Methods in molecular biology (Clifton, N.J.)Samantha K D'Spain, María Eugenia Cortina, R Clayton Bishop, Isabelle Derré
Identification and characterization of essential Chlamydia-specific factors are vital for the generation of effective therapeutics and vaccines. Despite delays in the development of genetic tools for Chlamydia, significant advancements in Chlamydia genetic manipulation have been made over the last decade and a half, now allowing for the targeted interruption or deletion of chromosomal genes. However, because of the obligate intracellular nature of the bacteria and its unique biphasic developmental cycle, it remains challenging to inactivate essential genes and study the function of their encoded proteins. Here, we describe a method to generate conditional knockout Chlamydia mutants by introducing, prior to gene inactivation, a complementation plasmid containing the gene of interest under the control of an anhydrotetracycline (aTc)-inducible promoter. The chromosomal copy of the gene can then be selectively deleted to produce a conditional knockout mutant in which expression of the gene is controlled with aTc. We detail how this conditional knockout mutant can be used to determine if the targeted gene is essential for Chlamydia infection in cell culture and in vivo.
Methods in molecular biology (Clifton, N.J.)Jenniffer Ramos Martins, Viviane Lima Batista, Celso M Queiroz-Junior, Mauro Martins Teixeira, Vivian Vasconcelos Costa
Arboviruses still represent a major challenge to public health in many parts of the world. Despite shared epidemiological characteristics, each arboviral disorder displays distinct cellular tropism and activation patterns, progressing to immunopathological outcomes that remain not fully elucidated. A deeper understanding of these processes requires methodologies capable of characterizing the often-complex cellular responses. In this context, flow cytometry emerges as a valuable tool, enabling the characterization of heterogeneous cell populations, the assessment of activation and proliferation markers, the quantification of inflammatory mediators, and the detection of cell death pathways. By integrating these parameters, flow cytometry can enhance our understanding of arbovirus pathogenesis, clarify their cellular tropism, and understand immune responses to infection. This methodological article outlines the main steps for applying the technique to different target tissues infected by chikungunya, dengue, yellow fever, and Zika viruses, emphasizing the most effective strategies.
Methods in molecular biology (Clifton, N.J.)Colleen C Monahan, Nicole A Grieshaber, Scott S Grieshaber, Anders Omsland
Due to genome streamlining, many genes in the ~1 Mb genome of Chlamydia trachomatis are essential and cannot be deleted. Ectopic overexpression and CRISPRi-based knockdown are complementary genetic approaches to study the function of genes in C. trachomatis, including essential genes. When used in combination with inducible promoters, these tools allow researchers to determine how precise regulation of gene expression contributes to proper progression of the chlamydial developmental cycle. This chapter includes protocols which detail ectopic overexpression of a protein or conversely, transcriptional inhibition of a specific gene in C. trachomatis serovar L2/434/Bu.
Methods in molecular biology (Clifton, N.J.)Nicole A Grieshaber, Scott S Grieshaber
The obligate intracellular bacterium Chlamydia employs a unique, asynchronous biphasic developmental cycle characterized by distinct morphological forms: the replicative reticulate body (RB), the infectious elementary body (EB), and an intermediate body (IB). The simultaneous presence of these phenotypically distinct cell types throughout infection complicates the study of gene expression regulation during development. Conventional population-level assays are inadequate for dissecting the regulatory mechanisms within this mixed population. This chapter describes a robust method utilizing hybridization chain reaction (HCR) RNA fluorescence in situ hybridization (FISH) in conjunction with dual-promoter reporter Chlamydia strains to determine cell-form-specific transcript expression. We leverage dual-reporter strains to provide visual identification of RBs and EBs. Probes targeting genes of interest are designed to interact with spectrally distinct fluorescent HCR amplifiers. This versatile, multiplexing system allows for precise spatial and temporal localization of specific mRNAs within identified Chlamydial cell forms, offering a powerful tool to overcome current limitations in Chlamydia research and elucidate the regulatory underpinnings of its complex life cycle.
Methods in molecular biology (Clifton, N.J.)Alexander W E Franz
Chikungunya virus (CHIKV) is a human pathogenic, mosquito-borne virus (arbovirus), which is causing epidemic outbreaks among human populations in Africa, Asia, Europe, and South- and Central America including the Caribbean. The development of novel approaches to prevent mosquito transmission of the virus in the field requires a detailed study of CHIKV interactions with its mosquito vectors in the laboratory. In this chapter, we describe how to prepare Aedes aegypti mosquitoes for the infection with freshly cultivated CHIKV. We also describe quantitative and qualitative viral detection assays in mosquitoes based on plaque assays and the amplification of saliva samples in cell culture.
