Polymicrobial Infections: A Comprehensive Review on Current Context, Diagnostic Bottlenecks and Future Directions
Abstract
1. Introduction
2. Materials and Methods
3. Results
3.1. Status of PMIs: Brief Global Context and Indian Subcontinent Scenario
3.1.1. The Global Context: A Ubiquitous and Underappreciated Burden
Intra-Abdominal Infections (IAIs)
Diabetic Foot Infections (DFIs)
Pneumonia/Respiratory Infections
Biofilm-Associated Infections
Cystic Fibrosis (CF) Lung Infections
3.1.2. The Indian Subcontinent: A “Hub” for PMIs
High Burden of Community-Acquired Syndromes
Epicenter of AMR in HAIs
Chronic and Co-Morbid Infections
3.2. Current Diagnostic Approaches and Major Gaps
3.2.1. Culture-Based Methods
3.2.2. Molecular and Immunological Techniques
3.2.3. Biomarkers and Their Limitations
3.3. Diagnostic Challenges in Vulnerable Populations
3.3.1. Children (Neonates and Pediatrics)
3.3.2. The Elderly
3.3.3. The Immunocompromised
3.3.4. Marginalized Populations and LMICs
3.4. Emerging Methods to Detect Polymicrobial Infection
3.4.1. Molecular Assays (CRISPR-Based)
3.4.2. Microfluidic Chip-Based Approaches
3.4.3. Culturomics
3.4.4. Sequencing and AI/ML-Based Approaches
4. Discussion
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
- Berenji, F.; Zarrinfar, H.; Gholizadeh, A.; Sargazi, F.; Jamali, J.; Noghabi, M.P.; Sangani, G.S.; Farash, B.R.H. Co-Infection of Lophomonas Blattarum and Pneumocystis Jiro-Vecii in Patients with Respiratory Disorders in Northeastern Iran. Iran J. Parasitol. 2025, 20, 299–306. [Google Scholar] [CrossRef]
- Medina, N.; Soto-Debrán, J.; Seidel, D.; Akyar, I.; Badali, H.; Barac, A.; Bretagne, S.; Cag, Y.; Cassagne, C.; Castro, C.; et al. MixInYeast: A Multicenter Study on Mixed Yeast Infections. J. Fungi 2020, 7, 13. [Google Scholar] [CrossRef]
- Anju, V.T.; Busi, S.; Imchen, M.; Kumavath, R.; Mohan, M.S.; Salim, S.A.; Subhaswaraj, P.; Dyavaiah, M. Polymicrobial Infections and Biofilms: Clinical Significance and Eradication Strategies. Antibiotics 2022, 11, 1731. [Google Scholar] [CrossRef]
- Infectious Diseases Society of America (IDSA). Combating Antimicrobial Resistance: Policy Recommendations to Save Lives. Clin. Infect. Dis. 2011, 52, S397–S428. [Google Scholar] [CrossRef]
- Murray, C.J.L.; Ikuta, K.S.; Sharara, F.; Swetschinski, L.; Robles Aguilar, G.; Gray, A.; Han, C.; Bisignano, C.; Rao, P.; Wool, E.; et al. Global Burden of Bacterial Antimicrobial Resistance in 2019: A Systematic Analysis. Lancet 2022, 399, 629–655. [Google Scholar] [CrossRef]
- Farrell, J.M.; Brown, S.P. Microbial Primer: Challenges and Opportunities in the Treatment of Chronic Polymicrobial Infections—An Eco-Evolutionary Perspective. Microbiology 2025, 171, 001567. [Google Scholar] [CrossRef]
- Park, S.Y.; Goldman, J.D.; Levine, D.J.; Haidar, G. A Systematic Literature Review to Determine Gaps in Diagnosing Suspected Infection in Solid Organ Transplant Recipients. Open Forum Infect. Dis. 2024, 12, ofaf001. [Google Scholar] [CrossRef] [PubMed]
- Zhang, H.-M.; Ding, J.-C.; Tang, J.-X.; Dai, L.-T.; Ling, J.-H.; Zou, M.-X.; Cao, X.-W.; Lin, L.-J.; Liu, W.-T.; Yuan, P.-B.; et al. Polymicrobial Bloodstream Infections: A Retrospective Cohort Study on Clinical Manifestations, Co-Infection Patterns, and Survival Outcomes. Microb. Pathog. 2025, 206, 107774. [Google Scholar] [CrossRef] [PubMed]
- Brogden, K.A.; Guthmiller, J.M.; Taylor, C.E. Human Polymicrobial Infections. Lancet 2005, 365, 253–255. [Google Scholar] [CrossRef]
