Assessment of efficacy of palm polymerase chain reaction with microscopy, rapid diagnostic test and conventional polymerase chain reaction for diagnosis of malaria

Purpose: Sensitive, specific, rapid and cost-effective technique for malaria diagnosis is need of the hour. Microscopy has been the gold standard for malaria diagnosis, but its interpersonnel variability and lack of sensitivity make it subjective test. Conventional polymerase chain reaction (cPCR) has proven to be sensitive technique, but costly and time-consuming. Considering these factors, we have compared microscopy and cPCR with newly derives ultra-fast, portable PCR machine called Palm PCR. Materials and Methods: Palm PCR is arranged with three heat blocks precisely made for three stages of PCR cycles with 34 min for 1100 bp Plasmodium genus outer primer to amplify and 10 min each for Plasmodium falciparum and Plasmodium vivax inner primers of 120 bp and 205 bp, respectively. A total of 191 suspected samples were processed and evaluated using receiver operating characteristic (ROC) curve analysis. Results: The area under ROC curve analysis for Palm PCR with reference standard microscopy for P. falciparum, P. vivax and Plasmodium was 0.8969, 0.9121 and 0.9116, respectively, and with reference standard cPCR was 1.0 for all of them. ROC curve area close of suggests that Palm PCR can be as significant as cPCR in malaria diagnosis. In fact, ultra-rapid amplification with same precision makes Palm PCR better technique than cPCR. Conclusion: Palm PCR is sensitive, rapid and works on battery with simple laboratory facility requirements. Portable electrophoresis and transilluminator combined with Palm PCR could be implemented as an important diagnostic tool in resource-limited and rural areas. Similar studies with wider parameters in rural areas will help us evaluate and maybe establish Palm PCR as PCR platform of choice for such specific set-ups.

Seroepidemiological survey of health care workers in Maharashtra.

Context: HCWs all over the world carry occupational risk of getting infected with major blood borne infections through needle stick injuries (NSIs). As health care industry has been expanding, risk of nosocomial infections is increasing proportionately. Measures to prevent it and put in place a mechanism to control these injuries are needed urgently, especially in India where there is not only increase in domestic demand but impetus in health tourism. Aim: To determine HBs Ag, HBc IgM level and to assess anti-HBs level prevalence in HCWs, in a tertiary care hospital and to study the influence of factors like age and sex in the vaccinated HCWs and formulate mechanism to increase awareness to create a safe working environment in the hospitals. Settings and Design: 437 HCWs, working in Laboratories, Surgical, Medical or Dental departments in 11 Civil Hospitals and Sub-district Hospitals covering 8 circles of the State. Methods and Material: Qualitative and Quantitative estimation of HBs Ag and Anti-HBs by sandwich ELISA technique and qualitative HBc IgM level by antibody-capture, non-competitive test. Liver profile (SGPT, SGOT and Alkaline Phosphatase) by IFCC method done. Statistical Analysis Used: Tabulation and Pie Circle Result: 193 of the total 229 vaccinated HCWs tested positive for core antibody, meaning that they were infected prior to HBs Ag vaccination, leaving a total of 36 ‘truly’ vaccinated HCWs. 11 HBs Ag positive HCWs were tested for Liver Profile and all had ALAT, ASAT and ALP within normal range. Out of total number of 141 HCWs having 10 and below IU/L anti HBs, 5 HCWs were positive for HBS Ag, showing a positivity of 3.5%. Conclusion: Need of vaccination and for post-vaccination serological testing of all HCWs considering the high rates of non-responders and low responders (anti-HBs-34.2%). Importance of educating the HCWs of safety precautions while handling body fluids, and the management of ‘ sharps ‘ injuries.

A preliminary study of recombinant human interferon-α-2a activity against rabies virus in murine model.

