Table of Contents

2017 Month : November Volume : 6 Issue : 90 Page : 6277-6281

CRYPTOCOCCAL ANTIGENAEMIA IN ANTIRETROVIRAL THERAPY NAIVE PATIENTS WITH HUMAN IMMUNODEFICIENCY VIRUS INFECTION.

Dibya Prasana Mohanty1, Dharma Niranjan Mishra2, Dillip Kumar Pradhan3

1Assistant Professor, Department of Microbiology, Utkal University, SCB Medical College, Cuttack.
2Assistant Professor, Department of Anatomy, Utkal University, SCB Medical College, Cuttack.
3Senior Resident, Department of Medicine, Utkal University, SCB Medical College, Cuttack.

Corresponding Author:
Dharma Niranjan Mishra,
Flat No. 3-B, Neelamani Enclave,
Professor Pada, Post- College Square,
Cuttack-753003.
E-mail: dharmaniranjan.mishra08@gmail.com

ABSTRACT

BACKGROUND

Human Immunodeficiency Virus (HIV) related to cryptococcal meningitis in India is a leading cause of morbidity and mortality among severely immunocompromised patients.

The aim of our study was to determine the prevalence of and risk factors for Cryptococcal antigenaemia among HIV-infected adults attending ART clinic and medical emergency.

MATERIALS AND METHODS

This was a hospital-based cross-sectional and prospective study carried out among newly diagnosed and confirmed HIV-infected patients after taking written informed consent and due ethical approval. Results were presented in simple tables with distribution and percentages while P value ≤ 0.05 was considered as statistically significant.

RESULTS

Out of 100 patients, there were 65 (65%) males and 35 (35%) females in the study. The median age was being 35 years (range18-67) followed by BMI 20.271 m2 (range15.1-26.48) and CD4 count 196 (range 6 -780) cells/mm3. Out of 100 patients, seven (7%) were positive for cryptococcal antigen (CRAG). Six (85.71%) of them were CRAG positives with CD4+ cell count less than 100 cells, while 1 (14.28%) had count above 100 cells/mm3. There were 4 (23.5%) SCRAG+ out of 17 symptomatic cases and 3 (3.6%) were SCRAG+ out of 83 asymptomatic patients with statistical significance (p<0.015). The symptoms of fever, headache, vomiting and neck rigidity are significantly associated with Cryptococcal antigenaemia (p<0.05).

CONCLUSION

All ART naive adults having CD4 count < 100 cells/mm3 should be screened for serum Cryptococcal antigen followed by presumptive antifungal therapy if serum Cryptococcal antigen is positive.

KEYWORDS

Cryptococcal Antigenaemia (CRAG), CD4- Cluster Differentiation, Human Immunodeficiency Virus (HIV).

BACKGROUND

Human Immunodeficiency Virus (HIV) infection is a global pandemic, with cases reported from virtually every country. World Health Organization (WHO) has estimated 34.0 million (31.4-35.9) global people living with HIV in 2011. Based on HIV Sentinel Surveillance 2008-09, it is estimated that India has 23.9 lakh people infected with HIV. Human Immunodeficiency Virus infection was first reported in India in the state of Tamil Nadu in 1986, since then the cases of Cryptococcal Meningitis (CM) have also increased.1Morbidity and mortality in HIV patients is mostly caused by opportunistic infections (OI) that occur due to lowered immune defences of the patients associated with decreased CD4 counts. Among these, meningitis with HIV has an important impact causing considerable morbidity and mortality. Meningitis associated with HIV infection can be classified according to aetiologic agents as fungal, tubercular, syphilitic and pyogenic. The most common OI in HIV patients in India is either tubercular or fungal.2 Cryptococcosis is a fungal disease caused by Cryptococcus neoformans (CN), which begins as droplet infection in the respiratory tract and eventually spreads to central nervous system to produce meningitis (CM).

