Direct Immunofluorescence Assay Research Articles

Autoimmunity and epithelial dysplasia in patients with oral lichenoid diseases.

The primary objective of this study was to explore relationship between autoimmunity and epithelial dysplasia in patients with oral lichenoid diseases. A total of 66 patients with oral lichen planus (OLP), 35 with oral lichenoid lesion (OLL), and 85 with oral lichenoid drug reaction (OLDR) were enrolled. OLP, OLL, and OLDR were diagnosed following the definitions of the modified World Health Organization criteria, except for the absence of epithelial dysplasia. All patients underwent diagnostic incisional biopsy and adjunctive direct immunofluorescence assays. An indirect immunofluorescence assay was conducted to determine the antinuclear antibody (ANA) positivity. OLP and OLDR patients with epithelial dysplasia demonstrated higher prevalence of serum ANA positivity compared to those without epithelial dysplasia. Elevated serum levels of high sensitivity-C reactive proteins were observed in the OLP, OLL, and OLDR patients with epithelial dysplasia. In the DIF analysis, patients with epithelial dysplasia in the OLP exhibited a higher prevalence of C3 deposition in the basement membrane zone. This study proposed that autoimmunity may contribute to elevating levels of focal and chronic systemic inflammation, potentially influencing abnormal wound healing and development of dysplastic changes in the oral epithelium among patients with oral lichenoid disease.

A Case of Psoriasis and Pemphigus Foliaceous in a 55-year-old Filipino

Pemphigus foliaceous is a rare autoimmune blistering disease, while psoriasis is a common immune-mediated inflammatory skin disease. The coexistence of psoriasis and pemphigus foliaceous has rarely been reported. We report a case of a 55-year-old Filipino female with an 8-year history of chronic plaque-type psoriasis biopsy-proven. After 5 years, she developed generalized flaccid bullae and crusted erosions over the face, trunk, and extremities, with no mucous membrane involvement. Skin punch biopsy, direct immunofluorescence, and enzyme-linked immunosorbent assay were consistent with pemphigus foliaceous. The combination of topical corticosteroids and oral methotrexate was selected as the therapeutic approach, leading to a notable improvement in the patient’s condition. This case report underscores the significance of identifying the simultaneous presence of psoriasis alongside autoimmune blistering diseases like pemphigus foliaceous. Examining predisposing and triggering factors, performing re-biopsy, and further work-up as the disease evolves may yield more profound insights. Nonetheless, effectively managing this condition poses a significant challenge.

Validation of a New Duplex Real-Time Polymerase Chain Reaction for Chlamydia trachomatis DNA Detection in Ocular Swab Samples.

Trachoma is the world-leading infectious cause of preventable blindness and is caused by the bacteria Chlamydia trachomatis. In developing countries, diagnosis is usually based on clinical evaluation. Serological-based tests are cheaper than molecular-based ones, but the latter are more sensitive and specific. The present study developed a new duplex qPCR which concomitantly detects the C. trachomatis cryptic plasmid and the human 18S rRNA gene, with an LOD95% for C. trachomatis DNA of 13.04 genome equivalents per reaction. The new qPCR was tested using 50 samples from an endemic area and 12 from a non-endemic area that were previously characterized using direct immunofluorescence assay (DFA) and clinical evaluation. Among the 50 endemic samples, 3 were found to be positive by clinical evaluation (6%), 18 were found to be positive by DFA (36%), and 48 were found to be positive by qPCR (96%). Next, the new duplex qPCR was validated using 50 samples previously characterized by qPCR. Validation was carried out on a benchtop instrument (ABI7500) or on a portable point-of-care instrument (Q3-Plus), showing 95% specificity and 100% sensitivity. The ubiquitous presence of C. trachomatis DNA in samples from the endemic region confirms that constant monitoring is of paramount importance for the effective measurement of the elimination of trachoma. The newly developed duplex qPCR presented in this study, along with its validation in a portable qPCR system, constitutes important tools toward achieving this goal.

Investigation of Factors Associated with Subclinical Infections of Giardia duodenalis and Cryptosporidium canis in Kennel-Housed Dogs (Canis lupus familiaris).

