Usefulness of a centrifuged buffy coat smear examination for diagnosis of malaria.

Purpose: Malaria continues to be a global public health challenge. Microscopic examination of peripheral blood smear (PBS) is the standard method for malaria diagnosis, which is easily available and has low cost but its reliability is questionable at low level of parasitaemia. The present study was undertaken to assess the usefulness of a modifi ed centrifuged buffy coat smear (CBCS) technique for diagnosis of malaria and to compare it with conventional PBS examination and antigen detection test. Materials and Methods: The study was carried out over a 6-month period from July to December 2011. Blood samples (2–3 ml per patient) collected in EDTAvials from patients with a clinical suspicion of malaria were subjected to all three tests, that is PBS, CBCS and antigen test and results were compared with antigen test as the gold standard. Result: Of 1655 samples received, 394 (23.8%) samples were positive for infection with malaria parasites. All the three tests detected malaria infection equally in 279 samples, and gave varied results in the remaining 115 samples. Addition of centrifugation (i.e. CBCS) to the conventional method of PBS enabled detection of 80 more cases of plasmodia infection, especially (43, 53.7%) at low levels of parasitaemia (<200 parasites/μl). While both PBS and CBCS had excellent specifi city (99.7% and 99.2%, respectively), PBS examination had low sensitivity (72.9%) in detecting malaria parasites in comparison to CBCS. The sensitivity of CBCS in detecting malaria parasites was 91.9%. Conclusion: The development of easy, rapid and accurate tests for the reliable detection of plasmodia infection is highly desirable. The CBCS technique fulfi ls most of these criteria and may be adopted for rapid and reliable diagnosis of malaria in resource-limited settings.

Antigen sequence typing of outer membrane protein (fetA) gene of Neisseria meningitidis serogroup A from Delhi & adjoining areas.

Background & objectives: Meningitis caused by Neisseria meningitidis is a fatal disease. Meningococcal meningitis is an endemic disease in Delhi and irregular pattern of outbreaks has been reported in India. All these outbreaks were associated with serogroup A. Detailed molecular characterization of N. meningitidis is required for the management of this fatal disease. In this study, we characterized antigenic diversity of surface exposed outer membrane protein (OMP) FetA antigen of N. meningitidis serogroup A isolates obtained from cases of invasive meningococcal meningitis in Delhi, India. Methods: Eight isolates of N. meningitidis were collected from cerebrospinal fluid during October 2008 to May 2011 from occasional cases of meningococcal meningitis. Seven isolates were from outbreaks of meningococcal meningitis in 2005-2006 in Delhi and its adjoining areas. These were subjected to molecular typing of fetA gene, an outer membrane protein gene. Results: All 15 N. meningitides isolates studied were serogroup A. This surface exposed porin is putatively under immune pressure. Hence as a part of molecular characterization, genotyping was carried out to find out the diversity in outer membrane protein (FetA) gene among the circulating isolates of N. meningitidis. All 15 isolates proved to be of the same existing allele type of FetA variable region (VR) when matched with global database. The allele found was F3-1 for all the isolates. Interpretation & conclusions: There was no diversity reported in the outer membrane protein FetA in the present study and hence this protein appeared to be a stable molecule. More studies on molecular characterization of FetA antigen are required from different serogroups circulating in different parts of the world.

Corynebacterium striatum: An emerging nosocomial pathogen in a case of laryngeal carcinoma.

Corynebacterium striatum is an emerging nosocomial pathogen associated with wound infections, pneumonia and meningitis. It is also a multidrug-resistant pathogen causing high morbidity. This is a report of an unusual case of wound infection in a patient with laryngeal carcinoma. Accurate diagnosis of the infection and prompt management helped in a favourable outcome for the patient. This case highlights the role of C. striatum as an important nosocomial pathogen in immunocompromised patients.

Prevalence of intestinal parasitic infections in HIV-infected individuals and their relationship with immune status.

