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BACKGROUND Diagnosis of tuberculosis (TB) in children remains challenging due to the paucibacillary nature of the disease. Detection of TB using urine lipoarabinomannan (LAM) antigen was evaluated in children with presumed TB. MATERIAL and METHODS Children with presumed intrathoracic tuberculosis (ITTB) and lymph node TB (LNTB) were enrolled. Expectorated or induced sputum or gastric aspirates from ITTB patients and fine-needle cytological aspirates from LNTB patients were subjected to Ziehl-Neelsen staining, MGIT™960™ culture and Xpert® MTB/RIF testing. Urine samples were tested to detect LAM, and the sensitivity and specificity calculated. RESULTS Of 280 children with presumed ITTB and 101 with presumed LNTB, respectively 71 (25.3%) and 25 (24.7%) were categorised as 'confirmed TB', 70 (25%) and 33 (32.7%) as 'unconfirmed TB', and 139 (49.6%) and 43 (42.5%) as 'unlikely TB'. Respectively 8 (2.8%) children with ITTB and 3 (2.9%) with LNTB were positive on smear, 56 (20.0%) and 23 (22.7%) on Xpert, and 50 (17.8%) and 9 (8.9%) on culture. LAM assay sensitivity was 73.2% in confirmed ITTB cases, and 76% in confirmed LNTB cases; LAM assay specificity in children with ITTB and those with LNTB initiated on anti-tuberculosis treatment was respectively 92% and 93%. Detection of TB using the LAM assay was significantly better than detection using Xpert (P CONCLUSION Urinary LAM testing showed high specificity and sensitivity, was detected in more cases initiated on treatment than reference tests, and improved disease detection by 38.5% in ITTB patients and by 41.6% in LNTB patients. .

Studies on Pneumocystis jirovecii dihydropteroate synthase (DHPS) genotypes among non-HIV immunocompromised patients from developing countries are rare. In the present prospective investigation, 24 (11.8%) cases were found to be positive for Pneumocystis jirovecii out of 203 non-HIV patients with a clinical suspicion of Pneumocystis pneumonia (PCP). Dihydropteroate synthase (DHPS) genotype 1 (Thr55+Pro57) was noted in 95.8% P. jirovecii isolates in the present study in contrast to only 4.1% of patients with DHPS genotype 4 (Thr55Ala + Pro57Ser).

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).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.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.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.

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.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.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.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.

The past decade has seen a rapid expansion of directly observed treatment, short-course (DOTS) centers throughout India, under the guidance of the Revised National Tuberculosis Control Programme (RNTCP). While expansion has been rapid and extensive, few reports exist detailing individual DOTS centers' experiences, their challenges, and their successes. We present a brief report on the status of a DOTS center being run at a large tertiary care center in northern India for almost four years (2001-2005).The DOTS center followed RNTCP guidelines for the evaluation and treatment of suspected TB cases. A register carrying detailed information of all patients seen at the DOTS center was kept by the senior clinician. Data from this register were extracted and analyzed for descriptive measures.A total of 1490 patients were evaluated. Of the 768 patients with cough, 27 per cent (211) were found to be sputum positive for acid-fast bacilli (AFB). Among patients who were initiated on anti-tuberculosis medications, cure was achieved in 92 per cent (71 of 77) of new sputum smear positive patients; treatment completion was achieved in 91 per cent (91 of 100) of extrapulmonary TB (EPTB) and 75 per cent (46 of 61) of sputum-negative pulmonary TB patients. Overall treatment success was achieved in 86 per cent (229 of 266).Treatment results were in keeping with the RNTCP guidelines. Tertiary care centers appear to be excellent place for education of medical students and operational research. The latter is much needed, as HIV-TB co-infection, multi-drug resistant TB, and EPTB continue to be major public health threats even in the era of DOTS.

In 1993, Government of India started the Revised National Tuberculosis Control Programme (RNTCP). A model Directly Observed Therapy, Short-Course (DOTS) centre was established at the All India Institute of Medical Sciences (AIIMS) to (i) identify the challenges and opportunities in establishing DOTS centres at tertiary care facilities, (ii) to teach the strategies of RNTCP to medical and paramedical staff, and (iii) to undertake relevant operational research connected with tuberculosis (TB) treatment and control. In this communication, we describe the experience of establishing a DOTS centre at India's premier medical institute and discuss the lessons learned.Since September 2001 through November 2002 AIIMS employees and their dependants diagnosed with tuberculosis were enrolled for treatment at AIIMS DOTS centre. One hundred sixty-eight patients were diagnosed as suffering from tuberculosis. Of these 49 patients were referred out and remaining 119 patients were treated at AIIMS DOTS centre.Treatment success was achieved in 80% (20/25) of new smear positive cases and the DOTS centre achieved other targets set up by the RNTCP. As the results of the pilot study at AIIMS DOTS centre were favourable, the facilities of AIIMS DOTS centre were extended to the general public from September 2002 onwards.Despite tremendous patient load at tertiary care facilities, it is possible to achieve targets established by the RNTCP. However, additional research needs to be conducted especially relating to drug resistance and surrogate markers of failure under RNTCP.

