Infectious diseases cause global healthcare expenses, economic losses, and social suffering [1]. Many places have disproportionately high rates of bacterial, viral, fungal, and parasitic diseases, which affect millions of people annually, due to socioeconomic level, environmental circumstances, and healthcare availability [2]. High population density, inadequate sanitation, and limited healthcare access in low- and middle-income countries accelerate the development of infectious diseases [3]. New infections, antibiotic resistance, and other unusual pathogens, including viral pandemics, make infectious disease management difficult even in high-income countries [4]. Effective management and control of many disorders require therapeutic approaches and precise and timely diagnosis [5]. Early detection of infectious microorganisms helps clinicians treat patients, reduce sequelae, reduce transmission, and maximise public health activities. The importance of reliable diagnostic methods in clinical and epidemiological settings is highlighted by the fact that delays or errors in diagnosis can prolong sickness, inappropriate antibiotic use, resistance, and increased healthcare costs [6].

Many diagnostic procedures have been developed to identify infectious diseases, each with its own philosophy, pros, and cons. The accuracy of culture-based approaches for diagnosing bacterial and fungal diseases has long been superior [7].  These methods cultivate infections on media and identify them by morphology, biochemistry, or molecular properties.  Culture is helpful for germ identification and antibiotic susceptibility testing for focused treatment.  Some slow-growing organisms take days or weeks to culture.  Fastidious, non-viable, and intracellular bacteria can also cause diseases culture cannot detect [8].  Due to its specificity and clinical application, culture is essential to infectious illness diagnosis despite these limitations.

However, PCR has transformed infectious illness diagnostics.  These methods swiftly and sensitively identify microbial DNA or RNA.  To treat acute infections quickly, PCR may detect minuscule amounts of pathogen genetic material in hours.  Due to its specificity and sensitivity, PCR can detect hard-to-grow viruses and bacteria.  Multiplex PCR detects numerous diseases from one specimen, however molecular diagnostics has limits [9].  These include being more expensive, requiring specialised lab equipment and experts, and sometimes yielding false-positive results due to contamination.

Serology, which detects host antibodies or antigens in response to infection, is another common diagnostic approach. In persistent viral infections or prior exposure evaluations, serological methods like enzyme-linked immunosorbent tests (ELISA) and rapid diagnostic kits are useful when direct pathogen identification is impractical or too slow [10]. Serology can confirm diseases, assess immune responses, and evaluate vaccination efficacy while providing epidemiological data [11]. Serological assays are limited in early-stage diagnosis because they may not distinguish between previous and acute infections and antibody responses may take days to weeks [12].

Multiple diagnostic methods with benefits and cons necessitate treatment efficacy comparisons.  Clinicians can evaluate infectious situation diagnostics by examining culture, PCR, and serology's sensitivity, specificity, turnaround time, and practical constraints.  Comparison studies indicate which option enhances diagnostic precision and patient outcomes when two or more approaches are equivalent.  Three prominent infectious illness diagnosis methods are compared in this six-month PMCH sample of 100 patients.  In hospitals and communities, this study compares culture, PCR, and serology to improve clinical decision-making, early infection identification, and sickness treatment.

Objectives

1. To evaluate and compare the diagnostic accuracy of culture, PCR, and serology in detecting infectious diseases among patients at PMCH.
2. To assess the sensitivity, specificity, and turnaround time of each method to determine their clinical effectiveness.
3. To provide evidence-based recommendations for selecting appropriate diagnostic methods for different types of infectious diseases in a tertiary care setting.