Cryptococcal meningitis (CM) is one of the presenting manifestations of Acquired Immune Deficiency Syndrome (AIDS).3 Cryptococcal meningitis is one of the most common OI among HIV-infected individuals, with an estimated 10 lakh cases of HIV associated CM and 6 lakh deaths every year or more than 1700 deaths everyday.4 Despite cryptococcal disease accounting for 13-44% of deaths in HIV-infected patients, cryptococcal diseases continue to be grossly under-diagnosed.5 Recent data indicates that the incidence of cryptococcal infection is high in India.6 It is the leading infectious cause of meningitis in patients with AIDS and is the initial AIDS defining diagnosis in approximately 2% of patients, mostly occurring in those with CD4 counts less than 100 cells/mm.3.7

Cryptococcal antigen (CRAG), a biological marker for Cryptococcal infection, is detectable in sera a median of 3 weeks (range 5 – 234 days) before symptoms of meningitis appear.8 Positive Cryptococcal antigenaemia is an independent predictor of CM and death in patients with severe immunosuppression.9 This asymptomatic period before development of symptomatic meningitis provides a window of opportunity to treat patients and potentially prevent fatal Cryptococcal disease. CRAG detection is highly sensitive as compared with microscopy and culture.10 The best prophylaxis to prevent OI is an immune reconstitution with anti-retro viral therapy (ART). In areas of high prevalence, the CM screening prior to ART is necessary for potential early diagnosis and treatment. This could decrease the risk of Immune Reconstitution Inflammatory Syndrome (IRIS). The WHO has recently released “Rapid Advice” guidelines for Cryptococcal disease among persons living with HIV. Early diagnosis is key to reduce mortality due to Cryptococcal disease. A major WHO recommendation is implementation of SCRAG screening and presumptive anti-fungal therapy (typically oral fluconazole) in those with a positive diagnostic test among ART naïve adults with a CD4 count less than 100 cell/mm3 in areas with a high prevalence of Cryptococcal disease (> 3%).

One limitation to implementing the WHO guidelines is that currently very less data exists on the extent of Cryptococcal infection in India. Hence, this prompted the study which aims at finding occurrence of and risk factors associated with Cryptococcal antigenaemia in ART naïve patients with HIV. The purpose of our study was to determine the prevalence of and risk factors for Cryptococcal antigenaemia among HIV-infected adults attending ART clinic and medical emergency at SCB Medical College and Hospital, Cuttack.

MATERIALS AND METHODS

Study Design: Cross-sectional observational study with asking research questionnaire developed for this purpose.

Study Location

This study was undertaken among newly diagnosed antiretroviral therapy naïve HIV/AIDS infected patients at the Postgraduate Department of Medicine, SCB Medical College, Cuttack after taking written informed consent. Hundred patients admitted in the medical wards and visiting ART clinic were considered for this study from May 2016 to Apr 2017. The screening test was done for serum cryptococcal antigen (SCRAG).

Inclusion Criteria

ART naïve patients ³ 18 years documented for HIV infection and confirmed by a series of 3 tests as per NACO Guidelines (First by dot immunoassay followed by two different immunochromatographic tests).

Exclusion Criteria

Previously diagnosed Cryptococcosis patients on fluconazole therapy and satisfying the above criteria.

Ethical Issues

This study confirms to the ethical principles of medical research developed by the World Medical Association Declaration of Helsinki. Ethical clearance was given by the Institutional Ethics Committee of SCB Medical College, Cuttack.

Data Analysis: All data obtained with questionnaire and microbiological culture analysis were analysed using the statistical software SSPS 16.0. The Chi-square test was used to compare between two groups. Statistical significance was accepted when P value is ≤ 0.05 and the Univariate and multivariate logistic regression analyses were done to assess risk factors for a positive cryptococcal antigenaemia. Risk factors with possible significance and known to be associated with cryptococcal disease were included in the present study.

RESULTS

The mean age of the study population was being 36.14 ± 10.42 years (ranges 18-67 years) and median was being 35 years. The mean body mass index (BMI) of our study population was 20.54 kg/m2 ± 2.72 (ranges 15.1 to 26.48 kg/m2). The present study population had mean cluster differentiation (CD4) count of 233 cells/mm3 ± 176 (ranges 6 to 780 cells/mm3) (Table 1).

Characteristic

No.

