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Bacterial and viral etiology of acute respiratory infection among the Forcibly Displaced Myanmar Nationals (FDMNs) in fragile settings in Cox’s Bazar- a prospective case-control study [1]

['Abu Bakar Siddik', 'Institute For Developing Science', 'Health Initiatives', 'Dhaka', 'Nabid Anjum Tanvir', 'International Centre For Diarrhoeal Disease Research', 'Bangladesh', 'Mohakhali', 'Golam Sarower Bhuyan', 'Md. Shahariar Alam']

Date: 2023-05

The leading infectious cause of death in children worldwide is lower acute respiratory infection (LARI), particularly pneumonia. We enrolled a total of 538 acute respiratory infection (ARI) cases according to WHO criteria and age-sex matched 514 controls in the Forcibly Displaced Myanmar National (FDMN) refugee camps in Cox’s Bazar, Bangladesh, between June 2018 and March 2020 to investigate the role of bacteria, viruses, and their co-infection patterns and observe Streptococcus pneumoniae (S. pneumoniae) serotype distribution. According to the etiological findings, children ≤5 years of age have a higher bacterial positivity (90%) and viral positivity (34%) in nasopharyngeal samples (NPS) compared to those >5 years of age, in both ARI cases as well as for the control group. Among the bacteria, S. pneumoniae was predominant in both cases and controls (85% and 88%). Adenovirus (ADV)(34), influenza virus A and B (IFV-A, B)(32,23), and respiratory syncytial virus (RSV)(26) were detected as the highest number among the viruses tested for the ARI cases. The total number of viruses was also found higher in ≤5 years of age group. Within this group, positive correlation was observed between bacteria and viruses but negative correlation was observed between bacteria. Both single and co-infection for viruses were found higher in the case group than the control group. However, co-infection was significantly high for Streptococcus aureus (S. aureus) and Haemophilus influenzae b (H. influenza b) (p<0.05). Additionally, semi-quantitative bacterial and viral load was found higher for the ARI cases over control considering Cycle threshold (Ct)≤30. Pathogen identification from blood specimens was higher by qRT-PCR than blood culture (16% vs 5%, p<0.05). In the S. pneumoniae serotype distribution, the predominant serotypes in ARI cases were 23F, 19A, 16F, 35B, 15A, 20 and 10F, while 11A, 10A, 34, 35A and 13 serotypes were predominant in the control group. Pathogen correlation analysis showed RSV positively correlated with human metapneumovirus (HMPV), S. aureus and H. influenza b while S. pneumoniae was negatively correlated with other pathogens in ≤5 years age group of ARI cases. However, in >5 years age group, S. aureus and H. influenza b were positively correlated with IFVs, and S. pneumoniae was positively correlated with HMPV and ADV. Logistic regression data for viruses suggested among the respondents in cases were about 4 times more likely to be RSV positive than the control. Serotype distribution showed 30% for PCV10 serotypes, 41% for PCV13 and 59% for other serotypes. Also, among the 40 serotypes of S. pneumoniae tested, the serotypes 22F, Sg24, 9V, 38, 8, and 1 showed strong positive correlation with viruses in the case group whereas in the control group, it was predominant for serotypes 14, 38, 17F and 39 ARI cases were prevalent mostly in monsoon, post-monsoon, and winter periods, and peaked in September and October. Overall these region-specific etiological data and findings, particularly for crisis settings representing the FDMNs in Cox’s Bazar, Bangladesh, is crucial for disease management and disease prevention control as well as immunization strategies more generally in humanitarian crisis settings.

Acute respiratory infections (ARIs) are one of the most common communicable diseases in crisis settings, causing significant morbidity and mortality. Non-comparable as well as little aetiological data from refugee camps and surveillance, make it difficult to compare the prevalence of ARI disease between crisis and non-crisis contexts. In the world’s largest refugee settlement situated in Cox’s Bazar, Bangladesh, very little is known about the etiology of ARIs. To our best knowledge, this is the first case-control prospective study to evaluate the level of involvement of each etiological agent in the onset of LARI etiology, and the association between respiratory viral infections and invasive pneumococcal infections, S. pneumoniae serotype distribution, antimicrobial resistance pattern both in case and control group. Our study showed that ≤5 years age group was the most vulnerable, having the highest number of bacteria as well as viruses both in the case and control group. S. pneumoniae was the predominant bacteria, whereas, among the viruses ADV, IFVs, and RSV were predominant. Semi-quantitative bacterial as well as viral load was found higher in the case group. Positive correlation between viruses and other pathogens was higher in cases of RSV than in the control group. However, single as well as co-infection between viruses were significantly high among the ARI case while for bacteria it was comparable between the case and control group. ARI case enrollment peaked in September and October in the FDMN camps in Bangladesh.

