International Journal for Sciences and Technology
Vol. 8, No. 1, March 2013 62
Prevalence of Neisseria meningitidis in Iraqi children presented with meningitis Atheer A. Razak (1), Harith J. F. Al-Mathkhury (2), Kifah A. Jasim (3), Modhafar Q. Saber (4) & Abdul Jabbar N.Al-Shammari (5) College of Veterinary Medicine, University of Baghdad(1), Dept. of Biology/College of Science/ University of Baghdad (2), Central Public Health Laboratory/ Baghdad (3), (4) - Iraq, Faculty of pharmacy/ Isra University/ Jordan (5) E-mail:
[email protected]
ABSTRACT Neisseria meningitidis is a leading cause of bacterial meningitidis and septicemia in children and young adults. Rapid and consistent identification of N. meningitidis serogroups is critical for thoughtful and convenient response to cases of meningococcal infections. N. meningeitidis isolates collected in Iraq through the Children Welfare Teaching Hospital in Baghdad and confirmed by the Central Public Health Laboratory (CPHL), Baghdad. Out of fifty suspected specimens of cerebrospinal fluid (CSF) two isolates of N. meningitidis were detected using culture and Real Time-PCR techniques (RT-PCR). Api NH confirmed the results of culture. Slide Agglutination Serogrouping (SASG) revealed that both isolates were belonging to the serogroup W135. This study compared the conventional methods with the RT-PCR for detecting N. meningitidis with specific primers targeting ctrA in short time with high specificity and quality.
ﺍﻝﻤﻠﺨﺹ ﺒﺎﻝﻠﻐﺔ ﺍﻝﻌﺭﺒﻴﺔ ﻭ ﻋﻠﻴـﻪ،ﺘﻌﺘﺒﺭ ﺍﻝﻨﺎﺴﻴﺭﻴﻪ ﺍﻝﻤﻨﻨﺠﺎﻴﺘﺱ ﻤﻥ ﺍﻻﺴﺒﺎﺏ ﺍﻝﺭﺌﻴﺴﻴﺔ ﺍﻝﺘﻰ ﺘﺅﺩﻯ ﺍﻝﻰ ﺍﻝﺘﻬﺎﺏ ﺍﻝﺴﺤﺎﻴﺎ ﺍﻝﺒﻜﺘﺭﻯ ﻭﺘﺠﺭﺜﻡ ﺍﻝﺩﻡ ﻋﻨﺩ ﺍﻻﻁﻔﺎل ﻭﺍﻝﺒﺎﻝﻐﻴﻥ ﺠﻤﻌﺕ ﺨﻤـﺴﻭﻥ.ﻓﺎﻥ ﺍﻴﺠﺎﺩ ﻁﺭﻴﻘﺔ ﺴﺭﻴﻌﺔ ﻭﻓﻌﺎﻝﺔ ﻝﺘﺸﺨﻴﺹ ﻫﺫﻩ ﺍﻝﺠﺭﺜﻭﻤﺔ ﻴﻌﺘﺒﺭ ﺒﺎﻝﻎ ﺍﻻﻫﻤﻴﺔ ﻝﻼﺴﺘﺠﺎﺒﺔ ﻤﺩﺭﻭﺴﺔ ﻭﺫﺍﺕ ﻤﺭﺩﻭﺩ ﻓﻌﺎل ﺒﻐﺩﺍﺩ/ﻋﻴﻨﺔ ﻤﻥ ﺍﻝﺴﺎﺌل ﺍﻝﻨﺨﺎﻋﻰ ﻤﻥ ﺍﻻﻁﻔﺎل ﻓﻰ ﻤﻨﻁﻘﺔ ﺒﻐﺩﺍﺩ ﻭﺠﺭﻯ ﺘﻭﺼﻴﻑ ﻭﺘﺸﺨﻴﺹ ﺍﻝﺒﻜﺘﺭﻴﺎ ﺍﻝﻤﺴﺒﺒﺔ ﻓﻰ ﻤﺨﺘﺒﺭ ﺍﻝﺼﺤﺔ ﺍﻝﻤﺭﻜﺯﻯ ﺘﻡ ﺘﻭﺼﻴﻑ ﻋﺯﺍﺘﻴﻥ ﻋﻠﻰ ﺍﻨﻬﻤﺎ ﺒﻜﺘﺭﻴﺎ ﺍﻝﻨﺎﺴﻴﺭﻴﺔ ﺍﻝﻤﻨﻨﺠﺎﻴﺘﺱ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺘﻔﺎﻋل ﺍﻝﺒﻠﻤﺭﺓ ﻓﻰ ﺍﻝﻭﻗﺕ ﺍﻝﺤﻘﻴﻘﻰ ﻭﻜﺫﻝﻙ ﺒﺎﻝﻤﻘﺎﺭﻨﺔ ﻤﻊ ﻓﺤـﺹ. ﻗﺎﺭﻨﺕ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﺒﻴﻥ ﺍﻝﻁﺭﻕ. 135 ﺘﻡ ﺘﺤﺩﻴﺩ ﺍﻝﻨﻤﻁ ﺍﻝﻤﺼﻠﻰ ﻭﺍﻝﺫﻯ ﻴﻌﻭﺩ ﺍﻝﻰ ﺍﻝﻨﻭﻉ ﺍﻝﻤﺼﻠﻰ ﺩﺒﻠﻴﻭ.ﺍﻻﻴﺒﻰ ﻭﺍﻝﺘﻼﺯﻥ ﻋﻠﻰ ﺍﻝﺸﺭﻴﺤﺔ .ﺍﻝﺘﻘﻠﻴﺩﻴﺔ ﻝﺘﺸﺨﻴﺹ ﻫﺫﻩ ﺍﻝﺒﻜﺘﺭﻴﺎ ﻤﻊ ﻓﺤﺹ ﺘﻔﺎﻋل ﺍﻝﺒﻠﻤﺭﺓ ﻓﻰ ﺍﻝﻭﻗﺕ ﺍﻝﺤﻘﻴﻘﻰ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺒﺎﺩﻯ ﺨﺎﺹ ﻴﻌﻁـﻰ ﻨﺘـﺎﺌﺞ ﺴـﺭﻴﻌﺔ ﻭﺩﻗﻴﻘـﺔ
