Alex Agyekum

Incidence and Characteristics of Bacteremia among Children in Rural Ghana

The objective of the study was to describe systemic bacterial infections occurring in acutely ill and hospitalized children in a rural region in Ghana, regarding frequency, incidence, antimicrobial susceptibility patterns and associations with anthropometrical data. Blood cultures were performed in all children below the age of five years, who were admitted to Agogo Presbyterian Hospital (APH), Asante Region, Ghana, between September 2007 and July 2009. Medical history and anthropometrical data were assessed using a standardized questionnaire at admission. Incidences were calculated after considering the coverage population adjusted for village-dependent health-seeking behavior. Among 1,196 hospitalized children, 19.9% (n = 238) were blood culture positive. The four most frequent isolated pathogens were nontyphoidal salmonellae (NTS) (53.3%; n = 129), Staphylococcus aureus (13.2%; n = 32), Streptococcus pneumoniae (9.1%; n = 22) and Salmonella ser. Typhi (7.0%; n = 17). Yearly cumulative incidence of bacteremia was 46.6 cases/1,000 (CI 40.9–52.2). Yearly cumulative incidences per 1,000 of the four most frequent isolates were 25.2 (CI 21.1–29.4) for NTS, 6.3 (CI 4.1–8.4) for S. aureus, 4.3 (CI 2.5–6.1) for S. pneumoniae and 3.3 (CI 1.8–4.9) for Salmonella ser. Typhi. Wasting was positively associated with bacteremia and systemic NTS bloodstream infection. Children older than three months had more often NTS bacteremia than younger children. Ninety-eight percent of NTS and 100% of Salmonella ser. Typhi isolates were susceptible to ciprofloxacin, whereas both tested 100% susceptible to ceftriaxone. Seventy-seven percent of NTS and 65% of Salmonella ser. Typhi isolates were multi-drug resistant (MDR). Systemic bacterial infections in nearly 20% of hospitalized children underline the need for microbiological diagnostics, to guide targeted antimicrobial treatment and prevention of bacteremia. If microbiological diagnostics are lacking, calculated antimicrobial treatment of severely ill children in malaria-endemic areas should be considered.

