- Volume 70, Issue 2, 2021

Cholera is a severe diarrhoeal disease that spreads rapidly and affects millions of people each year, resulting in tens of thousands of deaths. The disease is caused by Vibrio cholerae O1 and is characterized by watery diarrhoea that can be lethal if not properly treated. Cholera had not been reported in South America from the late 1800s until 1991, when it was introduced in Peru, wreaking havoc in one of the biggest epidemics reported to date. Within a year, the disease had spread to most of the Latin American region, resulting in millions of cases and thousands of deaths in all affected countries. Despite its aggressive entry, cholera virtually disappeared from the continent after 1999. The progression of the entire epidemic was well documented, making it an ideal model to understand cholera dynamics. In this review, we highlight how the synergy of socioeconomic, political and ecological factors led to the emergence, rapid spread and eventual disappearance of cholera in Latin America. We discuss how measures implemented during the cholera epidemic drastically changed its course and continental dynamics. Finally, we synthesize our findings and highlight potential lessons that can be learned for efficient and standardized cholera management programmes during future outbreaks in non-endemic areas.

Introduction. Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent threat in the USA and are associated with adverse clinical and economic outcomes. Several studies have evaluated risk factors for acquiring CRE versus carbapenem-susceptible Enterobacteriaceae , identifying antibiotic use and length of hospital stay as major players. However, no studies have compared risk factors for CRE colonization versus infection.

Hypothesis/Gap Statement. Patients with CRE infection will have different risk factors and worse clinical outcomes than patients with CRE colonization.

Aim. To assess clinical outcomes in patients with CRE infection versus CRE colonization.

Methodology. A retrospective cohort of adult patients admitted between 1 June 2013 and 31 July 2018 with the first positive CRE culture from any source was performed. Patients were divided into two groups: CRE infection versus CRE colonization. Data collected included demographics, comorbidities, past antimicrobial usage and clinical outcomes (length of stay, in-hospital mortality). The primary outcome was infection-related length of stay. Data analysis was performed utilizing SPSS with a two-sided P value of less than 0.05 considered statistically significant.

Results. A total of 56 patients were included (32 with infection; 24 with colonization). Baseline characteristics were similar between both groups. Infected patients were more likely to have higher actual body weight compared to colonized patients (P=0.03). CRE-infected patients had a longer infection-related hospital stay [12 days (5–20) and 7.5 days (1–13), respectively; P=0.08], but in-hospital mortality was similar between infected and colonized patients (37.5 and 29.2 %, respectively; P=0.30). Patients with infection were more likely to have previous exposure to levofloxacin (P=0.02) and trimethoprim/sulfamethoxazole (P=0.03) for a median of 9 days compared to those with colonization. The most common source of CRE in infected patients was the blood compared to respiratory sources in colonized patients.

Conclusion. CRE infection as opposed to colonization was more common in patients with previous exposure to levofloxacin and trimethoprim/sulfamethoxazole and those with higher actual body weight.

Saliva has recently been proposed as a suitable specimen for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Use of saliva as a diagnostic specimen may present opportunities for SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) testing in remote and low-resource settings. Determining the stability of SARS-CoV-2 RNA in saliva over time is an important step in determining optimal storage and transport times. We undertook an in vitro study to assess whether SARS-CoV-2 could be detected in contrived saliva samples. The contrived saliva samples comprised 10 ml pooled saliva spiked with gamma-irradiated SARS-CoV-2 to achieve a concentration of 2.58×104 copies ml SARS-CoV-2, which was subsequently divided into 2 ml aliquots comprising: (i) neat saliva; and a 1 : 1 dilution with (ii) normal saline; (iii) viral transport media, and (iv) liquid Amies medium. Contrived samples were made in quadruplicate, with two samples of each stored at either: (i) room temperature or (ii) 4 °C. SARS-CoV-2 was detected in all SARS-CoV-2 spiked samples at time point 0, day 1, 3 and 7 at both storage temperatures using the N gene RT-PCR assay and time point 0, day 1 and day 7 using the Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, USA) RT-PCR assay. The ability to detect SARS-CoV-2 in saliva over a 1 week period is an important finding that presents further opportunities for saliva testing as a diagnostic specimen for the diagnosis of SARS-CoV-2.

Introduction. Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species level

Gap Statement Available protocols for the molecular identification of fungi from formalin-fixed and paraffin-embedded (FFPE) tissues have limitations in terms of extraction and target selection, and standardisation.

Aim. Development of sequence-based fungal identification protocol after extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissues.

Methodology. A total of 63 FFPE tissues from histopathology proven IFI cases were used to standardize the DNA extraction (commercial QIAamp kit-based extraction and conventional phenol-chloroform-isoamyl alcohol [PCI] method) and sequence-based fungal identification protocols. The PCR targeted different ribosomal DNA (rDNA) regions including complete internal transcribed spacer (ITS1-5.8S-ITS2), separate ITS1 and ITS2, 18S and D1/D2 of 28S regions. Semi-nested PCR targeting Mucorales-specific 18S rDNA region was performed in tissues having aseptate hyphae. The optimized ITS1-PCR protocol was evaluated in 119 FFPE tissues containing septate hyphae or yeast, and Mucorales-specific semi-nested PCR in 126 FFPE tissues containing aseptate hyphae.

Results. The DNA yield by conventional PCI method was significantly higher (P<0.0001) than commercial kit, though the quality of DNA was similar by both protocols. The test accuracy was best while using ITS1 (61.9 %) as the target compared to 7.9, 29.9 and 22.2 % on targeting ITS1-5.8S-ITS2, ITS2, the D1/D2 region of 28S, respectively. The test accuracies of ITS1-PCR in tissues containing septate hyphae, aseptate hyphae and yeasts were 75.5, 18.7 and 100 %, respectively. The amplification (targeting ITS1 region) improved by increasing the thickness of tissue section (up to 50 µm) used for DNA extraction. ITS1-PCR protocol could amplify fungal DNA in 76 (63.8 %) tissues and Mucorales-specific semi-nested PCR in 86 (68.3 %) tissues.

