Development of In-House Multiplex PCR Assays to Detect Respiratory Microorganisms and Implementation using Nasopharyngeal Swabs from Rural Senegal (2025)

Introduction

Acute respiratory infections are a critical public health concern worldwide, particularly in countries with limited resources [1]. They are very common and ubiquitous, affecting adults and children alike [2]. The spectrum of microorganisms involved in acute respiratory infections is vast, including bacteria such as Streptococcus pneumoniae and viruses such as influenza viruses, respiratory syncytial viruses (RSV), rhinovirus, enteroviruses, metapneumovirus (hMPV), adenovirus (AdV), human bocavirus, parainfluenza (HPIV), MERS-CoV, coronaviruses (HCoV-E229, HCoV-OC43, HCoV-NL63, HCoV-HKU1), and SARS-CoV-2 since 2020 [3]. They are one of the main causes of consultations in health centres, morbidity, hospitalisation, and mortality [4], with impacts worldwide, as evidenced by the COVID-19 pandemic [5]. Infection with the influenza A virus also causes significant morbidity and mortality worldwide, despite vaccination and antiviral treatment [6]. Acute respiratory infections, particularly lower respiratory tract infections, are the leading cause of death among children under the age of five and are estimated to be responsible for between 1.9 million and 2.2 million childhood deaths globally every year. Forty-two percent of these acute respiratory infection-associated deaths occur in Africa [4]. In Senegal, they remain the leading cause of infectious mortality [7]. The diagnosis of infectious diseases currently relies on a range of laboratory methods, including culture, serological tests, nucleic acid amplification tests, antigen detection, and direct visualisation [8]. Isolating and identifying pathogens is a delicate task, and not within the reach capabilities of all healthcare facilities [9]. Moreover, a number of infectious diseases, many of them emerging and/or neglected, can only be reliably diagnosed by molecular biology [10]. Over the past fifteen years in Senegal, we have been studying the causes of non-malarial fevers using realtime PCR assays performed on blood extracts [11,12]. We first established a repertory of microorganisms, and then set up our first Point-Of-Care (POC) laboratory at the Dielmo-Ndiop dispensary, followed by another at the Niakhar dispensary. Several bacteria were identified by molecular biology, including Rickettsia species [13], Borrelia crocidurae [14], Tropheryma whipplei [11,15], and Coxiella burnetii [16]. With the onset of the COVID-19 pandemic, these POC laboratories began to contribute to the on-site molecular diagnosis and monitoring of SARS-CoV-2 in the rural areas of Niakhar, Dielmo, and Ndiop in Senegal [17].

Diagnostic methods have evolved significantly, with the advent of molecular biology techniques and, more particularly, multiplex molecular tests, which allow the simultaneous detection of a large number of infectious agents. These tests are, however, expensive and unsuited to low-income countries [2].

Our aim was to develop and evaluate in-house duplex and triplex PCR assays targeting a wide range of respiratory microorganisms, including bacteria and viruses, as well to complete the repertoire of respiratory microorganisms in the Niakhar area of rural Senegal.

Material and Methods

Origin of nasopharyngeal samples and ethical aspects

The specificity and sensitivity of the in-house duplexed and triplexed molecular systems were evaluated, firstly, on the basis of the “tube bottoms” of French samples, by comparing the results obtained with the same systems in simplex PCR and with a commercial kit used for routine diagnosis in our clinical microbiology laboratory. Then, the in-house duplexed and triplexed molecular systems were applied to Senegalese samples from a rural area of interest.

The French specimens corresponded to “tube bottoms” from nasopharyngeal swabs sampled as part of care and sent for the diagnosis of respiratory infections in our infectious agent diagnostic laboratory at the Marseille University Hospitals (APHM, Marseille). In accordance with French regulations, during their hospitalisation or consultation, patients are informed by the establishment of the possibility of the data or samples collected during routine care being reused for research purposes. The patient may object by writing a letter or email to the establishment’s Data Protection Officer. Patients who object to the use of their data or samples for research purposes are excluded. None of the patients for who nasopharyngeal “tube bottoms” had been used raised any objections to their reuse.

