Laboratory Infection Research Articles

Reciprocal hybrids of Atlantic salmon (Salmo salar) x brown trout (S. trutta) confirm a heterotic response to experimentally induced amoebic gill disease (AGD)

Amoebic gill disease (AGD) affects global marine aquaculture production of Atlantic salmon, requiring costly reiterative treatment and negatively impacting fish performance and welfare. Although there has been gradual and ongoing progress in genetic selection to improve host resistance to the causative agent (Neoparamoeba perurans) there has been little focus on the response of interspecific hybrids to AGD challenge. Previous research has demonstrated that Atlantic salmon x brown trout hybrids had reduced requirement for freshwater bathing (based on gross gill signs) during chronic natural AGD exposure, yet there has been no histological or molecular evidence confirming differential host response or parasite abundance. In this study we exposed Atlantic salmon (SS), brown trout (TT) and their reciprocal interspecific hybrids (TS and ST) to a single laboratory challenge (∼700 amoebae.l−1 for 18 days) and compared the resultant infection and disease signs characteristic of AGD. Gill score was significantly higher in SS than in TT and hybrids. Copy number quantification of amoebae on gills was higher in salmon than in hybrids or trout but did not differ between hybrids and trout. Gill histopathology confirmed both an increased amoebae abundance and higher expression of host tissue reaction in SS with significantly higher percentages of lesions harboring hyperplastic lesions. Mid-parent and best-parent heterosis of resistance to N. perurans and host response (gill lesions) was confirmed but did not differ between hybrid crosses. Although limited to a single laboratory infection event, our results support previously reported observations (from natural infection/reinfection) of reduced AGD signs in trout and interspecies hybrids. We conclude that interspecies hybrids have potential to improve our understanding of salmonid resistance to amoebic branchitis and support future gene-based research toward fish disease management.

Detection of Common Bacterial Pathogen in Hospital and Lab Settings and Their Anti-microbial Susceptibility Pattern in Various Medical Laboratories in Shendi Town, Sudan

Background: Laboratory infections can be classified as occupational and nosocomial infections. Laboratory-related infections are generally recognized as a potential risk for clinical laboratory workers. Some bacteria can survive longer on dry surfaces and more on wet surfaces that can infect others and also environments.
 Objective: To detect common bacterial pathogens in various medical laboratories in Shendi City.
 Materials and Methods: A cross-sectional analytical study was conducted in Shendi City (Sudan) from August to December 2021. This study included 17 laboratories and 50 samples collected by wet exchange from various locations including laboratory surfaces, microscopes, centrifuges, CBC devices, staining racks, and CBC devices.
 Results: This study included Staphylococcus aureus (No=11) (22%), Staphylococcus epidermidis (No=10) (20%), Escherichia coli (No=1) (2%), Klebsiella pneumonia (No=9) (18%), and Pseudomonas aeruginosa (No=2) (4%). Significant growth of pathogenic bacteria was recoded. Among all the organisms isolated, there was moderate resistance to antibiotics, some bacteria were very resistant, others were resistant, and some organisms were resistant to some antibiotic they were highly sensitive to the substance and resistant to other antibacterial agents. Bacterial isolates (39.4%) were resistant to Amoclane, (No=12) (36.4%) were resistant to gentamicin, and (No=11) (33.3%) were resistant to Ciprofloxacin and Imipenem.
 Conclusions: Contamination with pathogens was found on laboratory surfaces and equipment’s (approximately 66% of exchanged items contained pathogens), and dry surfaces may use these organisms as a source of laboratory infection.

BIOLOGICAL SAFETY AND BIOSECURITY SYSTEM IN RIBSP RESEARCH INSTITUTE FOR BIOLOGICAL SAFETY PROBLEMS

This article reflects the key aspects of the biological safety system and biological protection measures such as: biological risk assessment, engineering and technical means, personnel health control system, disinfection and disposal of solid and liquid waste, disinfection, disinfection and deratization, in industrial, scientific and experimental laboratories, insulators (vivarium’s) and facilities Research Institute for Biosafety Safety Problems in order to comply with the requirements of biosafety and biosecurity recommended by WHO, requirements of Sanitary rules and regulatory legal acts in the field of veterinary medicine. Search for information in Internet resources, for analysis, we used keywords in Russian and English. As a result, foreign and domestic literature sources on compliance with biological safety requirements and biological protection measures were analyzed.Analysis of literature sources has shown that failure to comply with these requirements and measures will lead to deliberate theft, leakage of strains of dangerous infectious diseases and failure to properly perform standard operating procedures, to an accident situation, laboratory infection of personnel. The measures taken to comply with the requirements of biosafety and biosecurity in the RIBSP significantly reduce the threat of infection of a person, the threat of the spread, penetration or theft of dangerous microorganisms.

