Molecular and microscopy detection of Pneumocystis jirovecii in hospitalized patients during the COVID-19 pandemic.

Introduction: Early detection of Pneumocystis jirovecii as an opportunistic pathogen that may endanger predisposed persons, including COVID-19 patients, may help to choose the optimal management. Methods: In this study, 585, including 530 COVID-19 patients, with clinical and radiological evidence of respiratory diseases, were investigated for P. jirovecii screening. Clinical specimens were examined by direct microscopy and PCR, and randomly selected positive PCR products were confirmed through DNA sequence analysis. Results: Thirty-one (5.3%) samples were positive in P. jirovecii-specific nested-PCR, while by direct microscopic tests, Pneumocystis was observed in 22 (3.76%) samples. Males (61.7%) and patients over 50 years old (75.6%) were more commonly affected than others, and malaise and fatigue (84%), and wheezing (75%) were the most common symptoms, followed by fever (40.48%) and dyspnea (39.51%). Among the Pneumocystis-positive patients, three cases had coinfection with Aspergillus fumigatus, A. flavus, and A. niger (each n = 1), as documented by direct microscopy, culture, and species identification by PCR-sequencing. Conclusion: Pneumocystis pneumonia is still a diagnostic challenge; therefore, additional large-scale studies are needed to clarify the epidemiology of the disease in immunocompromised or COVID-19 patients.

Development of a duplex real-time PCR assay for simultaneous detection and differentiation of Theileria equi and Babesia caballi.

Equine Piroplasmosis (EP) is a tick-borne disease caused by three apicomplexan protozoan parasites, Theileria equi (T. equi), Babesia caballi (B. caballi) and T. haneyi, which can cause similar clinical symptoms. There are five known 18S rRNA genotypes of T. equi group (including T. haneyi) and three of B. caballi. Real-time PCR methods for detecting EP based on 18S rRNA analysis have been developed, but these methods cannot detect all genotypes of EP in China, especially genotype A of T. equi. In this study, a duplex real-time PCR detection method was developed for the simultaneous detection and differentiation of T. equi and B. caballi. The primers and probes for this duplex real-time PCR assay were designed based on the conserved 18S rRNA gene sequences of all genotypes of T. equi and B. caballi including Chinese strain. Double-quenched probes were used in this method, which provide less background and more signal to decrease the number of false positives relative to single-quenched probes. The newly developed real-time PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. The real-time PCR assays were further validated by comparison with a nested PCR assay and a previous developed real-time PCR for EP and sequencing results in the analysis of 506 clinical samples collected from 2019 to 2020 in eleven provinces and regions of China. Based on clinical performance, the agreements between the duplex real-time PCR assay and the nPCR assay or the previous developed real-time PCR assay were 92.5% (T. equi) and 99.4% (B. caballi) or 87.4% (T. equi) and 97.2% (B. caballi). The detection results showed that the positivity rate of T. equi was 43.87% (222/506) (10 genotype A, 1 genotype B, 4 genotype C, 207 genotype E), while that of B. caballi was 5.10% (26/506) (26 genotype A), and the rate of T. equi and B. caballi co-infection was 2.40% (12/506). The established method could contribute to the accurate diagnosis, pathogenic surveillance and epidemiological investigation of T. equi and B. caballi infections in horses.

Establishment and application of semi-nest RT-PCR for detection of Transmissible gastroenteritis virus

In order to establish an efficient, sensitive and specific semi-nest RT-PCR method for the detection of Transmissible gastroenteritis virus (TGEV), three specific primers were designed according to the N gene published by GenBank, the reaction system was established and optimized, and specificity and sensitivity were detected. The results showed that the method could successfully amplify the bands of 483 bp and 338 bp, and had good specificity to TGEV, there is no cross reaction with PEDV, PRov, PBov and PDCov, and the lowest sensitivity was 1.86 x 10-1 pg/L. The semi-nest RT-PCR shown the positive rate was 36% in 50 samples of pig diarrhea, which was higher than that of common RT-PCR, and then the positive samples coincidence rate was 100%. This semi-nest RT-PCR method has high sensitivity and specificity, and can accurately diagnose TGEV infection, which provides an effective method for clinical detection and epidemiological investigation of TGEV.

Molecular Detection of Parvovirus in Captive Siberian Tigers and Lions in Northeastern China From 2019 to 2021.

The fact that wild felines are carriers of pernicious infectious viruses should be a major concern due to the potential cross-species transmission between the felines and human or domestic animals. However, studies on the virus in the captive wild felines, especially in tigers, are thin on the ground. In this study, we screened four infectious viruses, namely, feline parvovirus (FPV), feline coronavirus (FCoV), canine distemper virus (CDV), and influenza A virus (IAV), in the blood samples of 285 captive Siberian tigers (Panthera tigris altaica) and in the spleen samples of two deceased lions (Panthera leo), which were collected from 2019 to 2021 in three Siberian Tiger Parks from the northeast of China. Nucleic acids isolated from the blood samples collected from tigers and the spleen samples collected from two deceased lions were positive for FPV by PCR, and the positive rate was 4.6% (13/285) in tigers. Furthermore, the VP2 gene of FPV was amplified by nested PCR, and the sequences of the VP2 gene from these six FPV positive strains shared 98.3-99.9% homology with the reference. The key amino acid sites of VP2 protein were consistent with that of FPV reference strains. Phylogenetic analysis based on the VP2 gene showed that in this study, FPV-positive strains were grouped within the FPV clade and closely related to the Asian strains clade. The results of this study showed that FPV circulated in the captive Siberian tigers and lions in northeastern China and provided valuable information for the study of FPV epidemiology in wild felines. Therefore, we suggest that regular antibody monitoring and booster immunization for tigers should be performed.

