Refractory giardiasis: A molecular appraisal from a tertiary care centre in India.

Purpose: The intestinal flagellate Giardia lamblia includes many genetically distinct assemblages, of which assemblage A and B, predominantly infect humans. Nitroimidazoles derivatives (metronidazole and tinidazole) and nitazoxanide are some of the therapeutic agents for treatment of giardiasis. Nevertheless, some individuals with giardiasis are non‑responsive to standard therapy. The present study highlights cases of refractory giardiasis and attempts to elucidate if genetic heterogeneity in the parasite is associated with treatment failure. Materials and Methods: Three stool samples were obtained on three consecutive days from 4000 patients with diarrhoea and were microscopically examined for the detection of trophozoites, and/or cysts, using both normal saline and Lugol’s iodine. A hemi‑nested polymerase chain reaction (PCR) assay using triose phosphate isomerase (tpi) as the target gene was performed to determine the assemblages. Sequencing of the PCR products of the patients showing failure to treatment of giardiasis was also performed. Results: Two per cent (82/4000) of the total patients were microscopically positive for Giardia lamblia in the stool samples. All these patients were treated with metronidazole/tinidazole as per the standard regimens. However, eight patients showed treatment failure to giardiasis as stool examinations were repeatedly positive even after treatment with multiple courses of anti‑giardial therapy. Genetic characterisation of all eight Giardia isolates showed that they belonged to Assemblage B and had homogeneous sequences. These patients were either treated with extended regimens or with combination therapy of anti‑giardials. Conclusion: In our experience, combination of two or more drugs for a longer duration is the treatment modality to treat refractory giardiasis.

Molecular characterisation of Giardia intestinalis assemblages from human isolates at a tertiary care centre of India.

Purpose: The aim of the study was to determine the genetic heterogeneity of Giardia intestinalis isolates detected in stool samples of the study population using polymerase chain reaction assay and restriction fragment length polymorphism. We also tried to correlate the association/differences between the clinical symptomatology and infection by different assemblages (genotypes) of G. intestinalis. Materials and Methods: This cross-sectional study was conducted from April 2008 to June 2010. A total of 40 adults (n = 40) and 42 children (n = 42) below the age of 12 years with the clinical suspicion of giardiasis and with the onset of one or more of the following fi ve symptoms, i.e., loose stool, nausea, weight loss, fatigue and foul smelling faeces and confi rmed laboratory diagnosis of giardiasis at least once during the current episode of diarrhoea were included in this study. Results: Of the 82 patients (males 66) enrolled in the study, 70 (85%) presented with diarrhoea (56 males) and 12 (15%) without diarrhoea (10 males). Out of 70 diarrheic patients, 61 (87%) had chronic diarrhoea, 8 (11.5%) had acute diarrhoea and 1 (1.5%) had persistent diarrhoea. Of the total patients, 63 (77%) were clinically assessed and were apparently immunocompetent, whereas, 19 (23%) immunocompromised patients had different underlying conditions besides giardiasis. Genotyping identifi ed all 82 (100%) isolates as assemblage B. Conclusion: We found that assemblage B of G. intestinalis presents with all kinds of clinical features ranging from asymptomatic carriage to acute, persistent or chronic diarrhoea.

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.

Evaluation of small-subunit rRNA touchdown polymerase chain reaction for direct detection of Entamoeba histolytica in human pus samples from patients with amoebic liver abscess.

Purpose: The aim of the present study was to evaluate the use of touchdown polymerase chain reaction (TD-PCR) for the detection of Entamoeba histolytica in liver pus samples obtained from patients with a clinical diagnosis of amoebic liver abscess (ALA) using small-subunit rRNA (SSU rRNA) as the target gene. Materials and Methods: Microscopic examination in vitro culture and serological test for the detection of E. histolytica in 67 pus samples obtained from ALA patients was performed. Molecular studies were carried out by both conventional PCR and TD-PCR targeting the SSU rRNA gene using the same sets of primers and the results were compared. Results: TD-PCR detected the presence of E. histolytica in 86.5% of the liver pus samples within 2.5 h as compared with 82.08% by conventional PCR within 3.5-4 h. Conclusion: TD-PCR assay may serve as a relatively better detection method for E. histolytica over conventional PCR with respect to the turnaround time, increased sensitivity, specificity and yield.

Study of dihydropteroate synthase (DHPS) gene mutations among isolates of Pneumocystis jiroveci.

BACKGROUND & OBJECTIVES: Pneumocystis jiroveci (also known as P. carinii) causes fatal pneumonia in patients with AIDS and other immunocompromised patients. Co-trimoxazole (trimethoprim + sulphamethoxazole, TMP-SMZ) is the drug of choice for treatment and prophylaxis. Widespread use of sulpha medication has raised the possible selection of resistant P. jiroveci strains worldwide. Non-synonymous polymorphisms associated with sulpha resistance have been observed in P. jiroveci dihydropteroate synthase (DHPS) gene at codons 55 and 57. In view of this, we investigated mutation at DHPS locus amongst P. jiroveci isolates obtained at a tertiary care hospital in north India. METHODS: Microscopic examination of P. jiroveci in 69 clinical samples obtained from patients suspected to have P. carinii pneumonia (PCP), was performed by Grocott's Gomori methenamine silver and direct fluorescent antibody staining. Molecular studies were carried out by polymerase chain reaction (PCR) using major surface glycoprotein (MSG) as the target gene. Investigations for DHPS mutations were carried at specific 55th and 57th codon using PCR-RFLP (restriction fragment length polymorphism) assay. RESULTS: Microscopic examination detected P. jiroveci in four cases and MSG gene was amplified in five cases. Further, amplification of DHPS gene was successful in four of the five cases positive by MSG gene PCR. No point mutation was observed and all four isolates presented wild-type sequences at DHPS gene by RFLP analysis. INTERPRETATION & CONCLUSION: Although our findings suggest that in Indian subpopulation, point mutations in DHPS gene of P. jiroveci are not as common as in other parts of the developed world, further studies are needed.

