Screening of C. auris among Candida isolates from various tertiary care institutions in Lahore by VITEK 2 and real time PCR based molecular technique.

Candida auris is a multidrug-resistant pathogen, that is a well-known cause of nosocomial infections. This pathogen is being identified using advanced diagnostic approaches and epidemiological typing procedures. In underdeveloped nations, several researchers developed and validated a low-cost approach for reliably identifying Candida auris. The goal of this study was to assess the burden of Candida auris in different teaching hospitals of Lahore and to limit its spread to minimize hospital-related illnesses. Candida isolates were obtained from various tertiary care institutions in Lahore in the form of culture on various culture plates. Sabouraud agar culture plates were used to culture the Candida spp. Fluconazole-resistant Candida species were chosen for further identification using VITEK 2 Compact ID and molecular identification using species-specific PCR assay. The current study obtained 636 Candida samples from several tertiary care institutions in Lahore. Fluconazole resistance was found in 248 (38.9%) of 636 Candida samples. No isolate was identified as Candida auris by VITEK 2 Compact ID and real-time PCR-based molecular identification. Thus with limited resources, these two methods may serve as useful screens for Candida auris. However, it should be screened all over the country to limit its spread to break the chain of nosocomial infections.

Severity of Dengue Viral Infection Based on Clinical and Hematological Parameters among Pakistani Patients.

The global burden of dengue infections has increased dramatically. Early diagnosis of dengue infection is critical to proper medical management to avoid further complications in patients. This study was geared to assess the severity of dengue infections based on clinical and hematological examinations. A cross-sectional study was conducted among febrile patients with dengue infection in a teaching hospital in Pakistan. Blood samples were investigated for dengue-specific antibodies (IgM and IgG) and the nonstructural 1 antigen. The clinical findings of each subject were noted to assess the severity of the infection. Tests for hematological parameters were performed. Of 130 patients with confirmed dengue infection, 23 had severe and 107 had nonsevere dengue. Patients with severe dengue experienced mucosal bleeding (71.4%), fluid accumulation (57.1%), shock (35.7%), and gastrointestinal bleeding (28.6%). The most significant hematological findings among severe and nonsevere patients with dengue infection were thrombocytopenia, leukopenia, and a raised hematocrit level (P < 0.001). Patients with severe dengue infection showed marked thrombocytopenia, with a mean platelet count of 49.96 × 109 platelets/L. The clinical presentation of patients with dengue infection along with hematological markers are the most important clues for the diagnosis of, prognosis of, and therapy for dengue infection. Thrombocytopenia, leukopenia, and raised hematocrit levels were the most significant hematological parameters when assessing the severity of dengue infection.

Molecular Characterization of Extensively Drug Resistant Salmonella Enterica Serovar Typhi Clinical Isolates from Lahore, Pakistan.

Background: The emergence of extensively drug-resistant (XDR) typhoid in Pakistan has endangered the treatment options available to manage this infection. Third generation cephalosporin were the empiric choice to treat typhoid fever in Pakistan, but acquisition of ESBLs have knocked them out of the arsenal. The current empiric choice is azithromycin which is vulnerable to resistance too. This study aimed to assess the burden of XDR typhoid and the frequency of resistance determinants in blood culture samples collected from different hospitals in Lahore, Pakistan. Methods: A total of 835 blood cultures were collected from different tertiary care hospitals in Lahore during January 2019 to December 2021. Among 835 blood cultures, 389 Salmonella Typhi were identified, and 150 were XDR S. Typhi (resistant to all recommended antibiotics). Antibiotics resistance genes of the first-line drugs (blaTEM-1, catA1, sul1, and dhfR7) and second line drugs (gyrB, gyrA, qnrS, ParC and ParE) were investigated among XDR S. Typhi. There were different CTX-M genes isolated using the specific primers, blaCTX-M-U, blaCTX-M-1, blaCTX-M-15, blaCTX-M-2, blaCTX-M-8 and blaCTX-M-9. Results: Antibiotic resistant genes of the first-line drugs were isolated with different frequency, blaTEM-1 (72.6%), catA1 (86.6%), sul1 (70%), and dhfR7 (56%). Antibiotics resistance genes of second-line drugs were isolated as: gyrB (60%), gyrA (49.3%), qnrS (32.6%), parC (44%) and parE (28%). Among CTX-M genes, blaCTX-M-U (63.3%) was the most frequent followed by blaCTX-M-15 (39.3%) and blaCTX-M-1 (26%). Conclusion: Our study concluded that XDR isolates circulating in Pakistan have acquired first-line and second-line antibiotic resistant genes quite successfully along with CTX-M genes (ESBLs) rendering them resistant to the third generation cephalosporins as well. Emergence of azithromycin resistance in XDR S. Typhi which is currently used as an empiric treatment option is worrisome and needs to be monitored carefully in endemic countries like Pakistan.

