- Volume 3, Issue 3, 2021

Conventional cell-culture viral quantification methods, namely viral plaque and 50 % tissue culture infective dose assays, are time-consuming, subjective and are not suitable for routine testing. The viral plaque formation assay is the main method utilized for Rift Valley fever virus (RVFV) clone 13 quantification. The RVFV is a mosquito-borne RNA Phlebovirus belonging to the family Bunyaviridae. The virus comprises a single serotype and causes the zoonotic Rift Valley fever disease. The real-time cell analysis (RTCA) system has been developed for the monitoring of cell growth, cell adhesion, cell viability and mortality using electronic impedance technology. In this study, Vero cell growth kinetics and RVFV clone 13 replication kinetics were investigated in a roller bottle and RTCA systems. In roller bottles, Vero cell growth was measured by cell counts through trypan blue staining, whilst impedance expressed as the cell index (CI) was used for Vero growth measurement in the RTCA system. Similar growth patterns were observed in both roller bottle and RTCA systems. Exponential growth phase was observed between 48 and 100 h, followed by a stationary phase from 100 to 120 h, before cell death was observed. Viral plaque assay quantification of RVFV clone 13 in the roller bottle system and the time required for the CI to decrease 50 % after virus infection (CIT50) in the RTCA system were comparable. The highest RVFV clone 13 titre was obtained at 120 h in both roller bottle and RTCA systems. An increase in time for cytopathic effect (CPE) formation was observed with a decrease in the concentration of the virus used to infect the RTCA plates. A positive correlation was observed between the viral concentration and the time for a CPE and was used to calculate CIT50. A similar correlation was observed between the viral concentration and the time for a CPE in the roller bottle system. This study shows that the RTCA system can be used as an alternative method for conducting cell culture kinetics and viral quantification.

Many Australian native honeys possess significant antimicrobial properties due to the production of hydrogen peroxide (H2O2) by glucose oxidase, an enzyme derived from the honeybee. The level of H2O2 produced in different honey samples is highly variable, and factors governing its production and stability are not well understood. In this study, highly active Australian honeys that had been stored for >10 years lost up to 54 % of their antibacterial activity, although almost all retained sufficient activity to be considered potentially therapeutically useful. We used a simple colourimetric assay to quantify H2O2 production. Although we found a significant correlation between H2O2 production and antibacterial activity across diverse honey samples, variation in H2O2 only explained 47 % of the variation observed in activity, limiting the assay as a screening tool and highlighting the complexity of the relationship between H2O2 and the killing power of honey. To further examine this, we tested whether H2O2 detection in honey was being inhibited by pigmented compounds and if H2O2 might be directly degraded in some honey samples. We found no correlation between H2O2 detection and honey colour. Some honey samples rapidly lost endogenous and spiked H2O2, suggesting that components in honey, such as catalase or antioxidant polyphenols, may degrade or quench H2O2. Despite this rapid loss of H2O2, these honeys had significant peroxide-based antibacterial activity, indicating a complex relationship between H2O2 and other honey components that may act synergistically to augment activity.

Mycobacterium tuberculosis ranks among the top 10 causes of deaths in Nepal despite the country having a long history of national tuberculosis prevention programmes that have proved very successful in the control of tuberculosis. Several cases of active or latent tuberculosis are still missing despite that the number of infected individuals is increasing each year. Microscopy has its own limitations and factors like low bacterial load, quality of sample, quality of smear, experience of microscopist etc. influence the overall sensitivity of the test. The implementation of a molecular technique-based rapid, point-of-care testing system offers higher sensitivity in the early diagnosis of tuberculosis. Cepheid GeneXpert is the most commonly used molecular technology in Nepal. It is a cartridge-based semi-quantitative, nested real-time PCR-based diagnostic system. It detects mutations in the beta-subunit of RNA polymerase (rpoB) gene that lead to rifampicin resistance (RR) in M. tuberculosis complex. The present study aims to increase our understanding of the epidemiology of mutations in the rpoB gene in tuberculosis-positive patients by using the Xpert MTB/RIF assay in a rural setting in Pyuthan Hospital, Nepal. Sputum from 2733 patients was tested for the diagnosis of tuberculosis using the Cepheid GeneXpert system between July 2018 and January 2020 at Pyuthan Hospital. Two hundred and ninety-seven of these samples (10.86 %) were positive for M. tuberculosis , of which 3.3 % (10/297) were rifampicin-resistant. Among rifampicin-resistant tuberculosis (RR-TB) patients, 50.0 % (5/10) showed mutations located in codons 529–533 (probe E) of the rpoB gene, followed by others. The GeneXpert system can be a convenient, highly sensitive, rapid and accurate tool for the diagnosis of tuberculosis, also identifying RR-TB and at the same time determining the molecular epidemiology of rifampin resistance-associated mutations in rural and/or resource-limited laboratory settings.