Methods in molecular biology (Clifton, N.J.)Akino Emi, Youichi Suzuki
The cytopathic effect (CPE) is a key to understanding the pathogenicity of the chikungunya virus (CHIKV) and is useful for evaluating viral infectivity in vitro. The plaque assay, based on the CPE induced by viral infection, provides an infectious virus titer for test samples. Additionally, the assay is simple, quantitative, and cost-effective. Thus, the plaque assay remains the gold standard for determining the infectivity of CHIKV. However, manually counting of virus-induced plaques with the naked eye or under the microscope is often time-consuming and labor-intensive. Therefore, automated plaque-counting software would improve the consistency and efficiency of plaque counting. We recently developed plaQuest, a stand-alone Windows software that enables rapid, reliable plaque counting for CHIKV. In this chapter, we describe the basic procedure for detecting and counting the CHIKV-formed plaques in a 24-well plate.
Methods in molecular biology (Clifton, N.J.)Natália G Salomão, Kíssila Rabelo
Histopathological analysis of tissues infected with Chikungunya virus (CHIKV), including biopsy and autopsy specimens, can provide valuable insights into the pathogenesis of atypical and fatal cases. By examining tissue architecture and cellular alterations under the microscope, it is possible to identify patterns of injury, inflammation, and cellular degeneration. These morphological findings provide evidence of how the pathogen interacts with host cells and tissues. Moreover, immunohistochemistry may be performed in tissue sections for the detection of viral antigens. Importantly, this technique allows the characterization of diverse biologically relevant targets in tissue samples, such as distinct immune cell subsets, cytokines, and additional molecular markers. This is accomplished by the use of antibodies selected for their specificity toward the antigen of interest. In this chapter, we present a concise overview of how histological analysis and immunohistochemical approaches can enhance the understanding of CHIKV-associated pathological mechanisms.
Methods in molecular biology (Clifton, N.J.)David J Delgado Diaz, Katherine M Nelson, Vonetta L Edwards, Ian J Glomski, Filipa Ribeiro, Patrik M Bavoil, Alison K Criss, Jacques Ravel, Jason P Gleghorn, I…
Chlamydia trachomatis is an obligate intracellular bacterium that infects the columnar epithelium of the human endocervix. While conventional two-dimensional cell cultures and animal models have been instrumental in advancing our understanding of C. trachomatis biology, they are limited in capturing the multicellularity, architecture, and physiological microenvironment of the human cervix. This chapter describes the use of a three-dimensional (3D) microphysiologic model to study Chlamydia trachomatis infection. The model is inexpensively made without specialized equipment and is designed to recreate the epithelial-stromal interface. We outline procedures for coculturing cervical epithelial cells and fibroblasts, infecting epithelial cells with fluorescently labeled C. trachomatis, monitoring infection progression via fluorescent microscopy, and quantifying infectious progeny. The complete developmental cycle of C. trachomatis within this model provides a robust and accessible platform to investigate C. trachomatis-specific host-pathogen interactions, immune responses, and the influence of diverse physiological and environmental stimuli within a relevant cervical context.
Methods in molecular biology (Clifton, N.J.)Griselda Ines Oria
The processing of adult mosquitoes collected in the field for studies of natural infection with arboviruses of public health interest (genera: Flavivirus, Alphavirus, Orthobunyavirus) follows the assembly of homogenized mosquito pools and extraction of genetic material using commercial or manual techniques, and the detection of viral RNA by polymerase chain reaction (nested RT-PCR) [11]. This paper describes the procedures for capturing, taxonomic determination, obtaining genetic material, and molecular techniques for detecting alphaviruses.
Methods in molecular biology (Clifton, N.J.)Sebastiano Volpe, Claudia Filomatori
The plaque-forming assay is the gold-standard method for measuring infectious viral particles. In this technique, lytic viruses infect and destroy host cells, but their spread is restricted by a viscous overlay medium. As a result, new viral particles can only infect neighboring cells, leading to localized clear areas known as plaques, which become visible after staining the remaining living cells. The number of plaques reflects the number of infectious viral particles initially present in the sample and is reported as plaque-forming units (PFU) per sample volume. In this protocol, we describe step by step how to perform a plaque-forming assay to determine the concentration of chikungunya virus in a cell culture supernatant.
Methods in molecular biology (Clifton, N.J.)Aakriti Singh, Barbara S Sixt
The recent application of experimental and computational drug discovery workflows has identified hundreds of new molecules that display potent, and in some cases highly selective, activity against bacteria of the genus Chlamydia. The full value of these novel antichlamydial molecules, however, depends critically on our ability to determine their molecular targets and modes of action. A particularly powerful and widely applied strategy for identifying candidate targets involves selecting for bacterial mutants that have acquired resistance to a compound's inhibitory activity, followed by determining the mutations responsible for this resistance. Such mutations often pinpoint the compound's direct target or reveal cellular pathways that modulate target engagement. Here, we present a detailed, step-by-step protocol for applying this approach to the clinically important, human-pathogenic species Chlamydia trachomatis. The workflow includes: (1) determining compound potency by quantitative dose-response analysis, (2) generating a resistant C. trachomatis mutant through serial passaging under progressively increasing selective pressure, (3) confirming the resistance phenotype, and (4) isolating bacterial genomic DNA for whole-genome sequencing to identify resistance-associated mutations and facilitate downstream target discovery.