The Senegalese specimens were collected from the Fatick region. This community, located 155 km south-east of the capital, Dakar, has an estimated population of around 48,000, spread over 30 villages covering an area of 230 km2. Samples were collected at four health posts, including namely the Diohine Privé, Diohine Public, Toucar, and Ngayokheme health posts. Nasopharyngeal swabs were taken from febrile patients (temperature ≥ 38°C) who consulted one of the four health posts in 2020, 2021, and 2022. As soon as the sample was taken, the swab was introduced into a Virocult tube containing a biological preservation liquid (Medical Wire, Corsham, UK). The samples were first stored at -20°C in the field, then transported in an electric cooler at -20°C from Niakhar to Dakar, where they were stored at -80°C, before being transported in a polystyrene box containing ice packs (temperature around 4°C) by air to Marseille (France), where they were stored at -80°C before analysis. The entire study was approved by Senegal’s National Health Research Ethics Committee (declaration numbers #00087 and #00081). Each participant, or their legal guardian in the case of minors, also provided their written informed consent.

Nucleic acid extraction

Extraction of genetic material (DNA and RNA) from each sample was performed using a KingFisher system (Thermo Fisher Scientific, Illkirch-Graffenstaden, France) with the MacheryNagel “NucleoMag” kit (Macherey-Nagel, Hoerdt, France) following the manufacturer’s recommendations. A volume of 150 μL of nasopharyngeal sample was extracted and then eluted with 150 μL of elution buffer.

In-house multiplexed PCR tests

Primers and probes

All PCR systems that were multiplexed have already been published [16,18-35]. When planning a multiplex system, primers and probes should be tested in different combinations of fluorescence for probes and concentrations to achieve uniform performance. The complete list of primer sequences and fluorescent probes (FAM, VIC, and JUN) for all the microorganisms selected in this study, as well as the extract quality control (actin) and the final primer and probe concentrations used in PCR reactions are detailed in Supplementary Table 1.

Duplex and triplex PCR systems were designed to detect 23 microorganisms, including 18 viruses. Briefly, eleven systems, including nine duplexes (four targeting DNA microorganisms and five targeting RNA microorganisms), and two triplexes (targeting RNA microorganisms) were used. The combinations of duplex and triplex qPCR assays were as follows: (1) Staphylococcus aureus and human adenovirus (HAdV); (2) Haemophilus influenzae and Streptococcus pyogenes; (3) Streptococcus pneumoniae and Mycoplasma pneumoniae; (4) actin and human bocavirus (HBoV); (5) human metapneumovirus (hMPV) and human rhinovirus; (6) human parechovirus (HPeV) and respiratory syncytial virus (RSV); (7) human coronavirus OC43 (HCoV-OC43) and enterovirus; (8) human parainfluenza virus-2 (HPIV-2) and human parainfluenza virus-1 (HPIV-1); (9) human parainfluenza virus-3 (HPIV-3) and human parainfluenza virus-4 (HPIV-4); (10) human coronavirus HKU1 (HCoV-HKU1), human coronavirus 229E (HCoV-229E), and human coronavirus NL63 (HCoV-NL63); and (11) influenza A, influenza B, and SARS-CoV-2.

In-house multiplex DNA amplification assays

Each in-house multiplex DNA qPCR assay was performed in a final volume of 20 μL, including 5 μL of extract, 10 μL of 2x LightCycler 480 Probes Master (Roche Diagnostics, Meylan, France), each primer and probe at its specific concentration (Supplementary Table 1), and supplemented with water to reach the reaction volume. qPCR assays were performed on a LC480 thermal cycler (Roche).

Amplifications were performed using the following conditions: 95°C for five minutes followed by 40 cycles of denaturing at 95°C for three seconds, 60°C for 35 seconds, and finally a cooling step to 40°C for 40 seconds.

In-house multiplex RNA amplification assays

Each in-house multiplex qRT-PCR assay was performed in a final volume of 20 μL, including 5 μL of extract, 5 μL of 4x TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific, Grand Island, NY, USA), each primer and probe at its specific concentration (Supplementary Table 1), and supplemented with water to reach the reaction volume. qRT-PCR assays were performed on a LC480 thermal cycler (Roche) according to manufacturer’s recommendations. Amplifications were performed using the following conditions: reverse transcription at 50°C for five minutes, and 95°C for 20 seconds, followed by 40 cycles of denaturing at 95°C for 15 seconds, 60°C for 60 seconds and, finally, a cooling step to 40°C for 40 seconds. For DNA and RNA amplifications assays, negative and positive controls were systematically used for each run to guarantee the reliability and validation of the analyses.