Typhoid Fever among Children, Ghana

To the Editor: Typhoid fever (TF) remains a problem of concern in many low-income countries. Salmonella enterica serovar Typhi causes ≈22,000,000 symptomatic infections and 220,000 fatalities worldwide annually (1). However, the effect and incidence of TF in many parts of sub-Saharan Africa are largely unknown because diagnostic laboratories are lacking and fatal TF is frequently attributed to malaria (2,3). In Ghana, TF ranks among the leading 20 causes of outpatient illness, accounting for 0.92% of hospital admissions (4). We conducted our study at the rural Agogo Presbyterian Hospital in the Ashanti Region of Ghana. The percentage of residents of 99 villages and household clusters of buildings (population size 18–13,559 persons, median 277 persons) with access to the study hospital was assessed in a healthcare utilization survey. A proportional-to-size number of children were randomly selected in each village, and a standardized interview was conducted. TF incidences were calculated for September 2007–November 2008 (Table). A bacteriology laboratory with BACTEC 9050 automated blood culture system (Becton Dickinson, Sparks, MD, USA) was established in the study hospital and run to assess the number of admissions with TF, the incidence of TF in the adjoining community and S. enterica ser. Typhi resistance to a panel of antimicrobial drugs. Table Estimates of Salmonella enterica serovar Typhi incidence in children, Ghana, September 2007–November 2008 The study included 1,456 children <15 years of age who were admitted to the pediatric ward of Agogo Presbyterian Hospital over the 23-month study period. Overall, 52.1% were male; mean age of children was 32.2 months (SD ± 36.0 months; median 19 months, range 0–174 months). Blood was cultured by using a BACTEC 9050 blood culture system (Becton Dickinson), and positive samples were examined by standard methods. Antimicrobial drug susceptibility testing was performed on all serovar Typhi isolates by using the Kirby-Bauer disk-diffusion method for ampicillin, chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole, amoxicillin/clavulanic acid, gentamicin, ciprofloxacin, and ceftriaxone. Children <2 years of age had the highest proportion of positive blood cultures (164/1,456, 21.3%; Figure A1). Of 298 blood cultures yielding positive growth for bacterial pathogens or for Candida spp., 37 (12.4%) isolates (2.5% of the 1,456 hospitalized children) were positive for S. enterica ser. Typhi. The frequency of TF was low among children <2 years of age (7/1,018, 0.7%), increased among those 2 to <11 years of age (29/417, 7.0%), and decreased among children ≥11 years of age (1/22, 4.6%) (Figure A1). One (2.7%) child with TF died. Malaria parasites were detected in 2 children with S. enterica ser. Typhi. Pathogens other than S. enterica ser. Typhi were identified among 21.3% and 11.8% of children 0 to <2 years and 5 to <8 years of age, respectively. These pathogens included nontyphoidal salmonellae, Staphylococcus aureus, and Streptococcus pneumoniae. S. enterica ser. Typhi isolates were resistant to chloramphenicol (73%), trimethoprim/sulfamethoxazole (71%), ampicillin/amoxicillin (70%), tetracycline (64%), gentamicin (46%), and amoxicillin/clavulanic acid (24%) but susceptible to ciprofloxacin and ceftriaxone. TF incidence in children <5 years of age was ≈190 cases/100,000 population and highest in children 2–5 years of age (290/100,000 per year) and 5–8 years of age (200/100,000 per year) (Table). In children older than 8, incidence decreased continuously, and the number of cases was too low to enable precise age-stratified incidence calculations. The incidences in the study area point to a higher impact of TF than expected (4) and may reflect an underestimation of TF in other West African regions as well. Our high incidence figure may still underestimate the incidence because of a low sensitivity of standard microbiologic methods (up to 50%), which are prone to underdiagnose moderate bacteremia in Salmonella infections (5,6). Compared with Asia, only limited data are available from Africa on S. enterica ser. Typhi drug resistance. A study from Nigeria showed that, among serovar Typhi strains isolated from hospitalized patients in Lagos during 1997–2004, resistance rates reached 87% for ampicillin and were 0.7% for ciprofloxacin, compared with 70% and 0%, respectively, in the present study. Resistance to trimethoprim/sulfamethoxazole was 59% in Nigeria, compared with 71% in Ghana. In Togo, proportions of serovar Typhi strains resistant to chloramphenicol and trimethoprim/sulfamethoxazole were 33% and 46%, respectively, before 2002 and 73% and 79% in 2003–2004 (7) and thus similar to those in our study. In addition, resistances to ciprofloxacin and ceftriaxone were <10%. Multidrug resistance (resistance to ampicillin, trimethoprim/sulfamethoxazole, and chloramphenicol) was observed in 63% of children in our study, compared with 7% in India, 22% in Vietnam, and 65% in Pakistan (8–10). More effort is needed in Africa to enable reliable and standardized laboratory diagnoses of Salmonella infections and to sustain TF surveillance and drug sensitivity surveys. Moreover, introduction of a vaccination program should be discussed after more data are obtained from other areas in Ghana and West Africa. Such data currently are collected in an extensive standardized surveillance program across the continent performed by our group and others. In parallel, trials should be conducted to assess the effectiveness and cost-effectiveness of currently available and newly developed TF vaccines.

Phenotypic Detection of Carbapenemase-Producing Enterobacteriaceae by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and the Carba NP Test

ABSTRACT We compared the diagnostic accuracy of the Carba NP test with that of a straightforward matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) method for detecting carbapenemase-producing Enterobacteriaceae (CPE). Using PCR as the reference method, both tests demonstrated a sensitivity of 87% and a specificity of 100%. MALDI-TOF MS offers a potential alternative for the rapid detection of CPE in the clinical laboratory setting.

