In total, 684 study participants were included; 54.2% were male and the median age was 22 (interquartile range: 14-35) years. More than half of the participants were from rural areas and 167 (24.4%) could not read or write (Table 3).
Table 3. Sociodemographic characteristics of study participants
Socio-demographic characteristics
|
Numbers
|
Percentage (%)
|
Sex
|
|
|
|
|
Male
|
371
|
54.2
|
Age
|
|
|
1-15
|
190
|
27.8
|
|
16-30
|
286
|
41.8
|
|
31-45
|
110
|
16.1
|
|
46-60
|
51
|
7.5
|
|
>60
|
47
|
6.9
|
Area of residence
|
|
Rural
|
396
|
57.9
|
|
Urban
|
288
|
42.1
|
Educational status
|
|
Illiterate
|
167
|
24.4
|
|
Literate
|
434
|
63.5
|
|
Children before school age
|
83
|
12.1
|
Patient settings
|
|
|
|
Internal medicine ward
|
129
|
18.9
|
|
Pediatric ward
|
131
|
19.2
|
|
Surgical and Gynecology wards
|
47
|
6.9
|
|
Emergency OPD
|
347
|
50.7
|
|
ICU
|
30
|
4.4
|
ICU: intensive care unit; OPD: out-patients department
Bacterial cultures
The overall bacterial detection rate across all sample materials was 12.1% (83/684). In 9.6% (n=66) the isolated bacterium was considered a pathogen and in 2.5% (n=17) coagulase-negative staphylococci (CoNS) were isolated and considered as clinically irrelevant because of the high likelihood of contamination. The overall blood culture positivity rate was 5.4% (37/684), among which 51.4% (n=19) were GNB. The overall detection rate in other clinical samples was 60.6% (20/33), 20.8% (5/24), and 37.5% (3/8) in swabs/pus, urine, and other body fluids, respectively. Out of 12 cultured cerebrospinal fluid samples, only one sample revealed growth of a pathogen (N. meningitidis). Overall, 66 pathogenic bacteria were isolated from a total of 761 different clinical samples. Of those, 57.6% (n=38) were GNB, 39.4% (n=26) Gram-positive and 3% (n=2) Candida species. S. aureus and E. coli were the most prevalent isolates among Gram-positive and Gram-negative isolates, respectively. Among the 38 Gram-negative isolates, 42.1% (n=16) were E. coli, 23.7% (n=9) K. pneumoniae, and 10.5% (n=4) P. aeruginosa (for further details see Table 4).
Table 4. Frequencies of isolated GNB isolates from different clinical samples of the study participants (n=38)
Bacterial species
|
Number
|
%
|
E. coli
|
16
|
42.1
|
K. pneumonia
|
9
|
23.7
|
P. aeruginosa
|
4
|
10.5
|
Salmonella typhi
|
2
|
5.3
|
Enterobacter spp.
|
2
|
5.3
|
A. baumannii
|
1
|
2.6
|
N. meningitides
|
1
|
2.6
|
R. ornithinolytica
|
1
|
2.6
|
R. planticola
|
1
|
2.6
|
Serratia spp.
|
1
|
2.6
|
As indicated in Table 5, among all clinical samples bacterial growth was identified significantly more often among patients with skin and soft tissue infections (SSTI) (p<0.001). Although not statically significant, samples from patients with a leukocyte count >12,000 or <4000 cells /µL at the time of sampling tended to be more likely to reveal bacterial growth (p=0.07). On the other hand, blood cultures from patients with the diagnosis of acute febrile illness with unknown source were least likely to reveal bacteremia compared with other symptoms (p=0.005). Being HIV-positive did not have any impact on blood culture positivity rate (p=0.85). Equally the C-reactive protein (CRP) had no predictive value concerning blood culture positivity (blood culture positive versus blood culture negative, CRP mean 71.7 mg/L vs. 64.6 mg/L; p=0.20).
