Species identification, antifungal susceptibility profiles and biofilm formation attributes of Rhodotorula isolates from ocular infections.

BACKGROUND: Members of genus Rhodotorula are widely distributed in nature and have been traditionally considered non-pathogenic. Last few decades have seen the yeast as an emerging pathogen. We observed increase in numbers of Rhodotorula isolates from ocular infections in last few years, thus this prospective study was planned. OBJECTIVES: To identify the species of Rhodotorula isolates from ocular infections. To know the antifungal susceptibilities and study the biofilm formation attributes of the isolates. MATERIALS AND METHODS: Rhodotorula isolates were speciated using conventional methods, Matrix Assisted Laser Desorption and Ionisation - Time of Flight (MALDI- TOF) and sequencing of ITS region of ribosomal DNA. Antifungal susceptibility testing (AFST) was done using disc diffusion and E-test. Biofilm formation was studied using XTT [2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetra-zolium-5-carboxanilide] assay. RESULTS: Twenty four isolates (92.3%) were identified as R. mucilaginosa and two as R. Minuta. AFST showed high MICs against Fluconazole, Amphotericin-B, Caspofungin, Micafungin and Flucytosine; MIC distribution from low to very high against Voriconazole, Itraconazole and Natamycin; and very low MICs against Posaconazole 57.7% of isolates were strong biofilm producers, 23.1% were moderate, and 19.2% were non producers. CONCLUSIONS: This is the first prospective study on species distribution, antifungal susceptibility and biofilm production attributes of Rhodotorula isolates from ocular infections; also first time demonstrating the utility of proteomics based MALDI-TOF in diagnosing Rhodotorula up to species level. The study has shown high MICs against the conventional azoles, Amphotericin-B and Flucytosine. However, low MICs against Posaconazole and Natamycin give a hope for their possible therapeutic use.

Staphylococcus hominis subsp. novobiosepticus, an emerging multidrug-resistant bacterium, as a causative agent of septicaemia in cancer patients.

Staphylococcus hominis subsp. novobiosepticus is a new sub-species of S. hominis, thus dividing S. hominis into subsp. hominis and novobiosepticus. This study was designed to identify subsp. novobiosepticus isolates amongst the S. hominis isolated from blood samples of patients with malignancy and septicaemia and to study their resistance profile. The identification was performed by using three simple tests which differentiated between the two sub-species. It was found that 22.8 per cent of S. hominis isolates belonged to subsp. novobiosepticus.

Comparison of NS1 antigen detection ELISA, real time RT-PCR and virus isolation for rapid diagnosis of dengue infection in acute phase.

BACKGROUND & OBJECTIVES: Diagnosis of dengue infection in acute phase is important for clinical care, implementing control measures, surveillance and research. Currently, dengue fever is diagnosed by means of virus isolation, reverse transcriptase PCR or IgM and IgG based ELISA. Given the limitations of all the existing diagnostic methods, there is a need for rapid, sensitive and high throughput methods for detection of dengue virus in early stages of the disease. The study was conducted with the objectives to evaluate a dengue virus NS1 antigen detection ELISA and a TaqMan based real time RT-PCR for detection of all four serotypes of dengue virus, as diagnostic tools for acute dengue virus infection. METHODS: The acute phase serum samples of patients (n=153) presenting with dengue fever were subjected to NS1 antigen detection and real time RT-PCR. The results were compared to those of virus isolation in the C6/36 cell lines (n=55). RESULTS: The efficiency, sensitivity, specificity, positive and negative predictive values of NS1 Ag detection ELISA were 83.6, 73.5, 100, 100 and 70% respectively while for real time RT-PCR these were 87.3, 79.4, 100, 100 and 75% respectively. Maximum sensitivity of NS1 antigen detection ELISA was seen in two days of fever and that of real time RT-PCR in three days of fever. INTERPRETATION & CONCLUSION: NS1 antigen detection ELISA and real time RT-PCR were found to be rapid, convenient and efficient tests for diagnosing of dengue fever in acute phase and the diagnosis could be made as early as within three days of onset of fever.

Acanthamoeba keratitis: Experience from a tertiary eye care center in North India.

