rapid onsite whole-genome sequencing of two suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene diagnostic escape samples revealed a previously unreported N gene point mutation at genome position 29195. Because the G29195T mutation occurs within a region probed by a commonly referenced U.S. CDC N gene reverse transcription (RT)-PCR assay, we hypothesize that the G29195T mutation rendered the N gene target of a proprietary commercial assay undetectable. The putative diagnostic escape G29195T mutation demonstrates the need for nearly real-time surveillance, as emergence of a novel SARS-CoV-2 variant with the potential to escape diagnostic tests continues to be a threat. IMPORTANCE Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples. Potential diagnostic escapes, such as those of clinical samples harboring the G29195T mutation, may result in false-negative SARS-CoV-2 RT-PCR results. The rapid detection and sharing of potential diagnostic escapes are essential for diagnostic laboratories and manufacturers around the world, to optimize their assays as SARS-CoV-2 continues to evolve.

Immune Response in Myocardial Injury: In Situ Hybridization and Immunohistochemistry Techniques for SARS-CoV-2 Detection in COVID-19 Autopsies.

Coronavirus disease-19 (COVID-19) is caused by the newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the lung remains the primary target site of COVID-19 injury, damage to myocardium, and other organs also contribute to the morbidity and mortality of this disease. There is also increasing demand to visualize viral components within tissue specimens. Here we discuss the cardiac autopsy findings of 12 intensive care unit (ICU) naïve and PCR-positive COVID-19 cases using a combination of histological, Immunohistochemical/immunofluorescent and molecular techniques. We performed SARS-CoV-2 qRT-PCR on fresh tissue from all cases; RNA-ISH and IHC for SARS-CoV-2 were performed on selected cases using FFPE tissue from heart. Eight of these patients also had positive post-mortem serology for SARS-CoV-2. Histopathologic changes in the coronary vessels and inflammation of the myocardium as well as in the endocardium were documented which support the reports of a cardiac component to the viral infection. As in the pulmonary reports, widespread platelet and fibrin thrombi were also identified in the cardiac tissue. In keeping with vaccine-induced activation of virus-specific CD4+ and CD8+ T cells, and release of cytokines such as interferon-gamma (IFNγ), we observed similar immune cellular distribution and cytokines in these patients. Immunohistochemical and immunofluorescent localisation for the viral Spike (S-protein) protein and the nucleocapsid protein (NP) were performed; presence of these aggregates may possibly contribute to cardiac ischemia and even remodelling.

Saliva is more sensitive than nasopharyngeal or nasal swabs for diagnosis of asymptomatic and mild COVID-19 infection.

We aimed to test the sensitivity of naso-oropharyngeal saliva and self-administered nasal (SN) swab compared to nasopharyngeal (NP) swab for COVID-19 testing in a large cohort of migrant workers in Singapore. We also tested the utility of next-generation sequencing (NGS) for diagnosis of COVID-19. Saliva, NP and SN swabs were collected from subjects who presented with acute respiratory infection, their asymptomatic roommates, and prior confirmed cases who were undergoing isolation at a community care facility in June 2020. All samples were tested using RT-PCR. SARS-CoV-2 amplicon-based NGS with phylogenetic analysis was done for 30 samples. We recruited 200 subjects, of which 91 and 46 were tested twice and thrice respectively. In total, 62.0%, 44.5%, and 37.7% of saliva, NP and SN samples were positive. Cycle threshold (Ct) values were lower during the earlier period of infection across all sample types. The percentage of test-positive saliva was higher than NP and SN swabs. We found a strong correlation between viral genome coverage by NGS and Ct values for SARS-CoV-2. Phylogenetic analyses revealed Clade O and lineage B.6 known to be circulating in Singapore. We found saliva to be a sensitive and viable sample for COVID-19 diagnosis.

Design and Multicenter Clinical Validation of a 3-Dimensionally Printed Nasopharyngeal Swab for SARS-CoV-2 Testing.

