Multidrug-Resistant and Extensively Drug-Resistant Escherichia coli in Sewage in Kuwait: Their Implications.

In Kuwait, some sewage is discharged into the sea untreated, causing a health risk. Previously, we investigated the presence of pathogenic E. coli among the 140 isolates of E. coli cultured from the raw sewage from three sites in Kuwait. The aim of the current study was to characterize the antimicrobial resistance of these isolates and the implications of resistance. Susceptibility to 15 antibiotic classes was tested. Selected genes mediating resistance to cephalosporins and carbapenems were sought. ESBL and carbapenemase production were also determined. Two virulent global clones, ST131 and ST648, were sought. A total of 136 (97.1%), 14 (10.0%), 128 (91.4%), and 2 (1.4%) isolates were cephalosporin-resistant, carbapenem-resistant, multidrug-resistant (MDR), and extensively drug-resistant (XDR), respectively. Among the cephalosporin-resistant isolates, ampC, blaTEM, blaCTX-M, blaOXA-1, and blaCMY-2 were found. Eighteen (12.9%) samples were ESBL producers. All carbapenem-resistant isolates were negative for carbapenemase genes (blaOXA-48, blaIMP, blaGES, blaVIM, blaNDM, and blaKPC), and for carbapenemase production. Resistance rates in carbapenem-resistant isolates to many other antibiotics were significantly higher than in susceptible isolates. A total of four ST131 and ST648 isolates were detected. The presence of MDR and XDR E. coli and global clones in sewage poses a threat in treating E. coli infections.

Molecular fingerprinting by multi-locus sequence typing identifies microevolution and nosocomial transmission of Candida glabrata in Kuwait.

Backgrounds: Candida glabrata is a frequently isolated non-albicans Candida species and invasive C. glabrata infections in older patients are associated with high mortality rates. Opportunistic Candida infections in critically ill patients may be either endogenous or nosocomial in origin and this distinction is critical for effective intervention strategies. This study performed multi-locus sequence typing (MLST) to study genotypic relatedness among clinical C. glabrata isolates in Kuwait. Methods: Candida glabrata isolates (n = 91) cultured from 91 patients were analyzed by MLST. Repeat isolates (n = 16) from 9 patients were also used. Antifungal susceptibility testing for fluconazole, voriconazole, caspofungin and amphotericin B (AMB) was determined by Etest. Genetic relatedness was determined by constructing phylogenetic tree and minimum spanning tree by using BioNumerics software. Results: Resistance to fluconazole, voriconazole and AMB was detected in 7, 2 and 10 C. glabrata isolates, respectively. MLST identified 28 sequence types (STs), including 12 new STs. ST46 (n = 33), ST3 (n = 8), ST7 (n = 6) and ST55 (n = 6) were prevalent in ≥4 hospitals. Repeat isolates obtained from same or different site yielded identical ST. No association of ST46 with source of isolation or resistance to antifungals was apparent. Microevolution and cross-transmission of infection was indicated in two hospitals that yielded majority (57 of 91, 67%) of C. glabrata. Conclusion: Our data suggest that C. glabrata undergoes microevolution in hospital environment and can be nosocomially transmitted to other susceptible patients. Thus, proper infection control practices during routine procedures on C. glabrata-infected patients may prevent transmission of this pathogen to other hospitalized patients.

Fatal Lodderomyces elongisporus Fungemia in a Premature, Extremely Low-Birth-Weight Neonate.

Many rare yeasts are emerging as pathogens, causing invasive infections in susceptible hosts that are associated with poor clinical outcome. Here, we describe the first and fatal case of Lodderomyces elongisporus fungemia in a premature, extremely low-birth-weight neonate after spontaneous vaginal delivery. The bloodstream isolate was identified as C. parapsilosis by the VITEK 2 yeast identification system and as L. elongisporus by PCR-sequencing of the internal transcribed spacer (ITS) region of ribosomal DNA. Antifungal susceptibility testing data for the isolate, performed by the broth microdilution-based MICRONAUT-AM assay, showed susceptibility to all nine antifungal drugs tested. Despite the initiation of treatment with liposomal amphotericin B, the patient died on the same day that the blood culture yielded yeast growth. This is the first report of L. elongisporus bloodstream infection in a neonate as the previous nine cases reported in the literature occurred in adult patients. The crude mortality rate for invasive L. elongisporus infection is 50%, as only 5 of 10 patients survived.