The Effect of Virus-Specific Vaccination on Laboratory Infection Markers of Children with Rotavirus-Associated Acute Gastroenteritis

Rotavirus (RV) is one of the most common and important causes of acute gastroenteritis (AGE) in newborns and children worldwide. The aim of this study was to evaluate the effect of the RV vaccine on the natural history of RV infections using the neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and systemic immune inflammatory index (SII) as hematological indexes, clinical findings, and hospitalization. Children aged 1 month to 5 years who were diagnosed with RV AGE between January 2015 and January 2022 were screened, and 630 patients were included in the study. The SII was calculated by the following formula: neutrophil × platelet/lymphocyte. Fever and hospitalization were significantly higher and breastfeeding was significantly lower in the RV-unvaccinated group than in the RV-vaccinated group. The NLR, PLR, SII, and CRP were significantly higher in the RV-unvaccinated group (p < 0.05). The NLR, PLR, and SII were significantly higher both in the non-breastfed group than in the breastfed group and in the hospitalized group than in the not hospitalized group (p < 0.05). CRP was not significantly different in either the hospitalization group or the breastfeeding group (p > 0.05). SII and PLR were significantly lower in the RV-vaccinated group than in the RV-unvaccinated group in both the breastfed and non-breastfed subgroups. For NLR and CRP, while there was no significant difference according to RV vaccination status in the breastfed group, there was a significant difference in the non-breastfed group (p value: <0.001; <0.001). Despite the low level of vaccine coverage, the introduction of RV vaccination had a positive impact on the incidence of RV-positive AGE and related hospitalizations in children. These results showed that breastfed and vaccinated children were less prone to inflammation because their NLR, PLR, and SII ratios were lower. The vaccine does not prevent the disease 100%. However, it can prevent severe disease with exsiccation or death.

Predicting perioperative complications for partial versus radical nephrectomy in T1b-T2 renal cell carcinoma.

737 Background: The standard of care for larger localized RCC lesions is radical (RN) or partial (PN) nephrectomy. RN is increasingly utilized to maximize oncologic benefit in complex tumors. Although PN is a more technically complex procedure, its nephron-sparing nature confers lasting renal and cardiovascular benefits. We utilized the National Surgical Quality Improvement Program (NSQIP) database to elucidate predictors of perioperative morbidity in T1b-T2 RCC patients. Methods: Using the NSQIP Nephrectomy-Targeted PUF, 2,094 patients undergoing nephrectomy between 2019-2020 for localized T1b-T2 RCC were identified. Variables of interest included surgical procedure and approach, tumor stage, demographics, pre-operative laboratory values, comorbidities, infection and venothromboembolism (VTE) prophylaxis techniques, peri-operative complications, operative time, length of hospital stay, 30-day reoperations, and 30-day readmissions. Chi square test was used to analyze univariate associations between certain comorbidities and complications. Multivariate regression analysis was utilized to compare complication rates between PN and RN after adjusting for baseline characteristics and surgical approach. p&lt;0.05 was considered statistically significant. Results: 816 patients received PN while 1,278 received RN. PN patients had an increase in the following events: 30-day readmissions (7.0% vs. 4.7%, p=0.026), major bleeds (9.19% vs. 5.56%, p=0.001), renal failure requiring dialysis (1.23% vs. 0.31%, p=0.013), and urine leak or ureteric fistulae (1.10% vs. 0.31%, p=0.025). Open surgery was associated with increased VTE, renal failure, bleeds, urine leaks or ureteric fistulae, readmissions, and reoperations. Multivariate analysis revealed that PN remained predictive of all four aforementioned events, although further adjustment for robotic approach led to a loss of significance for renal failure and ureteric fistulae. Additional patient-specific predictors of relevant complications across procedure type included bleeding disorder and dialysis for bleeds, and renal failure, steroid use, and COPD for readmissions. Conclusions: This is the first study to evaluate the new NSQIP Nephrectomy-Targeted PUF. This population-based cohort provides unique insights into nephrectomy for pT1b-T2 localized RCC. We demonstrate significant associations between PN and specific complications, modulated by particular comorbidities, although both PN and RN were exceedingly safe. This analysis supports the development of novel risk stratification tools which account for specific patient comorbidities in predicting near term risk. Improved understanding of case-specific determinants of morbidity following PN or RN may facilitate shared decision making in localized RCC management.

Clinical Evaluation of Metagenomic Next-Generation Sequencing Method for the Diagnosis of Suspected Ascitic Infection in Patients with Liver Cirrhosis in a Clinical Laboratory.

