Crimean-Congo Hemorrhagic Fever Virus in Humans and Livestock, Pakistan, 2015-2017.

We detected Crimean-Congo hemorrhagic fever virus infections in 4 provinces of Pakistan during 2017-2018. Overall, seroprevalence was 2.7% in humans and 36.2% in domestic livestock. Antibody prevalence in humans was highest in rural areas, where increased contact with animals is likely.

Serologic Evidence of Severe Fever with Thrombocytopenia Syndrome Virus and Related Viruses in Pakistan.

We describe the seroprevalence of severe fever with thrombocytopenia syndrome virus (SFTSV) and the association of antibody occurrence with location, sex, and age among the human population in Pakistan. Our results indicate substantial activity of SFTSV and SFTSV-related viruses in this country.

Risk of infection with arboviruses in a healthy population in Pakistan based on seroprevalence.

Infectious diseases caused by arboviruses are a public health concern in Pakistan. However, studies on data prevalence and threats posed by arboviruses are limited. This study investigated the seroprevalence of arboviruses in a healthy population in Pakistan, including severe fever with thrombocytopenia syndrome virus (SFTSV), Crimean-Congo hemorrhagic fever virus (CCHFV), Tamdy virus (TAMV), and Karshi virus (KSIV) based on a newly established luciferase immunoprecipitation system (LIPS) assays, and Zika virus (ZIKV) by enzyme-linked immunosorbent assays (ELISA). Neutralizing activities against these arboviruses were further examined from the antibody positive samples. The results showed that the seroprevalence of SFTSV, CCHFV, TAMV, KSIV, and ZIKV was 17.37%, 7.58%, 4.41%, 1.10%, and 6.48%, respectively, and neutralizing to SFTSV (1.79%), CCHFV (2.62%), and ZIKV (0.69%) were identified, as well as to the SFTSV-related Guertu virus (GTV, 0.83%). Risk factors associated with the incidence of exposure and levels of antibody response were analyzed. Moreover, co-exposure to different arboviruses was demonstrated, as thirty-seven individuals were having antibodies against multiple viruses and thirteen showed neutralizing activity. Males, individuals aged ≤40 years, and outdoor workers had a high risk of exposure to arboviruses. All these results reveal the substantial risks of infection with arboviruses in Pakistan, and indicate the threat from co-exposure to multiple arboviruses. The findings raise the need for further epidemiologic investigation in expanded regions and populations and the necessity to improve health surveillance in Pakistan.

Insights into the Intersection of Biocide Resistance, Efflux Pumps, and Sequence Types in Carbapenem-Resistant Acinetobacter baumannii: A Multicenter Study.

Acinetobacter baumannii, a pathogenic bacterium acquired in hospitals, causes diverse infections in humans. Previous studies have reported resistance among A. baumannii strains, potentially selecting multi-drug-resistant variants. In Pakistan, research has primarily focused on carbapenem-resistant A. baumannii (CRAB) strains, overlooking the investigation of efflux pumps (EPs) and biocide resistance. This study aims to assess A. baumannii strains from five hospitals in Pakistan, focusing on antibiotic and biocide susceptibility, the impact of EP inhibitors on antimicrobial susceptibility, and the distribution of ARGs and STs. A total of 130 non-repeated Acinetobacter baumannii isolates were collected from five tertiary care hospitals in Pakistan and identified using API 20NE and multiplex PCR. Antimicrobial susceptibility testing utilized disc diffusion and broth microdilution assays, while biocide susceptibility was assessed with various agents. The impact of an efflux pump inhibitor (NMP) on antibiotic susceptibility was evaluated. PCR screening for ARGs and EPGs was followed by DNA sequencing validation. MLST was performed using the Pasteur scheme. Most isolates demonstrated resistance to tested antibiotics, with varying levels of susceptibility to biocides. All isolates exhibited the intrinsic class D ß-lactamase blaOXA-51, while acquired blaOXA-23 was present in all CRAB isolates. Among EPs, adeJ, abeD, amvA, and aceI were prevalent in almost all isolates, with adeB found in 93% of isolates and adeG, adeT1, adeT2, and qacEΔ1 displaying lower prevalence ranging from 65% to 79%. The most common STs were ST589 and ST2, accounting for 28.46% and 25.38% of isolates, respectively, followed by ST642 at 12.6%. These findings indicate that A. baumannii strains in Pakistan are resistant to antibiotics (excluding colistin and tigecycline) and may be developing biocide resistance, which could contribute to the selection and dissemination of multi-drug-resistant strains.