Pneumonia, a severe form of lower acute respiratory infection, continues to be a major cause of death among children under the age of five years globally and accounted for 14% of deaths in this age group in 2019 [ 1 ]. Both children and adults are affected by pneumonia, but mortality is highest in South Asia and Sub-Saharan Africa [ 1 ]. Pneumonia infections can be caused by bacteria, viruses, fungi, and parasites. However, S. pneumoniae, H. influenzae b and Staphylococcus aureus are the most commonly found bacteria associated with acute respiratory infections (ARI), particularly pneumonia [ 2 , 3 ]. Viruses associated with ARIs are Respiratory syncytial virus (RSV), influenza A virus (IFV-A) and influenza B virus (IFV-B), Human Parainfluenza Viruses 1, 2, 3 (HPIV1-3), adenovirus (ADV), and Human metapneumovirus (HMPV) are known to be a primary cause of ARI in children, as they can be attributed to over 60% of ARIs [ 4 – 6 ]. Several factors such as overcrowding, cold weather, and insufficient shelter all create an ideal condition for respiratory droplet transmission. Malnutrition, lack of sufficient sanitation, and stress can play a role in illness progression [ 1 , 7 ]. The clinical symptoms of viral and bacterial pneumonia are very similar irrespective of causative agents [ 8 ] which creates a challenge in diagnosing and managing patients with ARIs making it difficult for physicians to determine the best course of treatment, which is based solely on clinical presentation, can lead to the overuse or misuse of drugs. Another major issue around the world is the rise of drug-resistant bacteria as well as the lack of evidence of clinical benefit. Data on the causative agents, improved and accessible diagnostic techniques and the drug sensitivity/resistance spectrum of the organisms are critical for optimal prevention and treatment of ARIs and lack of these data leads to irrational and inappropriate antimicrobial use, resulting in an increase in multi-drug resistant bacteria [ 9 , 10 ]. Studies have documented the detection of respiratory infections that cause pneumonia throughout the previous decade, with wide variations in prevalence and pathogen spectrum seen among countries and regions, community demography, years, and seasons [ 11 – 14 ]. However, in everyday primary health care especially in refugee settings little is understood about the etiology of ARIs due to lack of data. Although it is difficult to compare the disease burden of ARIs in crisis and non-crisis settings due to data incompatibility, published evidence primarily from refugee camps and surveillance, suggests a very high morbidity and mortality (20-35%) percentage proportional mortality) as well as case fatality (up to 30-35%) due to ARIs [ 15 ]. Furthermore, data on pathogen detection in both symptomatic patients and healthy controls (without pneumonia) to differentiate between asymptomatic carriage and the presence of agents causing symptoms is also limited. Of bacterial pathogens responsible for ARIs. S. pneumoniae is a major pathogen causing pneumonia related deaths globally [ 2 , 16 , 17 ]. At least 98-100 pneumococcal capsular serotypes have been identified to date [ 18 , 19 ]. Currently, pneumococcal conjugate vaccines (PCV) are being used to prevent S. pneumoniae infections (PCV10 and PCV13). These vaccinations only protect against a limited number of pneumococcal serotypes and do not protect against non-vaccine serotypes or unencapsulated S. pneumoniae. It is believed that the number of antibiotic-resistant non-vaccine serotypes has risen dramatically. Innovative, effective, and economical pneumococcal vaccines that can cover a wide range of serotypes are urgently needed. Therefore, to minimize respiratory infection-related morbidity and mortality, continuous monitoring and updates on etiology, better diagnostics, and serotype distribution, antibiotic resistance profiling is required. However, to the best of our knowledge, there have not been investigations on bacterial and viral etiologies of ARIs and S. pneumonia serotype distribution patterns in crisis settings, particularly in the Forcibly Displaced Myanmar Nationals (FDMNs). Nowadays, traditional culture methodologies are applied in limited resource settings. Although culture remains the gold standard, it has significant drawbacks, including specimen collection and transport requirements, the danger of pathogen growth inhibition due to recent antibiotic treatment, and long time needed to obtain results [ 20 , 21 ]. Multiplexed molecular assays have recently emerged as a comprehensive diagnostic solution, providing laboratories with the necessary turnaround duration, sensitivity and specificity, and breadth of coverage of respiratory infections to support the physician’s decision-making needs [ 21 ]. To properly manage ARI cases, country-specific etiological and microbial sensitivity/resistance spectrum data are required. In Bangladesh, no study has been carried out in the FDMN camp settings focusing on pneumonia etiology. This study was driven by the understanding that ARI particularly pneumonia causes high mortality, particularly in crisis situations, and it intends to give data on ARI etiology. So, community-representative population studies are needed to better understand the true incidence of ARI infection. Moreover, the current new coronavirus disease (COVID-19) outbreak, which was first reported at the end of 2019 [ 22 ], is a public health emergency of international concern, prompting a growing interest in respiratory tract infections.