Importance: Three-dimensionally printed nasopharyngeal swabs (3DP swabs) have been used to mitigate swab shortages during the coronavirus disease 2019 (COVID-19) pandemic. Clinical validation for diagnostic accuracy and consistency, as well as patient acceptability, is crucial to evaluate the swab's performance. Objective: To determine the accuracy and acceptability of the 3DP swab for identifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Design, Setting, and Participants: A diagnostic study was conducted from May to July 2020 at 2 tertiary care centers in Singapore with different reference swabs (FLOQSwab [COPAN Diagnostics] or Dacron swab [Deltalab]) and swab processing techniques (wet or dry) to evaluate the performance of the 3DP swab compared with traditional, standard-of-care nasopharyngeal swabs used in health care institutions. The participants were patients with COVID-19 in the first 2 weeks of illness and controls with acute respiratory illness with negative test results for SARS-CoV-2. Paired nasopharyngeal swabs were obtained from the same nostril and tested for SARS-CoV-2 by reverse-transcriptase polymerase chain reaction. The sequence of swabs was randomized based on odd and even participant numbers. Main Outcomes and Measures: Primary outcome measures were overall agreement (OA), positive percentage agreement (PPA), and negative percentage agreement of the 3DP swab compared with reference swabs. Secondary outcome measures were the correlation of cycle threshold (Ct) values of both swabs. Results: The mean (SD) age of participants was 45.4 (13.1) years, and most participants were men (87 of 89 [97.8%]), in keeping with the epidemiology of the COVID-19 pandemic in Singapore. A total of 79 patients with COVID-19 and 10 controls were recruited. Among the patients with COVID-19, the overall agreement and PPA of the 3DP swab was 91.1% and 93.5%, respectively, compared with reference swabs. The PPA was 100% for patients with COVID-19 who were tested within the first week of illness. All controls tested negative. The reverse-transcriptase polymerase chain reaction Ct values for the ORF1ab and E-gene targets showed a strong correlation (intraclass correlations coefficient, 0.869-0.920) between the 3DP and reference swab on independent testing at each institution despite differences in sample processing. Discordant results for both gene targets were observed only at high Ct values. Conclusions and Relevance: In this diagnostic study of 79 patients with COVID-19 and 10 controls, the 3DP swab performed accurately and consistently across health care institutions and could help mitigate strained resources in the escalating COVID-19 pandemic.

Rapid detection of Staphylococcus aureus Panton-Valentine leukocidin in clinical specimens by enzyme-linked immunosorbent assay and immunochromatographic tests.

Staphylococcus aureus strains producing Panton-Valentine leukocidin (PVL) have been epidemiologically linked to specific human infections. To evaluate immunological tests that may be used to diagnose infections with PVL-producing strains, we prospectively collected pus, respiratory tract specimens, and joint fluid specimens from which S. aureus had been isolated in clinical laboratories in six countries. An enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT) targeting LukS-PV were performed directly with clinical samples for the detection of PVL. The same tests were applied to S. aureus culture supernatants. The corresponding S. aureus isolates were characterized by PCR for the presence of the PVL locus (lukS-PV and lukF-PV) and the mecA gene. A total of 185 samples from 144 skin infections, 23 bone and joint infections, and 18 lower respiratory tract infections were analyzed. By PCR, 72/185 S. aureus isolates were PVL locus positive (PVL(+)); 28 of these were also mecA positive. PVL was detected in the supernatants of all PVL(+) strains by both ELISA and an ICT, while no signal was observed with PVL-negative strains. The PVL concentrations in human clinical samples that grew PVL(+) strains ranged from 0 to 399 microg/ml by ELISA. By the use of 0.015 microg/ml of PVL as a cutoff value, PVL was detected in 65/72 (90%) of the clinical samples by ELISA. The sensitivity and specificity of the ELISA test were 90% and 100%, respectively. By the ICT, PVL was detected in 57/72 (79%) of the samples, and the sensitivity and specificity of ICT were 79% and 100%, respectively. PVL is expressed by S. aureus during human infection, and a PVL-specific ELISA and ICT could be reliable tests for the diagnosis of infections caused by PVL-producing strains.

Clinical utility of Epstein-Barr virus DNA and other liquid biopsy markers in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor ubiquitously associated with the Epstein-Barr virus (EBV), which is highly prevalent in South China, Southeast Asia, and North Africa. Despite being a highly radio-sensitive and treatable cancer, a majority of NPC patients are diagnosed in their advanced stage, and locoregional and distant relapses following definitive treatment contribute largely to cancer-specific mortality among these patients. Given that EBV-driven NPC is the predominant variant seen in endemic regions, various EBV detection methods have been developed and are utilized in screening, prognostication, and post-treatment surveillance of NPC patients. While the Immunoglobulin A (IgA) serology assay is the most extensively studied EBV detection method, the detection of plasma EBV DNA released during replication or cellular apoptosis has shown superior outcomes in endemic population screening, prognostication, and detection of distant relapse. Furthermore, there is emerging evidence on the use of circulating tumor cells, microRNAs, DNA hypermethylation, and combination assays in various clinical scenarios. Herein, this paper provides a comprehensive overview of the relevant studies using various EBV detection techniques in the management of NPC. Specifically, the recent advances, clinical evidence, and challenges associated with the clinical application of EBV liquid biopsies in population screening, prognostication, and surveillance of NPC are presented.

Rapid Progression to Acute Respiratory Distress Syndrome: Review of Current Understanding of Critical Illness from Coronavirus Disease 2019 (COVID-19) Infection

The coronavirus disease 2019 (COVID-19) outbreak that started in Wuhan, Hubei province, China in December 2019 has now extended across the globe with >100,000 cases and 3,000 deaths reported in 93 countries as of 7 March 2020. We report a case of COVID-19 infection in a 64-year-old man who developed rapidly worsening respiratory failure and acute respiratory distress syndrome (ARDS) that required intubation. As the clinical spectrum of COVID-19 ranges widely from mild illness to ARDS with a high risk of mortality, there is a need for more research to identify early markers of disease severity. Current evidence suggests that patients with advanced age, pre-existing comorbidities or dyspnoea should be closely monitored, especially at 1-2 weeks after symptom onset. It remains to be seen if laboratory findings such as lymphopenia or elevated lactate dehydrogenase may serve as early surrogates for critical illness or markers of disease recovery. Management of ARDS in COVID-19 remains supportive while we await results of drug trials. More studies are needed to understand the incidence and outcomes of ARDS and critical illness from COVID-19, which will be important for critical care management and resource planning.