Virulence and phylogenetic groups of Escherichia coli cultured from raw sewage in Kuwait.

In Kuwait, some untreated sewage is discharged into the sea which poses health risks. Therefore, we determined the virulence traits and the phylogenetic groups of E. coli cultured from raw sewage. Sewage was collected once every month for 12 months with culturing of a total of 140 E. coli isolates. E. coli was typed by the methods of Clermont. The five pathotypes of diarrheagenic E. coli (DEC), and extra-intestinal pathogenic E. coli (ExPEC) were detected by specific PCR assays. Four virulence genes which correlate with pathogenicity in animal models, were used for the first time for detection of ExPEC-vat (vacuolating auto-transporter toxin), fyuA (yersiniabactin receptor), chuA (heme-binding protein), and yfcV (major subunit of putative chaperon-usher fimbria). Most E. coli belonged to phylogenetic groups A (65[45%]) and B1 (34[24.3%]). Three (2.1%) isolates were DEC, while 14 (10%) isolates were ExPEC mostly in group B2 (57.1%). A relatively high prevalence of ExPEC in sewage has public health implications.

Preponderance of bla KPC-Carrying Carbapenem-Resistant Enterobacterales Among Fecal Isolates From Community Food Handlers in Kuwait.

Carbapenem-resistant Enterobacterales (CRE) are pathogens that have been found in several countries, with a significant public health concern. Characterizing the mode of resistance and determining the prevailing clones are vital to the epidemiology of CRE in our community. This study was conducted to characterize the molecular mode of resistance and to determine the clonality of the CRE fecal isolates among community food handlers (FHs) vs. infected control patients (ICPs) in Kuwait. Fecal CRE isolates obtained from FHs and ICPs from September 2016 to September 2018 were analyzed for their resistance genes. Gene characterization was carried out by polymerase chain reaction (PCR) assays and sequencing. Clonality of isolates was established by multilocus sequence typing (MLST). Of the 681 and 95 isolates of the family Enterobacterales isolated from FHs and ICPs, 425 (62.4%) and 16 (16.8%) were Escherichia coli, and 18 (2.6%) and 69 (72.6%) were Klebsiella pneumoniae, respectively. A total of 36 isolates were CRE with a prevalence of 5.3% among FH isolates and 87 (91.6%) among the ICPs. Of these, carbapenemase genes were detected in 22 (61.1%) and 65 (74.7%) isolates, respectively (p < 0.05). The detected specific genes among FHs and ICPs were positive for bla KPC 19 (86.4%) and 35 (40.2%), and bla OXA 10 (45.5%) and 59 (67.8%), in addition to bla NDM 2 (9.1%) and 32 (36.8%), respectively. MLST assays of the E. coli and K. pneumoniae isolates revealed considerable genetic diversity and polyclonality as well as demonstrated multiple known ST types and eight novel sequence types. The study revealed a relatively high number of CRE harboring predominantly bla KPC-mediated CRE among the community FH isolates vs. predominant bla OXA genes among the ICPs. Those heterogeneous CRE isolates raise concerns and mandate more efforts toward molecular surveillance. A multinational study is recommended to monitor the spread of genes mediating CRE in the community of Arabian Peninsula countries.

Epidemiology of Candidemia in Kuwait: A Nationwide, Population-Based Study.

The Candida species cause a majority of invasive fungal infections. In this article, we describe the nationwide epidemiology of candidemia in Kuwait in 2018. Yeast bloodstream isolates submitted from all major hospitals and identified by phenotypic MALDI-TOF MS and/or by molecular methods were studied. Susceptibility testing was performed by Etest. Out of 313 bloodstream yeasts, 239 Candida spp. isolates (excluding duplicate isolates) were obtained during 234 candidemic episodes among 223 patients. Mixed-species candidemia and re-infection occurred in 5 and 11 patients, respectively. C. albicans (n = 74), C. parapsilosis (n = 54), C. tropicalis (n = 35), C. auris (n = 33), C. glabrata (n = 32), other Candida spp. (n = 11), and other yeasts (n = 9) caused fungemia. Nearly 50% of patients were in intensive care units. Candida spp. isolates (except C. glabrata) were susceptible to caspofungin and 27% of C. auris were amphotericin B-resistant. Resistance to fluconazole was 100% in C. auris, 17% in C. parapsilosis, 12% in C. glabrata, and 1% in C. albicans. Mortality was 47% for other Candida/yeast infections. Nationwide candidemia incidence in 2018 was 5.29 cases/100,000 inhabitants. Changes in species spectrum, increasing fluconazole resistance in C. parapsilosis, and the emergence of C. auris as a major pathogen in Kuwait are noteworthy findings. The data could be of help in informing decisions regarding planning, in the allocation of resources, and in antimicrobial stewardship.