For all three major pathogens S. aureus (n = 60), H. influenza b (n = 57) and S. pneumoniae (n = 46) highest detection number reached the highest peak in September 2018. In 2018 case enrollment started in July when bacterial pathogens reached the highest peak in September followed by October and November. However, for the next two years in 2019 and 2020, the detection number for these three bacteria was highest during December (S. aureus 59, H. influenza b 61 and S. pneumoniae 68) and remained high between Septembers and February. In 2020, cases were enrolled until the month of March when January was the highest peak for the detection of all three bacterial pathogens. All eight viruses detected in the three-year period of the study are shown according to the month of sample collection. Low numbers of virus detections were seen compared to bacteria detection. However, RSV reached its highest peak in July (n = 20) and it was roughly constant for other months. Similarly, IFV-A detection was higher in May (n = 14) than in July (n = 11), IFV-B (n = 11) in September followed by August (n = 9). For HPIV-1 it was highest in January (n = 14) and HPIV-2 only 5 were detected, whereas 3 were in January. March was the highest time for HPIV-3 (n = 10) and HMPV was detected in highest number in September (n = 7) and then July (n = 6). ADV detection was highest in January (n = 13) and it was found in similar numbers for February (n = 6), March (n = 5), September (n = 6), and October (n = 4). Overall, the months of July, August, September and January, February, and March were the periods of most virus circulation found ( Fig 10 ).

Among the bacteria detected, S. pneumoniae was the most frequently detected bacterial pathogen both in case 80% (414/512) and control groups 82% (402/488) followed by S. aureus 21% (110/512) vs 24%(120/488) and H. influenza b (15% vs 13%). Moreover, high detection rate was observed for ≤5 years of age than >5 years of age group both in cases and control group for S. pneumoniae 85% (272/320) vs 65% (142/218), p<0.05 for cases and 88% (264/299) vs 64% (138/215), p<0.05 for controL, and H. influenza b (15% vs 13% for cases and 13% vs 11% for controls) where detection was found higher in control group for S. aureus 15% vs 27%, p<0.05 for cases and 16% vs 33%, p<0.05 for controls. For viruses, the overall detection number was higher in the case group than control group for all eight viruses tested by qRT-PCR from the NPS samples. In cases, number of detection was highest for ADV (34) followed by IFV-A (32), RSV (26), IFV-B (23), and HMPV (20). Within the genotypes of HPIV, the highest number was observed in HPIV1 (14), HPIV3 (13), and HPIV2 (2). For controls, the sequence of detection numbers for the viruses was ADV >IFV > HPIV and RSV. Some viruses were found significantly higher (p<0.05) for ≤5 years age group in cases than control like RSV (21 vs 6, p<0.05), IFV-B (15 vs 5, p<0.05), HPIV-3 (11 vs 3, p<0.05) and HMPV (13 vs 0, p<0.05). Also, ≤5 years age group was detected with highest number of viruses both in cases and control group. In case group, it was observed like ADV (25 vs 9), RSV (21 vs 5,p<0.05), IFV-a (17 vs 15), INF-B (15 vs 5), HMPV (13 vs 7), HPIV-3 (11 vs 2), HPIV-1 (7 vs 7) and HPIV-2 (1 vs 1). Similarly, in control group ≤5 years age group was also found with higher detection than >5 years age such as IFV-A (15 vs 9), IFV-B (8 vs 5), ADV (9 vs 8), HMPV (7 vs 1) and RSV (6 vs 0, p<0.05). Both in case and control group RSV was found significantly higher for ≤5 years age group. Within genotypes of HPIV number of detection were HPIV-1 (7 vs 6), HPIV-2 (1 vs 0), HPIV-2 (2 vs 0). Overall, ≤5 years age group was found at high risk both for case and control than the >5 years age group ( Fig 1 ).

