Clinical and mycological investigations of post-COVID-19 acute invasive fungal sinusitis.

Objectives: An increased incidence of acute invasive fungal sinusitis associated with the recent COVID-19 pandemic has been observed, which is considered a public health concern. This study aims to detect the incidence, risk factors, causative agents, clinical presentations, outcomes, and susceptibility rate of various antifungals. Methods: In this cross-sectional cohort study, a total of 30 patients showing acute invasive fungal rhinosinusitis following a COVID-19 infection were investigated. Histopathological biopsies, culture identification, and molecular confirmation of the causative agents were conducted. The demographic data, risk factors, clinical presentations, treatment regimen and its outcomes, and efficacy of antifungals were listed and analyzed. Results: A total of 30 cases with a mean age of 59.6 ± 11.9 years were included. Diabetes mellitus was the most recorded comorbidity with a rate of 86.7%, whereas most of the patients received corticosteroids. The mycological examination confirmed the existence of Mucor (Rhizopus oryzae) and Aspergillus (Aspergillus niger) in 96.7% and 3.3% of the cases, respectively. Various stages of sinonasal involvement (ethmoid, maxillary, sphenoid, and inferior turbinate) represented 100%, 83.3%, 66.7%, and 86.7% of the cases, respectively. Headache and facial pain, ophthalmoplegia, visual loss, and blindness represented 100%, 66.7%, 90%, and 53.3% of the cases, respectively. All the cases were simultaneously treated with surgical debridement and amphotericin B. Moreover, R. oryzae was susceptible to it, whereas A. niger was sensitive to voriconazole, resulting in a survival rate of 86.7% (26/30). The R. oryzae and A. niger isolates were proven to be sensitive to acetic acid, ethyl alcohol, formalin, and isopropyl alcohol. Conclusions: In patients with COVID-19, the diagnosis of acute invasive fungal sinusitis and prompt treatment with antifungal medicine and surgical debridement are important in achieving better outcomes and survival rates. Level of Evidence: 4.

Real-time PCR using atpE, conventional PCR targeting different regions of difference, and flow cytometry for confirmation of Mycobacterium bovis in buffaloes and cattle from the Delta area of Egypt.

BACKGROUND: Mycobacterium bovis notoriously causes detrimental infections in bovines and humans. In this study, 1500 buffaloes and 2200 cattle were tested by single intradermal comparative cervical tuberculin test and compared with the detection rates of M. bovis isolation, real-time and simplex PCR, and flow Cytometry. RESULTS: The tuberculin test is the reference test in Egypt, the positive rate was 54/3700 (1.5%) composed of 18/1500 (1.2%) buffaloes and 36/2200 (1.6%) cattle which were mandatorily slaughtered under the Egyptian legislation, after postmortem examination the non-visible-lesion proportion was 39/54 (72.2%) which surpassed the visible-lesion rate 15/54 (27.8%) with (p < 0.0001). The samples from each case were pooled into one sample representing the case, and the isolation rate of M. bovis was 25/54 (46.3%). Real-time PCR using atpE was positive for mycobacteria on the genus level in 18/18 (100%) and 5/5 (100%) of tissue samples and isolates, respectively; simplex PCR detected M. bovis in 44/54 (81.5%) and 25/25 (100%) of tissue samples and isolates, respectively. Flow Cytometry evaluation of the CD4+, CD8+, WC1+δγ, and CD2+ cell phenotypes showed increased counts in the tuberculin-positive cases compared with negative cases (p < 0.0001), and these phenotypes in the tuberculin-positive cases increased after antigen stimulation than in the negative cases (p < 0.0001). Detection rates of PCR techniques and flow Cytometry exceeded that of bacterial isolation (p < 0.0001) and exhibited a strong correlation. CONCLUSIONS: The skin test suffers from interference from non-tuberculous mycobacteria able to cause false-positive reactions in cattle and other species. Real-time PCR using atpE, conventional PCR targeting RDs, and flow Cytometry are rapid and accurate methods that correlate with the isolation and can be promising for detection and confirmation of infected live and slaughtered cases.

Genetic and antimicrobial resistance profiles of non-O157 Shiga toxin-producing Escherichia coli from different sources in Egypt.