Monitoring antibiotic resistance profiles of faecal isolates of Enterobacteriaceae and the prevalence of carbapenem-resistant isolates among food handlers in Kuwait.

OBJECTIVES: Carbapenem-resistant Enterobacteriaceae (CRE) have become one of the most challenging problems in infectious diseases worldwide. Unrecognised personnel such as food handlers (FHs) colonised with CRE serve as a reservoir for transmission. This study assessed the prevalence and susceptibility patterns of CRE isolates from FHs working in commercial eateries in the community (CFHs) and healthcare settings (HCFHs) in Kuwait over the period 2016-2018. METHODS: Representative colonies from faecal samples were identified by API 20E and a VITEK®2 ID System. Susceptibility testing against 21 antibiotics was performed by Etest and agar dilution. RESULTS: A total of 681 isolates of the family Enterobacteriaceae were isolated from 405 FHs, of which 425 (62.4%) were Escherichia coli and 126 (18.5%) were Klebsiella pneumoniae. The prevalence of CRE among FHs was 7.7% (31/405), comprising 32% CFHs (10/31) and 68% HCFHs (21/31). Ampicillin, tetracycline and cefalotin showed very poor activities against most isolates with resistance rates of 63.3%, 41.7% and 40.8%, respectively. The prevalence of multidrug-resistant (MDR) isolates was 30.5%, including 130 E. coli (30.6%) and 22 K. pneumoniae (17.5%). An alarming level of colistin resistance (11.3%) was noted. A significant proportion of FH isolates (13.2%) exhibited extended-spectrum ß-lactamases (ESBL) phenotypes, including 80 E. coli (18.8%) and 5 K. pneumoniae (4.0%). CONCLUSION: This study revealed that asymptomatic intestinal carriage of CRE, including MDR and ESBL isolates, was relatively common in our community. It is conceivable that FHs may pose a significant risk to consumers for the acquisition and spread of resistant strains.

Evaluating Food Safety Compliance and Hygiene Practices of Food Handlers Working in Community and Healthcare Settings in Kuwait.

Safe food handling and proper hygiene practices performed by food handlers (FHs) in catering establishments are fundamental elements in reducing foodborne diseases. This study aimed at assessing food safety knowledge and compliance of hygiene practices of FHs within food establishments (using a structured questionnaire). A cross-sectional study was carried out from May 2016 to March 2018 on FHs working in community and healthcare settings. A total of 405 FHs, including 44.9% and 55.1%, were working in community and healthcare settings, respectively. The majority, 84.7%, were males with a ratio of 5.5:1. Most of them, 84.4%, had a high school education and above. A greater number, 44%, of FHs were in the age bracket of 29-39 years. As high as 95.6% of them underwent a regular medical check-up. Unsafe attitudes were shown by 44.9%% who used the same hand gloves while handling raw meat and fresh food. Additionally, 42% went home with their uniforms. The hygiene assessment score was 95.8%. In general, FHs have adequate knowledge and compliance with food safety practices. It is recommended that regular and ongoing training on hygienic practices and proper food safety techniques must be given to all FHs to ensure food safety.

Papiliotrema laurentii fungemia in a premature, very low-birth-weight neonate in Kuwait successfully treated with liposomal amphotericin B.

Papiliotrema laurentii (formerly Cryptococcus laurentii) and Papiliotrema albidus (formerly Cryptococcus albidus) are yeast-like environmental fungi which are largely considered as non-pathogenic to humans. However, invasive infections caused by P. laurentii have recently been reported in some patients with an impaired immune system. Here, we describe the first case of P. laurentii fungemia in a premature, very low-birth-weight neonate in Kuwait and the Middle East. Repeated bloodstream isolates were obtained and were tentatively identified as P. laurentii by Vitek 2 yeast identification system. The identification of the yeast isolates as P. laurentii was confirmed by PCR-sequencing of ribosomal DNA (rDNA). Antifungal susceptibility testing data showed that the isolates were susceptible to amphotericin B, fluconazole and voriconazole but appeared resistant to caspofungin. The baby was successfully treated with liposomal amphotericin B.