Giardia duodenalis and Cryptosporidium spp. are zoonotic protozoal pathogens, spread by a fecal-oral route, which can infect a wide range of hosts including but not limited to dogs and humans. Giardia was recently estimated to be present in 37% to 50% of kennel-housed dogs. Cryptosporidium infections in kennel-housed dogs have been reported in 7% to 21% of the population. The goal of this study was to define demographic factors and fecal scores associated with positive screening test cases of Giardia and Cryptosporidium in kennel-housed laboratory dogs in the state of Texas. Fecal samples were collected from 153 clinically normal laboratory dogs at an academic research facility and a local laboratory dog supplier. We used 3 diagnostic tests evaluated in parallel to determine test positivity to each organism: a human point-of-care coproantigen test, a direct immunofluorescent assay, and an in-house polymerase chain reaction. Dogs were significantly more likely to test positive for Giardia (45%) than Cryptosporidium (7%) (P < 0.01). Dogs that were 18 mo of age or younger had 3 times the odds (P = 0.009) of subclinical Giardia infection compared with older dogs. We found no significant relationship between age and Cryptosporidium prevalence. Dogs with hard feces (fecal score 1-2) at the time of screening had 0.34 times lower odds ( P = 0.049) of testing positive for Giardia than dogs with normal feces, but no statistically significant relationship was found between fecal score and Cryptosporidium -positive test status. With these findings, we demonstrated the value of considering age and fecal score when choosing which dogs to screen for subclinical Giardia. Additional studies with larger sample sizes should be conducted to determine the relationship between age and fecal score and subclinical Cryptosporidium infection.

A two-year monitoring of Cryptosporidium spp. oocysts and Giardia spp. cysts in freshwater and seawater: A complementary strategy for measuring sanitary patterns of recreational tropical coastal areas from Brazil

Primary contact recreation is an important transmission route of gastroenteric protozoa such as Cryptosporidium and Giardia. However, data on the environmental epidemiology of both protozoa in Brazilian coastal areas still need to be available. The main goal of this study was to perform environmental monitoring and quantify the concentration of Cryptosporidium oocysts and Giardia cysts on the main beaches of the state of Paraná, Brazil, as a complementary tool for measuring the sanitary quality of bathing waters for two years. A total of 144 water samples were harvested monthly for two years from Guaratuba, Caiobá and Matinhos beaches (72 samples of seawater and 72 of freshwater) to search for pathogenic protozoa. Protozoa were concentrated by the membrane filtration technique and visualized by direct immunofluorescence assay (IFA). All positive samples by IFA were submitted for DNA extraction for PCR analysis. Fecal indicator bacteria (FIB) were enumerated by conventional microbiological techniques. Both protozoa were detected on all beaches during the two-year monitoring period. At least one protozoa were detected in 58.3% (n = 14) months of sampling, totaling 24 positive samples. Guaratuba was the most contaminated beach followed by Caiobá and Matinhos. Giardia spp. was detected in 15, Cryptosporidium spp. in seven and both parasites in two samples. Giardia DNA was confirmed on two beaches, and Cryptosporidium was detected for the first time through molecular assays in a coastal area of Brazil. An essential impact of FIB was observed in the first year and a reduction in contamination rates in the second year of monitoring. This is the first study conducted in the country in different environments that composes coastal areas to monitor together parasitological and microbiological quality for extended periods. The monitoring of coastal waters can guide environmental agencies aiming to improve the sanitary quality of beaches and safeguard public health.

Identification of Cryptosporidium parvum and Blastocystis hominis subtype ST3 in Cholga mussel and treated sewage: Preliminary evidence of fecal contamination in harvesting area

Cryptosporidium parvum and Blastocystis hominis are foodborne parasites known for causing diarrhea. They accumulate in mussels grown on contaminated water bodies, due to the discharge of treated sewage from sewage treatment plants (STP). Despite this, some countries like Chile do not include these parasites in the control or monitoring of sewage water. The objective of this research was to evaluate the contamination of C. parvum. and B. hominis from treated sewage (disinfected by chlorination) and Cholga mussels in a touristic rural cove from the bay of Concepción. Cholga mussels from commercial stores and a treated sewage sample were analyzed. Cryptosporidium spp. was identified by Ziehl-Neelsen-Staining (ZNS) and C. parvum by direct-immunofluorescence assay (IFA) from ZNS-positive samples. Blastocystis hominis was identified by PCR using locus SSU rDNA. C. parvum and B. hominis subtype ST3 were found in 40% and 45% of Cholga mussel samples, respectively, and both parasites were identified in the treated sewage. Blastocystis hominis SSU rDNA gene alignment from Cholga mussels and treated sewage showed 89% of similarity, indicating that could be the same parasite in both samples. We describe the first evidence of possible contamination with these parasites from treated sewage to Cholga mussel suggesting an environmental contamination with high human risk. Based on these results, further studies will consider all the rural coves and STP from the bay to prevent possible contamination of these parasites.