BACKGROUND: The Shiga toxin-producing Escherichia coli (STEC) represented a great risk to public health. In this study, 60 STEC strains recovered from broiler and duck fecal samples, cow's milk, cattle beef, human urine, and ear discharge were screened for 12 virulence genes, phenotypic and genotypic antimicrobial resistance, and multiple-locus variable-number tandem-repeat analysis (MLVA). RESULTS: The majority of strains harbored Shiga toxin 1 (stx1) and stx1d, stx2 and stx2e, and ehxA genes, while a minority harbored stx2c subtype and eaeA. We identified 10 stx gene combinations; most of strains 31/60 (51.7%) exhibited four copies of stx genes, namely the stx1, stx1d, stx2, and stx2e, and the strains exhibited a high range of multiple antimicrobial resistance indices. The resistance genes blaCTX-M-1 and blaTEM were detected. For the oxytetracycline resistance genes, most of strains contained tetA, tetB, tetE, and tetG while the tetC was present at low frequency. MLVA genotyping resolved 26 unique genotypes; genotype 21 was highly prevalent. The six highly discriminatory loci DI = 0.9138 are suitable for the preliminary genotyping of STEC from animals and humans. CONCLUSIONS: The STEC isolated from animals are virulent, resistant to antimicrobials, and genetically diverse, thus demands greater attention for the potential risk to human.

Virulence Determinants and Antimicrobial Profiles of Pasteurella multocida Isolated from Cattle and Humans in Egypt.

Pasteurella multocida is a Gram-negative bacterium that causes drastic infections in cattle and humans. In this study, 55 isolates were recovered from 115 nasal swabs from apparently healthy and diseased cattle and humans in Minufiya and Qalyubia, Egypt. These isolates were confirmed by kmt1 existence, and molecular classification of the capsular types showed that types B, D, and E represented 23/55 (41.8%), 21/55 (38.1%), and 11/55 (20.0%), respectively. The isolates were screened for five virulence genes with hgbA, hgbB, and ptfA detected in 28/55 (50.9%), 30/55 (54.5%), and 25/55 (45.5%), respectively. We detected 17 capsular and virulence gene combinations with a discriminatory power (DI) of 0.9286; the most prevalent profiles were dcbF type D and dcbF type D, hgbA, hgbB, and ptfA, which represented 8/55 (14.5%) each. These strains exhibited high ranges of multiple antimicrobial resistance indices; the lowest resistances were against chloramphenicol, ciprofloxacin, amoxicillin/clavulanic acid, and levofloxacin. The macrolide-lincosamide-streptogramin B methylase gene erm(Q), with erm(42) encoding MLSB monomethyltransferase, mph(E) encoding a macrolide efflux pump, and msr(E) encoding macrolide-inactivating phosphotransferase were present. The class 1 and 2 integrons and extended-spectrum ß-lactamase genes intl1, intl2, blaCTX-M, blaCTX-M-1, and blaTEM were detected. It is obvious to state that co-occurrence of resistance genes resulted in multiple drug-resistant phenotypes. The identified isolates were virulent, genetically diverse, and resistant to antimicrobials, highlighting the potential risk to livestock and humans.

Phenotypic and genotypic methods for identification of slime layer production, efflux pump activity, and antimicrobial resistance genes as potential causes of the antimicrobial resistance of some mastitis pathogens from farms in Menoufia, Egypt.

Mastitis caused by multi- or pan-drug resistant bacteria is a growing health concern. A total of 110 milk samples were collected: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus faecalis, and Escherichia coli were present in 54/110 (49.09%), 37/110 (33.63%), 25/110 (22.72%), 7/110 (6.36%), and 50/110 (45.45%) samples, respectively. A total of 20 methicillin-resistant S. aureus (MRSA) isolates, 19 Streptococcus sp. isolates, and 15 E. coli isolates were selected, and 100% were positive for (coagulase and hemolysins), streptokinase, and hemolytic activity, respectively. A number of 11 E. coli isolates were serotyped, and the serotypes were: O26, O55, O111, O119, O124, O125, O127, and O158. The antimicrobial resistance index ranges for MRSA, Streptococcus sp., and E. coli were 0.49-0.83, 0.39-0.83, and 0.56-1, respectively. The most effective antimicrobials on Gram-positive isolates were cephradine, ciprofloxacin, doxycycline, norfloxacin, and vancomycin, while doxycycline and norfloxacin were effective on E. coli serotypes. All of the selected isolates exhibited slime layer production. The efflux pumps of the 12 MRSA, 12 Streptococcus sp., and 11 E. coli isolates exhibited activity with ethidium bromide concentrations of 1, 1.5, and 0.5 µg/ml, respectively. There was a simultaneous antimicrobial activity of the efflux pump inhibitor chlorpromazine with amoxicillin/clavulanic acid, erythromycin, and oxacillin, to which the isolates were resistant. The 12 MRSA isolates harboured the methicillin resistance genes mec(A,A1, and A2), mecA1, and mecC at frequencies of 9/12 (75%), 9/12 (75%), and 8/12 (66.7%), respectively, and the penicillin resistance gene BlaZ was present at a frequency of 5/12 (41.7%). The distributions of erm(A), erm(B), erm(C), erm(F), erm(G), and erm(Q) were 8/12 (66.7%), 5/12 (41.7%), 12/12 (100%), 2/12 (16.7%), 0/12 (0.0%), and 8/12 (66.7%), respectively. The 12 Streptococcus sp. isolates harboured mec(A, A1, and A2), mecA1, mecC, and blaZ at rates of 4/12 (33.33%), 4/12 (33.33%), 5/12 (41.7%), and 4/12 (33.33%), respectively. The frequencies of erm(A) and erm(F) were 4/12 (33.33%), and 9/12 (75%), respectively. The 11 E. coli isolates harboured the extended-spectrum ß-lactamases integrase1, integrase2, blaCTX-M, blaCTX-M-1, and blaTEM at frequencies of 10/11 (90.90%), 11/11 (100%), 9/11 (81.81%), 6/11 (54.54%), and 10/11 (90.90%), respectively. Moreover, the frequencies of erm(A), erm(B), erm(C), erm(F), erm(G), and erm(Q) were 7/11 (63.63%), 4/11 (36.36%), 4/11 (36.36%), 5/11 (45.45%), 10/11 (90.90%), and 10/11 (90.90%), respectively. Our results demonstrated the high antimicrobial resistance of the investigated isolates and confirmed the existence of multiple mechanisms underlying multidrug resistance.

