Pediatric invasive disease due to Haemophilus influenzae serogroup A in Riyadh , Saudi Arabia : case series

We describe the first two cases of invasive disease caused by Haemophilus influenzae serotype A in Saudi Arabia. This is the first known reported invasive Haemophilus influenzae serotype A from Saudi Arabia. Case presentation: A ten-month-old and three-month-old male not known to have any past history of any medical illness and who had received H. influenzae type b (Hib) vaccine presented to our hospital mainly with fever of few days’ duration. A provisional diagnosis of meningitis with sepsis was made and laboratory tests were requested. The chest radiograph was normal. The laboratory results revealed leukocytosis, but leukopenia was noticed in the younger infant. Blood culture and cerebrospinal fluid specimens yielded a pure culture of Haemophilus influenzae and serotyping showed the isolates to be serogroup A. Both patients were started on vancomycin and third-generation cephalosporin. On receiving the blood culture result, vancomycin was stopped. Fever subsided after 48 hours, while in the second case, it continued for 12 days from the admission date. The repeat blood cultures were negative. Antibiotic therapy was given for 10 days for the first case with an unremarkable hospital course, while the second case was complicated by seizure and received a longer duration of antibiotics. Both infants were discharged home in good condition. Conclusions: Invasive non-typeable H. influenzae strains are emerging and there is a need for surveillance of this disease. This has implications in future vaccine development.


Introduction
Haemophilus influenzae is a small, non-motile, non-spore-forming, pleomorphic, Gram-negative coccobacillus; some strains possess a polysaccharide capsule.These strains are serotyped into six different types (A-F) based on the biochemical composition of their capsules.Some strains have no capsule and are termed non-encapsulated or non-typeable H. influenzae (NTHi).The different serotypes can be identified with slide agglutination or polymerase chain reaction (PCR) [1].
The most virulent strain is H. influenzae type b (Hib), with its polyribosylribitol phosphate capsule.It accounts for more than 80% of H. influenzae invasive diseases in children [2].The other encapsulated strains of H influenzae occasionally cause invasive disease similar to that of Hib.To a lesser extent, H. influenzae type a (Hia) has been reported to cause invasive disease (e.g., meningitis) clinically indistinguishable from that caused by Hib [3].
We report the first two cases of meningitis due to H. influenza serogroup A in infants in Riyadh, Saudi Arabia.

Case 1
H. influenzae serogroup A was isolated from blood culture and cerebrospinal fluid (CSF) samples from a 10-month-old boy admitted to King Fahad Medical City, Riyadh, Saudi Arabia on 18 April 2014 with fever and vomiting of two days' duration.The patient was not known to have any past history of any medical illness and had received two doses of Hib vaccine (information from parents and the vaccination card).The Hib vaccines were administered at two and four months in accordance with the Expanded Program of Immunization (EPI) schedule of the Kingdom of Saudi Arabia.The third dose of six months was not administered owing to some social problems encountered by the parents.
Examination revealed an ill-looking, lethargic, febrile child (body temperature 39°C, pulse rate 168, respiratory rate 60, blood pressure 101/57mmHg) with a bulging anterior fontanelle.Muscle tone, power, and reflexes were normal.All other systems were normal.A provisional diagnosis of meningitis with sepsis was made and blood specimens for full blood count (FBC), electrolytes, liver function, blood gases, coagulation profile, and blood culture together with cerebrospinal fluid and nasopharyngeal aspirate for respiratory viruses were submitted for analyses.The chest radiograph was normal.
The FBC profile revealed leukocytosis (31.4×10 9 /L; normal: 6-18×10 9 /L); the serum sodium was low (131 mmol/L; normal: 135-145mol/L), but all the other parameters were within the normal range.The CSF white cell count was high (1,215/mm 3 with 86% neutrophils), red cell count was 36/mm 3 , the protein value was also high 1.1g/L (normal: 0.15-0.45g/L), the glucose was low (2.9 mmol/L; serum glucose 5.9 mmol/L), and no organisms were seen on direct Gram stain.The CSF PCR for herpes simplex virus types 1 & 2 was negative.The PCR for respiratory viruses' panel (adenovirus, coronavirus, parainfluenza virus 1-4, respiratory syncytial virus [RSV] A&B, influenza A&B, metapneumovirus, enterovirus, human bocavirus) was also negative.Latex agglutination was negative on CSF.Blood culture and CSF specimens yielded a pure growth within 24 hours of incubation.Identification of bacterial species was performed using a Phoenix automated system (Becton Dickinson, San Diego, USA).Susceptibility was performed using Etest (AB Biodisk Solna, Sweden) according to the breakpoints identified by the Clinical Laboratory and Standards Institute (CLSI) breakpoints.Serotyping was performed by direct agglutination using specific rabbit antisera (BD Bioscience, Sparks, USA).Haemophilus influenzae was identified and serotyping revealed that it belonged to serogroup A. The isolate was betalactamase negative and susceptible to ampicillin (minimum inhibitory concentration [MIC] 0.38 µg/mL; breakpoint 1 µg/mL) and ceftriaxone (MIC 0.5 µg/mL; breakpoint 2 µg/mL).
The patient was admitted to the intensive care unit with a working diagnosis of meningitis with sepsis, and was started on vancomycin and ceftriaxone therapy (cefotriaxone: 100 mg per kg/day; cefotaxime: 200 mg per kg/day; vancomycin: 60 mg per kg/day).On receiving the blood culture result, vancomycin was stopped and the patient was transferred to the ward after 72 hours in a stable condition.The fever subsided after 48 hours from admission and the repeat blood culture after 48 hours was negative.Antibiotic therapy was given for 10 days and the infant was discharged home in a good condition.Immunological work-up, which included immunoglobulin assay and complement function performed during hospitalization, were normal.Hearing and visual assessment performed during the follow-up period one month after discharge was normal.Institutional approval from King Fahad Medical City Institution review board (IRB00008644) was obtained.