Discussion

To our knowledge, this is the first study conducted on a representative fragile population, i.e., FDMNs, which focused on both bacterial and viral etiology for ARI case group along with their matched control groups. This study also examined the level of involvement of each etiological agent in the onset of LARI as well as the association between respiratory viral and bacterial infections, single and co-infection frequency and their correlation pattern, distribution of S. pneumoniae serotypes, seasonality of isolated pathogens and the enrollment of LARI cases. The purpose of the control group was to examine the presence of bacteria and/or viruses in healthy participants in comparison to those with LARI cases and figure out whether there were any associations/differences etiologically. The patient enrollment data at different times of the year, pathogen identification seasonality pattern of LARI, S. pneumoniae serotypes distribution infections will help with different preventive measures, like vaccination. Sample collection from participants was carried out from June 2018 to March 2020, with modest enrollment of ARI cases between July to February, and the peak being in October. Pre-monsoon (March–May), monsoon (June–August), post-monsoon (September–November), and winter (December–February) are the four seasons of Bangladesh, where pre-monsoon, monsoon and post-monsoon seasons are basically the rainy seasons [29]. However, in our study, post-monsoon to winter covered 80% of ARI case enrollment, where as post-monsoon (October) was the highest peak. Seasonal fluctuation information might be useful in raising awareness among the general public and health professionals such as physicians and nurses. During the peak ARI season, preventive measures such as personal and in-house sanitation and cleanliness, avoiding mass gatherings, frequent hand washing as well as sufficient ventilation, may be implemented particularly in crisis settings where these factors might play crucial roles. Our study described that the majority of cases were observed between July to February corresponding to the monsoon and winter season which is a commonly known period for respiratory illness [30, 31]. Moreover, many studies have shown that winter is the peak season for respiratory infections in Bangladesh [32, 33]. However, no specific patterns of seasonal distribution were observed for viruses during the study period. Our study findings suggested that the most frequently detected viral pathogens ADV, RSV, and IFV-A had no distinguishable seasonal pattern and were detected almost throughout the year. However, ADV was detected in higher numbers from September to February and reached its peak circulation in January. Also, RSV detection remained high from July to November and highest in the month of July. For human parainfluenza virus (HPIV), detection was higher from July to November, HPIV-1 from December to January, HPIV-2 from December to January and HPIV-3 from February to March. For influenza viruses, IFV-A and IFV-B were found from April to October, which is similar to the previous study in Bangladesh [33]. Overall, during the months of April, May, and June viral pathogen detection was relatively low and circulation was higher only for influenza viruses, which circulated following no specific pattern rest of the months. The findings on viral pathogen seasonality in the monsoon and winter are similar to earlier studies in Bangladesh on respiratory viral pathogen detection [32]. All three bacteria in our study were found circulating throughout the year, particularly from September through December, with the peak months being December and January. However, in 2018 all these three bacteria S. pneumoniae, S. aureus and H. influenza b peaked during September. Throughout the post-monsoon and winter, particularly in this FDMNs crisis settings in Cox’s Bazar, Bangladesh, especially for children may need the proper preventive measures against ARI. However, because this study was for a short period, a longer study period, as well as sufficient data, are required to support the seasonality of the infections. Multiple virus detection is possible using real-time polymerase chain reaction (RT-PCR), which is more reliable and quicker than viral culture [34] and for different bacteria detection [35–37]. In our study pathogen identification using qRT-PCR enabled identification of pathogen proportion from NPS samples as well as blood specimens both in cases and control group. A total of 538 ARI cases were enrolled in this study and majority of them were from children less than 5 years of age (59%). In terms of bacteria detection from NPS samples by qRT-PCR, similar proportions were detected both in cases and control (80% vs 82%). Also, no difference was observed for sex and age in cases and control. The ≤5 years age group was found to have a significantly higher proportion of bacterial detection both in cases and control group than >5 years (p<0.05). Virus detection was significantly higher in the cases group compared to the control group (30% vs 16%, p<0.05) similar to previous studies [38, 39]. Also, both age groups ≤5 years and >5 years were found in higher proportion in cases than control group (p<0.05 and p<0.05). In case group, ≤5 years showed higher proportion than >5 years (p<0.05) whereas in control group no difference was found between age groups. In addition, virus detection was found significantly higher for both male and female groups in cases than control (p<0.05). Considering co-infection, a higher proportion was found for ≤5 years age than >5 years in cases (p<0.05) where in control group no age-specific difference was observed. Among the bacteria identified in our study S. pneumoniae was found in highest percentage both in ARI cases (80%) and control (82%) group followed by S. aureus and H. influenza b. S. pneumoniae may cause disease, but a robust immune system and a healthy balance between local flora and invaders can help to clean it out. The host is frequently and persistently colonized by S. pneumoniae due to weak defensive mechanisms, which might eventually cause illness [40, 41]. The longitudinal study by Lipsitch et al. implied that children were reservoirs because of the length of carriage and colonization [42, 43]. Althouse et al. came to the conclusion that infants play a far smaller part in transmission than toddlers and older kids do, despite the fact that infants account for a higher percentage of carriage [43]. However, these findings indicate that more study is necessary to properly understand the direction of transmission. Moreover, in our study, of bacterial detection, no significant differences were observed between cases and control which is corroborated with previous study [44]. Age-specific distribution suggested that in both age groups (≤5 years and >5 years) S. pneumoniae was found as most predominant among the three bacteria both in cases and control group. However, among 3 major bacteria S. pneumoniae was seen as most predominant for ≤5 years of age group than >5 years (85% vs 65%, p<0.05) in cases where H. influenza b were detected in a similar proportion with no significant difference. Where S. aureus was found in higher proportion for >5 years age both in case and control. Similarly, the PERCH multi-country case–control study showed that these same respiratory viruses and bacteria were frequently linked to ARI. Furthermore, ADV and influenza viruses were found more frequently in controls in our study who had no history of ARIs in the 14 days prior to recruitment and had no respiratory symptoms seven days after recruitment, according to participant or family reports. The exception was for S. aureus where it was detected in higher proportion in >5 years of age group both for cases and control which may suggest the opportunistic infection for older people.