Methods in molecular biology (Clifton, N.J.)Syed M A Rizvi, Owais R Hakiem, Ming Tan
Chromatin immunoprecipitation (ChIP) measures binding between a specific protein and its in vivo DNA-binding sites. It is challenging to perform ChIP for bacteria that reside inside a eukaryotic cell, but we have optimized this method for Chlamydia-infected cells and used it to identify genome-wide binding sites of chlamydial transcription factors and sigma factors. The ability to measure protein-DNA interactions in intracellular chlamydiae without the need to isolate bacteria from the host cell allows changes in DNA binding to be measured during the Chlamydia developmental cycle and under different growth conditions. In this chapter, we provide a detailed protocol for Chlamydia ChIP by describing how to cross-link proteins to DNA, shear chlamydial chromatin into DNA fragments, and immunoprecipitate protein-DNA complexes with specific antibodies against the protein of interest. After a decrosslinking step, DNA fragments are analyzed by qPCR to measure binding to individual DNA-binding sites, or with deep sequencing for unbiased identification of genome-wide binding sites.
Global genomic surveillance has emerged as a foundational pillar of public health in the twenty-first century, enabling real-time tracking of pathogen evolution and informing outbreak response. This chapter examines the strategic architecture of global genomic surveillance, focusing on its application to arboviruses such as chikungunya virus (CHIKV). It explores the integration of genomic data with epidemiological, clinical, and environmental information within a One Health framework, while addressing critical challenges in governance, equity, and interoperability. The discussion covers the entire genomic surveillance workflow, from sample collection and sequencing to bioinformatic analysis and phylogenetic inference, and highlights the transformative role of artificial intelligence (AI) in predictive surveillance. By analyzing global initiatives, operational barriers, and emerging technologies, this chapter underscores the necessity of sustainable, equitable, and interoperable genomic systems to proactively address current and future infectious disease threats.
The pathogenic bacterium Chlamydia replicates via an unusual developmental cycle that takes place within a membrane-bound inclusion inside an infected host cell. Immunofluorescence microscopy has revealed many details about the two specialized developmental forms and the recruitment of host organelles to the inclusion, but lacks the high resolution of electron microscopy (EM). In this chapter, we describe the use of an innovative microscopy method, called Tenfold Robust Expansion (TREx) microscopy, to visualize proteins in Chlamydia-infected cells at nanoscale resolution. We present a detailed protocol describing the steps of this expansion microscopy method, including immunostaining, anchoring, gelation, homogenization, expansion, and visualization with a standard confocal microscope.
Methods in molecular biology (Clifton, N.J.)Marcel Rühling, Thomas Rudel
The obligate intracellular bacterial pathogen Chlamydia trachomatis establishes a membrane-bound compartment called the inclusion, which serves as a replicative niche, within host cells. Sphingolipids, a major class of eukaryotic membrane lipids, are acquired from the host and incorporated into chlamydial membranes. Visualization of individual bacteria within inclusions or sub-bacterial structures by fluorescence microscopy remains challenging due to the limited resolution of conventional light microscopy. Expansion microscopy (ExM), a super-resolution imaging technique, enables nanoscale resolution on standard confocal microscopes and is an ideal tool to study Chlamydia infections. However, current ExM protocols are not compatible with sphingolipid visualization. Here, we present a strategy that enables ExM-based imaging of sphingolipids during C. trachomatis infection.
Methods in molecular biology (Clifton, N.J.)Jordan Wesolowski, Fabienne Paumet
Chlamydia subverts host cell pathways by secreting effector proteins into the membrane of its inclusion. These bacterial membrane proteins, called Incs, mediate interactions between the inclusion and the host cell. To study Inc protein dynamics in live cells, we have developed a methodology that relies on host cell expression of antibodies fused to fluorescent proteins, known as frankenbodies, which detect epitopes exposed to the cytosol. HeLa cells are first stably transfected with a plasmid encoding an mScarlet3-tagged anti-FLAG frankenbody. These cells are then infected with Chlamydia expressing a FLAG-tagged Inc protein, which is secreted into the inclusion membrane with the FLAG tag facing the cytosol. Recruitment of the fluorescent anti-FLAG frankenbody to the inclusion is monitored in real time using time-lapse confocal microscopy. This approach has been successfully employed to elucidate the dynamics of IncA on the inclusion membrane and can be expanded to simultaneously investigate multiple Incs, as well as Inc:Inc interactions, by using different tag-frankenbody combinations.
Methods in molecular biology (Clifton, N.J.)Rachel Weild, Richard D Hayward
This chapter describes methodological approaches for studying intracellular ion dynamics during Chlamydia trachomatis infection. It emphasizes the requirement for live-cell imaging to capture active and compartmentalized ion fluxes in infected cells precluded by fixed-cell techniques. Specifically, we present step-by-step protocols for labeling Chlamydia-infected cells with a fluorescent potassium ion (K+)-sensitive probe for performing live imaging with confocal microscopy or spinning disc confocal microscopy for higher resolution and procedures for image processing and quantification. We also describe time-lapse microscopy to monitor K+ dynamics over the extended time course of the infection. These advanced microscopy methods have the potential to visualize ion transport in real time at the host-pathogen interface.