Study Design and Setting A prospective observational study was undertaken at PMCH. The main goal was to assess the efficacy of culture, PCR, and serology in diagnosing infectious illnesses in individuals with clinical indications of infection. To minimise retroactive biases from medical record checks and test diagnostic performance in real time, a prospective methodology was selected. The study examines these approaches' practicality, turnaround time, and diagnosis accuracy in tertiary care hospitals. Study Period The study was carried out over a six-month period from January 2025 to June 2025, which included patient recruitment, specimen collection, laboratory testing, and data analysis. This duration allowed for the enrolment of a sufficient number of patients with diverse infectious conditions, ensuring variability in pathogen types and specimen sources. Study Population and Sample Size A total of 100 patients suspected of having infectious diseases were included in the study. The sample size was determined based on feasibility and the availability of patients presenting with clinical signs suggestive of infection during the study period. Inclusion Criteria • Adult patients (≥18 years) presenting with clinical signs or symptoms suggestive of infectious diseases. • Patients who provide informed consent for participation and specimen collection. Exclusion Criteria • Patients currently receiving antibiotic therapy or other treatments that could interfere with pathogen detection. • Immunocompromised individuals or patients with chronic conditions that may affect immune response and diagnostic accuracy. Specimen Collection Specimens were collected according to the type and suspected site of infection. Blood samples were obtained for systemic infections, urine samples for urinary tract infections, and swabs from throat, wound, or other affected sites as appropriate. All specimens were collected using aseptic techniques and processed immediately or stored under recommended conditions to preserve pathogen viability and nucleic acid integrity. Each specimen was divided into aliquots to allow simultaneous testing by culture, PCR, and serology without cross-contamination. Diagnostic Methods Culture: Medium prepared for the likely pathogen was used to grow specimens. Blood, MacConkey, and Sabouraud dextrose agars were used to grow bacteria and fungi, while selective media were used to cultivate species with specialised needs. After incubation cultures at optimal temperature and ambient conditions, to identified bacteria using morphological, biochemical, and biochemical-based automatic identification technologies. The gold standard for fungal and bacterial illnesses was culture positive. PCR: Molecular detection was done utilising pathogen-specific PCR assays targeting conserved genes in common bacterial and viral infections. Standardised kits extracted DNA or RNA from clinical specimens, which was amplified in an optimised thermocycler. Assay formats used gel electrophoresis or real-time fluorescence probes for detection. PCR was chosen because it can identify minimal pathogen nucleic acids and turns over quickly compared to traditional culture. Serology: Serological testing uses quick diagnostic test kits or ELISAs to identify pathogen-specific antigens or antibodies. IgM and IgG responses indicated recent or long-term infection. Serology can help diagnose infections when molecular testing isn't accessible or the pathogen is challenging to culture. Outcome Measures The primary outcomes assessed were sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy of each diagnostic method. Additionally, turnaround time was recorded for each method to evaluate the feasibility of rapid diagnosis in clinical settings. Statistical Analysis All data were entered and analyzed using SPSS. Categorical variables, including test positivity rates and demographic characteristics, were summarized as frequencies and percentages. The Chi-square test was applied to assess statistical significance between diagnostic methods, with a p-value <0.05 considered indicative of a significant difference. Comparative analyses focused on identifying which diagnostic method provided the highest sensitivity and specificity, while also considering practical factors such as test availability and time to result.

Patient Demographics

The study included 100 infectious disease suspects.  The age ranged from 18 to 72 years, with a mean of 42.5 ± 15.3 years.  The study had 56% (n=56) male participants and 44% (n=44) female participants, with a slightly larger number of male patients presenting with infections.  The types of infections observed included respiratory tract (35%), urinary tract (25%), gastrointestinal (20%), wound and soft tissue (15%), and febrile diseases of unknown origin (5%).  This varied infection type distribution permitted diagnostic technique evaluation across clinical situations.

Table 1 Patient Demographics

Diagnostic Yield

The diagnostic performance of culture, PCR, and serology was assessed by analyzing the number of positive cases detected, sensitivity, specificity, PPV, NPV, and turnaround time.

Culture yielded 62 positive cases from 100 specimens, the reference standard.  PCR detected 78 positive cases, including all culture-positive cases and 16 culture-negative instances, demonstrating its superior sensitivity.  Serology found 68 positive instances, mostly in viral or systemic diseases where pathogen identification was difficult.

 Most sensitive was PCR (95%), followed by serology (87%), and culture (79%).  Specificity was highest for culture (100%), PCR (92%), and serology (85%).  Culture had 100% PPV, suggesting no false positives, while PCR and serology had 89% and 82%, respectively.  PCR had the highest NPV (96%), compared to culture (76%) and serology (83%), indicating its capacity to detect infections in low-pathogen specimens.

 Turnaround time varies greatly among diagnostic procedures.  PCR findings were available within 4–6 hours, allowing speedy decision-making, while culture took 48–72 hours, depending on the pathogen. Serology results were available within 6–12 hours for ELISA-based tests or 30–60 minutes for rapid tests, providing an intermediate turnaround time.