Metagenomic next-generation sequencing (mNGS), mostly carried out in independent clinical laboratories, has been increasingly applied in clinical pathogen diagnosis. We aimed to explore the feasibility of mNGS in clinical laboratories and analyze its potential in the diagnosis of infectious ascites. Two reference panels composed of 12 strains commonly appearing in peritonitis were constructed to evaluate the performance metrics based on in-house mNGS protocols. The mNGS clinical detection value was analyzed in 211 ascitic samples and compared with culture and composite standards. Finally, eight patients with cirrhosis were prospectively enrolled to verify the clinical value of mNGS in peritoneal infection diagnosis. The mNGS analytical performance showed that the assay had great linearity, specificity, stability, interference, and limits of detection of 33 to 828 CFU/mL. The sensitivity and specificity of mNGS for bacterial or fungal detection using culture standards were 84.2% and 82.0%, respectively. After adjustment using digital PCR and clinical judgment, the sensitivity and specificity increased to 87.2% and 90.1%, respectively. Compared with culture, mNGS detected a broad range of pathogens and more polymicrobial infections (49% versus 9%, P < 0.05). The pathogen results were obtained within 24 h using mNGS in eight prospective cases, which effectively guided antibiotics therapy. mNGS testing in clinical laboratories affiliated with a hospital has certain advantages. It has unique superiority in pathogens detection, particularly in patients with polymicrobial infections. However, considering spectrum characteristics and test cost, pertinent pathogen panels should be developed in clinical practice. IMPORTANCE This study established and evaluated a complete metagenomics next-generation sequencing assay to improve the diagnosis of suspected ascitic infection in a clinical laboratory affiliated with a hospital. The assay is superior to traditional culture testing and will aid in the early and accurate identification of pathogens, particularly in patients with polymicrobial infections. This assay is also essential for precision therapy and can reduce the incidence of drug resistance stemming from irrational use of antibiotics.

The Importance of Gram-stained Smears in the Diagnosis of Nocardia Infections

Nocardia species are low virulence bacteria found in nature. They can be an infectious agent, especially in patients with risk factors such as underlying immunosuppression, chronic lung disease, and malignancy. They can be easily overlooked because they are not seen frequently and has no pathognomonic symptoms. With this study, it was aimed to draw attention to the importance of microscopic examination of Gram-stained smears in the diagnosis of Nocardia infections in routine microbiology laboratories. Cases in which Nocardia spp. were detected in their clinical samples between November 2014-December 2015 in Hacettepe University Medical Faculty Hospital were included in the study. In the direct microscopic examination of Gram-stained smears of the samples arriving to the laboratory, the incubation periods of the cultures of the samples compatible with Nocardia spp. were extended. Then relevant colonies were identified by conventional microbiological methods and also by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS, bioMerieux, France) automated system. Species-level identification of Nocardia isolates was performed by 16S rRNA gene sequence analysis. To demonstrate the genetic relationship between Nocardia isolates, pulsed-field gel electrophoresis (PFGE) was performed. In vitro susceptibility of the isolates against amoxicillin-clavulanate (AMC), linezolid, moxifloxacin, trimethoprim-sulfamethoxazole (TMP-SXT), amikacin, imipenem, clarithromycin, cefepime, cefotaxime, ceftriaxone, and ciprofloxacin was determined using the gradient strip method (E-test). A total of 19 Nocardia spp. strains were isolated from eight patients. Four cases exhibited repeated growth of Nocardia spp. up to a period of nine months. The most frequently isolated species was N.cyriacigeorgica, which was identified in four cases. Other species isolated from patients were N.asteroides, N.transvalensis, N.farcinicia, and N.asiatica/arthritidis. When the results obtained with DNA sequence analysis and MALDI-TOF MS were compared, 16 (84.2%) of 19 isolates were correctly identified to the genus level and 9 (47.4%) to the species level with MALDI-TOF MS, while three (15.8%) isolates could not be identified, and seven (36.8%) isolates were misidentified. According to the PFGE results, it was determined that the strains isolated from the same patient were genetically identical. All isolates were susceptible to amikacin, cefepime, cefotaxime, ceftriaxone, imipenem, linezolid, and except one isolate to TMP-SXT. Among the study isolates, the most common resistance was against ciprofloxacin (62.5%), followed by clarithromycin (37.5%). N.cyriacigeorgica was determined as the most frequently detected and the most resistant species to antibiotics in the study population. Direct microscopic examination of clinical specimens is one of the most valuable methods for the identification of Nocardia-type bacteria, which is difficult to isolate in microbiology laboratories. With this study, the importance of examining Gram-stained clinical samples was emphasized in the identification of Nocardia species, which can emerge with a wide variety of clinical forms and can be easily overlooked. In addition, antibiotic susceptibility profiles of the isolated bacteria were determinedto contribute to species-specific susceptibility profiles. Accurate identification of Nocardia species will contribute to clinical and epidemiological studies.