Methods in molecular biology (Clifton, N.J.)Robert Faris, Mary M Weber
To subvert host defenses and establish a replicative niche, obligate intracellular pathogens remodel the host cell using a discrete repertoire of effector proteins. For Chlamydia trachomatis, defining the repertoire of conventional type III secretion system effectors (cT3SS) has been exceedingly difficult as many of these proteins are present at low levels in the host cell and do not accumulate within a specific host compartment, necessitating signal amplification for accurate detection. Drawing on approaches widely used for assessing secretion of candidate T3SS and T4SS substrates, several enzymatic reporter tags have been adapted to evaluate secretion of putative C. trachomatis cT3SS substrates. Here we describe BlaM, CyaA, and GSK assays, which are three complementary assays that provide robust and reliable readouts of effector delivery into the host cell. This protocol describes each assay in detail, including the steps required to generate C. trachomatis transformants, confirm effector-reporter expression, and execute and analyze data for each assay.
Understanding Chlamydia trachomatis infection requires physiologically relevant models tailored to various research questions. One of the main sites of C. trachomatis infection is the columnar epithelium of the endocervix; therefore, a model that accurately mimics the environment of endocervical epithelium is essential. Polarized cell models provide a valuable solution, offering a cost-effective yet biologically relevant alternative to conventional 2D culture systems and more complex and costly 3D or animal models. Immortalized human endocervical epithelial cells (A2EN cells) form a polarized monolayer on transwell membranes, reproducing key in vivo features, including tight junctions, mucus secretion, proinflammatory cytokine production, and hormone receptor expression, when maintained at low passage. This chapter outlines protocols for generating a polarized A2EN culture and assessing polarization through morphological, biochemical, and functional assays. Furthermore, we describe how C. trachomatis development can be monitored in polarized cells through microscopy, flow cytometry, immunofluorescence, and reinfection assays.
Chikungunya virus (CHIKV) represents a significant global health threat due to its recent resurgence. This protocol highlights the use of capillary-modified visual loop-mediated isothermal amplification (LAMP) technology for the rapid detection of CHIKV. We employed Vero cells for virus propagation and utilized the MolPure Viral DNA/RNA Kit for nucleic acid extraction, successfully amplifying extracted viral RNA at 63°C for 25 min. The presence of CHIKV was indicated by a distinct yellowish-green color change, demonstrating the effectiveness and simplicity of this protocol. Our protocol underscores the potential of LAMP technology as a rapid diagnostic tool for CHIKV, enabling timely public health interventions.
Methods in molecular biology (Clifton, N.J.)Talita Émile Ribeiro Adelino, Joilson Xavier, Vagner Fonseca, Marta Giovanetti
Nanopore sequencing is transforming viral genomics through real-time, portable, long-read analysis of RNA and DNA. Unlike traditional short-read platforms, it detects nucleotide sequences by measuring ionic current changes as nucleic acids pass through nanoscale pores, enabling direct single-molecule sequencing and base modification detection. Its simplicity, flexibility, and capacity for ultra-long reads make it ideal for resolving complex genomic regions, structural variants, and full viral genomes. These advantages have accelerated its use in pathogen surveillance and outbreak response, especially in resource-limited settings. For chikungunya virus (CHIKV), nanopore sequencing allows rapid, culture-independent recovery of complete genomes from clinical and vector samples, enabling real-time tracking of viral diversity, evolution, and spread. Experiences from Ebola, Zika, and COVID-19 have demonstrated the power of portable sequencing, now applied to CHIKV monitoring. Advances in tools such as Guppy, Dorado, Minimap2, and Medaka enhance read quality, consensus accuracy, and downstream analyses. Despite challenges in basecalling and error correction, robust quality control pipelines ensure reliable results. Ongoing improvements in chemistry, flow cell design, and machine learning will further enhance fidelity and throughput, establishing nanopore sequencing as a cornerstone of CHIKV genomic surveillance and epidemic preparedness.
Methods in molecular biology (Clifton, N.J.)Chenyang Wu, Sara Ann Thannickal, Tommaso Cabani, Maria Gabriela Noval
Chikungunya virus (CHIKV) is a positive-sense RNA alphavirus transmitted to humans primarily by Aedes aegypti and Aedes albopictus mosquitoes. Its global circulation and significant public health impact underscore the need to better understand the molecular mechanisms driving CHIKV pathogenesis and transmission. Although robust molecular biology methods exist for CHIKV genome sequencing, a major limitation for surveillance and research is the inability to determine whether two nucleotide variations co-occur within the same viral genome when they are separated beyond the span of typical short-read designs. Here, we describe an optimized approach for processing CHIKV RNA samples that generates large amplicons suitable for long-read nanopore sequencing. This protocol enables amplification of the complete CHIKV genome in only two or three amplicons and facilitates detection of co-occurring nucleotide variations across 4-7.5 kb within the same molecule, thereby simplifying sequencing workflows and improving resolution in studies of viral evolution.
