Pathogens in the upper respiratory tract of Congolese children with radiologically confirmed pneumonia

Acute pneumonia remains a leading cause of death among children below 5 years of age in the Democratic Republic of the Congo (DR Congo), despite introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in 2013. Pathogens in the nasopharynx of hospitalised children with pneumonia have not been studied previously in DR Congo. Here we compare clinical characteristics, risk factors and nasopharyngeal occurrence of bacteria and viruses between children with severe and non-severe pneumonia Methods or Fisher’s exact test (n < 5). p < 0.2 were re-analysed by multivariate analysis. A p- value of < 0.05 considered statistically significant. Mann-Whitney U-test used for comparison of medians.


Introduction
Acute pneumonia remains a leading cause of childhood morbidity and mortality worldwide [1] although the estimated number of pneumonia episodes in young children decreased from 180 million in 2000 to 140 million in 2015 [2]. Pneumonia caused deaths in 0.9 million children below five years worldwide in 2015 [3] and is a leading cause of death in young children in the Democratic Republic of the Congo (DR Congo) [2]. The country has the fourth highest absolute number of pneumonia-related deaths worldwide and the second highest in Africa for children under 5 years of age [2]. The incidence of clinical childhood pneumonia decreased in the DR Congo from over 400 cases per 1000 children and year in 2000 to less than 300 in 2015 [2]. However, acute lower respiratory infections (ALRI) caused 20% of deaths among children aged between 1 and 59 months in 2017, with a death rate of 9.4 per 1,000 live births [4].
Approximately 60% of Congolese children with acute pneumonia are treated by inappropriate care providers, such as traditional practitioners, vendors of medicines, relatives or friends [5] and up to 40% do not have access to the correct antibiotic treatment [6]. Moreover, abuse of antibiotics is abundant due to self-medication or consulting a non-appropriate health care provider, as well as overprescription by clinicians [7,8].
The burden of bacterial pneumonia has been reduced significantly in children worldwide after the introduction of both conjugate Haemophilus influenzae type B and pneumococcal conjugate vaccines (PCVs) into the routine childhood immunisation programs [9]. However, Streptococcus pneumoniae (the pneumococcus) still remains an important cause of pneumonia in many settings [1,10]. The virulence of S. pneumoniae is largely due to its polysaccharide capsule which protects it from the host immune system and is the basis for epidemiological categorization of the pneumococci into almost 100 different serotypes [11,12]. In the Eastern part of DR Congo the 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in 2013.
Nowadays, with the more frequent use of molecular diagnostics, respiratory viruses are increasingly detected among children with acute respiratory disease [1,13]. Respiratory syncytial virus (RSV) has been reported as the most common virus in children below one year of age hospitalized with ALRI [14][15][16]; however, other respiratory viruses, such as influenza virus and enterovirus are frequently detected as well [1,14,16,17]. However, discerning the etiology of childhood pneumonia is complex because respiratory pathogens, including a wide range of bacteria and viruses, are frequently found in healthy children [14,16].
Increased knowledge surrounding the clinical presentation and pathogens present in children with acute pneumonia may improve future clinical management and prevention of the infection, not least in low-income settings such as DR Congo which not only has a high disease burden [2] but also high rates of antibiotic resistance in the pneumococci colonizing young children [18]. In DR Congo the antibiotic treatments available are entirely empiric due to insufficient microbiological diagnostics and, to our knowledge no studies exist which have assessed the burden of respiratory pathogens in children hospitalized due to acute respiratory infection. Our objective was to describe clinical characteristics and risk factors and to determine the occurrence of bacteria and viruses in the nasopharynx of hospitalised children with pneumonia in the Eastern part of DR Congo. We also aimed to relate these findings to the severity of the disease and outcomes.

Study site
The study was conducted at Panzi Hospital, a referral university hospital in Bukavu town in the South Kivu region of DR Congo, which served a population of 47,000 inhabitants including 89,000 children below five years in 2017. Panzi Hospital has 69 paediatric beds with an emergency ward which has 12 beds for acute and severe cases requiring nasal oxygen therapy. The department has two trained paediatricians and four resident doctors. The hospital has a radiology department run by a trained senior specialist in radiology.