Evaluation of the in vitro activity of ceftaroline, ceftazidime/avibactam and comparator antimicrobial agents against clinical isolates from paediatric patients in Kuwait: ATLAS data 2012-19.

Objectives: To report antimicrobial resistance data for Gram-positive and Gram-negative pathogens isolated from paediatric patients in three hospitals in Kuwait during 2012-19. Methods: In vitro activity of antimicrobials against isolates from documented infections was determined using CLSI broth microdilution method and breakpoints at a central laboratory. Enterobacterales and Pseudomonas aeruginosa isolates were screened for ß-lactamases using multiplex PCR assays. Phenotypic determination of resistance in Haemophilus influenzae and Gram-positive isolates was performed using standard methodologies. Results: Among 515 Enterobacterales isolates, 29.3% were ESBL-positive; susceptibility was highest to amikacin, ceftazidime/avibactam and meropenem (≥97.4%), regardless of ESBL status. CTX-M-15 was identified in 87.1% of ESBL-positive Escherichia coli and 84.2% of ESBL-positive Klebsiella pneumoniae isolates. Of 111 P. aeruginosa isolates, 9.9% were MDR and 12.6% meropenem-resistant (MEM-R). Amikacin and ceftazidime/avibactam had the highest susceptibility rates in the overall group (≥92.8%), with reduced rates among MDR and MEM-R isolates. All 269 MRSA and 180 MSSA isolates were susceptible to daptomycin, linezolid, teicoplanin, tigecycline and vancomycin. All MSSA and 99.3% of MRSA were ceftaroline susceptible. All 168 pneumococcal isolates were susceptible to ceftaroline, linezolid, tigecycline and vancomycin. H. influenzae and Streptococcus pyogenes ceftaroline susceptibility rates were ≥93.3% and ≥95.6%. Conclusions: Most isolates of Enterobacterales (including resistant phenotypes) and P. aeruginosa from Kuwait during 2012-19 were susceptible to ceftazidime/avibactam. Ceftaroline was active against most Gram-positive isolates, including resistant phenotypes, and ESBL-negative Enterobacterales. These results indicate that novel antibiotics such as ceftazidime/avibactam and ceftaroline represent valuable treatment options for paediatric infections, including those caused by MDR organisms.

Candida kefyr in Kuwait: Prevalence, antifungal drug susceptibility and genotypic heterogeneity.

OBJECTIVE: Candida kefyr causes invasive candidiasis in immunocompromised patients, particularly among those with oncohematological diseases. This study determined the prevalence of C. kefyr among yeast isolates collected during 2011-2018 in Kuwait. Antifungal susceptibility testing (AST) and genotypic heterogeneity among C. kefyr was also studied. METHODS: Clinical C. kefyr isolates recovered from bloodstream and other specimens during 2011 to 2018 were retrospectively analyzed. All C. kefyr isolates were identified by CHROMagar Candida, Vitek2 and PCR amplification of rDNA. AST was performed by Etest. Molecular basis of resistance to fluconazole and echinocandins was studied by PCR-sequencing of ERG11 and FKS1, respectively. Genotypic heterogeneity was determined with microsatellite-/minisatellite-based primers and for 27 selected isolates by PCR-sequencing of IGS1 region of rDNA. RESULTS: Among 8257 yeast strains, 69 C. kefyr (including four bloodstream) isolates were detected by phenotypic and molecular methods. Isolation from urine and respiratory samples from female and male patients was significantly different (P = 0.001). Four isolates showed reduced susceptibility to amphotericin B and one isolate to all (amphotericin B, fluconazole, voriconazole and caspofungin/micafungin) antifungals tested. Fluconazole-resistant isolate contained only synonymous mutations in ERG11. Echinocandin-resistant isolate contained wild-type hotspot-1 and hotspot-2 of FKS1. Fingerprinting with microsatellite-/minisatellite-based primers identified only three types. IGS1 sequencing identified seven haplotypes among 27 selected isolates. CONCLUSIONS: The overall prevalence of C. kefyr among clinical yeast isolates and among candidemia cases was recorded as 0.83% and 0.32%, respectively. The frequency of isolation of C. kefyr from bloodstream and other invasive samples was stable during the study period. The C. kefyr isolates grown from invasive (bloodstream, bronchoalveolar lavage, abdominal drain fluid, peritonial fluid and gastric fluid) samples and amphotericin B-resistant isolates were genotypically heterogeneous strains.

