Aetiological agents of pneumonia among HIV and non-HIV infected children in Ghana: A case-control study.

Pneumonia is the leading cause of death in children, however, the microbial aetiology of pneumonia is not well elucidated in low- and middle-income countries. Our study was aimed at determining the microbial aetiologies of childhood pneumonia and associated risk factors in HIV and non-HIV infected children. We conducted a case-control study that enrolled children with pneumonia as cases and non-pneumonia as controls from July 2017 to May 2020. Induced sputum and blood samples were investigated for microbial organisms using standard microbiological techniques. DNA/RNA was extracted from sputum samples and tested for viral and bacterial agents. Four hundred and four (404) subjects consisting of 231 (57.2%) cases and 173 (42.8%) controls were enrolled. We identified a significant (p = 0.011) proportion of viruses in cases (125; 54.1%, 95%CI: 47.4-60.7) than controls (71; 33.6%, 95%CI: 33.6-48.8) and these were mostly contributed to by Respiratory Syncytial Virus. Staphylococcus aureus (16; 4.0%), Klebsiella spp. (15, 3.7%) and Streptococcus pneumoniae (8, 2.0%) were the main bacterial agents identified in sputum or induced sputum samples. HIV infected children with viral-bacterial co-detection were found to have very severe pneumonia compared to those with only viral or bacterial infection. Indoor cooking (OR = 2.36; 95%CI:1.41-3.96) was found to be associated with pneumonia risk in patients. This study demonstrates the importance of various microbial pathogens, particularly RSV, in contributing to pneumonia in HIV and non-HIV paediatric populations. There is a need to accelerate clinical trials of RSV vaccines in African populations to support improvement of patient care.

Xpert MTB/RIF Ultra for the rapid diagnosis of extrapulmonary tuberculosis in a clinical setting of high tuberculosis prevalence country and interpretation of 'trace' results.

To evaluate the diagnostic performance of Xpert MTB/RIF Ultra (Ultra) for the diagnosis of extrapulmonary tuberculosis (EPTB) from different types of extrapulmonary specimens in comparison with culture and composite microbiological reference standard (CRS). A total of 240 specimens were prospectively collected from presumptive EPTB patients between July 2021-January 2022 and tested by Ultra, Xpert, culture and acid-fast bacilli (AFB) smear microscopy. Out of 240 specimens, 35.8 %, 20.8 %, 11.3 %, and 7.1 % were detected as Mycobacterium tuberculosis complex by Ultra, Xpert, culture and AFB microscopy, respectively. An additional 15.0 % cases were detected by Ultra compared to Xpert MTB/RIF (Xpert) assay. A total of 28 (11.7 %) cases were identified as 'trace' category by Ultra with indeterminate rifampicin resistance result; of which 36.4 % were clinically confirmed as EPTB. Compared to culture, the sensitivity and specificity of Ultra and Xpert were 100 % and 72.3 %; 92.6 % and 88.3 %, respectively. In comparison with CRS, these were respectively: 98.9 % and 100 %; 57.5 % and 100 %. For individual category of specimens, sensitivity of Ultra was 100 % with varying specificity. We found that Ultra was highly sensitive for the rapid diagnosis of EPTB and has extensive potential over current diagnostics in high TB burden countries, but 'trace' results should be interpreted with caution.

Diagnostic Yield of Xpert MTB/RIF Assay Using Bronchoalveolar Lavage Fluid in Detecting Mycobacterium tuberculosis among the Sputum-Scarce Suspected Pulmonary TB Patients.

Tuberculosis (TB) remains one of the leading causes of death worldwide and is caused by the single infectious agent Mycobacterium tuberculosis (Mtb). Although sputum is the most common specimen for pulmonary TB detection, some other respiratory specimens, such as bronchoalveolar lavage (BAL) fluid, gastric lavage (GL), and induced sputum (IS), are also collected from patients who are unable to deliver sputum. In this study, we aimed to evaluate the diagnostic performances of different test methods for TB diagnosis using BAL fluid specimens from sputum-scarce pulmonary TB patients. In this current study, a total of 210 BAL fluid specimens were collected and subjected to culture on Lowenstein-Jensen (L-J) medium, using an N-acetyl-L-cysteine-Sodium Hydroxide decontamination and digestion method, Xpert MTB/RIF (Xpert, Cepheid, Sunnyvale, CA, USA) assay, and acid-fast bacilli (AFB) microscopy with a Ziehl-Neelsen staining method for the detection of pulmonary TB. The sensitivity and specificity of these methods were then analyzed against the composite reference standard (CRS). Additionally, the receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of these assays. Among the 210 specimens, 39 (18.6%), 27 (12.8%), and 12 (5.7%) were found positive with Xpert assay, culture, and AFB microscopy, respectively. Considering the CRS, 42 (20%) were positive as the final diagnosis. The Xpert assay had a significantly higher sensitivity (92.9%, 95% CI: 80.5-98.5) compared to culture (64.3%, 95% CI: 48.0-78.4) and AFB microscopy (28.6%, 95% CI: 15.7-44.6) against the CRS. Additionally, the area under the ROC curve (AUC) for the Xpert assay, culture, and AFB microscopy accounted for 0.964, 0.821, and 0.655, respectively, when using CRS as the reference. In conclusion, our study findings demonstrated that the Xpert assay conferred a considerable diagnostic potential compared to other conventional methods for the diagnosis of pulmonary TB from BAL fluid specimens.