Moreover, no gender differences were seen in pathogen detection in cases or in control group suggesting that gender has no impact in the case of ARI infection. Similar results have been found in a previous study [45]. Viruses attributable to ARIs in our study were ADV in 6% of cases, influenza virus (IFV-A 6% and IFV-B 4%), RSV in 5% and HMPV in 4% of cases. These findings are similar to a recent study [44]. These findings are consistent with previous research, such as Shi et al who found that RSV, influenza virus and HMPV were all substantially linked to ARI [46]. Age distribution data showed that ADV is significantly higher among ≤5 years of age both in cases and controls than the other age group. RSV was found in a similar pattern where no significant differences were observed for other viruses in age distribution. So, age stratification revealed the highest-risk group where ≤5 years age group was found in a consistent pattern in case and control both for bacterial and viral infection, in line with previous studies for this age group [11]. Many types of organisms naturally colonize the nasopharynx. The majority of research focuses on bacteria, where sometimes the impact of viruses in the makeup of the respiratory microbiota is likely to be overlooked [47]. S. pneumonia, H. influenza b and S. aureus which was found in this study to be associated with controls where S. pneumonia, S. aureus were found in higher numbers in control and these are well-known contributors to acute respiratory infection. The fact that some of the microbes were identified more frequently in control does not rule out their involvement with ARI in some cases. Our study findings were similar to the PERCH study, where S. pneumoniae was detected significantly more frequently in controls than in patients. However, S. pneumoniae and H. influenzae b were strongly associated with lower respiratory tract infection [48]. However, a very low rate of blood culture positivity was found in our study, which is also similar to previous studies [49–51] and this finding may suggest bacterial pathogen identified from blood culture was only 5% hence indicating the true cause of infection rather than the carriage in the nasopharynx. However, antibiotic resistance was present for almost all the isolated pathogens from blood culture for different antibiotics (amoxicillin, ampicillin, chloramphenicol, erythromycin, oxytetracycline, ciprofloxacin, levofloxacin, doxycycline) where penicillin-G and cotrimoxazole were most predominant. S. pneumoniae was found resistant to penicillin-G, cotrimoxazole, and gentamycin. With the increased use of antibiotics, S. pneumoniae transforms and evolves, acquiring several genes for antibiotic resistance. Pneumococcus strains that are currently resistant to penicillin have spread throughout the world, and they are also resistant to other classes of antibiotics: tetracycline, chloramphenicol, and erythromycin corroborated with our study findings [52, 53]. In addition, in our study, qRT-PCR method was found more sensitive for bacterial detection (16%) including three bacteria (S. pneumonia, S. aureus, H. influenza b) from blood specimen following DNA extraction which may indicate the importance of RT-PCR method in terms of ARI pathogen detection, where a previous study also suggested the use of this technique [35, 37]. However, it is important to see how the presence of bacteria affects the co-infection, which may complicate the clinical symptoms and their management. We also analyzed co-infection data in terms of co-occurrences of at least two or more pathogens, where S. pneumoniae was found both in single and co-infection in a similar proportion, where co-infection was higher than single infection for S. aureus and H. influenza b were found both in cases and control group (p<0.05). For viruses, co-infection was higher for RSV, IFV-B, HPIV-3, ADV, and HMPV than single infection both in cases and control group. Where single infection was higher for the rest of the viruses. Moreover, the co-infection pattern is very important and can play an important role in causing disease and can alter the etiology. Our study suggested that the co-infection pattern is more common between bacteria and bacteria or viruses and bacteria but negligible numbers were found between viruses, as also found in a similar previous study [6, 54]. More specific analysis for correlation pairing between pathogens in case group showed the bacterial-viral synergistic effects were mainly from