Molecular assays for determining sulphadoxine-pyrimethamine drug resistance in India: a systematic review.

A plethora of studies analyse the molecular markers of drug resistance and hence help in guiding the evidence-based malaria treatment policies in India. For reporting mutations, a number of techniques including DNA sequencing, restriction-fragment length polymorphism and mutation-specific polymerase chain reaction have been employed across numerous studies, including variations in the methodology used. However, there is no sufficient data from India comparing these methods as well as report the prevalence of polymorphisms in SP drug resistance molecular markers independently using such methods. Therefore, all data from Indian studies available for molecular marker studies of Plasmodium falciparum drug resistance to sulphadoxine-pyrimethamine was gathered, and a systematic review was performed. This systematic review identifies the molecular methods in use in India and compares each method for detecting sulphadoxine-pyrimethamine drug resistance marker. To delay the spread of drug-resistant parasite strains, a simplified and standardized molecular method is much needed which can be obtained by analysing the performance of each method in use and answering the necessity of newer methodological approaches.

Determination of Etiology and Epidemiology of Viral Central Nervous System Infections by Quantitative Real-Time Polymerase Chain Reaction in Central India Population.

INTRODUCTION: Viral infections of the central nervous system (CNS) are the most common cause of hospital admission in worldwide and remain a challenging disease for diagnosis and treatment. The most common infectious agents associated with viral CNS infections are cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), Japanese encephalitis virus (JEV), Dengue virus (DENV),West Nile virus(WNV), and Chandipura virus(CHPV). The aim of the present work was to find the etiology of CNS viral infection in the Central India population by transcriptase PCR (RT-PCR) comparing real-time polymerase chain reaction (PCR) method [one-step and two-step reverse transcriptase (RT-PCR)] in cerebrospinal fluid (CSF) and blood samples of CNS viral infections patients. MATERIALS AND METHODS: One-step and two-step real-time PCR assays were evaluated in CSF and parallel blood samples from patients with viral CNS infections for detection of DNA and RNA viruses. A comparative analysis was also done between gDNA, gRNA, cDNA, and plasmid-based real-time PCR methods for an efficient quantitation of viral particles in clinical samples for determination of viral etiology. RESULT: On evaluation of 150 CSF and 50 parallel blood samples from suspected cases of viral CNS infections, a viral etiology was confirmed in 21 (14%) cases, including 3% for EBV, 1% of CMV, and 5% for VZV and JEV. The one-step RT-PCR has a higher detection limit for detection and quantitation of viral RNA in comparison to two-step RT-PCR. CONCLUSION: Our result reveals that VZV and JEV are the most usual cases of CNS viral infection in hospitalized patients in the Central India population and one-step RT-PCR shows higher viral load detection limits for quantitation of viral genome and more sensitivity in comparison to two-step RT-PCR.

Seroprevalence and clinical manifestations of chikungunya virus infection in rural areas of Chandrapur, Maharashtra, India.

BACKGROUND & OBJECTIVES: Chikungunya virus (CHIKV) infection has recently witnessed re-emergence, affecting rural areas of India with high morbidity rates. This prospective study was conducted to evaluate seroprevalence and clinical manifestation in targeted villages reporting cases of CHIKV infection. METHODS: A total of 482 patients were recruited from Kalmana and Kothari villages of Ballarpur; Chandrapur district of Maharashtra state, India during CHIKV outbreaks in 2011-12. The serum samples from infected CHIKV patients were simultaneously screened through ELISA for detection of antigen and antibodies (IgM and IgG). Chi-square analysis was used to evaluate differences in seropositivity between age, gender and clinical manifestations of CHIKV. RESULTS: Out of 482 enrolled participants, 197 (41%) males and 285 (59%) females were aged between 5 and 92 yr. The clinical manifestations such as small joint pain (80%), neck stiffness (75%), fever (49%) and large joint pain (47%) were observed amongst CHIKV infected subjects. Mucocutaneous rashes (91%) on knees (71%), feet (56%), fingers and palms (54%) were also observed. Overall, seroprevalence of CHIKV infection was found to be 46% in infected participants during the epidemic period. Among risk factors, ageing and female gender was strongly associated with a raised seroprevalence of CHIKV infection along with symptoms such as rashes, small joints pain and neck stiffness. INTERPRETATION & CONCLUSION: This study reported high seroprevalence rates of CHIKV infection in targeted popula- tions, suggesting its re-emergence in rural India. Proper surveillance is, therefore, necessary to minimize re-emergence and in controlling these impending and sporadic outbreaks.

