Abstract

Background: Infections of sterile body fluids are a global health problem, resulting in the deaths of an estimated 600,000 to 800,000 adults each year, according to World Health Organization estimates. The aim of this research was to present the microbiological, susceptibility and antimicrobial resistance profile of germs isolated from sterile body fluids during the study period. Methods: The study took place in the central region at the Centre Pasteur of Cameroon (CPC) from January 2010 to December 2019. Sterile body fluids (joint fluid, ascitic fluid, drain fluid, gastric fluid, hepatic fluid, cerebrospinal fluid, pericardial fluid, peritoneal fluid, pleural fluid and synovial fluid) were obtained by clinical specialists and samples were transferred to the CPC laboratory for analysis. India Ink and soluble antigen tests were carried out on cerebrospinal fluid (CSF) samples for Cryptococcus and Sabouraud chloramphenicol (Actidione-free) medium was used for culture. The search for other germs species was carried out on the appropriate culture media and incubations were carried out at 37°C under CO₂ between 18 and 24 hours. Other complementary tests, particularly biochemical tests, were carried out on the pure strains isolated. The Kirby-Bauer diffusion method was used to determine antimicrobial susceptibility, as well as the Vitek-2 Compact automated system. Results: From January 2010 to December 2019 a total of 2313 samples were analysed. The culture was positive for 1229 samples (53.1%). Men were more represented (55.0%) than women (41.1%). The average age of the infected participants was 33.4 ± 21.6 years with the <21 years group and the 41-60 years group being the most represented. Sample distributions by sex and age groups were significant (p<0.0001). The most represented bacterial and fungal species (>1%) were: Cryptococcus neoformans (13.8%) ; Streptococcus pneumoniae (6.8%) ; Staphylococcus aureus (4.8%); Escherichia coli (3.4%) ; Klebsiella pneumoniae (2.7%) ; Streptococcus sp. (2.1%) ; Haemophilus influenzae (1.5%) ; Staphylococcus sp. (1.4%) ; Streptococcus agalactiae (1.4%) and Pseudomonas aeruginosa (1.1%). The Pearson's Chi-squared test showed that a statistically significant association existed between the identified germs with sex (p<0.002) and age (p<0.0001). In addition, gender was a risk factor for CSF infection caused by Cryptococcus neoformans (OR: 2.07, 95% CI: 1.57-2.73, p<0.0001) with women having twice the risk of CSF infection compared to men. Antimicrobial susceptibility testing showed that resistance rates were higher for antibiotics in the penicillin, cephalosporin and sulfonamide families and that in terms of sensitivity, penicillins, cephalosporins, aminoglycosides, polypeptides, quinolones and oxazolidinones appeared to be effective against some of the bacterial species identified. As for fungi, Cryptococcus neoformans, Candida albicans and Candida sp. were sensitive to most of the antifungal agents tested. Conclusion: Infections of sterile body fluids are a health problem in both developed and developing countries. The alarming antimicrobial resistance results of the germs isolated in this research point to the need for increased surveillance investigations across the board to prevent neuromeningitis in our context.

1. Introduction

Infections of sterile body fluids are an urgent diagnostic and management emergency because they can be fatal if left untreated ¹. Among these infections, neuromeningeal cryptococcosis, pneumococcal meningitis and sepsis are responsible for 600,000 adult deaths every year according to World Health Organization (WHO) estimates ². Various types of microorganisms including bacteria, fungi, viruses and parasites are involved in sterile body fluids infections and are the major cause of mortality and morbidity worldwide ¹. The species Streptococcus pneumoniae (S. pneumoniae), Haemophilus influenzae type b (Hib) and Neisseria meningitidis (N. meningitidis) are the most common causes of bacterial meningitis worldwide ³ ; acute infections in which the meninges, subarachnoid space and brain parenchyma are all frequently subjected to inflammatory processes ⁴. In the youngest age group, i.e. newborns, it is estimated that about 3.6 million deaths were recorded worldwide in the context of neonatal meningitis in 2008, and that a third of these deaths were in Africa ⁵. In addition to the bacterial species mentioned above, other studies, notably that of Moosavian et al. ⁶, have shown that Acinetobacter baumannii (A. baumannii) is also becoming an increasingly important pathogen in hospital-acquired infections, including bloodstream and wound infections, pneumonia and meningitis. They also showed that the prevalence of A. baumannii in nosocomial meningitis was 4% with a predominance of infection in neurosurgical patients with cerebrospinal fluid (CSF) leakage ⁶. In addition to bacterial meningitis, fungal meningitis are also widespread worldwide with the encapsulated yeast of the genus Cryptococcus, of which Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii) are the main pathogenic species involved in cryptococcal meningitis ⁷. C. neoformans with its two serotypes C. neoformans var. grubii (serotype A), cosmopolitan and C. neoformans var. neoformans (serotype D), the most common in Europe is an encapsulated ubiquitous fungus that is mainly found in immunocompromised patients: carriers of the human immunodeficiency virus (HIV), suffering from a malignant hemopathy or under immunosuppressive therapy [7-10] ⁷. Since the 1980s, C. neoformans infections in immunocompromised individuals, especially those with HIV, have become a public health problem, and estimates show that HIV-associated cryptococcal meningitis is responsible for about 150,000 deaths per year, with a prevalence of 15% in those with Acquired Immunodeficiency Syndrome (AIDs) ¹¹. Other sterile body fluid infections are also a global health problem, including pleural effusions and empyema, which are primary manifestations of intrathoracic disease ^{12, 13}. Pleural effusion of a transudative and exudative nature is a manifestation of inflammation of the pleura characterized by increased vessel permeability and accumulation of fluid in the pleura ^{12, 14}. This pathology is a common health problem affecting about 60,000 patients in the United States (USA) and the United Kingdom (UK) ¹⁴. Empyema is a common complication of pneumonia, but can occur from infections at other biological sites ¹². Peritonitis, inflammation of the peritoneum, is also one of the major causes of treatment failure in patients who have undergone peritoneal fluid dialysis, with approximately 200,000 patients worldwide who are treated with peritoneal dialysis (PD) and who represent 11% of the global dialysis population ¹⁵. This can lead to the end of the dialysis procedure in 20% of cases with a mortality rate between 2˗6% ¹⁵.

