Haemophilus Influenzae Infections Workup

Updated: Aug 30, 2023
  • Author: Joseph Adrian L Buensalido, MD; Chief Editor: Pranatharthi Haran Chandrasekar, MBBS, MD  more...
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Laboratory Studies

Gram stain

Test results on body fluids from various sites of infection that reveal small, gram-negative, pleomorphic coccobacilli with polymorphonuclear cells are strong evidence of infection.

Bacterial culture

Detection of the organism in a blood culture or any other body fluid is the most confirmatory method of establishing the diagnosis. Optimal growth requires the use of chocolate agar and BVCCA, which is a selective media of chocolate agar that contains bacitracin, vancomycin, and clindamycin. BVCCA has been found superior to chocolate agar for isolation of NTHi from nasopharyngeal swabs. [47] Some H influenzae strains grow best in 5-10% carbon dioxide. [36]

The viability of H influenzae is lost rapidly, so clinical specimens should be inoculated to an appropriate culture media without delay. [36]

Seventy to 90% of patients with epiglottitis have positive blood culture results. However, to avoid laryngospasm, perform venipuncture and cultures of the inflamed epiglottitis only after the airway has been secured.


Slide agglutination with type-specific antisera is used for serotyping H influenzae. In one study, molecular typing with PCR was found to be more accurate than slide agglutination serotyping. [48]

Immunologic studies

Detection of the PRP polysaccharide capsule via countercurrent immunoelectrophoresis, latex particle agglutination, co-agglutination, and enzyme-linked immunosorbent assay is an important adjunct to culturing in establishing a rapid diagnosis.

Even if antibiotics were previously administered, the diagnosis can be confirmed based on the detection of the polysaccharide capsule in body fluids, including serum, CSF, urine, and pleural, pericardial, and articular fluid. False-positive results in CSF are rare but occur with serum or urine because of nonspecific agglutination and antigenic cross-reactivity with other bacteria.

CSF features

In meningitis, the CSF examination demonstrates pleocytosis (mean, 4000-5000 WBCs/µL) with a predominance of neutrophils.

Decreased CSF glucose levels are encountered in 75% of patients, increased CSF protein levels and detectable capsular antigen in 90%, and a positive CSF Gram stain result in 80%.

Prior antibiotic treatment significantly decreases the H influenzae type b (Hib) concentration in the CSF and decreases the sensitivity of the Gram stain; however, antibiotics do not substantially affect the total CSF blood cell count, differential, chemistries, and presence of the PRP capsule in pretreated patients.

Blood cell counts

Perform blood cell counts for anemia, leukocytosis, and thrombocytosis or thrombocytopenia.

Acute phase reactants

Elevated erythrocyte sedimentation rates (ESRs) and C-reactive protein levels are characteristically observed in patients with septic arthritis.

Nucleic acid amplification tests

Nucleic acid amplification diagnostics offer a rapid and accurate method of diagnosing microbial pathogens. Compared with conventional microbiological approaches, real-time PCR (rtPCR) has been shown to accurately differentiate NTHi from Haemophilus haemolyticus, a closely related species that is generally considered to be nonpathogenic. [49] Given the fastidious nature of H influenzae, PCR also has the advantage of detecting nonviable pathogens, especially among patients with previous antibiotic exposure. PCR-based methods are frequently used worldwide and are becoming the diagnostic method of choice in various clinical settings.

For H influenzae infection, PCR is more accurate than conventional methods of serotyping (slide agglutination method). The diagnostic sensitivity for Hib is 72-92%. [37]

In patients with pneumonia who are unable to provide lower respiratory tract samples, rtPCR of upper respiratory tract samples yields a sensitivity, specificity, PPV, and NPV of 75%, 80%, 45%, 94%, respectively, for H influenzae. [50]

Multiplex PCR and line probe assay

Multiplex PCR assay enables simultaneous detection of multiple pathogens, reducing analysis time and cost. It has been used for the diagnosis of multiple viral and bacterial pathogens, including H influenzae. Multiplex PCR assays are commonly used to evaluate CNS and respiratory tract infections.

Several in-house and commercial PCR-based assays have been described for the detection of meningitis caused by S pneumoniae, Neisseria meningitidis, and H influenzae. Sensitivity and specificity for detection of bacterial pathogens are high, ranging from 92.8-100% and 95.1-97.2%, respectively. [51, 52, 53]  A study looked at using real-time polymerase chain reaction (RT-PCR) of cerebrospinal fluid (CSF) as a rapid diagnostic test for bacterial meningitis and compared these with bacterial culture, and showed that sensitivity and specificity for RT-PCR were 100% and 95.46%, respectively. This becomes useful when antibacterial treatment was given before clinical samples were taken. [54]

For pneumonia, sensitivity (90%) and specificity (65%) are lower. [55] Nevertheless, multiplex PCR achieved greater pathogen detection(87%), roughly double that detected by culture-based methods (39%), even after antimicrobial administration (78% vs 32%). [56]  A 2023 study evaluating the clinical relevance of the Biofire©FilmArray pneumonia panel among hospitalized patients was done; the detection rate of Haemophilus influenzae (along with Staphylococcus aureus, and Moraxella catarrhalis) was high but not associated with pneumonia, and was much higher from bronchoalveolar lavage specimens. Further evaluation in randomized controlled trials was recommended. [57]

A line probe assay (LPA) that is based on multiplex PCR followed by reverse hybridization using sequence-specific oligonucleotide probes has been developed for the detection of bacterial pathogens that cause meningitis, specifically H influenzae, S pneumoniae, and N meningitidis. [58] Compared with standard PCR as reference, the overall sensitivity and specificity of LPA for the detection of bacterial pathogens were 76% and 88%, respectively. For Hib, LPA sensitivity and specificity were 88% and 96%.

