CMV testing was not done in cases where cytopenias occurred during ALL induction and those which could be directly attributed to chemotherapy or other drug toxicity (mercaptopurine, linezolid, etc.). Patients having cytopenia >10 days, but showing an improving trend in the cytopenia were also not tested for CMV.

All patients with ALL were treated with 4 drug induction (vincristine, L-asparginase, prednisolone, daunomycin, intrathecal methotrexate) followed by consolidation using moderate dose methotrexate and/or high dose cytarabine. Cranial radiation was given to children aged > 3 years who were not receiving high dose cytarabine. Maintenance chemotherapy consisted of oral 6-mercaptopurine and methotrexate with 3 monthly intensification with vincristine, daunomycin, L-asparginase and prednisolone. Patients having NHL were treated on MCP 842 protocol [10] with addition of moderate dose methotrexate in each cycle.

CMV testing: Testing for CMV were done by pp65 antigen analysis, qualitative or quantitative RT-PCR. Results were considered positive or negative based on laboratory determined cut-off values. If CMV positivity was documented, gancyclovir was given intravenously 5 mg/kg/dose twice a day for at least 10 days, followed by oral gancyclovir / valgancyclovir for total 21 days.

Treatment of neutropenic fever: Patients having neutropenic fever had a single baseline blood culture done. Blood cultures were repeated after every 48-72 hours if defervescence was not obtained. Patients having diarrhea were screened for cryptosporidium and isospora in their stools. Ceftriaxone and Amikacin were used as the initial empiric antibiotic combination. Switch to Piperacillin-Tazobactam, Carbapenems or Vancomycin were made if fever persisted or guided by culture reports. Amphoterocin-B was used as empiric antifungal therapy if fever persisted for more than 5-7 days.

Statistical analysis: CMV positive group was compared with CMV negative group using chi-square or Fisher’s exact rest for categorical variables. Duration of neutropenia was compared using Mann-Whitney U-test. P <0.05 was considered as statistically significant.

We identified 24 episodes of prolonged cytopenias, based on our definition (ALL-23, NHL-1). Of these 24 episodes, CMV infection was diagnosed by pp65 antigen analysis in 6 episodes, qualitative PCR in 10 and quantitative PCR in 8 episodes. CMV testing had been done after a median duration of 13 days of cytopenia (range 4-30 days). In 2 episodes, tests were done earlier than 10 days–one child had chronic diarrhea for 2 weeks prior to cytopenia, while the other child presented with respiratory distress along with cytopenia. CMV infection was detected in 13 (54%) out of the 24 episodes of prolonged cytopenia. Eight out of these 13 episodes occurred during or immediately after prolonged courses of steroids or moderate-dose methotrexate, while the rest occurred during maintenance treatment. Neutropenia and thrombocytopenia were present in 11 and 13 cases, respectively.

TABLE I  Clinical Characteristics of Cytopenia Episodes due to CMV Infections

Table I shows the clinical characteristics of the CMV-positive cytopenia episodes. Fever was present in 11 episodes (duration 2-20 days), which started at onset of cytopenia in 5 episodes. Web Table I shows details of blood culture results, other infections and antimicrobials used in the CMV positive episodes. Watery diarrhea with mucus was present in 9 out of 13 CMV positive episodes (duration 3-60 days). Lower respiratory tract infection (fever, tachypnea, bilateral diffuse infiltrates on X-ray chest) was seen in only 1 patient. In children chorioretinitis was seen about 3 months after the cytopenia episode. In both these episodes, decreased vision was the presenting complaint. Six out of the 13 episodes were associated with other co-infections (bacterial-3, Candidemia-1, Cryptosporidium parvum diarrhea-1, Varicella-1).

Table II shows comparison between CMV positive and negative episodes. Duration of cytopenia was not significantly different between the two groups. In the CMV positive group, in addition to the mortality during cytopenia episode described above, 2 more patients (patient 5 and 8 in Table I) died subsequently, outside the study period, as a result of CMV infection. First patient developed CMV colitis, which did not respond to gancyclovir therapy. Cidofovir could not be used due to financial constraints. The second patient developed recurrence of chorioretinitis and recurrent cytopenias towards the end of the maintenance chemotherapy. CMV PCR showed 3,72,000 copies/mL. Bone marrow was in remission. Parents refused further treatment and she died 3 weeks later. There has been no non-relapse mortality in the CMV negative group to date.

TABLE II Comparison Between CMV Positive and Negative Episodes of Cytopenia

Our study shows that a large proportion of patients having prolonged cytopenia after chemotherapy for ALL or NHL, which was not explainable on the basis of chemotherapy toxicity, had evidence of CMV infection. CMV related morbidity and mortality has been well described in the HSCT setting. Delayed engraftment, individual cytopenias, retinitis, pneumonitis and colitis have all been described [1-5,11].