The Molecular Detection of Class B and Class D Carbapenemases in Clinical Strains of Acinetobacter calcoaceticus-baumannii Complex: The High Burden of Antibiotic Resistance and the Co-Existence of Carbapenemase Genes.

The emergence of carbapenem-resistant Acinetobacter calcoaceticus-baumannii complex (CRACB) in clinical environments is a significant global concern. These critical pathogens have shown resistance to a broad spectrum of antibacterial drugs, including carbapenems, mostly due to the acquisition of various ß-lactamase genes. Clinical samples (n = 1985) were collected aseptically from multiple sources and grown on blood and MacConkey agar. Isolates and antimicrobial susceptibility were confirmed with the VITEK-2 system. The modified Hodge test confirmed the CRACB phenotype, and specific PCR primers were used for the molecular identification of blaOXA and blaNDM genes. Of the 1985 samples, 1250 (62.9%) were culture-positive and 200 (43.9%) were CRACB isolates. Of these isolates, 35.4% were recovered from pus samples and 23.5% from tracheal secretions obtained from patients in intensive care units (49.3%) and medical wards (20.2%). An antibiogram indicated that 100% of the CRACB isolates were resistant to ß-lactam antibiotics and ß-lactam inhibitors, 86.5% to ciprofloxacin, and 83.5% to amikacin, while the most effective antibiotics were tigecycline and colistin. The CRACB isolates displayed resistance to eight different AWaRe classes of antibiotics. All isolates exhibited the blaOXA-51 gene, while blaOXA-23 was present in 94.5%, blaVIM in 37%, and blaNDM in 14% of the isolates. The blaOXA-51, blaOXA-23, and blaOXA-24 genes co-existed in 13 (6.5%) isolates. CRACB isolates with co-existing blaOXA-23, blaOXA-24, blaNDM, blaOXA-51 and blaVIM genes were highly prevalent in clinical samples from Pakistan. CRACB strains were highly critical pathogens and presented resistance to virtually all antibacterial drugs, except tigecycline and colistin.

Extensively Drug-resistant Acinetobacter baumannii Belonging to International Clone II from A Pet Cat with Urinary Tract Infection; The First Report from Pakistan.

The carbapenem-resistant Acinetobacter baumannii (CRAB) has got global attention as a notorious nosocomial pathogen. This study describes a case of urinary tract infection in a 2-years old pet female cat infected with A. baumannii. The susceptibility profiling, screening for the resistance determinants, and the multilocus sequence typing was performed. The A. baumannii isolate was found to harbor the blaOXA23-like gene and corresponded to International clone II that has been widely reported to be involved in human infections. The study proposes that the pets may contribute towards the spread of clinically relevant antimicrobial-resistant pathogens.The carbapenem-resistant Acinetobacter baumannii (CRAB) has got global attention as a notorious nosocomial pathogen. This study describes a case of urinary tract infection in a 2-years old pet female cat infected with A. baumannii. The susceptibility profiling, screening for the resistance determinants, and the multilocus sequence typing was performed. The A. baumannii isolate was found to harbor the blaOXA23-like gene and corresponded to International clone II that has been widely reported to be involved in human infections. The study proposes that the pets may contribute towards the spread of clinically relevant antimicrobial-resistant pathogens.

Molecular mechanisms of antibiotic co-resistance among carbapenem resistant Acinetobacter baumannii.

INTRODUCTION: The spread of carbapenem-resistant Acinetobacter baumannii (CRAB) is difficult to control especially in the hospitals due to the successful mobilization and evolution of the genetic elements harboring the resistant determinants. The study was conducted to examine the distribution of aminoglycosides, tetracycline, and sulfonamide-resistant determinants among CRAB isolates that carry the blaOXA-23 gene. METHODOLOGY: For a total of 160 CRAB strains isolated at tertiary care hospitals of Pakistan that mainly carried blaOXA-23 gene were included in the study to evaluate the assortment of antibiotic resistance genes. RESULTS: The susceptibility rates of CRAB for other than beta-lactam drugs were 2.5% for both ciprofloxacin and aminoglycosides and 18% and 25% for sulfonamides and tetracyclines, respectively. Polymyxin B (MIC90, 1 g/mL) Colistin (MIC90, 1 g/mL) and Tigecycline (MIC90, 2 g/mL) were most active against these extensively drug-resistant CRAB isolates. The isolates were found to possess various genes mainly the tetB and sul2 for tetracycline and sulfonamide but the genes conferring resistance to aminoglycosides were varied with various combinations. CONCLUSION: Despite the CRAB clones containing blaOXA-23 have been previously reported in Pakistani hospitals, the screening of genetic determinants responsible for other antimicrobial agents is crucial for developing an effective surveillance and mitigation system for infection management.

