Effectiveness of Streptococcus Pneumoniae Urinary Antigen Testing in Decreasing Mortality of COVID-19 Co-Infected Patients: A Clinical Investigation.

BACKGROUND AND OBJECTIVES: Streptococcus pneumoniae urinary antigen (u-Ag) testing has recently gained attention in the early diagnosis of severe and critical acute respiratory syndrome coronavirus-2/pneumococcal co-infection. The aim of this study is to assess the effectiveness of Streptococcus pneumoniae u-Ag testing in coronavirus disease 2019 (COVID-19) patients, in order to assess whether pneumococcal co-infection is associated with different mortality rate and hospital stay in these patients. MATERIALS AND METHODS: Charts, protocols, mortality, and hospitalization data of a consecutive series of COVID-19 patients admitted to a tertiary hospital in northern Italy during COVID-19 outbreak were retrospectively reviewed. All patients underwent Streptococcus pneumoniae u-Ag testing to detect an underlying pneumococcal co-infection. Covid19+/u-Ag+ and Covid19+/u-Ag- patients were compared in terms of overall survival and length of hospital stay using chi-square test and survival analysis. RESULTS: Out of 575 patients with documented pneumonia, 13% screened positive for the u-Ag test. All u-Ag+ patients underwent treatment with Ceftriaxone and Azithromycin or Levofloxacin. Lopinavir/Ritonavir or Darunavir/Cobicistat were added in 44 patients, and hydroxychloroquine and low-molecular-weight heparin (LMWH) in 47 and 33 patients, respectively. All u-Ag+ patients were hospitalized. Mortality was 15.4% and 25.9% in u-Ag+ and u-Ag- patients, respectively (p = 0.09). Survival analysis showed a better prognosis, albeit not significant, in u-Ag+ patients. Median hospital stay did not differ among groups (10 vs. 9 days, p = 0.71). CONCLUSIONS: The routine use of Streptococcus pneumoniae u-Ag testing helped to better target antibiotic therapy with a final trend of reduction in mortality of u-Ag+ COVID-19 patients having a concomitant pneumococcal infection. Randomized trials on larger cohorts are necessary in order to draw definitive conclusion.

Lipoarabinomannan antigenic epitope differences in tuberculosis disease subtypes.

An accurate urine test for diverse populations with active tuberculosis could be transformative for preventing TB deaths. Urinary liporabinomannan (LAM) testing has been previously restricted to HIV co-infected TB patients. In this study we evaluate urinary LAM in HIV negative, pediatric and adult, pulmonary and extrapulmonary tuberculosis patients. We measured 430 microbiologically confirmed pretreatment tuberculosis patients and controls from Peru, Guinea Bissau, Venezuela, Uganda and the United States using three monoclonal antibodies, MoAb1, CS35, and A194, which recognize distinct LAM epitopes, a one-sided immunoassay, and blinded cohorts. We evaluated sources of assay variability and comorbidities (HIV and diabetes). All antibodies successfully discriminated TB positive from TB negative patients. ROAUC from the average of three antibodies' responses was 0.90; 95% CI 0.87-0.93, 90% sensitivity, 73.5% specificity (80 pg/mL). MoAb1, recognizing the 5-methylthio-D-xylofuranose(MTX)-mannose(Man) cap epitope, performed the best, was less influenced by glycosuria and identified culture positive pediatric (N = 19) and extrapulmonary (N = 24) patients with high accuracy (ROAUC 0.87, 95% CI 0.77-0.98, 0.90 sensitivity 0.80 specificity at 80 pg/mL; ROAUC = 0.96, 95% CI 0.92-0.99, 96% sensitivity, 80% specificity at 82 pg/mL, respectively). The MoAb1 antibody, recognizing the MTX-Man cap epitope, is a novel analyte for active TB detection in pediatric and extrapulmonary disease.

Metagenomic analysis of mycobacterial transrenal DNA in patients with HIV and tuberculosis coinfection.