- Clinical and Laboratory Standards Institute (CLSI). Guidance on the Identification of Polymicrobial Infections in Clinical Laboratories: New Protocols and Challenges; CLSI Document M100-S34; CLSI: Malvern, PA, USA, 2024. [Google Scholar]
- Huston, J.M.; Barie, P.S.; Dellinger, E.P.; Forrester, J.D.; Duane, T.M.; Tessier, J.M.; Sawyer, R.G.; Cainzos, M.A.; Rasa, K.; Chipman, J.G.; et al. The Surgical Infection Society Guidelines on the Management of Intra-Abdominal Infection: 2024 Update. Surg. Infect. 2024, 25, 419–435. [Google Scholar] [CrossRef] [PubMed]
- Alexiou, Z.W.; Hoenderboom, B.M.; Hoebe, C.J.P.A.; Dukers-Muijrers, N.H.T.M.; Götz, H.M.; van der Sande, M.A.B.; de Vries, H.J.C.; den Hartog, J.E.; Morré, S.A.; van Benthem, B.H.B. The Importance of Understanding Pelvic Inflammatory Disease as a Polymicrobial Infection-Authors’ Reply. Lancet Reg. Health Eur. 2024, 47, 101116. [Google Scholar] [CrossRef]
- Afonso, A.C.; Oliveira, D.; Saavedra, M.J.; Borges, A.; Simões, M. Biofilms in Diabetic Foot Ulcers: Impact, Risk Factors and Control Strategies. Int. J. Mol. Sci. 2021, 22, 8278. [Google Scholar] [CrossRef]
- Maity, S.; Leton, N.; Nayak, N.; Jha, A.; Anand, N.; Thompson, K.; Boothe, D.; Cromer, A.; Garcia, Y.; Al-Islam, A.; et al. A Systematic Review of Diabetic Foot Infections: Pathogenesis, Diagnosis, and Management Strategies. Front. Clin. Diabetes Healthc. 2024, 5, 1393309. [Google Scholar] [CrossRef] [PubMed]
- Yehya, A.; Ezzeddine, Z.; Chakkour, M.; Dhaini, Z.; Bou Saba, M.S.; Bou Saba, A.S.; Nohra, L.; Nassar, N.B.; Yassine, M.; Bahmad, H.F.; et al. The Intricacies of Acinetobacter Baumannii: A Multifaceted Comprehensive Review of a Multidrug-Resistant Pathogen and Its Clinical Significance and Implications. Front. Microbiol. 2025, 16, 1565965. [Google Scholar] [CrossRef]
- Centers for Disease Control and Prevention. Healthcare-Associated Infections (HAIs): Current HAI Progress Report. 2024. Available online: https://www.cdc.gov/healthcare-associated-infections/php/data/progress-report.html (accessed on 1 August 2025).
- Mayasari, E.; Utama, E.D. Epidemiology and Resistant Profile of Bacterial Pathogens in a Tertiary Health Care Hospital, Medan City: A Retrospective Study. PeerJ 2025, 13, e19510. [Google Scholar] [CrossRef]
- Cillóniz, C.; Calabretta, D.; Palomeque, A.; Gabarrus, A.; Ferrer, M.; Marcos, M.Á.; Torres, A. Risk Factors and Outcomes Associated with Polymicrobial Infection in Community-Acquired Pneumonia. Arch. Bronconeumol. 2025, 61, 408–416. [Google Scholar] [CrossRef]
- Lai, C.-C.; Wang, C.-Y.; Hsueh, P.-R. Co-Infections among Patients with COVID-19: The Need for Combination Therapy with Non-Anti-SARS-CoV-2 Agents? J. Microbiol. Immunol. Infect. 2020, 53, 505–512. [Google Scholar] [CrossRef]
- Carstens, G.; Kozanli, E.; Bulsink, K.; McDonald, S.A.; Elahi, M.; de Bakker, J.; Schipper, M.; van Gageldonk-Lafeber, R.; van den Hof, S.; van Hoek, A.J.; et al. Co-Infection Dynamics of SARS-CoV-2 and Respiratory Viruses in the 2022/2023 Respiratory Season in the Netherlands. J. Infect. 2025, 90, 106474. [Google Scholar] [CrossRef]
- Yan, X.; Li, K.; Lei, Z.; Luo, J.; Wang, Q.; Wei, S. Prevalence and Associated Outcomes of Coinfection between SARS-CoV-2 and Influenza: A Systematic Review and Meta-Analysis. Int. J. Infect. Dis. 2023, 136, 29–36. [Google Scholar] [CrossRef] [PubMed]
- Abbas, R.; Chakkour, M.; Zein El Dine, H.; Obaseki, E.F.; Obeid, S.T.; Jezzini, A.; Ghssein, G.; Ezzeddine, Z. General Overview of Klebsiella Pneumonia: Epidemiology and the Role of Siderophores in Its Pathogenicity. Biology 2024, 13, 78. [Google Scholar] [CrossRef] [PubMed]
