Molecular diagnosis of Pneumocystis pneumonia

The detection of Pneumocystis DNA in clinical specimens by using PCR assays is leading to important advances in Pneumocystis pneumonia (PcP) clinical diagnosis, therapy and epidemiology. Highly sensitive and specific PCR tools improved the clinical diagnosis of PcP allowing an accurate, early diagnosis of Pneumocystis infection, which should lead to a decreased duration from onset of symptoms to treatment, a period with recognized impact on prognosis. This aspect has marked importance in HIV-negative immunocompromised patients, who develop often PcP with lower parasite rates than AIDS patients. The specific amplification of selected polymorphous sequences of Pneumocystis jirovecii genome, especially of internal transcribed spacer regions of the nuclear rRNA operon, has led to the identification of specific parasite genotypes which might be associated with PcP severity. Moreover, multi-locus genotyping revealed to be a useful tool to explore person-to-person transmission. Furthermore, PCR was recently used for detecting P. jirovecii dihydropteroate synthase gene mutations, which are apparently associated with sulfa drug resistance. PCR assays detected Pneumocystis-DNA in bronchoalveolar lavage fluid or biopsy specimens, but also in oropharyngeal washings obtained by rinsing of the mouth. This non-invasive procedure may reach 90%-sensitivity and has been used for monitoring the response to treatment in AIDS patients and for typing Pneumocystis isolates.     

Multiple opportunistic fungal infections in an individual with severe HIV disease: A case report

BackgroundFungal infections have been commonly diagnosed in individuals with advanced HIV disease. Cryptococcosis, pneumocystosis, and histoplasmosis are the most frequent systemic mycoses in people suffering from HIV/AIDS.Case reportWe report a case of multiple fungal infections in an advanced AIDS-patient. A 33-year-old HIV-positive man from Brazil was hospitalized due to diarrhea, dyspnea, emaciation, hypoxemia, extensive oral thrush, and a CD4+ T lymphocyte count of 20 cells/mm³. Honeycombed-structures consistent with Pneumocystis jirovecii were observed by direct immunofluorescence in induced sputum. Cryptococcus neoformans was recovered from respiratory secretion and cerebrospinal fluid cultures. Histopathology of the bone marrow also revealed the presence of Histoplasma capsulatum. Molecular assays were performed in a sputum sample. Nested-PCR confirmed the presence of P. jirovecii and H. capsulatum; qPCR multiplex was positive for C. neoformans and H. capsulatum. With the treatment of antifungal drugs the patient progressed satisfactorily.ConclusionsThe diagnosis of several systemic mycoses demonstrates the vulnerability of advanced AIDS-patients. Thus, the detection of AIDS cases in the early stages of infection is necessary for a prompt and adequate introduction of HAART therapy, and the use of prophylaxis to control opportunistic infections.     

Genetic Polymorphisms of Pneumocystis Jirovecii in HIV-Positive and HIV-Negative Patients in Northern China

Pneumocystis jirovecii is an opportunistic fungus that can cause severe and potentially fatal Pneumocystis pneumonia (PCP) in immunodeficient patients. In this study, we investigated the genetic polymorphisms of P. jirovecii at eight different loci, including six nuclear genes (ITS, 26S rRNA, sod, dhps, dhfr and β-Tub) and two mitochondrial genes (mtLSU-rRNA and cyb) in three PCP cases, including two patients with HIV infection and one without HIV infection in Shanxi Province, P.R. China. The gene targets were amplified by PCR followed by sequencing of plasmid clones. The HIV-negative patient showed a coinfection with two genotypes of P. jirovecii at six of the eight loci sequenced. Of the two HIV-positive patients, one showed a coinfection with two genotypes of P. jirovecii at the same two of the six loci as in the HIV-negative patient, while the other showed a single infection at all eight loci sequenced. None of the three drug target genes (dhfr, dhps and cyb) showed mutations known to be potentially associated with drug resistance. This is the first report of genetic polymorphisms of P. jirovecii in PCP patients in Shanxi Province, China. Our findings expand our understanding of the genetic diversity of P. jirovecii in China. Open in a separate window     

<Emphasis Type="Italic">Pneumocystis jirovecii</Emphasis> and Cystic Fibrosis in Brittany,France