The existence of "transrenal" DNA (tr-DNA), i.e. cell-free DNA that has distributed through the renal barrier to the urine, was first shown from a pathogen in 2000 (Botezatu et al., 2000). However, a targeted search for tr-DNA from Mycobacterium tuberculosis (MBT) started relatively recently (Cannas et al., 2008; Green et al., 2009). While other MBT cellular components found in the urine, e.g. lipoarabinomannan, have been used as an enhanced diagnostic tool, tr-DNA has the potential for strain specific identification or a more persistent biomarker during treatment of active disease. We therefore sought to identify by high-throughput next generation sequencing (NGS) MBT genome fragments in the urine of people with human immunodeficiency virus and tuberculosis (HIV-TB) co-infection living in a co-epidemic setting, and to evaluate whether these DNA targets are suitable for the development a quantitative TaqMan polymerase chain reaction with real-time detection (rt-PCR). Selection and mapping to the reference MBT genome of strain H37Rv (NC_000962) revealed 158 fragments of mycobacterial DNA with length from 19 to 44 base pairs (bp) repeated in different DNA samples. Five targets were chosen for design of rt-PCR primers and probes. Comparative analysis of the newly developed tests that were based on the results of NGS did not reveal a significant increase in sensitivity and specificity relative to the previous empirically designed targets. Howver, highly reproducible NGS reads of mycobacterial tr-DNA were obtained. rt-PCR test development suitable for more practical clinical use was likely limited by the small size of the secreted DNA fragments. It is necessary to develop further molecular approaches for the detection of mycobacterial tr-DNA or rely on NGS techniques with inherent bioinformatics requirements.

Diagnostic value of the urine lipoarabinomannan assay in HIV-positive, ambulatory patients with CD4 below 200 cells/µl in 2 low-resource settings: A prospective observational study.

BACKGROUND: Current guidelines recommend the use of the lateral flow urine lipoarabinomannan assay (LAM) in HIV-positive, ambulatory patients with signs and symptoms of tuberculosis (TB) only if they are seriously ill or have CD4 count ≤ 100 cells/µl. We assessed the diagnostic yield of including LAM in TB diagnostic algorithms in HIV-positive, ambulatory patients with CD4 < 200 cells/µl, as well as the risk of mortality in LAM-positive patients who were not diagnosed using other diagnostic tools and not treated for TB. METHODS AND FINDINGS: We conducted a prospective observational study including HIV-positive adult patients with signs and symptoms of TB and CD4 < 200 cells/µl attending 6 health facilities in Malawi and Mozambique. Patients were included consecutively from 18 September 2015 to 27 October 2016 in Malawi and from 3 December 2014 to 22 August 2016 in Mozambique. All patients had a clinical exam and LAM, chest X-ray, sputum microscopy, and Xpert MTB/RIF assay (Xpert) requested. Culture in sputum was done for a subset of patients. The diagnostic yield was defined as the proportion of patients with a positive assay result among those with laboratory-confirmed TB. For the 456 patients included in the study, the median age was 36 years (IQR 31-43) and the median CD4 count was 50 cells/µl (IQR 21-108). Forty-five percent (205/456) of the patients had laboratory-confirmed TB. The diagnostic yields of LAM, microscopy, and Xpert were 82.4% (169/205), 33.7% (69/205), and 40.0% (84/205), respectively. In total, 50.2% (103/205) of the patients with laboratory-confirmed TB were diagnosed only through LAM. Overall, the use of LAM in diagnostic algorithms increased the yield of algorithms with microscopy and with Xpert by 38.0% (78/205) and 34.6% (71/205), respectively, and, specifically among patients with CD4 100-199 cells/µl, by 27.5% (14/51) and 29.4% (15/51), respectively. LAM-positive patients not diagnosed through other tools and not treated for TB had a significantly higher risk of mortality than LAM-positive patients who received treatment (adjusted risk ratio 2.57, 95% CI 1.27-5.19, p = 0.009). Although the TB diagnostic conditions in the study sites were similar to those in other resource-limited settings, the added value of LAM may depend on the availability of microscopy or Xpert results. CONCLUSIONS: LAM has diagnostic value for identifying TB in HIV-positive patients with signs and symptoms of TB and advanced immunodeficiency, including those with a CD4 count of 100-199 cells/µl. In this study, the use of LAM enabled the diagnosis of TB in half of the patients with confirmed TB disease; without LAM, these patients would have been missed. The rapid identification and treatment of TB enabled by LAM may decrease overall mortality risk for these patients.