Development of Nested PCR for SARS-CoV-2 Detection and Its Application for Diagnosis of Active Infection in Cats.

SARS-CoV-2 emerged in 2019 and found diagnostic laboratories unprepared worldwide. To meet the need for timely and accurate virus detection, laboratories used rapid Ag tests and PCR kits based on costly multi-channel real-time techniques. This study aimed to develop a conventional nested PCR based on the SARS-CoV-2 N gene, validate it against some approved assays, and apply it to samples from six cats with respiratory symptoms obtained in early 2020 during the first COVID-19 wave in humans in Bulgaria. The nested PCR technique showed 100% sensitivity and specificity; it could detect extracted SARS-CoV-2 RNA at concentrations as low as 0.015 ng/µL. The results identified the six tested cat samples as positive. Sequence analysis performed in two of them confirmed this. The presented technique is reliable, easy to implement and inexpensive, and can be successful in strategies for the prevention and control of SARS-CoV-2 in humans, cats and other susceptible species.

Field Evaluation of a Hemozoin-Based Malaria Diagnostic Device in Puerto Lempira, Honduras.

The diagnosis of malaria in Honduras is based mainly on microscopic observation of the parasite in thick smears or the detection of parasite antigens through rapid diagnostic tests when microscopy is not available. The specific treatment of the disease depends exclusively on the positive result of one of these tests. Given the low sensitivity of conventional methods, new diagnostic approaches are needed. This study evaluates the in-field performance of a device (Gazelle™) based on the detection of hemozoin. This was a double-blind study evaluating symptomatic individuals with suspected malaria in the department of Gracias a Dios, Honduras, using blood samples collected from 2021 to 2022. The diagnostic performance of Gazelle™ was compared with microscopy and nested 18ssr PCR as references. The sensitivity and specificity of Gazelle™ were 59.7% and 98.6%, respectively, while microscopy had a sensitivity of 64.9% and a specificity of 100%. The kappa index between microscopy and Gazelle™ was 0.9216 using microscopy as a reference. Both methods show similar effectiveness and predictive values. No statistical differences were observed between the results of the Gazelle™ compared to light microscopy (p = 0.6831). The turnaround time was shorter for Gazelle™ than for microscopy, but the cost per sample was slightly higher for Gazelle™. Gazelle™ showed more false-negative cases when infections were caused by Plasmodium falciparum compared to P. vivax. Conclusions: The sensitivity and specificity of Gazelle™ are comparable to microscopy. The simplicity and ease of use of the Gazelle™, the ability to run on batteries, and the immediacy of its results make it a valuable tool for malaria detection in the field. However, further development is required to differentiate Plasmodium species, especially in those regions requiring differentiated treatment.

High-resolution melting analysis after nested PCR for the detection of SARS-CoV-2 spike protein G339D and D796Y variations.

High-resolution melting (HRM) analysis was performed to detect G339D and D796Y variations in the SARS-CoV-2 Omicron variant spike protein. We employed two-step PCR consisting of the first RT-PCR and the second nested PCR to prepare the amplicon for HRM analysis. The melting temperatures (Tm) of the amplicon from the cDNA of the Omicron variant receptor binding domain (RBD) were 73.1 °C (G339D variation) and 75.1 °C (D796Y variation), respectively. These Tm values were clearly distinct from those of SARS-CoV-2 isolate Wuhan-Hu-1. HRM analysis after the two-step PCR was conducted on Omicron variant-positive specimens. The HRM curve and Tm value obtained with the Omicron variant-positive specimen were coincident with those of the amplicon from cDNA of the Omicron variant RBD. Our study demonstrates the utility of HRM analysis after two-step PCR for the detection of mutations in SARS-CoV-2 gene.

Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater.

Considering the lack of effective treatments against COVID-19, wastewater-based epidemiology (WBE) is emerging as a cost-effective approach for real-time population-wide SARS-CoV-2 monitoring. Here, we report novel molecular assays for sensitive detection and mutational/variant analysis of SARS-CoV-2 in wastewater. Highly stable regions of SARS-CoV-2 RNA were identified by RNA stability analysis and targeted for the development of novel nested PCR assays. Targeted DNA sequencing (DNA-seq) was applied for the analysis and quantification of SARS-CoV-2 mutations/variants, following hexamers-based reverse transcription and nested PCR-based amplification of targeted regions. Three-dimensional (3D) structure models were generated to examine the predicted structural modification caused by genomic variants. WBE of SARS-CoV-2 revealed to be assay dependent, and significantly improved sensitivity achieved by assay combination (94%) vs. single-assay screening (30%-60%). Targeted DNA-seq allowed the quantification of SARS-CoV-2 mutations/variants in wastewater, which agreed with COVID-19 patients' sequencing data. A mutational analysis indicated the prevalence of D614G (S) and P323L (RdRP) variants, as well as of the Β.1.1.7/alpha variant of concern, in agreement with the frequency of Β.1.1.7/alpha variant in clinical samples of the same period of the third pandemic wave at the national level. Our assays provide an innovative cost-effective platform for real-time monitoring and early-identification of SARS-CoV-2 variants at community/population levels.