Table 5. Culture positivity rate according to clinical diagnosis of source of infection and laboratory parameters among study participants blood
Infectious focus / laboratory parameters
|
Culture positive (N=66)
|
Culture negative (N=618)
|
p-value
|
|
% (n)
|
% (n)
|
|
Pneumonia / RTI (n=165)
|
19.7 (13)
|
24.6 (152)
|
0.38
|
Urinary tract infections (n=30)
|
7.6 (5)
|
4.4 (25)
|
0.20
|
Meningitis / encephalitis (n=57)
|
6.1 (4)
|
8.6 (53)
|
0.48
|
GITI / hepatitis (n=43)
|
6.1 (4)
|
6.3 (39)
|
0.94
|
SSTIs (n=52)
|
28.8 (19)
|
5.3 (33)
|
<0.001
|
Acute febrile illness with unknown source (n=264)
|
22.7 (15)
|
40.3 (249)
|
0.005
|
Sepsis* (n=32)
|
4.5 (3)
|
4.7 (29)
|
0.96
|
HIV sero-positivity (n=58)
|
9.1 (6)
|
8.4 (52)
|
0.85
|
Leukocytes count >12,000 or <4000 /µL (n=218)
|
42.4 (28)
|
30.7 (190)
|
0.07
|
|
Mean
|
Mean
|
|
CRP in mg/L
|
71.7
|
64.6
|
0.20
|
RTIs: Respiratory tract infection, SSTI = skin and soft tissue infection; GITI= gastrointestinal tract infection, CRP=C-reactive protein
* According to clinician’s diagnosis, regardless of the focus of the infection
Regarding culture positivity rate among different clinical samples, there was no significant difference between the positivity rates of Gram-positive or Gram-negative isolates. See Table 6 for details.
Table 6. Culture positivity rate among the different clinical samples
Clinical sample
|
Culture negative
|
Culture positive
|
Gram-negative isolate % (n)
|
Gram-positive isolate % (n)
|
All samples (n=761)
|
91.3 (695)
|
5.0 (38)
|
3.7 (28)
|
Blood (n=684)
|
94.6 (647)
|
2.9 (20)
|
2.5 (17)
|
Urine (n=24)
|
79.2 (19)
|
12.5 (3)
|
8.3 (2)
|
Swab or pus of infected skin lesion or abscess (n=33)
|
33.3 (11)
|
36.4 (12)
|
24.2 (8)
|
CSF (n=12)
|
91.7 (11)
|
8,3 (1)
|
-
|
Other body fluids (n=8)
|
62.5 (5)
|
25.0 (2)
|
(1)
|
Antibiotic susceptibility testing
In total, 27 Gram-negative isolates were available for susceptibility testing with VITEK® 2. Among those, the resistance rates against commonly used antibiotics at the study site were: ampicillin/sulbactam 93.3% (n=21), cefotaxime 88.9% (n=24), ceftazidime 74.1% (n=20), cefipime 74.1% (n=20), ciprofloxacin 70.4% (n=19), and gentamicin 63.0% (n=17). Resistance rates against rarely used antibiotics was 48.1% (n=13) for piperacillin/tazobactam, and 7.4% (n=2) for meropenem. Only one case of amikacin resistance was detected among all 27 isolates (Table7).
Resistance genes
The overall frequencies of ESBL and carbapenemase genes detection among the isolated GNB were 81.5% (22/27) and 7.4% (2/27), respectively. In 55.6% of cases (n=15), more than one ESBL gene was detected in the isolated GNB. The different ESBL were characterized as TEM (n=17, 77.3%), CTX-M-1-group (n=15, 68.2%), SHV-group (n=6, 27.3%) and CTX-M-9-group (n=2, 9.1%). Both, CTX-M-1-type and SHV-type were detected from 5/6 (83.3%) of the K. pneumoniae isolates, whereas SHV-group was only detected in 1/13 of the E. coli isolates. In E. coli, only the CTX-M-1-group was common (see Table 8). Regarding carbapenemases, a single blaNDM-1 from one isolated K. pneumoniae and a combination of blaNDM-1 plus blaOXA-51 from an isolate of A. baumannii were detected.
