This retrospective study used clinical microbiology testing records from the Department of Clinical Laboratory at the Affiliated Hospital of Zunyi Medical University. Consecutive bacterial and fungal testing results collected between January 1, 2016 and December 31, 2022 were extracted and anonymized under institutional ethics approval. A total of 367,824 eligible specimens were screened, yielding 74,838 positive isolates. The analysis focused on the 51 most frequently detected microbial species, which together represented 97.35% (72,862/74,838) of all positive specimens.
Specimens were collected from sites selected by treating clinicians according to infectious manifestations. Non-open site specimens included whole blood, pleural effusion, cerebrospinal fluid, puncture and drainage fluids, bile, tissue, bone marrow, dialysate and amniotic fluid. Open-site specimens included sputum, secretions, urine, feces, bronchoalveolar lavage fluid, cervical secretions, catheter specimens, throat swabs and other surface-source samples.
Primary culture methods were matched to microorganism types: aerobic and anaerobic bacteria on appropriate agar media, fungi on Sabouraud Dextrose Agar and CHROMagar, and Mycobacterium tuberculosis on modified Lowenstein-Jensen and MGIT 960 liquid media. Isolates were identified by biochemical assays and mass spectrometry (VITEK MS); nucleic acid amplification (Xpert MTB/RIF) was used for M. tuberculosis confirmation. Laboratory testing followed Chinese national standards and ISO 15189 accreditation; positive and negative quality controls were maintained.
To maximize statistical reliability, the study restricted analysis to the 51 species with highest detection frequencies. Patients were categorized into four age groups: children (0–14 years), young adults (15–47 years), middle-aged adults (48–63 years), and elderly (≥64 years). Seasons were defined per Chinese standards: spring (March–May), summer (June–August), autumn (September–November), and winter (December–February of the following year).
Comparisons used chi-square tests and goodness-of-fit tests; significance was set at P < 0.05. Analyses included stratification by pathogenic type (primary vs. opportunistic) and by Gram staining classification (Gram-positive vs. Gram-negative).
Across the study period 168 bacterial and fungal species were detected among the specimens. The top 51 species, which formed the analytic cohort, accounted for 72,862 of 74,838 positive isolates (97.35%). This selection was intended to ensure representativeness while maintaining statistical power for subgroup comparisons.
Among the 51 analyzed species, 40 showed statistically significant differences in detection rates between male and female patients. When stratified by pathogenic type, sex differences were present in 4 of 5 primary pathogens, 33 of 41 opportunistic pathogens, and 3 of 5 fungal species; however, the proportion of species with sex differences did not differ significantly across these three microbial groups.
By Gram staining, Gram-positive bacteria exhibited sex-related differences in 15 of 22 species (68.18%), whereas Gram-negative bacteria did so in 22 of 24 species (91.67%); this intergroup difference was statistically significant (χ2 = 4.02, P = 0.045). Of the 40 species with sex differences, 38 reached peak detection percentages in male patients and only 2 peaked in females (χ2 = 32.4, P < 0.001).
Age-associated differences in positive detection rates were observed for 48 of the 51 species. Stratified results showed 4 of 5 primary pathogens, 39 of 41 opportunistic pathogens, and all 5 fungal species had significant age-related variation. There was no significant difference in the prevalence of age disparities among primary pathogens, opportunistic pathogens and fungi.
By Gram classification, significant age differences were found in 20 of 22 Gram-positive species (90.91%) and 23 of 24 Gram-negative species (95.83%), with no significant difference between the two groups (χ2 = 0.46, P = 0.499). For species with notable age-related peaks, counts were: 8 species peaking in children, 18 in young adults, 11 in middle-aged adults, and 11 in elderly individuals. These peak distributions across age groups did not differ significantly (χ2 = 4.50, P = 0.212).
Seasonal variation in detection rates was significant for 32 of the 51 species. Seasonal differences appeared in 3 of 5 primary pathogens, 25 of 41 opportunistic pathogens, and 4 of 5 fungal species. The proportion of Gram-negative species with seasonal differences (18/24, 75.00%) was greater than that for Gram-positive species (10/22, 45.45%), and this difference was significant (χ2 = 5.26, P = 0.022).
Among species with seasonal peaks, counts by season were: 3 peaking in spring, 20 peaking in summer, 7 in autumn and 2 in winter. The predominance of summer peaks was statistically significant (χ2 = 25.75, P < 0.001), indicating a concentration of increased detection for many pathogens during warmer months.
Overall, the study identified stronger associations of Gram-negative species with both sex and seasonal variation compared with Gram-positive species. Age-related variation was widespread across both Gram categories without a significant intergroup difference. Comparisons among primary pathogens, opportunistic pathogens and fungi showed high frequencies of sex, age and seasonal differences, but the proportions of species affected did not differ significantly between those three broad microbial categories.
This large single-center analysis describes consistent sex, age, and seasonal distribution patterns among the 51 most commonly detected bacterial and fungal species. Key findings are that females had lower detection rates for most species, Gram-negative bacteria were more sensitive to sex and seasonality than Gram-positive bacteria, and many pathogens peaked in summer, underscoring environmental influences on infection frequencies.
Authors suggest that awareness of these distribution patterns can inform infection prevention strategies, diagnostic prioritization and treatment planning. They note that integrating sex, age and seasonal attributes into clinical decision-making and public health measures may improve targeting of surveillance and control efforts. The study’s methods, large sample size and alignment with national surveillance data support the general applicability of the reported epidemiological patterns.
Note: The manuscript states that raw original data are available on request to the local ethics committee under specified restrictions; individual-level raw data were not provided in the article text or supplements.