Applicability of using 15 MIRU-VNTR loci for genotyping of Mycobacterium avium subsp. paratuberculosis from two cattle farms in Egypt.

Mycobacterium avium subspecies paratuberculosis (MAP) is a notorious infectious agent that causes Johne's disease which leads to serious economic losses in cattle farms all over the world. The Lack of accurate epidemiological and molecular data is a major barrier to the implementation of disease control strategies. Basically, the tracing of infections requires rapid detection of the widely spreading genotypes with the ability to determine isolates from common and different sources. This study aimed to evaluate the applicability and discriminatory power of 15 mycobacterial interspersed repetitive unit (MIRU)-variable number tandem repeat (VNTR) loci of M. tuberculosis for MAP genotyping. Additionally, detection of the most efficient loci combinations for molecular epidemiological investigations of MAP isolates. The discriminatory capacity and applicability of 15 known loci [2 exact tandem repeat (ETR) loci, 6 MIRU loci, 4 Mtub loci, and 3 Queen's University of Belfast (QUB) group loci] were assessed using 26 isolates from two cattle herds (Holstein Frisian) in El buhaira and Giza Governorates at north of Egypt. The results proved the presence of 12 different genotypes. All the used loci gave Hunter-Gaston discrimination index of DI = 0.963 while the ten loci (Mtub04, MIRU10, QUB11b, MIRU26, QUB26, QUB4156, MIRU04, ETRC, Mtub30, and Mtub39) were highly discriminating with DI = 0.956. Moreover, the five loci (Mtub21, MIRU31, MIRU16, MIRU40, and ETRA) gave moderate discriminatory power with DI = 0.839. The MIRU31 locus expressed no polymorphism among strains. MIRU-VNTR typing generally proved applicability and high discriminatory power with DI = 0.963. The ten highly discriminating DI = 0.956 proved to be the most suitable for the first-line genotyping of MAP from cattle, with nearly similar resolving ability as all the 15 loci. MIRU-VNTR proved fastness, efficiency, and feasibility in genotyping of MAP from cattle in Egypt.

The rapid detection and differentiation of Mycobacterium tuberculosis complex members from cattle and water buffaloes in the delta area of Egypt, using a combination of real-time and conventional PCR.