Case 2
H. influenzae serogroup A was isolated from blood culture and cerebrospinal fluid samples from a threemonth-old boy who presented at King Fahad Medical City, Saudi Arabia in September 2014.The patient had received one dose of Hib vaccine (information from parents and the vaccination card).The Hib vaccines were administered at two months of age in accordance with the EPI schedule of the Kingdom of Saudi Arabia.
The infant presented to the emergency room with fever and irritability of five days' duration.Examination revealed an ill-looking, lethargic, febrile child (body temperature 39°C, pulse rate 210, respiratory rate 44, blood pressure 90/50mmHg) with a bulging anterior fontanelle.Muscle tone, power, and reflexes were normal.All other systems were normal except for the cardiovascular exam, which revealed a systolic murmur clinically.A ventricular septal defect was detected by echocardiogram; however, there was no vegetation.A provisional diagnosis of meningitis with sepsis was made and blood specimens for FBC, electrolytes, liver function, coagulation profile, and blood culture together with cerebrospinal fluid and nasopharyngeal aspirate for respiratory viruses were submitted for analyses.
The FBC profile revealed leukopenia (2.89×10 9 /L; normal: 6-18×10 9 /L); electrolytes were within the normal range.The CSF white cell count was high (1,224/mm 3 with 89% neutrophils), red cell count was 8/mm 3 , the protein value was also high (1.3 g/L; normal: 0.15-0.45g/L), the glucose was low (2.5 mmol/L; serum glucose 6 mmol/L), and no organisms were seen on the Gram stain.PCR for respiratory viruses' panel (adenovirus, coronavirus, parainfluenza virus 1-4, RSV A&B, influenza A&B, metapneumovirus, enterovirus, human bocavirus) was also negative.Direct latex agglutination on CSF was negative.Blood culture and CSF specimens yielded a pure growth within 24 hours of incubation.Identification of bacterial species was performed using a Phoenix automated system (Becton Dickinson, San Diego, USA).Susceptibility was performed using Etest (AB Biodisk, Solna, Sweden) according to the breakpoints identified by the CLSI.Serotyping was performed by direct agglutination using specific rabbit antisera (BD Bioscience, Sparks, USA).Haemophilus influenzae was identified and serotyping revealed that it belonged to serogroup A. The isolate was betalactamase negative and susceptible to ampicillin (MIC 0.5 µg/mL; breakpoint 1 µg/mL) and ceftriaxone (MIC 0.19 µg/mL; breakpoint 2 µg/mL).
The patient was admitted to the pediatric ward with a working diagnosis of meningitis with sepsis and was started on vancomycin and cefotaxime therapy.On receiving the blood culture result, vancomycin was discontinued.The repeat blood and CSF cultures after 72 hours were negative.The fever continued for a total of 12 days.The infant developed four attacks of generalized prolonged seizure, which lasted up to 30 minutes, with left-sided focality on two occasions.A CT scan of the head was done twice and revealed a normal result.Electroencephalography was also done with normal findings.Repeat blood workup revealed leukocytosis and thrombocytosis, improving over time, and a negative culture result as well.Antibiotic therapy was given for 14 days.The patient was discharged home in good condition on phenobarbitone.Hearing and visual assessment performed during the follow-up period one month after discharge was normal.

Discussion
Prior to the introduction of the Hib conjugate vaccine, Hib was the commonest cause of bacterial meningitis in children under five years of age.[7] Severe Hia invasive disease from Utah, USA Meningitis and bacteremia in 4 (3 females ages 6, 7, 12 months & 1 male 13 months), and a fifth case of pneumonia.