Exploring mosquito abundance and Plasmodium infection through nested-PCR: implications for disease surveillance and control.

The Plasmodium is responsible for malaria which poses a major health threat, globally. This study is based on the estimation of the relative abundance of mosquitoes, and finding out the correlations of meteorological parameters (temperature, humidity and rainfall) with the abundance of mosquitoes. In addition, this study also focused on the use of nested PCR (species-specific nucleotide sequences of 18S rRNA genes) to explore the Plasmodium spp. in female Anopheles. In the current study, the percentage relative abundance of Culex mosquitoes was 57.65% and Anopheles 42.34% among the study areas. In addition, the highest number of mosquitoes was found in March in district Mandi Bahauddin at 21 °C (Tmax = 27, Tmin = 15) average temperature, 69% average relative humidity and 131 mm rainfall, and these climatic factors were found to affect the abundance of the mosquitoes, directly or indirectly. Molecular analysis showed that overall, 41.3% of the female Anopheles pools were positive for genus Plasmodium. Among species, the prevalence of Plasmodium (P.) vivax (78.1%) was significantly higher than P. falciparum (21.9%). This study will be helpful in the estimation of future risk of mosquito-borne diseases along with population dynamic of mosquitoes to enhance the effectiveness of vector surveillance and control programs.

Situation analysis of research ethics governance in Pakistan.

Background: Mapping of ethical governance structures is very useful in identifying strengths and weaknesses in order to uphold integrity and ensure standardization. However, reliable countrywide data about ethical review committees (ERCs) is unavailable in Pakistan. Aims: To evaluate the research ethics governance mechanisms at national level and at key healthcare institutions in Pakistan. Methods: This pilot mapping exercise used a mixed-methods approach, involving a cross-sectional survey of 19 key healthcare research institutions, and structured in-depth interviews with the chairs of the National Bioethics Committee and the Drug Regulatory Authority of Pakistan. Results: Eighteen institutions responded to the ethics mapping survey. Twelve public sector ERCs had a permanent structure and 17 had formal terms of reference. Seven ERCs claimed accreditation, although no central accreditation agency exists in Pakistan. Eight ERCs were chaired by the heads of the institutions. There was no fixed tenure for the heads in 13 committees, and 14 committees allowed multiple terms. Six ERCs had follow-up mechanisms for ethical approvals, and 6 took punitive actions in response to any deviation from an approved protocol, or to a scientific misconduct. Two respondents recalled situations where applicants pressured committee members for favourable approvals. Survey respondents mentioned the lack of central research ethics guidelines as a weakness of the national governance system. Structured interviews revealed the need for formal training of committee members and capacity strengthening, particularly for administrative staff. Conclusion: There is a need to develop guidelines for local ethics governance in Pakistan, and ensure accreditation of ERCs through the National Bioethics Committee to uphold the integrity of the ethics governance structure.

Diagnosis of Acute Dengue Virus Infection Using Enzyme-Linked Immunosorbent Assay and Real-Time PCR.

Dengue fever is a viral infection caused by the dengue virus and is a growing concern for public health worldwide, particularly in tropical and subtropical regions. This study aimed to assess the diagnostic accuracy of a commercially available NS1 ELISA kit for dengue fever in Pakistan using multiplex qRT-PCR as the gold standard. The study recruited 1236 suspected cases of dengue fever admitted to public sector hospitals in Lahore, Pakistan. Of the suspected cases, 610 (49.3%) were confirmed positive for DENV infection through qRT-PCR, with all four serotypes detected. DENV-2 was the most prevalent serotype, detected in 95.7% of cases. The NS1 ELISA kit detected 71.1% of the positive cases. However, the diagnostic accuracy of the NS1 ELISA kit was found to be only 64.89%. Of the 610 confirmed cases, 68% were male and 32% were female, with a median age of 30 years. Dengue fever was diagnosed in 91.8% of cases, while 8.2% were diagnosed with dengue hemorrhagic fever (DHF). DHF patients had a higher prevalence of abdominal pain, hemorrhagic manifestations, and thrombocytopenia. The cocirculation of all four DENV serotypes in Lahore is concerning and could lead to more severe forms of the disease, such as DHF or dengue shock syndrome, in the future. The study highlights the low diagnostic accuracy of commercially available NS1 ELISA kits and emphasizes the importance of using molecular methods to confirm acute dengue infections. Given the increasing prevalence of dengue fever in developing countries like Pakistan, more accurate and reliable diagnostic tools are needed for effective disease management and control.