Detection and molecular characterization of Cryptosporidium spp. in dairy calves in Lisbon and Tagus Valley, Portugal

Cryptosporidium is a protozoan parasite with worldwide distribution, infecting a wide range of hosts with some zoonotic species. Calves have been identified as one of the most common reservoirs of this parasite. However, little is known about the genetics of Cryptosporidium in calves in Portugal. This study aimed to molecularly characterize infections of Cryptosporidium in pre-weaned calves from the Lisbon and Tagus Valley (LTV) in Portugal. Fifty-two samples were collected from calves from eight dairy and two beef farms in LTV, Portugal. Cryptosporidium oocysts were detected by Modified Ziehl-Neelsen staining (MZN) and direct immunofluorescent assay (DFA). MZN and DFA revealed the presence of Cryptosporidium oocysts in 40.4% (21/52) and 67.3% (35/52) samples, respectively. Positive samples were analyzed by PCR-RFLP of the 18 s rRNA gene for species identification. DNA amplification of the 18S rRNA gene was successful for 88.6% (31/35) of samples. Cryptosporidium parvum was identified in 96.8% (30/31) of the samples, and from one sample Cryptosporidium bovis was identified. Cryptosporidium parvum positive samples were subtyped by sequencing the PCR product of a partial fragment of the 60 kDa glycoprotein (gp60) gene. Subtype analysis of the C. parvum isolates revealed that all isolates belonged to subtype family IIa. Four subtypes were recognized within this subtype family, including the hyper-transmissible IIaA15G2R1 subtype that is the most frequently reported worldwide (27/30), IIaA14G2R1 (1/30), IIaA16G2R1 (1/30) and IIaA19G2R1 (1/30). To our knowledge, this is the first report of C. bovis, and C. parvum subtypes IIaA14G2R1 and IIaA19G2R1 in cattle in LTV, Portugal. The presence of the zoonotic C. parvum subtype in this study suggests that pre-weaned calves are likely to be a significant reservoir of zoonotic C. parvum, highlighting the importance of animal-to-human infection transmission risk. Further molecular studies are required to better understand the epidemiology of cryptosporidiosis in Portugal.

Risk Factors for Pneumocystis jirovecii Pneumonia in Non-HIV Patients Hospitalized for COVID-19: A Case-Control Study.

Very few cases of Pneumocystis jirovecii pneumonia (PJP) have been reported in COVID-19 so far, and mostly in patients with concomitant HIV infection or in solid-organ transplant recipients. Despite COVID-19 being associated with lymphopenia and the use of steroids, there are no studies specifically aimed at investigating the risk factors for PJP in COVID-19. A retrospective case-control study was performed. We matched PJP cases with controls with a 1:2 ratio, based on age ± 10 years, solid-organ transplantation (SOT), hematological malignancies, and in the setting of PJP development (ICU vs. non-ICU). A direct immunofluorescence assay on bronchoalveolar lavage fluid was used to diagnose PJP. We enrolled 54 patients. Among 18 cases of PJP, 16 were diagnosed as "proven". Seven of the eighteen cases were immunocompromised, while the other patients had no previous immunological impairment. Patients with PJP had significantly lower median lymphocyte values (p = 0.033), longer COVID-19 duration (p = 0.014), a higher dose of steroid received (p = 0.026), higher CRP values (p = 0.005), and a lower SARS-CoV-2 vaccination rate than the controls (p = 0.029). Cumulative steroid dose is the independent risk factor for PJP development (OR = 1.004, 95%CI = 1-1.008, p = 0.042). PJP develops in COVID-19 patients regardless of immunosuppressive conditions and the severity of disease, and it is correlated to the corticosteroid dose received.

Intracellular presence and genetic relationship of Helicobacter pylori within neonates' fecal yeasts and their mothers' vaginal yeasts.