- Xu, J.; Yuan, Y.; Wang, B.; Zhang, Q.; Wang, J.; Wang, S.; Li, Y.; Yan, W. Microbiological Analysis and Mortality Risk Factors in Patients with Polymicrobial Bloodstream Infections. Infect. Drug Resist. 2023, 16, 3917–3927. [Google Scholar] [CrossRef] [PubMed]
- Mishra, A.; Aggarwal, A.; Khan, F. Medical Device-Associated Infections Caused by Biofilm-Forming Microbial Pathogens and Controlling Strategies. Antibiotics 2024, 13, 623. [Google Scholar] [CrossRef] [PubMed]
- Baker, E.J.; Allcott, G.; Cox, J.A.G. Polymicrobial Infection in Cystic Fibrosis and Future Perspectives for Improving Mycobacterium Abscessus Drug Discovery. NPJ Antimicrob. Resist. 2024, 2, 38. [Google Scholar] [CrossRef]
- Berinson, B.; Both, A.; Berneking, L.; Christner, M.; Lütgehetmann, M.; Aepfelbacher, M.; Rohde, H. Usefulness of BioFire FilmArray BCID2 for Blood Culture Processing in Clinical Practice. J. Clin. Microbiol. 2021, 59, e0054321. [Google Scholar] [CrossRef]
- Asma, S.T.; Imre, K.; Morar, A.; Imre, M.; Acaroz, U.; Shah, S.R.A.; Hussain, S.Z.; Arslan-Acaroz, D.; Istanbullugil, F.R.; Madani, K.; et al. Natural Strategies as Potential Weapons Against Bacterial Biofilms. Life 2022, 12, 1618. [Google Scholar] [CrossRef]
- Lee, S.Y.; Park, M.H.; Oh, D.K.; Lim, C.-M.; Hong, S.-B.; Suh, G.Y.; Jeon, K.; Ko, R.-E.; Cho, Y.-J.; Lee, Y.J.; et al. Polymicrobial Bloodstream Infections per Se Do Not Increase Mortality Compared to Monomicrobial Bloodstream Infections in Sepsis Patients: A Korean Nationwide Sepsis Cohort Study. BMC Infect. Dis. 2024, 24, 285. [Google Scholar] [CrossRef]
- Lalremruata, R.; Prakash, S. Prevalence of Community-Acquired Methicillin-Resistant Staphylococcus Aureus in Patients with Skin and Soft Tissue Infections. Community Acquir. Infect. 2014, 1, 21. [Google Scholar] [CrossRef]
- Vaishnav, B.; Wadivkar, A.; Pailla, R.; Mondkar, S. Clinical and Microbiological Profile of Gram-Negative Infections in Critically Ill Diabetic Patients. Cureus 2024, 16, e65955. [Google Scholar] [CrossRef] [PubMed]
- Crowley, P.; Streck, N.; Challener, D.; Abu Saleh, O.M. P-1477. In Vitro Efficacy of Minocycline on Skin, Soft Tissue and Musculoskeletal Gram Negative Bacterial Isolates. Open Forum Infect. Dis. 2025, 12, ofae631.1647. [Google Scholar] [CrossRef]
- Ravishankar, A.; Singh, S.; Rai, S.; Sharma, N.; Gupta, S.; Thawani, R. Socio-Economic Profile of Patients with Community-Acquired Skin and Soft Tissue Infections in Delhi. Pathog. Glob. Health 2014, 108, 279–282. [Google Scholar] [CrossRef]
- Sondhiya, G.; Manjunathachar, H.V.; Singh, P.; Kumar, R. Unveiling the Burden of Scrub Typhus in Acute Febrile Illness Cases across India: A Systematic Review & Meta-Analysis. Indian J. Med. Res. 2024, 159, 601. [Google Scholar] [CrossRef]
- Jose, P.; Rajan, N.; Kommu, P.K.; Krishnan, L. Dengue and Scrub Typhus Co-Infection in Children: Experience of a Teaching Hospital in an Endemic Area. Indian J. Public Health 2022, 66, 292. [Google Scholar] [CrossRef]
- Virk, H.S.; Biemond, J.J.; Earny, V.A.; Chowdhury, S.; Frölke, R.I.; Khanna, S.M.; Shanbhag, V.; Rao, S.; Acharya, R.V.; Balakrishnan, J.M.; et al. Unraveling Sepsis Epidemiology in a Low- and Middle-Income Intensive Care Setting Reveals the Alarming Burden of Tropical Infections and Antimicrobial Resistance: A Prospective Observational Study (MARS-India). Clin. Infect. Dis. 2025, 80, 101–107. [Google Scholar] [CrossRef]