Pneumocystis jirovecii is a transmissible fungus with a high pulmonary tropism. The prevalence of P. jirovecii in patients with cystic fibrosis (CF) has been estimated in Germany at 7.4%, in Spain at 21.5% and in Brazil at 38.2%. Data on the prevalence of P. jirovecii in CF patients in France remain scarce, particularly in Brittany, where the prevalence of CF is high (from 1/1600 to 1/4500). Our objectives were to determine the prevalence of colonization of the airways by P. jirovecii in Brittany in CF patients monitored at the “Centre de Ressources et de Compétences de la Mucoviscidose (CRCM)” of Rennes compared to that previously observed at the CRCM of Roscoff–Brest. Sputa from 86 patients (178 specimens) followed in Rennes were analyzed retrospectively. The detection of P. jirovecii was performed using real-time PCR targeting the gene encoding the mitochondrial large subunit of ribosomal RNA. Pneumocystis jirovecii DNA was detected in 3/86 patients (3.5%) monitored at Rennes, whereas it had previously been detected in 1/76 patients (1.3%) monitored at Roscoff–Brest, thus showing an overall prevalence of 2.5% in Brittany. These results obtained from two Breton centers taken together show that P. jirovecii prevalence in patients with CF in Brittany is lower than those observed in Germany, Spain, Brazil or in other regions of France. This study is a preliminary step in determining the risk factors for P. jirovecii acquisition, its epidemiological and clinical significance in CF patients through a prospective multicenter study.     

New Short Tandem Repeat-Based Molecular Typing Method for Pneumocystis jirovecii Reveals Intrahospital Transmission between Patients from Different Wards

Pneumocystis pneumonia is a severe opportunistic infection in immunocompromised patients caused by the unusual fungus Pneumocystis jirovecii. Transmission is airborne, with both immunocompromised and immunocompetent individuals acting as a reservoir for the fungus. Numerous reports of outbreaks in renal transplant units demonstrate the need for valid genotyping methods to detect transmission of a given genotype. Here, we developed a short tandem repeat (STR)-based molecular typing method for P. jirovecii. We analyzed the P. jirovecii genome and selected six genomic STR markers located on different contigs of the genome. We then tested these markers in 106 P. jirovecii PCR-positive respiratory samples collected between October 2010 and November 2013 from 91 patients with various underlying medical conditions. Unique (one allele per marker) and multiple (more than one allele per marker) genotypes were observed in 34 (32%) and 72 (68%) samples, respectively. A genotype could be assigned to 55 samples (54 patients) and 61 different genotypes were identified in total with a discriminatory power of 0.992. Analysis of the allelic distribution of the six markers and minimum spanning tree analysis of the 61 genotypes identified a specific genotype (Gt21) in our hospital, which may have been transmitted between 10 patients including six renal transplant recipients. Our STR-based molecular typing method is a quick, cheap and reliable approach to genotype Pneumocystis jirovecii in hospital settings and is sensitive enough to detect minor genotypes, thus enabling the study of the transmission and pathophysiology of Pneumocystis pneumonia.     

Multicenter Study of Trimethoprim/Sulfamethoxazole-Related Hepatotoxicity: Incidence and Associated Factors among HIV-Infected Patients Treated for Pneumocystis jirovecii Pneumonia

The incidence of hepatotoxicity related to trimethoprim/sulfamethoxazole (TMP/SMX) administered at a therapeutic dose may vary among study populations of different ethnicities and hepatotoxic metabolites of TMP/SMX may be decreased by drug-drug interaction with fluconazole. We aimed to investigate the incidence of hepatotoxicity and the role of concomitant use of fluconazole in HIV-infected patients receiving TMP/SMX for Pneumocystis jirovecii pneumonia. We reviewed medical records to collect clinical characteristics and laboratory data of HIV-infected patients who received TMP/SMX for treatment of P. jirovecii pneumonia at 6 hospitals around Taiwan between September 2009 and February 2013. Hepatotoxicity was defined as 2-fold or greater increase of aminotransferase or total bilirubin level from baselines. Roussel UCLAF Causality Assessment Method (RUCAM) was used to analyze the causality of drug-induced liver injuries. NAT1 and NAT2 acetylator types were determined with the use of polymerase-chain-reaction (PCR) restriction fragment length polymorphism to differentiate common single-nucleotide polymorphisms (SNPs) predictive of the acetylator phenotypes in a subgroup of patients. During the study period, 286 courses of TMP/SMX treatment administered to 284 patients were analyzed. One hundred and fifty-two patients (53.1%) developed hepatotoxicity, and TMP/SMX was considered causative in 47 (16.4%) who had a RUCAM score of 6 or greater. In multivariate analysis, concomitant use of fluconazole for candidiasis was the only factor associated with reduced risk for hepatotoxicity (adjusted odds ratio, 0.372; 95% confidence interval, 0.145–0.957), while serostatus of hepatitis B or C virus, NAT1 and NAT2 acetylator types, or receipt of combination antiretroviral therapy was not. The incidence of hepatotoxicity decreased with an increasing daily dose of fluconazole up to 4.0 mg/kg. We conclude that the incidence of TMP/SMX-related hepatotoxicity was 16.4% in HIV-infected Taiwanese patients who received TMP/SMX for pneumocystosis. Concomitant use of fluconazole was associated with decreased risk for TMP/SMX-related hepatotoxicity.     

