Application Of Resuscitation-promoting Factors In Contact Investigation And Clinical Diagnosis Of Tuberculosis

Tuberculosis (TB) remains a major killer and a great challenge to global health and caused by Mycobacterium tuberculosis (Mtb) infection. This is largely due to a sizeable reservoir of latent TB infection (LTBI) individuals,10%of them relapse into active disease decades after first acquiring the infection. The distinction between subsequent active tuberculosis and stable LTBI has encouraged many studies to identify the factors involved. The likelihood of infection depends on the intensity, frequency, and duration of exposure, intrinsic and acquired conditions of the contact affect the likelihood of TB disease progression after infection. Contacts of patients with TB active case are at higher risk than members of the general population. After exposure to airborne droplets containing Mtb, some contacts will be infected and some of these will progress to active disease. The onset of disease may occur early, within6weeks, or many years later. If we can identify the LTBI individuals who are at higher risk of progression to active TB, it will benefit the disease control because the sustained treatment of LTBI is effective.The transmission of TB occurs via aerosols containing infectious bacilli that are released from patients with active TB. Effective and rapid treatment renders patients non-contagious, and thus is very important to the control of TB. Treatment and control depend on timely and accurate diagnosis. However, due to the slow growth of the bacteria, conventional Mtb culture normally takes4-8weeks.Resuscitation-promoting factors (Rpfs) are secreted bacterial proteins that act as bacterial cytokines. Mtb Rpfs represent a family of autocrine growth factors that are mainly expressed in actively growing cultures of Mtb. Rpfs were initially characterized through their resuscitating effect on non-replicating cells in vitro and in vivo through lysozyme and peptidoglycan hydrolase effects.Existing results indicated that LTBI are able to recognise Rpf, even if TB exposure occurred a long period before. However, the response to Rpf in LTBI who have community exposure (CE) is still unknown. It is worth to study the differences responses to Rpf between LTBI caused by different proximity of TB exposure, and the potential of the responses to Rpf in predicting progression from LTBI to active TB.Rpf also have the potential in enhancing culture-based Mtb tests since an occult population in sputum was shown to be dependent on Rpfs for growth. But it is still not confirmed. So it is worth to study whether Rpf can shorten the time to positivity of culture-based Mtb test and satisfy the clinical needs. This project mainly contains two subjects:1. Application of Rpf in tuberculosis contact investigation.2. The effects of Rpf in culture-based Mtb test.Application of Rpf in contact investigation of tuberculosisWe selected LTBI caused by different proximity of TB exposure and divided into4groups:pulmonary tuberculosis patients (PTB), CE, household contacts (HHC) and healthy control (HC). Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with RpfA or RpfD, cultural supernatant were collected and used for interferon-γ (IFN-y), tumor necrosis factor-a (TNF-α) and superoxide anion (O2-) detection by enzyme-linked immuno sorbent assay (ELISA), nitric oxide (NO) detectiong by Griess. Cells were collected for transcriptional level detection of IFN-y and TNF-a by real time quantity polymerase chain reaction (RT-qPCR), frequency of CD4+IFN-y+cells by flow cytometry.We identified LTBI by interferon-y release assays (IGRAs) and Mtb-IgG detection in group CE and HHC, and grouped as LTBI-CE and LTBI-HHC. When we analysed the IFN-y concentrations of TB-IGRA, we observed no statistically significant differences between the LTBI-HHC and LTBI-CE (P>0.05). In this study, significant differences of IFN-y production were observed among PTB, LTBI-HHC, LTBI-CE and HC (for RpfA, LTBI-HHC and other groups P<0.01; PTB and LTBI-CE P<0.05; for RpfD, LTBI-HHC and other groups P<0.01). No significant difference was found of TNF-α,O2-and NO release (P>0.05). Significant differences of IFN-y transcriptional level were observed (for RpfA, LTBI-HHC and other groups P<0.01; for RpfD, LTBI-HHC and LTBI-CE P<0.01, LTBI-HHC and HC P<0.01, PTB and HC P<0.05). No significant difference was found of TNF-a transcriptional level (P>0.05). Significant differences were observed when we compared the CD4+IFN-γ+cells responses among PTB, LTBI-HHC, LTBI-CE and HC (for RpfA, LTBI-HHC and LTBI-CE and LTBI-HHC and HC P<0.05; for RpfD, LTBI-HHC and LTBI-CE and LTBI-HHC and PTB P<0.05, LTBI-HHC and HC P<0.01). No significant difference was found when we compared the CD4+TNF-a+cells responses (P>0.05). We compared the IFN-y production response to RpfA and RpfD between individuals displayed positive TB-IGRA results in PTB, HHC and CE. Results demonstrated that higher IFN-y production in TB-IGRA positive individuals from the HHC compared to the CE and PTB groups. The results were as follows:PTB and HHC-IGRA (+), HHC-IGRA(+) and CE-IGRA (+)(P<0.01) for RpfA and RpfD. Although there was a clear similar tendency for the CD4+IFN-γ+T-cells frequency these differences failed to reach statistical significance.In conclusion, TB is endemic in China, and potential TB exposure can occur anywhere. Our results showed that lower exposure level may not be sufficient to drive RpfA and RpfD immune responses. In LTBI-HHC with more intense exposure, stronger responses to RpfA and RpfD can be induced. LTBI-CE can induce ESAT-6and CFP-10reponses because they have a stronger immunogenicity than RpfA and RpfD. For this reason, however, RpfA and RpfD may be able to distinguish different exposure level between LTBI-HHC and LTBI-CE, as we document in our study. Intrinsic and acquired conditions of the contact affect the likelihood of TB disease progression after infection; we therefore believe it is worth to study the potential of the IFN-y responses to RpfA and RpfD in predicting progression from LTBI to active TB in further research.The effects of Rpf in culture-based Mtb testFirstly, we acquired soluble recombinant RpfB and RpfE proteins. We added Rpf proteins (RpfB, RpfE) to sputum based culture system and compared the time to positivity. We utilized Auramine-Nile red labeling to detect the Mtb that survived after heat treatment of sputum samples, and compared the time to positivity between culture systems with or without Rpf added after heat treatment.In this study, we found that time to positivity is not correlated with the Ziehl-Neelsen staining scores in a routine diagnostic liquid-culture system (R2=0.35). The time to positivity was only shortened in samples that demonstrated longer times to positivity (over20days in this study) under standard procedures (P<0.05). Samples were heated for10min at60,63, or70℃, after treatment at63or70℃; all of the bacteria were killed and could not be detected by Auramine-Nile red staining. A higher proportion of LBP-Mtb was found in sputum treated at60℃for10min compared to the untreated sample (P<0.01) and demonstrated weak acid-fastness. None of the heat treated samples were detectable by standard culture and some special samples became positive following RpfB or RpfE supplementation. We cultivated replicating Mtb with or without RpfB and RpfE (2nM,20nM,200nM) at104CFU/ml,106CFU/ml and108CFU/ml, and found that RpfB and RpfE have no effect on the time to positivity in a rapidly replicating clinical strain of Mtb.In conclusion, persister-like and dormant LBP-Mtb are present in clinical sputum samples. Treatment at60℃for10min reduces the proportion of the fast replicating Mtb, but does not affect LBP-Mtb. RpfB and RpfE can shorten the time to positivity in samples with longer time to positivity and make the samples cultures positive after heat treatment, most likely by resuscitating the LBP-Mtb that survived in the treated samples. We provide evidence that Rpfs have the potential to improve the sensitivity of culture-based Mtb tests in samples that require long culture times when using the standard procedure. Further investigation with a larger cohort is still required.