CD4+, CD8+, CD3+ cell counts and CD4+/CD8+ ratio among patients with mycobacterial diseases (leprosy, tuberculosis), HIV infections, and normal healthy adults: a comparative analysis of studies in different regions of India.

In this study, we estimated the CD4+, CD8+, CD3+ cell counts and the CD4/CD8 ratio among normal healthy controls (adults and children), leprosy patients (without any complications and during reactional states), TB patients (with and without HIV), and HIV-positive patients (early infection and full-blown AIDS) and correlated the changes with disease progression. In our study, it was observed that among adults, CD4+ cell counts ranged from 518-1098, CD8+ from 312-952, whereas CD4/CD8 ratio from 0.75-2.30. Among children, both CD4+ and CD8+ cells were more and the CD4/CD8 ratio varied from 0.91-3.17. With regard to leprosy patients, we observed that CD4+ and CD8+ cell counts were lower among PB (pauci-bacillary) and MB (multi-bacillary) patients. CD4/CD8 ratio was 0.99 ± 0.28 among PB patients while the ratio was lower, 0.78 ± 0.20, among MB patients. CD4+ cell counts were raised during RR (reversal reactions) and ENL (erythema nodosum leprosum) among the PB and MB patients whereas the CD8+ cell counts were lower among PB and MB patients. CD4/CD8 ratio doubled during reactional episodes of RR and ENL. Among the HIV-negative tuberculosis (TB) patients, both the CD4+ and CD8+ cell counts were found to be less and the CD4/CD8 ratio varied between 0.53-1.75. Among the HIV-positive TB patients and HIV-positive patients, both the CD4+ and CD8+ cells were very less and ratio drops significantly. In the initial stages of infection, as CD4+ counts drop, an increase in the CD8+ cell counts was observed and the ratio declines. In full-blown cases, CD4+ cell counts were very low, 3-4 to 54 cells, CD8+ cells from 12-211 and the ratio drops too low. This study is the first of its kind in this region of the country and assumes importance since no other study has reported the values of CD4+ and CD8+ T-lymphocyte counts among patients with mycobacterial diseases (leprosy and TB), HIV infections along with normal healthy individuals of the region, and correlation with clinical presentations of patients.

Detection of viable Mycobacterium leprae in soil samples: insights into possible sources of transmission of leprosy.

Leprosy has ceased to be a public health problem world wide, after the successful implementation of effective chemotherapy (MDT) and use of control measures. However, new cases of leprosy continue to occur. Mycobacterium leprae cannot be grown in any acceptable culture medium and besides the wild armadillos, there is no known animal reservoir for leprosy. The transmission of leprosy is believed to be due to a large extent by droplet discharge of bacilli through nose and mouth and to a lesser extent by direct contact of susceptible host with a patient for long duration. The exact role of the environment in the transmission dynamics is still speculative. In the present study, we have tried to detect viable M. leprae from soil samples in endemic areas by using molecular methods. Eighty soil samples were collected from villages of this area, DNA and RNA of M. leprae extracted and identified using specific M. leprae primers. PCR amplification was done and real-time RT-PCR was used to detect viable M. leprae. DNA targeting the 16S region of M. leprae was detected in 37.5%, whereas M. leprae RNA targeting the same region was detected in 35% of these samples. Of the total 80 samples, 40 were collected from residential areas of leprosy patients whereas 40 samples were from no-patient areas. Fifty-five percent positivity for 16S rRNA of M. leprae was observed from the "patient" area in comparison to 15% positivity from the "no-patient" area (p < 0.001). This study thus provides valuable information of presence of viable M. leprae in soil specimens, which would be of use in investigating the transmission dynamics in leprosy.

Development and evaluation of real-time RT-PCR assay for quantitative estimation of viable Mycobacterium leprae in clinical samples.