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.

Human pneumocystosis.

Pneumocystis is an atypical fungus causing pneumonia in immuno-compromised individuals. Though previously termed as Pneumocystis carinii, the recent taxonomy has considered human derived Pneumocystis to be a different species Pneumocystis jiroveci. The organism is the most common cause of opportunistic infections among patients with acquired immunodeficiency syndrome (AIDS) in developed countries. Incidence of Pneumocystis pneumonia or pneumocystosis in developing countries including India continues to be low. Microscopy of appropriate clinical samples has been the mainstay of diagnosis of pneumocystosis. Amplification techniques are now being evaluated for detection of P. jiroveci. This review attempts to give a recent update on P. jiroveci with special focus on epidemiology, taxonomy, current diagnostic modalities and recommended immuno-prophylaxis.

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.

Evaluation of quantitative buffy coat (QBC) assay and polymerase chain reaction (PCR) for diagnosis of malaria.

A prospective study was undertaken to compare the Polymerase Chain Reaction (PCR) and Quantitative Buffy Coat (QBC) assay with conventional Giemsa technique for diagnosis of malaria. A total of 104 samples were taken for the purpose. They comprised of fever cases suggestive of malaria (n=74) and control group, fever cases other than malaria (n=30). Peripheral blood smears were prepared by Giemsa staining and QBC assay was performed as per standard protocol. From the stored blood samples, parasite DNA was extracted and PCR was performed using P. falciparum and P. vivax specific sets of primers. The QBC assay was 100% in agreement with the Giemsa stain. Specificity of the PCR detection of P. falciparum parasites was 100%. However, sensitivity for detection of P. falciparum and P. vivax by PCR was 64.28% and 82.35% respectively. In mixed cases of malaria (n=2), PCR results were in 100% agreement with that of Giemsa. The lower sensitivity of PCR for P. falciparum could probably be due to inaccessibility of target DNA, presence of PCR inhibitors in samples and parasite strain variations.

Echinococcosis: presenting as palpable lumps of breast.

While hydatid disease is common in developing countries, yet occurrence of echinococcal cysts in breast are rare. Our experience encountered in two patients with hydatidosis diagnosed on fine needle aspiration (FNA) is presented because it may cause a clinical dilemma with other cystic lesions of breast. No complications were observed after FNA in both the cases. The helpful role of indirect haemagglutination (IHA) test in evaluation of the disease is highlighted.

Comparative yield of different respiratory samples for diagnosis of Pneumocystis carinii infections in HIV-seropositive and seronegative individuals in India.

Respiratory specimens were prospectively examined for Pneumocystis carinii from 53 patients. The majority of specimens were comprised of expectorated sputum, induced sputum, broncho-alveolar lavage (BAL), and tracheal aspirates. In only four patients Pneumocystis carinii (P. carinii) was detected. All the samples were produced by broncho-alveolar lavage. Candida spp and Aspergillus spp were also identified in a small number of patients. Acid-fast-bacilli were not detected in any of the cases under study. There were no sex-related differences in distribution. The present prospective study was undertaken in order to determine P. carinii infections in human immunodeficiency virus (HIV) seropositive and seronegative individuals. Expectorated sputum samples were probably the major limiting factor in low positivity for detection of P. carinii and study of BAL specimens would be more useful for better results.

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.

Quantitative buffy coat: a special adjunct for diagnosis of malaria.

Quantitative buffy coats (QBC) technique was compared with conventional blood film technique for the diagnosis of malaria in a tertiary care hospital. The QBC technique was found to be a rapid technique with a sensitivity of 83% and specificity of 94%. Malaria species identification was also possible. It was essentially very useful to detect parasites below < or = 100 parasites/ul of blood by QBC technique. However, quantification of parasitaemia could not be made using this technique. Many cases of carriers having very few gametocytes in their blood were also identified. It is therefore, concluded that the QBC technique, may be appropriate for screening populations for malaria and for detection of asymptomatic carriers to control further transmission of the disease in the community.

Bone marrow examination for identifying malaria in fever of unknown origin.

To determine the utility of bone marrow examination for the diagnosis of malaria in patients with persistent fever for prolonged duration, we prospectively studied individuals undergoing diagnostic bone marrow examinations between January 1992 to December 1996. All marrow examinations of patients were examined microbiologically and resulted in diagnosis of malaria in 6.6% of the total patients studied. No case of bacterial, mycobacterial or fungal infection was diagnosed. The diagnostic efficacy of bone marrow for evidence of malaria was very useful in febrile individuals for whom the diagnosis was otherwise unknown.