Introduction

Acute respiratory infections (ARIs) remain one of the leading causes of morbidity and mortality worldwide, particularly affecting vulnerable populations such as children, older adults, and individuals with chronic illnesses [1]. According to the World Health Organization (WHO), ARIs account for a substantial proportion of healthcare visits and hospitalizations during seasonal peaks, especially in winter [2]. Viral pathogens such as rhinoviruses, influenza viruses, and respiratory syncytial virus (RSV) are frequently detected in ARIs. Bacterial pathogens, including Streptococcus pneumoniae and Staphylococcus aureus, may also be identified as primary pathogens or in mixed detections [3]. Rapid and accurate identification of respiratory pathogens is important for clinical management and infection-control strategies [4].

In Kazakhstan, ARIs represent a persistent public health burden, with seasonal outbreaks placing considerable strain on healthcare systems [5]. Although ARIs are known to impose a substantial disease burden, regional epidemiological surveillance studies characterizing both viral and bacterial detections remain limited. The prevalence and clinical implications of simultaneous detection of multiple respiratory pathogens are also insufficiently characterized in the local context, despite evidence that clinically confirmed co-infections may be associated with increased disease severity and prolonged hospitalization [6, 7].

Conventional diagnostic approaches, including culture-based methods and serology, can be time-consuming and may have limited sensitivity for some pathogens [8]. Multiplex real-time polymerase chain reaction (PCR), in contrast, enables simultaneous detection of multiple respiratory targets within a short time frame. This technology has been widely used for clinical diagnostics and epidemiological monitoring of respiratory pathogens, particularly during influenza and COVID-19 epidemics [9-15].

The 2023–2024 respiratory season in Kazakhstan was marked by increased ARI activity [5]. Despite the growing use of multiplex PCR in surveillance systems, regional data comprehensively describing circulating viral and bacterial respiratory targets and multi-pathogen detections during epidemic periods remain limited. Such data can support diagnostic strategies, antimicrobial stewardship, and public health responses [7].

This study aimed to analyze the distribution of viral and bacterial respiratory pathogens among patients with ARIs in Almaty during the 2023–2024 epidemic season using a multiplex real-time PCR panel capable of detecting 30 respiratory pathogens. Age-specific detection patterns and the frequency of simultaneous detection of multiple pathogens were also assessed.

Materials and Methods

Study design and setting. This descriptive laboratory-based study included patients with clinically diagnosed ARIs tested in Almaty, Kazakhstan, between November 2023 and January 2024. A total of 374 patients were included.

Laboratory testing. Respiratory specimens were tested at the OHKZ laboratory using Allplex™ Respiratory Panel 1–4 kits (Seegene, Republic of Korea) together with an OHKZ-developed assay for four additional bacterial targets. The combined testing scheme comprised 30 respiratory targets, described in the source manuscript as 19 viral and 11 bacterial targets (Table 1).

Variables and definitions. Outcomes included detection of at least one viral target, detection of at least one bacterial target, age-specific pathogen detection rates, and simultaneous detection of multiple targets. Multi-pathogen detections were categorized as two, three, or four or more detected targets per patient. Age categories were 0–1, 2–5, 6–10, 11–20, 21–30, 31–40, 41–50, 51–60, and ≥61 years, consistent with Figures 3 and 4.

Statistical analysis. Data were summarized descriptively using absolute counts and percentages. No inferential statistical testing is reported; therefore, between-month and between-age comparisons are interpreted descriptively rather than as statistically significant differences.

Reporting completeness. The source manuscript does not specify the respiratory specimen type(s), sampling and transport procedures, nucleic acid extraction method, PCR instrument, Ct cut-off/interpretation criteria, positive, negative and internal controls, or detailed quality-control procedures. These parameters were not reconstructed and should be supplied from the laboratory protocol by the authors before submission to ensure reproducibility.

Table 1 - Respiratory pathogens targeted by Allplex™ Respiratory Panel Kits (Seegene, Republic of Korea)

Results

I. Respiratory viral detections

Among 374 examined patients, at least one viral pathogen was detected in 195 (52.1%). The most frequently detected viruses were rhinovirus (24.3%), influenza A virus (14.7%), and adenovirus (5.9%). In December 2023, relatively high detection rates were observed for influenza A virus and respiratory syncytial virus A (RSV A), whereas in January 2024 the detection rates of rhinovirus, adenovirus, and RSV A increased (Figure 1).

Figure 1 - Detection rates of respiratory viruses among ARI patients in Almaty, Kazakhstan,

from November 2023 to January 2024 using multiplex real-time PCR

II. Respiratory bacterial detections

Respiratory bacterial targets were detected in 240 of 374 patients (64.2%). The most common bacterial detections were Haemophilus influenzae (32.9%), Streptococcus pneumoniae (24.9%), Moraxella catarrhalis (23.8%), Staphylococcus aureus (12.8%), Klebsiella pneumoniae (3.5%), and Pseudomonas aeruginosa (1.6%). Mycoplasma pneumoniae was not detected. Bordetella pertussis was detected in 2.4% of cases (Figure 2).

Figure 2 - Detection rates of respiratory bacterial pathogens among ARI patients in Almaty, Kazakhstan,

from November 2023 to January 2024.

III. Positivity rates of respiratory pathogens by age

Positivity rates of respiratory viruses by age

Age-specific viral detection rates were evaluated descriptively. Most viral targets were detected more frequently in children younger than 5 years than in adults, with the exception of influenza A virus. In the 0–1-year age category, adenovirus (14.3%), RSV A (11.4%), and RSV B (14.3%) showed higher positivity rates than in several older age categories (Figure 3). The overall positivity rate for influenza A virus was 14.7%, with the highest observed rate in the 31–40-year age group; among patients aged ≥61 years, the rate was 15.9%. In children aged ≤10 years, the average positivity rate for rhinovirus was 35.2%, the highest among the reported viral detections (Figure 3).

Figure 3 - Positivity rates of respiratory viruses by age

Positivity rates of respiratory bacteria by age

In children younger than 10 years, H. influenzae, S. pneumoniae, and M. catarrhalis were among the most frequently detected bacterial targets, whereas H. influenzae and S. aureus predominated among adults. B. pertussis was detected across several age categories (Figure 4).

Figure 4 - Positivity rates of respiratory bacteria by age

IV. Multiple respiratory pathogen detections

Among the 374 patients tested, a single viral target was detected in 33 patients (8.8%), a single bacterial target in 63 patients (16.8%), and no investigated target was detected in 76 patients (20.3%). Multiple positive assay targets were recorded in 202 patients (54.0%). Among the 202 patients with multiple positive targets, two targets were detected in 94 patients (46.5%), three in 67 patients (33.2%), and four or more in 41 patients (20.3%) (Figure 5).

Among the bacterial targets represented in the multiple-detection profiles, Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Moraxella catarrhalis, and Klebsiella pneumoniae were among the most frequently detected (Table 2).

Figure 5 - Distribution of single and multiple respiratory pathogen detections among ARI patients

Table 2 - Combinations of multiple respiratory pathogen targets detected by multiplex real-time PCR