Antimicrobial resistance of germs involved in infections of sterile body fluids is a global health problem and recent studies have, for example, shown geographical differences in susceptibility of C. neoformans to antifungal agents. Resistance to antifungal agents used in the treatment of cryptococcosis remains uncommon in isolates obtained from regions around the world, but resistance to fluconazole remains relatively high in some countries such as Spain and Cambodia ⁸. Information reported by other researchers, notably by Bongomin et al. ¹⁶ and Nishikawa et al. ¹¹, indicates that fluconazole inhibits fungal ergosterol biosynthesis and has fungicidal activity against Cryptococcus spp. when administered at a dose above 800 mg/d and that the combination of amphotericin B with flucytosine for induction followed by fluconazole for consolidation and suppression therapy is recommended as an antifungal regimen for the management of cryptococcal meningitis (CM). However, fluconazole-resistant Cryptococcus isolates increase in situations of therapeutic failure, especially in patients with AIDs, with the long-term use of fluconazole as suppressive monotherapy, the low bioavailability of the drugs in infected tissues, the fungistatic action of azoles and the intrinsic resistance i.e., heteroresistance observed in C. neoformans and C. gattii to azoles ¹¹. Apart from Cryptococcus spp., resistance of other species involved in sterile body fluids infections is also a problem with S. pneumoniae, which is increasingly resistant to antimicrobials such as penicillins, cephalosporins and macrolides ¹⁷. Klebsiella pneumoniae (K. pneumoniae), the pathogenic bacterium highly implicated in neonatal meningitis has also shown high levels of antimicrobial resistance with the classical non-virulent extended spectrum β-lactamase (ESBL) strain associated with hospital-acquired infections especially in newborns and the hypervirulent K. pneumoniae strains associated with infections in previously healthy outpatients also producing ESBL and registered in different geographical areas around the world ¹⁸.

In Cameroon, work on meningitis germs, particularly C. neoformans, has revealed that C. neoformans var. grubii (serotype A) is most commonly found in HIV-positive subjects, with relatively low minimal inhibitory concentrations (MICs) for most of the antifungal agents tested (posaconazole, amphotericin B, itraconazole, ketoconazole and voriconazole) ¹⁹. Kammalac Ngouana et al. ⁹ also revealed that cryptococcal meningitis remains a health problem in HIV-positive subjects in Yaounde, with the emergence of reduced sensitivity to fluconazole. In order to provide more precise information on the prevalence of germs responsible for infections of sterile body fluids and to contribute to the improvement of treatment protocols in people at risk, this research aimed firstly to present the bacterial and fungal species most represented in sterile body fluids: CSF, pleural fluid, ascitic fluid, joint fluid, peritoneal fluid etc. ; to evaluate the association between these germs with age and sex as risk factors for infection and to present the antimicrobial susceptibility and resistance patterns of the most represented pathogens during the study period.

2. Materials and Methods

This observational study was carried out from January 04, 2010 to December 29, 2019 in Yaounde, capital of the Center region and more precisely at the Centre Pasteur of Cameroon (CPC). The CPC, a reference and public health laboratory is a technical body of the Ministry of Public Health of Cameroon and Member of the International Network of Pasteur Institutes. For this purpose, it receives samples from several hospital in the city of Yaounde but also from other regions.

Samples of sterile body fluids (CSF, ascitic fluid, joint fluid, peritoneal fluid, pleural fluid etc.) were taken by the specialist physicians and the samples were transferred to the CPC in strict compliance with the delivery times. Each sample was first labelled and recorded by the sampling service and transferred to the Bacteriology laboratory at room temperature in clean, dry containers, accompanied by information or medical prescription sheets.

