Comprehensive proteomics investigation of P. vivax-infected human plasma and parasite isolates.

BACKGROUND: In recent times, Plasmodium vivax (P. vivax) has become a serious threat to public health due to its ability to cause severe infection with fatal outcomes. Its unique biology makes it resilient to control measures that are otherwise effective against P. falciparum. A deeper understanding of P. vivax biology and pathogenesis is, therefore, essential for developing the right control strategies. Proteomics of P. falciparum has been helpful in studying disease biology and elucidating molecular mechanisms involved in the development of disease. However, unlike P. falciparum, proteomics data for P. vivax infection is minimal due to the absence of a continuous culture system. The dependence on clinical samples and animal models has drastically limited P. vivax research, creating critical knowledge gaps in our understanding of the disease. This study describes an in-depth proteomics analysis of P. vivax-infected human plasma and parasite isolates, to understand parasite biology, pathogenesis, and to identify new diagnostic targets for P. vivax malaria. METHODS: A mass-spectrometry- (MS) based proteomics approach (Q Exactive) was applied to analyze human plasma and parasite isolates from vivax malaria patients visiting a primary health centre in India. Additionally, a targeted proteomics assay was standardized for validating unique peptides of most recurring parasite proteins. RESULTS: Thirty-eight P. vivax proteins were detected in human plasma with high confidence. Several glycolytic enzymes were found along with hypothetical, cytoskeletal, ribosomal, and nuclear proteins. Additionally, 103 highly abundant P. vivax proteins were detected in parasite isolates. This represents the highest number of parasite proteins to be reported from clinical samples so far. Interestingly, five of these; three Plasmodium exported proteins (PVX_003545, PVX_003555 and PVX_121935), a hypothetical protein (PVX_083555) and Pvstp1 (subtelomeric transmembrane protein 1, PVX_094303) were found in both plasma and parasite isolates. CONCLUSIONS: A parasite proteomics investigation is essential to understand disease pathobiology and design novel interventions. Control strategies against P. vivax also depend on early diagnosis. This work provides deeper insights into the biology of P. vivax by identifying proteins expressed by the parasite during its complex life-cycle within the human host. The study also reports antigens that may be explored as diagnostic candidates.

Pulmonary cystic echinococcosis: A case report from tertiary care hospital.

Human echinococcosis is a zoonotic infection transmitted by dogs in livestock-raising areas. We present a case of a 30-year-old female with respiratory symptoms.

Performance of Xpert MTB/RIF for detection of Mycobacterium tuberculosis and rifampicin resistance in pus aspirates.

INTRODUCTION: WHO endorsed Xpert MTB/RIF assay has proven to be rapid with results obtained within 2h. The evidence base regarding the use of Xpert MTB/RIF in pulmonary TB is strong. Relatively few performance data have been published to date on detection of Mycobacterium tuberculosis in aspirated pus specimens from abscesses. OBJECTIVES: The aim of the study was to determine the sensitivity and specificity of Xpert MTB/RIF assay for the detection of M. tuberculosis and rifampicin resistance in aspirated pus specimens using culture on Lowenstein Jensen (LJ) medium and economic variant of proportion method (PM) for drug susceptibility testing (DST) as the reference standard. RESULTS: Xpert MTB/RIF assay in comparison to conventional reference method showed sensitivity and specificity of 76.19% and 68.75% for detection of M. tuberculosis and 71.4% and 100% for detection of rifampicin resistance respectively. CONCLUSION: The simplicity, sensitivity, speed and automation makes this assay a very promising diagnostic test for detection of M. tuberculosis and rifampicin resistance in aspirated pus specimens.

Diagnostic accuracy of Xpert MTB/RIF assay in extrapulmonary tuberculosis.