The free-living amebae of genus Acanthamoeba are an important cause of microbial keratitis. The clinical appearance of Acanthamoeba keratitis (AK) usually mimics viral or fungal keratitis. Thus, microbiological workup plays a significant role in the diagnosis and timely treatment of such cases. We report a retrospective case series of seven culture-confirmed AK cases from a tertiary eye care center in North India. Various risk factors and triggers of infection, clinical presentations, microbiological findings, and management of AK are elucidated.

Evaluation of antifungal susceptibility and clinical characteristics in fungal keratitis in a tertiary care center in North India.

Purpose: To study the antifungal susceptibility of common corneal pathogenic fungi to antifungal agents in the North Indian population. Methods: Prospective study of the antifungal sensitivity testing (natamycin, amphotericin B, voriconazole, itraconazole, fluconazole, posaconazole, caspofungin, micafungin) of fungal isolates from 50 cases of culture positive fungal keratitis by using E test method. Details noted included demographic data, visual acuity, clinical details, grade of keratitis, healing time, and success in medical management. Results: Of 50 patients with fungal keratitis (mean age: 40.28 ± 16.77 years), 12 eyes healed within 3 weeks, 14 had a delayed healing response, and 24 had chronic keratitis. Among the 15 cases of Fusarium isolates, 93.3% were sensitive to natamycin, while 40% to amphotericin B; 66.6% to voriconazole, 13.4% to itraconazole and fluconazole each. 80% of Fusarium cases (n = 12) showed susceptibility to posaconazole. Among Aspergillus flavus isolates, 53.4% (n = 8) were sensitive to natamycin, with only 40% (n = 7) showing sensitivity to amphotericin B and good susceptibility to azoles. MIC against susceptible Fusarium spp. for natamycin was 3-16 µg/mL, amphotericin B: 1-8 µg/mL, voriconazole: 0.5-1.5 µg/mL, itraconazole: 0.5-12 µg/mL, posaconazole: 0.094-1.5 µg/mL. MIC against Aspergillus flavus was natamycin: 8-32 µg/mL, amphotericin B: 0.5-16 µg/mL, voriconazole: 0.025-4 µg/mL, itraconazole: 0.125-8 µg/mL, posaconazole: 0.047-0.25 µg/mL; against Aspergillus niger isolates, to natamycin was 6 µg/mL (n=1), amphotericin B 8-12 µg/mL (n = 3), voriconazole: 0.125-0.19 µg/mL (n = 3), itraconazole: 0.38-0.75 µg/mL, posaconazole: 0.064-0.19 µg/mL and against Aspergillus fumigatus (n = 1), was natamycin4 µg/mL, amphotericin B - 8 µg/mL, voriconazole 0.25 µg/mL, itraconazole 1 µg/mL, and posaconazole 0.19 µg/mL. MIC against susceptible Acremonium spp. for natamycin was 1.5-16 µg/mL, amphotericin B: 0.5-8 µg/mL, voriconazole: 0.19-3 µg/mL, itraconazole: 0.125 µg/mL, posaconazole: 0.125-0.5 µg/mL and against susceptible Curvularia was natamycin 0.75-4 µg/mL, amphotericin B 0.5-1 µg/mL, voriconazole 0.125-0.19 µg/mL, itraconazole 0.047-0.094 µg/mL, posaconazole 0.047-0.094 µg/mL. MIC against Mucor spp.+ Rhizopus spp. (n = 1) was natamycin: 8 µg/mL, amphotericin B: 0.75 µg/mL, posaconazole: 1.5 µg/mL. MIC against of Alternaria (n = 1) was voriconazole: 0.19 µg/mL, posaconazole: 0.094 µg/mL. MIC against Penicillium (n=1) was natamycin: 8 µg/mL, voriconazole: 0.25 µg/mL, itraconazole: 0.5 µg/mL, and Posaconazole: 0.125 µg/mL. Conclusion: Our observations highlight the variations in susceptibility to antifungal agents. Posaconazole seems to be effective with low MIC against common corneal pathogenic fungal isolates.

A Comprehensive Assessment of Self-Reported Post COVID-19 Symptoms Among Beneficiaries of Hospital Employee Scheme at a Tertiary Healthcare Institution in Northern India.