In the laboratory, the samples were previously recorded on the benchtop sheet and the analysis began with a macroscopic examination (colour, turbidity), followed by a cell count (fluid cytology) and slide smears were taken for Gram staining and leucocyte count. The search for bacteria and yeasts (aerobic and anaerobic) was carried out by inoculation on appropriate culture media (Sabouraud + chloramphenicol agar, PolyViteX chocolate agar, blood agar, Mac Conkey agar, Chapman agar, Eosin Methylene Blue agar, Bromocresol Purple lactose agar, Drigalski agar, Hektoen agar, etc.). India Ink test was also carried out for the detection of Cryptococcus in case of suspicion on direct examination or in case of a cloudy sample. The incubation period was 24 or 48 hours depending on the media used at 37°C under CO₂. The biochemical tests on API 20E ²⁰ and API 20C ²¹ galleries were used to characterise the bacterial or fungal species identified on the culture media. After identification, antimicrobial susceptibility tests were performed using the Mueller-Hinton agar (MH) diffusion method and Mueller-Hinton horse blood medium with the addition of β-NAD (MH-F). The Sabouraud medium was used for antifungal susceptibility testing. In addition to the diffusion method, the automated method (using the Vitek 2-compact automaton) was also used for antimicrobial susceptibility testing.

The antibiotics used in this study were: PEN: penicillin (6 µg); AMP: ampicillin (10 µg); AMO/ACM: amoxicillin (25 µg); AMC: amoxicillin + clavulanic acid (20 µg/10 µg); TIC: ticarcillin (75 µg); TCC: ticarcillin + clavulanic acid (75 µg/10 µg); PIC: piperacillin (100 µg); AME: enterococcal ampicillin (10 µg); PIT/TZP: piperacillin + tazobactam (100 µg/10 µg); SAM/FAM: ampicillin + sulbactam (10 µg/10 µg); OXA: oxacillin (1 µg); ETP: ertapenem (10 µg); IMI: imipenem (10 µg); MER: meropenem (10 µg); CFT: cephalotin (30 µg); CXM:cefuroxime (30 µg); CXT: cefoxitin (30 µg); CTX/CCM: cefotaxime (30 µg); CAZ: ceftazidime (30 µg); CFM: cefixime (5 µg); FEP: cefepime (30 µg); CRO: ceftriaxone (30 µg); ATM/AZT: aztreonam (30 µg); GEN: gentamicin (10 µg); SPT: spectinomycin (10 µg); KAN: kanamycin (30 µg); TOB: tobramycin (10 µg); AKN: amikacin (30 µg); NET: netilmicin (30 µg); KAH: kanamycin high load (1 mg); GEH: gentamicin 500; GE2: gentamicin 250; S: streptomycin (10 µg); STH: streptomycin HC (300 µg); CMP: chloramphenicol (30 µg); TET: tetracycline (30 µg); MIN: minocycline (30 µg); DOT: doxycycline (30 µg); TGC: tigecycline (15 µg); ERY: erythromycine (15 µg); LIN: lincomycin (15 µg); CLI/CM: clindamycin (2 µg); QDA: quinupristin + dalfopristin (15 µg); PRI: pristinamycin (15 µg); COL: colistin (50 µg); VAN: vancomycin (30 µg); TEC: teicoplanin (30 µg); PB: polymixin (300 UI); SSS: sulfonamide (250 µg); TSU/SXT: trimethoprim + sulfamethoxazole (1.25 µg + 23.75 µg); FUR: nitrofurantoin (300 µg); NAL: nalidixic acid (30 µg); OFL: ofloxacin (5 µg); PEF: pefloxacin (5 µg); NOR: norfloxacin (10 µg); CIP: ciprofloxacin (5 µg); LEV: levofloxacin (5 µg); MXF: moxifloxacin (5 µg); RFA: rifampicin (5 µg); FUC: fusidic acid (10 µg); FOS: fosfomycin (200 µg); LIZ: linezolid (30 µg) and TEL: telithromycin (15 µg). In the context of antifungals, those used in this study were: miconazole, econazole, ketoconazole, fluconazole, amphotericin B, nystatin, 5-fluorocytosine, voriconazole and clotrimazole. The reading and interpretation of the results were in accordance with the Clinical Laboratory Standard Institute [22-25] ²². The quality control strains used for antimicrobial susceptibility testing were: Escherichia coli ATCC 25922, Streptococcus pneumoniae ATCC 49619, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Haemophilus influenzae ATCC 49247 and Candida albicans ATCC 90028.

The data were retrieved from the CPC GLIMS data management software. The database contained the variables date of collection, sex, origin of the sample, germs identified, age of patients (years) and the antibiotics and antifungals tested (represented by their 3-letter codes). After extraction of the data from the GLIMS software, the database was first cleaned with Microsoft Office Excel 2019 and the statistical analyses were carried out using R language version 3.6.1 (2019-07-05) ²⁶. Within the framework of data presentation, the finalfit package was used to produce the tables ²⁷. The statistical tests used in this research were: The Pearson’s Chi-squared test and the Fischer exact test for the comparison of proportions and associations between qualitative variables; the non-parametric Kruskal-Wallis test for comparing the mean age of patients by age group and sex. The logistic regression model was used to assess the association between the identified germs and sex with the Odds-ratio (OR) values that were determined to see whether sex is a risk factor for infection. The level of significance was set at p<0.05.