Helicobacter pylori are transmissible from person to person and among family members. Mother-to-child transmission is the main intrafamilial route of H. pylori transmission. However, how it transmits from mother to child is still being determined. Vaginal yeast often transmits to neonates during delivery. Therefore, H. pylori hosted in yeast might follow the same transmission route. This study aimed to detect intracellular H. pylori in vaginal and fecal yeasts isolates and explore the role of yeast in H. pylori transmission. Yeast was isolated from the mothers' vaginal discharge and neonates' feces and identified by internal transcribed spacer (ITS) sequencing. H. pylori 16S rRNA and antigen were detected in yeast isolates by polymerase chain reaction and direct immunofluorescence assay. Genetic relationships of Candida strains isolated from seven mothers and their corresponding neonates were determined by random amplified polymorphic DNA (RAPD) fingerprinting and ITS alignment. The Candida isolates from four mother-neonate pairs had identical RAPD patterns and highly homologous ITS sequences. The current study showed H. pylori could be sheltered within yeast colonizing the vagina, and fecal yeast from neonates is genetically related to the vaginal yeast from their mothers. Thus, vaginal yeast presents a potential reservoir of H. pylori and plays a vital role in the transmission from mother to neonate.

Pembrolizumab-induced erosive lichenoid reaction in a patient with metastatic lung adenocarcinoma: A case report

Introduction: Immune-related adverse events induced by immune checkpoint inhibitors are quite common. Cutaneous lichenoid immune-related adverse events are among the most frequent. However, oral lichenoid adverse reactions are extremely rare. We herein describe a patient who was treated with pembrolizumab for metastatic lung cancer and developed an erosive oral lichenoid reaction induced by immunotherapy. Case presentation: An 87-year-old man treated with pembrolizumab for metastatic lung adenocarcinoma (stage IVa based on AJCC 2018) developed multifocal erosions of the oral mucosa mainly located on the buccal mucosa, palate, and inner portion of the lips with multiple small, irregular, hyperkeratotic areas. Histopathological examination showed epithelial necrosis and a dense band-like layer of an inflammatory infiltrate of lymphocytes and histiocytes within the upper dermis. Direct immunofluorescence was negative for both IgG and C3. A diagnosis of erosive oral lichenoid reaction of the mucosa induced by immunotherapy was established. Given the severity of the condition, pembrolizumab treatment was withheld and concomitant topical and systemic steroids were started. After 1 month, the drug-related toxicity was ameliorated and immunotherapy was re-introduced. Discussion: Only one other case of pembrolizumab-induced erosive lichen planus of the oral mucosa has been described to date. Previously reported drug-induced lichenoid rashes were mainly localized on the skin. Clinically, the main differential diagnoses of lichenoid erosive lesions are bullous immune-related disorders and should be excluded. In our patient, histological examination combined with negative results of both direct immunofluorescence and enzyme-linked immunosorbent assays confirmed the diagnosis of erosive lichenoid drug reaction. Conclusion: Clinicians should be aware of lichenoid involvement of the oral mucosa because related pain and food intake difficulties may seriously compromise treatment compliance. Prompt treatment of oral drug-related reactions may prevent interruption of immunotherapy and improve patients’ quality of life.

Pemphigus vulgaris mimicking Steven-Johnson syndrome/toxic epidermal necrolysis: report of an unusual case.

Pemphigus vulgaris (PV) is an autoimmune blistering disease affecting the skin and mucosa. It clinically presents as painful erosions, mainly in the oral cavity, and flaccid blisters and erosions on the skin. Steven-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) is a severe mucocutaneous drug hypersensitivity reaction characterized by painful, dusky, erythematous eruptions that often progress to blisters, erosions, and extensive epidermal detachment accompanied by systemic symptoms. Herein, we report the case of a 48-year-old man who presented with extensive skin and mucosal lesions following the ingestion of an unknown medication. The patient's symptoms clinically mimicked SJS, and accordingly, a preliminary diagnosis of SJS/TEN was made. However, clinical investigation of skin biopsy and direct immunofluorescence assay results indicated PV, and a final diagnosis of PV was established.

Epidemiological Characteristics of Respiratory Syncytial Virus Infection Among Hospitalized Children With Acute Respiratory Tract Infections From 2014 to 2022 in a Hospital in Hubei Province, China: Longitudinal Surveillance Study.