Background. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for burn-wound infection. High incidence, infection severity and increasing resistance characterize P. aeruginosa -induced burn infection.

Purpose. To estimate quorum-sensing (QS)-dependent virulence factors of P. aeruginosa isolates from burn wounds and correlate it to the presence of QS genes.

Methods. A cross-sectional descriptive study included 50 P . aeruginosa isolates from burn patients in Mansoura University Plastic and Burn Hospital, Egypt. Antibiotic sensitivity tests were done. All isolates were tested for their ability to produce biofilm using a micro-titration assay method. Protease, pyocyanin and rhamnolipid virulence factors were determined using skimmed milk agar, King’s A medium and CTAB agar test, respectively. The identity of QS lasR and rhlR genes was confirmed using PCR.

Results. In total, 86 % of isolates had proteolytic activity. Production of pyocyanin pigment was manifested in 66 % of isolates. Altogether, 76 % of isolates were rhamnolipid producers. Biofilm formation was detected in 96 % of isolates. QS lasR and rhlR genes were harboured by nearly all isolates except three isolates were negative for both lasR and rhlR genes and two isolates were positive for lasR gene and negative for rhlR gene. Forty-nine isolates were considered as extremely QS-proficient strains as they produced QS-dependent virulence factors. In contrast, one isolate was a QS deficient strain.

Conclusions. QS affects P. aeruginosa virulence-factor production and biofilm in burn wounds. Isolates containing lasR and rhlR seem to be a crucial regulator of virulence factors and biofilm formation in P. aeruginosa whereas the lasR gene positively regulates biofilm formation, proteolytic activity, pyocyanin production and rhamnolipid biosurfactant synthesis. The QS regulatory RhlR gene affects protease and rhamnolipid production positively.

Urogenital Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection throughout the world. While progress has been made to better understand how type strains develop and respond to environmental stress in vitro, very few studies have examined how clinical isolates behave under similar conditions. Here, we examined the development and persistence phenotypes of several clinical isolates, to determine how similar they are to each other, and the type strain C. trachomatis D/UW-3/Cx. The type strain was shown to produce infectious progeny at a higher magnitude than each of the clinical isolates, in each of the six tested cell lines. All chlamydial strains produced the highest number of infectious progeny at 44 h post-infection in the McCoy B murine fibroblast cell line, yet showed higher levels of infectivity in the MCF-7 human epithelial cell line. The clinical isolates were shown to be more susceptible than the type strain to the effects of penicillin and iron deprivation persistence models in the MCF-7 cell line. While subtle differences between clinical isolates were observed throughout the experiments conducted, no significant differences were identified. This study reinforces the importance of examining clinical isolates when trying to relate in vitro data to clinical outcomes, as well as the importance of considering the adaptations many type strains have to being cultured in vitro.