Rabies remains an important public health problem in the world due to uncontrolled enzootic rabies. Although rabies associated fatalities may be prevented with timely immunoprophylaxis, but till date a therapeutic molecule has remained elusive. We investigated the role of rhuIFN α-2a in murine model challenged with rabies virus. Titre of 104.25 LD50/0.03 ml of 10% w/v RV CVS stock suspension were obtained. Based on 1LD50 titre, challenge dose of 50 LD50 was administered along with rhuIFN α-2a with pre-exposure (primed) and post-exposure with the rabies virus. Both showed increased survival time as compared with the virus controls. These fi ndings suggest that the rhuIFN α-2a might have some anti-viral activity, which can be used for the treatment of rabies infection. Further research on the effi cacy of interferon along with anti-viral drugs for the treatment will be helpful in designing combination therapy against the disease.

Surveillance of Acute Respiratory Infections in Mumbai during 2011-12.

Purpose: Acute respiratory infections (ARIs) are a leading cause of morbidity and mortality in individuals aged less than 5 years. ARI often leads to hospitalisation, and it has been indicated that causative viral and bacterial infections go undetermined and results in the occurrence of resistant strains. The objective of the study was to assess the prevalence of various viral and bacterial infections in patients with ARIs. Materials and Methods: Two hundred samples were collected from July 2011 to July 2012 with patients suffering from ARI. Viral and bacterial infections were determined by real time reverse transcriptase polymerase chain reaction. Results: Infl uenza-like illness (ILI) consisted of 109 patients and ARI consisted of 91 patients. Pandemic infl uenza A H1N1 was the major viral infection with 21 (19.2%) patients in ILI as compared with 16 (17.4%) patients in ARI. Respiratory syncytial virus (RSV) was found to be 1 (0.9%) in ILI and ARI. Viral co-infections were 16 (14.4%) in ILI and 4 (4.37%) in ARI where pandemic infl uenza A H1N1 and infl uenza type B were major contributors. In bacterial infections, Streptococcus pneumoniae with 11 (10.9%) cases were predominant in both the groups. Bacterial co-infection accounted for only 1 (1.09%) case in both the groups but the most signifi cant fi nding was the viral-bacterial co-infection in which Haemophilus infl uenzae was the major co-infecting bacteria with the infl uenza viruses with 4 (4.36%) cases as compared with Streptotoccus pneumoniae. Conclusion: This data indicate the need to undertake continued surveillance that will help to better defi ne the circulation of respiratory viruses along with the bacterial infections.

Prevalence of leptospirosis among dogs and rodents and their possible role in human leptospirosis from Mumbai, India.

A total of 100 blood and 18 urine samples of rodents and suspected dogs were collected from Mumbai, India during 2006-2008. In order to determine the role of animals in transmission of the disease to humans, all the samples were screened retrospectively by real-time polymerase chain reaction for leptospiral DNA and antibodies were detected using microscopic agglutination test. Leptopsiral DNA was detected from two blood and fi ve urine samples from rodents. Of a total of 71 rodent and dog samples investigated for anti-Leptospira antibodies, 14 (19.7%) were positive. Pyrogenes was the predominant serovar found in 100.0% (7/7) and 85.7% (6/7) from suspected canine cases and rodents, respectively; followed by Icterohemorrhagiae, which was found in one rodent sample 14.28% (1/7). The study proves that there is high prevalence of leptospirosis in rodents and dogs in this region, which proves possible role of these animals in transmission of leptospires to humans. Hence it is imperative to necessary control measures to prevent human leptospirosis.

Comparison of Different Methods of Detection of Enteric Pathogenic Protozoa.