A K-17 serotype specific Klebsiella phage JKP2 with biofilm reduction potential.

Klebsiella pneumoniae is an opportunistic pathogen responsible for nearly one-third of all Gram-negative infections. Increasing antibiotic resistance has pushed scientists to look for alternative therapeutics. Bacteriophages have emerged as one of the promising alternatives. In the current study, the Klebsiella phage JKP2 was isolated from a sewage sample and characterized against the K-17 serotype of K. pneumoniae. It produced bulls-eye-shaped clear plaques and has a latent period of 45 min with a burst size of 70 pfu/cell. It remained stable at tested pH (5 to 10) and temperatures (37 to 60 °C). Its optimum temperature for long-term storage is 4 °C and -80 °C. The JKP2 showed its infectivity against the K. pneumoniae K-17 serotype only. It controlled planktonic cells of K. pneumoniae 12 h post-incubation. At MOI-1, it efficiently eliminated 98% of 24 and 96% of 48-hour-old biofilm and 86% and 82% of mature biofilm of day 3 and 4, respectively. The JKP2 has an icosahedral capsid of 54 ± 0.5 nm with a short, non-contractile tail, measuring 12 ± 0.2 nm. It possesses a double-stranded DNA genome of 43.2 kbp with 54.1% GC content and encodes 54 proteins, including 29 with known functions and 25 with unknown functions. JKP2 was classified as Drulisvirus within the Autographiviridae family. It uses a T7-like direct terminal repeat strategy for genome packaging. JKP2 can be applied safely for therapeutic purposes as it does not encode an integrase or repressor genes, antibiotic resistance genes, bacterial virulence factors, and mycotoxins.

Expression of a mosquito larvicidal gene in chloroplast and nuclear compartments of Chlamydomonas reinhardtii.

As a part of the search for environment-friendly biocontrol of mosquito-borne diseases, mosquito larvicidal potential of Bacillus thuringiensis subsp. jegathesan (Btj) Cry toxins is explored for toxins with increased toxicity. Safe delivery of the Cry toxins to mosquito larvae in aquatic habitats is a major concern. This is because in water bodies Bacillus thuringiensis (Bt) protein formulations degrade by sunlight, can sink down and get adsorbed by the silt. So, because of its short persistence the toxin requires repeated applications at the given site. Therefore, an upcoming approach is incorporating the Bt toxins in Chlamydomonas reinhardtii (C. reinhardtii) because it is a food of mosquito larvae in water and its molecular toolkit is well investigated for foreign gene expression. The present work aimed to compare the feasibility of C. reinhardtii chloroplast and nuclear compartments for stable expression of Cry11Ba toxin as this is the most toxic Btj protein to date, lethal to different mosquito species. With chloroplast expression of cry11Ba gene we were able to generate marker-free C. reinhardtii strain stably expressing Cry11Ba protein and demonstrating mortality against Aedes aegypti larvae. Moreover, for nuclear expression linking the cry11Ba gene to zeocin via foot and mouth disease virus (FMDV) 2A peptide resulted in the selection of transformants with increased cry11Ba mRNA expression levels by semi-quantitative reverse transcriptase PCR. Obtained results lay a foundation for the C. reinhardtii chloroplast expression system to be used for genetic engineering with Bt toxins which possess enhanced toxicity.

Dengue and Chikungunya virus co-infection in major metropolitan cities of provinces of Punjab and Khyber Pakhtunkhwa: A multi-center study.