Leishmania Antigenuria to Predict Initial Treatment Failure and Relapse in Visceral Leishmaniasis/HIV Coinfected Patients: An Exploratory Study Nested Within a Clinical Trial in Ethiopia.

Background: Biomarkers predicting the risk of VL treatment failure and relapse in VL/HIV coinfected patients are needed. Nested within a two-site clinical trial in Ethiopia (2011-2015), we conducted an exploratory study to assess whether (1) levels of Leishmania antigenuria measured at VL diagnosis were associated with initial treatment failure and (2) levels of Leishmania antigenuria at the end of treatment (parasitologically-confirmed cure) were associated with subsequent relapse. Methods:Leishmania antigenuria at VL diagnosis and cure was determined using KAtex urine antigen test and graded as negative (0), weak/moderate (grade 1+/2+) or strongly-positive (3+). Logistic regression and Kaplan-Meier methods were used to assess the association between antigenuria and (1) initial treatment failure, and (2) relapse over the 12 months after cure, respectively. Results: The analysis to predict initial treatment failure included sixty-three coinfected adults [median age: 30 years interquartile range (IQR) 27-35], median CD4 count: 56 cells/µL (IQR 38-113). KAtex results at VL diagnosis were negative in 11 (17%), weak/moderate in 17 (27%) and strongly-positive in 35 (36%). Twenty (32%) patients had parasitologically-confirmed treatment failure, with a risk of failure of 9% (1/11) with KAtex-negative results, 0% (0/17) for KAtex 1+/2+ and 54% (19/35) for KAtex 3+ results. Compared to KAtex-negative patients, KAtex 3+ patients were at increased risk of treatment failure [odds ratio 11.9 (95% CI 1.4-103.0); P: 0.025]. Forty-four patients were included in the analysis to predict relapse [median age: 31 years (IQR 28-35), median CD4 count: 116 cells/µL (IQR 95-181)]. When achieving VL cure, KAtex results were negative in 19 (43%), weak/moderate (1+/2+) in 10 (23%), and strongly positive (3+) in 15 patients (34%). Over the subsequent 12 months, eight out of 44 patients (18%) relapsed. The predicted 1-year relapse risk was 6% for KAtex-negative results, 14% for KAtex 1+/2+ and 42% for KAtex 3+ results [hazard ratio of 2.2 (95% CI 0.1-34.9) for KAtex 1+/2+ and 9.8 (95% CI 1.8-82.1) for KAtex 3+, compared to KAtex negative patients; P: 0.03]. Conclusion: A simple field-deployable Leishmania urine antigen test can be used for risk stratification of initial treatment failure and VL relapse in HIV-patients. A dipstick-format would facilitate field implementation.

Performance of point-of-care urine test in diagnosing tuberculosis suspects with and without HIV infection in selected peripheral health settings of Addis Ababa, Ethiopia.