Reporting of Azithromycin Activity against Clinical Isolates of Extensively Drug-Resistant Salmonella enterica Serovar Typhi.

This study aimed to evaluate the minimum inhibitory concentration (MIC) of azithromycin (AZM) in clinical isolates of extensively drug-resistant (XDR) Salmonella Typhi (i.e., resistant to chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole, fluoroquinolones, and third-generation cephalosporin) using the E-test versus the broth microdilution method (BMD). From January to June 2021, a retrospective cross-sectional study was carried out in Lahore, Pakistan. Antimicrobial susceptibility was performed initially by the Kirby-Bauer disk diffusion method for 150 XDR Salmonella enterica serovar Typhi isolates, and MICs of all the recommended antibiotics were determined by the VITEK 2 (BioMérieux) fully automated system using Clinical Laboratory Standard Institute (CLSI) 2021 guidelines. The E-test method was used to determine AZM MICs. These MICs were compared with the BMD, which is the method recommended by the CLSI but not adopted in routine laboratory reporting. Of 150 isolates, 10 (6.6%) were resistant by disk diffusion. Eight (5.3%) of these had high MICs against AZM by the E-test. Only three isolates (2%) were resistant by E-test, having an MIC of 32 µg/mL. All eight isolates had a high MIC by BMD with different MIC distributions, but only one was resistant, having an MIC of 32 µg/mL by BMD. The sensitivity, specificity, negative predictive value, positive predictive value, and diagnostic accuracy of the E-test method versus BMD were 98.65%,100%, 99.3%, 33.3%, and 98.6%, respectively. Similarly, the concordance rate was 98.6%, negative percent agreement was 100%, and positive percent agreement was 33%. The BMD is the most reliable approach for reporting AZM sensitivity in XDR S. Typhi compared with the E-test and disk diffusion methods. Potentially, AZM resistance in XDR S. Typhi is around the corner. Sensitivity patterns should be reported with MIC values, and if possible, higher values should be screened for the presence of any potential resistance genes. Antibiotic stewardship should be strictly implemented.

Staphylococcal Cassette Chromosome mec Typing and Multilocus Variable Number Tandem Repeat Analysis of Methicillin Resistant Staphylococcus aureus Clinical Isolates with Vancomycin Creep Phenomenon.