- Balasubramanian, R.; Van Boeckel, T.P.; Carmeli, Y.; Cosgrove, S.; Laxminarayan, R. Global Incidence in Hospital-Associated Infections Resistant to Antibiotics: An Analysis of Point Prevalence Surveys from 99 Countries. PLoS Med. 2023, 20, e1004178. [Google Scholar] [CrossRef]
- Sharma, K.; Tak, V.; Nag, V.L.; Bhatia, P.K.; Kothari, N. An Observational Study on Carbapenem-Resistant Enterobacterales (CRE) Colonisation and Subsequent Risk of Infection in an Adult Intensive Care Unit (ICU) at a Tertiary Care Hospital in India. Infect. Prev. Pract. 2023, 5, 100312. [Google Scholar] [CrossRef] [PubMed]
- Lathakumari, R.H.; Vajravelu, L.K.; Thulukanam, J.; Nair, D.M.; Vimala, P.B.; Panneerselvam, V. Prevalence and Molecular Insights into Carbapenem Resistance: A 2-Year Retrospective Analysis of Superbugs in South India. Front. Med. 2025, 12, 1571231. [Google Scholar] [CrossRef] [PubMed]
- Kataria, S.; Galhotra, S.; Kumar, M.; Thandi, P.; Jindal, N. Comparison of Prevalence of BlaNDM-1 Gene among Metallo-Beta Lactamase-Producing Multidrug-Resistant Fermenters and Nonfermenters in a Tertiary Care Hospital. Arch. Med. Health Sci. 2024, 12, 13–19. [Google Scholar] [CrossRef]
- Wu, W.; Feng, Y.; Tang, G.; Qiao, F.; McNally, A.; Zong, Z. NDM Metallo-β-Lactamases and Their Bacterial Producers in Health Care Settings. Clin. Microbiol. Rev. 2019, 32, e00115-18. [Google Scholar] [CrossRef] [PubMed]
- Dwibedy, S.K.; Padhy, I.; Panda, A.K.; Mohapatra, S.S. Prevalence of Polymyxin-Resistant Bacterial Strains in India: A Systematic Review and Meta-Analysis. J. Antimicrob. Chemother. 2024, 79, 1762–1774. [Google Scholar] [CrossRef]
- Basu, S.; Chakraborty, S. A Comprehensive Review of the Diagnostics for Pediatric Tuberculosis Based on Assay Time, Ease of Operation, and Performance. Microorganisms 2025, 13, 178. [Google Scholar] [CrossRef]
- Kim, H.-J.; Na, S.W.; Alodaini, H.A.; Al-Dosary, M.A.; Nandhakumari, P.; Dyona, L. Prevalence of Multidrug-Resistant Bacteria Associated with Polymicrobial Infections. J. Infect. Public Health 2021, 14, 1864–1869. [Google Scholar] [CrossRef] [PubMed]
- Dincer, C.; Bruch, R.; Kling, A.; Dittrich, P.S.; Urban, G.A. Multiplexed Point-of-Care Testing—XPOCT. Trends Biotechnol. 2017, 35, 728–742. [Google Scholar] [CrossRef] [PubMed]
- Khan, A.R.; Hussain, W.L.; Shum, H.C.; Hassan, S.U. Point-of-Care Testing: A Critical Analysis of the Market and Future Trends. Front. Lab Chip Technol. 2024, 3, 1394752. [Google Scholar] [CrossRef]
- Swain, J.; Singh, J.; Manglunia, A.; Jena, S.; Sravya, S.L. Epidemiology of Infections in Diabetes, Pre and Post-COVID Era in India. Chron. Diabetes Res. Pract. 2022, 1, 114–120. [Google Scholar] [CrossRef]
- Ledeboer, N.A.; Caldwell, J.M.; Boyanton, B.L. Review: Diagnostic Potential for Collaborative Pharyngitis Biomarkers. J. Infect. Dis. 2024, 230, S190–S196. [Google Scholar] [CrossRef] [PubMed]
- World Health Organization. Obesity and Overweight; WHO: Geneva, Switzerland, 2025. [Google Scholar]
- Zhang, Y.; Hu, A.; Andini, N.; Yang, S. A ‘Culture’ Shift: Application of Molecular Techniques for Diagnosing Polymicrobial Infections. Biotechnol. Adv. 2019, 37, 476–490. [Google Scholar] [CrossRef]
- Doualeh, M.; Payne, M.; Litton, E.; Raby, E.; Currie, A. Molecular Methodologies for Improved Polymicrobial Sepsis Diagnosis. Int. J. Mol. Sci. 2022, 23, 4484. [Google Scholar] [CrossRef]