Detection of live organisms by molecular methods has special significance in leprosy where causative organism can not be cultivated in vitro. Such techniques would be especially important for monitoring the progress of the disease. While real-time RT- PCR technology will be appropriate for this purpose, there is very little experience of use of such tools in leprosy. This study describes the development of a quantitative RT-PCR targeting 16S rRNA based on primers used in a semi quantitative RT-PCR and its application on clinical samples including slit scraping and biopsies. RNA was extracted from biopsies from 3 lepromatous leprosy (LL) cases and standard curve was generated by plotting crossing over point against the dilutions of input RNA quantity (number of bacilli used for RNA extraction). Real-time RT-PCR was performed for quantitative detection of live M. leprae in 28 slit (13/28 smear positive) scrappings and 32 biopsies (22/32 smear positive). Number of viable bacteria as estimated by solid stained bacilli and real-time PCR correlated (no difference p>0.05). The test achieved a theoretical analytical sensitivity limit of up to single live bacillus even considering 11.3% efficiency of RNA preparation which was calculated by spiking of known number of leprosy bacilli in non leprosy skin biopsies (PCR negative). All smear positive cases were positive by this assay. This assay appears to be a promising tool for detection and quantification of viable bacilli in selected clinical situations and should be of use even in smear negative cases also.

Leprosy scenario in Agra: epidemiological observations from rapid population survey 2004-06.

This study based on rapid survey methodology examining 17.86 lakhs population revealed that leprosy prevalence in Agra District is 6.1/10,000 during 2004-06, with 97.2% of the cases detected for the first time. Although leprosy is still endemic but all the leprosy indicators have shown positive change since last survey in 2001-03. The results seem promising to achieve elimination target (<1/10,000) since prevalence/10,000 population declined from 16.4 in 2001-03 to 6.1 in 2004-06, MB rate from 22.3 to 17.1, Mean duration of disease at detection (months) from 32.3 to 22.9, per cent new cases increased from 88.2% to 97.2% and visible disability (Grade > or = 2) rate declined from 4.8% to 2.36% over this period. The data on patients with incomplete history of treatment (prevalent) but having active disease indicate that only about 3% (31/1090) had approached the health center for treatment. Of these 31 patients, 29% defaulted from treatment and still have active disease and 75.2% (23/31) had MB disease indicating a pattern of late reporting to health system. This study suggests that repeat surveys are useful to detect cases for treatment and seems the key to achieve leprosy elimination or even eradication at district level in all endemic districts.

Changing profile of disease in leprosy patients diagnosed in a tertiary care centre during years 1995-2000.

A hospital based retrospective study was carried out to determine change in the profile of disease in leprosy patients taking 1995 as baseline and compared with the profile seen in year 2000. A total of 2149 and 1703 cases were studied respectively of year 1995 and 2000. Male to female ratio slightly increased from 2.95:1 in year 1995 to 3.4:1 in year 2000. Majority of patients were of borderline type in both years. Proportion of cases with MB leprosy was nearly same in females (60.8%) and males (63.1%) in year 1995 and in year 2000 (64.8% females and 67.6% males). Proportion of highly bacillary cases has decreased over the years in females (from 20.95% in 1995 to 11.7% in year 2000, p=0.03) as well as in males (from 25% in 1995 to 15.5% in year 2000, p=0.001). Incidence of total reactions increased from 27.6% to 35.4% over the years which is significant (p<0.01). Proportion of type 1 reactions were more in reproductive age group in females in both years (p<0.05) and of type 2 reactions were significantly (p > or = 0.05) more in males in both years. Incidence of disability (both grade 1 and grade 2) was significantly more in males than in females in both years (p > or = 0.04). Grade 1 disability has significantly increased over years in females from 10.11% to 14.8%(p<0.03) as well as in males from 13.27% to 21.3%(p<0.001). Onset of reactions was associated with pregnancy/lactation in 62% of cases and with menopause in 21% of cases in 2000, which suggests strong correlation with hormonal imbalance. To conclude while leprosy incidence has declined after MDT, recognition and management of reactions in women around changes in their hormonal levels should be properly monitored for early and effective management.

Serum samples from patients with mycobacterial infections cross-react with HIV structural proteins Gp41, p55 and p18.

