A Sputum Screening Test to Rule-out Pneumonia at an Early Stage

A lower respiratory tract infection is a serious situation that can abruptly become complicated by sepsis, respiratory failure, lung tissue necrosis, and multiple organ dysfunction. Hence, empiric antibiotic treatment is commonly initiated as soon as infection is suspected and cultures and/or other diagnostic tests are procured, representing a major reason for antibiotic prescription. Widespread antibiotic overuse and misuse have led to the emergence of multiple resistant bacterial strains, posing a major health threat. There exists a critical need for practical solutions to prevent antibiotic overuse, especially in communities where antibiotics are available without prescription.

Pathophysiologic studies show that infection does not result from bacterial or viral overgrowth alone, but rather from the microorganisms' penetration beyond the host immune system. Therefore, infection assessment requires investigation of both microorganism presence and the defense mechanisms activated within the patient's body. Bacteria overgrowth leads to lung tissue inflammation, recruitment of white blood cells to the infected area, and chemokine and cytokine production and release, which can cause the alveoli to become filled with fluid, leading the patient to develop a cough with phlegm or pus, fever, chills, and dyspnea.

Microbiological diagnosis of lower respiratory tract infections requires assessment of the invading microorganism by examination of sputum using microscopy, quantitative culture, and PCR. However, such microbiological investigations have limited value in pneumonia management. Polymicrobial flora make it difficult to interpret cultures from patients with chronic bronchitis. It is also difficult to evaluate cultures from nosocomial pneumonia since the pathogenic bacteria are often identical to those appearing in the throat flora. Immunosuppressed patients frequently produce sputum containing low number of white blood cells. The patient's clinical signs, such as respiratory and circulatory status, are the most reliable markers for determining treatment efficacy.

In practice, most physicians choose to examine additional markers at early stages to closely monitor the effects of therapy, particularly in high-risk patients who have been admitted to intensive care units. Commonly used systemic markers include body temperature, C-reactive protein (CRP), procalcitonin (PCT), and interleukin 6 (IL-6). Hepatocyte growth factor (HGF) concentration reportedly increases during organ damage, such as that caused by infectious diseases. Studies show increased HGF concentrations in serum and exhaled-breath condensate from patients with pneumonia, with HGF presence being significantly correlated with survival. Moreover, HGF levels markedly decrease within 48 hours of initiating appropriate antibiotic therapy. Surface plasmon resonance (SPR) results indicate that HGF produced during acute infection shows high affinity for the extracellular matrix component heparan sulfate proteoglycan (HSPG). These findings suggest that HGF assessment in sputum could be a tool for detecting bacterial infection at the site of injury.

Proteins can be detected based on their specific interaction with a corresponding antibody. However, this measurement system relies on specialized resources, limiting its usefulness in non-equipped centers or as a self-test. Metachromasia is a characteristic color change exhibited by certain aniline dyes upon binding to chromotropic substances. This phenomenon has been widely used in histology. Methylene blue (O-Toluidine) is an excellent metachromatic dye that changes from blue to pink upon binding to high-molecular-weight polysaccharides, such as sulfated glycan. The pink dye will then quickly turn back to blue following addition of a proportional amount of a protein with high affinity to sulfated glycan (inverted metachromacia).

Here the investigators used this approach to develop a new strip test-referred to herein as the index text-to assess the presence of dextran-sulfate-binding proteins in sputum. The investigators then assessed the accuracy of this strip test for detecting bacterial infection in sputum, by analyzing leftover sputum samples that were sent for examination to the Department of Microbiology, University hospital in Linköping.

Inclusion criteria

• Sputum samples collected for routine diagnostic and reached the laboratory within 12 hours after Collection and considered as representative by microscopy and kept 4-8 C after Culture.

Exclusion criteria

• Samples not collected as above