Mean ± SD

Median

Range Min-Max

I Q Range

Age in years

100

36.14 ± 10.42

35

18-67

30.0-42

BMI(kg/m2)

100

20.54 ± 2.72

20.271

15.1-26.48

19.13-22.36

CD4 count cells/mm3

100

233.06 ± 176.13

196

6-780

70.5-355.5

I .Q .R -Interquartile range, CD4 -cluster differentiation (cells/mm3) and BMI- body mass index

Table 1. Age, BMI and CD4 Count Characteristics Distribution of Study Population

 

 

The maximum number of patients were from age group of 30-39 years (42%) followed by 18-30 years (24%), 40-50 years (20%) and >50 years (14%). There were 65% males (65 out of 100) and 35% (35 out of 100) females in our study and the male to female ratio was being 1.86. Maximum patients had BMI in between 18.5 to 25 kg/m2 followed by 20% with < 18.5 kg/m2 and 6% with >25 kg/m2. In the present study, 31% of individuals had CD4 count <100 cells/mm3 and 69% >100 cells/mm3 were observed. Signs and symptoms of meningitis were found in 17% and others were asymptomatic (Table 2).

Age in years

Number(n)

Percentage

18-29

24

24%

30-39

42

42%

40-49

20

20%

³ 50

14

14%

Male

65

65%

Female

35

35%

BMI (kg/m2)<18.5

20

20%

BMI (kg/m2)18.5-25

74

74%

BMI (kg/m2)>25

06

6%

CD4 count<100 (cells/mm3)

31

31%

CD4 count >100 (cells/mm3)

69

69%

Signs and symptoms Meningitis

17

17%

Asymptomatic

83

83%

BMI- body mass index, CD-cluster differentiation

Table 2. Demographic and Clinical Characteristics of the Study Population

 

The mean age among patients with serum cryptococcal antigen positive (SCRAG +) was 43.71 years as compared to 35.57% in cryptococcal antigen negative (SCRAG-) and this difference was statistically significant (p-0.046). The mean BMI of SCRAG+ patients and SCRAG- were 20.22 ± 2.64 kg/m2 and 20.56 ± 2.74 respectively, which is statistically insignificant. The mean CD4 count of SCRAG- patients (246 ± 175.0) cells/mm3 was higher than SCRAG+ patients (56.57 ± 56.0). The median CD4 count was being forty one (41 cells/mm3) in SCRAG+ and 203 cells/mm3 in SCRAG- patients. The difference was statistically significant (p=0.001)                 (Table 3).

 

 

Type

SCRAG Positive(n=7)

SCRAG Negative(n=93)

Characteristic

Age in years

BMI

(kg/m2)

CD4 count (cells/mm3)

Age in years

BMI (kg/m2)

CD4 count (cells/mm3)

Mean ± S.D

43.71 ± 13.94

20.22 ± 2.64

56.57 ± 56.0

35.57 ± 9.978

20.56 ± 2.74

246.34 ± 175.0

Median

40

19.1

41

35

20.5

203

Range

26 - 67

16.9 -23.9

6 - 168

18- 64

15.1-26.5

15 - 780

I Q Range

35.5 -52.25

18.7-22.8

19.0- 78.3

29.75-42.0

19.3-22.4

91- 370.0

P value

0.046

0.750

0.001

0.046

0.750

0.001

SCRAG - serum cryptococcal antigen, I Q Range -Interquartile range, CD4 count-cluster differentiation count (cells/mm3), P value

Table 3. Age, BMI and CD4 Count Characteristics Distribution of SCRAG Positive and SCRAG Negative Patients

 

Out of 65 males, 5 (7.7%) were positive for SCRAG and 2 (5.7%) were positive among 35 female patients. Majority of the study population were literate (77%) and doing unskilled work (63%). Maximum cases (88, 88%) of the study population were married. The sexual transmission route for HIV infection was found to be 99% where the duration of illness is longer. The majority of SCRAG+ (6 out of 7) patients had BMI between 18.5 to 25 kg/m2 as compared to the other groups, but the difference was not statistically significant. Out of 33 patients who had CD4 count <100 cells/mm3, 6 (19.35%) were positive for SCRAG and significant statistically (p< 0.003). Of sixty-nine patients who had CD4 count >100 cells/mm3, 1 (1.45%) was positive for SCRAG having statistically significance (p<0.012). Patients with cryptococcal antigenaemia were more prone to have CD4 count <100 cells/mm3 (Table 4).