Effectiveness of empirical antibiotic treatment
The results of the locally performed Kirby-Bauer disc diffusion test were available from 25 study participants with E. coli and K. pneumoniae isolates. Eighteen of these 25 study participants received an empirically initiated antibiotic treatment at the time of sampling. The Kirby-Bauer AST revealed high levels of resistance against commonly used antibiotics, rendering 72.2% (13/18) of the initiated antibiotic treatments ineffective. In particular, 72.0% (18/25) of the isolated GNB were resistant to 3GC, 60.0% (15/25) to fluoroquinolones and 48.0% (12/25) to gentamicin. The results of the initial clinical evaluation, empirical antibiotic treatment and AST result according to Kirby-Bauer disc diffusion test are summarized below (Table 9).
Table 7. Antibiotic resistance rate of Gram-negative isolates
Bacterial species
|
Ampicillin
|
Ampicillin/ sulbactam
|
Piperacillin
|
Piperacillin/ tazobactam
|
Cefotaxime
|
Ceftazidime
|
Cefepime
|
Meropenem
|
Amikacin
|
Gentamicin
|
Ciprofloxacin
|
Escherichia coli (n=13)
|
92.3% (12/13)
|
92.3%
(12/13)
|
92.3%
(12/13)
|
69.2%
(9/13)
|
84.6%
(11/13)
|
84.6%
(11/13)
|
84.6%
(11/13)
|
0%
(0/13)
|
0%
(0/13)
|
53.8%
(7/13)
|
23.1%
(3/13)
|
Klebsiella pneumoniae (n=6)
|
100%
(6/6)
|
100%
(6/6)
|
100%
(6/6)
|
50.0%
(3/6)
|
100%
(6/6)
|
100%
(6/6)
|
100%
(6/6)
|
16.7%
(1/6)
|
16.7%
(1/6)
|
83.3%
(5/6)
|
50.0%
(3/6)
|
Pseudomonas aeruginosa (n=4)
|
-
|
-
|
50.0%
(2/4)
|
0%
(0/4)
|
100%
(4/4)
|
50.0%
(2/4)
|
50.0%
(2/4)
|
0%
(0/4)
|
0%
(0/4)
|
25.0%
(1/4)
|
75.0%
(3/4)
|
Other (n=4)
|
75.0%
(3/4)
|
75.0%
(3/4)
|
75.0%
(3/4)
|
25.0%
(1/4)
|
75.0%
(3/4)
|
25.0%
(1/4)
|
25.0%
(1/4)
|
25.0%
(1/4)
|
0%
(0/4)
|
50.0%
(2/4)
|
75.0%
(3/4)
|
Total (n=27)
|
91.3%
(21/23)
|
91.3%
(21/23)
|
85.2%
(23/27)
|
48.1%
(13/27)
|
88.9%
(24/27)
|
74.1%
(20/27)
|
74.1%
(20/27)
|
7.4%
(2/27)
|
3.7%
(1/27)
|
63.0%
(17/27)
|
70.4%
(19/27)
|
Others: Raoultella ornithinolytica (n=1); Raoultella planticola (n=1); Salmonella typhi (n=1); Acinetobacter baumannii (n=1)
Table 8. Characterization and frequency of detected ESBL and carbapenemase genes among the Gram-negative isolates
Bacterial species
|
Resistance genes % (n)
ESBL genes
|
|
|
CP genes
|
|
All ESBL
|
CTX-M-1 group
|
TEM-group
|
SHV-group
|
CTX-M-9 group
|
All CP
|
NDM-1
|
OXA-51
|
E. coli (n=13)
|
92.3 (12)
|
69.2 (9)
|
76.9 (10)
|
7.7 (1)
|
0
|
0
|
0
|
0
|
K. pneumoniae (n=6)
|
100 (6)
|
83.3 (5)
|
50.0 (3)
|
83.3 (5)
|
0
|
16.7 (1)
|
16.7 (1)
|
0
|
P. aeruginosa (n=4)
|
50.0 (2)
|
25.0 (1)
|
50.0 (2)
|
0
|
0
|
0
|
0
|
0
|
Other* (n=4)
|
75.0 (3)
|
0
|
50.0 (2)
|
0
|
50.0 (2)
|
25.0 (1)
|
25.0 (1)
|
25.0 (1)
|
All GNB (n=27)
|
81.5 (22)
|
55.6 (15)
|
63.0 (17)
|
22.2 (6)
|
7.4 (2)
|
7.4 (2)
|
7.4 (2)
|
3.7 (1)
|
*Other isolates: R. planticola (n=1) and R. ornithinolytica (n=1): both positive for CTX-M-9 group; Salmonella Typhi (n=1): no ESBL- or CPE-production; and A. baumannii (n=1): positive for OXA-51 and NDM-1
CP, carbapenemase
Table 9. Clinical-evaluation, Kirby-Bauer AST result and empirical treatment of participants with E. coli or K. pneumoniae
Participant No.