3. Results

The total number of samples analysed during the study period was 2313 for a prevalence of infection of 53.13% (1229 samples contaminated by a bacterial or fungal germ). The most represented sex among the contaminated samples was male with 676 samples (55.0%) against 505 samples for the female sex (41.1%). The difference in the distribution of positive samples according to sex was statistically significant (p<0.0001). The mean age of infected participants was 33.4 years ± 21.6. The distribution of the age variable by group showed that the youngest (< 21 years) were the most represented (34.01%) followed by adults aged between 41˗60 years (28.56%). The distribution of contaminated samples according to age groups was also significant (p<0.0001). The results in Table 1 showed that CSF sampling was the most represented (50.7%), followed by pleural fluid (28.9%), joint fluid (7.4%), ascitic fluid (5.7%) and peritoneal fluid (3.3%). Other methods of sampling were much less represented (< 1.5%).

The data in Table 2 showed that among the bacteria isolated from the contaminated samples, Cocci Gram + were the most represented with the species S. pneumoniae and S. aureus with prevalences of 6.8% and 4.8% respectively. The distribution of the other bacteria isolated from the contaminated samples is presented in Table 2. For fungi, C. neoformans was the most represented with a prevalence of 13.8% followed by C. albicans and Candida sp. much less represented in the present study (0.4% and 0.3% respectively).

The distribution of germs isolated during the study period according to the sampling sites showed that in CSF, C. neoformans followed by S. pneumoniae were the most abundant (51.0% and 14.4% respectively in relation to the total samples of this fluid). In pleural fluid, S. aureus and S. pneumoniae were the most represented with very similar prevalences (16.3% and 16.1% respectively). In joint fluid, the previous species (S. aureus and S. pneumoniae) were also the most represented with a predominance in favour of S. aureus (44.0%). For the ascitic and peritoneal fluid samples, Escherichia coli (E. coli) was the most abundant bacterial species with percentages of 34.3% and 27.5% respectively in relation to the total of these samples. The distribution of the other germs according to the sampling site is shown in Table 3.

A statistically significant association between the identified germs and age groups was found in the present study (p<0.0001) with statistically significant distributions in < 21 years for: H. influenzae (p = 0.00044), Micrococcus sp. (p<0.0001), S. aureus (p = 0.04933), Staphylococcus sp. (p<0.0001), S. agalactiae (p<0.0001) and S. pneumoniae (p<0.0001). In adults (21˗40 years, 61˗80 years), the difference in distribution was significant in favour of Enterococcus faecalis (p = 0.008825) for both age groups and Streptococcus sp. (p<0.0001) for those of 61˗80 years. C. neoformans, a fungus highly represented in the CSF was more abundant in the subjects of 41˗60 years old (p<0.0001). The different distribution of the other germs can be found in Table 4.

Sex was a protective factor against S. aureus infection (OR: 0.60; 95% CI: 0.39˗0.94; p = 0.01938) and S. pneumoniae (OR: 0.68; 95% CI: 0.46˗0.98; p = 0.032). For CSF infection caused by C. neoformans, gender was a risk factor for infection (OR: 2.07; 95% CI: 1.57˗2.73; p<0.0001) with women who had twice as likely to get C. neoformans infection in their CSF than men. The data in Table 5 showed that the association between isolated germs and sex was statistically significant (p<0.002).

Antibiotic susceptibility tests showed that in the penicillin family, the highest resistance was in favour of ticarcillin with 98.4% resistance for K. pneumoniae, 88.6% resistance for E. coli, 83.3% resistance for Citrobacter freundii (C. freundii), 66.7% resistance for Enterobacter cloacae (E. cloacae), 64.0% resistance for P. aeruginosa and 62.5% resistance for Morganella morganii (M. morganii). Piperacillin resistance was also high for M. morganii (62.5%) and K. pneumoniae (58.7%). Serratia marcescens (S. marcescens) was the only one in this family of antibiotics to show high resistance rates for amoxicillin + clavulanic acid (83.3%) and amoxicillin (66.7%). S. aureus was the single most resistant germ to penicillin (92.7%). In the cephalosporin family, resistance was highest for C. freundii with 83.3% resistance to cephalotin and 66.7% resistance to cefoxitin ; E. cloacae with 100% resistance to cephalotin and 66.7% resistance to cefoxitin ; E. coli with 65.8% resistance to cephalotin ; K. pneumoniae with 66.7% resistance to cephalotin and 65.1% resistance to cefotaxime ; M. morganii with 100% resistance to cephalotin and 62.5% resistance to cefotaxime and S. marcescens with 83.3% resistance to cephalotin. For aminoglycosides, the two most resistant bacterial species were K. pneumoniae with 61.9% resistance to gentamicin and M. morganii with 87.5% resistance to gentamicin and 62.5% resistance to tobramycin. In the tetracycline family, Cocci Gram + were the most represented in terms of tetracycline resistance with 84.4% resistance to S. agalactiae, 80.0% resistance to Streptococcus pyogenes (S. pyogenes) and 60.0% resistance to E. faecalis. For streptogramines family, E. faecalis was the most resistant with 70.0% resistance to quinupristin + dalfopristin and 50.0% resistance to pristinamycin. In the sulfonamide family, the species most resistant to trimethoprim + sulfamethoxazole (Cotrimoxazole) were: K. pneumoniae (81.0%), M. morganii (87.5%), P. mirabilis (66.7%), P. aeruginosa (60.0%), A. baumannii (60.0%), E. cloacae (77.8%), E. coli (84.8%) and H. influenzae (97.1%).