Methods in molecular biology (Clifton, N.J.)Eliana F Castro, Claudia Filomatori
Chikungunya virus (CHIKV), an arthropod-borne alphavirus within the Togaviridae family, is transmitted primarily by Aedes aegypti and Aedes albopictus. The virus causes an acute febrile illness characterized by severe, often bilateral polyarthralgia, with potential progression to chronic musculoskeletal pain and rare systemic complications involving cardiovascular and neurological systems. CHIKV exhibits a spherical, enveloped virion (~70 nm) with T = 4 icosahedral symmetry, incorporating E1/E2 glycoprotein heterodimers that mediate receptor binding and membrane fusion. Its positive-sense RNA genome (~11.8 kb) encodes nonstructural proteins for replication and structural proteins for virion assembly. Replication occurs in cytoplasmic spherules, involving synthesis of genomic and subgenomic RNAs, followed by glycoprotein maturation and budding at the plasma membrane. Epidemiologically, CHIKV has expanded beyond Africa and Asia, with major outbreaks driven by adaptive mutations enhancing transmission via A. albopictus. Since introduction to the Americas in 2013, the global incidence remains high, with >180,000 confirmed cases reported in 2025. Preventive strategies rely on vector control and vaccination; VLP-based vaccines (e.g., Vimkunya) show promise, while live-attenuated formulations face safety concerns. No licensed antivirals exist; current management is supportive, though investigational therapies targeting viral replication and immune modulation are under development.
We adapted the plaque reduction neutralization test (PRNT) to detect chikungunya virus (CHIKV) neutralizing antibodies in liver homogenates instead of serum. This approach enables serological investigation using tissues collected from animal carcasses as nonhuman primate ones, during arbovirus surveillance, expanding opportunities for CHIKV monitoring and strengthening epidemiological surveillance efforts.
Methods in molecular biology (Clifton, N.J.)Eugenia S Bardossy, Claudia Filomatori
Chikungunya virus (CHIKV) is a positive-sense single-stranded RNA virus, which poses challenges for its study and genetic manipulation. Because direct mutagenesis of viral RNA genomes is technically impractical, reverse genetics systems are essential tools for investigating viral biology. To enable such approaches, infectious clones containing a full-length cDNA copy of the viral genome are constructed. The cDNA is positioned under the control of a bacteriophage RNA polymerase promoter, allowing commercial RNA polymerases to use the linearized plasmid as a template for the in vitro transcription of full-length viral genomic RNA (gRNA). Importantly, positive-sense viral genomes serve as mRNAs for the translation of viral proteins in a cellular environment, meaning that these transcripts contain all the information required to initiate viral replication. Following transfection into permissive cultured cells, viral proteins are expressed, enabling genome replication and, ultimately, the recovery of infectious particles from the cell supernatant. Here, we describe a detailed procedure for generating CHIKV particles through plasmid linearization, in vitro transcription, and subsequent RNA transfection.
Arboviral diseases represent a major public health challenge, especially in tropical regions where environmental conditions may favor the proliferation and spread of mosquito vectors. Thus, early and accurate detection of chikungunya virus (CHIKV) in mosquito populations can be a valuable tool for effective surveillance of circulating variants and for identifying new viral introductions. Given the challenges of detecting arboviruses in field-captured mosquitoes, we describe an integrated workflow for CHIKV molecular detection and whole-genome sequencing. This protocol includes mosquito homogenization using a bead-based mechanical disruptor, RNA extraction using TRIzol reagent with minor modifications, molecular screening using CHIKV-specific RT-qPCR, and whole-genome amplification followed by sequencing on Illumina platforms. Despite the protocol being optimized for individual mosquitoes, it results in high-quality RNA suitable for both entomological surveillance and genomic analysis. As this protocol allows recovery of complete CHIKV genomes from mosquito specimens, it can serve as a basis for genomic epidemiology studies, enabling monitoring of viral diversity and lineage dynamics, and facilitating early detection of emerging variants to support timely and targeted public health interventions in endemic and at-risk regions.
Methods in molecular biology (Clifton, N.J.)Xipeng Yan, He Xie, Jujun Sun, Limin Chen
RT-qPCR is characterized by high sensitivity, strong specificity, excellent reproducibility, and operational simplicity, enabling rapid quantification of viral RNA within a short timeframe. The real-time PCR system relies upon the measurement of a fluorescent reporter during PCR, in which the amount of emitted fluorescence is directly proportional to the amount of the PCR product in a reaction. Here, we describe the use of TaqMan® probe-based one-tube Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) for the detection and quantification of the Chikungunya virus (CHIKV) RNA.
Methods in molecular biology (Clifton, N.J.)Kacy S Yount
High-resolution characterization of antigen-specific T cell responses is essential for understanding cellular immunity to Chlamydia trachomatis infection in humans. This chapter describes two complementary flow cytometry-based approaches for measuring human antigen-specific T cell responses: intracellular cytokine staining (ICS) and activation-induced marker (AIM) staining. Both assays enable single-cell-level analysis following antigen stimulation but differ in their readouts. ICS quantifies functional cytokine production in response to short- or long-term stimulation, whereas the AIM assay identifies antigen-specific T cells based on transient surface expression of activation markers without fixation or permeabilization, preserving cells for downstream applications. Detailed protocols are provided for peripheral blood mononuclear cell (PBMC) thawing and preparation, short-term or long-term antigen stimulation, and flow cytometry staining, as well as practical considerations for assay selection, experimental controls, optimization, and data acquisition.