Detection of CMV antigen or DNA in blood is an indicator of CMV infection. To diagnose CMV disease, symptoms consistent with CMV disease together with detection of CMV in an appropriate specimen from the involved tissue is necessary (e.g. Broncho-alveolar lavage for CMV pneumonitis, CMV inclusions on colonic biopsy, etc.) [1,2,12-14]. In the allogeneic transplant setting, weekly screening for CMV is recommended for the first 100 days (longer if GVHD present or prior CMV reactivation documented) [1,3]. There is a strong co-relation between detection of CMV viremia and subsequent development of CMV disease in allogeneic HSCT patients [15,16]. Hence, pre-emptive treatment with gancyclovir is recommended if antigenemia or PCR is positive, even in the absence of overt symptoms related to CMV disease [1,3,17]. There are no universally accepted cutoffs for PCR positivity, which vary between centers and may range from >100 copies/mL to > 1000 copies/mL based on the clinical situation and treatment center [17]. In contrast to the data and guidelines available regarding CMV prophylaxis, screening and treatment for allogeneic HSCT patients, there are very few reports regarding the incidence, risk factors and outcome of CMV infection in patients having ALL or NHL receiving conventional chemotherapy [6-9]. Our study shows that CMV infection was present in a large proportion of patients having prolonged ‘unexplained’ cytopenia after chemotherapy for ALL or NHL. Other manifestations of CMV disease such as colitis, chorioretinitis and pneumonitis were also simultaneously or sequentially present in many patients.

The prompt recovery of cytopenias in a majority of our patients after starting gancyclovir also supports the causative role played by CMV infection. Although our patients benefited due to gancyclovir therapy, our study is not designed to address the issue of treatment for CMV in this patient population.

Unlike in the developed nations, where CMV seropositivity is seen in 30-40% of the population, the incidence of CMV seropositivity in India is very high, reaching upto 95% by about 5 years of age [18]. CMV infection, once acquired, remains latent in the leukocytes. Reactivation would occur in cases of severe and prolonged suppression of T-cell immunity. Myelosuppression due to CMV infection occurs either by direct infection of progenitor cells or by infection of stromal cells leading to decreased growth factor production [19,20]. Most of the prolonged cytopenia episodes in our series have occurred following prolonged courses of steroids or moderate-dose methotrexate. Both these agents have significant immunosuppressive activity. With use of more aggressive "western" protocols for treatment of ALL in Indian children, increase in the incidence of CMV re-activation and disease is likely to occur.

Fever was present in a majority, either due to CMV infection or to secondary bacterial or fungal infection. CMV infection in the HSCT setting has been associated with increased mortality due to bacterial or fungal pathogens [21-22]. In our study, 46% of CMV positive patients had evidence of other co-existing pathogens. The increased non-relapse mortality in the CMV positive group as compared to the CMV negative group indicates the seriousness of the problem even in patients receiving conventional chemotherapy. Increased risk of other pathogens along with CMV reactivation may be due to an overall immunosuppression contributing to both CMV reactivation as well as other opportunistic bacterial or fungal pathogens, or due to an immunomodulatory effect of CMV infection itself [21-22].

Two out of the three patients who had chorioretinitis developed it few months after other manifestations had been successfully treated. No other evidence of CMV infection was present in these patients at the time of detection of chorioretinitis. Similar late occurrence of CMV chorioretinitis has been described in the HSCT setting [1, 11]. Our study is likely to underestimate the incidence of CMV chorioretinitis. Milder cases with spontaneous resolution may have been undetected. Also, fundoscopy data was not available in a majority of our cases. Patients having non-ocular CMV disease should have periodic screening for next few months for detection of chorioretinitis.

In conclusion, our study shows that CMV infection can cause prolonged cytopenias even after conventional chemotherapy for ALL or NHL. Serious morbidity or treatment delays can occur as a result. This finding is important in the Indian context, where CMV sero-positivity is almost universal among children. With increasing use of more aggressive chemotherapeutic regimens for treatment of childhood ALL or NHL in India, we are likely to see a higher incidence of CMV reactivation. In the absence of clear-cut screening guidelines in the non-HSCT setting, early suspicion of CMV disease is necessary. Our study suggests that cytopenia that is prolonged or not explainable on the basis of chemotherapy toxicity should be evaluated for CMV infection, so that antiviral treatment can be instituted early and chemotherapy delays avoided.

Acknowledgments: Dr Atul Mulay and KRNST for their valuable inputs regarding data analysis and manuscript preparation.

Contributors: SK: study design, data collection, analysis and interpretation, drafting the manuscript; PB: data analysis and interpretation, drafting the manuscript; SP: data analysis and interpretation, drafting the manuscript. All authors approved the final manuscript. SK will be the guarantor for this study.

Funding: None; Competing interests: None stated.

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