David M. Lowe

Neutrophils in tuberculosis: friend or foe?

Neutrophils are rapidly recruited to sites of mycobacterial infection, where they phagocytose bacilli. Whether neutrophils can kill mycobacteria in vivo probably depends on the tissue microenvironment, stage of infection, individual host, and infecting organism. The interaction of neutrophils with macrophages, as well as the downstream effects on T cell activity, could result in a range of outcomes from early clearance of infection to dissemination of viable bacteria together with an attenuated acquired immune response. In established disease, neutrophils accumulate in situations of high pathogen load or immunological dysfunction, and are likely to contribute to pathology. These activities may have clinical importance in terms of new treatments, targeted interventions and vaccine strategies.

Pneumocystis jirovecii Pneumonia in Tropical and Low and Middle Income Countries: A Systematic Review and Meta-Regression

Objective Pneumocystis jirovecii pneumonia (PCP), the commonest opportunistic infection in HIV-infected patients in the developed world, is less commonly described in tropical and low and middle income countries (LMIC). We sought to investigate predictors of PCP in these settings. Design Systematic review and meta-regression. Methods Meta-regression of predictors of PCP diagnosis (33 studies). Qualitative and quantitative assessment of recorded CD4 counts, receipt of prophylaxis and antiretrovirals, sensitivity and specificity of clinical signs and symptoms for PCP, co-infection with other pathogens, and case fatality (117 studies). Results The most significant predictor of PCP was per capita Gross Domestic Product, which showed strong linear association with odds of PCP diagnosis (p<0.0001). This was not explained by study design or diagnostic quality. Geographical area, population age, study setting and year of study also contributed to risk of PCP. Co-infection was common (444 episodes/1425 PCP cases), frequently with virulent organisms. The predictive value of symptoms, signs or simple tests in LMIC settings for diagnosis of PCP was poor. Case fatality was >30%; treatment was largely appropriate. Prophylaxis appeared to reduce the risk for development of PCP, however 24% of children with PCP were receiving prophylaxis. CD4 counts at presentation with PCP were usually <200×103/ml. Conclusions There is a positive relationship between GDP and risk of PCP diagnosis. Although failure to diagnose infection in poorer countries may contribute to this, we also hypothesise that poverty exposes at-risk patients to a wide range of infections and that the relatively non-pathogenic P. jirovecii is therefore under-represented. As LMIC develop economically they eliminate the conditions underlying transmission of virulent infection: P. jirovecii, ubiquitous in all settings, then becomes a greater relative threat.

Neutrophilia independently predicts death in tuberculosis

To the Editor: Experimental animal work indicates that neutrophils play a key role in the immune response to mycobacteria [1, 2]. They appear protective against early infection [3] but in established disease, neutrophilia associates with pathology [1, 4]. In humans, higher neutrophil counts at tuberculosis diagnosis predict slower sputum conversion to negative during therapy [5, 6], but the overall prognostic significance of neutrophilia in human tuberculosis remains elusive. We therefore aimed to analyse this phenomenon in a study powered to detect an independent relationship with mortality. Tuberculosis patients were identified by database/case-note review at Newham University Hospital Trust and King’s College Hospital, London, UK. All patients diagnosed between 1999 and 2006 were eligible for inclusion in an analysis of neutrophilia at baseline; those with a recorded outcome of successfully completing treatment or death were included in an analysis of determinants of mortality. Healthy contacts of tuberculosis cases were recruited from the same hospitals. Data were extracted on patient age, sex, ethnicity, comorbidity, use of immunosuppressive medication, HIV status and site of disease. Laboratory data were collected from samples taken on the date of tuberculosis diagnosis: serum sodium, bilirubin and albumin concentrations; peripheral blood haemoglobin concentration; and peripheral blood neutrophil, monocyte, lymphocyte and platelet counts. Blood culture results were recorded where performed. Protocols were approved by the Barking and Havering NHS Research Ethics Committee (REC 08/H0702/25) and North East London Research Ethics Committee (REC P/02/146). We calculated that 584 patients (34 deaths and 550 survivors) would be required to detect a three-fold difference in mortality in the presence of neutrophilia with 80% power (5% significance level), assuming a 15% prevalence of neutrophilia and a death/survival …

Blood Neutrophil Counts in HIV-Infected Patients with Pulmonary Tuberculosis: Association with Sputum Mycobacterial Load.