S. pneumoniae with other bacteria and viruses and the co-infection number was higher in ≤5 years of age. In our study, among the ≤5 years group highest co-infection number was found mainly from S. pneumoniae with H. influenza b followed by S. aureu s, RSV, IFVs, ADV. RSV was found with other bacterial pathogens in the highest number followed by ADV, IFVs, HMPV and HPIV-2. Number of co-infections was less in >5 years group than the other age group where all the viruses interact with S. pneumoniae to a similar extent except for HPIV-2 and HPIV-3 and correlation findings corroborated with the previous study. In the case group, the correlation pattern (positive and/or negative) was observed for bacteria and viruses for both age groups, whereas in the control group, no such correlation was found. In the case group, the correlation pattern of co-infection in ≤5 years age was different from >5 years age group. In ≤5 years of age, most predominant co-infection was between RSV and S. aureus, RSV and H. influenza b, RSV, and ADV where the correlation was positively correlated and a negative correlation was observed for RSV and IFV-A. Similarly, a previous study showed when RSV infection rates are high, influenza infection rates are low due to viral competition, and vice versa [55]. In >5 years age, the numbers of co-infection between bacteria and viruses were higher than ≤5 years group. Positive interactions were between influenza viruses and S. aureus. Also, S. pneumoniae was positively correlated with ADV, HMPV and HPIV viruses. However, no positive correlation was observed between bacteria where they were negatively correlated, suggesting that S. pneumoniae less likely to coexist with other bacteria rather than viruses. Also, some previous studies suggested S. pneumoniae colonization may enhance during viral co-infection and increased colonization during viral infection may induce the transmission [56, 57]. Moreover, very few numbers of co-infection were detected between viruses (RSV and HMPV) where a higher number of bacterial-viral infections may correlate with the previous findings and their proposed mechanisms where a wide range of pathogens interact with viruses through resource competition, immune response or interaction between viral protein, etc. [58, 59]. Overall, co-infection pattern and their number may suggest their interaction and that they can facilitate secondary infection for viruses or bacteria and this phenomenon is common in respiratory infection [60]. Our current findings may suggest, either bacterial or viral infection in combination with either pathogen can trigger secondary optimistic infection, which perhaps suggests future research. Moreover, there is growing evidence suggesting an association between bacterial colonization and future ARI occurrence [61], which is related to our bacterial interaction findings. In addition, viral infection may enhance bacterial superinfection by promoting bacterial attachment sites on nasopharyngeal epithelial cells and increased mucous production may further enhance bacterial growth [62]. Also, sub-clinical infection in asymptomatic cases may act as a source of pathogen transmission [44]. Furthermore, a logistic regression analysis showed that case groups were more likely to be positive for RSV four times, HPIV-3 four times, HMPV twenty times and IFV-B two times more than the control group. Moreover, ≤5 years group was likely to be positive for RSV, HMPV, and IFV-B in a higher percentage than the other age groups. Prior investigations of bacterial and viral loads in the upper respiratory tracts of young Congolese children suggested the importance of the load pathogens [63] and to our best knowledge there is no study conducted among the FDMNs focusing on the viral and bacterial load in ARI cases and healthy individuals. Cycle threshold (Ct) values are continuous, semi-quantitative assessments of viral load used in qRT-PCR [63, 64]. However, our study evaluated the bacterial and viral prevalence according to Ct value detected by RT-PCR where the detection number was higher for all the bacteria considering Ct≤30 in the case group than the control group. However, the percentage was higher in control group when a lower load (Ct>30) was considered. In addition, viral load (Ct≤30 and Ct>30) was found always high in case group for both the age (≤5 years and >5 years) than the control group for all the viruses except for ADV and HMPV-1 corroborated with previous study [65]. However, understanding the complicated