Mycobacterium tuberculosis complex (MTBC) has the potential to cause infections in animals and human beings. The combination of real-time PCR targeting atpE or lpqT and RD1, and conventional PCR targeting regions of difference (RD) was rigorously evaluated as a descriptive molecular epidemiology tool. A total of 2100 cattle and buffaloes from the Menoufia, Sharkia, Gharbia, Dakahlia, Elbuhaira, and Cairo Governorates were tested by single intradermal comparative cervical tuberculin test (SICCT). The frequency was 74/2100 (3.5%); thereafter, on post-mortem examination (PM), 49/74 (66.21%) showed visible lesions, while only 25/74 (33.78%) were non-visible with a significant difference of (p < .0001). Real-time PCR using atpE or lpqT and RD1 similarly detected the frequency of infection, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy, which represented 73/74 (98.65%), 98.65, 100, 100, 90.91, and 98.81%, respectively. Multiplex conventional PCR targeting RD1, 4, 9, and 12 confirmed that 49/74 (66.21%) were M.bovis, while the simplex conventional PCR targeting RD4 and RD9 confirmed mycobacteria in 71/74 (95.94%) samples, which included 61/74 (82.4%) M.bovis and 2/74 (2.7%) M.tuberculosis. Additionally, 8/74 (10.8%) exhibited mixed patterns of M.bovis and M.tuberculosis, and 3/74 (4.05%) were negative. There was a significant difference between the results of simplex and multiplex conventional PCR (p < .0001). Moreover, simplex conventional PCR targeting RD4 and RD9 proved higher sensitivity, specificity, positive predictive value, negative predictive value, and accuracy, which were 95.95, 100, 100, 76.92, and 96.43%, respectively, when compared with the values of multiplex conventional PCR targeting RD1,4,9, and 12 which were 66.22, 100, 100, 28.57, and 70.24%, respectively. The repeatability results of real-time PCR using atpE or lpqT and RD1, and simplex conventional PCR targeting RD4 and RD9 were acceptable. In conclusion, a combination of real-time PCR using atpE or lpqT and RD1 as the first step with simplex conventional PCR targeting RD4 and RD9 as the second step was reliable as a diagnostic tool.

A first insight into the application of high discriminatory MIRU-VNTR typing using QIAxcel technology for genotyping Mycobacterium bovis isolated from the Delta area in Egypt.

Mycobacterium bovis is a notorious infectious agent leading to serious economic losses for cattle farms worldwide. Analysis of the widely spreading genotypes is vital for tracing infections, understanding transmission dynamics, and controlling the cluster growth. This study aimed to evaluate the discrimination ability of 15 mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR) loci and to assess the extremely efficient loci subset for molecular epidemiological investigations of M.bovis from farms in the Delta area of Egypt. The discriminating ability of MIRU-VNTR genotyping using 15 loci {2 exact tandem repeat (ETR) loci, 6 MIRU loci, 4 Mtub loci, and 3 Queen's University of Belfast (QUB) group loci)} were evaluated on 61 M.bovis isolates from cattle (Holstein Frisian) and buffaloes. The results indicate that there are 48 genotypes: 3 unique genotypes and 45 genotypes with shared similarities. Using the MIRU-VNTRplus database, M.bovis ID 7540/01 and ID 5346/02 were the nearest lineages to both groups. Six loci (MIRU10, QUB11b, QUB26, ETRA, Mtub30, and Mtub39) were highly discriminating, seven other loci (Mtub21, MIRU26, QUB4156, MIRU04 (ETRD), MIRU16, MIRU 40, and ETRC) gave moderate discriminatory power, and the last two loci (Mtub04 and MIRU31) were poorly discriminative. MIRU-VNTR typing generally proved efficacy and high discriminatory power, with a collective allele's diversification of 0.9641. Both the six highly discriminating (DI = 0.9492) and the seven moderately discriminating loci (DI = 0.9269) evidenced to be suitable for M.bovis first-step initial genotyping from cattle herds in Egypt. MIRU-VNTR is rapid and effective in the genotyping of M.bovis from cattle and buffaloes in Egypt.

LCD array and IS900 efficiency in relation to traditional diagnostic techniques for diagnosis of Mycobacterium avium subspecies paratuberculosis in cattle in Egypt.

This study aimed to compare traditional tests (Johnin test, fecal staining and fecal culture) with advanced laboratory tests (ELISA, LCD array and IS900 PCR) for detection of Johne's disease. A total of 365 Holstein-Friesian dairy cattle (40 express profuse diarrhea unresponsive to treatment and 325 contacting them) tested with Johnin test, blood collected for ELISA and fecal samples for fecal staining as well as fecal culture, application of LCD array and PCR using IS900 on DNA extracted from Mycobacterium paratuberculosis bacilli (from feces and culture). Johnin test was 40/40 (100%) and 25/325 (7.69%), fecal staining was 13 (37.1%) and 2 (50%), ELISA was 35/40 (87.5%) and 4/25 (16%) for clinical cattle and apparently healthy contacting them respectively. Isolation was 12/13 (92.3%) of the (Johnin test +ve, ELISA +ve and Acid Fast Bacilli +ve) from the clinically positive cattle and 1/2 (50%) of the (Johnin test +ve, ELISA +ve and Acid Fast Bacilli +ve) from apparently healthy contacting them while LCD array and IS900 gave 100% confirming the isolation results. In conclusion, LCD array depending on 16S RNA and DNA hybridization with specific probes for detection of M. paratuberculosis are fast, sensitive and labor-saving when combined with IS900.