Background Increasing evidence suggests that neutrophils play a role in the host response to Mycobacterium tuberculosis. We determined whether neutrophil counts in peripheral blood are associated with tuberculosis (TB) and with mycobacterial load in sputum in HIV-infected patients. Methodology/Principal Findings Adults enrolling in an antiretroviral treatment (ART) clinic in a Cape Town township were screened for TB regardless of symptoms. Paired sputum samples were examined using liquid culture, fluorescence microscopy, and the Xpert MTB/RIF assay. Absolute neutrophil counts (ANC) were measured in blood samples. Of 602 HIV-infected patients screened, 523 produced one or more sputum samples and had complete results available for analysis. Among these 523 patients, the median CD4 count was 169×109/L (IQR, 96–232) and median ANC was 2.6×109/L (IQR, 1.9–3.6). Culture-positive pulmonary tuberculosis was diagnosed in 89 patients. Patients with TB had a median ANC of 3.4×109/L (IQR, 2.4–5.1) compared to 2.5×109/L (IQR, 1.8–3.4) among those who were culture negative (p<0.0001). In multivariable analyses, having pulmonary TB was associated with an adjusted risk ratio (aRR) of 2.6 (95%CI, 1.5–4.5) for having an ANC level that exceeded the median value (ANC ≥2.6×109/L; p = 0.0006) and an aRR of 6.8 (95%CI, 2.3–20.4) for having neutrophilia defined by a neutrophil count exceeding the upper limit of the normal range (ANC >7.5×109/L; p = 0.0005). Patients were then classified into four mutually exclusive groups with increasing sputum mycobacterial load as defined by the results of culture, Xpert MTB/RIF and sputum smear microscopy. Multivariable analyses demonstrated that increasing sputum mycobacterial load was positively associated with blood ANC ≥2.6×109/L and with neutrophilia. Conclusions/Significance Increased blood neutrophil counts were independently associated with pulmonary TB and sputum mycobacterial burden in this HIV-infected patient group. This observation supports the growing body of literature regarding the potential role for neutrophils in the host response to TB.

The erythrocyte sedimentation rate in HIV: a neglected parameter?

Accepted wisdom in the assessment of a person with HIV is that the Erythrocyte Sedimentation Rate (ESR) is not a useful test. Although initially considered as an indicator of disease progression [1, 2], later studies disagreed [3] or demonstrated only a negligible fall in CD4 count with rising ESR [4].

A novel assay of antimycobacterial activity and phagocytosis by human neutrophils

Summary Despite abundant evidence that neutrophils arrive early at sites of mycobacterial disease and phagocytose organisms, techniques to assay phagocytosis or killing of mycobacteria by these cells are lacking. Existing assays for measuring the antimycobacterial activity of human leukocytes require cell lysis which introduces new bioactive substances and may be incomplete. They are also time-consuming and carry multiple risks of inaccuracy due to serial dilution and organism clumping. Flow cytometric techniques for measuring phagocytosis of mycobacteria by human cells have failed to adequately address the effects of organism clumping, quenching agents and culture conditions on readouts. Here we present a novel in-tube bioluminescence-based assay of antimycobacterial activity by human neutrophils. The assay yields intuitive results, with improving restriction of mycobacterial bioluminescence as the ratio of cells to organisms increases. We show that lysis of human cells is not required to measure luminescence accurately. We also present a phagocytosis assay in which we have minimised the impact of mycobacterial clumping, investigated the effect of various opsonisation techniques and established the correct usage of trypan blue to identify surface-bound organisms without counting dead cells. The same multiplicity of infection and serum conditions are optimal to demonstrate both internalisation and restriction of mycobacterial growth.

Brief Report: HIV-1 Infection Impairs CD16 and CD35 Mediated Opsonophagocytosis of Mycobacterium tuberculosis by Human Neutrophils.