Comparison of the Novel Partec Rapid Malaria Test to the Conventional Giemsa Stain and the Gold Standard Real-Time PCR

ABSTRACT Malaria remains the single most frequent cause of death in Africa, killing one child every 30 s, but treatment decisions are often made only on clinical diagnosis, as laboratory techniques to confirm the clinical suspicion are labor intensive and costly. In this study, we evaluated the recently developed Partec rapid malaria test (PM) for the detection of Plasmodium spp. in human blood from patients in an area where malaria is endemic and compared the results with those of thick blood film Giemsa stain (GS) in terms of its performance and operational characteristics, using real-time (RT) PCR as the gold standard. The sensitivities of the PM and the GS were 62.2% (95% CI, 56.3 to 67.8) and 61.8% (95% CI, 55.9 to 67.4), respectively, while the specificities were 96.0% (95% CI, 92.3 to 98.3) and 98% (95% CI, 95.0 to 99.5), respectively. There was an excellent agreement between the results for the PM and those of the GS (k [level of agreement] = 0.96; P < 0.001). The results for the PM were obtained more quickly and at less cost than those for the GS. The performance characteristics of the PM were almost equal to those of the GS, but the operational characteristics were better, and the PM can therefore be considered as an alternative method for GS.

blaCTX-M-15 carried by IncF-type plasmids is the dominant ESBL gene in Escherichia coli and Klebsiella pneumoniae at a hospital in Ghana.

Escherichia coli and Klebsiella pneumoniae producing extended-spectrum β-lactamases (ESBLs) are among the most multidrug-resistant pathogens in hospitals and are spreading worldwide. Horizontal gene transfer and spread of high-risk clones are involved in ESBL dissemination. Investigation of the resistance phenotypes of 101 consecutive clinical E. coli (n=58) and K. pneumoniae (n=43) isolated at the Komfo Anokye Teaching Hospital in Ghana over 3 months revealed 63 (62%) with an ESBL phenotype. All 63 had a blaCTX-M gene, and sequence analysis showed that 62 of these were blaCTX-M-15. blaCTX-M-15 was linked to ISEcp1 and orf477Δ in all isolates, and most isolates also carried blaTEM, aac(3)-II, aacA4cr, and/or blaOXA-30 genes on IncF plasmids. XbaI/pulsed-field electrophoresis showed heterogeneity among isolates of both species, suggesting that blaCTX-M-15 dissemination is caused by horizontal gene transfer rather than clonal spread of these species in Ghana.

Predictability of phenotype in relation to common β-lactam resistance mechanisms in Escherichia coli and Klebsiella pneumoniae.

ABSTRACT The minimal concentration of antibiotic required to inhibit the growth of different isolates of a given species with no acquired resistance mechanisms has a normal distribution. We have previously shown that the presence or absence of transmissible antibiotic resistance genes has excellent predictive power for phenotype. In this study, we analyzed the distribution of six β-lactam antibiotic susceptibility phenotypes associated with commonly acquired resistance genes in Enterobacteriaceae in Sydney, Australia. Escherichia coli (n = 200) and Klebsiella pneumoniae (n = 178) clinical isolates, with relevant transmissible resistance genes (bla TEM, n = 33; plasmid AmpC, n = 69; extended-spectrum β-lactamase [ESBL], n = 116; and carbapenemase, n = 100), were characterized. A group of 60 isolates with no phenotypic resistance to any antibiotics tested and carrying none of the important β-lactamase genes served as comparators. The MICs for all drug-bacterium combinations had a normal distribution, varying only in the presence of additional genes relevant to the phenotype or, for ertapenem resistance in K. pneumoniae, with a loss or change in the outer membrane porin protein OmpK36. We demonstrated mutations in ompK36 or absence of OmpK36 in all isolates in which reduced susceptibility to ertapenem (MIC, >1 mg/liter) was evident. Ertapenem nonsusceptibility in K. pneumoniae was most common in the context of an OmpK36 variant with an ESBL or AmpC gene. Surveillance strategies to define appropriate antimicrobial therapies should include genotype-phenotype relationships for all major transmissible resistance genes and the characterization of mutations in relevant porins in organisms, like K. pneumoniae.

Clinical indicators for bacterial co-infection in Ghanaian children with P. falciparum infection.