Multi-country evaluation of RISK6, a 6-gene blood transcriptomic signature, for tuberculosis diagnosis and treatment monitoring.

There is a crucial need for non-sputum-based TB tests. Here, we evaluate the performance of RISK6, a human-blood transcriptomic signature, for TB screening, triage and treatment monitoring. RISK6 performance was also compared to that of two IGRAs: one based on RD1 antigens (QuantiFERON-TB Gold Plus, QFT-P, Qiagen) and one on recombinant M. tuberculosis HBHA expressed in Mycobacterium smegmatis (IGRA-rmsHBHA). In this multicenter prospective nested case-control study conducted in Bangladesh, Georgia, Lebanon and Madagascar, adult non-immunocompromised patients with bacteriologically confirmed active pulmonary TB (ATB), latent TB infection (LTBI) and healthy donors (HD) were enrolled. ATB patients were followed-up during and after treatment. Blood RISK6 scores were assessed using quantitative real-time PCR and evaluated by area under the receiver-operating characteristic curve (ROC AUC). RISK6 performance to discriminate ATB from HD reached an AUC of 0.94 (95% CI 0.89-0.99), with 90.9% sensitivity and 87.8% specificity, thus achieving the minimal WHO target product profile for a non-sputum-based TB screening test. Besides, RISK6 yielded an AUC of 0.93 (95% CI 0.85-1) with 90.9% sensitivity and 88.5% specificity for discriminating ATB from LTBI. Moreover, RISK6 showed higher performance (AUC 0.90, 95% CI 0.85-0.94) than IGRA-rmsHBHA (AUC 0.75, 95% CI 0.69-0.82) to differentiate TB infection stages. Finally, RISK6 signature scores significantly decreased after 2 months of TB treatment and continued to decrease gradually until the end of treatment reaching scores obtained in HD. We confirmed the performance of RISK6 signature as a triage TB test and its utility for treatment monitoring.

Clinical Evaluation of a Multiplex PCR for the Detection of Salmonella enterica Serovars Typhi and Paratyphi A from Blood Specimens in a High-Endemic Setting.

Enteric fever is a major public health concern in endemic areas, particularly in infrastructure-limited countries where Salmonella Paratyphi A has emerged in increasing proportion of cases. We aimed to evaluate a method to detect Salmonella Typhi (S. Typhi) and Salmonella Paratyphi A (S. Paratyphi A) in febrile patients in Bangladesh. We conducted a prospective study enrolling patients with fever > 38°C admitted to two large urban hospitals and two outpatient clinics located in Dhaka, Bangladesh. We developed and evaluated a method combining short culture with a new molecular assay to simultaneously detect and differentiate S. Typhi and S. Paratyphi A from other Salmonella directly from 2 to 4 mL of whole blood in febrile patients (n = 680). A total of 680 cases were enrolled from the four participating sites. An increase in the detection rate (+38.8%) in S. Typhi and S. Paratyphi A was observed with a multiplex polymerase chain reaction (PCR) assay, and absence of non-typhoidal Salmonella detection was reported. All 45 healthy controls were culture and PCR negative, generating an estimated 92.9% of specificity on clinical samples. When clinical performance was assessed in the absence of blood volume prioritization for testing, a latent class model estimates clinical performance ≥ 95% in sensitivity and specificity with likelihood ratio (LR) LR+ > 10 and LR- < 0.1 for the multiplex PCR assay. The alternative method to blood culture we developed may be useful alone or in combination with culture or serological tests for epidemiological studies in high disease burden settings and should be considered as secondary endpoint test for future vaccine trials.

Investigating Pneumonia Etiology Among Refugees and the Lebanese population (PEARL): A study protocol.