Longitudinal studies characterizing the epidemic trend of respiratory syncytial virus (RSV) in Hubei Province are scarce. We aimed to depict the dynamics of the RSV epidemic among hospitalized children with acute respiratory tract infections (ARTIs) during 2014 to 2022 in the Maternal and Child Health Hospital of Hubei Province and investigate the influence of the 2-child policy and the COVID-19 pandemic on RSV prevalence. The medical records and testing results of hospitalized children with ARTI from January 2014 to June 2022 were extracted. Nasopharyngeal samples were tested with direct immunofluorescence assay. Detection rates of RSV were categorized according to the diagnosis of patients: (1) overall, (2) upper respiratory tract infection (URTI), and (3) lower respiratory tract infection (LRTI). Poisson regression models were used to investigate the association between RSV detection rate and age, gender, or diagnosis. The detection rates of RSV before and after the implementation of the universal 2-child policy were compared using a Poisson regression model. Multiple comparisons of RSV detection rates were conducted among 3 stages of the COVID-19 pandemic using chi-square tests. Seasonal autoregressive integrated moving average was performed to predict RSV behaviors from February 2020 to June 2020 under the assumption of a non-COVID-19 scenario. Among 75,128 hospitalized children with ARTI, 11.1% (8336/75,128) were RSV-positive. Children aged <1 year had higher detection rates than older children (4204/26,498, 15.9% vs 74/5504, 1.3%; P<.001), and children with LRTI had higher detection rates than children with URTI (7733/53,145, 14.6% vs 603/21,983, 2.7%; P<.001). Among all the children, a clear seasonal pattern of the RSV epidemic was observed before 2021. Most of the highest detection rates were concentrated between December and February. The yearly detection rate of RSV remained at a relatively low level (about 8%) from 2014 to 2017, then increased to 12% and above from 2018. The highest monthly detection rate was in December 2018 (539/1493, 36.1%), and the highest yearly rate was in 2021 (1372/9328, 14.7%). There was a moderate increase in the RSV detection rate after the 2-child policy was implemented (before: 860/10,446, 8.2% vs after: 4920/43,916, 11.2%; P<.001). The largest increase, by 5.83%, occurred in children aged <1 year. The RSV epidemic level decreased sharply in the short term after the COVID-19 outbreak (detection rate before: 1600/17,010, 9.4% vs after: 32/1135, 2.8%; P<.001). The largest decrease, by 12.0%, occurred in children aged <1 year, but a rebounding epidemic occurred after 2020 (680/5744, 11.8%; P<.001). Children have been experiencing increased prevalence of RSV since 2018 based on surveillance from a hospital in Hubei Province with a large sample size. The 2-child policy might have increased the RSV prevalence, and the COVID-19 epidemic had a temporary inhibitory effect on RSV transmission. Vaccines against RSV are urgently needed.

Epidemiological characteristics of respiratory viruses in hospitalized children during the COVID-19 pandemic in southwestern China.

Multinational studies have reported that the implementation of nonpharmaceutical interventions (NPIs) to control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission coincided with the decline of other respiratory viruses, such as influenza viruses and respiratory syncytial virus. To investigate the prevalence of common respiratory viruses during the coronavirus disease 2019 (COVID-19) pandemic. Respiratory specimens of children with lower respiratory tract infections (LRTIs) hospitalized at the Children's Hospital of Chongqing Medical University from January 1, 2018 to December 31, 2021 were collected. Seven common pathogens, including respiratory syncytial virus (RSV), adenovirus (ADV), influenza virus A and B (Flu A, Flu B), and parainfluenza virus types 1-3 (PIV1-3), were detected by a multiplex direct immunofluorescence assay (DFA). Demographic data and laboratory test results were analyzed. 1) A total of 31,113 children with LRTIs were enrolled, including 8141 in 2018, 8681 in 2019, 6252 in 2020, and 8059 in 2021.The overall detection rates decreased in 2020 and 2021 (P < 0.001). The detection rates of RSV, ADV, Flu A, PIV-1, and PIV-3 decreased when NPIs were active from February to August 2020, with Flu A decreasing most predominantly, from 2.7% to 0.3% (P < 0.05). The detection rates of RSV and PIV-1 resurged and even surpassed the historical level of 2018-2019, while Flu A continued decreasing when NPIs were lifted (P < 0.05). 2) Seasonal patterns of Flu A completely disappeared in 2020 and 2021. The Flu B epidemic was observed until October 2021 after a long period of low detection in 2020. RSV decreased sharply after January 2020 and stayed in a nearly dormant state during the next seven months. Nevertheless, the detection rates of RSV were abnormally higher than 10% in the summer of 2021. PIV-3 decreased significantly after the COVID-19 pandemic; however, it atypically surged from August to November 2020. The NPIs implemented during the COVID-19 pandemic affected the prevalence and seasonal patterns of certain viruses such as RSV, PIV-3, and influenza viruses. We recommend continuous surveillance of the epidemiological and evolutionary dynamics of multiple respiratory pathogens, especially when NPIs are no longer necessary.