BACKGROUND: Infection with Mycobacterium leprae is associated with a high frequency of false positive results in a variety of serological assays. Our studies have found cross-reactivity to HIV structural proteins in serum samples from leprosy patients, irrespective of the type of disease, treatment duration, age and gender and from a few patients with active TB disease. METHODS: Western blot (WB) analysis revealed that sera from HIV negative leprosy patients across the spectrum showed high reactivity with p18, Gp41 and p55 and lower reactivity with other HIV proteins. The reactivity appeared to be specific; western blot-positive samples were negative in ELISA and in several rapid tests for HIV. Cross-reactivity was not found in sera from patients with leishmaniasis or from normal healthy individuals. RESULTS: None of the WB reactive leprosy patients seroconverted to HIV positivity within 6 months to 1 year after Western blot testing. BLAST analysis revealed that envelope antigens of HIV (Gp41, Gp120 and Gp160) contained amino acid sequences similar to M. leprae ML0470, putative integral membrane protein, Rv0740, mmpL9 (M. tuberculosis). Core (gag) antigens (p18) had similarities to ML0406, but polymerase antigens (p52) had similarities to PE_PGRS (M. tuberculosis, H37Rv). Nucleotide sequence analysis, on the other hand, did not reveal any significant homology between M. leprae or M. tuberculosis and HIV. CONCLUSIONS: The occurrence of these high false-positive rates in M. leprae-infected individuals suggests a possible complication of serodiagnosis of HIV in regions where mycobacterial infections are endemic. There is a need for caution in reporting HIV infection among leprosy patients. Our observations emphasise the value of the various rapid assay kits for HIV, where this false positivity is not observed.

Leprosy situation in the slums of Agra City--epidemiological findings.

The present article is the result of a study of the leprosy caseload in Agra City and is based on a house-to-house survey conducted during April-July 2003 in 5 areas. During the survey 198,150 persons were examined, and 287 cases were detected, giving a prevalence rate of 14.5/10,000. A majority of them (92%) were new cases, detected and diagnosed for the first time. The patient load was found to be unevenly distributed with comparatively more number of patients in areas such as Jamuna Kinara, Shah Ganj and Lohamandi. Among the 264 newly detected cases, 14.8% were of MB type. Overall deformity of grade > or = 2 was seen in 2.8% of patients--0.4% in PB and significantly high at 18% in MB leprosy. The observations reveal that leprosy is endemic in slum areas of Agra City.

Seroprevalence of HIV infection among leprosy patients in Agra, India: trends and perspective.

This study compares the results of HIV seroprevalence, which was carried out in two phases, i.e., 1989 to 1993 and 1999 to 2004. Although the number of leprosy patients screened for HIV infection in the second phase is less (2125) as compared to those screened during the first phase (4025), a rise in HIV infection from 0.12% to 0.37% is certainly disturbing since this area appears to be endemic for both the infections. During the study period, the Out Patient department attendance of a few types of leprosy patients like borderline and borderline lepromatous have risen, whereas others like borderline tuberculoid and polar tuberculoid have declined in the second phase as compared to that of the first phase. The trend over a decade suggests that HIV infection is low among the leprosy patients when compared with other risk groups. Follow-up of these patients at an interval of six months, revealed that none of them downgraded into a severe form of leprosy nor developed ARC or AIDS. In this study, it appears that neither infection precipitated the other. The occurrence of downgradation as well as reversal reactions and neuritis (both chronic and acute) was not observed among the leprosy patients. None of them developed erythema nodosum leprosum reactions. Similarly, the HIV-positive leprosy cases did not develop either AIDS related complex (ARC) or full blown case of AIDS.

Ulnar and median nerves in paucibacillary leprosy--a follow-up study of electrophysiological functions in patients before and after nerve trunk decompression.