International Journal for Sciences and Technology
INTRODUCTION Neisseria meningitidis is a Gram-negative diplococcal bacterium which considered as an important cause of morbidity and mortality worldwide. Additionally, it is the leading cause of bacterial meningitis and septicemia (1). There are 13 clinically significant serogroups in this species, which are classified according to the antigenic structure of their polysaccharide capsule. Five serogroups (A, B, C, Y and W135) are responsible for nearly all cases of the disease. However, serogroup B is the predominant one (2,3). Changes in the epidemiology of meningococcal disease have imposed important implications for vaccination and on the prevention strategies (4). Up to 36 hr is needed to identify N. meningitidis by conventional culture methods. What's more, starting antimicrobial treatment prior to specimen collection will decrease the capacity to confirm detection of the causative agent of bacterial meningitis and septicemia (5). Unlike biochemical tests and slide agglutination serogrouping, polymerase chain reaction (PCR) does not require viable bacteria and can be used to identify and characterized even non groupable N. meningitidis meningococci (6,7). Additionally, it is necessary to detect small numbers of N. meningitidis in clinical specimens; bacterial loads in cerebrospinal fluid (CSF) of patients range from 3×10¹ to 4×10³ CFU/ml (8,9). TagMan RT-PCR has been shown to detect as few as 8 meningococcal genomes per reaction (10,11). The capsular transport protein (ctrA) gene is unique to N. meningitidis and can be found in all meningococcal serogroups (12). Therefore it may be the most frequently targeted gene to detect N. meningitidis using PCR (13). This study aimed to detect of N. meningitidis isolated from CSF by RT-PCR assay in Iraqi children.
MATERIALS AND METHODS Isolation and identification Fifty CSF specimens from young children (114 year) presented with meningitis were cultured as soon as possible after collection on blood agar and chocolate agar and incubated for 24-48h at 37ºC with 10% CO2.