- Rieber, H.; Frontzek, A.; Heinrich, S.; Breil-Wirth, A.; Messler, J.; Hegermann, S.; Ulatowski, M.; Koutras, C.; Steinheisser, E.; Kruppa, T.; et al. Microbiological Diagnosis of Polymicrobial Periprosthetic Joint Infection Revealed Superiority of Investigated Tissue Samples Compared to Sonicate Fluid Generated from the Implant Surface. Int. J. Infect. Dis. 2021, 106, 302–307. [Google Scholar] [CrossRef]
- Tseng, W.-L. Challenges in the Diagnosis and Management of Polymicrobial Infections. J. Clin. Microbiol. Antimicrob. 2025, 9, 1–2. [Google Scholar] [CrossRef]
- Srinivasan, A.; Sajeevan, A.; Rajaramon, S.; David, H.; Solomon, A.P. Solving Polymicrobial Puzzles: Evolutionary Dynamics and Future Directions. Front. Cell Infect. Microbiol. 2023, 13, 1295063. [Google Scholar] [CrossRef]
- Atallah, J.; Mansour, M.K. Implications of Using Host Response-Based Molecular Diagnostics on the Management of Bacterial and Viral Infections: A Review. Front. Med. 2022, 9, 107. [Google Scholar] [CrossRef]
- Woodhouse, E.W.; McClain, M.T.; Woods, C.W. Harnessing the Host Response for Precision Infectious Disease Diagnosis. Clin Microbiol. Rev. 2024, 37, e00078-24. [Google Scholar] [CrossRef]
- Hung, S.-K.; Lan, H.-M.; Han, S.-T.; Wu, C.-C.; Chen, K.-F. Current Evidence and Limitation of Biomarkers for Detecting Sepsis and Systemic Infection. Biomedicines 2020, 8, 494. [Google Scholar] [CrossRef]
- Gleeson, B.; Ferreyra, C.; Palamountain, K.; Jacob, S.T.; Spotswood, N.; Kissoon, N.; Nisar, Y.B.; Fitzgerald, F.; Murless-Collins, S.; Okomo, U.; et al. A Call to Bridge the Diagnostic Gap: Diagnostic Solutions for Neonatal Sepsis in Low- and Middle-Income Countries. BMJ Glob. Health 2024, 9, e015862. [Google Scholar] [CrossRef] [PubMed]
- Raturi, A.; Chandran, S. Neonatal Sepsis: Aetiology, Pathophysiology, Diagnostic Advances and Management Strategies. Clin. Med. Insights Pediatr. 2024, 18, 11795565241281337. [Google Scholar] [CrossRef]
- Iroh Tam, P.-Y.; Bendel, C.M. Diagnostics for Neonatal Sepsis: Current Approaches and Future Directions. Pediatr. Res. 2017, 82, 574–583. [Google Scholar] [CrossRef]
- Debonera, F.; Simmons, B. Infections in Older Adults: The Art of Early Recognition. Ann. Long-Term Care 2021. [Google Scholar] [CrossRef]
- Theodorakis, N.; Feretzakis, G.; Hitas, C.; Kreouzi, M.; Kalantzi, S.; Spyridaki, A.; Kollia, Z.; Verykios, V.S.; Nikolaou, M. Immunosenescence: How Aging Increases Susceptibility to Bacterial Infections and Virulence Factors. Microorganisms 2024, 12, 2052. [Google Scholar] [CrossRef] [PubMed]
- Esme, M.; Topeli, A.; Yavuz, B.B.; Akova, M. Infections in the Elderly Critically-Ill Patients. Front. Med. 2019, 6, 118. [Google Scholar] [CrossRef]
- Wang, Y.; Zhang, H.; Miao, C. Unraveling Immunosenescence in Sepsis: From Cellular Mechanisms to Therapeutics. Cell Death Dis. 2025, 16, 393. [Google Scholar] [CrossRef]
- Dey, M.; Jain, S.; Shah, I. Polymicrobial Infection in Immunocompromised Host-How to Manage? Pediatr. Oncall 2024, 21, 134–137. [Google Scholar] [CrossRef]
- Higgins, E.; Gupta, A.; Cummins, N.W. Polymicrobial Infections in the Immunocompromised Host: The COVID-19 Realm and Beyond. Med. Sci. 2022, 10, 60. [Google Scholar] [CrossRef] [PubMed]
- Wu, X.; Sun, T.; Cai, Y.; Zhai, T.; Liu, Y.; Gu, S.; Zhou, Y.; Zhan, Q. Clinical Characteristics and Outcomes of Immunocompromised Patients with Severe Community-Acquired Pneumonia: A Single-Center Retrospective Cohort Study. Front. Public Health 2023, 11, 581. [Google Scholar] [CrossRef]
- Lee, S.; Chintalapudi, K.; Badu-Tawiah, A.K. Clinical Chemistry for Developing Countries: Mass Spectrometry. Annu. Rev. Anal. Chem. 2021, 14, 437–465. [Google Scholar] [CrossRef]