Synthetic Peptide-Based Antibody Detection for Diagnosis of Chikungunya Infection with and without Neurological Complications.

Synthetic peptide-based diagnosis of Chikungunya can be an efficient and more accessible approach in immunodiagnostics. Here, we describe the identification of Chikungunya-specific 40 kD protein for development of synthetic peptide-based enzyme-linked immunosorbent assay for the detection of Chikungunya virus-specific antibodies in the patient's sample. The total sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile of the patient's sample can be done to identify specific protein bands. The identified proteins can be subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) for characterization. After characterization, immunogenic peptides can be designed using softwares and subsequently synthesized chemically. The peptides can be used to develop more specific, sensitive, and simpler diagnostic assay.

Diagnosis of Herpes Simplex Encephalitis by ELISA Using Antipeptide Antibodies Against Type-Common Epitopes of Glycoprotein B of Herpes Simplex Viruses.

Herpes simplex encephalitis (HSE) represents one of the most severe infectious diseases of the central nervous system (CNS). As effective antiviral drugs are available, an early, rapid, and reliable diagnosis has become important. The objective of this article was to develop a sensitive ELISA protocol for herpes simplex viruses (HSV) antigen detection and quantitation by assessing the usefulness of antipeptide antibodies against potential peptides of HSV glycoprotein B (gB). A total of 180 cerebrospinal fluid (CSF) samples of HSE and non-HSE patients were analyzed using a panel of antipeptide antibodies against synthetic peptides of HSV glycoprotein gB. The cases of confirmed and suspected HSE showed 80% and 51% positivity for antipeptide against synthetic peptide QLHDLRF and 77% and 53% positivity for antipeptide against synthetic peptide MKALYPLTT, respectively for the detection of HSV antigen in CSF. The concentration of HSV antigen was found to be higher in confirmed HSE as compared to suspected HSE group and the viral load correlated well with antigen concentration obtained using the two antipeptides in CSF of confirmed HSE group. This is the first article describing the use of antibodies obtained against synthetic peptides derived from HSV in diagnostics of HSE using patients' CSF samples.

Rapid diagnosis and simultaneous identification of tuberculous and bacterial meningitis by a newly developed duplex polymerase chain reaction.

The present study describes the development and evaluation of a duplex polymerase chain reaction (D-PCR) for diagnosis and simultaneous identification of tuberculous meningitis (TBM) and bacterial meningitis (BM) in a single reaction. A D-PCR with primers amplifying portions of the Mycobacterium tuberculosis IS6110 and the eubacteria 16SrDNA sequence in a same reaction mix was developed and tested on DNA extracted from 150 clinical CSF samples from different categories (TBM = 39, BM = 26, control infectious and non-infectious category = 85). The results indicate a clear differentiation between bands for eubacteria and M. tuberculosis with an analytical sensitivity of 10(3) cfu/ml for eubacteria and 10(2) cfu/ml for M. tuberculosis. When evaluated in clinical samples, D-PCR overall diagnosed 100 % confirmed TBM and 100 % confirmed BM cases with overall specificity of 96.5 %. D-PCR can be an effective tool for diagnosis and simultaneous identification of TBM or BM in a single PCR reaction. It saves time, cost, labour and sample amount and help in administration of appropriate antimicrobial therapy. The proposed diagnostic assay would be helpful in correct and rapid management of TBM and BM patients.

Genome Sequence of Mycobacterium tuberculosis C2, a Cerebrospinal Fluid Clinical Isolate from Central India.

We report the annotated genome sequence of a Mycobacterium tuberculosis clinical isolate from the cerebrospinal fluid of a tuberculous meningitis patient admitted to the Central India Institute of Medical Sciences, Nagpur, India.

Determination of Toll-like receptor-induced cytokine profiles in the blood and cerebrospinal fluid of Chikungunya patients.