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Identification was achieved by api NH (bioMérieux -France). Serogrouping The sero- grouping was done with 50µl of each biological sample by using commercial kit (BD UK) according to the manufacturer's instructions. Antibiotic susceptibility test (AST) Neisseria meningitidis isolates were test for AST using Amoxicillin (20µg), sulfamethoxazole-trimethoprim (10µg), ceftriaxone (30µg), cefotaxime (30µg), and gentamicin (10µg) on chocolate agar and incubated at 37ºC with 10%CO2. Standardization of bacterial culture Clinical isolates of pure culture was emulsified in 2ml of sterile injectable water in a microbiological class 2 safety cabinet. Using a spectrophotometer (Pharmacia, St. Albans, England) set at 650 nm, the bacterial suspension was standardized to an optical density of 0.1 and adjusted to a concentration of approximately 2×104 cell/ml, which represents 40 cells per 2µl of inoculum. DNA extraction peqGOLD bacterial DNA kit was used for the isolation of up to30µg genomic DNA from CSF specimens and bacterial cultures following the manufacturer's instructions. The extracted DNA was stored at −20°C. RT-PCR A partial region of ctrA (110bp) was amplified using 300nM (each) specific primers. The bacterial isolates and CSF samples were analyzed by real-time PCR (RT -PCR) as reported by Corless et al. (14). Briefly, based on a 25-ml reaction volume, the master mixture was prepared from the (Taqman, Applied Biosystem, USA), this comprises a 300 nM concentration of each oligonucleotide 617 primer (ctrA Forward 635 and ctrA GCTGCGGTAGGTGGTTCAAReverse 727-TTGTCGCGGATTTGCAACTA708 ); 25 nM 6-carboxyfluorescein-labeled probe (6-FAM–680CATTGCCACGTGTCAGCTGCACAT-657); 200 mM (each) 5.5 mM MgCl2; deoxynucleoside triphosphates dATP, dCTP,
International Journal for Sciences and Technology dGTP, and dUTP; and 0.125 U of Taq DNA polymerase. A negative (no-template) control and control DNA preparations (2 ml) for each of the bacterial pathogens were included in every run. DNA was amplified with the Applied Biosystem 7500 system (Applied Biosystem Inc.,USA) using the following cycling parameters: heating at 95°C for 10 min followed by 40 cycles of a two-stage temperature profile of 95°C for 15 s and 60°C for 1 min. RT-PCR results were based on the fluorescence readings, which are used to calculate a baseline reading for each reaction. The cycle threshold (CT) value is the PCR cycle number (out of 40) at which the measured fluorescent signal exceeds a calculated background threshold identifying amplification of the target sequence. If no increase in fluorescent signal is observed after 40 cycles, the sample is assumed to be negative.
RESULTS Isolation and identification Fifty CSF specimens were collected and cultured on blood agar and chocolate agar incubated for 24-48 hours at 37ºC with 10% CO2. On a blood agar, colonies of N. meningitidis were grey, unpigmented round, smooth, moist, glistening, and convex, with a clearly defined edge. On chocolate agar they appeared large, colorless and opaque. The results revealed that two bacterial isolates (N6 and N23) were identified as N. meningitidis by api NH. Serogrouping were employed to identify the type of N. meningitidis by using N. meningitidis antisera, the results showed that both of them were belonging to the serogroup W135.
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Fig(1): Antibiotic susceptibility of isolated N. meningitides.
RT-PCR Results from real-time PCR assays using nucleic acids extracted from CSF specimens and bacterial cultures are given in Fig.(2). Real-time PCR using primers and probes targeting the capsule transport gene, ctrA, of N meningitidis confirmed the presence of N meningitidis in all positive samples. Serogroup-specific, real-time PCR assays were able to determine the N meningitidis serogroup W135. Of the 50 cases of N meningitidis serogroupW135, were confirmed using Aggultination test and RT-PCR.
Antibiotic susceptibility AST illustrated that the two isolates were sensitive to amoxicillin, cefotaxime, ceftriaxone, chloramphenicol, and resistant to sulfamethoxazole trimethoprime (fig.1). Fig(2): RT-PCR analysis revealing the presence of ctrA of N. meningitidis
International Journal for Sciences and Technology
DISCUSSION The prevalence of N. meningitides was relatively high (4%) in Iraqi children in comparison to global incidence of meningococcal carriage which is shown to be