Pneumocystis Jirovecii detection and comparison of multiple diagnostic methods with quantitative real-time PCR in patients with respiratory symptoms

Pneumocystis jirovecii (PCP) remains a significant cause of mortality and morbidity in patients with respiratory infections. Accurate diagnosis of PCP is still a diagnostic challenge. Hence, the main objectives were to study the incidence of Pneumocystis Jirovecii pneumonia infection among respiratory problems patients and to compare the real-time quantitative PCR technique with various diagnostic methodologies. Patients who have respiratory symptoms of PCP like breathlessness, cough, and fever were enrolled. Bronchoalveolar lavage (BAL) samples were collected and homogenized, and then smears were prepared for examination by Gomorimethanamine silver staining (GMSS), Immunofluorescent staining (IFAT), Toludine blue O (TBO), and Giemsa staining. Further, RT-PCR was also performed for the detection of PCP. The mean patients’ age was 52 (SD ± 16) years. 41% were female, and 59% of the patients were male. Weight loss (80%), fever (92%), cough (100%), and dyspnea (76%) were the most common complaints. Twenty-eight patients have been diagnosed with pulmonary infiltrates using chest X-ray. Out of 100 patients, 35% were positive for PCP. The organism was detected using IFAT in all the 35 specimens, 15 of 35 (42.86%) by GMSS, 8 of 35 (17.6%) by Giemsa stain, and 1 of 35 (2.8%) was detected by TBO stains. RT-PCR showed that 39 patients was found to be positive for PCP. Thirty-five of these 39 patients had a positive IFAT (89.74%); the IFAT was negative or undefined in 4 samples. All 39 patients (100%) had signs and symptoms for PCP. Our results suggest that RT-PCR is still the most highly sensitive method for Pneumocystis Jirovecii detection. In poor resource settings where RT-PCR and IFAT is not available, diagnosis of Pneumocystis jirovecii pneumonia remains a complicated issue. In settings where RT-PCR & IFAT are not available, GMSS staining may be the next best choice to detect PCP.     

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 <200×10^3/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.     

Low Prevalence of Pneumocystis pneumonia (PCP) but High Prevalence of Pneumocystis dihydropteroate synthase (dhps) Gene Mutations in HIV-Infected Persons in Uganda

Pneumocystis jirovecii pneumonia (PCP) is an important opportunistic infection in patients infected with HIV, but its burden is incompletely characterized in those areas of sub-Saharan Africa where HIV is prevalent. We explored the prevalence of both PCP in HIV-infected adults admitted with pneumonia to a tertiary-care hospital in Uganda and of putative P. jirovecii drug resistance by mutations in fungal dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr). In 129 consecutive patients with sputum smears negative for mycobacteria, 5 (3.9%) were diagnosed with PCP by microscopic examination of Giemsa-stained bronchoalveolar lavage fluid. Concordance was 100% between Giemsa stain and PCR (dhps and dhfr). PCP was more prevalent in patients newly-diagnosed with HIV (11.4%) than in patients with known HIV (1.1%; p = 0.007). Mortality at 2 months after discharge was 29% overall: 28% among PCP-negative patients, and 60% (3 of 5) among PCP-positive patients. In these 5 fungal isolates and an additional 8 from consecutive cases of PCP, all strains harbored mutant dhps haplotypes; all 13 isolates harbored the P57S mutation in dhps, and 3 (23%) also harbored the T55A mutation. No non-synonymous dhfr mutations were detected. PCP is an important cause of pneumonia in patients newly-diagnosed with HIV in Uganda, is associated with high mortality, and putative molecular evidence of drug resistance is prevalent. Given the reliability of field diagnosis in our cohort, future studies in sub-Saharan Africa can investigate the clinical impact of these genotypes.     