Table 2 Detection Rates and Diagnostic Performance

PCR has the highest sensitivity and NPV, making it the ideal technique to detect infectious agents early on when cultures fail due to a low pathogen load or selective organisms. Due to its perfect specificity and PPV, culture was the gold standard for antimicrobial susceptibility testing and pathogen identification. Serology is a reliable adjunct for viral infections or when culture and PCR are unavailable. Culture takes longer, but PCR and serology are the fastest approaches for rapid diagnosis. These findings show that a variety of diagnostic methods may best assess infectious illnesses in tertiary care settings like PMCH, where speed, sensitivity, and specificity are crucial for clinical decision-making.

This study examined the sensitivity and specificity of culture, PCR, and serology in diagnosing infectious diseases in 100 PMCH patients. PCR detected 78 positive cases (including 16 culture-negative cases), making it the most sensitive diagnostic method. Since PCR can amplify even little amounts of pathogen DNA or RNA, it can identify them in specimens with low microbial burden, making it sensitive. Culture, with 100% specificity but lower sensitivity (62 positive cases), proved its gold standard status. With 68 confirmed cases, serology showed moderate sensitivity and specificity. It worked well in viral and systemic disorders where culture is often insufficient or nonviable. The results reveal that PCR is best for early detection, culture is best for diagnosis and antibiotic resistance screening, and serology is a great adjunct.

Comparison with Existing Literature

This study supports previous findings that PCR is the best way to diagnose infectious illnesses. Multiple investigations have demonstrated that PCR is better than conventional culture at detecting even very low amounts of bacterial or virus nucleic acids in acute infections or finicky species. In bloodstream infections, PCR identified bacteria 20-30% more often than culture. Study 1 [13] showed that PCR may detect respiratory viral infections in 4-6 hours and with high sensitivity. Culture is essential for targeted therapy and maintains its excellent specificity when testing for antimicrobial susceptibility, according to standard microbiology literature, including study 2 [14]. According to study 3 [15], serology provides epidemiological insights and validates prior exposure, making it valuable for monitoring immune responses and diagnosing illnesses when direct pathogen detection is difficult. Serology is less sensitive in early infection. These studies complement PMCH findings and show how different diagnostic methods function together.

Advantages and Limitations of Each Method

Culture is crucial to clinical decision-making because it can detect infections and determine drug susceptibility. Its downsides include poorer sensitivity for fragile or intracellular organisms and a 48-72-hour turnaround time. Culture's low false-positive rate and excellent specificity provide clinicians confidence in validated findings.

Due to its speed and sensitivity, PCR can detect pathogens early in patients with low pathogen loads or partial antibiotic therapy. PCR's downsides are higher costs, specialised equipment, and skilled people. Non-viable organisms or pollutants can also cause PCR to yield false-positive findings, which do not always indicate an infection. Despite these limitations, PCR's speed is a major benefit in emergency and epidemic situations.

Serology is useful for monitoring immune responses to viruses and persistent infections, where pathogen isolation is challenging. ELISA and fast tests are useful in resource-constrained situations since they are simple. Cross-reactivity with related organisms may reduce specificity, and delayed seroconversion might produce early infection false negatives.

100 PMCH patients were tested for infectious disease diagnosis using culture, PCR, and serology. The results reveal that PCR is best for early detection and intervention because to its high sensitivity and speed. Serology can supplement culture, which is still the gold standard for viral and chronic infections because to its excellent specificity and antibiotic susceptibility data. The findings emphasise the importance of selecting the proper diagnostic method based on infection type, time to diagnosis, and resources. Multiple diagnostic approaches in clinical settings improve accuracy, treatment speed, and patient outcomes. Future studies with more samples, multicenter participation, and diverse illnesses should validate and optimise diagnostic approaches. Rapid molecular methods and point-of-care testing may improve hospital and community-based infectious illness treatment. Limitations of the Study This research has some limitations. Since the sample size was 100 patients, the results may not apply to larger populations. PMCH alone conducted this study, hence its conclusions may only apply to local pathogen prevalence and laboratory capabilities, not to other hospitals or locations. Due to the minimal number of infectious agents included, this study may have eliminated rare or developing infections from diagnostic performance evaluation. Excluding antibiotic-taking or immune-compromised patients may have caused selection bias.

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