Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry.

Purpose: The purpose of this study was to evaluate the identification of Mycobacterium tuberculosis which is often plagued with ambiguity. It is a time consuming process requiring 4-8 weeks after culture positivity, thereby delaying therapeutic intervention. For a successful treatment and disease management, timely diagnosis is imperative. We evaluated a rapid, proteomic based technique for identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Materials and Methods: Freshly grown mycobacterial isolates were used. Acetonitrile/trifluoroacetic acid extraction procedure was carried out, following which cinnamic acid charged plates were subjected to identification by MALDI-TOF MS. Results: A comparative analysis of 42 clinical mycobacterial isolates using the MALDI-TOF MS and conventional techniques was carried out. Among these, 97.61% were found to corroborate with the standard methods at genus level and 85.36% were accurate till the species level. One out of 42 was not in accord with the conventional assays because MALDI-TOF MS established it as Mycobacterium tuberculosis (log (score) >2.0) and conventional methods established it to be non-tuberculous Mycobacterium. Conclusions: MALDI-TOF MS was found to be an accurate, rapid, cost effective and robust system for identification of mycobacterial species. This innovative approach holds promise for early therapeutic intervention leading to better patient care.

A rare case of cutaneous larva migrans due to Gnathostoma sp.

A 28-year-old lady presented with recurrent erythematous skin lesions in different parts of the body for 3 months. There were several episodes of worm coming out of the lesions. Examination of the worms in the parasitology laboratory revealed it to be a larva of Gnathostoma sp. She was advised treatment with Albendazole for 21 days, and there was no recurrence of lesions.

Dirofilariasis: A rare case report.

Human dirofilariasis is a zoonotic infection most commonly caused by Dirofilaria repens. It has not been widely recognized in India. There is probably a focus of human infection with D. repens in Kerala. We report the first case of dirofilariasis, from the Eastern-part of India, to the best of our knowledge. Among the documented cases of human dirofilariasis caused by D. repens, recorded in India, most of them had ocular infections and few had subcutaneous involvement of the face. This is the first case report of human dirofilariasis from India involving the lower part of human body.

Acanthamoeba keratitis with Curvularia co-infection.

We report a case of Acanthamoeba keratitis with Curvularia co-infection. Acanthamoeba and fungal co-infection have been uncommonly reported in literature, worldwide. A classical history with a strong clinical suspicion and experienced laboratory personnel with systematic examination of corneal scrapings for bacterial, viral, parasitic and fungal causes are imperative for accurate diagnosis. Early diagnosis of Acanthamoeba keratitis or fungal infection followed by aggressive and appropriate treatment with effective agents is critical for the retention of good vision. Acanthamoeba keratitis is difficult to diagnose and, despite improvement in treatment options, may culminate in prolonged morbidity and significant loss of visual acuity. This case emphasizes the important role played by clinical microbiologists in making prompt diagnosis which can ultimately reduce visual morbidity.

Fatal haemophagocytic syndrome and hepatitis associated with visceral leishmaniasis.

Haemophagocytic syndrome (HPS) secondary to infections occurs due to excessive, non-malignant proliferation of histiocytes, with resultant haemophagocytosis. The syndrome is essentially treatable, provided timely etiological diagnosis is achieved. In this report, we present a rare case of a child who hailed from Uttaranchal and presented with severe hepatitis. Bone marrow examination revealed an unexpected diagnosis of HPS secondary to visceral leishmaniasis. Despite initiating appropriate antileishmanial treatment, the child had a fatal outcome.

Profile of fatal visceral leishmaniasis at an Indian tertiary care center.

Visceral leishmaniasis is a highly morbid and incapacitating infection, which usually presents with prolonged fever, weight loss and hepato-splenomegaly. Despite the availability of effective treatment, the disease can have a high mortality even at referral centers. A case series of fatal visceral leishmaniasis, encountered during a prospective, two year period is presented. All the patients died due to multisystem organ failure. However, delayed diagnosis due to atypical manifestations was an important factor contributing to the fatal outcome of the patients. Instead of relying solely on the classical clinical features of visceral leishmaniasis, simple laboratory findings like pancytopenia, altered albumin/globulin ratio and appositive aldehyde and rK 39 dipstick tests can help in making an early diagnosis even in atypical cases, thereby reducing the mortality of visceral leishmaniasis.

Chronic diarrhea and malabsorption caused by Leishmania donovani.