BACKGROUND: Chikungunya infection caused by Chikungunya virus (CHIKV) is an inflammatory disease affecting the joints and may also lead to neurological complications. We investigated a panel of human Toll-like receptor (TLR)-induced cytokines in Chikungunya patients with and without neurological complications. METHODS: In a case-control study, a panel of 12 cytokines and chemokines, TNF-α, IFN-α, IL-1ß, IL-6, IL-12, IL-17A, IL-8, monocyte chemotactic protein (MCP)-1, RANTES, interferon (IFN)-γ-induced protein (IP)-10, monokine induced by IFN-γ (MIG) and thymus and activation-regulated chemokine (TARC), was analysed using a conventional ELISA protocol in the serum samples of Chikungunya patients without neurological complications and in the cerebrospinal fluid (CSF) and paired serum samples of Chikungunya patients with neurological complications. RESULTS: The levels of 3 cytokines, IL-1ß, IL-17A and IL-8, and 4 chemokines, MCP-1, RANTES, IP-10 and TARC, were raised in serum samples of Chikungunya patients without neurological complications, whereas, 4 cytokines, TNF-α, IFN-α, IL-6 and IL-8, and 4 chemokines, MCP-1, RANTES, MIG and TARC, were elevated in CSF samples of Chikungunya patients with neurological complications. Moreover, the levels of IL-6 and IL-8 cytokines were significantly elevated in the CSF compared to paired serum samples in Chikungunya patients with neurological complications. CONCLUSIONS: In CHIKV infection, multiple cytokines are elevated in serum and CSF. The elevation in IL-6 and IL-8 cytokines in CSF correlates with neurological involvement.

Determination of viral load by quantitative real-time PCR in herpes simplex encephalitis patients.

OBJECTIVE: Human herpesviruses cause various acute, subacute, and chronic disorders of the central nervous system and peripheral nervous systems in adults and children. The objective of the present study is to summarize the experience gained with the estimation of viral load in the central nervous system of children and adults with herpes simplex encephalitis (HSE) admitted to a neurological institute at Nagpur, India, by quantitative real-time PCR (qPCR) assay within the past 4 years. METHODS: The qPCR assay was evaluated retrospectively in 242 cerebrospinal fluid (CSF) samples from patients. Evaluation of possible relationships was done between the herpes simplex virus (HSV) DNA concentration in CSF with that of patients' clinical and laboratory manifestations. The prevalence of the type of HSV in the study population was also determined using type-specific real-time PCR analysis. RESULTS AND CONCLUSIONS: Real-time analysis using type-specific primers revealed the presence of predominantly HSV-1 genotype in the study population. The qPCR results show that in patients with higher viral loads in their CSF, a greater number of cases were associated with the presence of lesions in the brain as revealed by computed tomography/magnetic resonance imaging scan. They required acyclovir therapy for a longer duration and had a poorer clinical outcome than the patients with lower viral loads in their CSF.

Protein A-based ELISA: its evaluation in the diagnosis of herpes simplex encephalitis.

Herpes simplex encephalitis (HSE) represents one of the most severe infectious diseases of the central nervous system. As effective antiviral drugs are available, early rapid and reliable diagnosis has become important. The objective of the present study was to develop a sensitive enzyme-linked immunosorbent assay (ELISA) protocol for herpes simplex virus (HSV) antigen detection by assessing the usefulness of hyperimmune sera isolated from HSV-seropositive patients. A total of 52 cerebrospinal fluid (CSF) and 62 serum samples of HSE patients and non-HSE persons were analyzed. An in-house ELISA protocol utilizing hyperimmune sera was developed for HSV antigen detection. To improve the specificity of the method, protein A was incorporated into the protocol for ELISA. The sensitivity (70% and 90%) of antigen detection was high in CSF and serum samples, respectively, of confirmed HSE patients. However, lower specificity (52.3% and 42.3%), respectively, was obtained, which was improved by using protein A in the ELISA protocol. The modification in the method yielded good sensitivity (80% and 70%) and specificity (85.7% and 88.4%) of HSV antigen detection in the CSF and sera, respectively, of the HSE and non-HSE groups. The ELISA method utilizing hyperimmune sera along with protein A for HSV antigen detection yielded good sensitivity and specificity in both CSF and sera, and hence can be useful for the diagnosis of HSE.