Study patients
Out of a total of 2,322 children between 2 months and 5 years of age treated for any disease at the Emergency and admissions Unit, Paediatric Department, between June 2015 and June 2017, 184 were diagnosed with acute lower respiratory infections. Of these, 116 cases had pneumonia that was radiologically confirmed and these children were thus included in the study. Children admitted for severe malaria with pneumonia as complication were not included due to difficulties differentiating infection from pulmonary oedema. The parents or guardians of five HIV-positive children declined participation in the study and were thus not included while another three HIV-positive children were included. Four patients referred from health centers to Panzi hospital had X-ray confirmed pneumonia but were not treated at the hospital because of the cost of care that was regarded as too high and were therefore not included. The direct cost of five days' treatment for pneumonia at Panzi hospital could amount to 150 US dollars.
Data were collected on age, sex, dates of admission, duration of hospital care, as well as underlying conditions, including malnutrition, HIV, sickle cell disease, cerebral palsy, post-neonatal anoxia and congenital diseases. The WHO child growth standards were used to evaluate the nutritional status of children by computing the Z-scores of weight-for-age and height-for-age [19].
Patients were classified according to the revised WHO classification for pneumonia. This encompasses non-severe pneumonia with fast breathing and/or chest indrawings. Rapid breathing was present when the respiratory rate was ≥ 50 breaths per minute for infants between 2 and 12 months of age and ≥ 40 breaths per minute for children between 12 months and five years of age [19]. Children were considered to have severe pneumonia when there were any additional danger signs (e.g. not able to drink or breastfeed, persistent vomiting, convulsions, lethargy or loss of consciousness, stridor in a calm child or severe malnutrition) [20]. Data on pre-hospitalisation use of antibiotics were obtained from the parent or guardian, or from the referral note from any first-level health care facility. The use of antibiotics and nasal oxygen therapy during hospitalisation was also recorded.
Patient outcomes were grouped into the following three categories: survived without complications, survived with complications (e.g. pneumothorax, empyema, or pleural effusion) and death. The group surviving without complications included patients discharged with improvements and who had switched to oral antibiotic treatment. This group included those discharged against medical advice but who were receiving oral antibiotic treatment. Nasopharyngeal specimen collection A nasopharyngeal specimen was obtained from all included children using an Eswab (Copan Diagnostics Inc., Murrieta, CA), following a standardised procedure as previously described [18]. The samples were shipped immediately to the Clinical Laboratory at Panzi Hospital for subsequent pneumococcal culture as described earlier [18]. The nasopharyngeal samples were thereafter stored frozen at -20 °C before shipment to the Department of Infectious Diseases, University of Gothenburg, Gothenburg, Sweden, for further molecular analyses (see below).

Data management and statistical analysis
Descriptive analysis was performed using the SPSS package (version 24.0) for logistic regression analysis of the relationship between nucleic acid identification and symptoms, medical conditions or outcome. Prevalence rates and the 95% CI were calculated. Potential variables associated with identified bacteria or viruses were assessed by odds ratios (OR) with 95% CI and were tested by univariate analysis with the Pearson's chi-squared or Fisher's exact test (n < 5). Associations with p < 0.2 were re-analysed by multivariate analysis. A p-value of < 0.05 was considered statistically significant. The Mann-Whitney U-test was used for comparison of medians.