Direct immunofluorescence cannot be used solely to differentiate among oral lichen planus, oral lichenoid lesion, and oral epithelial dysplasia

Background/purposeSome red and white lesions may have similar manifestations, making them difficult to be diagnosed. A direct immunofluorescence (DIF) assay can assist in making a final diagnosis of oral lichen planus (OLP). The aim of this study was to evaluate and compare the DIF profile in patients who had the clinical presentations of OLP and were histopathologically diagnosed with OLP, OLL (oral lichenoid lesion), or OED (oral epithelial dysplasia). Materials and methodsThe data were obtained from the medical records of 136 patients with the clinical presentations of OLP. Demographic information, histopathological diagnosis, malignant transformation, and DIF results were collected and analyzed. ResultsIn this study, 117 patients (86.0%) were DIF-positive, while 19 patients (14.0%) were DIF-negative. The highest DIF-positivity rate was in the OLP group (88.9%) followed by the OLL (83.7%), and the OED groups (81%). There were no significant differences in DIF-positivity rate, type of immunoreactants, location, or interpretation among these groups. Shaggy fibrinogen at the basement membrane zone (BMZ) was the most common DIF pattern in all groups. ConclusionThe DIF assay alone cannot be regarded as sufficient evidence for OLP, OLL, and OED differentiation. A histopathological examination is required to determine the presence of epithelial dysplasia or malignancy. To diagnose dysplastic lesions with the clinical manifestations of OLP, careful clinicopathologic correlation is mandatory. Due to the lack of scientific evidence to identify the primary pathology and the ongoing malignancy risk of epithelial dysplasia, meticulous long-term follow-up plays a crucial role in patient management.

Comparison of the clinical features of human bocavirus and metapneumovirus lower respiratory tract infections in hospitalized children in Suzhou, China.

We compared the clinical data of hospitalized children with lower respiratory tract infections caused by human bocavirus (HBoV) and human metapneumovirus (hMPV). In total, 8,430 children admitted to the Department of Respiration, Children's Hospital of Soochow University for lower respiratory tract infections from January 2017 to October 2021 were enrolled. Seven common respiratory viruses, including respiratory syncytial virus, influenza virus A, influenza virus B, parainfluenza virus (PIV) I, PIV II, PIV III, and adenovirus, were detected by direct immunofluorescence assay, whereas human rhinovirus and hMPV were detected by reverse transcription-polymerase chain reaction. Mycoplasma pneumoniae (MP) and HBoV were detected by real-time fluorescence quantitative polymerase chain reaction. Bacteria was detected in blood, nasopharyngeal secretion, bronchoalveolar lavage specimen or pleural fluid by culture. In parallel, MP was detected by enzyme-linked immunosorbent assay. In addition, we performed metagenomic testing of alveolar lavage fluid from some of the patients in our study. The detection rate of HBoV was 6.62% (558/8430), whereas that of hMPV was 2.24% (189/ 8430). The detection rate of HBoV was significantly higher in children aged 1 to <3 years than in other age groups, but there were no significant differences in positivity rates for hMPV by age. Before 2020, the incidence of HBoV infection peaked in summer and autumn, whereas that of hMPV peaked in spring. The epidemiology of both HBoV and hMPV has changed because of the impact of the novel coronavirus. Among the positive cases, the HBoV mixed infection rate was 51.6%, which was similar to that for hMPV mixed infection (44.4%). Comparing clinical characteristics between HBoV and hMPV single infection, the median age of children was 17 months in the HBoV group and 11 months in the hMPV group. In the HBoV single infection group, 31 patients (11.5%) had pulse oxygen saturation of less than 92% on admission, 47 (17.4%) had shortness of breath, and 26 (9.6%) presented with dyspnea. Meanwhile, four patients (3.8%) in the hMPV single infection group had pulse oxygen saturation of less than 92% on admission, eight (7.6%) displayed shortness of breath, and three (2.9%) had dyspnea. The proportion of patients requiring mechanical ventilation and the rate of PICU admission were higher in the HBoV group than in the hMPV group. The prevalence of HBoV infection is higher than that of hMPV infection in children with lower respiratory tract infection in Suzhou, and HBoV is more likely to cause severe infection than hMPV. Public health interventions for COVID-19 outbreaks have affected the prevalence of HBoV and hMPV.