Background and Objectives: Intestinal parasitic infection is a common entity in patients infected with human immunodeficiency virus (HIV). These infections may lead to fatal complications in the immuno suppressed individuals. The aim of the present study was to determine the prevalence of intestinal parasitic infections in HIV sero-positive patients and their relationship with the immune status of individuals. Materials and Method s: Fecal samples from 100 HIV sero-positive and an equal number of HIV sero-negative individuals were collected and examined for enteric parasites by direct microscopy. CD4 counts were carried out in only HIV sero-positive patients. Prevalence of intestinal parasites in patients with CD4 count <200 cells/μl, 200-499 cells/μl, and ≥500 cells/μl in HIV-infected patients were compared. Results: Enteric parasites were detected in 59.3% HIV-infected patients with CD4 count <200 cells/μl as compared with 23.5% in patients with CD4 count >200 cells/μl (P < 0.01). Prevalence of coccidian parasites was significantly (P < 0.01) higher (14%) in HIV sero-positive subjects compared with HIV sero-negative subjects (2%). Isospora belli (25%) was the most common parasite with CD4 count <200 cells/μl, followed by Cryptosporidium parvum (12.5%). Prevalence of intestinal parasitic infections was significantly higher in patients with diarrhea, 73.6% than without diarrhea, 25.9%, (P < 0.05). The mean CD4 count of HIV sero-positive patients presenting with diarrhea was significantly (P < 0.01) lower (181.26 ± 135.14) than without diarrhea (352.02 ± 204.03). Conclusion: This study emphasizes the need for routine screening of parasites especially in patients with lower CD4 count so as to decrease the morbidity by ensuring the early treatment of the cases.

Evaluation of Nucleic Acid Probe for Rapid Identification of Mycobacterium tuberculosis Complex in Extra-Pulmonary Culture Isolates.

Patients infected with Non-tuberculous mycobacteria (NTM) usually do not respond to conventional anti-tubercular treatment and are misdiagnosed as infection with multi-drug resistant strains of mycobacterium tuberculosis (M.tb) due to lack of correct species identification, particularly in the developing countries like India. One of the challenges faced by clinicians in the treatment of tuberculosis is the absence of an easy, reliable and rapid identification tool that can accurately differentiate disease caused by M.tb complex from NTM. Keeping this in consideration, the performance of species specific nucleic acid probe i.e. Accuprobe was assessed and compared with conventional niacin production, nitrate reductase assay techniques for identification of M.tb complex in 80 mycobacterial isolates obtained from different extra - pulmonary sites. Accuprobe identified 62 isolates (77.5%) as M. tuberculosis complex and remaining 18 isolates (22.5%) as NTM whereas 64 isolates (80%) were identified as M.tb and rest 16 (20%) were interpreted as NTM by conventional biochemical techniques. The overall agreement between both techniques was 96.9% The sensitivity, specificity, positive predictive value(PPV) and negative predictive value (NPV) shown by accuprobe were 96.9%, 100%, 96.9%, and 88.9% respectively. Thus, accuprobe has showed impressive sensitivity and specificity giving results in <3 hrs from culture-positive isolates and have sure edge over conventional biochemical methods which are, nonetheless, labour intensive and cumbersome to perform thus delaying prompt mycobacterial identification.

The use of E-test for the drug susceptibility testing of Mycobacterium tuberculosis - A solution or an illusion.

Aim: To evaluate E-test as a tool for rapid determination of drug susceptibility against the conventional LJ method focusing on reliability, expense, ease of standardization and performance of the technique in low resource settings. Materials and Methods: A total of 74 clinical isolates (2004-2005) of Mycobacterium tuberculosis were tested using E-test for susceptibility to streptomycin (STM), isoniazid (INH), rifampicin (RIF) and ethambutol (EMB) by E-strip and LJ (LJPM) proportion methods. Results: The LJPM method, the gold standard, detected resistance against STM in 16.2%, INH in 40.5%, RIF in 18.9% and EMB in 27% cases. In comparison, the resistance values showed by E-test was 66.67% for STM, 57.14% for INH 71.43% for RIF and 80% for EMB. The susceptible correlation was 90.32% for STM, 73.91% for INH, 93.33% for RIF and 59.26% for EMB. E-test correctly identified only eight of the 12 (66.6%) MDR isolates and wrongly identified four isolates which were not MDR. The overall agreement between the two methods was only 48.6%. Resistant isolates showed false positive resistance observed while using E-strip towards all the drugs. Conclusion: E-strips are not quite feasible as a replacement for LJ-proportion method on a large scale due to high risk of cross contamination, laboratory infection, expense associated with it and high false positive resistance observed to all first line drugs. However, the good correlation observed for RIF between the two methods indicates that E-test could contribute to the role in rapid screening of MDR TB isolates as rifampicin mutations are invariably observed in MDR TB isolates.

Clinical and mycological profile of cryptococcosis in a tertiary care hospital.