Biosafety of personnel of microbiological laboratories in the context of the Federal Law of the Russian Federation № 492-FZ of December 30, 2020 «On the biological safety of the Russian Federation».

One of the most important requirements for the personnel of microbiological laboratories working with pathogenic and infectious agents is the observance of precautionary measures and the implementation of a set of preventive measures, collectively interpreted as biological safety (biosafety). To a large extent, biosafety problems are also relevant for all clinical laboratories working with biosubstrates, with the potential threat of containing pathogens of bloodborne infections in them. On December 30, 2020, the President of the Russian Federation signed Federal Law № 492 «On the Biological Safety of the Russian Federation» (№ 492-FZ), which regulates the basic legal norms and regulation of biosafety issues, as well as a list of measures to prevent the risks of the spread of infections due to accidents, bioterrorist acts and sabotage. The current pandemic of the coronavirus infection COVID-19 has demonstrated, on the one hand, the epidemiological vulnerability of the single world space, and on the other hand, the decisive influence of biological emergencies on the emergence of negative political and economic processes in the world community. In this regard, the issues of ensuring biosafety in the work of microbiological laboratories in the context of protecting personnel and the environment from accidental or unintentional spread of infections are relevant. Working with pathogenic biological agents in microbiological laboratories is constantly associated with the risk of accidents and possible laboratory infection (laboratory-acquired infections) of employees, environmental pollution if the requirements of regulatory documents on biological safety are not met. In accordance with the requirements of № 492-FZ, in order to prevent biological threats, it is necessary to create a system for monitoring biological risks in microbiological laboratories when working with any infected material.

Antimicrobial susceptibility pattern of extended-spectrum beta-lactamase-producing uropathogens in aminu Kano teaching hospital, Northwestern Nigeria.

UTI is the most common nosocomial infection among hospitalized patients. Emerging increasing resistance has been observed among uropathogens of the family enterobacterales. Extended spectrum beta lactamase genes encode for multidrug resistance and unrestricted use of antibiotics in hospitals provides an environment for spread of infections with limited treatment options. This necessitates therapies based on culture and antimicrobial sensitivity to improve patients' outcomes We aimed to determine susceptibility pattern of ESBL uropathogens among hospitalized patients in Aminu Kano Teaching Hospital, Kano. Three hundred and eighty-nine urine samples were obtained from in-patients with UTI between April 2020 and April 2021. Five samples were rejected and remaining analyzed. Susceptibility testing was done by modified Kirby Bauer technique. Clinical Laboratory Standards Institute guidelines 2019 (CLSI 2019) and the European Committee on Antimicrobial Susceptibility testing guidelines version 2 (EUCAST version 2) were used for screening and confirmation of ESBL production respectively. Of the 384-urine processed, 105 (27.3%) were gram negatives and 81 were Enterobacterales, Isolation rates were E. coli-55.6%, K. pneumoniae-29.6%, Citrobacter spp.-12.3%, P. mirabilis-1.2% and Morganella spp.-1.2%. Among Enterobacterales, 32 (39.5%) were ESBL producers. Prevalence of ESBL were 62.5% for Escherichia coli, 28.1% for Klebsiella pneumoniae and 9.4% for Citrobacter species. Susceptibility showed that ESBL-producing Isolates were highly susceptible to amikacin (96.9%). Resistance to other antibiotics varied from 3.1% to 100%. We recommend strengthening laboratory capacity, antibiotics stewardship and Infection control to prevent spread of resistant pathogens including ESBLs.

Another decade ofTrichuris murisresearch: An update and application of key discoveries.

The mouse whipworm, Trichuris muris, has been used for over 60 years as a tractable model for human trichuriasis, caused by the related whipworm species, T. trichiura. The history of T. muris research, from the discovery of the parasite in 1761 to understanding the lifecycle and outcome of infection with different doses (high versus low dose infection), as well as the immune mechanisms associated with parasite expulsion and chronic infection have been detailed in an earlier review published in 2013. Here, we review recent advances in our understanding of whipworm biology, host-parasite interactions and basic immunology brought about using the T. muris mouse model, focussing on developments from the last decade. In addition to the traditional high/low dose infection models that have formed the mainstay of T. muris research to date, novel models involving trickle (repeated low dose) infection in laboratory mice or infection in wild or semi-wild mice have led to important insights into how immunity develops in situ in a multivariate environment, while the use of novel techniques such as the development of caecal organoids (enabling the study of larval development ex vivo) promise to deliver important insights into host-parasite interactions. In addition, the genome and transcriptome analyses of T. muris and T. trichiura have proven to be invaluable tools, particularly in the context of vaccine development and identification of secreted products including proteins, extracellular vesicles and micro-RNAs, shedding further light on how these parasites communicate with their host and modulate the immune response to promote their own survival.