INTRODUCTION: The WHO endorsed Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay, has been evaluated for pulmonary TB in a number of studies but very few have investigated it for extrapulmonary specimens. The present study evaluates the performance of Xpert MTB/RIF assay in the diagnosis of extrapulmonary TB (EPTB). AIM AND OBJECTIVES: The aim of the study is to determine sensitivity and specificity of Xpert MTB/RIF assay for diagnosis of EPTB and RIF resistance in comparison to culture on Lowenstein-Jensen (LJ) medium and proportion method (PM), respectively. MATERIALS AND METHODS: A total of 738 specimens from clinically suspected cases of EPTB were subjected to Ziehl-Neelsen staining, Xpert MTB/RIF assay and culture on LJ medium. PM was done on MTB isolates. RESULTS: The sensitivity, specificity of Xpert MTB/RIF assay for diagnosis of EPTB were 84.91% (95% confidence interval [CI] 72.41%-93.25%) and 86.72% (95% CI 83.94%-89.17%) and for RIF resistance detection were 60.00% (95% CI 32.29%-83.66%) and 94.74% (95% CI 73.97%-99.87%), respectively. Among culture-positive cases, the sensitivity of Xpert MTB/RIF assay was 94.12% in smear positive and 80.56% in smear-negative cases. Xpert MTB/RIF showed maximum sensitivity of MTB detection from lymph node specimens (100% [95% CI 54.07%-100.00%]) and other body fluids (100% [95% CI 15.81%-100.00%]). CONCLUSION: The present study establishes Xpert MTB/RIF assay as a promising tool in the rapid diagnosis of EPTB and detection of RIF resistance.

Identification of Highly Expressed Plasmodium Vivax Proteins from Clinical Isolates Using Proteomics.

Plasmodium vivax is the most geographically widespread species responsible for malaria in humans. Our study focused on identifying highly expressed parasite proteins using a shotgun proteomics approach. Parasites (P. vivax) are isolated from seven patient samples using saponin lysis. Protein extracts from these parasites are processed and subjected to LC-MS/MS analysis. An overall proteome coverage of 605 P. vivax proteins along with 1670 human host proteins are obtained upon combining the data from LC-MS/MS runs. While a major proportion of the P. vivax proteins are either hypothetical or involved in basic cellular activities, few proteins such as tryptophan-rich antigen (Pv-fam-a; PVX_090265), Pv-fam-d protein (PVX_101520), Plasmodium exported protein (PVX_003545), Pvstp1 (PVX_094303) and hypothetical protein (PVX_083555) are detected in more than 80% of the clinical isolates and found to be unique to P. vivax without orthologs in P. falciparum. Our proteomics study on individual parasite isolates reveals highly expressed P. vivax proteins, few of which may be good candidates for vivax malaria diagnosis due to their abundance and absence in P. falciparum. This study represents the first step towards the identification of biomarkers for P. vivax malaria. In future, their clinical diagnostic values must be explored and validated on large patient cohorts.

Detection of ofloxacin resistance by nitrate reductase assay in Mycobacterium tuberculosis isolates from extrapulmonary tuberculosis.

CONTEXT: Increased use of fluoroquinolones to treat community-acquired infections has led to the decreased susceptibility to Mycobacterium tuberculosis. There is a paucity of data on ofloxacin (OFX) resistance detection by nitrate reductase assay (NRA). Hence, the present study was carried out to find the efficacy of NRA for detection of OFX resistance in M. tuberculosis isolated from extrapulmonary tuberculosis (EPTB) cases. AIMS: (1) To compare sensitivity, specificity and median time required to obtain results by NRA with economic variant proportion method (PM) for detection of OFX resistance.(2) To determine the extent of OFX resistance in clinical isolates of M. tuberculosis. SETTINGS AND DESIGN: Seventy-three M. tuberculosis isolates from cases of EPTB were subjected to economic variant of PM for isoniazid, rifampicin and OFX. NRA was done for detection of OFX resistance. SUBJECTS AND METHODS: Seventy-three isolates from clinical samples of suspected EPTB received in the Department of Microbiology were included in the study. Drug susceptibility test was performed on Lowenstein-Jensen medium with and without drugs. STATISTICAL ANALYSIS USED: Of turnaround time was done by Mann-Whitney test on SPSS (version 19, released in 2010, IBM Corp, Armonk NY),P < 0.05. RESULTS: OFX resistance was seen in nine isolates. The sensitivity and specificity of OFX resistance by NRA was 100% and 96.87%, respectively. Median time required to obtain results by NRA was 10 days as compared to 28 days by PM. CONCLUSIONS: NRA is a specific and sensitive method for detection of OFX resistance in resource-restricted settings.