BACKGROUND: There are few rapid point-of-care tests (POCT) for tuberculosis (TB) for use in resource-constrained settings with high levels of human immunodeficiency virus (HIV). This hinders early tuberculosis (TB) treatment. This cross-sectional study evaluates the recently developed urine Determine tuberculosis lipoarabinomannan (TB LAM) antigen test. A total of 122 participants with signs and symptoms of TB, including 21 (17.1%) participants positive for HIV, were enrolled from September 2011 to March 2012 at three selected health centers in Addis Ababa, Ethiopia. Blood, sputum and urine samples were collected. Löwenstein-Jensen (LJ) solid culture was used as a gold standard to evaluate the performance of the Determine TB LAM antigen test. Data were analyzed using STATA (Statacorp LP, USA). RESULTS: Of the 122 participants with suspected TB, 35 (28.7%) had TB confirmed bacteriologically by LJ culture. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Determine TB LAM (for both HIV-positive and HIV-negative participants) was 37.1% (95% CI 21.5-55.1), 97.7% (95% CI 91.9-99.7), 86.7% (95% CI 59.5-98.3) and 79.4% (95% CI 70.5-86.6), respectively. However, in participants who were co-infected with TB and HIV, sensitivity, specificity, PPV and NPV were 55.6% (95% CI 21.2-86.3), 100% (95% CI 73.5-100), 100% (95% CI 47.8-100) and 75.0% (95% CI 47.6-92.7). Moreover, the level of immunosuppression of the HIV-infected TB patients was found to have a significant association with the performance of Determine TB LAM (χ2 = 7.89, p = 0.002). CONCLUSIONS: The Determine TB LAM test is a potential alternative in peripheral health settings for TB diagnosis in patients who are co-infected with HIV, with advanced immunosuppression.

Time to clearance of Chlamydia trachomatis RNA and DNA after treatment in patients coinfected with Neisseria gonorrhoeae - a prospective cohort study.

BACKGROUND: Performing a test of cure (TOC) could demonstrate success or failure of antimicrobial treatment of Chlamydia trachomatis infection, but recommendations for the timing of a TOC using nucleic acid amplification tests (NAATs) are inconsistent. We assessed time to clearance of C. trachomatis after treatment, using modern RNA- and DNA-based NAATs. METHODS: We analysed data from patients with a C. trachomatis and Neisseria gonorrhoeae coinfection who visited the STI Clinic Amsterdam, The Netherlands, from March through October 2014. After treatment with ceftriaxone plus either azithromycin or doxycycline, patients self-collected anal, vaginal or urine samples during 28 consecutive days. Samples were analysed using an RNA-based NAAT (Aptima Combo 2) and a DNA-based NAAT (Cobas 4800 CT/NG). We defined clearance as three consecutive negative results, and defined "blips" as isolated positive results following clearance. RESULTS: We included 23 patients with C. trachomatis and N. gonorrhoeae coinfection. All patients cleared C. trachomatis during follow-up, and we observed no reinfections. The median time to clearance (range) was 7 days (1-13) for RNA, and 6 days (1-15) for DNA. Ninety-five per cent of patients cleared RNA at day 13, and DNA at day 14. The risk of a blip after clearance was 4.4 % (RNA) and 1.7 % (DNA). CONCLUSIONS: If a TOC for anogenital chlamydia is indicated, we recommend performing it at least 14 days after initiation of treatment, when using modern RNA- and DNA-based assays. A positive result shortly after 14 days probably indicates a blip, rather than a treatment failure or a reinfection.

Use of a Chagas Urine Nanoparticle Test (Chunap) to Correlate with Parasitemia Levels in T. cruzi/HIV Co-infected Patients.