Metronidazole of the nitroimidazole family showed higher resistance for Peptococcus sp. (87.5%). Among the quinolones, M. morganii was the most resistant with 87.5% resistance to ofloxacin and 62.5% resistance to ciprofloxacin followed by E. coli which was more resistant to nalidixic acid (62.5%). For the various antibiotics, the highest resistance was in favour of A. calcoace with 62.5% resistance to fosfomycin, Morganella morganii with 75.0% resistance to fosfomycin and P. aeruginosa with 68.0% resistance to rifampicin and 68.0% resistance to fosfomycin.

Despite the resistance observed in this study, most of the antibiotics tested also showed an effective action against the majority of bacterial germs isolated from sterile body fluids. The results in Table 6 showed that for most of the families of antibiotics represented, high levels of sensitivity were observed. For the penicillin family, the most effective antibiotics were: ticarcillin with 62.5% sensitivity for A. calcoace; enterococcal ampicillin with 70.0% sensitivity for E. faecalis; piperacillin + tazobactam with 66.7% and 60.0% sensitivity respectively for P. mirabilis and A. baumannii; amoxicillin + clavulanic acid with 62.5% sensitivity for Peptococcus sp. 62.5% sensitivity for Peptostreptococcus sp. 83.3% sensitivity for P. mirabilis, 62.5% sensitivity for Salmonella sp., 83.3% sensitivity for Salmonella typhimurium (S. typhimurium) and 65.2% sensitivity for S. pneumoniae; penicillin with 93.7% and 90.0% sensitivity respectively for S. agalactiae and S. pyogenes; ampicillin with 71.9% sensitivity for S. agalactiae, 85.7% sensitivity for Streptococcus oralis and 70.0% sensitivity for S. pyogenes ; amoxicillin with 84.8% sensitivity for Streptococcus pneumoniae; oxacillin with 77.3% sensitivity for S. aureus.

In the carbapenem family, imipenem was more effective against A. baumannii (70.0%), A. calcoace (87.5%), E. cloacae (61.1%), E. coli (70.9%), K. pneumoniae (73.0%), M. morganii (87.5%), P. mirabilis (66.7%), P. aeruginosa (68.0%), S. typhimurium (83.3%) and S. marcescens (66.7%). For cephalosporins that inhibit bacterial envelope synthesis, the most sensitive germs were: A. baumannii (80.0% sensitivity to ceftazidime), A. calcoace (75.0% sensitivity to ceftazidime), E. coli (62.0% sensitivity for ceftazidime and cefotaxime), H. influenzae (100.0% sensitivity to cefotaxime), P. mirabilis (66.7% sensitivity to cephalotin and cefoxitin and 83.3% sensitivity to cefotaxime and ceftazidime), P. aeruginosa (88.0% sensitivity to ceftazidime), Salmonella sp. (100.0% sensitivity for cefotaxime and ceftazidime), S. typhimurium (100.0% sensitivity for cefotaxime and ceftazidime), S. agalactiae (81.3% sensitivity to cefotaxime), S. oralis (85.7% sensitivity to cefotaxime), S. pyogenes (90.0% sensitivity to cefotaxime) and Streptococcus sp. (63.3% sensitivity to cefotaxime). Other families of antibiotics for which high sensitivities were obtained were: aminoglycosides, macrolides, streptogramins, polypeptides, quinolones and oxazolidinones. The profiles of the most sensitive germs in these families are presented in Table 6.

With regard to fungi, Table 7 presents the antifungal agents tested during the study period. C. neoformans which was most represented in CSF was more sensitive to azole family antifungal agents with higher sensitivities to fluconazole (57.1%), miconazole (48.3%), ketoconazole (47.0%) and econazole (45.1%). Concerning polyenes, the sensitivity was higher for amphotericin B (51.4%) compared to nystatin (50.2%). Sensitivity patterns for C. albicans and Candida sp. are shown in Table 7.

4. Discussion

The sterile body fluids infections that are the subject of this research are increasingly a public health problem worldwide with high mortality and morbidity rates in both developed and developing countries ¹. This study showed that out of 2313 samples analysed, 1229 samples were positive for infection, for a prevalence of 53.13%. This result is different from that of Sharma et al. ¹ who obtained a germ growth rate in sterile body fluids of 30%. In other studies, notably Sujatha et al. ²⁸ and Sorlin et al. ²⁹, the prevalences of infection in sterile body fluids were 31% and 24%, respectively, lower than in the present study. This difference could be explained by the study site, the study period or the population under consideration. This research also showed that the age groups most affected by the infection were patients under 21 years of age and those between 41-60 years of age, with men being the most contaminated. These results are close to those obtained in other studies [13,28,30-33] where males were the most infected along with the youngest and adults who were most concerned by the infection of sterile body fluids.