Human noroviruses (HuNoVs) circulate globally, affect all age groups and place a substantial burden upon health services. High genetic diversity leading to antigenic variation plays a significant role in HuNoV epidemiology, driving periodic global emergence of epidemic variants. Studies have suggested that immunocompromised individuals may be a reservoir for such epidemic variants, but studies investigating the diversity and emergence of HuNoV variants in immunocompetent individuals are underrepresented. To address this, we sequenced the genomes of HuNoVs present in samples collected longitudinally from one immunocompetent (acute infection) and one immunocompromised (chronic infection) patient. A broadly reactive HuNoV capture-based method was used to concentrate the virus present in these specimens prior to massively parallel sequencing to recover near complete viral genomes. Using a novel bioinformatics pipeline, we demonstrated that persistent minor alleles were present in both acute and chronic infections, and that minor allele frequencies represented a larger proportion of the population during chronic infection. In acute infection, minor alleles were more evenly spread across the genome, although present at much lower frequencies, and therefore difficult to discern from error. By contrast, in the chronic infection, more minor alleles were present in the minor structural protein. No non-synonymous minor alleles were detected in the major structural protein over the short sampling period of the HuNoV chronic infection, suggesting where immune pressure is variable or non-existent, epidemic variants could emerge over longer periods of infection by random chance.

HSV-1 envelope glycoprotein E (gE) is important for viral egress and cell-to-cell spread but the host protein(s) involved in these functions have yet to be determined. We aimed to investigate a role for the Arp2/3 complex and actin regulation in viral egress based on the identification of a WAVE Regulatory Complex (WRC) Interacting Receptor Sequence (WIRS) in the cytoplasmic tail (CT) of gE. A WIRS-dependent interaction between the gE(CT) and subunits of the WRC was demonstrated by GST-pulldown assay and a role for the Arp2/3 complex in cell-to-cell spread was also observed by plaque assay. Subsequent study of a recombinant HSV-1 gE WIRS-mutant found no significant changes to viral production and release based on growth kinetics studies, or changes to plaque and comet size in various cell types, suggesting no function for the motif in cell-to-cell spread. GFP-Trap pulldown and proximity ligation assays were unable to confirm a WIRS-dependent interaction between gE and the WRC in human cell lines though the WIRS-independent interaction observed in situ warrants further study. Confocal microscopy of infected cells of neuronal origin identified no impairment of gE WIRS-mutant HSV-1 anterograde transport along axons. We propose that the identified gE WIRS motif does not function directly in recruitment of the WRC in human cells, in cell-to-cell spread of virus or in anterograde transport along axons. Further studies are needed to understand how HSV-1 manipulates and traverses the actin cytoskeleton and how gE may contribute to these processes in a WIRS-independent manner.

Introduction. Empirical evidence suggests that Portulaca oleracea L. treats enteric infections, including dysentery, cholera, and acute infectious gastroenteritis.

Aim. The aim of this study is to clarify the growth-inhibitory effects of Portulaca oleracea L. extract against 56 strains of intestinal pathogens.

Methodology. ‘Gogyo-so-cha (GSC)’ was used as the P. oleracea L. formulation. A growth curve analysis was used to measure the growth-inhibitory effects of GSC, and Shiga toxin induction was measured using the latex agglutination test.

Results. GSC demonstrated strong bactericidal effects against Shigella dysenteriae and Vibrio cholerae strains from various isolates. GSC demonstrated weak or no bactericidal effects against intestinal commensal bacteria, including Enterococcus spp. and Escherichia coli . GSC did not induce Shigella toxins.

Conclusion. GSC significantly inhibited the growth of intestinal pathogens, including S. dysenteriae and V. cholerae , without adversely affecting the intestinal flora, supporting the usage of GSC in traditional Chinese medicine. Taken together, GSC would be of immense value in the developing world, where diarrhoeal infectious diseases continue to pose a major health risk.

Background. Since the work of Band et al. in 1990 (Proc. Natl. Acad. Sci. USA 87:463–467), several studies have suggested a possible link between the pathogenesis of breast cancer and viral infection. Infection with oncogenic HPV has been one of the viruses implicated in breast cancer cases worldwide.

Objective. To investigate the presence of HPV DNA in archived paraffin-embedded breast cancer cases at the University Hospital of Brazzaville and to assess the association between viral HPV infections and clinicopathological features.

Methods. A total of 40 formalin-fixed paraffin-embedded (FFPE) biopsies were retrospectively collected and available information was recorded. HPV detection and genotyping were performed by real-time PCR by GeneXpert technology (Cepheid, Sunnyvale, USA).