The recent emergence of dengue hemorrhagic fever/dengue shock syndrome (DHF/ DSS) in India has been a source of concern. In the present study a quantitative comparison of 406 nucleotide long sequence from the capsid-premembrane junction region (C-PrM) of 9 dengue virus type 2 (DEN-2) isolates from Delhi with 10 DEN-2 isolates from diverse geographic areas provided sufficient information for estimating genetic relationships. The data indicated that the 1996 epidemic of DHF in Delhi was caused by genotype IV strains of DEN-2. This genotype, perhaps, displaced genotype V strains of DEN-2, which was circulating genotype in 1967. The period during which this displacement had occurred is not clear from the present study. Nonetheless, similar experience in four countries in Latin America and in Sri Lanka suggest that the introduction of new genotypes of DEN-2 displacing the circulating genotype may be associated with the appearance of DHF/DSS. More work is required to elucidate this hypothesis. Transitions at nucleotide positions 406 and 431 resulted in amino acid substitutions near (aa position 104, methionine --valine) and at the hinge region (aa position 112, valine --alanine) of C-PrM, respectively in all/most genotypes of group III and IV DEN-2 viruses analysed. Most of these virus strains have been isolated from DHF/DSS outbreaks. Significance of this observation is discussed. The data presented in this study suggest the utility of C-PrM sequence analysis for molecular epidemiology of dengue viruses.

1. The production rates of bacteria in the rumen of buffalo (Bos bubalis) calves were estimated using an isotope-dilution technique. A series of fifteen experiments was done with animals given green maize and nine experiments with animals given cowpea (Vigna unguiculata).2. The turnover time ranged from 205 to 567 min in the group given green maize and from 330 to 648 min in animals offered cowpea. The production rates of bacteria were (mean±SE; g/d) 145.77±7.240 and 237.09±11.847 in animals given green maize and cowpea respectively.3. There was a significant correlation between bacterial production rates and dry matter intake, digestible organic matter and total volatile fatty acids formed in the rumen.4. Regression equations obtained for the two foodstuffs were different suggesting that the bacterial growth rate may vary depending upon the quantity and quality of foodstuff digested and possibly the ratio nitrogen: energy of the foodstuff.

The testicular seminal pathway in the camel is divided into two portions: an intratesticular and an extratesticular portion. The intratesticular seminal pathway consists of three portions: a 'tapering terminal segment' of the seminiferous tubules opening into the receptacle, the tubuli recti with three parts and the rete testis. The extratesticular portion consists of the rete testis, as an extension of the intratesticular portion and an elongated sac from which 6-7 ductuli efferentes originated. The tapering terminal segments are lined by modified Sertoli cells. Both, the tubuli recti and rete testis, are lined by simple cuboidal to columnar cells. The epithelium of the ductuli efferentes which, at its origin from the sac is simply cuboidal, gradually changes to an epithelium that consists of alternating groups of high-columnar ciliated and low-columnar non-ciliated cells. The strongly PAS-positive basement membrane of the ductuli efferentes is surrounded by a thin circular layer of smooth muscle fibres. The pseudo-stratified columnar epithelium of the epididymis is supported by an intensely PAS-reactive basement membrane. Morphological changes in the epithelium of the head and body regions of the epididymis due to seasonal variations are well marked in the camels of all age groups.

Histological and a few histochemical studies were done on the testes of 34 camels between 4 and 20 years of age, during three seasons: the tunica albuginea of the testes was composed of collagenous and a few elastic fibers. Isolated bundles of the smooth muscle fibers were observed in the capsule. The mediastinum testis and its radiating septula into the parenchyma also consisted of mainly collagenous and a few elastic fibers. The interstitial tissue was composed of Leydig cells and a network of reticular fibers. These cells increased in volume in the rutting period from December to March, and were distorted and reduced in volume in the nonrutting period from April to November. The seminiferous tubules were 113--250 micrometer in diameter, and were lined by many layers of spermatogenic cells and a single layer of Sertoli cells. The spermatogenesis was observed as a continuous process throughout the year, particularly in the camels between 4 and 15 years of age. Almost in all the testes studied, three types of spermatogonia, two to three layers of primary spermatocytes, rarely secondary spermatocytes and spermatids in two stages were observed. The spermatozoa were present attached to the Sertoli cells as well as free in the lumen of the tubules. The oval head of the spermatozoa was characterized by the presence of an elongated pointed, strongly PAS-positive acrosome.

SummaryThe rates of production of protozoa in the rumen of buffalo calves have been estimated using a single-injection isotope dilution technique. The calves were fed at 2 levels of crude protein, namely, 13% lower and 19% higher than that recommended by the National Research Council. The animals were given their rations at 2-h intervals for 3 weeks. Thereafter 14C-labelled rumen protozoa were injected into the rumen of each calf in a single injection. Samples of the rumen liquor were drawn at various time-intervals for 10 h and were analysed for the concentration of protozoa and radioactivity. The decline in the specific radioactivity of protozoa cells in the rumen as a function of time was used for calculating half life (t½). A mathematical equation was used to calculate the rate of production of protozoa. The average t½ was 1077 min for both groups and the rates of production of protozoa were 73·9 and 92·1mg/min in groups fed on low and high planes of crude protein respectively, showing that the production of protozoa is significantly (P < 0·01) higher in animals consuming a ration high in crude protein.