Chlamydia trachomatis (CT) causes genital tract infection in humans, but the clinical significance of C. trachomatis detection in the gastrointestinal (GI) tract is an area of active research. Chlamydia muridarum (CM) is a mouse-adapted species used to investigate chlamydial biology and pathogenicity in both the genital and gastrointestinal tracts. Since chlamydial colonization of different segments of the GI tract may lead to distinct consequences, quantifying chlamydial yields across these regions will facilitate understanding of chlamydial biology and pathogenicity. This chapter describes a step-by-step protocol to isolate and process GI tract tissue in a CM mouse model of infection and quantify the infectious yields of chlamydia.
Methods in molecular biology (Clifton, N.J.)Pooja Bhardwaj, Gaurav Raj Dwivedi, Hari Shanker Joshi, Rajeev Singh
Chikungunya virus (CHIKV) is transmitted through the bite of Aedes mosquitoes, specifically A. aegypti and A. albopictus. CHIKV belongs to the alphavirus with a positive-sense ssRNA genome of 11-12 kb size. The virus has been reported from various geographical regions across the globe. Chikungunya fever is an acute febrile illness, which, if left untreated, may develop into chronic arthralgia that may persist for several months or acute encephalitis syndrome. Therefore, early diagnosis of CHIKV is crucial to initiate prompt supportive treatment. Laboratory diagnosis of CHIKV typically relies on serological tests such as IgM antigen capture ELISA and molecular methods including RT-PCR or qRT-PCR. However, both these methods are not viable in peripheral settings. This chapter highlights recent advancements in molecular detection techniques for CHIKV, specifically isothermal detection methods that eliminate the requirement for complex instruments. The detection is facilitated by RT-RPA and CRISPR/Cas12a endonuclease. The assay offers advantages over existing methods such as rapid and early detection, and eliminates cross-over contamination, ultra-sensitivity, high specificity, and ease of result interpretation.
Methods in molecular biology (Clifton, N.J.)Eliana F Castro, Diego Alvarez
Chikungunya virus (CHIKV) causes a disease characterized by chronic musculoskeletal inflammation for which specific antivirals are not yet available. Currently, a supportive therapy to alleviate fever and pain is used, but it does not limit viral replication or the persistence of chronic arthritis symptoms. Thus, the identification and development of new active molecules against CHIKV is urgently needed. Here, we present a cell-based methodology that enables the implementation of a rapid and cost-effective strategy for high- and medium-throughput screening (HTS) of compounds, including repurposed drugs or novel molecules. This methodology allows for the identification of novel antiviral hits with a good activity and selectivity profile against CHIKV.
Methods in molecular biology (Clifton, N.J.)Lana H Jachmann, Barbara S Sixt
Chlamydia trachomatis, an obligate intracellular pathogen responsible for trachoma and widespread urogenital infections, replicates exclusively within the confines of a membrane-bound vacuole known as the inclusion. This compartment shields the bacterium from host cellular immune detection; thus, clarifying how inclusion integrity is maintained and protected from vacuole-destabilizing host attacks may identify new therapeutic strategies. To enable such insights, we recently developed split-GFP-based microscopic reporters that can detect early forms of membrane damage and track individual bacteria released from compromised inclusions. These reporters rely on infecting GFP1-10-expressing cells with C. trachomatis strains engineered to express either a GFP11-tagged outer membrane protein, exposing GFP11 on the bacterial surface, or a GFP11-tagged inclusion membrane protein, exposing GFP11 to the inclusion lumen. Damage to the inclusion allows GFP1-10 to access the bacteria and inner surface of the inclusion membrane, leading to GFP reconstitution and fluorescence at either cytosol-exposed bacteria or damaged membrane sites. Here, we provide detailed protocols for applying these reporter systems, including the generation of the required bacterial strains, development of a GFP1-10-expressing cell line, and execution of a representative infection experiment.
Chikungunya virus (CHIKV) is an emerging arbovirus lacking widely effective antiviral therapies, highlighting the need to characterize molecular interactions essential for its replication and pathogenesis. Surface plasmon resonance (SPR) is a sensitive, label-free technique that enables real-time analysis of biomolecular interactions by detecting refractive index changes at a sensor surface. SPR provides precise kinetic and affinity measurements for protein-protein, protein-RNA, and protein-ligand interactions using minimal sample quantities and supporting high-throughput screening. In CHIKV research, it has been applied to evaluate antiviral compounds, antibodies, receptor binding, and viral protein complexes. This chapter outlines key methodological considerations, including ligand preparation, immobilization strategies, assay optimization, and kinetic analysis.
This protocol provides a comprehensive, step-by-step workflow for whole-genome sequencing of Chikungunya virus (CHIKV) using an amplicon-based strategy optimized for Oxford Nanopore Technologies (ONT) platforms. The procedure includes detailed instructions for sample handling, viral RNA extraction, quality control, cDNA synthesis, multiplex PCR amplification, library preparation, sequencing, and primary bioinformatic processing. The protocol is designed to maximize reproducibility across laboratories and is suitable for genomic surveillance applications, including outbreak investigation and molecular epidemiology, even when working with low-to-moderate viral loads.