Serum antibody levels to the Pneumocystis jirovecii major surface glycoprotein in the diagnosis of P. jirovecii pneumonia in HIV+ patients

Background

Pneumocystis jirovecii remains an important cause of fatal pneumonia (Pneumocystis pneumonia or PcP) in HIV+ patients and other immunocompromised hosts. Despite many previous attempts, a clinically useful serologic test for P. jirovecii infection has never been developed.

Methods/Principal Findings

We analyzed serum antibody responses to the P. jirovecii major surface glycoprotein recombinant fragment C1 (MsgC1) in 110 HIV+ patients with active PcP (cases) and 63 HIV+ patients with pneumonia due to other causes (controls) by an enzyme-linked immunosorbent assay (ELISA). The cases had significantly higher IgG and IgM antibody levels to MsgC1 than the controls at hospital admission (week 0) and intervals up to at least 1 month thereafter. The sensitivity, specificity and positive predictive value (PPV) of IgG antibody levels increased from 57.2%, 61.7% and 71.5% at week 0 to 63.4%, 100%, and 100%, respectively, at weeks 3–4. The sensitivity, specificity and PPV of IgM antibody levels rose from 59.7%, 61.3%, and 79.3% at week 0 to 74.6%, 73.7%, and 89.8%, respectively, at weeks 3–4. Multivariate analysis revealed that a diagnosis of PcP was the only independent predictor of high IgG and IgM antibody levels to MsgC1. A high LDH level, a nonspecific marker of lung damage, was an independent predictor of low IgG antibody levels to MsgC1.

Conclusions/Significance

The results suggest that the ELISA shows promise as an aid to the diagnosis of PCP in situations where diagnostic procedures cannot be performed. Further studies in other patient populations are needed to better define the usefulness of this serologic test.     

A Case of Pneumonia Caused by Pneumocystis jirovecii Resistant to Trimethoprim-Sulfamethoxazole

A 50-year-old male visited the outpatient clinic and complained of fever, poor oral intake, and weight loss. A chest X-ray demonstrated streaky and fibrotic lesions in both lungs, and chest CT revealed multifocal peribronchial patchy ground-glass opacities with septated cystic lesions in both lungs. Cell counts in the bronchoalveolar lavage fluid revealed lymphocyte-dominant leukocytosis, and further analysis of lymphocyte subsets showed a predominance of cytotoxic T cells and few T helper cells. Video-assisted wedge resection of the left upper lobe was performed, and the histologic examination was indicative of a Pneumocystis jirovecii infection. Trimethoprim-sulfamethoxazole (TMP-SMX) was orally administered for 3 weeks; however, the patient complained of cough, and the pneumonia was aggravated in the follow-up chest X-ray and chest CT. Molecular studies demonstrated mutations at codons 55 and 57 of the dihydropteroate synthase (DHPS) gene, which is associated with the resistance to TMP-SMX. Clindamycin-primaquine was subsequently administered for 3 weeks replacing the TMP-SMX. A follow-up chest X-ray showed that the pneumonia was resolving, and the cough was also alleviated. A positive result of HIV immunoassay and elevated titer of HCV RNA indicated HIV infection as an underlying condition. This case highlights the importance of careful monitoring of patients with P. jirovecii pneumonia (PCP) during the course of treatment, and the molecular study of DHPS mutations. Additionally, altering the anti-PCP drug utilized as treatment must be considered when infection with drug-resistant P. jirovecii is suspected. To the best of our knowledge, this is the first case of TMP-SMX-resistant PCP described in Korea.     

Rapid Detection and Immune Characterization of Mycobacterium abscessus Infection in Cystic Fibrosis Patients

Cystic fibrosis patients are highly susceptible to infections with non-tuberculous mycobacteria. Especially Mycobacterium abscessus infections are common but reliable diagnosis is hampered by non-specific clinical symptoms and insensitive mycobacterial culture. In the present study we established novel methods for rapid detection and immune characterization of Mycobacterium abscessus infection in cystic fibrosis patients. We performed Mycobacterium abscessus specific DNA-strip- and quantitative PCR-based analyses of non-cultured sputum samples to detect and characterize Mycobacterium abscessus infections. Concomitantly in vitro T-cell reactivation with purified protein derivatives (PPDs) from different mycobacterial species was used to determine Mycobacterium abscessus specific T-cell cytokine expression of infected cystic fibrosis patients. Four of 35 cystic fibrosis patients (11.4%) were Mycobacterium abscessus culture positive and showed concordant DNA-strip-test results. Quantitative PCR revealed marked differences of mycobacterial burden between cystic fibrosis patients and during disease course. Tandem-repeat analysis classified distinct Mycobacterium abscessus strains of infected cystic fibrosis patients and excluded patient-to-patient transmission. Mycobacterium abscessus specific T-cells were detected in the blood of cystic fibrosis patients with confirmed chronic infection and a subgroup of patients without evidence of Mycobacterium abscessus infection. Comparison of cytokine expression and phenotypic markers revealed increased proportions of CD40L positive T-cells that lack Interleukin-2 expression as a marker for chronic Mycobacterium abscessus infections in cystic fibrosis patients. Direct sputum examination enabled rapid diagnosis and quantification of Mycobacterium abscessus in cystic fibrosis patients. T-cell in vitro reactivation and cytokine expression analyses may contribute to diagnosis of chronic Mycobacterium abscessus infection.     