Purpose: The study was conducted to compare different methods of detection of pathogenic protozoan parasites in stool specimens of People Living with HIV/AIDS (PLHA). Materials and Methods: Stool specimens of 242 HIV sero-positive patients were examined using the wet mount technique, modified Ziehl-Neelsen's (ZN) staining, auto-fluorescence and auramine fluorescence staining. Patient specimens, 94 and 40 out of 242, were also subjected to Giardia antigen detection using an enzyme immunoassay and Cryptosporidium antigen detection by immuno-chromatography, respectively. For calculation of sensitivity, specificity, positive and negative predictive values, light microscopy of wet mounts and modified ZN stained smears for Giardia and Coccidia, respectively, were considered as gold standards. Results: Sensitivity of auto-fluorescence, auramine-O staining and antigen detection techniques was found to be 100% as compared to the routine standards. The specificity of auto-fluorescence was 90.6% and 100% for Cyclospora and Isospora, respectively; that of auramine-O staining was 98.9% for Cryptosporidium, 99.30% for Cyclospora and 100% for Isospora; and that of antigen detection was 90.6% and 97.7% for Cryptosporidium and Giardia, respectively. Conclusion: In laboratories requiring screening of large number of stool specimens for detection of protozoan parasites, fluorescence microscopy and antigen detection can be useful techniques. Confirmation of positive results, however, needs to be done with the standard techniques.

Prevalence of influenza virus among the paediatric population in Mumbai during 2007-2009.

Purpose: Influenza has a major impact on public heath, annually affecting 15-20% of the global population. Information on the activity of influenza virus in Mumbai is limited. The present study was carried out to determine the prevalence of influenza viruses causing acute respiratory infections in children by molecular methods. Objective: To study the prevalence of influenza viruses among the paediatric population in Mumbai by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR). Materials and Methods: From July 2007 to July 2009, 100 respiratory samples (nasal and throat swabs) were collected from paediatric patients with acute respiratory symptoms. attending out patients department, and admitted to the paediatric wards of B. J. Wadia Hospital for Children, Mumbai. The samples were collected and processed as per World Health Organization (WHO) guidelines. Viral RNA was extracted and one-step rRT-PCR was performed to detect influenza type A (H1 and H3) and influenza type B virus. Results: Out of 100 samples processed by rRT-PCR, a total of 11 samples (11%) were positive for influenza virus. The typing for influenza A subtypes showed 1% (1) positivity for H1 and 5% (5) positivity for H3 subtypes and 5% (5) samples tested positive for influenza type B virus. Conclusion: It was observed that both influenza type A and B viruses were prevalent in Mumbai during the study period. Such surveillance data are important in the early detection of any antigenic variants that may be helpful in global influenza vaccine preparation and for any pandemic preparedness activity.

Utility of in vitro proton magnetic resonance spectroscopy in aetiological characterisation of brain abscesses.

Purpose: Brain abscesses often present an aetiological dilemma. Microscopy is insensitive and culture techniques are time consuming. Hence, a new rapid technique in vitro Proton Magnetic Resonance Spectroscopy ( 1 HMRS) was evaluated for its usefulness in the identification of aetiology of brain abscesses. Materials and Methods: A total of 39 pus specimens from brain abscesses were subjected to in vitro 1 HMRS. These pus specimens were also processed by conventional culture methods. The spectral patterns generated by in vitro 1 HMRS were further correlated with culture results. Results: Pus specimens which showed the presence of anaerobes on culture revealed the presence of multiplet at 0.9 ppm (100%), lactate-lipid at 1.3 ppm (100%), acetate at 1.92 ppm (100%) and succinate at 2.4 ppm (75%). Pus specimens that revealed the presence of facultative anaerobes on culture showed a pattern B, i.e., the presence of lactate-lipid at 1.3 ppm (100%), acetate at 1.92 ppm (88.88%) along with the multiplet at 0.9 ppm (100%). Pattern C was seen in aerobic infection which showed the presence of lactate-lipid at 1.3 ppm (100%) along with the multiplet at 0.9 ppm. Pus from two tuberculous abscesses showed the complete absence of multiplet at 0.9 ppm. Conclusions: We observed in this study that it was possible to differentiate bacterial and tuberculous brain abscesses using in vitro 1 HMRS. Further, it was also possible to distinguish between aerobic and anaerobic brain abscesses on the basis of spectral patterns. In vitro 1 HMRS of fungal and actinomycotic brain abscess are also presented for its unusual spectra.