Multi-strain compatibility polymorphism between a parasite and its snail host, a neglected vector of schistosomiasis in Africa

Interactions between Schistosoma mansoni and its snail host are understood primarily through experimental work with one South American vector species, Biomphalaria glabrata. However, 90% of schistosomiasis transmission occurs in Africa, where a diversity of Biomphalaria species may serve as vectors. With the long-term goal of determining the genetic and ecological determinants of infection in African snail hosts, we developed genetic models of Biomphalaria sudanica, a principal vector in the African Great Lakes. We determined laboratory infection dynamics of two S. mansoni lines in four B. sudanica lines. We measured the effects of the following variables on infection success and the number of cercariae produced (infection intensity): (i) the combination of parasite and snail line; (ii) the dose of parasites; and (iii) the size of snail at time of exposure. We found one snail line to be almost completely incompatible with both parasite lines, while other snail lines showed a polymorphism in compatibility: compatible with one parasite line while incompatible with another. Interestingly, these patterns were opposite in some of the snail lines. The parasite-snail combination had no significant effect on the number of cercariae produced in a successful infection. Miracidia dose had a strong effect on infection status, in that higher doses led to a greater proportion of infected snails, but had no effect on infection intensity. In one of the snail-schistosome combinations, snail size at the time of exposure affected both infection status and cercarial production in that the smallest size class of snails (1.5–2.9 mm) had the highest infection rates, and produced the greatest number of cercariae, suggesting that immunity increases with age and development. The strongest predictor of the infection intensity was the size of snail at the time of shedding: 1 ​mm of snail growth equated to a 19% increase in cercarial production. These results strongly suggest that infection status is determined in part by the interaction between snail and schistosome genetic lines, consistent with a gene-for-gene or matching allele model. This foundational work provides rationale for determining the genetic interactions between African snails and schistosomes, which may be applied to control strategies.

Infection Characteristics and Transcriptomics of African Swine Fever Virus in Bama Minipigs.

Animal experiments on African swine fever virus (ASFV) are vital to the study of ASFV; however, ASFV can only infect pigs, and animal experiments need to be performed in animal biosafety level 3 (ABSL-3) laboratories, meaning that many small ABSL-3 laboratories are unable to carry out in vivo ASFV experiments. Therefore, miniaturized experimental animals for ASFV infection are urgently needed. Here, we successfully isolated genotype II of ASFV SY-1 from wild boars and evaluated ASFV-infected Bama minipigs in a negative-pressure isolator of a small ABSL-3 laboratory. The pathological changes of ASFV-infected Bama minipigs were consistent with characteristic lesions of ASFV-infected domestic pigs and wild boars. All pigs died 5 to 14 days postinfection (dpi) through intramuscular injection. Viral genomic DNA from nasal, oral, and rectal swab samples was first detectable at 2 to 4 dpi. The common differentially expressed genes were clustered in the immune-related, metabolic, and inflammatory response pathways from the spleen and inguinal lymph node samples comparing infected to mock. In summary, these results demonstrated that the Bama minipig was an appropriate model for ASFV infection in small ABSL-3 laboratories that can accelerate the research of vaccines and antiviral drugs and uncover pathogenic mechanisms of ASFV infection. IMPORTANCE African swine fever virus (ASFV) can only infect pigs rather than other animals. However, the domestic pigs cannot be kept in small ABSL-3 laboratories for a long time due to the characteristics of rapid growth and large size, which hinder ASFV research, including research of vaccines, antiviral drugs, and mechanisms. In contrast, Bama minipigs have unique advantages consisting of low growth and small size. In the research, Bama minipigs were used to evaluate the characteristics of ASFV infection in small ABSL-3 laboratories. The pathological changes, viral shedding, and gene regulation were consistent with those of domestic pigs infected with ASFV. Therefore, Bama minipigs can be a suitable model for ASFV infection in small ABSL-3 laboratories.

The Epidemiology of Healthcare-Associated Bloodstream Infection in an Adult Intensive Care Unit: A Retrospective Cohort Study in a Single Tertiary Care Hospital in Hanoi, Vietnam.