A Dynamic Immune Response Shapes COVID-19 Progression.

The inflammatory response to SARS-coronavirus-2 (SARS-CoV-2) infection is thought to underpin COVID-19 pathogenesis. We conducted daily transcriptomic profiling of three COVID-19 cases and found that the early immune response in COVID-19 patients is highly dynamic. Patient throat swabs were tested daily for SARS-CoV-2, with the virus persisting for 3 to 4 weeks in all three patients. Cytokine analyses of whole blood revealed increased cytokine expression in the single most severe case. However, most inflammatory gene expression peaked after respiratory function nadir, except expression in the IL1 pathway. Parallel analyses of CD4 and CD8 expression suggested that the pro-inflammatory response may be intertwined with T cell activation that could exacerbate disease or prolong the infection. Collectively, these findings hint at the possibility that IL1 and related pro-inflammatory pathways may be prognostic and serve as therapeutic targets for COVID-19. This work may also guide future studies to illuminate COVID-19 pathogenesis and develop host-directed therapies.

Emerging prevalence of microsporidial keratitis in Singapore: epidemiology, clinical features, and management.

OBJECTIVE: To investigate the incidence and epidemiologic factors involved in the development of microsporidial keratitis. The association of host immune status and clinical pattern, clinical features, and the role of fluoroquinolone monotherapy in treatment are also examined. DESIGN: Retrospective, noncomparative case series. PARTICIPANTS: All cases (124 patients, 134 eyes) of microsporidial keratitis confirmed with modified trichrome stain positive of corneal scrape over a 4-year period. METHODS: Epidemiologic factors were observed. Host immune status with human immunodeficiency virus (HIV) serology and CD4/CD8 analysis was performed when consent was obtained. Visual acuity (VA) and slit-lamp examination throughout the course of keratitis was recorded. Treatment used included topical fluoroquinolones (ciprofloxacin 0.3%, moxifloxacin 0.5%, gatifloxacin 0.5%, levofloxacin 0.5%, or norfloxacin 0.3%) as monotherapy or in combination with topical fumagillin and/or systemic albendazole. Where corneal edema developed, ultrasound corneal pachymetry was recorded. MAIN OUTCOME MEASURES: Demographic features and epidemiologic factors, including host immune status. Clinical features and disease course, including the response to different therapeutic regimes. RESULTS: Patients ranged in age from 11 to 68 years (mean, 31.9; median, 30) with a male:female ratio of 8:1 (females n = 17 [13.7%]). We performed HIV serology and CD4/CD8 in 45.9% of cases (n = 57); all the cases tested were negative with normal T-cell indices. Epidemiologic factors included soil exposure (50%), contact lens wear (21.1%), and topical steroid treatment (17.1%). The VA on presentation ranged from 20/20 to 20/100 (median, 20/30) with no loss in lines of VA on resolution. Common features were follicular papillary conjunctivitis and coarse punctate epithelial lesions in 3 patterns--diffuse, peripheral, and paracentral--evolving into nummular keratitis before resolution. Resolution occurred in 99% of cases on topical fluoroquinolone monotherapy. Four patients had recurrent disease that resolved with repeat fluoroquinolone or fluoroquinolone/oral albendazole combination. Two new clinical features were identified--diffuse endotheliitis (19.4%) with corneal edema and limbitis. CONCLUSIONS: This study identifies an increasing incidence of microsporidial keratitis in Singapore with a strong correlation with prior soil exposure. Diffuse endotheliitis and limbitis have not been described and resolves with topical steroid therapy. Topical fluoroquinolone monotherapy is a valid treatment option.

Case Report: Diagnosis of Cryptosporidiosis in Renal Transplantation in a Low-Prevalence Setting.

In high prevalence settings, cryptosporidiosis is commonly implicated as a cause of a gastroenteritis syndrome in the organ transplant population. Stool microscopy is predominant diagnostic modality. Therapeutic options in this group of patients are limited, making their management exceptionally challenging. We describe a case of a renal transplant recipient with cryptosporidiosis confirmed by the stool FilmArray gastrointestinal panel (GIP) nucleic acid-based assay and stool microscopy, describe our institutional experience in diagnosing cryptosporidiosis in a low-prevalence setting, and review the available literature on management of this condition in the organ transplant population. In a low-prevalence setting, the GIP can serve as a rapid screening tool in the diagnosis of cryptosporidiosis.