This study examined the extent of cryptococcosis in clinically diagnosed cases of meningitis in HIV-1 seropositive and apparently immunocompetent patients. One hundred and forty-six samples, obtained from 126 chronic meningitis patients comprised of cerebrospinal fluid (CSF), blood, sputum and urine. The samples were processed by standard microbiological procedures. Cryptococcal isolates were identified by microscopy, cultural characteristics, melanin production on niger seed agar and hydrolysis of urea. The isolates were further speciated on cannavanine glycine bromothymol blue (CGB) media. Cryptococcal antigen detection of CSF samples was performed by latex agglutination test (LAT). Minimum inhibitory concentration (MIC) of amphotericin B for the isolates was also tested. Cryptococcosis was diagnosed in 13 patients (eight HIV-1 seropositive and five apparently immunocompetent). Cryptococcus neoformans var. neoformans was the predominant isolate. Cryptococcal antigen was detected in all, whereas microscopy could detect yeast cells in nine patients. The isolates were sensitive to amphotericin B. CD4 cell counts ranged from 8 to 96/cu mm. The study concludes that all CSF samples with clinical diagnosis of subacute and chronic meningitis should be subjected to tests for detection of Cryptococcus in clinical laboratory irrespective of the immune status.

Use of rK39 for diagnosis of post kala-azar dermal leishmaniasis in Nepal.

A recently developed nitrocellulose-based dipstick test, rK39, has been widely used for the diagnosis of kala-azar. In this study, we evaluated its use for the diagnosis of post kala-azar dermal leishmaniasis (PKDL). We also investigated the time taken by patients to develop PKDL after apparent cure of kala-azar (visceral leishmaniasis, VL) and the time taken by patients to come to the hospital after the appearance of symptoms of PKDL. A majority of patients developed the disease within three years after the apparent cure of kala-azar (KA). A majority of patients sought treatment within five years after the onset of PKDL. The amastigotes of Leishmania donovani bodies (LDBs) were demonstrated in 70, 20, and 20% of slit-skin smears (SSS) prepared, respectively, from nodular, papular, and macular forms. The presence of highest density (6+) LDBs in the SSS of 20% of nodular PKDL patients indicated that they may have acted as reservoir in the community. Other reservoirs are not known in Nepal. Only 8% cases were detected by aldehyde test. Although this test is obsolete it is still used in rural parts of Nepal. The dipstick (rK39) was 96% sensitive and 100% specific to diagnose PKDL. Its positive predictive value, negative predictive value, and diagnostic efficacy were 100, 91, and 97% respectively. Due to the advantage of cost compared with the direct agglutination test (DAT), and being easy to use and store in field conditions, rK39 is a good tool to diagnose PKDL in rural situations. All the PKDL patients were cured of the disease after treatment by SAG.

Isospora belli infection in a malnourished child.

Isospora belli, a coccidian parasite usually causes a self limiting illness of acute onset with fever, diarrhoea and colicky pain in a normal host. In immunocompromised patients human isosporiasis becomes chronic. We report a case of a malnourished 9 year old female child who presented with complaints of loose stools, nausea, vomiting and weight loss for the past three months. Stool examination revealed immature oocysts of Isospora belli. The patient was successfully treated with TMP-SMX.

Eumycetoma pedis due to Exophiala jeanselmei.

A case of eumycetoma of foot in an 8-year old male child was clinically diagnosed as chronic osteomyelitis and was microbiologically confirmed as eumycetoma. The case is being reported for its uncommon clinical presentation and etiological agent, Exophiala jeanselmei. The patient recovered completely after treatment with ketoconazole.

A study of diarrhoea among children in eastern Nepal with special reference to rotavirus.

PURPOSE: To examine the incidence of Rotavirus infection in children below five years of age. METHODS: Faecal samples from 160 children under five years of age with acute gastroenteritis were collected over a period of one year from July 1999 to June 2000. These were studied for the presence of Rotavirus antigen by enzyme immuno assay (EIA). RESULTS: Rota antigen could be detected in 62 (38.7%) samples. Co-infection with other parasites or bacterial pathogens in presence of Rota antigen was also demonstrated. Forty one (66.4%) children were admitted for hospital care. Forty two samples positive by EIA were further tested by latex agglutination (LA) to consider introducing this test routinely in clinical laboratory. Although a rapid and convenient test, LA failed to demonstrate antigen in 15 (35.6%) of the samples. CONCLUSIONS: Rotavirus infection of children in Nepal is reported for the first time. EIA was found to be more sensitive than LA for the detection of Rotavirus antigen in faecal samples.