Background: The association of treatment failure and mortality with vancomycin minimum inhibitory concentration creep (MIC) is a matter of serious concern in patients with severe methicillin resistant Staphylococcus aureus (MRSA) infections. The purpose of the study was to identify and characterize staphylococcal cassette chromosome mec (SCCmec) and clonal types of MRSA strains, exhibiting the vancomycin MIC creep phenomenon. Methods: A total of 3305 S. aureus strains were isolated from various clinical samples of Lahore General Hospital, Lahore, Pakistan. MRSA strains were identified by cefoxitin resistant (≤21mm) followed by mecA and mecC gene genotyping. Vancomycin MIC creep was determined by E-test. Isolates having MIC values >1.5 µg/mL were further subjected for SCCmec typing (I-V and XI) and multiple-locus variable number tandem repeat analysis (MLVA) by amplification of spa, sspA, clfA, clfB, and sdrCDE genes. A dendrogram was created based on the similarity index using bioneumerics software. Results: About 13.3% (440/3305) isolates were MRSA with 99.3% (437/440) and 0.7% (3/440) carried mecA and mecC genes, respectively. In 120 MRSA isolates, the MIC of vancomycin was >1.5µg/mL. In MRSA isolates with high vancomycin MIC (>1.5µg/mL), the most common SCCmec type was SCCmec III (38.3%), followed by SCCmec IVa (15.8%), SCCmec IIIa (13.3%,), SCCmec IVc (7.5%), SCCmec IVe (5.8%), SCCmec IVd (5.8%), SCCmec IVb (4.2%), SCCmec II (2.5%), SCCmec V (1.7%), SCCmec I (1.7%) and SCCmec XI (1.7%). MLVA revealed 60 genotypic groups of MRSA isolates having a 92% similarity index. Conclusion: SCCmec III was the most common type in genetically related MRSA isolates showing vancomycin MIC creep. The presence of SCCmec XI may further add burden to infection control measures.

Effect of different doses of Manuka honey in experimentally induced mouse typhoid.

Typhoid fever is a major cause of morbidity and mortality in the developing world. Data from World Health Organization (WHO) shows that 21 million cases of typhoid occur globally every year and over 200,000 die each year; most of them at a very young age. The situation in Pakistan is similar. Typhi and other typhoidal salmonellae have developed resistance to chloramphenicol and other first line anti-typhoid. There is a rapid increase in multi-drug resistance (MDR) throughout the world. There is an urgent need to find out alternative medicine to sort out this problem. This study was conducted to establish preventive as well as therapeutic potential of Manuka honey. A total of eighty pathogen free BALB/C mice between 8 weeks to 12 weeks of age, weighing 25-30 grams were taken and divided into 4 groups. Group A, B and C were infected through oral route with 10(8) colony forming unit (CFU) of Salmonella typhimurium ATCC 14028 to produce typhoid like disease in mice. Group A, which comprised of 20 mice was further divided in A1 and A2 given Manuka honey at a dose of 15ml/kg and 20 ml/kg respectively. Group B, which comprised of 20 mice was further divided in B1 and B2 was given Manuka honey at dose of 20ml/kg and 25ml/kg respectively. Clinical features of mouse typhoid, like body temperature, respiratory rate, number of stools and general behavior were recorded twice daily. Blood cultures of mice in different groups were taken at different days to evaluate the establishment of infection as well as to observe the therapeutic and preventive potential of Manuka honey in mouse typhoid. Fisher's Exact, Chi- Square and t-test were used to analyze the data. Significant association was observed in the ultimate fate of mice in Group A1 and Group A2 (P<0.001), showing that from a total of 20 mice in both groups, 10 mice fall in Group A1 of which 10 (100%) developed infection as it was not prevented by honey at a dose of 15ml/kg body weight (15.00±0.00) in Group A1 and ten mice fall in Group A2 of which 10(100%) did not developed an infection as it was prevented by honey at a dose of 20ml/kg body weight (20.00±0.00) in Group A2. Significant association was observed in the ultimate fate of mice in Group B1 and Group B2 (P<0.001) showing that from a total of 20 mice in both groups, 10 mice fall in Group B1 of which 10 (100%) had an infection, which was not treated by honey at a dose of 20 ml/kg body weight. Ten mice fall in Group B2 of which 10 (100%) had an infection, which was treated by honey at a dose of 25 ml/kg body weight (25.00±0.00). Results of the present study suggest that Manuka honey (UMF25±) has a potent anti-typhoid activity in vivo as well. There is an intense need for a carefully designed clinical trial in which this therapeutic potential of Manuka honey should be further evaluated. There is also need for the search of local honeys comparable to Manuka honey as a therapeutic option for typhoid fever.