- Fall, B.; Lo, C.I.; Samb-Ba, B.; Perrot, N.; Diawara, S.; Gueye, M.W.; Sow, K.; Aubadie-Ladrix, M.; Mediannikov, O.; Sokhna, C.; et al. The Ongoing Revolution of MALDI-TOF Mass Spectrometry for Microbiology Reaches Tropical Africa. Am. Soc. Trop. Med. Hyg. 2015, 92, 641–647. [Google Scholar] [CrossRef]
- Zhang, M.; Zhang, H.; Yan, B.; Ren, M.; Wang, W.; Zhang, T. Diagnostic Performance of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in Bronchoalveolar Lavage Fluid for Pulmonary Tuberculosis in HIV-Infected Patients. J. Clin. Tuberc. Other Mycobact. Dis. 2024, 36, 100459. [Google Scholar] [CrossRef] [PubMed]
- Suneja, M.; Beekmann, S.E.; Dhaliwal, G.; Miller, A.C.; Polgreen, P.M. Diagnostic Delays in Infectious Diseases. Diagnosis 2022, 9, 332–339. [Google Scholar] [CrossRef]
- Shen, Y.; Liu, Y.; Krafft, T.; Wang, Q. Progress and Challenges in Infectious Disease Surveillance and Early Warning. Med. Plus 2025, 2, 100071. [Google Scholar] [CrossRef]
- Sahel, D.K.; Giriprasad, G.; Jatyan, R.; Guha, S.; Korde, A.; Mittal, A.; Bhand, S.; Chitkara, D. Next-Generation CRISPR/Cas-Based Ultrasensitive Diagnostic Tools: Current Progress and Prospects. RSC Adv. 2024, 14, 32411–32435. [Google Scholar] [CrossRef]
- Lou, H.; Wang, X.; Jiang, Q.; Li, X.; Yao, Y.; Chen, Q.; Chen, L.; Zhang, S.; Yu, Y.; Liu, C.; et al. Clinical Evaluation of a Highly Multiplexed CRISPR-Based Diagnostic Assay for Diagnosing Lower Respiratory Tract Infection: A Prospective Cohort Study. Infect Dis. 2025, 57, 167–177. [Google Scholar] [CrossRef]
- Chen, S.-J.; Rai, C.-I.; Wang, S.-C.; Chen, Y.-C. Point-of-Care Testing for Infectious Diseases Based on Class 2 CRISPR/Cas Technology. Diagnostics 2023, 13, 2255. [Google Scholar] [CrossRef]
- Lamb, C.H.; Kang, B.; Myhrvold, C. Multiplexed CRISPR-Based Methods for Pathogen Nucleic Acid Detection. Curr. Opin. Biomed. Eng. 2023, 27, 100471. [Google Scholar] [CrossRef]
- Zhang, L.; Wang, H.; Yang, S.; Liu, J.; Li, J.; Lu, Y.; Cheng, J.; Xu, Y. High-Throughput and Integrated CRISPR/Cas12a-Based Molecular Diagnosis Using a Deep Learning Enabled Microfluidic System. ACS Nano 2024, 18, 24236–24251. [Google Scholar] [CrossRef]
- Zhang, J.; Fu, Y.; Fong, C.Y.; Hua, H.; Li, W.; Khoo, B.L. Advancements in Microfluidic Technology for Rapid Bacterial Detection and Inflammation-Driven Diseases. Lab Chip 2025, 25, 3348–3373. [Google Scholar] [CrossRef] [PubMed]
- Treffon, J.; Isserstedt-John, N.; Klemm, R.; Gärtner, C.; Mellmann, A. Evaluation of a Microfluidic-Based Point-of-Care Prototype with Customized Chip for Detection of Bacterial Clusters. Microbiol. Spectr. 2024, 12, e0086224. [Google Scholar] [CrossRef]
- Agnihotri, S.N.; Fatsis-Kavalopoulos, N.; Windhager, J.; Tenje, M.; Andersson, D.I. Droplet Microfluidics–Based Detection of Rare Antibiotic-Resistant Subpopulations in Escherichia coli from Bloodstream Infections. Sci. Adv. 2025, 11, eadv4558. [Google Scholar] [CrossRef] [PubMed]
- Dubourg, G.; Baron, S.; Cadoret, F.; Couderc, C.; Fournier, P.-E.; Lagier, J.-C.; Raoult, D. From Culturomics to Clinical Microbiology and Forward. Emerg. Infect. Dis. 2018, 24, 1683–1690. [Google Scholar] [CrossRef]
- Zhao, Y.; Zhang, W.; Zhang, X. Application of Metagenomic Next-Generation Sequencing in the Diagnosis of Infectious Diseases. Front. Cell Infect. Microbiol. 2024, 14, 8316. [Google Scholar] [CrossRef] [PubMed]