Rapid identification of non-sporing anaerobes using nuclear magnetic resonance spectroscopy and an identification strategy.

PURPOSE: The non-sporing anaerobes cause a wide spectrum of infections. They are difficult to culture and their identification is tedious and time-consuming. Rapid identification of anaerobes is highly desirable. Towards this end, the potential of nuclear magnetic resonance (NMR) spectroscopy for providing a fingerprint within the proton spectrum of six genera belonging to anaerobes reflecting their characteristic metabolites has been investigated. METHODS: NMR analysis was carried out using Mercury plus Varian 300 MHz (7.05 T) NMR spectrophotometer on six different anaerobes. These included Bacteroides fragilis, Prevotella melaninogenica, Prevotella denticola, Fusobacterium necrophorum, Peptococcus niger and Peptostreptococcus spp. After the NMR analysis (256/512 scans), the different peaks were noted. The eight pus specimens, which yielded pure culture of anaerobe, also were analysed similarly. RESULTS: The major resonances of multiplex of amino acids/lipid at 0.9 ppm along with lactate/lipid at 1.3 ppm, acetate at 1.92 ppm and multiplex of lysine at 3.0 ppm remained constant to label the organism as an anaerobe. There was a difference found in the MR spectra of different genera and species. A simple algorithm was developed for the identification of the six different anaerobes studied. The MR spectra of the pure culture of the organism matched the MR spectra of pus from which the organism was isolated. CONCLUSIONS: MR-based identification was of value in the identification of anaerobes. However, a larger database of the peaks produced by anaerobes needs to be created for identification of all genera and species. It could then have the potential of diagnosing an anaerobic infection in vivo and thus expedite management of deep-seated abscesses.

Disseminated crytococcosis with extensive cutaneous involvement in AIDS.

Cutaneous infections is observed in 15% of patients with disseminated cryptococcosis with AIDS. We present here a case of a 34 years old female with AIDS. She presented with multiple skin coloured umbilicated over face, neck, trunk and limbs, which mimicked molluscum contagiosum and kaposi sarcoma. The tissue from cutaneous lesions was collected by excision biopsy and processed by standard mycological methods. Cryptococcus neoformans was isolated and identified. Cerebrospinal fluid (CSF) also yielded the growth of C. neoformans . Cryptococcal antigen was detected with a titre of 1024 by Latex agglutination, is serum and CSF. Her serum was reactive for HIVI and 2 antibodies. The CD4 lymphocytes count was 80/cmm. The HIV viral load was 2,48,084 copies/mL. She was treated with amphotericin B injectable and oral fluconazole. She responded well and lesions regressed.

Carniofacial Microsomia

Cranoifacial microsomia is an unique clinical presentation of '1st and 2nd arch syndrome' with asymmetrical craniofacial development alongwith conductive hearing loss. A series of 11 patients (4 males, 7 females) is presented which include two patients of 'Goldenhar's variant' with epibulbar dermoids. Two patients had no response on pure tone audiometry (blank audiograms) and underwent CT scan of temporal bone which revealed 'Michel's aplasia' (complete labyrinthine agenesis), rarely reported in the literature.

Surgical site infection in clean and clean-contaminated cases.

The rate of surgical site infections and the frequency of various pathogens causing surgical site infection with their antibiotic resistance pattern in general surgery units were studied. In the period from May 2001 to July 2002, 190 patients admitted for surgery (clean and clean-contaminated elective cases) were assessed preoperatively, intraoperatively and postoperatively. Normal microbial flora was studied within 24 to 48 hours of admission and patients were followed up to 30 days postoperatively. Infected wounds were studied bacteriologically and clinically. The overall infection rate was 8.95%. Surgical site infection rate was 3.03% in clean surgeries and 22.41% in clean-contaminated surgeries. Significant increase was seen in surgical site infection rate with an increase in preoperative stay. The increase in duration of surgery was associated with a significant rise in the rate of surgical site infection. Surgical site infection rate was much higher (22.41%) in cases where a drain was used than in non-drained wounds (3.03%). The most common isolate was Staphylococcus aureus followed by Pseudomonas aeruginosa.