Conclusion. Conventional PCI-based DNA extraction from thick tissue (50 µm) may be used until optimal commercial fungal DNA extraction kit is developed. Subsequent ITS1-PCR for septate fungi and yeast, and semi-nested PCR targeting 18S rDNA for Mucorales are recommended to identify the fungus in FFPE tissues.

Introduction. Chlamydia psittaci is primarily a pathogen of birds but can also cause disease in other species. Equine reproductive loss caused by C. psittaci has recently been identified in Australia where cases of human disease were also reported in individuals exposed to foetal membranes from an ill neonatal foal in New South Wales.

Hypothesis/Gap Statement. The prevalence of C. psittaci in association with equine reproductive over time and in different regions of Australia is not known.

Aim. This study was conducted to detect C. psittaci in equine abortion cases in Australia using archived samples spanning 25 years.

Methodology. We tested for C. psittaci in 600 equine abortion cases reported in Australia between 1994 to 2019 using a Chlamydiaceae real-time quantitative PCR assay targeting the 16S rRNA gene followed by high-resolution melt curve analysis. Genotyping and phylogenetic analysis was performed on positive samples.

Results. The overall prevalence of C. psittaci in material from equine abortion cases was 6.5 %. C. psittaci -positive cases were detected in most years that were represented in this study and occurred in Victoria (prevalence of 7.6 %), New South Wales (prevalence of 3.9 %) and South Australia (prevalence of 15.4 %). Genotyping and phylogenetic analysis showed that the C. psittaci detected in the equine abortion cases clustered with the parrot-associated 6BC clade (genotype A/ST24), indicating that infection of horses may be due to spillover from native Australian parrots.

Conclusion. This work suggests that C. psittaci has been a significant agent of equine abortion in Australia for several decades and underscores the importance of taking appropriate protective measures to avoid infection when handling equine aborted material.

Introduction. Serratia marcescens is a bacterial pathogen that causes ventilator-associated pneumonia and ocular infections. The FlhD and FlhC proteins complex to form a heteromeric transcription factor whose regulon, in S. marcescens , regulates genes for the production of flagellum, phospholipase A and the cytolysin ShlA. The previously identified mutation, scrp-31, resulted in highly elevated expression of the flhDC operon. The scrp-31 mutant was observed to be more cytotoxic to human airway and ocular surface epithelial cells than the wild-type bacteria and the present study sought to identify the mechanism underlying the increased cytotoxicity phenotype.

Hypothesis/Gap Statement. Although FlhC and FlhD have been implicated as virulence determinants, the mechanisms by which these proteins regulate bacterial cytotoxicity to different cell types remains unclear.

Aim. This study aimed to evaluate the mechanisms of FlhDC-mediated cytotoxicity to human epithelial cells by S. marcescens .

Methodology. Wild-type and mutant bacteria and bacterial secretomes were used to challenge airway and ocular surface cell lines as evaluated by resazurin and calcein AM staining. Pathogenesis was further tested using a Galleria mellonella infection model.

Results. The increased cytotoxicity of scrp-31 bacteria and secretomes to both cell lines was eliminated by mutation of flhD and shlA. Mutation of the flagellin gene had no impact on cytotoxicity under any tested condition. Elimination of the phospholipase gene, phlA, had no effect on bacteria-induced cytotoxicity to either cell line, but reduced cytotoxicity caused by secretomes to airway epithelial cells. Mutation of flhD and shlA, but not phlA, reduced bacterial killing of G. mellonella larvae.

Conclusion. This study indicates that the S. marcescens FlhDC-regulated secreted proteins PhlA and ShlA, but not flagellin, are cytotoxic to airway and ocular surface cells and demonstrates differences in human epithelial cell susceptibility to PhlA.

Introduction. The widespread of hepatitis B virus is a severe global public problem, and the infant hepatitis B vaccine has been proved effective. But the failure of the immune response was reported in studies, and boosters were recommended. There were few studies about the effect of hepatitis B vaccine boosters in different levels of the epidemic area.

Hypothesis. Booster immunization is recommended because there may be a lack of immunization in infants vaccinated with the hepatitis B vaccine. In order to verify the effectiveness of booster immunization, this study hypothesized that it worked well in different levels of endemic areas.

Aim. To evaluate the effects of hepatitis B vaccine boosters on children from the areas with different prevalence of hepatitis B whose hepatitis B surface antibody (anti-HBs) were negative (<10 mIU ml−1).

Methodology. A total of 940 children were initially enrolled in screening; however, 421 were excluded. The participants were divided into three groups according to the different areas they come from: group I, low epidemic area; group II, middle epidemic area; and group III, high epidemic area. In total, 519 subjects were administered three doses of booster hepatitis B vaccine (0–1–6 months, 10 µg). The antibody titre changes were examined at four time points: 1 month after dose 1, 1 month, 1 year and 5 years after dose 3.

Results. The protective seroconversion rates in three groups were 96.30, 97.16, 96.63% at 1 month after dose 1, and 100.00, 100.00, 100.00% at 1 month after dose 3, and 97.79, 100.00, 98.50% at 1 year after dose 3, and 90.77, 93.67, 93.59% at 5 years after dose 3 (P>0.05).

Conclusions. This study demonstrates that three doses of booster vaccination have a longtime effect, no matter whether it is in low, middle or high prevalence areas in which subjects live.