Detection of West Nile virus lineage 1 sequences in blood donors, Punjab Province, Pakistan.

OBJECTIVES: This study was performed to determine the presence of West Nile virus (WNV) in mosquito specimens and human blood donors in Pakistan. METHODS: A total of 4150 mosquito specimens were collected using CO2-baited traps from five selected districts of Punjab Province, Pakistan. The mosquitoes were taxonomically identified using standard morphological keys, resulting in 166 pools. In addition, 1070 serum samples were collected from human blood donors. RNA was extracted from mosquito and human samples and screened for WNV using a reverse transcriptase PCR (RT-PCR). RESULTS: None of the mosquito pools tested positive for WNV, whereas three samples from asymptomatic humans tested positive. To determine the WNV strains, partial sequences were compared against a global representation of 23 WNV sequences. The study strains were determined to come from WNV lineage 1. CONCLUSIONS: This study is novel in reporting the circulation of lineage 1 WNV in Pakistan. Given its ability to transmit from human to human via blood transfusion, this highlights the urgent need for nationwide surveillance to assess the distribution and impact of WNV in Pakistan. Determining the source of human infection will require more extensive mosquito sampling.

In vitro and in vivo activity of Manuka honey against NDM-1-producing Klebsiella pneumoniae ST11.

AIM: To determine the therapeutic potential of Manuka honey against New Delhi metallo-ß-lactamase-1-producing Klebsiella pneumoniae ST11 in vitro and in vivo. MATERIALS & METHODS: Carbapenamases and metallo-ß-lactamases-producing K. pneumoniae ST11 isolated from blood culture was confirmed by VITEK-2® system, matrix-assisted laser desorption ionization-time of flight and multilocus sequence typing, followed by determination of minimum inhibitory concentration (µg/ml) using VITEK-2 system. Genetic analysis of bla NDM-1 was done by PCR, pulsed-field gel electrophoresis and DNA hybridization. In vitro and in vivo efficacy of Manuka honey was performed by microbroth dilution assay and BALB/c mice model respectively. RESULTS: K. pneumoniae ST11 displayed resistance to commonly used antibiotics. bla NDM-1 was located on 150 and 270kb plasmids. Minimum inhibitory concentration and minimum bactericidal concentration of Manuka honey was 30% (v/v) and substantial reduction of bacterial mean log value (>1 log) was observed in mice. Histological analysis of mice liver and kidneys demonstrated mild to moderate inflammation. CONCLUSION: Manuka honey can be used as an alternate therapeutic approach for management of New Delhi metallo-ß-lactamase-producing pathogens.

Countrywide Survey for MERS-Coronavirus Antibodies in Dromedaries and Humans in Pakistan.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a zoonotic pathogen capable of causing severe respiratory disease in humans. Although dromedary camels are considered as a major reservoir host, the MERS-CoV infection dynamics in camels are not fully understood. Through surveillance in Pakistan, nasal (n = 776) and serum (n = 1050) samples were collected from camels between November 2015 and February 2018. Samples were collected from animal markets, free-roaming herds and abattoirs. An in-house ELISA was developed to detect IgG against MERS-CoV. A total of 794 camels were found seropositive for MERS-CoV. Prevalence increased with the age and the highest seroprevalence was recorded in camels aged > 10 years (81.37%) followed by those aged 3.1-10 years (78.65%) and ≤ 3 years (58.19%). Higher prevalence was observed in female (78.13%) as compared to male (70.70%). Of the camel nasal swabs, 22 were found to be positive by RT-qPCR though with high Ct values. Moreover, 2,409 human serum samples were also collected from four provinces of Pakistan during 2016-2017. Among the sampled population, 840 humans were camel herders. Although we found a high rate of MERS-CoV antibody positive dromedaries (75.62%) in Pakistan, no neutralizing antibodies were detected in humans with and without contact to camels.