Dengue has become endemic in Pakistan with annual recurrence. A sudden increase in the dengue cases was reported from Rawalpindi in 2016, while an outbreak occurred for the first time in Peshawar in 2017. Therefore, a multi-center study was carried out to determine the circulating dengue virus (DENV) serotypes and Chikungunya virus (CHIKV) co-infection in Lahore, Rawalpindi, and Peshawar cities in 2016-18. A hospital-based cross-sectional study was carried out in Lahore and Rawalpindi in 2016-18, while a community-based study was carried out in Peshawar in 2017. The study participants were tested for dengue NS1 antigen using an immunochromatographic device while anti-dengue IgM/IgG antibodies were detected by indirect ELISA. All NS1 positive samples were used for DENV serotyping using multiplex real-time PCR assay. Additionally, dengue samples were tested for CHIKV co-infection using IgM/IgG ELISA. A total of 6291 samples were collected among which 8.11% were NS1 positive while 2.5% were PCR positive. DENV-2 was the most common serotype (75.5%) detected, followed by DENV-1 in 16.1%, DENV-3 in 3.9% and DENV-4 in 0.7% while DENV-1 and DENV-4 concurrent infections were detected in 3.9% samples. DENV-1 was the predominant serotype (62.5%) detected from Lahore and Rawalpindi, while DENV-2 was the only serotype detected from Peshawar. Comorbidities resulted in a significant increase (p-value<0.001) in the duration of hospital stay of the patients. Type 2 diabetes mellitus substantially (p-value = 0.004) contributed to the severity of the disease. Among a total of 590 dengue positive samples, 11.8% were also positive for CHIKV co-infection. Co-circulation of multiple DENV serotypes and CHIKV infection in Pakistan is a worrisome situation demanding the urgent attention of the public health experts to strengthen vector surveillance.

Dengue seroprevalence and its socioeconomic determinants in Faisalabad, Pakistan: a cross-sectional study.

INTRODUCTION: Socioeconomic disparities in the community make some groups more vulnerable to dengue infection. METHODS: Fourteen dengue cases (IgM positive) served as index cases for the positive geographic cluster investigations. RESULTS: Of 292 individuals, the overall dengue seroprevalence was 22.9% (IgM positive 4.8%; IgG positive 18.1%). The highest (45%) seroprevalence was reported in the most socioeconomically vulnerable lower class, followed by the middle class (39%). Orthogonal comparisons showed that socioeconomic factors play a significant role in the prevalence of dengue. CONCLUSIONS: An integrated approach is required to control the menace through vector control strategies and improvement of socioeconomic conditions.

Dengue seroprevalence and its socioeconomic determinants in Faisalabad, Pakistan: a cross-sectional study

Abstract INTRODUCTION: Socioeconomic disparities in the community make some groups more vulnerable to dengue infection. METHODS: Fourteen dengue cases (IgM positive) served as index cases for the positive geographic cluster investigations. RESULTS: Of 292 individuals, the overall dengue seroprevalence was 22.9% (IgM positive 4.8%; IgG positive 18.1%). The highest (45%) seroprevalence was reported in the most socioeconomically vulnerable lower class, followed by the middle class (39%). Orthogonal comparisons showed that socioeconomic factors play a significant role in the prevalence of dengue. CONCLUSIONS: An integrated approach is required to control the menace through vector control strategies and improvement of socioeconomic conditions.

Demographic and clinico-epidemiological features of dengue fever in Faisalabad, Pakistan.

This cross-sectional study was carried out to explore the epidemiological and clinical features of dengue fever in Faisalabad, Pakistan during 2011 and 2012. During the study period, anti-dengue IgM positive cases were reported in the post-monsoon period during the months of August-December. Certain hotspots for the dengue infection were identified in the city that coincide with the clusters of densely populated urban regions of the city. Out of total 299 IgM positive patients (male 218 and female 81); there were 239 dengue fever (DF) and 60 dengue hemorrhagic fever (DHF) patients. There was decrease in the median age of dengue patients from 31 years in 2011 to 21.5 years in 2012 (p<0.001). Abdominal pain was seen in 35% DHF patients followed by nausea in 28.3%, epistaxis in 25% and rash in 20% patients (p<0.05). Patients reported to be suffering from high-grade fever for an average of 8.83 days in DHF as compared to 5.82 days in DF before being hospitalized. Co-morbidities were found to be risk factor for the development of DHF in dengue patients. Clinical and laboratory features of dengue cases studied could be used for the early identification of patients at risk of severe dengue fever.