Epidemiologic profile of hard ticks and molecular characterization of Rhipicephalus microplus infesting cattle in central part of Khyber Pakhtunkhwa, Pakistan.

Tick infestation is a major public and animal health concern causing significant financial losses, especially in tropical and subtropical regions of the world. This study aimed at investigating the epidemiologic profile of ticks infesting cattle and molecular identification of R. microplus in the centrally ignored part of Khyber Pakhtunkhwa, Pakistan. A total of 600 cattle from 20 farms were examined for the tick infestation, among them 358 (59.7%) cattle were infested with ticks. A total of 2118 nymph, larvae and adult tick stages were collected and morphologically identified followed by molecular confirmation of Rhipicephalus microplus. Host-based demographic and ecological parameter analysis revealed significantly higher tick infestation in adult, female, exotic, freely grazing, and with irregular/no acaricides treated cattle. The univariate logistic analysis showed that host age, gender, breed, acaricides use, and feeding method were significantly (P < 0.05) associated, whereas multivariate analysis revealed only host breed and feeding method were potential risk factors (P < 0.05) for tick infestation. Microscopy-based examination identified four different species of ticks including R. microplus (44.5%), Hyalomma anatolicum (38.5%), and Hyalomma marginatum (10.5%) and Hyalomma excavatum (6.5%). Tick infestation pattern showed that 55.9% of cattle was found co-infested with R. microplus and H. anatolicum followed by R. microplus and H. anatolicum and H. marginatum (29.3%) then R. microplus, H. anatolicum, H. marginatum, and H. excavatum (11.2%). Sequencing of the second internal transcribed spacer (ITS2-) and 16S rRNA gene fragments also confirmed the molecular identification of Rhipicephalus microplus. Phylogenetic analysis of ITS-2 revealed all sequences clustered in single clade of the R. microplus while the 16S rRNA nucleotide sequences showed that R. microplus in this study was clustered together in clade A along with other isolates from Pakistan, China, and India. The high tick infestation suggests the need for designing strategic and integrated control measures for ticks in order to ensure good health of domestic animals in this region of Pakistan.

Correction: A cross-sectional survey of hard ticks and molecular characterization of Rhipicephalus microplus parasitizing domestic animals of Khyber Pakhtunkhwa, Pakistan.

[This corrects the article DOI: 10.1371/journal.pone.0255138.].

Antimicrobial Resistance and Associated Risk Factors for Clostridium difficile in Patients Attending Tertiary Care Settings.

To determine the incidence of antimicrobial-resistant emerging pathogens, Clostridium difficile, and its associated risk factors in tertiary care setups of Pakistan. This cross-sectional prospective study was conducted from January 2019 to December 2020, to determine the prevalence and antimicrobial resistance patterns of C. difficile strains isolated from 450 stool specimens of patients suffering from diarrhea hospitalized in tertiary care hospitals in Peshawar, Pakistan. The stool samples of the patients were processed for culture and detection of toxin A and toxin B by enzyme-linked immunosorbent assay (ELISA) and tpi PCR. The drug sensitivity test was performed for antibiotics including ampicillin, cefixime, cefepime, amoxicillin, nalidixic acid, sulpha/TMP (SXT), chloramphenicol, metronidazole, vancomycin, ciprofloxacin, levofloxacin, and imipenem. Of 450 stool specimens, 108 (24%) were positive for C. difficile by stool culture, whereas 115 (25.5%) were only positive for C. difficile toxins based on ELISA and PCR (128 (28.6%). Of 108, 90.7% (n = 98) isolates were resistant to one antibiotic, and 90 (83.4%) were resistant to three or more antimicrobials. The highest resistance rates were found against penicillin (83.3%) followed by amoxicillin (70%), nalidixic acid (61%), and metronidazole (38%), and the lowest resistance was found against vancomycin (6.4%) and imipenem (3.7%). CDI was statistically significantly correlated with increased age, use of antibiotics, abdominal surgeries, use of proton pump inhibitors and H2a, and presence of comorbidities. The high frequency of C. difficile in Peshawar, Pakistan, indicates that CDI is an important nosocomial infection in different hospitals. The results will be helpful for clinicians to redesign control and therapeutic strategies in hospitals.

A cross-sectional survey of hard ticks and molecular characterization of Rhipicephalus microplus parasitizing domestic animals of Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: In tropical and subtropical countries, tick infestation causes major public health problems and considerable financial losses to the livestock industry. This study was aimed to assess the species composition of richness and analyze the phylogeny of Rhipicephalus microplus in the District Bannu of Khyber Pakhtunkhwa, Pakistan. METHODS: Collected ticks were identified morphologically and DNA extracted from R. microplus was amplified and subjected to sequencing. RESULTS: A total of 3,600 animals were examined among them 1,494 animals were found to be infested with ticks, including 669 cows, 476 buffaloes, 163 goats, and 186 sheep (p = 0.001). Tick infestation was significantly high (43.58%) in animals of age group (<1 year) (p-value = 0.027). Female animals were more (44.05%) infested with ticks than males (34.43%) (p = 0.001). The intensity of infestation was significantly higher in summer (77.49%) (p = 0.001). A total of 5,557 ticks were collected comprising three genera and six species. R. microplus was predominantly prevalent (n = 1,474; 26.52%), followed by Rhipicephalus annulatus (n = 1,215; 21.86%), Hyalomma anatolicum (n = 1,139; 20.49%), Hyalomma marginatum (n = 1,086; 19.54%), and Rhipicephalus turanicus (n = 761; 13.69%), while the least common was Haemaphysalis aciculifer (n = 80; 1.43%) (p = 0.001). Morphologically identified R. microplus species were also analyzed genetically by using two genetic markers 16S ribosomal RNA (16S rRNA) and internal transcribed spacer 2 (ITS2) genes. The phylogenetic study revealed that R. microplus is genetically diversified and clustered in clade B with R. microplus species from China, India, and Pakistan. CONCLUSION: Ticks infestation was significantly correlated with various factors including age, sex, season, and animal type. R. microplus genetically resembled species reported from India and China. However, major knowledge gaps concerning various species of ticks exist and many areas are still unexplored in Pakistan. Therefore, it is necessary to explore the epidemiological and molecular aspects of various tick species in other regions of southern Khyber Pakhtunkhwa.