Evaluation of in-house multiplexed PCR tests

The results obtained with in-house multiplex PCR assays were systematically compared with those obtained using the same PCR systems but in simplex mode and with those obtained with a commercial system, using the FTD^® Respiratory Pathogens 21 kit (Fast-Track Diagnostics, Esch-sur-Alzette, Luxembourg), used according to the manufacturer’s recommendations. The performance of the different duplex and triplex PCR assays was thus evaluated in several ways. A first series of samples which were positive for the various microorganisms and human actin, as well as negative samples, were analysed in parallel by simplex PCR assay. A second series of samples which were positive for the various microorganisms and human actin, as well as negative samples, were analysed in parallel by the commercial FTD^® kit. It was not possible to test the same samples with the PCR simplex assay and the FTD^® commercial kit, as these were tube bottoms and quantities were insufficient to test everything in parallel. The cost of reagents and the duration of duplex and triplex qPCR assays were also estimated.

Statistical analysis

Categorical variables were reported as frequencies and percentages. Fisher’s exact test and phi coefficient (Φ) were used to measure the strength of association between microorganisms. A two-sided P-value of less than .05 was considered statistically significant. Analyses were carried out using SAS 9.4 statistical software (SAS Institute, Cary, North Carolina, USA).

Results

Evaluation of duplex and triplex PCR assays

Overall, to evaluate duplex and triplex PCR assays, data obtained from 965 different DNA and RNA extracts including 408 samples which were positive for a targeted respiratory microorganism or for human actin were analysed.

Comparison with system using simplex qPCR

To compare duplex and triplex qPCR assays with simplex qPCR assays, 445 DNA/RNA extracts, including 203 positive samples, were analysed with the different strategies. Among the 445 DNA/ RNA extracts analysed, here were 14 for S. aureus (five of which were positive), 14 for adenovirus (seven of which were positive), 13 for H. influenzae (six of which were positive), 13 for S. pyogenes (six of which were positive), 15 for S. pneumoniae (five of were positive), 15 for M. pneumoniae (eight of which were positive), 48 human actin (42 of which were positive), 48 for bocavirus (23 of which were positive), 16 for metapneumovirus (five of which were positive), 16 for rhinovirus (nine of which were positive), 17 for parechovirus (eight of which were positive), 17 for RSV (seven of which were positive), 14 for HCoV-OC43 (five of which were positive), 14 for enterovirus (seven of which were positive), 15 for HPIV-2 (seven of which were positive), 15 for HPIV-1 (seven of which were positive), 15 HPIV-3 (eight of which were positive), 15 for HPIV-4 (six of which were positive), 21 for HCoV-NL63 (eight of which were positive), 21 for HCoV-HKU1 (five of which were positive), 21 for HCoV-229 (six of which were positive), 16 for influenza A (five of which were positive), 16 for influenza B (four of which were positive), and 16 for SARS-CoV-2 (five of which were positive). The data obtained showed excellent sensitivity and specificity of the duplex and triplex qPCR assays, and are summarised in Supplementary Table 2. More precisely, all the results which were expected to be negative were indeed negative, and all the results which were expected to be positive were positive.

The Cycle Thresholds (CTs) were almost identical for most of the targets. For a few others, there were no more than two CTs differences between the different molecular assays.