Characteristics of the included children
Two years after the introduction of PCV13 in the infant immunization program in the eastern DR Congo, 125/2,322 (5.4%) of all children admitted to Panzi hospital, DR Congo were diagnosed with radiologically confirmed pneumonia. Of these children, 116 were included in the present study (median age 12.5 months). Ninety-five (82%) of the included children came from Bukavu town while 21 came from rural areas (Table 1).  (1) 0--1Bukavu urban area (n = 95): Ibanda area, n = 80; Kadutu area, n = 13; and Bagira, n = 2 2Undernutrition defined as weight for age or weight for height as a Z score ≤ -2 standard deviations, determined by ENA for smart software 2011 3Other diseases (Congenital and others): sickle cell disease (n = 2), congenital cardiac disorder (n = 1), cerebral palsy (n = 2), Down syndrome (n = 1), and HIV (n = 3) 4PCV13 = the 13-valent conjugate pneumococcal vaccine 5Gastro-intestinal symptoms: diarrhoea or vomiting 6The normal range of White cells count is 4,000-10,000 cells/µL 7 (The normal value for C-Reactive Protein was < 10 mg/dL 7Fast breathing: Respiratory rate ≥ 50 breaths per minute for infants of 2 to 12 months of age and ≥ 40 breaths per minute for children between 12 months to five years of age 8Other antibiotics (pre-hospitalisation antibiotic treatment): erythromycin (n = 5), ciprofloxacin (n = 3) or cloxacillin (n = 2) 9Others (antibiotic treatment during hospital stay): ciprofloxacin (n = 3) and cloxacillin (n = 5) Almost all the children 101 (87%) were treated with antibiotics before hospital admission, according to the parent or guardian (n = 39) or the referral notes (n = 62). The most common pre-hospitalisation antibiotics were either amoxicillin/ampicillin/penicillin or trimethoprim-sulfamethoxazole (Table 1).
Two thirds (67%) of the children had received two or three doses of PCV13. The main signs and symptoms noted during the physical examination at admission were fever or recent history of fever (96%), abnormal lung auscultation (99%), cough or recent history of cough (100%), and rapid or difficult breathing (94%). Malnutrition was present in 11 (9.5%) of the patients. During hospital stay 53% were treated with ceftriaxone and gentamicin whilst 40% received ampicillin and gentamicin (Table 1).

Differences in clinical characteristics between children with severe and nonsevere pneumonia
According to the WHO pneumonia classification, 85/116 (73%) of the children met the criteria for having severe pneumonia at admission whereas 31 (27%) had non-severe pneumonia. Between the two groups, there was no statistically significant difference in sex, age, living area or immunisation status, signs or symptoms including fever, cough or abnormal auscultation (data not shown).
However, a white cell count of over 20,000/µL was significantly associated with severe pneumonia (OR 4.75; 95%CI 1.0-21.6, p = 0.043) ( Table 1). CRP levels above 75 mg/dL were also significantly associated with severe pneumonia at admission (OR 38.9; 95%CI 5.1-299, p = 0.0004) while CRP levels below 25 mg/dL were negatively associated with severe disease (OR 0.19; 95% CI 0.08-0.48, p = 0.0003) ( Table 1). As expected, nasal oxygen therapy was more often used in children with severe pneumonia as compared to children with less severe disease (Table 1). Overall, pre-hospitalisation antibiotic treatment was more common among children with severe pneumonia than in children with non-severe disease. While penicillin and the penicillin derivatives amoxicillin or ampicillin were equally common in the two groups, trimethoprim-sulfamethoxazole was more commonly used in children with severe pneumonia (OR 4.75; 95%CI 1.04-21.65, p = 0.043) ( Table 1). During hospital stay, ceftriaxone combined with gentamicin was more frequently used to treat the severe pneumonia cases than the children with non-severe disease (Table 1).

Pathogens found in the nasopharyngeal secretions
From only one nasopharyngeal sample pneumococci could be isolated by culture. However, by realtime PCR, performed directly on the nasopharyngeal sample, pneumococci could be detected in almost all children (96%), whereas H. influenzae was detected in 54% and B. pertussis in 10% (Table 2). When employing a more stringent cut-off level (Ct < 30), bacteria were found in 62% of the samples; S. pneumoniae in 53% of the cases, and H. influenzae in 20% (Table 2). The most frequently detected virus was rhinovirus (73%), followed by enterovirus (17%), while RSV was found in only 7% of the cases and influenza virus was rare ( serotypes were 15BC, 10A and 11A (Fig. 1). Two samples were found to be negative for the CpsA pneumococcal capsule gene but were strongly positive for the LytA gene (CT < 30), indicating nonencapsulated non-typeable pneumococci.