Nonpharmaceutical interventions for COVID-19 disrupt the dynamic balance between influenza A virus and human immunity.

During the COVID-19 epidemic, nonpharmaceutical interventions (NPIs) blocked the transmission route of respiratory diseases. This study aimed to investigate the impact of NPIs on the influenza A virus (IAV) outbreak. The present study enrolled all children with respiratory tract infections who came to the Children's Hospital of Zhejiang University between January 2019 and July 2022. A direct immunofluorescence assay kit detected IAV. Virus isolation and Sanger sequencing were performed. From June to July 2022, in Hangzhou, China, the positive rate of IAV infection in children has increased rapidly, reaching 30.41%, and children over 3 years old are the main infected population, accounting for 75% of the total number of infected children. Influenza A (H3N2) viruses are representative strains during this period. In this outbreak, H3N2 was isolated from a cluster of its own and is highly homologous with A/South_Dakota/22/2022 (2021-2022 Northern Hemisphere). Between isolated influenza A (H3N2) viruses and A/South_Dakota/22/2022, the nucleotide homology of the HA gene ranged from 97.3% to 97.5%; the amino acid homology was 97%-97.2%, and the genetic distance of nucleotides ranged from 0.05 to 0.052. Compared with A/South_Dakota/22/2022, the isolated H3N2 showed S156H, N159Y, I160T, D186S, S198P, I48T, S53D, and K171N mutations. There was no variation in 13 key amino acid sites associated with neuraminidase inhibitor resistance in NA protein. Long-term NPIs have significantly affected the evolution and transmission of the influenza virus and human immunity, breaking the dynamic balance between the IAV and human immunity.

Nasopharyngeal aspirates in children with severe community-acquired pneumonia collected within 3 days before bronchoscopy can partially reflect the pathogens in bronchoalveolar lavage fluids

BackgroundThere is little evidence about consistency between nasopharyngeal and pulmonary pathogens in children with severe pneumonia. This study aims to compare the difference of pathogens between nasopharyngeal aspirates (NPAs) collected before bronchoscopy and bronchoalveolar lavage fluids (BALFs) in children with severe community-acquired pneumonia (SCAP).MethodsNPAs and BALFs were collected form pediatric SCAP cases hospitalized from January 2018 to March 2019. NPAs were colleced within 3 days before bronchoscopy. Samples were detected by direct immunofluorescence assay (DFA) for seven respiratory viruses and by routine bacterial culture in the clinical microbiology laboratory. Respiratory syncytial virus (RSV), Adenovirus (ADV), Influenza virus types A, B (IV-A and IV-B), Parainfluenza virus 1–3 (PIV1-3) were detected with a commercial assay. The virological and bacteriological detention results of NPAs were compared with the results of BALFs.ResultsIn total 204 cases with mean age of 3.4 ± 2.8 years (IQR, 1 month-14 years) were included in the study. Both NPA and BALF were collected from those cases. The positive rates of pathogen in NPAs and BALFs were 25.0% (51/204) and 36.7% (75/204), respectively (x2 = 6.614, P = 0.010). Respiratory viruses were found in 16.1% (33/204) from NPAs and 32.3% (66/204) from BALFs (x2 = 14.524, P < 0.001). RSV and ADV were the two most frequent detected viruses in NPAs and BALFs. High consistentcy of pathogens between NPAs and BALFs was observed, and 96.9% (32/33) viruses detected in NPAs were also found in BALFs. While bacteria were isolated from 12.7% (26/204) and 10.7% (22/204) of the two kinds of samples, respectively (x2 = 0.378, P = 0.539). In addition, Haemophilus influenzae (HI) was the dominant germ in both samples.ConclusionThe DFA method used to detect seven respiratory viruses from NPAs collected within 3 days before bronchoscopy can partially reflect the pathogens in the lungs in children with SCAP.

Diagnostic Role of Direct Immunofluorescence Assay in Determining The Etiology of Erythroderma: Experience in a Tertiary Referral Hospital.