Abstract:Using a flow cytometric assay, we investigated neutrophil–Mycobacterium tuberculosis opsonophagocytosis and the impact of HIV-1–infected serum on this process. The mean (±SD) percentage of neutrophils internalizing bacilli after 30 minutes incubation was significantly reduced by pretreatment with anti-CD16 (18.2% ± 8.1%, P < 0.001) or anti-CD35 antibody (23.2% ± 10.6%, P < 0.05) versus anti-CD4 controls (29.9% ± 8.1%). Blocking CD88 or CD11a did not affect internalization. Using heat-inactivated serum, maximal internalization was lower using HIV-1–infected serum versus HIV-1–uninfected. Using non–heat-inactivated serum, internalization decreased more rapidly with sequential dilutions of HIV-1–infected versus HIV-1–uninfected serum. CD16 and CD35 are important for neutrophil internalization of M. tuberculosis, whereas HIV-1 infection adversely affects opsonophagocytosis.

Neutrophil activation and enhanced release of granule products in HIV-TB immune reconstitution inflammatory syndrome.

Background: Tuberculosis immune reconstitution inflammatory syndrome (TB-IRIS) remains incompletely understood. Neutrophils are implicated in tuberculosis pathology but detailed investigations in TB-IRIS are lacking. We sought to further explore the biology of TB-IRIS and, in particular, the role of neutrophils. Setting: Two observational, prospective cohort studies in HIV/TB coinfected patients starting antiretroviral therapy (ART), 1 to analyze gene expression and subsequently 1 to explore neutrophil biology. Methods: nCounter gene expression analysis was performed in patients with TB-IRIS (n = 17) versus antiretroviral-treated HIV/TB coinfected controls without IRIS (n = 17) in Kampala, Uganda. Flow cytometry was performed in patients with TB-IRIS (n = 18) and controls (n = 11) in Cape Town, South Africa to determine expression of neutrophil surface activation markers, intracellular cytokines, and human neutrophil peptides (HNPs). Plasma neutrophil elastase and HNP1-3 were quantified using enzyme-linked immunosorbent assay. Lymph node immunohistochemistry was performed on 3 further patients with TB-IRIS. Results: There was a significant increase in gene expression of S100A9 (P = 0.002), NLRP12 (P = 0.018), COX-1 (P = 0.025), and IL-10 (P = 0.045) 2 weeks after ART initiation in Ugandan patients with TB-IRIS versus controls, implicating neutrophil recruitment. Patients with IRIS in both cohorts demonstrated increases in blood neutrophil count, plasma HNP and elastase concentrations from ART initiation to week 2. CD62L (L-selectin) expression on neutrophils increased over 4 weeks in South African controls whereas patients with IRIS demonstrated the opposite. Intense staining for the neutrophil marker CD15 and IL-10 was seen in necrotic areas of the lymph nodes of the patients with TB-IRIS. Conclusions: Neutrophils in TB-IRIS are activated, recruited to sites of disease, and release granule contents, contributing to pathology.

Endothelial Dysfunction in HIV

Vani Subbarao1, David Lowe2,3, Reza Aghamohammadzadeh4,5 and Robert J. Wilkinson2,3,6 1Madwaleni Hospital, Elliotdale, Eastern Cape 2Clinical Infectious Disease Research Initiative, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town 3Wellcome Trust Centre for Research in Clinical Tropical Medicine, Department of Medicine, Imperial College London 4British Heart Foundation Clinical Research Fellow 5NIHR Manchester Biomedical Research Centre Clinical Research Fellow 6Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London 1,2South Africa 3,4,5,6UK

An undiagnosed stupor in the acute medical unit: a case of malignant catatonia

### Learning Point for Clinicians Malignant catatonia is a rare cause of a reduced Glasgow Coma Score. It is difficult to diagnose as it has no specific investigation findings, and it may mimic a number of other conditions. A dramatic response to benzodiazepines however is characteristic and may assist in the diagnosis. A 45-year-old man was brought to our hospital by Ambulance having been found lying unconscious on the pavement. On admission he was obtunded with a Glasgow Coma Score (GCS) of 6 (E1, V1, M4), but maintaining his airway. He was pyrexial (temperature 38.5°C) and tachycardic with a pulse rate of 106 beats per minute. His blood pressure was 142/96 mmHg and capillary blood glucose 6.2 mmol/l. There was no evidence of trauma or needle marks. Neurological examination revealed mildly increased tone and brisk …