BACKGROUND: Early diagnosis of reactivated Chagas disease in HIV patients could be lifesaving. In Latin America, the diagnosis is made by microscopical detection of the T. cruzi parasite in the blood; a diagnostic test that lacks sensitivity. This study evaluates if levels of T. cruzi antigens in urine, determined by Chunap (Chagas urine nanoparticle test), are correlated with parasitemia levels in T. cruzi/HIV co-infected patients. METHODOLOGY/PRINCIPAL FINDINGS: T. cruzi antigens in urine of HIV patients (N = 55: 31 T. cruzi infected and 24 T. cruzi serology negative) were concentrated using hydrogel particles and quantified by Western Blot and a calibration curve. Reactivation of Chagas disease was defined by the observation of parasites in blood by microscopy. Parasitemia levels in patients with serology positive for Chagas disease were classified as follows: High parasitemia or reactivation of Chagas disease (detectable parasitemia by microscopy), moderate parasitemia (undetectable by microscopy but detectable by qPCR), and negative parasitemia (undetectable by microscopy and qPCR). The percentage of positive results detected by Chunap was: 100% (7/7) in cases of reactivation, 91.7% (11/12) in cases of moderate parasitemia, and 41.7% (5/12) in cases of negative parasitemia. Chunap specificity was found to be 91.7%. Linear regression analysis demonstrated a direct relationship between parasitemia levels and urine T. cruzi antigen concentrations (p<0.001). A cut-off of > 105 pg was chosen to determine patients with reactivation of Chagas disease (7/7). Antigenuria levels were 36.08 times (95% CI: 7.28 to 64.88) higher in patients with CD4+ lymphocyte counts below 200/mL (p = 0.016). No significant differences were found in HIV loads and CD8+ lymphocyte counts. CONCLUSION: Chunap shows potential for early detection of Chagas reactivation. With appropriate adaptation, this diagnostic test can be used to monitor Chagas disease status in T. cruzi/HIV co-infected patients.

Evaluation of chromium and manganese in biological samples (scalp hair, blood and urine) of tuberculosis and diarrhea male human immunodeficiency virus patients.

BACKGROUND: The consequence of trace elements deficiency has been associated with an increased risk of human immunodeficiency virus type 1 (HIV-1) disease progression and mortality. This study examined the association between high concentrations of chromium (Cr) and manganese (Mn) in scalp hair, blood, and urine and opportunistic infections in hospitalized patients with the acquired immune deficiency syndrome (AIDS). METHODS: The study was performed on 62 male HIV+ patients (HIV-1) from different cities of Pakistan. The patients were divided in two groups according to secondary infections (tuberculosis, diarrhea, or high fever). The biological samples (scalp hair, blood and urine) were collected from AIDS patients, and for comparative study 120 healthy subjects (males) of same age group (31 - 45 years), socio-economic status, localities, and dietary habits were also included. The elements in the biological samples were analyzed by electrothermal atomic absorption spectrophotometry after microwave-assisted acid digestion. The validity and accuracy of the methodology was checked by using certified reference materials (CRMs) and with the values obtained by conventional wet acid digestion method on the same CRMs. RESULTS: The results indicated significantly lower concentrations of Cr and Mn in the biological samples (scalp hair, blood, and urine) of male HIV-1 patients, compared with control subjects. It was observed that the lower levels of these trace elements may be predictors for secondary infections in HIV-1 patients. There was a significant decrease in mean values of Cr and Mn in whole blood and scalp hair, whilst higher concentrations were observed in urine samples of the three groups of AIDS patients as compared to a controlled healthy male group (p < 0.001). CONCLUSIONS: Low Cr and Mn levels may be due to increased Cr and Mn losses. These data present guidance to clinicians and other professional investigating deficiencies of Cr and Mn in biological samples of AIDS patients.

Proteomic analysis of serum and urine of HIV-monoinfected and HIV/HCV-coinfected patients undergoing long term treatment with nevirapine.