International journal of psychology : Journal international de psychologieQiandong Wang, Yawen Li, Yuxuan Liang, Sio Pan Hoi, Li Yi
Individuals from regions affected by infectious disease outbreaks may be stigmatized because others hold negative mental images-or mental representations-of them. This study investigated whether such outbreaks lead to the formation of negative mental representations of residents in affected areas and explored the role of disease severity in this process. Study 1 revealed that during the early stages of the COVID-19 epidemic, the public's mental representations of residents from Wuhan were perceived as less attractive than those of Chengdu residents and associated with disgust and behavioural avoidance. However, differences disappeared after the restrictions of the epidemic were lifted. The results indicate that the COVID-19 outbreak triggered stigmatization of residents in Wuhan. In Study 2, disease severity and perceived threat were manipulated, and results show that participants formed less attractive face representations of residents from areas where severe infectious diseases broke out than those from areas affected by mild infectious diseases. This proved that the severity of infectious diseases affected the extent of stigmatization towards residents in outbreak areas. These results further our understanding of the mechanisms and factors underlying stigmatization associated with affected regions and may inform effective public health strategies to mitigate stigma and support disease control efforts.
Sexual healthHarlan Smith, Josephine Wen, Zohal Rashidzada, Jenna Maleki, Xin Fang, Karin Thursky, Rodney James, Courtney Ierano
Sexually transmitted infections (STIs) place a substantial burden on outpatient health care and are a key focus for antimicrobial stewardship due to rising antimicrobial resistance. Suboptimal treatment can result in ongoing transmission, reinfection and complications, such as pelvic inflammatory disease. Evaluating prescribing quality is essential to improve patient care and support stewardship efforts. This review synthesises evidence on antimicrobial prescribing quality for STIs and identifies gaps to inform targeted interventions for this unique but critical setting. This PRISMA-ScR scoping review searched Ovid MEDLINE and Embase for English-language studies published from 2015 to May 2025 assessing antimicrobial prescribing quality for STIs in outpatient settings. Results were summarised descriptively; no formal critical appraisal was undertaken. Fifteen studies assessing antimicrobial prescribing quality for STIs in outpatient settings were included, predominantly from high-income countries, and focused on Neisseria gonorrhoeae and Chlamydia trachomatis. Substantial heterogeneity was observed in analytic units, denominators and outcome definitions, limiting comparability across studies. Prescribing quality was primarily assessed against national guidelines, although approaches varied from antimicrobial choice alone to multi-criteria and all-or-nothing assessments, with inconsistent terminology used throughout. Reported guideline concordance varied widely, with generally higher concordance for chlamydia than gonorrhoea. Antimicrobial prescribing for STIs in outpatient settings is both understudied and highly variable. Despite increasing global policy emphasis on antimicrobial stewardship, there are no standardised frameworks to support routine auditing in this setting. Developing standardised auditing approaches would improve surveillance, support stewardship efforts and identify opportunities to optimise prescribing practices across a broader range of STIs.
Sexual healthDaniela Eassey, Jason J Ong, Ken Koh, David Nolan, David Baker, Mark O'Reilly, Beng Eu, Jillian Lau, Fiona Bisshop, Jessica Kent, Andrés Doblado-Maldonado, Dan…
BACKGROUND: Although clinical trials and real-world studies demonstrate the efficacy and tolerability of dolutegravir and lamivudine (DTG + 3TC), Australian real-world evidence remains limited despite differences in access to health care and prescribing context. Here, we present the motivations for switching to DTG/3TC (a fixed-dose, single-tablet regimen) and treatment outcomes. METHODS: We performed a retrospective, observational analysis of individuals with HIV with an undetectable viral load (VL; <50 copies/mL) who switched to DTG/3TC during a 24-month inclusion window from 1 December 2020 to 1 December 2022, in nine Australian clinics. Healthcare providers completed an electronic survey using baseline demographics of people with HIV and other clinical information gathered from participant medical records. Data were collected after December 2023 to allow a 12-month follow-up. Primary endpoints were baseline demographics, clinical characteristics, and motivations for switching to DTG/3TC. Secondary endpoints included virologic outcomes and rates and reasons for DTG/3TC discontinuation. RESULTS: Overall, 276 individuals with HIV who switched to DTG/3TC were included. Most were male (97%) and White (76%), with a median (interquartile range) age of 54 (45-61) years. The most common antiretroviral therapy before DTG/3TC switch was abacavir/dolutegravir/lamivudine (54%). The most common reason for switching to DTG/3TC was clinician preference for two-drug regimen (43%). Most individuals (98%) maintained virologic suppression (VL <50 copies/mL), and none experienced virologic failure. Through Month 12, one (<1%) individual discontinued DTG/3TC. CONCLUSIONS: These real-world data support the use of DTG/3TC as a viable treatment strategy in this Australian population of individuals with HIV.