Increasing Trends of Reduced Susceptibility to Antifungal Drugs Among Clinical Candida glabrata Isolates in Kuwait.

Among non-albicans Candida species, Candida glabrata is the leading cause of invasive infections in critically ill patients. It is intrinsically less susceptible to fluconazole/other azoles that limits therapeutic options. This study determined distribution of C. glabrata in clinical specimens and determined their susceptibility to fluconazole, caspofungin, and amphotericin B by E test. During 8-year period (2011-2018), 1,410 isolates were obtained from 1,410 patients including 600, 409, and 131 isolates from respiratory, urine, and bloodstream specimens, respectively. Proportion of C. glabrata isolates was nearly the same during the two 4-year periods. Demographic details were available from 731 patients and susceptibility data for 1,225 isolates. C. glabrata isolation from bloodstream, respiratory, and urine specimens was higher from elderly (>60 years) versus younger patients. More bloodstream and urine isolates were obtained from female patients, however, more respiratory isolates were recovered from male patients (p = <0.05). Resistance to all three drugs increased during 2015-2018 compared with 2011-2014 but was more pronounced for fluconazole (p = 0.001). More isolates with reduced susceptibility to fluconazole/amphotericin B were obtained from elderly patients versus younger subjects and urine versus respiratory samples (p = <0.05). Our data show increasing trends of reduced susceptibility to antifungals, particularly fluconazole, among clinical C. glabrata isolates in Kuwait. Most isolates with reduced susceptibility to fluconazole/amphotericin B were obtained from elderly patients and urine/respiratory samples with urinary tract appearing as the most favorable niche for antifungal drug resistance development. The study also highlights the need for continued surveillance and better antifungal drug stewardship to control resistance development in C. glabrata.

Candida auris in various hospitals across Kuwait and their susceptibility and molecular basis of resistance to antifungal drugs.

BACKGROUND: Candida auris, a multidrug-resistant species, has the propensity of nosocomial transmission despite normal decontamination procedures. Here, we describe the isolation of C auris from patients in various hospitals in Kuwait during 2014-2018. Susceptibility to antifungal drugs and molecular basis of resistance to fluconazole, voriconazole and micafungin were also studied. METHODS: Candida auris (n = 314) obtained from 126 patients in eight hospitals were studied. All isolates were identified by PCR amplification and/or PCR-sequencing of ribosomal DNA (rDNA). Antifungal susceptibility was determined by Etest. Molecular basis of resistance to fluconazole and micafungin was studied by PCR-sequencing of ERG11 and FKS1 genes, respectively. FINDINGS: Bloodstream (n = 58), urine (n = 124), respiratory (n = 98) and other (n = 34) specimens yielded 314 C auris isolates. The proportion of bloodstream C auris among all yeast isolates was higher (42 of 307, 13.7%) in 2018 as compared to 2014-2017 (16 of 964, 1.7%) (P = .001). More bloodstream isolates (42 of 139) were cultured in 2018 than during 2014-2017 (16 of 175) (P = .001). Resistance to amphotericin B, fluconazole, voriconazole and micafungin was detected in 27.1%, 100%, 41.1% and 1.7% isolates, respectively. Fluconazole-resistant isolates contained either Y132F or K143R mutation in ERG11. Isolates with K143R mutation were additionally resistant to voriconazole. Micafungin-resistant isolates contained S639F mutation in hot spot 1 of FKS1. CONCLUSIONS: Our study highlights spreading of C auris in major hospitals across Kuwait and its increasing role as a bloodstream pathogen in 2018. Cross-resistance to voriconazole was also seen in isolates with K143R mutation in ERG11, while micafungin-resistant isolates harboured S639F mutation in hot spot 1 of FKS1.

Lack of detection of Candida nivariensis and Candida bracarensis among 440 clinical Candida glabrata sensu lato isolates in Kuwait.

