Kapil Goyal

NS1 antigen as an early diagnostic marker in dengue: report from India

Detection of specific IgM antibodies by ELISA forms the mainstay for diagnosis of dengue infection. However, IgM antibodies develop after 4 to 5 days of infection. The methods for early diagnosis include virus isolation and reverse transcriptase polymerase chain reaction (RT-PCR) which need a sophisticated laboratory. Another alternative that has recently come up is NS1 antigen detection. The present study compared IgM antibody detection with NS1 antigen for the diagnosis of acute dengue in 87 samples. NS1 antigen could be detected with good sensitivity (71-100%) till day 3 of fever, whereas IgM had a sensitivity of 0% to 50% at this time. On day 4 of illness, both the tests had comparative sensitivity. Beyond day 4, IgM antibody detection was superior to NS1. Both these diagnostic modalities were also compared with RT-PCR in 40 acute samples. NS1 detected additional 15 samples, which were missed by PCR. NS1 antigen is an early diagnostic marker that is feasible in a routine diagnostic laboratory.

Neurocysticercosis: A disease of neglect

Neurocysticercosis (NCC) is a neglected tropical disease caused by larval forms of the parasite Taenia solium lodging in central nervous system (CNS). There is a huge morbidity and debilitation due to CNS manifestations of NCC in developing and underdeveloped regions of the globe, mainly Asian, African and Latin American countries. It is the cause of epilepsy in about 1% of the population of endemic countries and is the underlying etiology in about 15-50% persons with epilepsy, depending upon the geographical region. There is no perfect diagnostic method and the diagnosis relies on a combination of clinical, radio-imaging, immunologic and epidemiologic data. Treatment includes anti-parasitic treatment by cysticidal drugs and management of associated symptoms and complications. The disease is eradicable and control depends on an integrated and coordinated involvement of international bodies like the World Health Organization along with scientific institutions and political and administrative strata of the endemic countries to provide the essential tools such as adequate sanitation, live-stock management, health education and improved socio-economic conditions.

Multiplex PCR for rapid diagnosis of gastrointestinal tuberculosis.

Background: Rapid and specific diagnosis of gastrointestinal tuberculosis (GITB) is of utmost importance. Aim: To evaluate Multiplex PCR (MPCR) using MPB64 and IS6110 primers specific for M. tuberculosis for rapid diagnosis of GITB. Materials and Methods: MPCR was performed on colonoscopy biopsy specimens on 11 GITB confirmed (culture/AFB/histopathology was positive), 29 GITB suspected and 30 Non GITB (control group) patients. Results: MPB64 PCR had sensitivity and specificity of 90% and 100% for confirmed GITB cases. In 29 clinically diagnosed but unconfirmed GITB cases, MPCR was positive in 72.41%. MPCR was negative in all control group patients. The overall sensitivity and specificity of microscopy, culture, histopathology and MPCR was 5%, 2% 20% and 77.5% and 100%, 100%, 100% and 100% respectively. Conclusion: MPCR has good sensitivity and specificity in diagnosing gastrointestinal tuberculosis.

Keeping parasitology under the One Health umbrella

The One Health concept is no longer new, but remains an accepted concept in modern disease control – where the interactions between animal health, human health, and the environment in which we live are recognised as being of importance. However, emerging infectious diseases often garner the greatest attention and resources. Parasitic infections, many of which are zoonotic but cannot truly be considered as emerging, must ensure that they retain their place under the One Health umbrella.

Trichomoniasis and Lactoferrin: Future Prospects

Trichomonas vaginalis is a parasitic protozoan which infects the urogenital tract and requires iron as an essential nutrient. Iron is known to upregulate various adhesins required for cytoadherance and other factors involved in pathogenesis. At mucosal surfaces, iron is chelated by lactoferrin resulting in low levels of free iron. However, pathogens have evolved mechanisms for an increased uptake of iron. The present review highlights the role of iron in survival of Trichomonas during fluctuating concentrations of iron at mucosal surfaces during the menstrual cycle. Future prospects in terms of new drug and vaccine targets related to iron and its receptors have also been described.

RealAmp Loop-Mediated Isothermal Amplification as a Point-of-Care Test for Diagnosis of Malaria: Neither Too Close nor Too Far

TO THE EDITOR—Patel et al [1] investigated the usefulness of the real-time fluorescence loop-mediated isothermal amplification (LAMP) assay, RealAmp, as a point-of-care test in diagnosing malaria. In an accompanying editorial, Hsiang et al [2] highlighted some advantages and limitations of LAMP as a point-ofcare test. Molecular techniques are well known for their high sensitivity and specificity [3]. The same characteristics apply in the diagnosis of submicroscopic malaria by molecular techniques, compared with conventional microscopy or antigen-detection methods [4]. LAMP provides results rapidly and uses relatively inexpensive equipment for amplification, compared with conventional polymerase chain reaction (PCR) [5]. However, for validation of DNA extraction methods, it is essential to amplify housekeeping genes and to include positive and negative controls during each run. Because LAMP amplifies genomic DNA several fold, slight contamination during processing may result in false-positive findings, as highlighted byHsiang et al [2]. Thus, it is essential to analyze the results of RealAmp, along with the quality-control results, with caution. Another important factor that requires consideration is the DNA-extraction method of boil and spin, which has been used by Patel et al for onsite extraction of DNA. Briefly, this method requires boiling and centrifugation steps. Therefore, certain equipment and uninterrupted electricity supply are required if many samples have to be screened. Although the authors mentioned that equipment used for performing RealAmp can be run with the help of a rechargeable battery, in remote areas the supply of electricity may be insufficient for recharging the battery [6]. Moreover, whether a rechargeable battery can be used for long periods to power the equipment required for DNA extraction has not been investigated. For performing good-quality molecular work without contamination, separate workstations, along with sterile autoclaved tips and PCR tubes, are required. Before the use of PCR workstations, UV light exposure is required for decontamination. Thus, a regular electricity supply is essential to run an autoclave and for performing RealAmp, which may be the most limiting factor in remote areas. Innovations are required whereby DNA can be isolated by enzymes or reagents without sophisticated equipment, and LAMP can be performed in the same vial by adding reaction mixtures to it so that chances of contamination are minimized and amplification of DNA is achievable [7]. Further, automated systems can be developed in which all steps, starting with DNA extraction and isothermal amplification and continuing through the analysis of results, can be performed quickly, so that contamination can be avoided and the assay made more user friendly [8]. Thus, we are today neither too far nor too close to using LAMP as a point-ofcare test for the diagnosis of malaria.

