Case reports
Three cases of laboratory-acquired Salmonella Typhi infection occurred over the period 2012 to 2016 in South Africa. We describe these as follows.
Case one
In September 2012, a laboratory technologist working at Laboratory-A presented to her clinician with signs of severe malaise, fever and mild diarrhoea. The technologist had six years of working experience in a clinical microbiology laboratory. Blood cultures were collected for laboratory testing. The diagnostic laboratory identified Salmonella using the MALDI Biotyper and the isolate failed to agglutinate in Salmonella Typhi-specific antisera. Subsequently, the isolate was reported to the clinician as NTS, susceptible to fluoroquinolones. The patient was treated with seven days of oral ciprofloxacin. The isolate was referred to the Centre for Enteric Diseases (CED) at the National Institute for Communicable Diseases (NICD), for confirmation of identification. The CED confirmed Salmonella Typhi with intermediate resistance to fluoroquinolones [ciprofloxacin minimum inhibitory concentration (MIC), 0.25 μg/ml] and susceptibility to azithromycin (MIC, 4 μg/ml). The patient, though recovered, was still excreting Salmonella Typhi in her stool and was then treated with a combination of azithromycin and fluoroquinolone for 14 days. Subsequent stools tested negative for Salmonella Typhi. The patient, being a laboratory technologist with frequent exposure to Salmonella Typhi, had received Salmonella Typhi vaccination nine months earlier as part of an occupational health program. On investigation, it was found that this vaccine was part of a batch recalled by the manufacturer, because of potentially low (below specification) antigen content [19] .
Case two
In February 2016, a trainee clinical pathology resident working on the stool bench at a clinical microbiology laboratory (Laboratory-B), presented to a physician with fever, nausea and vomiting. The patient was admitted to hospital and discharged three days later. Salmonella Typhi was cultured from microbiological cultures of blood taken on admission. The isolate was referred to the CED for confirmation of identification. The CED confirmed Salmonella Typhi with susceptibility to fluoroquinolones (ciprofloxacin MIC, 0.008 μg/ml) and to azithromycin (MIC, 8 μg/ml). The patient responded well to levofloxacin therapy. Further investigation revealed that the patient had recently serotyped a clinical isolate of Salmonella Typhi in the laboratory and had not worn gloves at the time. The patient had one year of working experience in a clinical microbiology laboratory and had not been vaccinated against Salmonella Typhi.
Case three
In October 2016, a laboratory technologist working in a clinical microbiology laboratory (Laboratory-C) started complaining of headaches at work. His colleagues offered him an over the counter analgesic but his symptoms persisted. He reported to a hospital emergency room with symptoms of diarrhoea, worsening headaches, temperatures of 39 °C and severe malaise; he was admitted into hospital. Blood chemistry revealed raised liver enzymes and a C-reactive protein (CRP) reading of 90 mg/L. Blood cultures were collected for laboratory testing; Salmonella Typhi was cultured. The diagnostic laboratory determined that the isolate was susceptible to ceftriaxone and the fluoroquinolones (Kirby-Bauer disk susceptibility testing method - pefloxacin disk (5-μg) screening zone size of 25 mm). The patient was commenced on intravenous ciprofloxacin by the treating physician. He received seven days of intravenous ciprofloxacin followed up by a seven-day course of oral ciprofloxacin on discharge. The isolate was referred to the CED for confirmation of identification. The CED confirmed the Salmonella Typhi identification and determined that the isolate was intermediately-resistant to fluoroquinolones (ciprofloxacin MIC, 0.25 μg/ml) and susceptible to azithromycin (MIC, 4 μg/ml). Salmonella Typhi with the same MICs was isolated from subsequent stool samples, collected 16 days and 19 days after the blood culture sample tested positive. The patient was contacted by the treating private physician and readmitted to hospital where he received five days of intravenous azithromycin. On discharge he was prescribed combination therapy comprising of oral ciprofloxacin and azithromycin for one month. Subsequent stools tested negative for Salmonella Typhi. The technologist had 28 months working experience in a clinical microbiology laboratory and had no history of vaccination against typhoid fever. Epidemiological investigation determined that the technologist had been exposed to Salmonella Typhi while processing a culture of Salmonella Typhi.
Identification of typhoid fever cases and public health response
Typhoid fever is a notifiable disease in South Africa. Based on laboratory diagnosis, typhoid fever is relatively uncommon in South Africa. Laboratory networks report the isolation of Salmonella Typhi to the Outbreak Response Unit of the NICD, who in turn notify the district and provincial communicable diseases coordinators. Cases are investigated through a home visit, interviewed and a case investigation form is completed. Cases are later followed up and include further testing of stool samples. For persons who have had contact with cases, their symptoms are assessed and stool samples are also collected for testing. Details of all cases, including patient occupation are reported to the NICD and recorded in a database. Databases are reviewed to identify cases whose occupation includes laboratory work; data collection includes demographic details, clinical and treatment history and outcome data. For the currently described three cases: all patients came from relatively affluent and hygienic home circumstances, and there was no suggestion of family members being ill or other likely sources of infection; laboratory infection control procedures and laboratory safety policies were reviewed and refresher staff training was conducted; we contacted patients and obtained written informed consent to describe their case.