Purpose: With millions of people being affected by COVID-19, people living with post COVID-19 clinical symptoms (PCS) are expected to rise further. The primary aim of the study was to comprehensively assess self-reported PCS and its associated risk factors among beneficiaries of Hospital Employee Scheme of a tertiary healthcare institution in Delhi. Patients and Methods: An online cross-sectional study was conducted using a semi-structured questionnaire developed by employing nominal group technique among individuals aged 18 years and above who were novel SARS-CoV-2 positive from January to April 2021. Participants were telephoned first, before sending the online survey link. Socio-demographic data, information on PCS along with potential risk factors, pre-existing morbidities, vaccination status, severity of acute illness and management were collected between June and July 2021. PCS was presented as relative frequency; Chi-Square test and odds ratio; adjusted values were used to rule out any association between PCS and predictors. Results: In total, 773 of 1801 eligible participants responded to the survey (completion rate 42.9%), with a median age of 34 years (IQR 27-44). Males accounted for 56.4% and PCS was present in 33.2%. The most prevalent symptoms were fatigue (79.3%), arthralgia (33.4%), myalgia (29.9%), hair loss (28.0%), headache (27.2%), breathlessness (25.3%), and sleep disturbance (25.3%). The prevalence of PCS was reduced to 12.8% at 12 weeks. Female gender, older age, oxygen supplementation, severity of acute illness, and pre-existing co-morbidities were positively associated with PCS. Vaccination (second dose) reduced the odds of developing PCS by 39% compared to unvaccinated participants (aOR 0.61; 95% CI 0.40-0.96). Conclusion: PCS affects almost all organ systems of the body, regardless of the severity of acute COVID-19 illness. Two doses of vaccine hel reduce the development of PCS.

Recent Perspectives in the Management of Fungal Keratitis.

Mycotic keratitis is common in warm, humid regions with a varying profile of pathogenic fungi according to geographical origin, socioeconomic status, and climatic condition. Clinical diagnosis can be challenging in difficult cases and those refractory to treatment. Fungal hyphae on microscopic examination and culture isolation have been the gold standard in the laboratory diagnosis of fungal keratitis. A culture isolate of the aetiological fungus is essential to perform antifungal susceptibility testing. As the culture isolation of fungi is time-consuming, causing delays in the initiation of treatment, newer investigative modalities such as in vivo confocal microscopy and molecular diagnostic methods have recently gained popularity. Molecular diagnostic techniques now help to obtain a rapid diagnosis of fungal keratitis. Genomic approaches are based on detecting amplicons of ribosomal RNA genes, with internal transcribed spacers being increasingly adopted. Metagenomic deep sequencing allows for rapid and accurate diagnosis without the need to wait for the fungus to grow. This is also helpful in identifying new emerging strains of fungi causing mycotic keratitis. A custom-tear proteomic approach will probably play an important diagnostic role in future in the management of mycotic keratitis. Positive repeat cultures are being suggested as an important gauge indicative of a poor prognosis. Positive repeat fungal cultures help to modify a treatment regimen by increasing its frequency, providing the addition of another topical and oral antifungal agent along with close follow-up for perforation and identifying need for early therapeutic keratoplasty. The role of collagen crosslinking in the treatment of fungal keratitis is not convincingly established. Rapid detection by multiplex PCR and antifungal susceptibility testing of the pathogenic fungi, adopted into a routine management protocol of fungal keratitis, will help to improve treatment outcome. Early therapy is essential in minimizing damage to the corneal tissue, thereby providing a better outcome. The role of conventional therapy with polyenes, systemic and targeted therapy of antifungal agents, newer azoles and echinocandins in fungal keratitis has been widely studied in recent times. Combination therapy can be more efficacious in comparison to monotherapy. Given the diversity of fungal aetiology, the emergence of new corneal pathogenic fungi with varying drug susceptibilities, increasing the drug resistance to antifungal agents in some genera and species, it is perhaps time to adopt recent molecular methods for precise identification and incorporate antifungal susceptibility testing as a routine.

Evaluation of 16S rRNA broad range PCR assay for microbial detection in serum specimens in sepsis patients.