Background: Community-acquired pneumonia (CAP), a leading cause of mortality, mainly affects children in developing countries. The harsh circumstances experienced by refugees include various factors associated with respiratory pathogen transmission, and clinical progression of CAP. Consequently, the etiology of CAP in humanitarian crisis situations may differ to that of settled populations, which would impact appropriate case management. Therefore, the Pneumonia Etiology Among Refugees and the Lebanese population (PEARL) study was initiated with the objective of identifying the causal pathogenic microorganisms in the respiratory tract of children and adults from both the refugee and host country population presenting with signs of CAP during a humanitarian crisis. Methods: PEARL, a prospective, multicentric, case-control study, will be conducted at four primary healthcare facilities in Tripoli and the Bekaa valley over 15 months (including two high-transmission seasons/winters). Sociodemographic and medical data, and biological samples will be collected from at least 600 CAP cases and 600 controls. Nasopharyngeal swabs, sputum, urine and blood samples will be analyzed at five clinical pathology laboratories in Lebanon to identify the bacterial and viral etiological agents of CAP. Transcriptomic profiling of host leukocytes will be performed. Conclusions: PEARL is an original observational study that will provide important new information on the etiology of pneumonia among refugees, which may improve case management, help design antimicrobial stewardship interventions, and reduce morbidity and mortality due to CAP in a humanitarian crisis.

Viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances IP-10: a translational study.

Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.

Viral and bacterial pathogens identification in children hospitalised for severe pneumonia and parapneumonic empyema.

Pneumonia is caused by respiratory bacteria and/or viruses. Little is known if co-infections are an aggravating factor in hospitalised children with severe pneumonia. We studied the impact of respiratory pathogens on the severity of pneumonia. Between 2007 and 2009, 52 children hospitalised with a well-documented diagnosis of community-acquired pneumonia (CAP), with or without parapneumonic empyema (PPE), were enrolled in the study. The patients were classified into 2 groups: CAP + PPE (n = 28) and CAP (n = 24). The identification of respiratory viruses and bacteria in nasopharyngeal aspirates and pleural effusion samples were performed using conventional bacterial techniques and molecular assays. Using real-time multiplex PCR and antigen detection, Streptococcus pneumoniae was the main agent identified in 76% of the cases by molecular tests and BinaxNOW® in pleural fluid. A total of 8% of pleural fluid samples remained undiagnosed. In nasopharyngeal aspirates, rhinovirus, parainfluenza viruses, human metapneumovirus, and respiratory syncytial virus were detected in both CAP and CAP + PPE populations; however, the percentage of viral co-detection was significantly higher in nasopharyngeal aspirates from CAP + PPE patients (35%) compared with CAP patients (5%). In conclusion, viral co-detection was observed mainly in patients with more severe pneumonia. Molecular biology assays improved the pathogens detection in pneumonia and confirmed the S. pneumoniae detection by BinaxNOW® in pleural effusion samples. Interestingly, the main S. pneumoniae serotypes found in PPE are not the ones targeted by the heptavalent pneumococcal conjugate vaccine.

False-positive results in a recombinant severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid-based western blot assay were rectified by the use of two subunits (S1 and S2) of spike for detection of antibody to SARS-CoV.

To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any cross-reactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV.

Expression of hepatitis C virus proteins in epithelial intestinal cells in vivo.

Previous work on hepatitis C virus (HCV) led to the discovery of a new form of virus particle associating virus and lipoprotein elements. These hybrid particles (LVP for lipo-viro-particles) are enriched in triglycerides and contain at least apolipoprotein B (apoB), HCV RNA and core protein. These findings suggest that LVP synthesis could occur in liver and intestine, the two main organs specialized in the production of apoB-containing lipoprotein. To identify the site of LVP production, the genetic diversity and phylogenetic relationship of HCV quasispecies from purified LVP, whole serum and liver biopsies from chronically infected patients were studied. HCV quasispecies from LVP and liver differed significantly, suggesting that LVP were not predominantly synthesized in the liver but might also originate in the intestine. The authors therefore searched for the presence of HCV in the small intestine. Paraffin-embedded intestinal biopsies from 10 chronically HCV-infected patients and from 12 HCV RNA-negative controls (10 anti-HCV antibody-negative and two anti-HCV antibody-positive patients) were tested for HCV protein expression. HCV NS3 and NS5A proteins were stained in small intestine epithelial cells in four of the 10 chronically infected patients, and not in controls. Cells expressing HCV proteins were apoB-producing enterocytes but not mucus-secreting cells. These data indicate that the small intestine can be infected by HCV, and identify this organ as a potential reservoir and replication site. This further emphasizes the interaction between lipoprotein metabolism and HCV, and offers new insights into hepatitis C infection and pathophysiology.