Electrophysiological functions of ulnar and median nerves in paucibacillary leprosy patients were studied. Patients who showed deterioration of sensory motor functions in spite of steroid therapy were offered nerve decompression together with oral steroids. On periodic follow-up of those who opted for surgery, it was observed, in general, that NCV and amplitude remained reduced even though clinical recovery occurred. Only 80% recovery of electrophysiological functions was seen (as compared to control levels), even in cases that showed good results. Motor function recovered better than sensory function. Complete electrophysiological recovery, if it occurs at all, takes much longer than clinical recovery.

Some epidemiological observations on leprosy in India

This population sample survey conducted in rural and urban areas of the Agra District in India showed an active leprosy caseload of 60.1/10,000 in the rural and 39.1/10,000 in the urban areas against a targeted prevalence of < 1/10,000. The disease appeared to be widespread since almost 65% of the villages or urban pockets surveyed had at least one prevalent case of leprosy. Significantly larger numbers of leprosy patients were found among males, agricultural/manual workers, persons with no formal schooling, individuals living in unkept households with dirty surroundings, and among those living in dark and poorly ventilated houses. The epidemiological significance of this study reveals the endemic nature of leprosy in Agra and suggests the need to intensify and widen case-detection activities to achieve leprosy control.

Some epidemiological observations on leprosy in India.

This population sample survey conducted in rural and urban areas of the Agra District in India showed an active leprosy caseload of 60.1/10,000 in the rural and 39.1/10,000 in the urban areas against a targeted prevalence of < 1/10,000. The disease appeared to be widespread since almost 65% of the villages or urban pockets surveyed had at least one prevalent case of leprosy. Significantly larger numbers of leprosy patients were found among males, agricultural/manual workers, persons with no formal schooling, individuals living in unkept households with dirty surroundings, and among those living in dark and poorly ventilated houses. The epidemiological significance of this study reveals the endemic nature of leprosy in Agra and suggests the need to intensify and widen case-detection activities to achieve leprosy control.

Response of leprosy patients with single lesions to MDT.

This study reports the clinical profile and therapeutic response of seventy-two mono-lesions leprosy cases. These 72 cases were among 578 paucibacillary (PB) cases classified according to WHO (1982) and were followed-up on multidrug therapy (MDT). Of these 72 mono-lesion cases, 46 (64%) were tuberculoid (TT) cases, 24 (33%) were Indeterminate (Ind) cases and 2 (3%) were of borderline tuberculoid (BT) types. While 37.5% of these cases presented as macular patches, the remaining 62.5% had raised erythematous lesions. In majority of cases (94%), the lesions were present on the exposed parts like legs and feet, arms and hands, face, whereas only 6% presented on covered areas of trunk and buttocks. These cases were treated with dapsone 100 mg daily for 12 months and rifampicin 600 mg once a month for 6 months. After 6 months of MDT, lesions in 81% of the patients regressed clinically and by one year of therapy 96% of cases had regressed. Treatment was stopped in all cases by one year of therapy. There were no relapse or late reaction in the 5 years of post treatment follow-up. The response of mono-lesion PB cases was better than the multi-lesions PB cases at 6 months and during the post treatment follow-up period.

Treatment of paucibacillary leprosy with a regimen containing rifampicin, dapsone and prothionamide.

Ninety paucibacillary leprosy patients having indeterminate (I), tuberculoid (TT) and borderline tuberculoid (BT) type of leprosy with bacterial index (BI) of less than two on the Ridley scale were treated with rifampicin (RFM) 600 mg once a month, dapsone (DDS) 100 mg daily and prothionamide (PTH) 250 mg daily. Treatment was stopped at the end of six months. The patients tolerated the drugs fairly well and in only two patients the drugs had to be stopped (in one due to jaundice and in the other due to gastric intolerance). About 6% of patients had early reactions which subsided with additional steroid therapy. The inactivity rate was 60% at six months and this improved to 96% at 12 months. No cases of late reactions and relapses were encountered in the limited follow-up period of six months; and a longer follow-up is necessary for ascertaining the relapse rates. The preliminary results however suggest that the addition of prothionamide to the standard WHO paucibacillary regimen is well-tolerated with increased inactivity rate and fewer instances of late reactions.