Occurrence of Candida nivariensis and Candida bracarensis, two species phenotypically similar to Candida glabrata sensu stricto, in human clinical samples from different geographical settings remains unknown. This study developed a low-cost multiplex PCR (mPCR) and three species-specific singleplex PCR assays. Reference strains of common Candida species were used during development and the performance of mPCR and singleplex PCR assays was evaluated with 440 clinical C. glabrata sensu lato isolates. The internal transcribed spacer (ITS) region of rDNA was also sequenced from 85 selected isolates and rDNA sequence variations were used for determining genetic relatedness among the isolates by using MEGA X software. Species-specific amplicons for C. glabrata (~360 bp), C. nivariensis (~250 bp) and C. bracarensis (~180 bp) were obtained in mPCR while no amplicon was obtained from other Candida species. The three singleplex PCR assays also yielded expected results with reference strains of Candida species. The mPCR amplified ~360 bp amplicon from all 440 C. glabrata sensu lato isolates thus identifying all clinical isolates in Kuwait as C. glabrata sensu stricto. The results of mPCR were confirmed for all 440 isolates as they yielded an amplicon only in C. glabrata sensu stricto-specific singleplex PCR assay. The rDNA sequence data identified 28 ITS haplotypes among 85 isolates with 18 isolates belonging to unique haplotypes and 67 isolates belonging to 10 cluster haplotypes. In conclusion, we have developed a simple, low-cost mPCR assay for rapid differentiation of C. glabrata sensu stricto from C. nivariensis and C. bracarensis. Our data obtained from a large collection of clinical C. glabrata sensu lato isolates show that C. nivariensis and C. bracarensis are rare pathogens in Kuwait. Considerable genetic diversity among C. glabrata sensu stricto isolates was also indicated by rDNA sequence analyses.

Changing trends in epidemiology and antifungal susceptibility patterns of six bloodstream Candida species isolates over a 12-year period in Kuwait.

Changing trends in incidence and antifungal susceptibility patterns of six Candida species causing candidemia in Kuwait between 2006-2017 are reported. A total of 2075 isolates obtained from 1448 patients were analyzed. Identity of Candida species isolates was determined by phenotypic methods and confirmed by PCR amplification/PCR-sequencing of rDNA and/or MALDI-TOF MS. Antifungal susceptibility was determined by Etest. C. albicans accounted for 539 (37.22%) cases followed by C. parapsilosis (n = 502, 34.67%), C. tropicalis (n = 210, 14.5%), C. glabrata (n = 148, 10.22%), C. krusei (n = 27, 1.81%) and C. dubliniensis (n = 22, 1.5%). The comparative percent distribution of Candida species causing candidemia between 2006-2011 and 2012-2017 was as follows: C. albicans 41.8% and 33.1%, C. parapsilosis complex 32.01% and 37.04%, C. tropicalis 13.59% and 15.31%, and C. glabrata 8.77% and 11.51%, C. krusei 2.0% and 1.7%, and C. dubliniensis 1.75 and 1.3%, respectively. Three of 371 C. albicans isolates during 2006-2011 and five of 363 during 2012-2017 were resistant to fluconazole. Among C. parapsilosis isolates, one of 310 during 2006-2011 and 21 of 446 during 2012-2017 were resistant to this drug. Furthermore, at an epidemiologic cutoff value (ECV) of ≤0.5 µg/ml, 70.1% C. albicans isolates were wild-type for fluconazole during 2006-2011 as compared to 58.1% during 2012-2017. Likewise, at an ECV of ≤2 µg/ml, 98.0% of C. parapsilosis isolates were wild-type during 2006-2011 as compared to 93.4% during 2012-2017. Clonal spread of fluconazole-resistant C. parapsilosis in one major hospital was documented. An 8.8% shift in favor of non-albicans Candida species with concomitant increase in MICs between the two periods preludes emergence of fluconazole-resistant candidemia cases in Kuwait.

Candida lusitaniae in Kuwait: Prevalence, antifungal susceptibility and role in neonatal fungemia.