BACKGROUND: Early and accurate laboratory diagnosis and appropriate management of infection improves the survival rate in sepsis. In this study we evaluated broad range 16S rRNA and 16 S-23 S intergenic spacer region (ISR) PCR assays followed by nucleotide sequencing directly from patients' serum and automated blood culture for laboratory diagnosis in admitted sepsis patients. METHODS: A broad range 16S rRNA PCR and 16 S-23 S ISR PCR assay followed by nucleotide sequencing was used directly from patients' serum in hospital admitted patients in 62 sepsis and 16 suspected blood stream infection (sBSI) patients. Automated blood culture was also used in the same patients. Nucleotide sequences were analyzed against NCBI Genbank database and organisms were identified using CLSI MM18A guidelines. RESULTS: Bacterial culture were positive in 10/62 (16.12%) sepsis and 3/16 (18.75%) suspected BSI patients along with 3 detected fungi (2 in sepsis and 1 in suspected BSI group). PCR assay was positive in 36/62 (58.06%) sepsis and 6/16 (37.5%) suspected BSI patients respectively. All but 2 bacteria (both from culture negative patients) detected by PCR assay could be identified from nucleotide sequencing. Survival in sepsis patients was 77%. PCR assay could detect bacteria in 9/14 (64.28%) of sepsis patients with death. CONCLUSION: Broad range PCR assay was far superior for early diagnosis of infection. The bacteria which could not be detected by culture and were not commonly reported from this centre, were detected by the broad range PCR assays. Detection of these rare bacteria/fungi had significant clinical correlation with patient's underlying clinical conditions, immune status and prognosis. The tests could provide definitive diagnosis of infection in >58% of sepsis patients, which helped in patient management and better survival.

Utility of broad-range 16S rRNA PCR assay versus conventional methods for laboratory diagnosis of bacterial endophthalmitis in a tertiary care hospital.

BACKGROUND: Endophthalmitis, a sight-threatening intraocular infection, can be of postsurgical, post-traumatic or endogenous origin. Laboratory diagnosis-based appropriate therapy can be vision-saving. Conventional culture-based laboratory diagnosis takes time and lacks sensitivity. In this study a broad-range PCR assay was assessed against conventional and automated culture methods in vitreous specimens for accurate microbiological diagnosis. AIMS: To use broad-range PCR assay targeting 16S ribosomal RNA (rRNA) region of bacteria and to assess its performance vis-à-vis conventional and automated culture methods in the laboratory diagnosis of endophthalmitis. METHODS: Vitreous specimens from 195 patients with clinically diagnosed endophthalmitis were processed for classical and automated culture methods, antimicrobial sensitivity and broad-range PCR assay targeting 762 bp region of 16S rRNA followed by nucleotide sequencing by Sanger's method. Causative agents were identified from the nucleotide sequences analysed against the GenBank database, and organisms were identified using the Clinical and Laboratory Standards Institute (CLSI) MM18A guidelines. RESULTS: Bacteria could be detected from 127 (65.13%) of the 195 vitreous specimens by broad-range PCR assay; bacterial isolation was possible from 17 (8.7%) and 60 (30.76%) of these specimens by conventional and automated culture methods, respectively (p<0.0001). PCR assay could detect two uncultured bacterium, and in five cases the bacterial identity could not be determined from NCBI database matching. CONCLUSION: Broad-range PCR assay could provide definitive microbial diagnosis within 24 hours in significantly more patients (p<0.0001). Some rare organisms could be detected, useful in treatment modalities. Automated culture was significantly more sensitive than conventional culture.

Chlamydia trachomatis Antigen Positivity in Patients with Different Ocular Manifestations over 8 Years.

Laboratory confirmation of chlamydial antigen in clinically suspected cases of chlamydial eye infections is important, as similar clinical picture can be presented by different infective or noninfective causes. We retrospectively analyzed the presence of Chlamydia trachomatis antigen in 690 clinically suspected patients over the last 8 years (2009-2016). The chlamydial antigen was detected using direct immunofluorescence assay. Overall, Chlamydia-specific antigen positivity was 45.5%. The highest positivity was seen in 2014 (68.6%) and the least in 2016 (9.4%). The antigen positivity in years 2015 (13.4%) and 2016 (9.4%) was significantly less than in all the previous study years (P < 0.0001). Antigen positivity in patients having clinical diagnosis of trachoma was significantly higher than those having other eye manifestations suggestive of chlamydial infections (P = 0.0274). Stringent surveillance both at community level and in hospital attendees is required to know the actual load of this pathogen.