The Impact of HIV Co-Infection on the Genomic Response to Sepsis

HIV patients have an increased risk to develop sepsis and HIV infection affects several components of the immune system involved in sepsis pathogenesis. We hypothesized that HIV infection might aggrevate the aberrant immune response during sepsis, so we aimed to determine the impact of HIV infection on the genomic host response to sepsis. We compared whole blood leukocyte gene expression profiles among sepsis patients with or without HIV co-infection in the intensive care unit (ICU) and validated our findings in a cohort of patients admitted to the same ICUs in a different time frame. To examine the influence of HIV infection per se, we also determined the expression of genes of interest in a cohort of asymptomatic HIV patients. We identified a predominantly common host response in sepsis patients with or without HIV co-infection. HIV positive sepsis patients in both ICU cohorts showed overexpression of genes involved in granzyme signaling (GZMA, GZMB), cytotoxic T-cell signaling (CD8A, CD8B) and T-cell inhibitory signaling (LAG3), compared to HIV negative patients. Enhanced expression of CD8A, CD8B and LAG3 was also unmasked in asymptomatic HIV patients. Plasma levels of granzymes in sepsis patients were largely below detection limit, without differences according to HIV status. These results demonstrate that sepsis is characterized by a massive common response with few differences between HIV positive and HIV negative sepsis patients. Observed differences in granzyme signaling, cytotoxic T-cell signaling and T-cell inhibitory signaling appear to be changes commonly observed in asymptomatic HIV patients which persist during sepsis.     

A new luciferase immunoprecipitation system assay provided serological evidence for missed diagnosis of severe fever with thrombocytopenia syndrome

Severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV) infection, was first reported in 2010 in China with an initial fatality of up to 30%. The laboratory confirmation of SFTSV infection in terms of detection of viral RNA or antibody levels is critical for SFTS diagnosis and therapy. In this study, a new luciferase immunoprecipitation system (LIPS) assay based on pREN2 plasmid expressing SFTSV NP gene and tagged with Renilla luciferase (Rluc), was established and used to investigate the levels of antibody responses to SFTSV. Totally 464 serum samples from febrile patients were collected in the hospital of Shaoxing City in Zhejiang Province in 2019. The results showed that 82 of the 464 patients (17.7%) had antibody response to SFTSV, which were further supported by immunofluorescence assays (IFAs). Further, qRT-PCR and microneutralization tests showed that among the 82 positive cases, 15 patients had viremia, 10 patients had neutralizing antibody, and one had both (totally 26 patient). However, none of these patients were diagnosed as SFTS in the hospital probably because of their mild symptoms or subclinical manifestations. All the results indicated that at least the 26 patients having viremia or neutralizing antibody were the missed diagnosis of SFTS cases. The findings suggested the occurrence of SFTS and the SFTS incidence were higher than the reported level in Shaoxing in 2019, and that LIPS may provide an alternative strategy to confirm SFTSV infection in the laboratory.     

A Comparative Study of Peripheral Immune Responses to Taenia solium in Individuals with Parenchymal and Subarachnoid Neurocysticercosis

Background

The ability of Taenia solium to modulate the immune system likely contributes to their longevity in the human host. We tested the hypothesis that the nature of the immune response is related to the location of parasite and clinical manifestations of infection.

Methodology

Peripheral blood mononuclear cells (PBMC) were obtained from untreated patients with neurocysticercosis (NCC), categorized as having parenchymal or subarachnoid infection by the presence of cysts exclusively within the parenchyma or in subarachnoid spaces of the brain, and from uninfected (control) individuals matched by age and gender to each patient. Using multiplex detection technology, sera from NCC patients and controls and cytokine production by PBMC after T. solium antigen (TsAg) stimulation were assayed for levels of inflammatory and regulatory cytokines. PBMC were phenotyped by flow cytometry ex vivo and following in vitro stimulation with TsAg.