OBJECTIVES: Candida lusitaniae is an opportunistic yeast pathogen in certain high-risk patient populations/cohorts. The species exhibits an unusual antifungal susceptibility profile with tendency to acquire rapid resistance. Here, we describe prevalence of C. lusitaniae in clinical specimens in Kuwait, its antifungal susceptibility profile and role in neonatal fungemia. METHODS: Clinical C. lusitaniae isolates recovered from diverse specimens during 2011 to 2017 were retrospectively analyzed. All isolates were identified by germ tube test, growth on CHROMagar Candida and by Vitek 2 yeast identification system. A simple species-specific PCR assay was developed and results were confirmed by PCR-sequencing of ITS region of rDNA. Antifungal susceptibility was determined by Etest. Minimum inhibitory concentrations (MICs) were recorded after 24 h incubation at 35°C. RESULTS: Of 7068 yeast isolates, 134 (1.89%) were identified as C. lusitaniae including 25 (2.52%) among 990 bloodstream isolates. Species-specific PCR and PCR-sequencing of rDNA confirmed identification. Of 11 cases of neonatal candidemia, 9 occurred in NICU of Hospital A and are described here. Eight of 9 neonates received liposomal amphotericin B, which was followed by fluconazole in 7 and additionally by caspofungin in 2 cases as salvage therapy. Three of 8 (37.5%) patients died. No isolate exhibited reduced susceptibility to amphotericin B, fluconazole, voriconazole, caspopfungin, micafungin and anidulafungin. The MIC ± geometric mean values for amphotericin B, fluconazole, voriconazole, and caspofungin were as follows: 0.072 ± 0.037 µg/ml, 2.32 ± 0.49 µg/ml, 0.09 ± 0.01 µg/ml and 0.16 ± 0.08 µg/ml, respectively. Only two isolates exhibited reduced susceptibility to fluconazole. CONCLUSIONS: This study describes the prevalence and antifungal susceptibility profile of clinical C. lusitaniae isolates in Kuwait. No isolate showed reduced susceptibility to amphotericin B. The study highlights the emerging role of C. lusitaniae as a healthcare-associated pathogen capable of causing fungemia in preterm neonates and causing significant mortality.

High-resolution fingerprinting of Candida parapsilosis isolates suggests persistence and transmission of infections among neonatal intensive care unit patients in Kuwait.

Candida parapsilosis causes ~35% of all candidemia cases in neonates. High-resolution fingerprinting of C. parapsilosis isolates from neonatal intensive care unit (NICU) patients in Maternity Hospital (MH) was performed to identify epidemiologically related strains. Sixty-eight bloodstream/colonizing strains isolated from 59 NICU patients, two isolates from health care workers (HCWs) from MH and 18 bloodstream isolates from two other hospitals were used. Six microsatellite markers were employed, isolates were assigned a numerical microsatellite genotype (MSG), dendrogram was constructed and similarities between genotypes were visualized by minimum spanning tree. Fifty bloodstream isolates from MH yielded 37 MSGs with 20 isolates clustering in 7 MSGs. Duplicate isolates and colonizing strains yielded same/highly similar MSG as bloodstream isolates. Colonizing strains from two non-candidemia patients yielded unique MSGs while others belonged to a cluster. All isolates from HCWs and from two other hospitals belonged to unique MSGs. Cluster isolates came from patients in NICU-1 or from neonates in NICU-1 and other NICUs. Clonal complexes comprising closely related genotypes indicative of microevolution were also detected. Our data show that some C. parapsilosis strains have persisted in MH environment over several years and these endemic genotypes were transmitted to other patients in NICU-1 and/or other nearby NICUs.

Cyberlindnera fabianii fungaemia outbreak in preterm neonates in Kuwait and literature review.

BACKGROUND: Cyberlindnera fabianii has rarely been reported as a human pathogen. Here, we describe an outbreak of C. fabianii fungaemia involving 10 preterm neonates during a seven-month period in Kuwait and review the published reports. METHODS: Blood cultures were processed, and yeast isolates were initially identified by ID 32 C and/or VITEK 2. Molecular identification was done by PCR sequencing of internally transcribed spacer (ITS) region and D1/D2 domains of rDNA. Fingerprinting was performed with microsatellite-based and minisatellite-based primers to examine genetic relatedness among the isolates. Antifungal susceptibility testing of the isolates was done by Etest. FINDINGS: All infected neonates were preterm, received prior antibiotics and had an intravascular catheter in place. All bloodstream isolates were initially identified as Candida utilis by ID 32 C and/or VITEK 2 and showed reduced susceptibility to triazoles. PCR sequencing of rDNA identified all isolates as Cyberlindnera fabianii. Fingerprinting studies yielded identical patterns indicating clonality. One neonate died before treatment, one died during treatment, and eight neonates survived treatment with amphotericin B with/without fluconazole or caspofungin. Source of infection remained unknown despite surveillance cultures. CONCLUSION: The outbreak highlights emergence of C. fabianii as a neonatal pathogen and reinforces importance of molecular methods in its accurate identification.