Comparison with the FTD^® Respiratory Pathogens 21 kit

To compare the duplex and triplex qPCR assays with a commercial kit (the FTD^® Respiratory Pathogens 21 kit), 520 DNA/RNA extracts, including 205 positives, were analysed with the different strategies. Of the 520 DNA/RNA extracts analysed, there were 23 for S. aureus (including 11 positive), 23 for adenovirus (including nine positive), 24 for H. influenzae (including 16 positive), 24 for S. pyogenes (including six positive), 17 for S. pneumoniae (including seven positive), 17 for M. pneumoniae (including eight positive), 15 human actin (including five positive), 15 for bocavirus (including five positive), 23 for metapneumovirus (including nine positive), 23 for rhinovirus (including 12 positive), 23 for parechovirus (including five positive), 23 for RSV (including 17 positive), 19 for HCoV-OC43 (including seven positive), 19 for enterovirus (including ten positive), 18 for HPIV-2 (including six positive), 18 for HPIV-1 (including ten positive), 17 HPIV-3 (including six positive), 17 for HPIV-4 (including six positive), 22 for HCoVNL63 (including eight positive), 22 for HCoV-HKU1 (including six positive), 22 for HCoV-229 (including six positive), 32 for influenza A (including 12 positive), 32 for influenza B (including seven positive), and 32 for SARS-CoV-2 (including 11 positive). The data obtained showed excellent sensitivity and specificity of the duplex and triplex qPCR assays, and are summarised in Supplementary Table 3. More precisely, all the results which were expected to be negative were indeed negative, and all the results which were expected to be positive were positive. The CTs were almost identical for most of the targets. For a few others, there were no more than two CT differences between the different molecular assays.

Cost and length of duplex and triplex qPCR analyses

The cost of analyses was estimated on the basis of the pathogens and reagent costs applied at Marseille University Hospitals (France) (Table 1). DNA/RNA extraction using the KingFisher device and the Macherey-Nagel “NucleoMag” kit was estimated at €2.79 excluding value-added tax (VAT) per sample. Each DNA duplex qPCR targeting two DNA pathogens was estimated at €0.35 excluding VAT. Each RNA duplex qPCR targeting two RNA pathogens was estimated at €1.28 excluding VAT.

Table 1: Estimated costs of in-house multiplex PCR assays compared to commercial kits used in our clinical microbiology diagnostic laboratory.

The RNA triplex qPCR targeting three RNA pathogens was estimated at €1.34 excluding VAT. The lengths of analysis were estimated taking into account the various steps involved in extraction, amplification, and handling. Extraction time was 25 minutes. DNA qPCR time was one hour, and RNA qPCR time was one hour 25 minutes, with an additional ten minutes of handling. The estimated time for DNA qPCR was one hour 25 minutes, and two hours for RNA qPCR.

Senegalese patients and respiratory microorganisms

A total of 500 nasopharyngeal samples were analysed from 500 Senegalese patients. These 500 samples are part of a larger series of 2000 samples. Only a quarter of the samples had previously been analysed by a commercial kit, due to the excessive cost [36]. Of the 500 patients, 146 (29.2%) were aged between one month and five years, 154 (30.8%) between six and ten years, 111 (22.2%) between 11 and 20 years, and 89 (17.8%) were 21 years and over. The mean age of the patients was 12 years (youngest one month, oldest 73 years). Of these 500 samples, 261 (52.2%) were from women, 235 (47%) from men and this information was not specified for four patients (0.8%).

Prevalence of respiratory microorganisms

A respiratory microorganism was detected in at least 294 of the 500 nasopharyngeal samples (58.8%). Of these, 54.2% (271/500) were infected with at least one bacterium and 20.2% (101/500) were infected with a virus (Table 2). S. pneumoniae was the most prevalent bacterium detected (36.2%, 181/500), followed by S. aureus (21.6%, 108/500) and H. influenzae (20.2%, 101/500). Only four nasopharyngeal samples were positive for S. pyogenes (0.8%). No samples were positive for M. pneumoniae. Adenovirus was the most prevalent virus detected (5.2%, 26/500), followed by RSV (3.0%, 15/500), SARS-CoV-2 (2.2%, 11/500), rhinovirus (2.0%, 10/500) and influenza A virus (1.8%, 9/500) (Table 2). Enterovirus, HCoV-OC43, and human bocavirus were each detected in five patients (1.0%), HPIV-1 and influenza B virus each in four patients (0.8%), and HPIV-3 and HCoV-HKU1 each in three patients (0.6%). Human metapneumovirus was observed in two patients (0.4%). Finally, HPIV-2 and HCoV-NL63 were each detected in one patient (0.2%). No samples were positive for HPIV-4, parechovirus, or HCoV-229E.