Chlamydial eye infections: Current perspectives.

Chlamydia trachomatis, an obligate intraocular bacteria causing trachoma, adult and neonatal inclusion conjunctivitis, was the leading cause of blindness in the last century worldwide. Improvement in socioeconomic and living conditions, availability of antibiotics, and introduction of National Trachoma Control Programmes reduced the prevalence in developed countries, but it persisted in resource-poor settings of Africa and Asia, including India. In 2016, as per the WHO report, trachoma is restricted to 42 countries, causing blindness/visual impairment in ~1.9 million people. India is one of the five countries with nearly half of total active trachoma patients. Introduction of Global Elimination of Trachoma 2020 program by the WHO, using SAFE strategy (surgery for trachomatous trichiasis; Antibiotics for C. trachomatis; Facial cleanliness; and environmental improvement) greatly reduced the prevalence, but trachoma still persists in India. Global increase in the reproductive tract infection by C. trachomatis urogenital serotypes (D-K) has led to concurrent increase in C. trachomatis eye infections. Therefore, kerato eye infections due to chlamydial infections continue to be seen in hospitals. Over the years, there have been advances in laboratory diagnostics, in understanding the pathogenesis, tissue tropism, C. trachomatis genomics, and treatment modalities. Due attention and research is still needed for the study of C. trachomatis eye infections.

Pattern of co-infection by enteric pathogenic parasites among HIV sero-positive individuals in a Tertiary Care Hospital, Mumbai, India.

INTRODUCTION: One of the major medical concerns in people living with HIV/AIDS (PLHA) is management of diarrhea that can lead to severe morbidity and mortality. Such clinical scenario warrants an analysis of intestinal parasites, which are important opportunistic pathogens in PLHA. Owing to the scarcity of recent pattern of intestinal opportunistic infections from this region, the study was designed to determine the opportunistic parasites causing diarrhea in PLHA; and to find out whether there is any significant difference in the enteric parasitic pathogens in patients with different immunological status and in those on highly active anti retro-viral therapy (HAART). MATERIALS AND METHODS: Analysis of the spectrum of intestinal parasites was carried out with 192 subjects in two groups (142 HIV sero-positive patients having diarrhea and 50 HIV sero-negative patients having diarrhea). The routine light microscopic examination was carried out to determine the infection and CD4+ T-Lymphocyte count was estimated using flow cytometry. RESULTS: Enteric parasites were detected in 35.9% of HIV sero-positive patients having diarrhea and 18% of HIV sero-negative patients having diarrhea. Most common opportunistic enteric parasite was Isospora belli (11.5%); others were Entamoeba histolytica (4.7%), Cryptosporidium sp. (3.6%), Strongyloides stercoralis (3.1%), Giardia intestinalis (3.1%) and Cyclospora cayatanenesis (1.6%). Opportunistic enteric parasites were detected in significantly low numbers in patients with CD4+ T-Lymphocyte counts >500 cells/ml; and in those taking HAART.

Dengue Fever outbreak in delhi, north India: a clinico-epidemiological study.