- Ma, R.; Yin, Y.; Zhang, J.-P.; Zhang, M.-X.; Zhou, J.-R.; He, Y.; Gai, W.; Zhang, X.-H.; Wang, Y.; Xu, L.-P.; et al. Metagenomic Next-Generation Sequencing Compared with Blood Culture as First-Line Diagnostic Method for Bloodstream Infection in Hematologic Patients with Febrile Neutropenia: A Multicenter, Prospective Study. Open Forum Infect. Dis. 2025, 12, ofaf288. [Google Scholar] [CrossRef]
- Kalantar, K.L.; Carvalho, T.; de Bourcy, C.F.A.; Dimitrov, B.; Dingle, G.; Egger, R.; Han, J.; Holmes, O.B.; Juan, Y.-F.; King, R.; et al. IDseq—An Open Source Cloud-Based Pipeline and Analysis Service for Metagenomic Pathogen Detection and Monitoring. Gigascience 2020, 9, giaa111. [Google Scholar] [CrossRef]
- Tournoud, M.; Ruppé, E.; Perrin, G.; Schicklin, S.; Guigon, G.; Mahé, P.; Lazarevic, V.; Hauser, S.; Mirande, C.; Levrat, A.; et al. Clinical Metagenomics Bioinformatics Pipeline for the Identification of Hospital-Acquired Pneumonia Pathogens Antibiotic Resistance Genes from Bronchoalveolar Lavage Samples. bioRxiv 2020. preprint. [Google Scholar] [CrossRef]
- Signoroni, A.; Ferrari, A.; Lombardi, S.; Savardi, M.; Fontana, S.; Culbreath, K. Hierarchical AI Enables Global Interpretation of Culture Plates in the Era of Digital Microbiology. Nat. Commun. 2023, 14, 6874. [Google Scholar] [CrossRef]
- MacLean, A.R.; Gunson, R. Automation and Standardisation of a Quantitative Multiplex PCR Assay Using PCR.Ai. J. Virol. Methods 2024, 329, 114981. [Google Scholar] [CrossRef]
- Schwengers, O.; Hoek, A.; Fritzenwanker, M.; Falgenhauer, L.; Hain, T.; Chakraborty, T.; Goesmann, A. ASA3P: An Automatic and Scalable Pipeline for the Assembly, Annotation and Higher-Level Analysis of Closely Related Bacterial Isolates. PLoS Comput. Biol. 2020, 16, e1007134. [Google Scholar] [CrossRef]
- Basu, S.; Ramaiah, S.; Anbarasu, A. In-Silico Strategies to Combat COVID-19: A Comprehensive Review. Biotechnol. Genet. Eng. Rev. 2021, 37, 64–81. [Google Scholar] [CrossRef] [PubMed]
- Tillotson, G.S. Trojan Horse Antibiotics–A Novel Way to Circumvent Gram-Negative Bacterial Resistance? Infect. Dis. Res. Treat. 2016, 9, 45–52. [Google Scholar] [CrossRef]
- Ezzeddine, Z.; Ghssein, G. Towards New Antibiotics Classes Targeting Bacterial Metallophores. Microb. Pathog. 2023, 182, 106221. [Google Scholar] [CrossRef]
- Al-Fadhli, A.H.; Jamal, W.Y. Recent Advances in Gene-Editing Approaches for Tackling Antibiotic Resistance Threats: A Review. Front. Cell Infect. Microbiol. 2024, 14, 115. [Google Scholar] [CrossRef] [PubMed]
- Jeff, C.; Quarton, S.; Hatton, C.; Parekh, D.; Thickett, D.; McNally, A.; Sapey, E. Metagenomics in the Diagnosis and Treatment of Urinary Tract Infections: A Systematic Review and Meta-Analysis. Diagn. Microbiol. Infect. Dis. 2025, 113, 116995. [Google Scholar] [CrossRef] [PubMed]
Feature | Global Context (High-Income Countries) | Indian Subcontinent Context | Diagnostic Implication | References |
---|---|---|---|---|
Pathogen Spectrum | PMIs: 15–25% with anaerobes/atypicals | >30% polymicrobial complexity | Diagnostics require better multiplex panels including tropical, resistant pathogens | [3,35] |
AMR | MRSA prevalence: 1–5% community-acquired | Carbapenem-resistant Gram-negative pathogen in ICUs | Rapid multiplex genotypic tests (include beta-lactamase genes) | [3,35,43] |
VRE prevalence: 3–7% in hospital settings | MDR strains in critical care; blaNDM-1 plasmid in Enterobacterales | |||
Healthcare Infrastructure | MALDI-TOF in most clinical labs | Culture reliance in ~60% healthcare facilities | Need point-of-care testing: <$50/test, <1 h, multiplexed detection tailored to local epidemiology | [5,6,44,45] |