Rapid and Accurate Identification of Candida albicans and Candida dubliniensis by Real-Time PCR and Melting Curve Analysis.

OBJECTIVE: Candida albicans and Candida dubliniensis are germ tube-positive pathogenic yeast species. Accurate identification of these two species is warranted since C. albicans is a highly pathogenic species while C. dubliniensis exhibits increased adherence to buccal epithelial cells, reduced susceptibility to azoles and resistance to flucytosine. We have developed a duplex real-time PCR assay for rapid detection and differentiation between clinical C. albicans and C. dubliniensis isolates. MATERIALS AND METHODS: A duplex real-time PCR assay was developed by using two species-specific primer pairs and SYBR Green dye to differentiate C. albicans and C. dubliniensis isolates via melting curve analysis of real-time PCR amplicons. Amplification products were also analyzed by agarose gel electrophoresis to confirm real-time PCR results. RESULTS: Melting temperatures (Tm) for reference strains of C. albicans and C. dubliniensis were 86.55 and 82.75°C, respectively. No amplicon was obtained with DNA from reference strains of 8 other common Candida spp. When real-time PCR was applied on 226 clinical isolates previously identified by the Vitek 2 system and/or PCR sequencing of rDNA, Tm values for C. albicans (n = 113) and C. dubliniensis (n = 98) were 86.68 ± 0.529 and 82.616 ± 0.535°C, respectively. The results were confirmed by agarose gel electrophoresis. No amplicon was obtained from 15 isolates belonging to 9 other Candida spp. CONCLUSIONS: The real-time PCR assay described here does not require prior identification of clinical yeast isolates as C. albicans/C. dubliniensis by germ tube formation and accurately reports results within 2 h. Detection of amplicons by agarose gel electrophoresis is also suitable for resource-poor settings devoid of real-time PCR facilities.

Increasing prevalence, molecular characterization and antifungal drug susceptibility of serial Candida auris isolates in Kuwait.

Candida auris is an emerging yeast pathogen of global significance. Its multidrug-resistant nature and inadequacies of conventional identification systems pose diagnostic and therapeutic challenges. This study investigated occurrence of C. auris in clinical specimens in Kuwait and its susceptibility to antifungal agents. Clinical yeast strains isolated during 3.5-year period and forming pink-colored colonies on CHROMagar Candida were studied by wet mount examination for microscopic morphology and Vitek 2 yeast identification system. A simple species-specific PCR assay was developed for molecular identification and results were confirmed by PCR-sequencing of rDNA. Antifungal susceptibility testing of one isolate from each patient was determined by Etest. The 280 isolates forming pink-colored colonies on CHROMagar Candida, were identified by Vitek 2 as Candida haemulonii (n = 166), Candida utilis (n = 49), Candida kefyr (n = 45), Candida guilliermondii (n = 9), Candida famata (n = 6) and Candida conglobata (n = 5). Species-specific PCR and PCR-sequencing of rDNA identified 166 C. haemulonii isolates as C. auris (n = 158), C. haemulonii (n = 6) and Candida duobushaemulonii (n = 2). C. auris isolates originated from diverse clinical specimens from 56 patients. Of 56 C. auris isolates tested, all were resistant to fluconazole, 41/56 (73%) and 13/56 (23%) were additionally resistant to voriconazole and amphotericin B, respectively. Eleven (20%) isolates were resistant to fluconazole, voriconazole and amphotericin B. One isolate was resistant to caspofungin and micafungin. Increasing isolation of C. auris in recent years from diverse clinical specimens including bloodstream shows that C. auris is an emerging non-albicans Candida species in Kuwait causing a variety of infections. Inability of conventional identification methods to accurately identify this pathogen and multidrug-resistant nature of many strains calls for a greater understanding of its epidemiology, risk factors for acquiring C. auris infection and management strategies in high-risk patients. This is the first comprehensive study on the emergence of this multidrug-resistant yeast from Kuwait and the Middle East.