BACKGROUND: Dengue viruses, single-stranded positive polarity ribonucleic acid (RNA) viruses of the family Flaviviridae, are the most common cause of arboviral disease in the world. We report a clinico-epidemiological study of the dengue fever outbreak of 2010 from a tertiary care hospital in Delhi, North India. OBJECTIVES: Objectives of the study were to know the incidence of laboratory-confirmed dengue cases among the clinically suspected patients; to study the clinical profile of dengue-positive cases; and to co-relate the above with the prevalent serotype and environmental conditions. MATERIALS AND METHODS: Four thousand three hundred and seventy serum samples from clinically suspected cases of dengue infection were subjected to µ-capture enzyme-linked immunosorbent assay (ELISA) for detection of dengue-virus-specific IgM antibodies. Virus isolation was done in 55 samples on C6/36 cell mono-layers. Clinical and demographic details of the patients were obtained from requisition forms of the patients or from treating clinicians. RESULTS: Out of the 4,370 serum samples, 1,700 were positive for dengue-virus-specific IgM antibodies (38.9%). Prevalent serotype was dengue virus type-1. Thrombocytopenia and myalgia was seen in 23.1% and 18.3% of the 1,700 dengue IgM-positive patients, respectively. Also, 10.3% of 1,700 were dengue hemorrhagic fever (DHF) patients; and the mortality in serologically confirmed dengue fever cases was 0.06%. CONCLUSIONS: A change in the predominant circulating serotype, unprecedented rains, enormous infrastructure development, and increased reporting due to improved diagnostic facilities were the factors responsible for the unexpected number of dengue fever cases confronted in 2010.

Antimicrobial resistance of bacterial isolates from respiratory secretions of ventilated patients in a multi-specialty hospital.

CONTEXT: Ventilator-associated pneumonia (VAP) is a common nosocomial infection occurring in intensive care unit (ICU) settings. VAP occurs due to interplay of three factors - impaired host defense, access of large numbers of pathogenic bacteria to the lower respiratory tract and the virulence of the organism. Knowledge of colonizing microbial flora and their antibiogram in ventilated patients is of great importance in timely institution of empirical therapy, so that mortality and morbidity due to VAP can be reduced. SUBJECTS AND METHODS: A prospective study was performed over a period of 6 months in a multi-specialty hospital to determine the various pathogens in respiratory secretions and to determine the prevalence of multidrug resistance (MDR). RESULTS: Pseudomonas aeruginosa (26%), Acinetobacter (26%), Klebsiella pneumoniae (26%), followed by Escherichia coli (15%), Staphylococcus aureus (6%) and Citrobacter spp. (1.5%) were the common pathogens isolated in our study. In all, 72.73% (48/66) bacterial isolates were isolated from medical ICU, while 25.76% (17/66) were isolated from surgical ICU. Only one strain (Acinetobacter) was isolated from pediatric ICU. Fifty-seven (86.36%) of the 66 pathogens in our study were MDR. CONCLUSION: There is increasing colonization of pathogenic bacteria in ventilated patients admitted in ICUs, which are predominantly MDR. These colonizers may cause infection resulting in VAP. Judicious use of antibiotics, guided by local antibiotic resistance profile coupled with strict infection control practices alongside application of VAP bundle are important measures to prevent these pathogens from causing VAP in ICU patients.

Antibiotic Resistance Pattern of Uropathogens: An Experience from North Indian Cancer Patient.

Empirical treatment of urinary tract infections (UTIs) can be made evidence based if it is governed by the resistance pattern of common uropathogens. A retrospective study was carried out at a tertiary care cancer institute to identify the common uropathogens and to know their resistance profile. 20.82% of the outpatients' urine samples (community-acquired urinary tract infection (CA-UTI)) and 24.83% of the indoor patients' urine samples (hospital-acquired urinary tract infection (HA-UTI)) grew uropathogens. Escherichia coli was the predominant pathogen both in CA-UTI (68%) and HA-UTI (45%) followed by Klebsiella spp and Enterococcus spp. High level of resistance to fluoroquinolones and third generation cephalosporins was noted. Nitrofurantoin was found to be a reliable oral drug for treatment of most of the uropathogens.

Surgical Site Infection Caused by Aeromonas hydrophila in a Patient with Underlying Malignancy.

Aeromonas skin and soft tissue infections in cancer patients can lead to serious life threatening conditions such as cellulitis, necrotizing fasciitis and myonecrosis. We report here a case of surgical site infection, post radical mastectomy, in a 58-year-old female with carcinoma breast. Cultures of exudates from the wound grew Aeromonas hydrophila on repeated occasions. Recovery was uneventful following targeted antimicrobial therapy and regular dressing of the wound. Early suspicion, diagnosis, and treatment with potent antibiotics are needed to prevent any further complications resulting from infection by this emerging pathogen.