interactions between viruses, bacteria, and viral–bacterial interactions could contribute to the understanding of respiratory pathogen epidemiology and planning public health strategies. S. pneumoniae is the leading cause of child mortality especially for younger children of ≤5 years despite the vaccination program and it is responsible for 33% death worldwide [66]. Some of the discrepancies were most likely due to methodological differences, whereas others were most likely due to actual demographic disparities. Bangladesh and different NGOs introduced PCV-10 vaccination among the FDMNs and current PCV-13 replaced previous PCV-7 and PCV-10 which is protective against 13 serotypes [53, 67]. In our study, we identified 40 serotypes from NPS using RT-PCR where few serotypes/serogroups were found in higher proportions 6AB, 14, 23F, Sg18, and 5. Serotypes/serogroup 6AB (12% and 13%) and 23F (10% and 9%) were found in higher proportion under PCV-10 in cases and control. However, serotypes 16F, 35B, 15A, 20, and 10F were predominant in case group whereas 11A, 10A, 34, 35A, and 13 were predominant in control group in higher proportions. Among the nonPCV-10 serotypes/serogroups higher proportion (>5%) were found for 19A, 11A, 16F, 35B, 15B/C, 15A, 20, 10F both for the case and control groups. Even if the 10- or 13-valent vaccines now being developed are only effective against half of the strains that cause invasive pneumococcal illness, their introduction into the huge number of FDMNs population might be beneficial. However, there are a number of strains that aren’t covered by the current vaccine candidates. Here, several co-infection patterns and their positive/negative interaction between 41 serotypes and all other pathogens tested might help in vaccination strategy planning in the future. Correlations between viruses and serotypes were observed in higher numbers than the bacterial interactions with serotypes which are predominant in cases over the control group. Overall, this study suggests that S. pneumoniae is the most predominant bacteria followed by S. aureus and H. influenza b in ARI patients. Among the viruses, RSV was the highest detected followed by ADV, IFVs and HMPV, and their seasonal pattern was mostly during rainy monsoon and cold dry winter seasons in Bangladesh. Age group distribution showed ≤5 years age was found as the highest risk group. Moreover, single and co-infection were found similar for S. pneumoniae where the co-infection number is significantly higher for S. aureus and H. influenza b. For viruses, RSV, HMPV, IFV-B and HPIV-3 were found in high proportions for co-infection than single infection, both in cases and control group. Also, the co-infection pattern showed the predominant interaction occurred between bacteria and viruses and very limited extent for virus and virus. Besides, high viral and bacterial loads were present in the ARI case group compared with the control for most of the pathogens.

However, this study has several limitations, like it was not possible for us to collect sputum samples since most of the cases were children. Also, in the context of this study, X-rays were not available and decisional tree for disease presentation was established based on clinical symptoms and examination findings only as our study was in such humanitarian crisis settings. Also, only very severe cases were hospitalized as per suggestion by the physician or referred to the tertiary hospitals. Our study did not follow up with the participants for tertiary hospitalization. Moreover, our study period was not long enough to determine the actual seasonality of ARI pathogens. Besides, we were unable to differentiate the S. pneumoniae serotypes like 6A and 6B, 15B and 15C, 9N and 9L or detect serogroups 18 and 24 because of the limitations of the RT-PCR method we used. Despite this limitation, we observed ARI etiology related to age, gender, single and co-infection pathogen spectra, S. pneumoniae serotype distribution pattern, which may help in public health strategies like identifying the predominant respiratory pathogen, identifying the highest risk age group, and vaccine administration in such humanitarian settings.

Importantly, the prevalence of viral infections data may play a crucial role in preventing the unnecessary use of antibiotics. Also, S. pneumoniae serotype distribution data may also be useful in determining disease-causing serotypes and future vaccine development.

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