Background Healthcare-associated infections (HAIs), including bloodstream infections (BSIs) in the intensive care unit (ICU), are growing global public health problems. While high-income countries have reported the burden of HAIs precisely, low- and middle-income countries (LMICs), including Vietnam, often lack surveillance systems for HAIs. In Vietnam, few reports described HAI-associated BSIs. Therefore, in this study, we aim to clarify the characteristics of HAI-associated BSIin an adult ICU. Materials and methods We conducteda retrospective cohort study of HAI-associated BSIin the adult ICU of Bach Mai Hospital (BMH), Vietnam, between December 2013 and August 2015. For every case identified with bacteremia, we collected characteristics and laboratory findings of the case andfollowed the length of hospital stay and seven-day and 30-day survival. Predictors of 30-day mortality were analyzed using univariate and multivariate analyses. Results Among the 90 cases identified, the median age of the study cohort was 57 (range: 18-90) years, and 59 (65.6%) were male. Chronic heart disease was the most frequent comorbidity (n = 26, 28.9%). The pathogens isolated were mostly Candida spp. (n = 26, 26.3%) and Enterococcus spp. (n = 19, 19.2%). Among the 90 patients with confirmed HAI-associated BSI, 34 (37.8%) patients survived, while 31 (34.4%)patients died in 30 days. In multivariate analysis, chronic heart disease tended to increase with 30-day all-cause mortality (odds ratio (OR) = 3.5, 95% confidence interval (CI) = 1.0-11.9, p = 0.051). Conclusions Our retrospective cohort study is the largest investigation to describe HAI-associated BSI in an adult ICU in a tertiary care hospital in Vietnam. Improved laboratory detection and infection surveillance systems are needed to reduce HAI-associated BSI.

Evaluation of Various Alternative Economical and High Throughput SARS-CoV-2 Testing Methods within Resource-Limited Settings.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak posed a challenge for diagnostic laboratories worldwide, with low-middle income countries (LMICs) being the most affected. The polymerase chain reaction (PCR) is the gold standard method for detecting SARS-CoV-2 infection. However, the challenge with this method is that it is expensive, which has resulted in under-testing for SARS-CoV-2 infection in many LMICs. Hence, this study aimed to compare and evaluate alternative methods for the mass testing of SARS-CoV-2 infection in laboratories with limited resources to identify cost-effective, faster, and accurate alternatives to the internationally approved kits. A total of 50 residual nasopharyngeal swab samples were used for evaluation and comparison between internationally approved kits (Thermo Fisher PureLink™ RNA Isolation Kit and Thermo Fisher TaqPath™ COVID-19 Assay Kit) and alternative methods (three RNA extraction and four commercial SARS-CoV-2 RT-PCR assay kits) in terms of the cost analysis, diagnostic accuracy, and turnaround time. In terms of performance, all of the alternative RNA extraction methods evaluated were comparable to the internationally approved kits but were more cost-effective (Lucigen QuickExtract™ RNA Extraction Kit, Bosphore EX-Tract Dry Swab RNA Solution and Sonicator method) and four commercial SARS-CoV-2 RT-PCR assay kits (Nucleic Acid COVID-19 Test Kit (SARS-CoV-2), abTESTM COVID-19 qPCR I Kit, PCL COVID19 Speedy RT-PCR Kit, and PCLMD nCoV One-Step RT-PCR Kit) with a sensitivity range of 76-100% and specificity of 96-100%. The cost per sample was reduced by more than 50% when compared to internationally approved kits. When compared to the Thermo Fisher PureLink™ Kit and Thermo Fisher TaqPath™ COVID-19 Assay Kit, the alternative methods had a faster turnaround time, indicating that laboratories with limited resources may be able to process more samples in a day. The above-mentioned cost-effective, fast, and accurate evaluated alternative methods can be used in routine diagnostic laboratories with limited resources for mass testing for SARS-CoV-2 because these were comparable to the internationally approved kits, Thermo Fisher PureLink™ Kit and Thermo Fisher TaqPath™ COVID-19 Assay Kit. The implementation of alternative methods will be the most cost-effective option for testing SARS-CoV-2 infection in LMICs.

Evaluating the Clinical and Immune Responses to Spotted Fever Rickettsioses in the Guinea Pig-Tick-Rickettsia System.