Chronic diarrhea and malabsorption are uncommon in immunocompetent patients with visceral leishmaniasis. We report two immunocompetent patients with visceral leishmaniasis where the predominant presentation was chronic diarrhea. One of them had clinically overt malabsorption and duodenal mucosa was loaded with Leishmania donovani bodies. The other patient had diffuse colonic aphthous and discrete ulcerations and Leishmania donovani bodies were seen in the crush smears of the colonic mucosa. With amphotericin B, there was reversal of malabsorption and healing of colonic ulcers.

Detection of Entamoeba histolytica using polymerase chain reaction in pus samples from amebic liver abscess.

BACKGROUND AND OBJECTIVE: Direct demonstration of Entamoeba histolytica by conventional microscopy and in vitro culture in pus obtained from amebic liver abscess (ALA) is often unsuccessful. We evaluated polymerase chain reaction (PCR) for detection of E. histolytica DNA in such pus. METHODS: Species-specific primers were used for the amplification of E. histolytica DNA from liver pus obtained from 30 patients with ALA. Patients with pyogenic liver abscess and sterile (autoclaved) pus spiked with Entamoeba dispar and bacteria (Escherichia coli, Klebsiella spp. and Bacteroides spp.) were used as negative controls. RESULTS: PCR was positive in 83% of pus specimens from patients with ALA, and was negative in all 25 pus specimens obtained from pyogenic abscess and autoclaved pus spiked with known bacteria. Sensitivity and specificity of PCR were 83% and 100%, respectively. The overall positivity of PCR was higher compared to serological tests. CONCLUSION: PCR may be a more reliable and better alternative diagnostic modality for ALA.

Refractory isosporiasis.

The authors describe a case of severe debilitating diarrhea due to isosporiasis in a two year old child, a known case of systemic vasculitis receiving prolonged corticosteroids therapy, an association rarely reported previously. It was refractory to treatment with dihydrofolate reductase inhibitor combined with sulfonamide such as cotrimoxazole to which isosporiasis usually responds well and is being described here for clinical interest and uniqueness of its presentation and laboratory findings.

Plasmodium lactate dehydrogenase assay to detect malarial parasites.

BACKGROUND: Microscopic examination of blood smears remains the gold standard for the diagnosis of malaria. However, it is labour-intensive and requires skilled operators. Immunochromatographic dipstick assays provide a potential alternative. One such dipstick, the Plasmodium lactate dehydrogenase assay (pLDH), is based on detection of the Plasmodium intracellular metabolic enzyme, LDH. The differentiation of malarial parasites is based on the antigenic differences between the pLDH isoforms. This study was designed to assess the sensitivity and specificity of pLDH assays in detecting and differentiating between various malarial species compared with microscopy. METHODS: Blood samples (n = 124) submitted to our laboratory for routine diagnosis of malaria were included in this study. From each blood sample, two thin films and a quantitative buffy coat (QBC) were made for microscopy. Thin films were stained with Giemsa and acridine orange. The pLDH assay was performed on all the samples according to the manufacturer's instructions. RESULTS: Of the 124 blood samples, 84 were negative by all methods (Giemsa, acridine orange, QBC and pLDH assay). Of the 38 samples positive for Plasmodium falciparum on microscopy, pLDH assay correctly identified 36 at parasite counts as low as < 40 parasites/microl and had a sensitivity and specificity of 94.3% and 97.6%, respectively. Of the 21 samples positive for Plasmodium vivax, pLDH assay correctly identified 19 at parasite counts as low as < 80/microl, and had a sensitivity and specificity of 90.4% and 100%, respectively. However, it failed to identify two Plasmodium vivax infections at parasite counts of 5000/microl and > 200/microl, suggesting that plasmodial gene deletions could be responsible for non-expression of pLDH. CONCLUSIONS: Our data demonstrate that pLDH assay, given its accuracy, rapidity (10-15 minutes), ease of performance and interpretation, can be a useful tool for the detection of malaria in countries where both plasmodial species are co-endemic and where laboratory support is limited.

Transthoracic lung aspiration in etiology of pneumonia.