Trichosporon asahii as an emerging etiologic agent of disseminated trichosporonosis: A case report and an update.

PURPOSE: To report a fatal case of disseminated trichosporonosis caused by Trichosporon asahii in a patient with acute myeloblastic leukemia (AML) and to present an update on systemic trichosporonosis with special reference to India. METHODS: The diagnosis was based on repeated demonstration of budding yeast cells and arthroconidia by direct microscopic examination of sputum and by isolation of T. asahii in culture of sputum and blood. The update is largely based upon literature search in Medline and Review of Medical and Veterinary Mycology. RESULTS: A 41-year-old male presented with acute myeloblastic leukemia, cough and fever. He had received cytotoxic drug therapy, broad spectrum antibiotics and was neutropenic. Trichosporon asahii was repeatedly demonstrated in his sputum by direct microscopy and culture, and also isolated from blood. It was identified by appropriate morphological and physiological characteristics viz., arthroconidium formation, diazonium blue B reaction, urease activity and assimilation of carbon and nitrogen compounds. The identification was confirmed by PCR amplification and direct DNA sequencing of internally transcribed spacer (ITS) 1 and ITS2 of rDNA. CONCLUSION: With greater awareness of etiologic significance of T.asahii, trichosporonosis is likely to be recognised more frequently than apparent from the available published reports.

Parasitic diarrhoea in patients with AIDS.

BACKGROUND: Diarrhoea is a common clinical manifestation of HIV infection regardless of whether or not patients have AIDS. Two newly recognized opportunistic coccidial protozoa are parasitic pathogens in AIDS patients. We attempted to determine the common parasites in Indian patients with AIDS. METHODS: Between October 1994 and December 1996, a total of 110 stool specimens from 94 AIDS patients with acute or chronic diarrhoea were examined by microscopy of wet mounts and smears stained by a modified Ziehl-Neelsen's (cold) staining method. RESULTS: Isospora belli was the most frequently encountered parasite in 17% of patients, followed by Entamoeba histolytica in 14.9% and Cryptosporidium in 8.5%. Strongyloides stercoralis and Giardia lamblia were detected in 5.3% and 4.3% of patients, respectively. CONCLUSION: The presence of different parasites in 56.4% of stool specimens of patients with AIDS indicates that their specific diagnosis is essential. This will help initiate therapy to reduce the morbidity and mortality among such patients due to these pathogens.

Giardiasis--clinical and diagnostic perspective.

Fifty patients of chronic diarrhoea in the pediatric age group admitted in Kalawati Saran Children's Hospital were studied. Thirty cases were diagnosed to be suffering from giardiasis based on microscopy of fecal and/or duodenal fluid specimens. Fecal specimen microscopy missed two cases which were diagnosed by duodenal aspirate microscopy and vice-versa. All fecal specimens were negative on culture, while duodenal aspirate culture gave large number of false negative results. Serum immunoglobulin levels did not show significant changes. Thus, routine microscopic testing is presently the best means for early diagnosis of giardiasis.

Screening for chlamydial infections in women with pelvic inflammatory diseases.

One hundred female patients clinically diagnosed as pelvic inflammatory disease (PID) were studied for the presence of chlamydial infection by cytology and antigen detection. Cervical smears stained by Giemsa revealed inclusion bodies, only in 3 percent of cases. While using Immunocomb Enzyme Immunoassay (EIA) test, antigen was detected in 13% of cases, thereby showing that antigen detection is a better method than cell cytology. A significant correlation with the low socioeconomic status and younger age group was seen in patients showing presence of Chlamydia trachomatis antigen.