Assessment of dried blood samples as an alternative less invasive method for detection of Hepatitis E virus marker in an outbreak setting.

Hepatitis E virus (HEV) is the causative agent of hepatitis E. It can be asymptomatic, associated with acute self‐limiting hepatitis or acute liver failure. The conventional diagnosis of HEV infection relies on anti‐HEV IgM serology. The collection of blood samples by venepunture for laboratory confirmation is often difficult during an outbreak. Thus, testing the specimens of dried blood spots (DBS) on filter papers can prove to be a feasible alternative. The present study aimed to evaluate the applicability of anti‐HEV IgM detection from DBS samples and the stability of anti‐HEV IgM detection at varied time interval, at various storage temperatures. Paired blood and DBS sample were collected from 44 jaundiced patients and eight healthy controls during HEV outbreaks. The DBS were tested for anti‐HEV IgM by available ELISA kit with in‐house modifications. Three cut offs were determined, that is, the CO1: kit cut‐off, CO2: mean of negative controls above 3SD and CO3: area under Receiver operating Curve. The sensitivity of anti‐HEV IgM detection ranged from 86–91%. The maximum sensitivity (91%) and specificity (100%) was obtained using CO3. Maximum stability of anti‐HEV IgM antibodies (100%) was observed till 65 days at 4°C. Storage at 37°C significantly reduced anti‐HEV IgM positivity, wherein 42.85% sample became negative by 45 days. DBS showed good sensitivity and specificity for detecting anti‐HEV IgM and can be considered an alternate to serum sample. Moreover, anti‐HEV IgM was stable at 4°C, which makes DBS a preferred method for storage and transportation of the sample to reference laboratory. J. Med. Virol. 86:713–719, 2014.

Shigellemia in a post renal transplant patient: a case report and literature review.

Shigellemia is a complication of shigellosis that occurs generally in malnourished children. In adults, shigellemia is usually seen in immunocompromised individuals. Here we report the first case of shigellemia in a renal transplant patient from India. The patient had history of diarrhea, which was treated symptomatically. Subsequently, the patient developed high-grade fever and blood culture was positive for Shigella flexneri. Recovery was uneventful after the initiation of antimicrobial therapy. In a country like India with high prevalence of shigellosis, screening for Shigella in the pre-transplant period may minimize the morbidity and prolonged hospital stay associated with the complication of septicemia.

Investigation of suspected viral hepatitis outbreaks in North West India

Hepatitis E (HEV) infection is diagnosed on the basis of serum anti-HEV IgM detection. In outbreaks, early diagnostic method is important for prompt control measures. This study compared 3 diagnostic methods in 60 serum samples collected in suspected HEV outbreaks. The suitability of saliva samples for antibody detection was also evaluated in 21 paired serum saliva samples. The anti-HEV IgM, HEV-Ag, and HEV-RNA were detected in serum samples of 52 (86.66%), 16 (26.66%), and 18 (30%) patients, respectively. The concordance between serum and saliva IgM was found to be 76.91%. The positivity of PCR and HEV-Ag detection was 100% within 1 week of illness which declined to 5-10% thereafter. The outbreak was attributed to HEV genotype 1, subtype 1a, and the clinical and environmental strains clustered together. HEV-antigen and RNA were an early diagnostic marker with 96.66% concordance. Saliva samples can be used as an alternative in outbreak setting.

Rubella seronegativity among health care workers in a tertiary care north Indian hospital: Implications for immunization policy

BACKGROUND Rubella is traditionally considered a childhood disease but has the potential to cause outbreaks in hospital set ups. It is important to know the susceptibility status of health care workers (HCWs) as to frame guidelines for their immunization and thus prevent hospital outbreaks. PARTICIPANTS The rubella susceptibility status of 313 HCWs working in the institute was assessed. This study was initiated after we reported an outbreak due to rubella among HCWs of our institute. MATERIALS AND METHODS The serum samples were tested to determine Rubella IgG titres by enzyme linked immunosorbent assay (ELISA). RESULTS Overall, 48 (15.3%) subjects were found to be negative, thereby indicating their susceptibility to infection. Out of them, 29 (60.5%) were in contact with pregnant women during the course of their employment. There is a risk of nosocomial transmission of rubella from affected HCWs to their contacts especially pregnant women as many of the rubella infections are asymptomatic. CONCLUSION Hence, we stress the need for vaccinating the HCWs at the start of their employment to contain the spread of infection and also to reduce the risk of outbreaks in work place.