Sexual healthKudzai Hlahla, Moni Neradilek, Morgan Garcia, Felix Mhlanga, Thesla Palanee-Phillips, Krishnaveni Reddy, Nishanta Singh, Carolyne A Akello, Samantha Siva, Leil…
BACKGROUND: Access to novel HIV prevention technologies often raise concerns of risk compensation. This analysis examined changes in the sexual behaviors of MTN 020/ASPIRE participants, a placebo controlled randomized control trial, who subsequently enrolled in MTN-025/HOPE, an open-label extension using the dapivirine vaginal ring. METHODS: Both studies enrolled healthy, sexually active, HIV-negative women from Malawi, South Africa, Uganda and Zimbabwe. Longitudinal data on participants' sexual behaviors, specifically sex with a nonprimary partner (past 3 months), and use of male or female condoms at the last vaginal sex act were compared between ASPIRE and HOPE. Conditional and mixed ordinal logistic regression models evaluated associations between study and sexual behaviors at enrollment, and quarterly over the first 12 months. RESULTS: Of the 2629 individuals enrolled in ASPIRE, 1456 (55%) participated in HOPE. At enrollment, the proportion of participants who reported sex with a nonprimary partner and condom use at last vaginal sex act did not differ by study. Across all quarterly follow-up visits, sex with a nonprimary partner was more common in HOPE (13.9-18.7%) than ASPIRE (10.7-12.6%); adjusted OR 1.47, 95% CI 1.24-1.75, P < 0.001. There was no difference in condom use at the last vaginal act across all quarterly follow-up visits between ASPIRE (36.5-42.7%) and HOPE (43.6-46.7%); adjusted OR 1.05, 95% CI 0.94-1.18. CONCLUSION: More women reported sex with nonprimary partners in HOPE, with little change in condom use over time between the two studies. Understanding behavior changes during PrEP use allows for tailored, holistic reproductive health programming.
Sexual healthRebecca Houghton, Anik Ray, Alison Mahony, Lisa McCann, Brent Mackie, Joubert van der Merwe, Rick Varma, Arthur Wong
BACKGROUND: Overseas-born gay, bisexual, and other men who have sex with men (GBMSM) experience higher HIV notification rates compared with Australian-born GBMSM. This disparity is perpetuated by delayed access to HIV testing and biomedical prevention driven by a range of factors including Medicare eligibility, language and cultural barriers, and unfamiliarity with the health system. Community-based services offer an alternative pathway to HIV testing that may better serve migrant GBMSM facing challenges accessing mainstream healthcare settings. METHODS: We conducted a cross-sectional study comparing settings of first HIV test within our service, either peer-led community-based testing or traditional clinic-based testing for overseas-born GBMSM who arrived in Australia between January 2022 and January 2024. Participants were grouped by time of arrival to first HIV test (either less than 6 months or more than 6 months). Comparative analysis and multivariable logistic regression were used to identify characteristics associated with attendance at the community-based setting. RESULTS: A total of 1204 GBMSM were included with 424 (35.2%) attending community-based setting for their first HIV test after arrival. Those who attended community-based testing were more likely to have arrived in Australia more than 6 months ago (66.8% vs 26.9%) and never previously tested for HIV (37.5% vs 17.2%). These factors were also associated with higher likelihood of community-based testing in the multivariable model (aOR 5.27, 95% CI 3.98-6.97 and aOR 2.13, 95% CI 1.57-2.91 respectively). CONCLUSION: Community-based, peer-led HIV testing services play an important role in engaging overseas-born GBMSM who may be under-served by traditional clinic-based models, particularly those with longer residence in Australia and no prior HIV testing.
Sexual healthWondimeneh Shiferaw, Fan Yu, Colleen L Lau, Luis Furuya-Kanamori, Judith A Dean
BACKGROUND: Australia is a popular destination for international students. However, being away from their usual environment potentially increases their risk of sexually transmissible infections. We explored the awareness and barriers to accessing sexual health information and services before departure and within the first year of arrival among international students in Australia and identify ways of improving access to information and services. METHODS: Semi-structured interviews was conducted on international students enrolled at The University of Queensland in 2025, which were audio-recorded, transcribed, and imported into NVivo 14 for analysis. The interviews were synthesised thematically using an inductive approach. RESULTS: Fifteen students were interviewed. Most were unaware of pre-travel sexual health information and services. Barriers to accessing sexual health information and services were identified both before departure and post-arrival, and clustered into five themes. Pre-departure barriers included: (1) awareness and knowledge; (2) socio-cultural and religious; and (3) psychological barriers. Post-arrival barriers included: (1) challenges in navigating the Australian healthcare system; and (2) reshaping of pre-departure barriers in the post-arrival context. Despite the challenges, students recommended possible strategies to improve pre-departure sexual health access in their country of origin, including incorporating sexual education into the curriculum, pre-departure briefings, and providing sexual health services through ongoing systems. CONCLUSION: Most students were not aware of pre-travel sexual health services. Limited awareness, socio-cultural, and psychological barriers were identified as challenges to accessing pre-travel sexual health information and services. Providing culturally-sensitive services, delivering clear information about available services and how to access them, and strengthening supportive structures are crucial steps toward overcoming these barriers.