Differentiation of infectious causes in severely ill children is essential but challenging in sub- Saharan Africa. The aim of the study was to determine clinical indicators that are able to identify bacterial co-infections in P. falciparum infected children in rural Ghana. In total, 1,915 severely ill children below the age of 15 years were recruited at Agogo Presbyterian Hospital in Ghana between May 2007 and February 2011. In 771 (40%) of the children malaria parasites were detected. This group was analyzed for indicators of bacterial co-infections using bivariate and multivariate regression analyses with 24 socio-economic variables, 16 terms describing medical history and anthropometrical information and 68 variables describing clinical symptoms. The variables were tested for sensitivity, specificity, positive predictive value and negative predictive value. In 46 (6.0%) of the children with malaria infection, bacterial co-infection was detected. The most frequent pathogens were non-typhoid salmonellae (45.7%), followed by Streptococcus spp. (13.0%). Coughing, dehydration, splenomegaly, severe anemia and leukocytosis were positively associated with bacteremia. Domestic hygiene and exclusive breastfeeding is negatively associated with bacteremia. In cases of high parasitemia (>10,000/μl), a significant association with bacteremia was found for splenomegaly (OR 8.8; CI 1.6–48.9), dehydration (OR 18.2; CI 2.0–166.0) and coughing (OR 9.0; CI 0.7–118.6). In children with low parasitemia, associations with bacteremia were found for vomiting (OR 4.7; CI 1.4–15.8), severe anemia (OR 3.3; CI 1.0–11.1) and leukocytosis (OR 6.8 CI 1.9–24.2). Clinical signs of impaired microcirculation were negatively associated with bacteremia. Ceftriaxone achieved best coverage of isolated pathogens. The results demonstrate the limitation of clinical symptoms to determine bacterial co-infections in P. falciparum infected children. Best clinical indicators are dependent on the parasitemia level. Even with a moderate sensitivity of >60%, only low positive predictive values can be obtained due to low prevalence of bacteremia. Rapid testing for distinguishing parasitemia and bacteremia is essential.

Host adaptation and convergent evolution increases antibiotic resistance without loss of virulence in a major human pathogen

As human population density and antibiotic exposure increases, specialised bacterial subtypes have begun to emerge. Arising among species that are common commensals and infrequent pathogens, antibiotic-resistant ‘high-risk clones’ have evolved to better survive in the modern human. Here, we show that the major matrix porin (OmpK35) of Klebsiella pneumoniae is not required in the mammalian host for colonisation, pathogenesis, nor for antibiotic resistance, and that it is commonly absent in pathogenic isolates. This is found in association with, but apparently independent of, a highly specific change in the co-regulated partner porin, the osmoporin (OmpK36), which provides enhanced antibiotic resistance without significant loss of fitness in the mammalian host. These features are common in well-described ‘high-risk clones’ of K. pneumoniae, as well as in unrelated members of this species and similar adaptations are found in other members of the Enterobacteriaceae that share this lifestyle. Available sequence data indicates evolutionary convergence, with implications for the spread of lethal antibiotic-resistant pathogens in humans. Author summary Klebsiella pneumoniae is a Gram-negative enterobacteria and a significant cause of human disease. It is a frequent agent of pneumonia, and systemic infections can have high mortality rates (60%). OmpK35 and OmpK36 are the major co-regulated outer membrane porins of K. pneumoniae. OmpK36 absence has been related to antibiotic resistance but decreased bacterial fitness and diminished virulence. A mutation that constricts the porin channel (Gly134Asp135 duplication in loop 3 of the porin, OmpK36GD) has been previously observed and suggested as a solution to the fitness cost imposed by loss of OmpK36. In the present study we constructed isogenic mutants to verify this and test the impact of these porin changes on antimicrobial resistance, fitness and virulence. Our results show that loss of OmpK35 has no significant cost in bacterial survival in nutrient-rich environments nor in the mammalian host, consistent with a predicted role outside that niche. When directly compared with the complete loss of the partner osmoporin OmpK36, we found that isogenic OmpK36GD strains maintain high levels of antibiotic resistance and that the GD duplication significantly reduces neither gut colonisation nor pathogenicity in a pneumonia mouse model. These changes are widespread in unrelated genomes. Our data provide clear evidences that specific variations in the loop 3 of OmpK36 and the absence of OmpK35 in K. pneumoniae clinical isolates are examples of successful adaptation to human colonization/infection and antibiotic pressure, and are features of a fundamental evolutionary shift in this important human pathogen.