Among the species isolated from sterile body fluids, fungi were the most represented with C. neoformans being the most important species in CSF (51.0%) as well as among all germs isolated during the study period (13.8%). This result is in agreement with those obtained in other studies ^{8, 9, 34, 35} where C. neoformans was highly represented in the CSF. This result could be explained by the fact that this fungus is quite frequent in cases of meningitis. In addition, research has shown that it is very often found in the CSF of immunocompromised patients, particularly those carrying the HIV with a CD4 level of less than 100 mm³ as stated by Bertout et al. ¹⁹, Trpković et al. ⁸, or Kammalac Ngouana et al. ⁹. Apart from C. neoformans which was the fungal species most represented among all the germs identified and more specifically in the CSF, this study showed that the other bacteria frequently encountered in the most important samples (CSF, pleural fluid, joint fluid, ascitic fluid and peritoneal fluid) were S. pneumoniae, S. aureus, E. coli, K. pneumoniae, Streptococcus sp., H. influenzae, Staphylococcus sp., S. agalactiae and P. aeruginosa. Similar results have also been found in other works [1,4,5,28,36-38] in which most of the germs mentioned were found in sterile body fluids. In pleural fluid, which was the second most represented sample in the present study, Abdollahi et al. ¹⁴ showed that the most represented species were S. aureus and E. coli with similar results to those of the present study where these species were highly represented. Further investigations by Muley et al. ², who also worked on germs isolated from sterile body fluids, showed that S. pneumoniae was very abundant in CSF and pleural fluid, with Gram-positive Cocci also being highly represented in these fluids as shown in the present study. Another study, notably that of Shaifali et al. ¹² showed that in pleural fluid the most identified organisms were Gram-positive with S. aureus being the most important. This study also showed that in the peritoneal fluid, the most represented bacterial species were E. coli and K. pneumoniae, a result similar to that of Kumar-M et al. ³⁰ who obtained a higher proportion of E. coli and K. pneumoniae in the peritoneal fluid (47.9% and 12.5% respectively). In the ascites fluid, as in the peritoneal fluid, E. coli was also the most represented followed by Streptococcus sp. and K. pneumoniae. Other research teams including Reginato et al. ³⁹, Oey et al. ⁴⁰, Purohit et al. ³¹ and do Amaral Ferreira et al. ⁴¹ also obtained a similar result to those of the present study with E. coli which was highly represented in ascitic fluid. Saad et al. ⁴² who worked in a tertiary care hospital in Karachi also obtained a predominance of E. coli in peritoneal fluid and ascitic fluid as in the present study.

This research showed that both age and gender were associated with sterile body fluids infection with significant Chi-squared p-values (p<0.0001 and p<0.002 respectively). Regarding the age variable, group distribution showed that the distributions were significant for E. faecalis (p = 0.008825) more abundant in patients 21-40 years old and those 61-80 years old; H. influenzae (p = 0.00044), Micrococcus sp. (p<0.0001), S. aureus (p = 0.04933), Staphylococcus sp. (p<0.0001), S. agalactiae (p<0.0001) and S. pneumoniae (p<0.0001) were more represented in patients under 21 years of age; as for Streptococcus sp. (p<0.0001), its presence was more effective in adult subjects and the elderly (21-80 years); C. neoformans for its part was more encountered in patients aged 21-40 years and those aged 41-60 years with a significant difference in distribution according to age group (p<0.0001). For infections caused by S. aureus and S. pneumoniae, very abundant in the CSF, pleural fluid and joint fluid, the genus was a protective factor against infection with S. pneumoniae (OR: 0.68, CI: 0.46-0.98, p = 0.032) and S. aureus (OR: 0.60, CI: 0.39-0.94, p<0.01938) which were more abundant in men than in women. As for C. neoformans, sex was a risk factor for its presence in the CSF (OR: 2.07, CI: 1.57-2.73, p<0.0001) with women having twice the risk of infection with this fungus compared to men. Irfan et al. ³³, who worked on antimicrobial susceptibility patterns, demographics and risk factors in patients with and without invasive meningitis with S. pneumoniae in Karachi, Pakistan also found that a significant association was established between age groups and patient status (with and without meningitis) with 5-15 year old subjects who were significantly associated with meningitis strains. Furthermore, in the study, after univariate and multivariate analyses, male gender and other predictors of mortality (severe diseases requiring referral to the emergency department and staying in intensive care units) were significantly associated with patient death after age adjustment [OR of the multivariate analysis, CI (p-value) for male gender: 5.3, 1.5-18.7 (p = 0.009)]. In another study carried out in the adult population in India, Jayaraman et al. ⁴³ also showed that in invasive disease caused by S. pneumoniae, age was associated with a fatality rate of 34.9% in individuals aged 66 years with a significant Chi-squared p-value (p = 0.002). However, in a study in the United States where meningitis cases were more likely to be caused by C. neoformans in HIV patients, Vigil et al. ⁴⁴ found different results from this study with age and sex not being independent risk factors for worse outcome in all patients with meningitis [for males, OR (95% CI, p-value): 1.0 (0.6-1.4, p = 1.0), for females > 60 years of age, OR (95% CI, p-value): 1.7 (0.8-3.5, p = 0.1)]. In a cohort study of patients with HIV associated with Cryptococcal meningitis, Jarvis et al. ⁴⁵ found an association between age and risk of mortality after 2 weeks [Multivariate AOR (95% CI, p-value): 3.9 (1.4-11.1, p = 0.02)] and after 10 weeks [Multivariate AOR (95% CI, p-value): 4.0 (1.4-11.4, p = 0.009)] with patients aged ≥ 50 years who were 4 times more likely to die than those aged < 50 years. This significant association of gender and age with bacterial and fungal infection of sterile body fluids underlines or shows the need for attention to the management of these risk groups.