The guinea pig was the original animal model developed for investigating spotted fever rickettsiosis (SFR). This model system has persisted on account of the guinea pig's conduciveness to tick transmission of SFR agents and ability to recapitulate SFR in humans through clinical signs that include fever, unthriftiness, and in some cases the development of an eschar. The guinea pig is the smallest animal model for SFR that allows the collection of multiple blood and skin samples antemortem for longitudinal studies. This unit provides the basic protocols necessary to establish, maintain, and utilize a guinea pig-tick-Rickettsia model for monitoring the course of infection and immune response to an infection by spotted fever group Rickettsia (SFGR) that can be studied at biosafety level 2 (BSL-2) and arthropod containment level 2 (ACL-2); adaptations must be made for BSL-3 agents. The protocols cover methods for tick feeding and colony development, laboratory infection of ticks, tick transmission of Rickettsia to guinea pigs, and monitoring of the course of infection through clinical signs, rickettsial burden, and immune response. It should be feasible to adapt these methods to study other tick-borne pathogens. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tick transmission of SFGR to guinea pigs Support Protocol 1: Laboratory infection of ticks by injection Alternate Protocol 1: Needle inoculation of SFGR to guinea pigs Basic Protocol 2: Monitoring the course of guinea pig rickettsial infection: clinical signs Basic Protocol 3: Monitoring the course of guinea pig rickettsial infection: collection of biological specimens Support Protocol 2: Guinea pig anesthesia Basic Protocol 4: Monitoring rickettsial burden in guinea pigs by multiplex qPCR Basic Protocol 5: Monitoring guinea pig immune response to infection: blood leukocytes by flow cytometry Basic Protocol 6: Monitoring immune response to guinea pig rickettsial infection: leukocyte infiltration of skin at the tick bite site by flow cytometry Basic Protocol 7: Monitoring the immune response to guinea pig rickettsial infection: antibody titer by ELISA Support Protocol 4: Coating ELISA Plates Alternate Protocol 2: Monitoring immune response to guinea pig rickettsial infection: antibody titer by immunofluorescence assay.

Infection Experiments Indicate That Common Florida Anurans and Lizards May Serve as Intermediate Hosts for the Invasive Pentastome Parasite, Raillietiella orientalis

Raillietiella orientalis, an invasive pentastome parasite, inhabits the lungs of snakes as adults and is rapidly spreading in Florida and Australia. The species that serve as intermediate hosts are currently unknown. We conducted laboratory infection experiments with insects (Blaberus discoidalis), lizards (Anolis sagrei), and anurans (Anaxyrus terrestris, Lithobates sphenocephalus, Osteopilus septentrionalis) to develop an understanding of which species may serve as intermediate hosts and to determine the fitness consequences of infection by R. orientalis. Lizards and insects, but not anurans, were readily infected by consuming food that was contaminated with pentastome eggs. Anolis sagrei and L. sphenocephalus were both infected after eating a single roach that was infected with R. orientalis larvae. Comparison with uninfected control animals revealed that pentastome infection did not significantly affect survival or growth in roaches, lizards, or anurans. The life cycle of R. orientalis in Florida is likely to involve a sequence of three hosts, with eggs hatching in coprophagous insects that infect lizards and anurans that infect the definitive host (snakes) after they are consumed. Our results indicate that the native species that serve as intermediate hosts are unlikely to experience major negative consequences from R. orientalis, unlike the native snake species that serve as the definitive hosts. The diversity of species that can serve as intermediate hosts and the potential for vehicular rafting by infected roaches and anoles indicate that the rapid geographic range expansion of R. orientalis will probably continue.

Genetic variability in the susceptibility of immature peach fruit to Monilinia laxa is associated with surface conductance but not stomatal density

Monilinia laxa is a fungus that causes brown rot in stone fruit. Immature green fruits in the first stage of fruit development (stage I) are generally susceptible. To investigate the relationship between the physical characteristics of immature fruit and susceptibility to M. laxa, we characterized the progeny, derived from a clone of wild peach (Prunus davidiana) crossed with two commercial nectarines (Prunus persica) varieties, through laboratory infection, transpiration monitoring and stomata counting. Two types of fruit infections were observed - ‘classic’ brown rot and ‘clear spot’ symptoms - which have not previously been described in the literature. The number and density of stomata did not explain the observed variability of infection in the progeny. However, surface conductance was positively correlated with infection level. This study provides experimental evidence partially linking physical fruit characteristics to brown rot infection at the immature fruit stage. The role of delayed cuticle deposition in susceptibility to brown rot of immature fruit is discussed.

Determination of etiology in patients admitted due to isolated leukopenia.