Referral of bacterial isolates to the CED
The CED is the national reference centre in South Africa for human infections due to enteric pathogens including: Salmonella species, Shigella species, Campylobacter species, diarrhoeagenic Escherichia coli, Vibrio cholerae and Listeria monocytogenes. Isolates from across South Africa are voluntarily submitted to the CED through national laboratory-based surveillance from >200 clinical microbiology laboratories across the country, in relation to potential outbreaks. The CED proceeds with phenotypic and genotypic characterization of isolates. If required, molecular subtyping of isolates is performed. To determine if these cases of typhoid fever in laboratory-workers were acquired from the isolates to which they were exposed in the laboratory, we identified isolates of Salmonella Typhi that were submitted to the CED from the laboratory in which the patient worked during the two months prior to onset of illness and subjected these isolates and the patient’s isolate to phenotypic and genotypic characterization, as described below.
Phenotypic characterization of bacteria
Bacteria were received on Dorset-Egg transport media [Diagnostic Media Products (DMP), National Health Laboratory Service, Johannesburg, South Africa] and sub-cultured onto 5% Blood Agar (DMP), to check for viability and purity. Cultures were identified using standard phenotypic microbiological identification and serotyping techniques, briefly described as follows. As required, bacterial colonies were identified using the VITEK-2 COMPACT 15 automated microbial identification system (bioMérieux, Marcy-l’Étoile, France). Serotyping was performed according to the White-Kauffmann-Le Minor Scheme. Antimicrobial susceptibility testing was performed using the VITEK-2 COMPACT 15 system (bioMérieux) and the Etest method (bioMérieux). Interpretation of antimicrobial susceptibility data was done in accordance with the Clinical and Laboratory Standards Institute (CLSI) [20].
PCR for the H58 haplotype of Salmonella Typhi
PCR to determine whether Salmonella Typhi isolates belonged to the H58 haplotype were performed according to the methodology described by Murgia and coworkers [21].
Pulsed-field gel electrophoresis (PFGE) analysis of bacteria
PFGE analysis of XbaI digested genomic DNA was performed using a Bio-Rad CHEF-DR III electrophoresis system (Bio-Rad Laboratories, Hercules, USA), following a PulseNet protocol [22]. PFGE patterns were analyzed using BioNumerics (version 6.5) Software (Applied Maths, Sint-Martens-Latem, Belgium) with dendrograms of the patterns created using the unweighted pair group method with arithmetic averages, with analysis of banding patterns incorporating the Dice-coefficient at an optimization setting of 1.5% and a position tolerance setting of 1.5%.
Whole-genome sequencing (WGS) analysis of bacteria
Genomic DNA was isolated from bacteria using the Qiagen QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). DNA libraries were prepared using a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA), followed by a 2 × 300 paired-end sequencing runs with 100× coverage using Illumina MiSeq equipment. Raw data generated on the MiSeq was further analyzed using tools available in the CLC Genomics Workbench Software, version 8.5 (Qiagen). Using the ‘Trim Sequences Tool’, sequence reads were trimmed to include quality trimming and ambiguity trimming, and length trimming to discard reads below a length of 50 bases. Trimmed reads were assembled using the ‘De novo Assembly Tool’; the assembly algorithm works by using de Bruijn graphs to produce contiguous (contig) sequences (minimum contig length was set at 200 bases).
Multilocus sequence typing (MLST) of bacteria
Assembled genome data was analyzed using the ‘multilocus sequence typing (MLST)’ on-line analysis pipeline available at the Center for Genomic Epidemiology (CGE) of the Technical University of Denmark [23]. MLST produces sequence types (STs) based on sequence analysis of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA and thrA), as described at the Salmonella MLST database [24].
Single nucleotide polymorphism (SNP) profiles and phylogenetic analysis of bacteria
Assembled genome data was analyzed using the ‘CSIPhylogeny 1.4’ on-line analysis pipeline available at the CGE [25]. The CSIPhylogeny pipeline uses various publicly available programs and the analysis steps are briefly described as follows: assembled genome data is aligned against a reference genome and single nucleotide polymorphisms (SNPs) are called; SNPs are filtered and qualified; final qualified SNPs for each genome is concatenated to an alignment; phylogeny is then inferred based on a comparison of SNP alignments of strains. SNPs were called by alignment and referencing against a South African strain isolated in 2016 (reference number TCD981492). SNP alignments were analyzed with iTOL software [26] to generate phylogenetic maximum-likelihood trees.