Cultivation of parasites.

Parasite cultivation techniques constitute a substantial segment of present-day study of parasites, especially of protozoa. Success in establishing in vitro and in vivo culture of parasites not only allows their physiology, behavior and metabolism to be studied dynamically, but also allows the nature of the antigenic molecules in the excretory and secretory products to be vigorously pursued and analyzed. The complex life-cycles of various parasites having different stages and host species requirements, particularly in the case of parasitic helminths, often make parasite cultivation an uphill assignment. Culturing of parasites depends on the combined expertise of all types of microbiological cultures. Different parasites require different cultivation conditions such as nutrients, temperature and even incubation conditions. Cultivation is an important method for diagnosis of many clinically important parasites, for example, Entamoeba histolytica, Trichomonas vaginalis, Leishmania spp., Strongyloides stercoralis and free-living amoebae. Many commercial systems like InPouch TV for T. vaginalis, microaerophilous stationary phase culture for Babesia bovis and Harada-Mori culture technique for larval-stage nematodes have been developed for the rapid diagnosis of the parasitic infections. Cultivation also has immense utility in the production of vaccines, testing vaccine efficacy, and antigen - production for obtaining serological reagents, detection of drug-resistance, screening of potential therapeutic agents and conducting epidemiological studies. Though in vitro cultivation techniques are used more often compared with in vivo techniques, the in vivo techniques are sometimes used for diagnosing some parasitic infections such as trypanosomiasis and toxoplasmosis. Parasite cultivation continues to be a challenging diagnostic option. This review provides an overview of intricacies of parasitic culture and update on popular methods used for cultivating parasites.

Quantitative buffy coat analysis-an effective tool for diagnosing blood parasites.

Quantitative buffy coat (QBC) analysis, which is based on principle of centrifugal stratification of blood components, is a well-known and a very sensitive technique which can be used for the detection of malarial parasites in peripheral blood. In our experience, this technique is also highly specific for doing speciation of malarial parasite in Indian set up. In addition, this technique was also found to be a sensitive and specific tool for diagnosing filariasis. Lastly, the cellular pattern of buffy coat in QBC, together with other non-specific findings, has many times aided in making correct diagnoses in difficult cases of visceral Leishmaniasis.

Non-pigmented strain of serratia marcescens: an unusual pathogen causing pulmonary infection in a patient with malignancy.

Serratia marcescens is a member of the family Enterobacteriaceae. It has emerged in recent years as an opportunistic pathogen of nosocomial infections. Some biotypes of Serratia marcescens produce the non-diffusible red pigment prodigiosin. Though both pigmented and non-pigmented biotypes may be pathogenic for humans, the non-pigmented biotypes are more virulent due to cytotoxin production and presence of plasmids mediating antibiotic resistance. However in India only one study done 31 years back has reported on infections caused by non-pigmented strains of Serratia marcescens. We present a case of a patient with squamous cell carcinoma of the left retromolar trigone, soft palate and buccal mucosa, who developed pulmonary infection with non-pigmented strain of Serratia marcescens. According to the available literature, this is the second report on infection with non-pigmented strain of Serratia marcescens from India. It is imperative to accurately detect the non-pigmented biotypes due to their tendency to cause serious and difficult to treat infections.

Multidrug-resistant Staphylococcus hominis subsp. novobiosepticus causing septicemia in patients with malignancy.

A new subspecies of Staphylococcus hominis described by Kloos et al. in 1998 and named S. hominis subsp. novobiosepticus (SHN) has been implicated in nosocomial outbreaks. Multidrug resistance, including resistance to novobiocin and oxacillin, is a particularly important feature of SHN. In our institute, we encountered 13 cases of S. hominis subsp. hominis in cancer patients with septicemia, of which seven were methicillin resistant. The isolates were identified by VITEK ® 2 compact automated system, using GP REF 21342 identification card and antimicrobial susceptibility testing card P-628. The biochemical reactions and antibiotic susceptibility pattern of the seven methicillin-resistant isolates were re-analyzed and patient details were re-checked to finally identify them as SHN. The increasing number of cases reporting isolation of SHN from biological specimens point to potential virulence and clinical importance of this bacterium.