Results. The mean age was 51.1±11.4 years (range 22–75 years; median was 47). Overall, HPV DNA was detected in six (15%) breast carcinoma samples. HPV-16, the most common genotype was identified in 83.7 % of all samples. HPV porting with clinicopathological features showed no significant difference (P>0.05). However, a statistically significant difference was observed between HPV infection and SBR grade (P=0.05).

Conclusion. Our study described a high prevalence of HPV-HR in breast cancer cases in the Congolese woman. Future type case-control studies are necessary to better describe the potential role of HPV in the occurrence of breast cancer in Congo.

The aim of this prospective study was to investigate the serotypes and antibiotic susceptibility of S. pneumoniae carried by children infected with HIV before and after vaccination with the seven-valent pneumococcal conjugate vaccine in Jakarta, Indonesia in 2013. We collected nasopharyngeal swab specimens from 52 children pre-vaccination and 6 months post-vaccination. Serotyping was performed by conventional multiplex polymerase chain reaction and Quellung reaction. The antibiotic susceptibility profile was obtained by disc diffusion. We determined that 27 (52%) and 24 (46%) of the 52 children carried S. pneumoniae during pre- and post-vaccination periods, respectively with the majority of the isolates being non-vaccine type strains (85% pre-vaccination and 75% post-vaccination). Serotypes 34, 6C, and 16F (two strains each) were the most commonly identified serotypes at pre-vaccination. Serotypes 23A (three strains) and 19F (two strains) were the most commonly identified serotypes post-vaccination. In general, isolates were most commonly susceptible to chloramphenicol (88%) and clindamycin (88%), followed by erythromycin (84%), trimethoprim-sulphamethoxazole (69%), tetracycline (61%), and penicillin (59%). In conclusion, serotypes of S. pneumoniae isolated from the nasopharynges of children infected with HIV varied and were more likely to be non-vaccine type strains both before and after vaccination.

Segmented filamentous bacteria (SFB) are thought to play a role in small intestine immunological maturation. Studies in weanling mice have shown a positive correlation between ileal SFB abundance and plasma and faecal interleukin 17 (IL-17) and immunoglobulin A (IgA) concentrations. Although the first observation of SFB presence was reported in rats, most studies use mice. The size of the mouse ileum is a limitation whereas the rat could be a suitable alternative for sufficient samples. Changes in SFB abundance over time in rats were hypothesized to follow the pattern reported in mice and infants. We characterized the profile of SFB colonization in the ileum tissue and contents and its correlation with two immune markers of gastrointestinal tract (GIT) maturation. We also compared two published ileum collection techniques to determine which yields data on SFB abundance with least variability. Whole ileal tissue and ileal mucosal scrapings were collected from 20- to 32-day-old Sprague-Dawley rats. SFB abundance was quantified from proximal, middle and distal ileal tissues, contents and faeces by quantitative PCR using SFB-specific primers. Antibody-specific ELISAs were used to determine IL-17 and IgA concentrations. Significant differences in SFB abundance were observed from whole and scraped tissues peaking at day 22. Variability in whole ileum data was less, favouring it as a better collection technique. A similar pattern of SFB abundance was observed in ileum contents and faeces peaking at day 24, suggesting faeces can be a proxy for ileal SFB abundance. SFB abundance at day 26 was higher in females than males across all samples. There were significant differences in IgA concentration between days 20, 30 and 32 and none in IL-17 concentration, which was different from reports in mice and infants.

Enterococcus faecalis and E. faecium are Gram-positive bacteria that normally inhabit the human gastrointestinal tract. They are also opportunistic pathogens and can cause nosocomial infection outbreaks. To prevent the spread of nosocomial infections, hospitals may rely on screening methods to identify patients colonized with multidrug-resistant organisms including vancomycin-resistant enterococci (VRE). Spectra VRE agar (Remel) contains vancomycin and other medium components that select for VRE and phenotypically differentiate between E. faecalis and E. faecium by colony colour. We obtained 66 de-identified rectal swab cultures on Spectra VRE agar that were obtained during routine patient admission surveillance at a hospital system in Dallas, Texas, USA. We analysed 90 presumptive VRE from 61 of the Spectra VRE agar cultures using molecular and culture methods. Using ddl typing, 55 were found to be E. faecium and 32 were found to be E. faecalis . While most of the E. faecium were positive for the vanA gene by PCR (52 of 55 strains), few of the E. faecalis were positive for either vanA or vanB (five of 32 strains). The 27 E. faecalis vanA- and vanB-negative strains could not be recultured on Spectra VRE agar. Overall, we found that Spectra VRE agar performed robustly for the identification of vancomycin-resistant E. faecium , but presumptive false positives were obtained for vancomycin-resistant E. faecalis .