Fatal outcome
The overall case fatality rate among the hospitalized children with radiological confirmed pneumonia was 9.5% (11 children (Table 3). Treatment at the hospital, including nasal oxygen therapy or antibiotic regimens, did not differ between the children who died, compared to children who recovered. Fatal outcome was, however, associated with high nucleic acid levels (Ct < 30) of pneumococci or RSV in the upper respiratory tract of the children (Table 3). The two RSV-positive cases with fatal outcome were also positive for pneumococci. Two different pneumococcal serotypes could be determined in the nasopharyngeal secretions from the children with fatal outcome; serotype 19F in four cases and 15BC in one case. In the remaining five children with pneumococci detected, no serotype could be identified. 1Nasal oxygen treatment = 0.5-2L/minutes 2Congenital diseases = sickle cell disease (n = 2), congenital cardiac disorder (n = 1), cerebral palsy (n = 2), Down syndrome (n = 1) and HIV (n = 3) 3Malnutrition defined as weight for age or weight for height as a Z score ≤ -2 standard deviations, determined by ENA for smart software 2011

Discussion
This is the first study on hospitalised children with radiologically confirmed pneumonia in DR Congo in the PCV13 post-vaccine era. It includes both clinical and microbiological aspects. A majority (73%) of the children were diagnosed as having severe pneumonia. The most common symptoms for both severe and non-severe pneumonia cases were fever, cough or abnormal auscultation, similar to that described in a Vietnamese [24] and a Tanzanian study [25]. High white cell counts (> 20,000 cells/µL) and high CRP levels (> 75 mg/dL) were associated with severe pneumonia at admission. Similar findings of elevated white cell counts in hospitalised children with pneumonia were reported from Senegal [26], as well as high CRP levels (> 80 mg/dL) also being associated with radiologically confirmed pneumonia in Tanzanian children [16].
The case fatality rate in our study was 9.5%, similar to findings from a Tanzanian district hospital (11%) [25] but higher than those reported from hospitalized children in Cambodia (3.2%) [27]. Up to 80% of deaths in children with severe respiratory infections may occur outside hospitals in lowincome countries [28]. It is possible that some children first receive self-medication by parents or guardians, first seeking care at a private pharmacy, traditional practitioner [29] or at any other nonappropriate health care provider before attending professional health care facilities [5, 28,30]. This is supported by the fact that less than half (42%) of under-five children with suspected pneumonia in DR Congo were found to have been treated by a trained health care provider [8].
Eighty-seven percent of the children included in our study received pre-hospitalisation antibiotic treatment, in most cases amoxicillin, ampicillin or penicillin (59%) followed by trimethoprimsulfamethoxazole (20%). In 34% of the cases, information about pre-hospitalisation medication was found that pre-hospitalisation medication by the peroral broad spectrum antibiotic trimethoprimsulfamethoxazole was more common in children with severe pneumonia than in children with nonsevere disease, and pre-hospitalisation treatment with trimethoprim-sulfamethoxazole was also associated with fatal outcome.
Recently the DR Congo introduced clinical guidelines for the management of pneumonia. However, the antibiotic management of severe pneumonia was not included. For optimizing antibiotic treatment regimens antimicrobial resistance testing and surveillance are needed, especially considering the high level of resistance to commonly used antibiotics in the country [18]. In the present study, ceftriaxone and gentamicin were the main antibiotics used for treating severe pneumonia. This corroborates the findings of a study in Senegal where more than 55% of pneumonia cases were treated with ceftriaxone [26,38].
The low isolation rate of live pneumococcus (0.9%) by culture in the present study may be explained by the high frequency of pre-hospitalisation antibiotic treatment. The pneumococcal isolation rate was higher (21%) among the 794 healthy Congolese children included in our previous study; Also, 7% of these children had been treated with antibiotics in the last month according to their parents or guardians [18]. If carried out before specimen collection, this is known to reduce the culture isolation rate of bacteria but has less impact on the detection rate by PCR [36,39]. Here we detected pneumococci by PCR in almost all (96%) nasopharyngeal secretions and H. influenzae in 54%. This result corroborates findings from Zanzibar, Tanzania, in which pneumococci were found in 87% and H.