Nevirapine (NVP) is an effective nonnucleoside reverse transcriptase inhibitor (NNRTI) of particular interest as it is often used in resource limited countries. However, one of the main concerns with the use of NVP is hepatotoxicity and elevation of liver enzymes as a consequence of highly active antiretroviral therapy (HAART) containing NVP is more often reported in HIV patients coinfected with hepatitis C virus than in HIV-monoinfected patients. To discover possible markers of NVP induced hepatotoxicity, serum and urine samples from twenty-five HIV or HIV/HCV patients, all of whom had received NVP continuously for at least four months, and healthy controls were subjected to in-solution or in-gel proteomic analysis. A total of 83 differentially regulated proteins consisted of 34 proteins identified in serum by in-solution analysis, 2 proteins identified from serum in a 2D gel electrophoresis analysis, and 47 proteins identified in urine in an in-solution analysis. Three proteins, namely, haptoglobin, Rho-related BTB domain containing protein 3, and death-associated protein kinase 3, were selected for further validation by Western blot analysis and results showed that haptoglobin has potential for further development as an additional marker of NVP induced hepatotoxicity.

Human papillomavirus detection from human immunodeficiency virus-infected Colombian women's paired urine and cervical samples.

Infection, coinfection and type-specific human papillomavirus (HPV) distribution was evaluated in human immunodeficiency virus (HIV)-positive women from paired cervical and urine samples. Paired cervical and urine samples (n = 204) were taken from HIV-positive women for identifying HPV-DNA presence by using polymerase chain reaction (PCR) with three generic primer sets (GP5+/6+, MY09/11 and pU1M/2R). HPV-positive samples were typed for six high-risk HPV (HR-HPV) (HPV-16, -18, -31, -33, -45 and -58) and two low-risk (LR-HPV) (HPV-6/11) types. Agreement between paired sample results and diagnostic performance was evaluated. HPV infection prevalence was 70.6% in cervical and 63.2% in urine samples. HPV-16 was the most prevalent HPV type in both types of sample (66.7% in cervical samples and 62.0% in urine) followed by HPV-31(47.2%) in cervical samples and HPV-58 (35.7%) in urine samples. There was 55.4% coinfection (infection by more than one type of HPV) in cervical samples and 40.2% in urine samples. Abnormal Papanicolau smears were observed in 25.3% of the women, presenting significant association with HPV-DNA being identified in urine samples. There was poor agreement of cervical and urine sample results in generic and type-specific detection of HPV. Urine samples provided the best diagnosis when taking cytological findings as reference. In conclusion including urine samples could be a good strategy for ensuring adherence to screening programs aimed at reducing the impact of cervical cancer, since this sample is easy to obtain and showed good diagnostic performance.

Severe falciparum malaria with dengue coinfection complicated by rhabdomyolysis and acute kidney injury: an unusual case with myoglobinemia, myoglobinuria but normal serum creatine kinase.

BACKGROUND: Acute kidney injury (AKI) is a complication of severe malaria, and rhabdomyolysis with myoglobinuria is an uncommon cause. We report an unusual case of severe falciparum malaria with dengue coinfection complicated by AKI due to myoglobinemia and myoglobinuria while maintaining a normal creatine kinase (CK). CASE PRESENTATION: A 49-year old Indonesian man presented with fever, chills, and rigors with generalized myalgia and was diagnosed with falciparum malaria based on a positive blood smear. This was complicated by rhabdomyolysis with raised serum and urine myoglobin but normal CK. Despite rapid clearance of the parasitemia with intravenous artesunate and aggressive hydration maintaining good urine output, his myoglobinuria and acidosis worsened, progressing to uremia requiring renal replacement therapy. High-flux hemodiafiltration effectively cleared his serum and urine myoglobin with recovery of renal function. Further evaluation revealed evidence of dengue coinfection and past infection with murine typhus. CONCLUSION: In patients with severe falciparum malaria, the absence of raised CK alone does not exclude a diagnosis of rhabdomyolysis. Raised serum and urine myoglobin levels could lead to AKI and should be monitored. In the event of myoglobin-induced AKI requiring dialysis, clinicians may consider using high-flux hemodiafiltration instead of conventional hemodialysis for more effective myoglobin removal. In Southeast Asia, potential endemic coinfections that can also cause or worsen rhabdomyolysis, such as dengue, rickettsiosis and leptospirosis, should be considered.