A novel technology (VAPChip assay) currently under development combines bacterial species identification and detection of resistance genes associated with beta-lactam resistance encoding carbapenemases, extended-spectrum beta-lactamases, and penicillin-binding protein 2a. It was designed to detect nosocomial pneumonia pathogens, including H influenzae. The VAPChip system showed sensitivity and specificity of 72.9% and 99.1%, respectively, for the identification of respiratory pathogens. Detection of resistance genes, however, needs improvement. [59]

Isothermal NAT

Because PCR testing requires expensive thermocycler equipment and a skilled operator, it cannot be used as a point-of-care test or in resource-limited settings. Isothermal nucleic acid amplification methods provide a cheaper alternative to PCR, as they do not require thermocycler equipment. Duplex recombinase polymerase amplification (RPA) is a novel isothermal NAT method used to detect pathogens that cause meningitis, specifically H influenzae, S pneumoniae, and N meningitidis. [60] It exhibited 100% sensitivity and 100% specificity for the diagnosis of H influenzae meningitis.

Proteomic profiling

Proteomic profiling via matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an alternative to biochemical and molecular methods of species identification. Studies have shown that MALDI-TOF MS accurately differentiates between NTHi and H haemophilus, a respiratory tract commensal. [61, 62] Apart from species identification, the sensitivity and specificity of MALDI-TOF MS for capsule typing was found to be 100% and 92.2%, respectively. [63] In another study, the sensitivity and specificity for identification of type b, type e, and type f capsular serotypes and NTHi were 100% and 94.3%, 94.7% and 97.9%, 97.4% and 97.9%, and 85.5% and 99.2%, respectively. [64] MALDI-TOF MS is quicker to perform than PCR and conventional serotyping (slide agglutination) methods and has a lower cost per sample, making it a useful tool for H influenzae surveillance and outbreak investigations.

Whole-genome–sequencing–based tools

Whole–genome–sequencing (WGS)–based tools identify species by comparing sample genomes against a reference collection of representative genomes. Owing to long computational runtime, WGS-based tools once were considered impractical for routine clinical use. An innovation that addressed this limitation was developed in BMScan, [65] which allows rapid and accurate identification of bacterial meningitis pathogens, including H influenzae and related species, N meningitidis and related species, S pneumoniae, Listeria monocytogenes, and Escherichia coli. Topaz et al demonstrated that BMScan accurately identified 99.97% of species of interest within an average of 16 minutes and 47 seconds, meaning it has potential for clinical use. This is a proof-of-concept study that can be extended to other pathogens and clinical settings. In the future, this could lead to transition from the traditional phenotypic detection method to genome–sequencing–based methods of identification.


Imaging Studies

CT scanning

In meningitis, a CT scan of the head is not required routinely.

In infants and children with suspected bacterial meningitis, CT scanning of the head is recommended before lumbar puncture in patients with the following [6] :

  • Immunocompromise
  • History of selected CNS disease, particularly those with CSF shunts, hydrocephalus, space-occupying lesions, history of trauma, post-neurosurgery
  • Papilledema
  • Selected focal neurologic deficits, but not cranial nerve palsy of VI or VII
  • Delayed diagnostic lumbar puncture

In adults with suspected bacterial meningitis, CT scanning is recommended before lumbar puncture in patients with the following [6] :

  • Immunocompromise
  • History of CNS disease
  • Newly onset seizure
  • Papilledema
  • Altered consciousness
  • Focal neurologic deficit
  • Delayed diagnostic lumbar puncture

In addition, a head CT scan may help identify subdural effusion.

In patients with orbital cellulitis, a CT scan of the head is useful in delineating the extent of the lesion.

Chest radiography

Patients with Hib pneumonias tend to have more pleural and pericardial involvement (50% of patients) than those with other bacterial pneumonias.

Community-acquired pneumonias due to NTHi are characterized by alveolar infiltrates in patchy or lobar distributions.

Lateral neck radiography

In epiglottitis, a lateral neck radiograph reveals dilatation of the hypopharynx and a swollen epiglottis (termed the thumbprint sign). In addition, the cervical spine is usually straightened.

If epiglottitis is clinically suspected, obtain radiography only if a functional airway is guaranteed.


Obtain echocardiography when pericarditis is suspected.


Other Tests

In patients with cellulitis, direct aspiration of the soft tissue or aspiration after injecting the subcutaneous tissue with sterile nonbacteriostatic solution can be used to detect the organisms via Gram stain and culture.



Perform a lumbar puncture when meningitis is suspected. CT scanning should be performed prior to lumbar puncture in certain cases (see Imaging Studies).

The following invasive procedures can be used to obtain appropriate fluid and to establish an etiologic diagnosis:

  • Bronchoscopy
  • Joint, lung, sinus, and soft-tissue aspiration
  • Transtracheal aspiration
  • Tympanocentesis
  • Pericardiocentesis

In women, obtain tubal cultures via laparoscopy and peritoneal fluid cultures by culdocentesis for NTHi.

In patients with epiglottitis, use endotracheal intubation or tracheostomy to secure an airway.