|
Bacterial isolate
|
Resistance against
|
Clinical diagnosis
|
Empirical antibiotic treatment
|
Effectiveness of antibiotic treatment
|
3GC
|
Fluoroquinolones
|
Gentamicin
|
20
|
E. coli
|
Yes
|
Yes
|
No
|
UTI
|
Ciprofloxacin
|
Ineffective
|
56
|
E. coli
|
Yes
|
No
|
Yes
|
RTI
|
Ceftriaxone, cloxacillin
|
Effective
|
258
|
E. coli
|
No
|
Yes
|
Yes
|
AFI
|
None
|
-
|
259
|
E. coli
|
Yes
|
Yes
|
Yes
|
AFI
|
None
|
-
|
264
|
E. coli
|
Yes
|
No
|
No
|
SSTI
|
Ceftriaxone, metronidazole
|
Ineffective
|
272
|
E. coli
|
Yes
|
Yes
|
Yes
|
SSTI
|
Cloxacillin
|
Ineffective
|
275
|
E. coli
|
Yes
|
No
|
No
|
SSTI
|
Ceftriaxone, metronidazole
|
Ineffective
|
351
|
E. coli
|
Yes
|
Yes
|
Yes
|
SSTI
|
Ceftriaxone metronidazole
|
Ineffective
|
411
|
E. coli
|
Yes
|
Yes
|
Yes
|
AFI
|
None
|
-
|
423
|
E. coli
|
No
|
Yes
|
Yes
|
RTI
|
None
|
-
|
432
|
E. coli
|
Yes
|
Yes
|
Yes
|
RTI
|
Azithromycin, ceftriaxone
|
Ineffective
|
440
|
E. coli
|
Yes
|
Yes
|
Yes
|
UTI
|
Ceftriaxone
|
Ineffective
|
483
|
E. coli
|
Yes
|
Yes
|
No
|
AFI
|
Azithromycin, ceftriaxone
|
Ineffective
|
503
|
E. coli
|
No
|
No
|
No
|
RTI
|
None
|
-
|
568
|
E. coli
|
No
|
No
|
No
|
AFI
|
None
|
-
|
639
|
E. coli
|
Yes
|
No
|
No
|
UTI
|
None
|
-
|
42
|
K. pneumoniae
|
No
|
No
|
No
|
AFI
|
Ceftriaxone, gentamicin
|
Effective
|
59
|
K. pneumoniae
|
No
|
No
|
No
|
RTI
|
Ceftriaxone, metronidazole
|
Effective
|
64
|
K. pneumoniae
|
No
|
Yes
|
No
|
Meningitis / encephalitis
|
Ceftriaxone
|
Effective
|
278
|
K. pneumoniae
|
Yes
|
Yes
|
Yes
|
SSTI
|
Ceftriaxone, cloxacillin
|
Ineffective
|
314
|
K. pneumoniae
|
Yes
|
Yes
|
Yes
|
SSTI
|
Ceftriaxone, vancomycin
|
Ineffective
|
332
|
K. pneumoniae
|
Yes
|
Yes
|
Not
|
AFI, sepsis
|
Ceftriaxone, gentamicin
|
Effective
|
545
|
K. pneumoniae
|
Yes
|
Yes
|
Yes
|
UTI
|
Tuberculostatic treatment
|
Ineffective
|
677
|
K. pneumoniae
|
Yes
|
Not
|
No
|
RTI
|
Ceftazidime, vancomycin
|
Ineffective
|
681
|
K. pneumoniae
|
Yes
|
Yes
|
No
|
RTI
|
Ceftriaxone, vancomycin
|
Ineffective
|
3GC: 3rd generation cephalosporin; UTI: Urinary tract infection; RTI: respiratory tract infection; AFI: Acute febrile illness (febrile disease with unknown source); SSTI: skin and soft tissue infection;