 

 

Parameters

SCRAG+ (n=7)

SCRAG-  (93)

Total

P Value

Male

5 (7.7)

60 (92.3%)

65 (100%)

1.00

Female

2 (5.7%)

33 (94.3%)

35 (100%)

1.00

Illiterate

2 (8.7%)

21 (91.3%)

23 (100%)

0.660

Education <10th standard

2 (4.5%)

42 (95.5%)

44 (100%)

0.660

Education >10th standard

3 (9.1%)

30 (90.9%)

33 (100%)

0.660

Skilled worker

3 (8.1%)

34 (91.9%)

37 (100%)

0.708

Unskilled worker

4 (6.3%)

59 (93.7%)

63 (100%)

0.708

Married

6 (6.8%)

82 (93.2%)

88 (100%)

1.00

Unmarried

1 (8.3%)

11 (91.7%)

12 (100%)

1.00

Recent onset of the disease

7 (9.9%)

64 (90.1%)

71 (100%)

0.104

Past history of the disease

0 (0%)

29 (100%)

29 (100%)

0.104

BMI<18.5 kg/m2

1 (4.5%)

19 (95.5%)

20 (100%)

0.700

BMI 18.5-25.0 kg/m2

6 (8.2%)

68 (91.8%)

74 (100%)

0.700

BMI >25.1 kg/mm2

0 (0%)

6 (100%)

6 (100%)

0.700

CD4<100 cells/mm3

6 (19.35%)

27 (80.65%)

33 (100%)

0.001

CD4>100 cells/mm3

1 (1.45%)

68 (98.55%)

69 (100%)

0.001

Symptomatic Cr Ag

4 (23.5%)

13 (76.5%)

17 (76.5%)

0.015

Asymptomatic Cr Ag

3 (3.6%)

80 (96.4%)

83 (96.4%)

0.015

CD –cluster differentiation, SCRAG- serum cryptococcal antigen

Table 4. Comparison of Different Parameters in SCRAG+ and SCRAG- Study Group

 

 

Univariate analysis showed fever (p<0.005, OR 23.368, CI 2.652-205.398), headache (p<0.010, OR 8.205, CI 1.644-40.950), vomiting (p<0.004, OR 13.200, CI 2.300-75.750), neck rigidity (p<0.014, OR 7.969, CI 1.510-42.044) and CD4 count <100 cells/mm3 (p<0.012, OR 16.320, CI 1.871-142.374) were significantly associated with Cryptococcal antigenaemia. However, age, sex, socioeconomic status, marital status, altered mental status, duration of HIV infection, BMI and CD4 count <200 cells/mm3 were not significantly associated with cryptococcal antigenaemia (Table 5).

 

Risk Factor

P value

Odds Ratio

95% CI for Odds Ratio

 

 

 

Lower

Upper

Age in years

0.055

1.072

0.999

1.151

Male sex

0.713

0.727

0.134

3.957

Education < 8th standard

0.489

0.488

0.064

3.712

Education > 8th standard

0.913

1.086

0.167

7.085

Married

0.847

0.805

0.088

7.237

BMI kg/m2

0.747

0.954

0.718

1.268

CD4 count <50cells/mm3

0.001

20.750

3.548

121.385

CD4 count <100 cells/mm3

0.012

16.320

1.871

142.374

CD4 count <200 cells/mm3

0.053

17.414

0.967

313.749

Fever

0.005

23.368

2.652

205.368

Headache

0.010

8.205

1.644

40.950

vomiting

0.004

13.200

2.300

75.750

Neck rigidity

0.014

7.969

1.510

42.044

Altered mental status

0.617

1.771

0.189

16.595

C.I-95% Confidence Intervals of Odds Ratios

Table 5. Univariate Analysis of Risk Factors for Cryptococcal Antigenaemia among HIV-infected Patients

 

In multivariate analysis, CD4 count <50 cells/mm3 was acting as independent risk factor for cryptococcal antigenaemia (p <0.019, OR 17.769, CI 1.594-198.042) (Table 6). However, the other factors did not contribute to independent risk factors in the present study.