With regard to antimicrobial susceptibility and resistance testing, this research showed that the most represented bacterial and fungal germs (>1.0%) were more resistant to antibiotics of the penicillin, cephalosporin and sulfonamide family. E. coli more represented in ascitic and peritoneal fluid was more resistant to amoxicillin (82.3%), ticarcillin (88.6%), cephalotin (65.8%), trimethoprim + sulfamethoxazole (84.8%) and nalidixic acid (59.5%); H. influenzae was more resistant to cotrimoxazole (97.1%); Klebsiella pneumoniae present in the five most represented samples was more resistant to amoxicillin (71.4%), ticarcillin (98.4%), piperacillin (58.7%), cephalotin (66.7%), cefotaxime (65.1%), gentamicin (61.9%) and cotrimoxazole (81.0%); P. aeruginosa was more resistant to ticarcillin (64.0%), ticarcillin + clavulanic acid (60.0%), cotrimoxazole (60.0%), rifampicin (68.0%) and fosfomycin (68.0%); S. aureus more represented in pleural fluid and joint fluid was more resistant to penicillin (92.7%). As for S. pneumoniae and S. agalactiae represented in CSF and Streptococcus sp. represented in pleural fluid and ascitic fluid, their resistance was higher for tetracycline (48.1%, 84.4% and 57.1% respectively). C. neoformans which was most represented during the study period showed lower resistance rates than the previously mentioned germs with 14.7% resistance to amphotericin B, 7.2% resistance to 5-fluorocytosine and 4.4% resistance to fluconazole; with higher sensitivity rates for most of the antifungal agents tested. These results are close to those of Mengistu et al. ⁴, who obtained a higher resistance of the germs most represented in the CSF to sulfonamides, in particular to trimethoprim + sulfamethoxazole (cotrimoxazole). Apart from E. coli and K. pneumoniae, another germ less represented in the study, notably E. cloacae, was particularly resistant to antibiotics of the penicillin family, cephalosporins and sulfonamides with: 77.8% resistance to amoxicillin, 100% resistance to amoxicillin + clavulanic acid, 66.7% resistance to ticarcillin, 100% resistance to cephalotin, 66.7% resistance to cefoxitin and 77.8% resistance to cotrimoxazole. These results are close to those of Shi et al. ³⁸, who obtained higher rates of resistance of Enterobacteriaceae to antibiotics of the penicillin family (> 85% for ampicillin). Abdollahi et al. ¹⁴, who presented the microbiological profile of the germs present in the pleural fluid also obtained high resistance rates for E. coli, K. pneumoniae, P. aeruginosa, S. aureus and S. pneumoniae to penicillin, cephalosporin and sulfonamide antibiotics. Another research, by Shaifali et al. ¹², on samples from pleural fluid showed that Gram-positive bacteria (GPB) were resistant to penicillin, azithromycin and clindamycin and Gram-negative bacteria (GNB) more resistant to cephalosporins (cephalotin and ceftazidime), ciprofloxacin, trimethoprim + sulfamethoxazole and amoxicillin + clavulanic acid. Similarly, Ahmed et al. ⁴⁶, found higher resistances of GPB and GNB to penicillins, cephalosporins, tetracyclines and sulfonamides. For C. neoformans, the results obtained by Kammalac Ngouana et al. ⁹, were similar to those of the present study with 92.7% of the strains of C. neoformans being sensitive to fluconazole (MIC < 32 µg/mL) and only 7.3% of the strains resistant to this antifungal agent (MIC > 32 µg/mL). Bertout et al. ¹⁹, who worked on the genotyping and antifungal susceptibility of C. neoformans in HIV-positive Cameroonian patients, obtained very low MICs for all antifungal drugs tested. For Bongomin et al. ¹⁶, the mean resistance of Cryptococcus isolates to fluconazole was also low (12.1% for n = 4995 strains tested). In Serbia, Trpković et al. ⁸ also obtained high susceptibility profiles of C. neoformans to fluconazole, itraconazole, 5-fluorocytosine and amphotericin B. The multi-resistance observed in this study shows the interest of carrying out investigations at the molecular level in order to know the genes associated with resistance to antibiotics commonly used in our context of germs isolated from sterile body fluids.