Principal Findings

Sera from patients with parenchymal NCC demonstrated significantly higher Th1 (IFN-γ/IL-12) and Th2 (IL-4/IL-13) cytokine responses and trends towards higher levels of IL-1β/IL-8/IL-5 than those obtained from patients with subarachnoid NCC. Also higher in vitro antigen-driven TNF-β secretion was detected in PBMC supernatants from parenchymal than in subarachnoid NCC. In contrast, there was a significantly higher IL-10 response to TsAg stimulation in patients with subarachnoid NCC compared to parenchymal NCC. Although no differences in regulatory T cells (Tregs) frequencies were found ex vivo, there was a trend towards greater expansion of Tregs upon TsAg stimulation in subarachnoid than in parenchymal NCC when data were normalized for the corresponding controls.

Conclusions/Significance

T. solium infection of the subarachnoid space is associated with an enhanced regulatory immune response compared to infection in the parenchyma. The resulting anti-inflammatory milieu may represent a parasite strategy to maintain a permissive environment in the host or diminish inflammatory damage from the host immune response in the central nervous system.     

TLTF in Cerebrospinal Fluid for Detection and Staging of T. b. gambiense Infection

Background

Trypanosome-derived lymphocyte triggering factor (TLTF) is a molecule released by African trypanosomes that interacts with the host immune system, resulting in increased levels of IFN-γ production.

Methodology/Principal findings

TLTF and anti-TLTF antibodies were assessed in sera and cerebrospinal fluid (CSF) from patients infected with Trypanosoma brucei gambiense (T. b. gambiense) in an attempt to identify alternative markers for diagnosis and stage determination of human African trypanosomiasis or sleeping sickness. Seventy-four serum and sixty-one CSF samples from patients with parasitologically confirmed infection and known disease stage along with 13 sera and CSF from uninfected controls were tested. In serum the levels of anti-TLTF antibodies were unrelated to the disease stage. In contrast, levels of anti-TLTF antibodies in CSF were higher in intermediate/late stages than in early stage disease patients. Specificity of the detected antibodies was assessed by inhibition of TLTF bioactivity as represented by its ability to induce IFN-γ production. Additionally, TLTF was detected in CSF from late stage patients by Western blotting with the anti-TLTF specific monoclonal antibody MO3.

Conclusions/Significance

These findings suggest a new possibility for disease diagnosis with focus on involvement of the CNS through detection of TLTF and anti-TLTF antibodies in the CSF.     

Clinical validation of integrated nucleic acid and protein detection on an electrochemical biosensor array for urinary tract infection diagnosis

Background

Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management.

Methodology/Principal Findings

The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both).

Conclusion/Significance

We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.     

Development of Ss-NIE-1 Recombinant Antigen Based Assays for Immunodiagnosis of Strongyloidiasis

Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG₄ specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG₄. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG₄ ELISA were 95% and 93%, respectively. For the IgG₄ Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG₄ ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States.     

Innate immune recognition of,and response to,Clostridium sordellii

Clostridium sordellii, an anaerobic pathogen, has recently been associated with rapidly fatal infections following medically induced abortions and injecting drug use. Patients with C. sordellii infection display few signs of inflammation such as fever, or redness and pain at the site of infection. We hypothesized that this could be due to reduced recognition of the organism by Toll-like receptors (TLRs) of the innate immune system. An ELAM-NF-κB luciferase reporter system in TLR-transfected HEK cells was used to measure TLR-dependent recognition of washed, heat-killed C. sordellii and other pathogenic clostridial species. Results demonstrated that all clostridia were well recognized by TLR2 alone and that responses were greatest when TLR2 was co-expressed with TLR6. Further, isolated human monocytes produced the pro-inflammatory cytokine TNFα and the immunoregulator IL-10 in response to C. sordellii. In addition, C. sordellii-stimulated monocytes produced 30% less TNFα following treatment with an anti-TLR2 blocking antibody. These data demonstrate that innate immune recognition of, and response to, cell-associated components of C. sordellii and other clostridial pathogens are mediated by TLR2 in combination with TLR6. We conclude that the characteristic absence of inflammatory signs and symptoms in C. sordellii infection is not related to inadequate immune detection of the organism, but rather is attributable to a species-specific immune system dysfunction that remains to be elucidated.