 

Risk factor

P value

Odds Ratio

95% C.I for Odds Ratio

Fever

0.074

12.736

0.780

208.014

Headache

0.991

1.022

0.022

47.211

vomiting

0.293

4.474

0.274

73.150

Neck rigidity

0.568

17.761

1.594

198.042

CD4 count <50 cells/mm3

0.019

17.769

1.594

198.042

95% Confidence Intervals of Odds Ratios

Table 6. Multivariate Analysis of Risk Factors for Cryptococcal Antigenaemia Among HIV-infected Patients

 

DISCUSSION

In the present study, overall prevalence of Cryptococcal antigenaemia is found to be 7%, which is comparable to the studies in Uganda (5-10%),11 South Africa (7%)12 and Kenya(7%),13 which confirms that India has high rates of cryptococcal disease in HIV-infected patients in comparison to tuberculosis. The prevalence of patients with CD4 count less than 100 cells/mm3 is 19.35% in the present study, which coincides with the study of Otella et al13 in Uganda. Out of 17 meningitis cases, 4 (23.52%) cases are having cryptococcal meningitis as compared with Gomerep et al.14 We observed the overall mean age being 36 years (range 18-67 years) and 43 years in SCRAG positive group as compared to 35 years in SCRAG negative group (p<0.046). On univariate analysis, we did not find advanced age as a risk factor.15 There are 65 males and 35 females and the M: F ratio being 1.86:1 as compared with earlier studies.15 Most of the study population has average income and literate doing unskilled work but they do not have any significant difference with positivity of SCRAG (p>0.05).

In the present study, all patients with SCRAG+ are recently diagnosed for HIV and 14 (19.71%) out of 71 have advanced disease and 25 (35.21%) patients have CD4 count less than 100 cells/mm,3 which may be due to lack of IEC (information, education and communication) activities to reach all section of population of our country.

There are 17 symptomatic patients out of which 4 (23.5%) are SCRAG+ and out of 83 asymptomatic patients 3 (3.6) are SCRAG+, which is statistically significant (p<0.015).10 The symptoms of fever, headache, vomiting and neck rigidity are significantly associated with Cryptococcal antigenaemia (p<0.05).16

The mean BMI of the study population is 20.54 kg/m2. The SCRAG + has showed BMI 20.22 kg/m2 whereas SCRAG- cases 20.56 kg/m2, which is definitely higher than the previous researchers (<15.4 kg/m2)Oyella et. al and Micol                et al.13,17 They have included patients with more advanced disease and lesser CD4 counts.

The median CD4 count of SCRAG+ individuals is 41 cells/mm3 (mean 56, range 6-168, IQR 19.000-78.250) as compared to 203 cells/mm3 (mean 246, range 15-780, IQR 91-370) in SCRAG– individuals (p<0.001) in Andama et al study.18 The CD4 count <100 cells/mm3 is found in 31 patients out of which 6 (19.35%) are SCRAG+ in comparison to CD4 count >100 cells/mm3 in which 1.45% are SCRAG+. On univariate analysis, CD4 count <100 cells/mm3 is significantly associated with positive SCRAG (p< 0.012, OR- 16.320, 95% CI 1.871-142.374) as studied by Tenna et al,11 but 67 cases in multivariate analysis do act as independent risk factor for Cryptococcal antigenaemia in Oyella et al study.13 We observed high prevalence of subclinical infection 3 (3.6%) in the present study irrespective of CD4 count. Antigenaemia is not only predictive of the development of cryptococcal meningitis but also an independent predictor of mortality.12

 

CONCLUSION

There is a high prevalence of symptomatic Cryptococcal antigenaemia (7%) and asymptomatic Cryptococcal antigenaemia (3.6%) in ART naïve HIV patients irrespective of CD4 count. There is a high prevalence of Cryptococcal antigenaemia (19.35%) in ART naïve patients having CD4 count <100 cells/mm3. Symptoms like fever, headache, vomiting and neck rigidity have been observed in patients having CD4 count <100 cells/mm3, significant association with Cryptococcal antigenaemia on univariate analysis seen. Cryptococcal antigenaemia is not only predictive of the development of cryptococcal meningitis in HIV patients but also an independent predictor of mortality. All ART naïve adults having CD4 count <100 cells/mm3 should be screened for serum Cryptococcal antigen followed by presumptive anti-fungal therapy if serum Cryptococcal antigen is positive. There is a need to strengthen IEC (information, education and communication) activities and increase routine counselling and testing of the patients attending ART clinics in Odisha.

Abbreviations

SCRAG- serum cryptococcal antigenaemia, CD4- cluster differentiation, HIV- Human Immunodeficiency Virus, WHO- World Health Organization, CM– Cryptococcal meningitis , OI- opportunistic infections , AIDS- Acquired Immune Deficiency Syndrome, IQR- Interquartile range, CD4- cluster differentiation (cells/mm3) and BMI- body mass index and C.I-95% Confidence Intervals of Odds Ratios.

 

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