Despite the multidrug resistance observed during the study period, the results of this research also showed that antibiotics from the family of penicillins, carbapenems, cephalosporins, aminosides, macrolides, streptogramins, polypeptides, quinolones and oxazolidinones were effective against most of the germs identified. The highest efficacy rates were in favour of imipenem with 70.9% sensitivity for E. coli and 73.0% sensitivity for K pneumoniae; amikacin with 87.3% sensitivity for E. coli, 87.3% sensitivity for K. pneumoniae and 76.0% sensitivity for P. aeruginosa; cefotaxime with 100% sensitivity for H. influenzae and 81.3% sensitivity for S. agalactiae; oxacillin with 77.3% sensitivity for S. aureus; gentamicin with 90.0% sensitivity for S. aureus; minocycline with 74.5% sensitivity for S. aureus; erythromycin with 75.5% sensitivity for S. aureus and 84.4% sensitivity for S. agalactiae; lincomycin with 83.6% sensitivity to S. aureus; quinupristin-dalfopristin with 93.6% resistance to S. aureus; pristinamycin with 86.4% sensitivity to S. aureus; vancomycin with 94.5% sensitivity to S. aureus, 90.6% sensitivity for S. agalactiae, 94.9% sensitivity for S. pneumoniae and 79.6% sensitivity for Streptococcus sp.; teicoplanin with 86.4% sensitivity for S. aureus, 93.8% sensitivity for S. agalactiae and 71.4% sensitivity for Streptococcus sp.; penicillin with 93.7% sensitivity for S. agalactiae; ampicillin with 71.9% sensitivity for S. agalactiae; cotrimoxazole with 71.9% sensitivity for S. agalactiae; levofloxacin with 87.5% sensitivity for S. agalactiae, 74.1% sensitivity for S. pneumoniae and 73.5% sensitivity for Streptococcus sp.; rifampicin with 71.9% sensitivity for S. agalactiae; linezolid with 90.6% sensitivity for S. agalactiae; amoxicillin with 84.8% sensitivity for S. pneumoniae; chloramphenicol with 70.9% sensitivity for S. pneumoniae and ceftazidime with 88.0% sensitivity for P. aeruginosa. Similar results have also been obtained in other studies ^{1, 2, 5, 13, 15, 28, 43, 47} with high sensitivity rates for most of the germs represented in sterile body fluids (CSF, ascitic fluid, pleural fluid, peritoneal fluid etc.). Indeed, as the investigators of these main studies have shown, the families of antibiotics presented were those preferentially used in therapy in meningitis or peritonitis situations in patients at risk of infection.

The results of this study, which took place in the Yaounde over a period of 10 years, show that cases of meningitis are the most frequent in the context of infections of sterile body fluids, especially the CSF, which is the preferred site for fungal and bacterial germs. In addition to the genotyping studies of Cryptococcus strains already carried out in Cameroon by Bertout et al. ¹⁹ and by Kammalac Ngouana et al. ³⁴, it is important and even crucial that a mapping of the genes associated with resistance to antifungal and antibiotics in our context be carried out in order to develop strategies to improve the surveillance of these multi-resistant germs in the most vulnerable subjects.

5. Conclusion

Infections of sterile body fluids are a global health problem and continue to cause the deaths of millions of people each year. The results of this research have shown that the prevalence of sterile body fluid infection is high in our context with age and gender being risk factors for infection. For gender, women are twice as likely to have C. neoformans infection in their CSF compared to men and for age, the youngest (< 21 years) followed by adults (41˗60 years) are the most vulnerable. Despite the resistance observed for most of the families of antibiotics tested, the families of penicillins, carbapenems, cephalosporins, aminoglycosides, polypeptides and quinolones were very effective against the bacterial species identified. As for antifungals, azoles and polyenes were also effective against Candida and C. neoformans species. The results of this study show us that the surveillance of multidrug resistance to antimicrobials of germs isolated from sterile body fluids must be extended throughout the country in order to reduce the prevalence of infection and improve diagnosis and management in patients at risk.

List of Abbreviations

AIDs Acquired Immunodeficiency syndrome

CLS IClinical and Laboratory Standards Institute

CM cryptococcal meningitidis

CPC Centre Pasteur of Cameroon

CSF Cerebrospinal fluid

ESBL Extended Spectrum β-Lactamase

GNB Gram negative bacteria

GPB Gram positive bacteria

HIV Human Immunodeficiency Virus

MIC minimal inhibitory concentrations

MRSA Methicillin-resistant Staphylococcus aureus

MSSA Methicillin-sensitive Staphylococcus aureus

PD peritoneal dialysis

WHO World Health Organisation

ACKNOWLEDGEMENTS

Thanks are due to all the individuals who participated in this study.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

References