Table 2: Prevalence of respiratory microorganisms detected using duplex and triplex qPCR assays in nasopharyngeal swabs from 500 febrile patients in the Niakhar area of Senegal.

Co-infection with respiratory microorganisms and their correlations

Co-infection was detected in 28.2% (141/500) of the nasopharyngeal samples (Table 3). Of the 141 co-infections, 82 (58.1%) involved two microorganisms. The combination of S. pneumoniae and H. influenzae was the most common (40.2%, 33/82), followed by S. pneumoniae and S. aureus (14.4%, 11/82), then S. pneumoniae and adenovirus (7.3%, 6/82) (Supplementary Table 4). Fifty-three out of 141 co-infections (37.6%) involved three microorganisms (Table 3). The three most frequent triple infections were as follows: (1) S. pneumoniae, H. influenzae, and S. aureus (26.4%, 14/53); (2) S. pneumoniae, H. influenzae, and adenovirus (15.1%, 8/53); and (3) S. pneumoniae, H. influenzae, and RSV (15.1%, 8/53) (Supplementary Table 4).

Table 3: Number of respiratory microorganisms identified by nasopharyngeal sampling in 500 febrile patients from the Niakhar region of Senegal.

Finally, six co-infections out of 141 (4.2%) involved four microorganisms, as follows: (1) S. pneumoniae, H. influenzae, S. aureus, and adenovirus; (2) S. pneumoniae, H. influenzae, S. aureus, and HCoV-HKU1; (3) S. pneumoniae, S. aureus, S. pyogenes, and influenza A virus; (4) S. pneumoniae, H. influenzae, S. aureus, and influenza B virus; (5) S. pneumoniae, H. influenzae, metapneumovirus, and adenovirus; (6) S. pneumoniae, H. influenzae, S. pyogenes, and HCoV-NL63 (Supplementary Table 4).

The correlation analysis for all respiratory microorganisms reveals a statistically significant positive association between the presence of H. influenzae and S. pneumoniae (Φ=0.46, P<0.001) (Supplementary Table 5). Similarly, a strong positive correlation was observed between HCoV-NL63 and S. pyogenes (Φ=0.50, P<0.001). Moderate positive correlations were also identified between HPIV-1 and bocavirus (Φ=0.22, P<0.001) and between RSV and S. pneumoniae (Φ=0.21, P<0.001).

Seasonality of respiratory microorganisms

The prevalence of each respiratory microorganism by month and season is illustrated in Figures 1, 2, and 3. S. pneumoniae, H. influenzae and S. aureus can be observed all year round, but have higher peaks during the dry season (Figure 1). A peak in cases of SARS-CoV-2 infection was detected in January (Figure 2). While sporadic cases of influenza A virus infection are observed throughout the year, a major peak was observed in October (Figure 2). Cases of influenza B virus infection were observed exclusively in November and December, with a major peak in December. Rhinovirus was mainly observed during the rainy season (Figure 2). Bocavirus was exclusively observed during the dry season from April to May (Figure 3), whereas RSV was observed almost exclusively during the rainy season, with a peak in September (Figure 3). Adenovirus circulates year-round with a higher prevalence during the dry season, and a peak in March (Figure 3). Circulation of HCoV-OC43 was mainly observed during the rainy season (June, July, August, and October). Low circulation of HCoV-HKU1 was also noted during the rainy season (June and July) (Figure 2).

Figure 1: Seasonal prevalence of S. pneumoniae, S. aureus, H. influenzae, and S. pyogenes from nasopharyngeal swabs of febrile patients in the Niakhar region of rural Senegal (number of tested samples in parentheses).

Figure 2: Seasonal prevalence of respiratory viruses, including SARS-CoV-2, influenza A, influenza B, HRV, HCoV-NL63, HCoVHKU1, and HCoV-OC43 from nasopharyngeal swabs of febrile patients in the Niakhar region of rural Senegal (number of tested samples in parentheses).

Figure 3: Seasonal prevalence of respiratory viruses, including HPIV-3, HPIV-2, HPIV-1, hMPV, hBoV, RSV, enterovirus (EV), and adenovirus (AdV) from nasopharyngeal swabs in 500 febrile patients from the Niakhar area in rural Senegal (number of samples tested in brackets).