Patients with isolated leukopenia pose difficulties in diagnosis because there is no related guideline in the literature. In this study, our aim was to evaluate the clinical and laboratory associations of isolated, nonspecific (not related to neutropenia) leukopenia.In this retrospective data review study, patients who were admitted to Hacettepe University Hematology Outpatient Clinic between 2014 and 2019 due to leukopenia were evaluated. The patients with anemia (other than iron deficiency) or thrombocytopenia were excluded. Clinical and laboratory data and the final diagnoses (if present) of the remaining cases and especially of those without neutropenia (the most difficult group to diagnose) were evaluated.One hundred sixty-nine patients were included in the study. One hundred forty-four (85.2%) patients were female and 25 (14.8%) were male. One hundred ten of them had 1500/µL or higher neutrophil count. In these nonneutropenic cases, the etiological factors contributing to leukopenia were as follows: iron deficiency anemia (21.8%), other autoimmune/autoinflammatory diseases (17.3%), autoimmune thyroid disease (21.8%), autoimmune laboratory tests (2.7%), drugs (12.7%), infection (5.5%), hematopoietic disorder (2.7%), hypersplenism (2.7%), radiotherapy sequel (1.8%), and B12 deficiency (1.8%). No etiology was recognized in 44 patients. On the other hand, the etiological factors in patients with neutrophil count <1500/µL were as follows; iron deficiency anemia (10.2%), other autoimmune/autoinflammatory diseases (17%), autoimmune thyroid disease (5.1%), autoimmune laboratory tests (8.5%), drugs (8.5%), infection (6.8%), hematopoietic disorder (11.9%), hypersplenism (1.7%), radiotherapy sequel (1.7%), and B12 deficiency (1.7%). No etiology was recognized in 25 patients. Physicians ordered bone marrow examination more frequently in patients with neutropenia. If isolated antinuclear antibody positivity was also considered in favor of autoimmunity, 91/169 (53.8%) cases had an autoimmune diagnosis or laboratory finding.In the present study, the most frequent reasons of isolated leukopenia in nonneutropenic patients are found as iron deficiency anemia, other autoimmune/autoinflammatory diseases, and autoimmune thyroid disease. In neutropenic patients, the most frequent reasons of isolated leukopenia are found as iron deficiency anemia, autoimmune/autoinflammatory diseases, and hematopoietic disorders. Therefore, autoimmunity is detected as an important factor leading to isolated leukopenia.

The diagnostic utility of IL-10, IL-17, and PCT in patients with sepsis infection.

ObjectiveThe purpose of this study is to determine the diagnostic value and net clinical benefit of interleukin-10 (IL-10), interleukin-17 (IL-17), procalcitonin (PCT), and combination tests in patients with sepsis, which will serve as a standard for sepsis early detection.Patients and methodsAn investigation of 84 sepsis patients and 81 patients with local inflammatory diseases admitted to the ICU of Tongji University Hospital in 2021. In addition to comparing inter-group variability, indicators relevant to sepsis diagnosis and therapy were screened.ResultsLASSO regression was used to examine PCT, WBC, CRP, IL-10, IFN-, IL-12, and IL-17. Multivariate logistic regression linked IL-10, IL-17, and PCT to sepsis risk. The AUC values of IL-10, IL-17, PCT, and the combination of the three tests were much higher than those of standard laboratory infection indicators. The combined AUC was greater than the sum of IL-10, IL-17, and PCT (P < 0.05). A clinical decision curve analysis of IL-10, IL-17, PCT, and the three combined tests found that the three combined tests outperformed the individual tests in terms of total clinical benefit rate. To predict the risk of sepsis using IL-10, IL-17, and PCT had an AUC of 0.951, and the model's predicted probability was well matched. An examination of the nomogram model's clinical value demonstrated a considerable net therapeutic benefit between 3 and 87%.ConclusionThe IL-10, IL-17, and PCT tests all have a high diagnostic value for patients with sepsis, and the combination of the three tests outperforms the individual tests in terms of diagnostic performance, while the combined tests have a higher overall clinical benefit rate.

Molecular Characterization of Enterobacter cloacae Isolated from Urinary Tract Infections

Background: Urinary tract infections represent a major expensive, common public health problem worldwide due to their high prevalence and the difficulties associated with their management. Objectives: This study aimed to characterize the Enterobacter cloacae strains isolated from urinary tract infections in the medical diagnostic laboratories of Shahrekord, Iran. Methods: Urine samples from patients with urinary tract infections from the Shahrekord medical diagnostic laboratories located in Chaharmahal and Bakhtiari Province, Iran, were collected from June 2019 to February 2020. When the samples were cultured, the different isolates of E. cloacae were identified by biochemical tests. Biofilm production capacity was evaluated. Bacterial susceptibility to antibiotics was determined using the Kirby Bauer method, and antibiotic resistance genes were researched by the multiplex PCR technique. Results: In this study, 65 isolates of E. cloacae were obtained. The highest percentage of resistance was observed for co-trimoxazole (84.62%), ampicillin (76.93%), tetracycline (73.85%), and above half of the E. cloacae strain isolates (53,85%) were strongly involved in biofilm production. Some genes, including qnr A, qnr B, qnr S, tetA, tet B, sul1, bla CTXM, bla SHV, and(2)la, ant(3)la, and aac(3)IIa, were detected in the genome of these isolates. Conclusions: The strains are multi-resistant, and their resistance has already reached the carbapenem class. This requires further investigation, and urgent measures must be adopted.