Direct detection of the multidrug resistance genome of Haemophilus influenzae in cerebrospinal fluid of children: implications for treatment of meningitis.
BACKGROUND
Multidrug resistance (MDR), specifically to ampicillin and chloramphenicol, has complicated the treatment of Haemophilus influenzae type b (Hib) meningitis. This is worsened by use of prior antibiotics, which limits identification of the causative agent by culture and increases reliance on antigen detection.


OBJECTIVE
We aimed to develop a PCR assay for detecting the family of Haemophilus integrating and conjugative elements (ICEs) represented by ICEHin1056 among antibiotic resistant Hib, and then apply this directly to CSF to diagnose Hib meningitis and predict organism susceptibility, irrespective of culture results.


METHODS
Primers specific for orf 51 of ICEHin1056 were designed and multiplexed with Bex primers, specific for H. influenzae, and tested on culture positive and negative cases.


RESULTS
Of 73 Hib isolates, orf 51 PCR amplicons, predicting the presence of ICEs, were found in all 33 MDR isolates while only in 1 of 33 sensitive strains. The remaining 7 ampicillin susceptible, chloramphenicol and tetracycline resistant strains did not produce a PCR product to orf 51. PCR amplification from CSF specimens of these culture positive cases produced identical results with 100% and 97% positive and negative predictive values, respectively. Multiplex PCR to detect Bex and orf 51 identified another 16 MDR Hib cases among 81 culture-negative CSF samples.


CONCLUSIONS
Direct PCR for orf 51 in CSF identified resistance pattern of 51% more Hib strains than culture alone (110 versus 73). The ability to detect MDR, in culture negative Hib meningitis cases has significant implications for better directing antibiotic treatment of meningitis cases and thus for preventing disability and death.
Saha
Samir K
SK
Department of Microbiology, Dhaka Shishu Children Hospital, Dhaka, Bangladesh. sksaha@bangla.net
Darmstadt
Gary L
GL
Baqui
Abdullah H
AH
Islam
Nurul
N
Qazi
Shamim
S
Islam
Maksuda
M
El Arifeen
Shams
S
Santosham
Mathuram
M
Black
Robert E
RE
Crook
Derrick W
DW
D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D000667:Ampicillin / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D002555:Cerebrospinal Fluid / Q000382:microbiology*; D002675:Child, Preschool; D002701:Chloramphenicol / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D019741:Haemophilus influenzae type b / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007223:Infant; D020071:Interspersed Repetitive Sequences / Q000235:genetics; D008583:Meningitis, Haemophilus / Q000175:diagnosis* / Q000188:drug therapy* / Q000382:microbiology; D008826:Microbial Sensitivity Tests; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D013752:Tetracycline / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C068101:bexA protein, Haemophilus influenzae; D002701:Chloramphenicol; D000667:Ampicillin; D013752:Tetracycline