mNGS TAT: 24–48 h; high cost: (>$80/test) | Limited accessibility to advanced diagnostics | |||
Host Factors | Obesity: ~30% adults; controlled diabetes: ~10% | Malnutrition: up to 40% (rural) | Diagnostics must incorporate host biomarkers to distinguish infection vs. colonization | [13,46,47,48] |
Uncontrolled diabetes: 15–20%; TB incidence | ||||
HIV prevalence: ~0.3% |
Pipeline | AI/ML Based | Main Features | Sensitivity | Source |
---|---|---|---|---|
IDseq | No | Cloud-based, mNGS data processing pipeline with host filtering, assembly-based alignment, taxonomic classification, reporting, and visualization. | High sensitivity for microbial pathogen detection; supports divergent/novel virus detection. | [83] |
MetaCherchant/TBwDM Pipeline | Partially | Bioinformatics pipeline for detection of pathogens and AMR genes (ARGs) from polymicrobial simulated reads; marker-based confirmation | High precision and recall (>97% for monomicrobial; ~78% overall in polymicrobial) | [84] |
BacPipe | No | User-friendly whole-genome sequencing pipeline for bacterial genome assembly, annotation, and outbreak detection with clinical application | Supports rapid clinical bacterial diagnostics; accuracy clinically validated | https://github.com/wholeGenomeSequencingAnalysisPipeline/BacPipe, accessed on 1 August 2025 |
DeepColony | Yes | AI-based hierarchical multi-network for automated culture plate interpretation, species ID, quantitation | >99% agreement for negative, >95% for positive cultures | [85] |
PCR.Ai | Yes | Machine learning-based automation of PCR data analysis; auto-interpretation and quality control | 100% concordance with manual interpretation; faster TAT | [86] |
NEKSUS (Oxford Ont Hybrid Assembly Tool) | No | Nanopore long-read hybrid assembler for genome surveillance in bloodstream infections | High completeness and accuracy | https://github.com/oxfordmmm/NEKSUS_ont_hybrid_assembly_comparison, accessed on 1 August 2025 |
ASA3P | No | Automatic and scalable pipeline for bacterial genome assembly, annotation, and analysis | Fully automatic, locally executable; suited for clinical bacterial genomics | [87] |
DMP (Dutch Microbiome Project pipeline) | Partially | Pipeline and workflows for microbiome profiling and analysis | Published in microbiome studies; quality-controlled profiling | https://github.com/GRONINGEN-MICROBIOME-CENTRE/DMP, accessed on 1 August 2025 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 by the authors. Published by MDPI on behalf of the Hellenic Society for Microbiology. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Patnaik, A.; Kayal, T.; Basu, S. Polymicrobial Infections: A Comprehensive Review on Current Context, Diagnostic Bottlenecks and Future Directions. Acta Microbiol. Hell. 2025, 70, 39. https://doi.org/10.3390/amh70040039
Patnaik A, Kayal T, Basu S. Polymicrobial Infections: A Comprehensive Review on Current Context, Diagnostic Bottlenecks and Future Directions. Acta Microbiologica Hellenica. 2025; 70(4):39. https://doi.org/10.3390/amh70040039
Chicago/Turabian StylePatnaik, Amit, Titirsha Kayal, and Soumya Basu. 2025. "Polymicrobial Infections: A Comprehensive Review on Current Context, Diagnostic Bottlenecks and Future Directions" Acta Microbiologica Hellenica 70, no. 4: 39. https://doi.org/10.3390/amh70040039
APA StylePatnaik, A., Kayal, T., & Basu, S. (2025). Polymicrobial Infections: A Comprehensive Review on Current Context, Diagnostic Bottlenecks and Future Directions. Acta Microbiologica Hellenica, 70(4), 39. https://doi.org/10.3390/amh70040039