influenzae in 77% of febrile children [40]. When employing a more stringent cut-off level, (i.e. Ctvalue < 30) in which only high amounts of microbial nucleic acids are identified, we found S. pneumoniae in 53%, and H. influenzae in 20% of the cases. We could not show any significant differences between the age groups and presence of pathogens as reported in other studies [41,42] nor an association between severity of disease and specific viruses or bacterial species as identified in the study. However, high levels of bacterial or viral nucleic acids in the nasopharynx (irrespective of the species or types) were associated with more severe pneumonia at admission, compared to less severe cases. This supports the importance of not only bacteria but also viruses in the development of severe pneumonia.
Surprisingly, our detection rate of 7% RSV was much lower than the 31% reported in a multicentre study in Africa and Asia [14]. This discrepancy might be explained by the geographic variability of pathogens [14]. However, although only a few cases were detected, RSV was more prevalent in children that died, also having high nucleic acid levels of pneumococci. In the two fatal RSV-positive cases, pneumococci were also detected, supporting the evidence of RSV being associated with pneumococci in critical cases.
The frequent co-occurrence of viruses and bacteria in childhood pneumonia [14] has been associated with disease severity [10,14]. Infection with RSV facilitates colonisation with bacteria such as S.
pneumoniae and H. influenzae in the nasopharynx of young children [43]. In addition, invasive pneumococcal infection increases during the seasonal peak of various respiratory viruses, including influenza virus and RSV [14].
Since we did not include a hospital based control group, and most of these pathogens are also prevalent in healthy children, it was not possible to discern the etiology of the pneumonia cases in the present study, not even when only microbial nucleic acids in large amounts were considered [16,40,44,45].
PCV13 serotypes/serogroups were more commonly identified than non-PCV13 serotypes/groups (63% versus 37%). This was in contrast to a study performed in Mozambique, in which half of the identified serotypes were included in the vaccine [46]. However, we may have under-estimated the frequency of non-PCV13 serotypes. This is because our method could neither distinguish between the 6ABCD serotypes, (of which only 6A and 6B are in PCV13), or between 9A and 9V (of which only 9V is in PCV13). Moreover, the assay measures all the serotypes included in the pneumococcal vaccines but few of the additional ones. This suggests that, because those 50 samples were positive for pneumococci according to our PCR assay, but were negative for the serotypes included in the detection panel, they may have contained non-vaccine serotype pneumococci. Thus, non-vaccine serotypes were most likely much more prevalent than we detected here. As seen in other studies nonvaccine serotypes are continually emerging after the introduction of PCV13 [47,48]. Here, serotype 19F was the most frequent PCV13 serotype, whereas serotypes 15BC, 11A and 10A were the most prevalent non-PCV13 serotypes. This was similar to our findings among the healthy Congolese children [18]. In this way, similar information about serotype distribution was obtained from healthy children as from children with pneumonia requiring hospital stay in the same area. However, the previous study was based on cultured pneumococcal isolates rather than direct detection in the nasopharyngeal sample as was done here [18].

Conclusions
We found an association between pre-hospitalisation use of trimethoprim-sulfamethoxazole and having severe pneumonia at admission, as well as fatal outcome in some of the children. Any high bacterial or viral nucleic acid levels were more often detected in children having severe pneumonia than in those with non-severe disease. RSV and influenza were rarely detected although RSV was associated with fatality, as were pneumococci at high levels. Out of all identified pneumococcal serotypes/serogroups 37% were not in PCV13.

Declarations
Ethics approval and consent to participate Informed oral and written consent was obtained from the accompanying parent or guardian of each child included in the study.

Consent for publication
Not applicable.

Availability of data and material
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.