Transthoracic lung aspiration was performed in 30 episodes of pneumonia in 27 children with malignancy on chemotherapy to assess etiology of pulmonary infections. Total of 22 organisms were isolated in 16/30 (53.3%) episodes. No acid fast bacilli or Pneumocystis carinii were seen. Organisms grown by blood culture correlated with that of lung puncture in 5 episodes, while throat culture and nasopharyngeal organisms correlated with that of lung puncture on one occasion each. Organisms isolated in 8/18 episodes (44.4%) of antemorten transthoracic aspiration included: Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Streptococcus faecalis and Diphtheroids. In 3/18 episodes, lung puncture results altered treatment and thus resulted in survival of the patients. Only one minor complication occurred in this study--pneumothorax that resolved spontaneously. Thus, transthoracic lung puncture is an useful and safe procedure in immunocompromised patients with pneumonia who do not respond to initial broad spectrum antibiotics.

Purification and partial characterization of PfHRP-II protein of Plasmodium falciparum.

The human malarial parasite Plasmodium falciparum secretes various intra-and extra-cellular proteins during its asexual life cycle in human RBC. Histidine rich protein-II (HRP-II) is one of the most prominent proteins, found to be secreted by P. falciparum throughout the asexual cycle with the peak during mature schizont stage of the parasite development in human IRBC. The high histidine content (35% of the total amino acids in protein) of this protein suggested the potential to bind divalent metal ions. We have demonstrated by metal chelate chromatography, an extraordinary capacity of HRP-II to bind nickel ions (Ni++) and employed this characteristic to purify the extra-cellular HRP-II protein secreted by P. falciparum from culture supernatant. The identity of the purified protein was verified by the relative molecular weight on SDS-PAGE, by reacting with polyclonal antibodies directed against it using Western blot technique.

Passive malaria surveillance in a low endemic area of India: validation of a clinical case definition.

BACKGROUND: In India, 2.55 million cases of malaria were reported during 1997; roughly one-third were due to Plasmodium falciparum. Malaria cases are identified by passive and active surveillance and all patients with fever are treated with chloroquine (10 mg/kg body weight). Since all fevers are not malaria, this results in overtreatment and has a bearing in terms of the parasites developing resistance. We aimed to test the validity of a clinical algorithm for passive malaria surveillance by primary care doctors (fever with pallor or splenomegaly) in a low endemic, Plasmodium vivax-predominant area of Ballabgarh block in Faridabad District, Haryana. METHODS: Passive surveillance was carried out at the general and paediatric outpatient departments (OPDs) of Ballabgarh hospital. All persons with fever attending the OPD were examined for the presence of fever, pallor and splenomegaly by the treating doctor. A blood smear was prepared and examined in all these cases. RESULTS: A total of 3119 slides for malaria were made at Ballabgarh hospital but clinical details in the requisition form were available for only 2616 patients who form the subjects of this analysis. A total of 59 malaria cases (30 P. vivax cases and 29 P. falciparum) were diagnosed. The presence of fever with pallor or splenomegaly had a sensitivity of 28.8% (95% CI: 18.1-42.3); specificity of 88.6% (95% CI: 87.3-89.8), positive predictive value of 5.5% (95% CI: 3.3-8.8) and negative predictive value of 98.2% (95% CI: 97.5-98.7). CONCLUSION: The algorithm did not have sufficient sensitivity to detect malaria cases by passive surveillance.

Seropositivity of Toxoplasma gondii in domestic animals.

Serum samples from 107 goats, 40 sheep, 50 cows were tested for Toxoplasma gondii antibody by indirect haemagglutination test (IHA) in dilutions of 1:10, 1:54, 1:162 and 1:486. Toxoplasma gondii-antibodies were found in 21 (19.6%), 10 (25%), 26 (52%), goat, sheep and cow sera respectively. No serum sample showed a titre higher than 1:162. All animals were kept in good hygienic condition. These results indicate that Toxoplasma gondii antibodies are widespread in animal populations which supports that toxoplasmosis is a widely spread zoonotic infection in this country.

Evaluation of a rapid antigen capture assay for the diagnosis of falciparum malaria.

A study was carried out to evaluate the accuracy of a dipstick antigen capture assay for the detection of Plasmodium falciparum histidine rich protein II antigen (Pf HRP-II) in peripheral blood to diagnose P. falciparum malaria. The sensitivity and specificity of the test was found to be 97 and 100 per cent respectively. The persistence of the antigen varied from 5 to 15 days after initiation of antimalarial therapy. The assay was simple to perform, rapid and easy to interpret. However, infection due to other species of human malaria parasites cannot be detected by this dipstick assay. Use of more sensitive technique like polymerase chain reaction (PCR) would help in diagnosing cases with low parasitaemia and infection due to other plasmodial species.