Erythroderma is a life-threatening dermatologic emergency which is characterized by diffuse erythema and exfoliation affecting more than 90% of the body surface area. Most common cutaneous diseases associated with erythroderma are systemic contact dermatitis, psoriasis, drug eruption and atopic dermatitis. Clinical-pathological correlation is used to determine the underlying disease. In addition, direct immunofluorescence (DIF) may provide significant clues for etiology of erythroderma especially in the case of autoimmune bullous skin diseases (ABSDs). In our study, we aimed to analyze the demographic data, clinical pre-diagnoses, final diagnosis, histopathological and DIF examination findings, accompanying systemic signs and laboratory abnormalities of erythrodermic patients. We conducted a retrospective study of 31 erythroderma patients in a referral hospital between 2014 and 2021. Cutaneous biopsies were taken from all patients for H&E and DIF examination. Average age was 54.6 ± 23 years, 48.4% of the patients were female (N = 15) whereas 51.6 % of the patients were male (N = 16). Average time between the onset of rash and biopsy was 18.8 days. DIF analysis showed immune deposits in 19.4% (N = 6) of the patients; whereas no immune deposits were detected in 80.6% (N = 25) of the patients. The most frequent final diagnosis was adverse cutaneous drug eruption followed by ABSDs. Our findings suggest that DIF may be used in conjunction with clinical-pathologic and clinical findings to reveal the associated skin diseases in erythrodermic patients. Erythrodermic patients presenting with clinical findings of ABSD should be considered for DIF examination.

Development of a diagnostic assay by three-tube multiplex real-time PCR for simultaneous detection of nine microorganisms causing acute respiratory infections

Acute respiratory infections are widespread in vulnerable populations of all ages and are characterized by a variety of symptoms. The underlying infection can be caused by a multitude of microorganisms, including viruses and bacteria. Early detection of respiratory infections through rapid pathogen screening is vital in averting infectious respiratory disease epidemics. This study utilized a multiplex real-time PCR system to develop a three-tube reverse transcription-PCR (RT-PCR) assay, enabling simultaneously detect nine respiratory pathogens, including: influenza A and B, adenovirus, respiratory syncytial virus (RSV), Streptococcus pneumoniae, Legionella pneumophila, Haemophilus influenzae, Chlamydia pneumoniae, and Mycoplasma pneumoniae. This technique utilizes a one-step assay, with specifically designed TaqMan primer–probe sets combined in the same tube. This assay provided rapid and simplified detection of the nine prevalent pathogens, as well as increased sensitivity and reduced cross-contamination. This assay was evaluated using 25 related viral/bacterial strains as positive references, the other 25 irrelevant strains as negative controls, and clinical specimens from 179 patients. All positive strains were detected with no amplification of the non-target microorganism mixtures and the assay’s detection limits ranged between 250–500 copies/ml (1.25–2.5 copies/reaction). A total of 167 (93.3%) samples tested positive for at least one of the pathogens identified; 109 of these samples were from patients confirmed to have RSV infections. The diagnostic accuracy of our assay was further confirmed by matching results from classical direct immunofluorescence assay and nucleotide sequencing. These data demonstrate the innovative multiplex real-time PCR assay as a promising alternative to the current approaches used for early screening of acute respiratory infections.

Imaging Leishmania major Antigens in Experimentally Infected Macrophages and Dermal Scrapings from Cutaneous Leishmaniasis Lesions in Tunisia.

Leishmania major cutaneous leishmaniasis (CL) lesions are characterized by an intense process of parasite destruction and antigen processing that could limit microscopic amastigote detection. The aim of our study was to develop a direct immunofluorescence (DIF) assay for in situ visualization of L. major antigens and access its reliability in the routine diagnosis of CL. The developed DIF assay used IgG polyclonal antibodies produced in rabbits by intravenous injections of live L. major metacyclic promastigotes chemically coupled to fluorescein isothiocyanate. Applied to L. major infected RAW macrophages, corresponding macrophage-derived amastigotes and dermal scrapings from CL lesions, the immunofluorescence assay stained specifically Leishmania amastigotes and showed a diffuse Leishmania antigen deposit into cytoplasm of phagocytic cells. Reliability of DIF in CL diagnosis was assessed on 101 methanol-fixed dermal smears from 59 positive and 42 negative CL lesions diagnosed by direct microscopy and/or kDNA real-time PCR. Sensitivity and specificity of DIF was 98.3% and 100%, respectively, being more sensitive than microscopy (p < 0.001) and as sensitive as ITS1-PCR. ITS1-PCR-RFLP allowed Leishmania species identification in 56 out of the 58 DIF-positive smears, identifying 52 L. major, two L. infantum and two L. tropica cases, which indicates antigenic cross-reactivity between Leishmania species.