Foodborne campylobacteriosis is the most common cause of human bacterial enteritis in Germany. Campylobacter jejuni and Campylobacter coli are the main causative agents for enteric disease, but a number of other species are involved, including rare ones. These rare Campylobacter spp. are emerging zoonotic pathogens in humans due to increasing international movement of supplies, livestock and people. Campylobacter lanienae was first isolated from healthy abattoir workers in Switzerland and at first its pathogenic potential for humans was considered to be low. Recently, the first case of Campylobacter lanienae -associated human enteritis was reported in Canada. Here, we describe a case of mild Campylobacter lanienae -associated enteritis with subsequent asymptomatic excretion in a butcher. The isolate is available at the TLV strain collection (no. TP00333/18). This first reported case of human Campylobacter lanienae campylobacteriosis in Germany demonstrates the agent’s likely zoonotic pathogenicity.

Streptococcus gallolyticus ssp pasteurianus (SGp) is an uncommon but increasingly recognized cause of neonatal sepsis and meningitis. Liver abscess in neonates is extremely rare. But liver abscess due to SG has never been reported in the literature. We present the first case of liver abscess due to SGp in a late preterm infant. A female infant was born at 36 weeks via normal vaginal delivery to a mother with unremarkable antenatal history. She had progressively worsening respiratory distress since birth and was intubated at 13 h of life. One dose of surfactant was delivered and ventilation continued. Parenteral crystalline Penicillin and Gentamicin were initiated and her blood culture at birth grew SGp. She had a spike of fever on day 5 of life. An ultrasound (US) scan of the abdomen was included in the septic work up. A multi-septated cystic liver abscess was noted in the right lobe of the liver. As there was inadequate response to appropriate intravenous antibiotics, needle aspiration and biopsy were performed on day 35 of life. Aspirate was sterile and histopathology confirmed a liver abscess. The patient continued to be treated with antibiotics for 8 weeks with serial US scans of the liver showing resolution of the abscess. Increasing awareness among paediatric and neonatal fraternity about these new emerging bacterial infections can facilitate early diagnosis and treatment.

Critically ill patients with coronavirus disease 2019 (COVID-19) are at risk of invasive pulmonary aspergillosis. The clinical manifestations of a superimposed fungal infection in those patients are difficult to underpin. This is compounded by the non-specific radiological findings associated with this infection and the challenges associated with performing bronchoscopy in COVID-19 patients. We would like to present two COVID-19 cases who developed secondary Aspergillus pulmonary infection in the intensive care unit as shown by respiratory cultures, serum galactomannan and radiological images. Despite advanced intensive care and use of antifungal agents, both patients died eventually. This report illustrates the negative impact of secondary Aspergillosis and calls for the need of increased awareness of COVID19-associated pulmonary aspergillosis.

Introduction. Bacillus clausii as a probiotic supplement is increasingly used in both adult and paediatric patient populations. There is limited awareness about potential adverse effects.

Case presentation. We report a case of prolonged (111 days) B. clausii bacteraemia after brief probiotic use in a 17-month-old immunocompetent child, without a definite focus of infection and in the absence of predisposing risk factors or underlying co-morbidities. We identified seven probiotic use-associated cases of prolonged B. clausii bacteraemia (mean duration [range] 64 days [14–93 days] where data were available) in the literature, all with underlying co-morbidities.

Conclusion. B. clausii probiotic preparations may cause prolonged bacteraemia, rendering patients with underlying co-morbidities as well as those with unrecognized risk factors vulnerable for significant infectious complications.